Embryonic Stem Cell Research Literatures

Mark Herbert, PhD

World Development Institute 39 Main Street, Flushing, Queens, New York 11354, USA, [email protected]

Abstract: Stem cells are derived from embryonic and non-embryonic tissues. Most stem cell studies are for animal stem cells and plants have also stem cell. Stem cells were discovered in 1981 from early mouse embryos. Stem cells have the potential to develop into all different cell types in the living body. Stem cell is a body repair system. When a stem cell divides it can be still a stem cell or become adult cell, such as a brain cell. Stem cells are unspecialized cells and can renew themselves by cell division, and stem cells can also differentiate to adult cells with special functions. Stem cells replace the old cells and repair the damaged tissues. Embryonic stem cells can become all cell types of the body because they are pluripotent. Adult stem cells are thought to be limited to differentiating into different cell types of their tissue of origin. This article introduces recent research reports as references in the related studies. [Mark H. Embryonic Stem Cell Research Literatures. Stem Cell 2019;10(4):38-217]. ISSN: 1945-4570 (print); ISSN: 1945-4732 (online). http://www.sciencepub.net/stem. 6. doi:10.7537/marsscj100419.06.

Key words: stem cell; life; research; literature

Introduction systems: suspension vs. adherent conditions. In the The stem cell is the origin of an organism’s life present study, an ES cell line derived from an Atoh1- that has the potential to develop into many different green fluorescent (GFP) transgenic mouse was types of cells in life bodies. In many tissues stem cells used to track the generation of otic progenitors, initial serve as a sort of internal repair system, dividing HCs and to compare these two differentiation systems. essentially without limit to replenish other cells as We used a two-step short-term differentiation method long as the person or animal is still alive. When a stem involving an induction period of 5 days during which cell divides, each new cell has the potential either to ES cells were cultured in the presence of remain a stem cell or become another type of cell with Wnt/transforming growth factor TGF-beta inhibitors a more specialized function, such as a red blood cell or and insulin-like growth factor IGF-1 to suppress a brain cell. This article introduces recent research mesoderm and reinforce presumptive ectoderm and reports as references in the related studies. otic lineages. The generated embryoid bodies were The following introduces recent reports as then differentiated in medium containing basic references in the related studies. fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture Abboud, N., et al. (2017). "Culture conditions methods. Upon completion of differentiation, have an impact on the maturation of traceable, quantitative polymerase chain reaction analysis and transplantable mouse embryonic stem cell-derived otic immunostaining monitored the expression of otic/HC progenitor cells." J Tissue Eng Regen Med 11(9): progenitor lineage markers. The results indicate that 2629-2642. cells differentiated in suspension cultures produced The generation of replacement inner ear hair cells cells expressing otic progenitor/HC markers at a (HCs) remains a challenge and stem cell therapy holds higher efficiency compared with the production of the potential for developing therapeutic solutions to these cell types within adherent cultures. Furthermore, hearing and balance disorders. Recent developments we demonstrated that a fraction of these cells can have made significant strides in producing mouse otic incorporate into ototoxin-injured mouse postnatal progenitors using cell culture techniques to initiate HC cochlea explants and express MYO7A after differentiation. However, no consensus has been transplantation. Copyright (c) 2016 John Wiley & reached as to efficiency and therefore current methods Sons, Ltd. remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors Abd Jalil, A., et al. (2017). "Vitamin E-Mediated from embryonic stem (ES) cells in two cell culture Modulation of Glutamate Expression in an

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Oxidative Stress Model of Neural Cells Derived from Embryonic Stem Cell Cultures." Evid Based Abdelbaset-Ismail, A., et al. (2016). "Vitamin D3 Complement Alternat Med 2017: 6048936. stimulates embryonic stem cells but inhibits migration Glutamate is the primary excitatory and growth of ovarian cancer and teratocarcinoma cell neurotransmitter in the central nervous system. lines." J Ovarian Res 9: 26. Excessive concentrations of glutamate in the brain can BACKGROUND: Deficiency in Vitamin D3 be excitotoxic and cause oxidative stress, which is (cholecalciferol) may predispose to some malignancies, associated with Alzheimer's disease. In the present including gonadal tumors and in experimental models study, the effects of vitamin E in the form of vitamin D3 has been proven to inhibit the growth of tocotrienol-rich fraction (TRF) and alpha-tocopherol cancer cells. To learn more about the potential role of (alpha-TCP) in modulating the glutamate receptor and vitamin D3 in cancerogenesis, we evaluated the neuron injury markers in an in vitro model of oxidative expression and functionality of the stress in neural-derived embryonic stem (ES) cell (VDR) and its role in metastasis of ovarian cancer cultures were elucidated. A transgenic mouse ES cell cells and of murine and human teratocarcinoma cell line (46C) was differentiated into a neural lineage in lines. METHODS: In our studies we employed murine vitro via induction with retinoic acid. These cells were embrynic stem cells (ESD3), murine (P19) and human then subjected to oxidative stress with a significantly (NTERA-2) teratocarcimona cells lines, human high concentration of glutamate. Measurement of ovarian cancer cells (A2780) as well as purified reactive oxygen species (ROS) was performed after murine and human purified very small embryonic like inducing glutamate excitotoxicity, and recovery from stem cells (VSELs). We evaluated expression of this toxicity in response to vitamin E was determined. Vitamin D3 receptor (VDR) in these cells as well as The expression levels of glutamate receptors and effect of vitamin D3 exposure on cell proliferation and neuron-specific enolase were elucidated using real- migration. RESULTS: We here provide also more time PCR. The results reveal that neural cells derived evidence for the role of vitamin D3 in germline- from 46C cells and subjected to oxidative stress derived malignancies, and this evidence supports the exhibit downregulation of NMDA, kainate receptor, proposal that vitamin D3 treatment inhibits growth and and NSE after posttreatment with different metastatic potential of several germline-derived concentrations of TRF and alpha-TCP, a sign of malignancies. We also found that the ESD3 murine neurorecovery. Treatment of either TRF or alpha-TCP immortalized embryonic stem cell line and normal, reduced the levels of ROS in neural cells subjected to pluripotent, germline-marker-positive very small glutamate-induced oxidative stress; these results embryonic-like stem cells (VSELs) isolated from adult indicated that vitamin E is a potent antioxidant. tissues are stimulated by vitamin D3, which suggests that vitamin D3 affects the earliest stages of Abdelalim, E. M. (2013). "Molecular embryogenesis. CONCLUSIONS: We found that mechanisms controlling the cell cycle in embryonic however all normal and malignant germ-line derived stem cells." Stem Cell Rev 9(6): 764-773. cells express functional VDR, Vitamin D3 differently Embryonic stem (ES) cells are originated from affects their proliferation and migration. We postulate the inner cell mass of a blastocyst stage embryo. They that while Vitamin D3 as anticancer drug inhibits can proliferate indefinitely, maintain an proliferation of malignant cells, it may protect normal undifferentiated state (self-renewal), and differentiate stem cells that play an important role in development into any cell type (pluripotency). ES cells have an and tissue/organ regeneration. unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S Abdelhady, S., et al. (2013). "Erg channel is checkpoint. Cell division and cell cycle progression critical in controlling cell volume during cell cycle in are controlled by mechanisms ensuring the accurate embryonic stem cells." PLoS One 8(8): e72409. transmission of genetic information from generation to The cell cycle progression in mouse embryonic generation. Therefore, control of cell cycle is a stem cells (mESCs) is controlled by ion fluxes that complicated process, involving several signaling alter cell volume [1]. This suggests that ion fluxes pathways. Although great progress has been made on might control dynamic changes in morphology over the molecular mechanisms involved in the regulation the cell cycle, such as rounding up of the cell at of ES cell cycle, many regulatory mechanisms remain mitosis. However, specific channels regulating such unknown. This review summarizes the current dynamic changes and the possible interactions with knowledge about the molecular mechanisms regulating actomyosin complex have not been clearly identified. the cell cycle of ES cells and describes the relationship Following RNAseq transcriptome analysis of cell existing between cell cycle progression and the self- cycle sorted mESCs, we found that expression of the renewal. K (+) ion channel Erg1 peaked in G1 cell cycle phase,

39 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ which was confirmed by immunostaining. Inhibition nephrons after birth is limited. Due to the medical and of Erg channel activity caused loss of G1 phase cells financial impact of chronic and end stage renal disease, via non-apoptotic cell death. Cells first lost the ability an improved understanding of nephron formation is of membrane blebbing, a typical feature of cultured necessary if regenerative or cell therapy are to be a embryonic stem cells. Continued Erg inhibition further feasible alternative to dialysis or renal transplant. In increased cell volume and the cell eventually ruptured. the study presented by Lusis et al., the presence of In addition, atomic force measurements on live cells metanephric mesenchymal stem cells is definitively revealed a decreased cortical stiffness after treatment, demonstrated. However, these "Nephrospheres" have suggesting alterations in actomyosin organization. characteristics of mesenchymal stem cells and When the intracellular osmotic pressure was substantially lack the ability to undergo an epithelial- experimentally decreased by hypertonic solution or to-mesenchyme transition or to form epithelial block of K (+) ion import via the Na, K-ATPase, cell elements otherwise necessary for building the viability was restored and cells acquired normal constituent cells of the nephron. Nevertheless, this volume and blebbing activity. Our results suggest that newly isolated cell population opens many Erg channels have a critical function in K (+) ion opportunities to investigate the consequences of homeostasis of mESCs over the cell cycle, and that normal and aberrant nephrogenesis, including Wilm's cell death following Erg inhibition is a consequence of tumor. the inability to regulate cell volume. Abu Khamidakh, A. E., et al. (2018). "Wound Aberdam, D. (2008). "Epidermal stem cell fate: healing of human embryonic stem cell-derived retinal what can we learn from embryonic stem cells?" Cell pigment epithelial cells is affected by maturation Tissue Res 331(1): 103-107. stage." Biomed Eng Online 17(1): 102. Because of its constant renewal and high BACKGROUND: Wound healing of retinal propensity for repair, the epidermis is, together with pigment epithelium (RPE) is a complex process that the gut and the hematopoietic system, a tissue of may take place in common age-related macular choice to explore stem cell biology. Previous research degeneration eye disease. The purpose of this study over many years has revealed the complexity of the was to evaluate whether wounding and wound healing epidermis: the heterogeneity of the stem cell has an effect on Ca (2+) dynamics in human compartment, with its rare, slowly cycling, multipotent, embryonic stem cell (hESC)-RPEs cultured different hair-follicle, "bulge" stem cells and the more restricted periods of time. METHODS: The 9-day-cultured or interfollicular, follicle-matrix, and sebaceous-gland 28-day-cultured hESC-RPEs from two different cell stem cells, which in turn generate the large pool of lines were wounded and the dynamics of spontaneous transit-amplifying progeny. Stem cell activity has been and mechanically induced intracellular Ca (2+) used for some considerable time to repair skin injuries, activity was measured with live-cell Ca (2+) imaging but ex-vivo keratinocyte amplification has its either immediately or 7 days after wounding. The limitations, and grafted skin homeostasis is not totally healing time and speed were analyzed with time-lapse satisfactory. Human embryonic stem cells raise the bright field microscopy. The Ca (2+) activity and hope that the understanding of the developmental steps healing speed were analysed with image analysis. In leading to the generation of epidermal stem cells and addition the extracellular matrix deposition was the characterization of the key signaling pathways assessed with confocal microscopy. RESULTS: The involved in skin morphogenesis (such as p63) will be Ca (2+) dynamics in hESC-RPE monolayers differed translated into therapeutic benefit. Our recent results depending on the culture time: 9-day-cultured cells suggest the feasibility not only of identifying but also had higher number of cells with spontaneous Ca (2+) of amplifying human ES cells, early ectodermal activity close to freshly wounded edge compared to progenitors with an intact multipotent potential that control areas, whereas in 28-day-cultured cells there might improve the quality and functionality of grafts, was no difference in wounded and control areas. The provided that preclinical in vivo studies confirm our 28-day-cultured, wounded and 7-day-healed hESC- expectations from in vitro analysis. RPEs produced wide-spreading intercellular Ca (2+) waves upon mechanical stimulation, while in controls Abraham, J. and C. Keller (2010). "Renal stem propagation was restricted. Most importantly, both cell biology starts to take spherical shape. wave spreading and spontaneous Ca (2+) activity of Commentary on: Lusis et al., Isolation of clonogenic, cells within the healed area, as well as the cell long-term self renewing embryonic renal stem cells." morphology of 28-day-cultured, wounded and Stem Cell Res 5(1): 1-3. thereafter 7-day-healed areas resembled the 9-day- The nephron is the fundamental unit of renal cultured hESC-RPEs. CONCLUSIONS: This acquired function, yet the ability of the kidney to regenerate knowledge about Ca (2+) dynamics of wounded

40 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ hESC-RPE monolayers is important for understanding Aflatoonian, B., et al. (2009). "In vitro post- the dynamics of RPE wound healing, and could offer a meiotic germ cell development from human reliable functionality test for RPE cells. The data embryonic stem cells." Hum Reprod 24(12): 3150- presented in here suggests that assessment of Ca (2+) 3159. dynamics analysed with image analysis could be used BACKGROUND: Investigating the mechanisms as a reliable non-invasive functionality test for RPE of human primordial germ cell (PGC) and gamete cells. development are important for understanding the causes of infertility and effects of environmental Adler, E. D., et al. (2009). "In vivo detection of chemicals on reproductive development. However, embryonic stem cell-derived cardiovascular progenitor there are practical and ethical difficulties associated cells using Cy3-labeled Gadofluorine M in murine with obtaining human tissue in early development. The myocardium." JACC Cardiovasc Imaging 2(9): 1114- aim of this study was to investigate whether human 1122. embryonic stem cell-hESC-generated germ cells could OBJECTIVES: The aim of the current study is to provide an in vitro model of gamete development. test the ability to label and detect murine embryonic METHOD: Human ESCs were differentiated as stem cell-derived cardiovascular progenitor cells (ES- embryoid bodies (EBs) in vitro. Gene and protein CPC) with cardiac magnetic resonance (CMR) using marker expression profiles of EBs in different periods the novel contrast agent Gadofluorine M-Cy3 (GdFM- of culture were analysed by quantitative polymerase Cy3). BACKGROUND: Cell therapy shows great chain reaction (Q-PCR) and immunolocalization to promise for the treatment of cardiovascular disease. monitor germ cell development. Secretion of An important limitation to previous clinical studies is hormones involved in germ cell maturation was the inability to accurately identify transplanted cells. measured, to detect the existence of a germ cell niche GdFM-Cy3 is a lipophilic paramagnetic contrast agent within EBs. RESULTS: Q-PCR revealed gene that contains a perfluorinated side chain and an expression profiles consistent with PGC formation and amphiphilic character that allows for micelle germ cell development. A small population of post- formation in an aqueous solution. Previous studies meiotic spermatid cells were identified using sperm- reported that it is easily taken up and stored within the specific antibodies (Protamine 1 and 1.97). Although cytosol of mesenchymal stem cells, thereby allowing profiles characteristic of oocyte for paramagnetic cell labeling. Investigators in our development and follicle-like structures were detected, laboratory have recently developed techniques for the a committed oocyte with extra-cellular zona pellucida robust generation of ES-CPC. We reasoned that was not recognized with zona pellucida-specific GdFM-Cy3 would be a promising agent for the in vivo monoclonal antibody. CONCLUSIONS: hESCs can detection of these cells after cardiac cell form PGCs and post-meiotic spermatids in vitro, transplantation. METHODS: ES-CPC were labeled however, there remains doubt about oocyte with GdFM-Cy3 by incubation. In vitro studies were development. Levels of steroid hormones produced by performed to assess the impact of GdFM-Cy3 on cell EBs were significant when compared with known function and survival. A total of 500,000 GdFM-Cy3- values for a similar quantity of human testis, labeled ES-CPC or control ES-CPC were injected into suggesting that hESC may intrinsically create a the myocardium of mice with and without myocardial favourable hormonal niche for spermatogenesis. infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Ahmad, S., et al. (2007). "Differentiation of Mice were then euthanized, and their hearts were human embryonic stem cells into corneal epithelial- sectioned for fluorescence microscopy. RESULTS: In like cells by in vitro replication of the corneal vitro studies demonstrated that GdFM-Cy3 was easily epithelial stem cell niche." Stem Cells 25(5): 1145- transfectable, nontoxic, stayed within cells after 1155. labeling, and could be visualized using CMR and Human embryonic stem cells (hESCs) are fluorescence microscopy. In vivo studies confirmed pluripotent cells capable of differentiating into any cell the efficacy of the agent for the detection of cells type of the body. It has long been known that the adult transplanted into the hearts of mice after myocardial stem cell niche is vital for the maintenance of adult infarction. A correspondence between CMR and stem cells. The cornea at the front of the eye is histology was observed. CONCLUSIONS: The results covered by a stratified epithelium that is renewed by of the current study suggest that it is possible to stem cells located at its periphery in a region known as identify and potentially track GdFM-Cy3-labeled ES- the limbus. These so-called limbal stem cells are CPC in murine infarct models via CMR. maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma.

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Because this niche is very specific to the stem cells subtractive hybridization." Stem Cells Dev 19(8): (rather than to the more differentiated cells) of the 1249-1256. corneal epithelium, it was hypothesized that Human embryonic stem cells (hESCs) are replication of these factors in vitro would result in pluripotent, self-renewing cells derived from the inner hESC differentiation into corneal epithelial-like cells. cell mass of human blastocysts. During normal Indeed, here we show that culturing of hESC on development, hESCs differentiate into 3 germ layers. collagen IV using medium conditioned by the limbal Cellular lineages differentiated from hESCs express a fibroblasts results in the loss of pluripotency and set of that are exclusive to these specialized cells. differentiation into epithelial-like cells. Further Therefore, we hypothesized that endothelial cells differentiation results in the formation of terminally derived from hESCs would express genes specific to differentiated epithelial-like cells not only of the endothelial cells. We previously isolated endothelial cornea but also of skin. Scanning electron microscopy cells from human embryonic stem cells (hESC-ECs) shows that some differences exist between hESC- using fluorescence-activated cell sorter (FACS). The derived and adult limbal epithelial-like cells, aim of the current study was to identify genes necessitating further investigation using in vivo animal associated with hESC-derived endothelial-like cells. models of limbal stem cell deficiency. Such a model of Using suppression subtractive hybridization (SSH), we hESC differentiation is useful for understanding the identified a set of genes specific to cells differentiated early events of epithelial lineage specification and to from hESC-ECs. We obtained 113 clones of expressed the eventual potential application of epithelium sequences that were more abundant in hESC-ECs differentiated from hESC for clinical conditions of compared with hESCs. Based on the NCBI GenBank epithelial stem cell loss. Disclosure of potential database, 56 of these clones were known genes, 13 conflicts of interest is found at the end of this article. clones corresponded to nucleotides, 2 clones showed homology with sequences, and 42 clones Ahn, J. I., et al. (2004). "Temporal expression showed no significant homology with any nucleotide changes during differentiation of neural stem cells sequences. These identified genes are related to derived from mouse embryonic stem cell." J Cell cytoskeleton and cell adhesion, development, heat Biochem 93(3): 563-578. shock protein, metabolism, signal transduction, and Temporal analysis in gene expression during transcription/nuclear-specific . We anticipate differentiation of neural stem cells (NSCs) was that further study of these genes will provide crucial performed by using in-house microarrays composed of insights into their specific roles in the development of 10,368 genes. The changes in mRNA level were endothelial cells from hESCs. measured during differentiation day 1, 2, 3, 6, 12, and 15. Out of 10,368 genes analyzed, 259 genes were up- Aiba, K., et al. (2009). "Defining developmental regulated or down-regulated by 2-fold or more at least potency and cell lineage trajectories by expression at one time-point during differentiation, and were profiling of differentiating mouse embryonic stem classified into six clusters based on their expression cells." DNA Res 16(1): 73-80. patterns by K-means clustering. Clusters characterized Biologists rely on morphology, function and by gradual increase have large numbers of genes specific markers to define the differentiation status of involved in transport and cell adhesion; those which cells. Transcript profiling has expanded the repertoire showed gradual decrease have much of genes in of these markers by providing the snapshot of cellular nucleic acid metabolism, cell cycle, transcription status that reflects the activity of all genes. However, factor, and RNA processing. In situ hybridization (ISH) such data have been used only to assess relative validated microarray data and it also showed that Fox similarities and differences of these cells. Here we M1, cyclin D2, and CDK4 were highly expressed in show that principal component analysis of global gene CNS germinal zones and ectonucleotide expression profiles map cells in multidimensional pyrophosphatase/phosphodiesterase 2 (Enpp2) was transcript profile space and the positions of highly expressed in choroid plexus where differentiating cells progress in a stepwise manner stem/progenitor cells are possibly located. Together, along trajectories starting from undifferentiated this clustering analysis of expression patterns of embryonic stem (ES) cells located in the apex. We functionally classified genes may give insight into present three 'cell lineage trajectories', which represent understanding of CNS development and mechanisms the differentiation of ES cells into the first three of NSCs proliferation and differentiation. lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural Ahn, J. S., et al. (2010). "Identification of ectoderm. The positions of the cells along these differentially expressed genes in human embryonic trajectories seem to reflect the developmental potency stem cell-derived endothelial cells using suppression of cells and can be used as a scale for the potential of

42 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cells. Indeed, we show that embryonic germ cells and carcinogenesis may prove crucial in developing novel induced pluripotent cells are mapped near the origin of therapeutics that specifically target cancer cells. the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the Akyash, F., et al. (2017). "Human embryonic trajectories. We suggest that this method can be used stem cells and good manufacturing practice: Report of as the non-operational semi-quantitative definition of a 1- day workshop held at Stem Cell Biology Research cell differentiation status and developmental potency. Center, Yazd, 27(th) April 2017." Int J Reprod Furthermore, the global expression profiles of cell Biomed (Yazd) 15(5): 255-256. lineages provide a framework for the future study of in This report explains briefly the minutes of a 1- vitro and in vivo cell differentiation. day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" Aktug, H., et al. (2016). "Comparison of cell held by Stem Cell Biology Research Center based in cycle components, apoptosis and cytoskeleton-related Yazd Reproductive Sciences Institute at Shahid molecules and therapeutic effects of flavopiridol and Sadoughi University of Medical Sciences, Yazd, Iran geldanamycin on the mouse fibroblast, lung cancer on 27(th) April 2017. In this workshop, in addition to and embryonic stem cells." Tumour Biol 37(9): 12423- the practical sessions, Prof. Harry D. Moore from 12440. Centre for Stem Cell Biology, University of Sheffield, Similarities and differences in the cell cycle UK presented the challenges and the importance of the components, apoptosis and cytoskeleton-related biotechnology of clinical-grade human embryonic molecules among mouse skin fibroblast cells (MSFs), stem cells from first derivation to robust defined mouse squamous cell lung carcinomas (SqCLCs) and culture for therapeutic applications. mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation Allison, T. F., et al. (2018). "Identification and capacity of these cells. To reveal apoptotic pathways Single-Cell Functional Characterization of an and to examine the distribution and the role of cell Endodermally Biased Pluripotent Substate in Human cycle-cell skeleton comparatively would necessitate Embryonic Stem Cells." Stem Cell Reports 10(6): tumour biology and stem cell biology to be assessed 1895-1907. together in terms of oncogenesis and embryogenesis. Human embryonic stem cells (hESCs) display The primary objectives of this study are to investigate substantial heterogeneity in gene expression, implying the effects of flavopiridol, a cell cycle inhibitor, and the existence of discrete substates within the stem cell geldanamycin, a heat shock protein inhibitor on mouse compartment. To determine whether these substates somatic, tumour and embryonic stem cells, by impact fate decisions of hESCs we used a GFP specifically focusing on alterations in cytoskeletal reporter line to investigate the properties of fractions proteins, cell polarity and motility as well as cell cycle of putative undifferentiated cells defined by their regulators. To meet these objectives, expression of differential expression of the endoderm transcription several genes, cell cycle analysis and factor, GATA6, together with the hESC surface immunofluorescence staining of intracellular marker, SSEA3. By single-cell cloning, we confirmed cytoskeletal molecules were performed in untreated that substates characterized by expression of GATA6 and flavopiridol- or geldanamycin-treated cell lines. and SSEA3 include pluripotent stem cells capable of Cytotoxicity assays showed that SqCLCs are more long-term self-renewal. When clonal stem cell sensitive to flavopiridol than MSFs and mESCs. colonies were formed from GATA6-positive and Keratin-9 and keratin-2 expressions increased GATA6-negative cells, more of those derived from dramatically whereas cell cycle regulatory genes GATA6-positive cells contained spontaneously decreased significantly in the flavopiridol-treated differentiated endoderm cells than similar colonies MSFs. Flavopiridol-treated SqCLCs displayed a slight derived from the GATA6-negative cells. We increase in several cell cytoskeleton regulatory genes characterized these discrete cellular states using as well as cell cycle regulatory genes. However, gene single-cell transcriptomic analysis, identifying a expression profiles of mESCs were not affected after potential role for SOX17 in the establishment of the flavopiridol treatment except the Cdc2a. Cytotoxic endoderm-biased stem cell state. concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased Almstrup, K., et al. (2006). "From embryonic gene in the geldanamycin-treated MSFs. However, stem cells to testicular germ cell cancer-- should we be expression levels of cell cytoskeleton-associated genes concerned?" Int J Androl 29(1): 211-218. were increased dramatically in the geldanamycin- Since the discovery of testicular carcinoma in treated SqCLCs. Our results revealing differences in situ (CIS) -- the precursor cell for the vast majority of molecular mechanisms between embryogenesis and germ cell tumours -- it has been proposed that CIS

43 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cells could be derived from transformed primordial review on current state-of-the-art techniques for germ cells or gonocytes. Here, we review recent harnessing hESC-based strategies toward development discoveries not only substantiating that initial of a stem cell therapeutic for PD. Importantly, we also hypothesis but also indicating that CIS cells have a briefly describe a novel genetic-programming striking phenotypic similarity to embryonic stem cells approach that may address many of the key challenges (ESC). Many cancers have been proposed to originate that remain in the field and that may hasten clinical from tissue-specific stem cells [so-called 'cancer stem translation. cells' (CSC)] and we argue that CIS may be a very good example of a CSC, but with exceptional features Amir, H., et al. (2017). "Spontaneous Single- due to the retention of embryonic pluripotency. In Copy Loss of TP53 in Human Embryonic Stem Cells addition, considering the fact that pre-invasive CIS Markedly Increases Cell Proliferation and Survival." cells are transformed from early fetal cells, possibly Stem Cells 35(4): 872-885. due to environmentally induced alterations of the niche, Genomic aberrations have been identified in we discuss potential risks linked to the uncontrolled many human pluripotent stem cell (hPSC) cultures. therapeutic use of ESC. Commonly observed duplications in portions of 12p and 17q have been associated with Amano, T., et al. (2001). "Full-term development increases in genetic instability and resistance to of enucleated mouse oocytes fused with embryonic apoptosis, respectively. However, the phenotypic stem cells from different cell lines." Reproduction consequences related to sporadic mutations have not 121(5): 729-733. been evaluated to date. Here, we report on the effects The developmental potential of enucleated mouse of a single-copy deletion of the chr17p13.1 region, a oocytes receiving embryonic stem cells from ten lines sporadic mutation that spontaneously arose with either the same or different genetic backgrounds independently in several subclones of a human using the cell fusion method was examined in vitro embryonic stem cell culture. Compared to cells with and in vivo. The development of nuclear-transferred two normal copies of chr17p13.1 ("wild-type"), the oocytes into blastocysts was high (34-88%). However, cells with a single-copy deletion of this region there was no clear correlation between development ("mutant") displayed a selective advantage when into blastocysts after nuclear transfer and the chimaera exposed to stressful conditions, and retained a higher formation rate of embryonic stem cells. The percentage of cells expressing the pluripotency marker development into live young was low (1-3%) in all cell POU5F1/OCT4 after 2 weeks of in vitro lines and 14 of 19 young died shortly after birth. Most differentiation. Knockdown of TP53, which is a gene of the live young had morphological abnormalities. Of encompassed by the deleted region, in wild-type cells the five remaining mice, two died at days 23 and 30 mimicked the chr17p13.1 deletion phenotype. Thus, after birth, but the other three mice are still active at sporadic mutations in hPSCs can have phenotypic days 359 (mouse 1) and 338 (mice 4 and 5) after birth, effects that may impact their utility for clinical with normal fertility. However, the reasons for the applications. Stem Cells 2017;35:872-885. abnormalities and postnatal death of embryonic stem cell-derived mice are unknown. Amirpour, N., et al. (2012). "Differentiation of human embryonic stem cell-derived retinal progenitors Ambasudhan, R., et al. (2014). "Potential for cell into retinal cells by Sonic hedgehog and/or retinal therapy in Parkinson's disease using genetically pigmented epithelium and transplantation into the programmed human embryonic stem cell-derived subretinal space of sodium iodate-injected rabbits." neural progenitor cells." J Comp Neurol 522(12): Stem Cells Dev 21(1): 42-53. 2845-2856. Transplantation of retinal cells has recently Neural transplantation is a promising strategy for provided a promising therapeutic approach for retinal restoring dopaminergic dysfunction and modifying degeneration. Here, we differentiated initially retinal disease progression in Parkinson's disease (PD). progenitors (RPs) from adherent feeder-free human Human embryonic stem cells (hESCs) are a potential embryonic stem cells (hESCs) with the use of defined resource in this regard because of their ability to media supplemented with a specific combination of provide a virtually limitless supply of homogenous growth factors. The differentiated RPs highly (>80%) dopaminergic progenitors and neurons of appropriate expressed related molecular features that included lineage. The recent advances in developing robust cell Six3 at an early stage in addition to Crx, Rx, Pax6, culture protocols for directed differentiation of hESCs Otx2, and Chx10 at later stage. Next, we examined the to near pure populations of ventral mesencephalic (A9- induction of photoreceptors by Shh and/or the type) dopaminergic neurons has heightened the coculture of rabbit retinal pigmented epithelium with prospects for PD cell therapy. Here, we focus our hESCs-derived RPs. The differentiation of retinal cells

44 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ was demonstrated by protein and gene expression in Anand, S., et al. (2013). "Quiescent very small all groups. However, S-Opsin, a cone photoreceptor embryonic-like stem cells resist oncotherapy and can marker, had higher expression in the presence of Shh, restore spermatogenesis in germ cell depleted whereas expressions of Gli and Hes1 decreased in the mammalian testis." Stem Cells Dev. same group. Finally, hESC-derived RPs were treated Adult mouse and human testes harbor relatively with Shh transplanted into the subretinal space of quiescent, pluripotent very small embryonic-like stem sodium iodate-injected albino-type adult rabbits and cells (VSELs), in addition to actively dividing analyzed 4 weeks later. Transplanted retinal cells spermatogonial stem cells (SSCs). Here we report that survived, migrated into retinal layers, and restored a various oncotherapy regimens in human cancer small but significant B-wave. The grafted cells patients (n=7) and busulphan treatment (25mg/Kg expressed photoreceptor markers, S-Opsin and body weight) in eight weeks old male mice (n=15) Rhodopsin. Our results indicate that putative hESC- selectively affects actively dividing SSCs, derived retinal cells express related genes and proteins. spermatogonia, haploid germ cells and somatic Further, our results show that retinal-like cells can be microenvironment resulting in germ cell aplasia, useful replacements for photoreceptors in retinal whereas VSELs are unaffected and persist in otherwise diseases. germ cell depleted testis. Testicular VSELs are 2-5 microm in size, have high nucleo-cytoplasmic ratio, Amit, M., et al. (2005). "No evidence for SCA-1+/CD45-/LIN- (mice), CD133+/CD45-/LIN- infection of human embryonic stem cells by feeder (human survivors of childhood cancer) and express cell-derived murine leukemia viruses." Stem Cells various pluripotent transcripts including OCT-4A. 23(6): 761-771. SCA-1 sorted cells from busulphan treated mice testes Until recently, culture and expansion of in vitro formed small clusters suggestive of self- nondifferentiated human embryonic stem cells (hESCs) renewal and differentiation into progenitors, which depended on coculture with murine embryonic divide rapidly. Inter-tubular random injections of fibroblasts. Because mice are known to harbor a syngeneic Sertoli cells (105 cells per testis, n=14) or variety of pathogens, such culture conditions implicate bone marrow derived mesenchymal cells (104 cells per the risk of xenozoonoses. Among these pathogens, testis, n=16) into the germ cell depleted busulphan endogenous retroviruses, including murine leukemia treated mice testes, were able to restore viruses (MuLVs), are of special importance. It is well spermatogenesis from persisting VSELs. Transplanted known that some strains cause pathogenic (e.g., Sertoli or mesenchymal cells possibly were a source of leukemic) effects and that xenotropic, polytropic, and growth factors essential for VSELs differentiation. amphotropic MuLVs are able to infect human cells. In Since sperm formation occurred in situ, various view of potential clinical applications of hESC lines, it epigenetic concerns associated with the 'synthetic is therefore imperative to investigate potential gametes' may be eliminated in our approach. Ability of infection of hESCs by mouse feeder cell-derived mesenchymal cells to restore spermatogenesis may viruses. As a first step towards a comprehensive benefit existing azoospermic survivors of childhood infection risk assessment, we have analyzed cancer who were otherwise deprived of testicular embryonic fibroblasts derived from different mouse tissue cryopreservation prior to oncotherapy. Further strains for expression and release of xenotropic, studies are warranted to delineate the underlying polytropic, and amphotropic MuLVs. Moreover, mechanisms and to study quality and potential of several hESC lines have been investigated for sperm generated by this approach. expression of specific receptors for xenotropic/polytropic MuLVs, as well as for MuLV Anand, T., et al. (2011). "Buffalo (Bubalus infection and expression. Evidence for expression of bubalis) embryonic stem cell-like cells and humantropic MuLVs was found in cultures of mouse preimplantation embryos exhibit comparable embryonic fibroblasts (MEFs). Moreover, expression expression of pluripotency-related antigens." Reprod of specific receptors for xenotropic/ polytropic MuLV Domest Anim 46(1): 50-58. on human HEK293 and hESC lines and infection after In this study, inner cell mass (ICM) cells were coculture with an MuLV-producing mink cell line isolated from in vitro produced buffalo blastocysts and could be demonstrated. In contrast, no evidence of were cultured on mitomycin-C treated buffalo foetal MuLV transmission from MEFs to human HEK293 fibroblast feeder layer for producing embryonic stem cells or to the hESC lines I-3, I-6, I-8, and H-9 has (ES) cells. Among different sources (hatched vs been obtained. Our results suggest that recently expanded blastocysts) or methods (enzymatic vs established hESC lines are free of MuLV infections mechanical), mechanical isolation of ICM from despite long-term close contact with MEFs. hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage

45 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ number up to which ES cells survived. Putative ES give rise to astroglia, oligodendroglia, and neurones cells expressed alkaline phosphatase and exhibited a was investigated. After intracerebral transplantation, normal karyotype up to passage 7. Putative ES cells long-lasting integration of precursor cells into the host and embryos at 2- to 4-cell, 8- to 16-cell, morula and tissue was observed, serving as a pool for successive blastocyst stages strongly expressed stage-specific neuronal and glial differentiation. EGFP expression by embryonic antigen (SSEA)-4 but lacked expressions of ES cell-derived neural precursor cells may be a SSEA-1 and SSEA-3. Putative ES cells also expressed valuable tool to optimize protocols for maintenance tumour rejection antigen (TRA)-1-60, TRA-1-81 and and expansion of these cells in vitro as well as in vivo Oct4. Whereas in all early embryonic stages, TRA-1- after intracerebral transplantation. In addition, 60 was observed only in the periplasmic space, and preparative fluorescence-activated cell sorting of TRA-1-81 expression was observed as small spots at a EGFP-labeled neural precursor cells should be useful few places inside the embryos, both these markers for standardization of a donor cell population for cell were expressed by ICM. Oct4 expression, which was replacement therapies. observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo Anzai, H., et al. (1999). "Self-renewal and putative ES cells possess a unique pluripotency-related differentiation of a basic fibroblast growth factor- surface antigen phenotype, which resembles that of the dependent multipotent hematopoietic cell line derived ICM. from embryonic stem cells." Dev Growth Differ 41(1): 51-58. Ando, Y., et al. (2017). "Can Human Embryonic Despite the accumulation of informat on on the Stem Cell-Derived Stromal Cells Serve a Starting origin of hematopoietic stem cells, it is still unclear Material for Myoblasts?" Stem Cells Int 2017: how these cells are generated in ontogeny. Isolation of 7541734. cell lines equivalent to early embryonic hematopoietic A large number of myocytes are necessary to progenitor cells can be helpful. A multipotent treat intractable muscular disorders such as Duchenne hematopoietic progenitor cell line, A-6, was isolated muscular dystrophy with cell-based therapies. from H-1 embryonic stem (ES) cells. The self-renewal However, starting materials for cellular therapy of A-6 cells was supported by basic-fibroblast growth products such as myoblasts, marrow stromal cells, factor (b-FGF) and their differentiation into definitive menstrual blood-derived cells, and placenta-derived erythroid cells, granulocytes and macrophages was cells have a limited lifespan and cease to proliferate in induced after co-culture with ST-2 stromal cells. A-6 vitro. From the viewpoints of manufacturing and cells were positive for the surface markers of quality control, cells with a long lifespan are more hematopoietic stem cell, c-kit, CD31, CD34, Flt3/Flk2, suitable as a starting material. In this study, we PgP-1, and HSA, but were negative for that of the generated stromal cells for future myoblast therapy differentiated cells. Reverse transcription-polymerase from a working cell bank of human embryonic stem chain reaction analysis showed that A-6 cells produced cells (ESCs). The ESC-derived CD105(+) cells with mRNA from SCL/tal-1 and GATA-2 genes. Among extensive in vitro proliferation capability exhibited various cytokines examined, on y stem cell factor myogenesis and genetic stability in vitro. These results (SCF) and Flt3/Flk2 ligand (FL) supported the imply that ESC-derived CD105(+) cells are another proliferation of A-6 cells instead of b-FGF. The FL, as cell source for myoblasts in cell-based therapy for well as b-FGF, supported the self-renewal of A-6 cells, patients with genetic muscular disorders. Since ESCs whereas SCF induced differentiation into myeloid are immortal, mesenchymal stromal cells generated cells. A-6 cells will be useful for the characterization from ESCs can be manufactured at a large scale in one of hematopoietic progenitor cells derived from ES lot for pharmaceutical purposes. cells and provide a model system to realize the control mechanisms between self-renewal and different ation Andressen, C., et al. (2001). "Nestin-specific of hematopoietic stem cells. green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for Aoki, H., et al. (2006). "Embryonic stem cells transplantation." Stem Cells 19(5): 419-424. that differentiate into RPE cell precursors in vitro Expression of the enhanced green fluorescent develop into RPE cell monolayers in vivo." Exp Eye protein (EGFP) under control of a thymidine kinase Res 82(2): 265-274. promoter/nestin second intron was specifically A culture system to generate eye-like structures detected in nestin immunoreactive neural precursor consisting of lens, neural retina, and retinal pigmented cells after selection of murine embryonic stem (ES) epithelium (RPE) cells from undifferentiated cells in chemically defined medium. Allowing embryonic stem cells has been established. Precursors differentiation in vitro, the capacity of these cells to of RPE cells that differentiated in the cultures were

46 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ responsive to Wnt2b signaling and identified organoids we examined RGC markers and surface retrospectively to form secondary colonies consisting antigen expression and made comparisons to human of only RPE-like cells in eye-like structures. These fetal retina. RGCs in both tissues exhibited CD184 and transplanted eye-like structures were capable of CD171 expression and distinct expression patterns of populating the developing chick eye as neuronal retina the RGC markers BRN3 and RBPMS. The retinal and RPE cells. The outgrowth of a single cell layer of progenitor cells (RPCs) of retinal organoids expressed mature RPE cells from the grafted eye-like structures CD184, consistent with its expression in the confirmed the existence of precursors for RPE cells. neuroblastic layer in fetal retina. In retinal organoids These results suggest that the eye-like structures CD184 expression was enhanced in RGC competent resulted from the normal developmental pathway RPCs and high CD184 expression was retained on responsible for generating eyes in vivo. If a functional post-mitotic RGC precursors; CD171 was detected on effect of these cells can be established, such eye-like maturing RGCs. The differential expression timing of structures may be potentially used to establish therapy CD184 and CD171 permits identification and models for various eye diseases. enrichment of RGCs from retinal organoids at differing maturation states from committed progenitors Aoyama, M., et al. (2010). "Resistance to to differentiating neurons. These observations will chemotherapeutic agents and promotion of facilitate molecular characterization of PSC-derived transforming activity mediated by embryonic stem RGCs during differentiation, critical knowledge for cell-expressed Ras (ERas) signal in neuroblastoma establishing the veracity of these in vitro produced cells." Int J Oncol 37(4): 1011-1016. cells. Furthermore, observations made in the retinal Neuroblastoma is a common childhood tumor organoid model closely parallel those in human fetal derived from neural crest precursor cells. In the retina further validating use of retinal organoid to present study, we investigated the expression and model early retinal development. function of embryonic stem cell-expressed Ras (ERas), a novel Ras family protein previously reported as the Araki, R., et al. (2017). "The Number of Point specific expression gene in embryonic stem cells (ES Mutations in Induced Pluripotent Stem Cells and cells), in neuroblastoma cell lines. Our results showed Nuclear Transfer Embryonic Stem Cells Depends on that the expressions of ERas were detected in the Method and Somatic Cell Type Used for Their neuroblastoma cell lines by RT-PCR and Western Generation." Stem Cells 35(5): 1189-1196. blotting. Therefore, we transfected a full length ERas Induced pluripotent stem cells hold great promise expression vector into the neuroblastoma cell line SH- for regenerative medicine but point mutations have SY5Y, which has weak endogenous expression of been identified in these cells and have raised serious ERas, and obtained clones with higher levels of concerns about their safe use. We generated nuclear expression. Overexpression of ERas did not increase transfer embryonic stem cells (ntESCs) from both the growth rate of the ERas transfectants but promoted mouse embryonic fibroblasts (MEFs) and tail-tip their transforming activity. The ERas transfectants fibroblasts (TTFs) and by whole genome sequencing were more resistant to all the chemotherapy agents found fewer mutations compared with iPSCs than the parental cell line. The ability of ERas to generated by retroviral gene transduction. Furthermore, rescue cells from the toxic effect of chemotherapeutic TTF-derived ntESCs showed only a very small agents was inhibited by the phosphatidylinositol 3'- number of point mutations, approximately 80% less kinase (PI3K) inhibitor PD294002. These results show than the number observed in iPSCs generated using that the ERas/PI3K pathway may provide resistance to retrovirus. Base substitution profile analysis confirmed chemotherapy and promote transforming activity in this greatly reduced number of point mutations. The neuroblastoma. point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to Aparicio, J. G., et al. (2017). "Temporal genome reprogramming. Moreover, the dramatic expression of CD184(CXCR4) and CD171(L1CAM) reduction in point mutations in ntESCs suggests that identifies distinct early developmental stages of human most are not essential for genome reprogramming. Our retinal ganglion cells in embryonic stem cell derived results suggest that it is feasible to reduce the point retina." Exp Eye Res 154: 177-189. mutation frequency in iPSCs by optimizing various Human retinal ganglion cells (RGCs) derived genome reprogramming conditions. We conducted from pluripotent stem cells (PSCs) have anticipated whole genome sequencing of ntES cells derived from value for human disease study, drug screening, and MEFs or TTFs. We thereby succeeded in establishing therapeutic applications; however, their full potential TTF-derived ntES cell lines with far fewer point remains underdeveloped. To characterize RGCs in mutations. Base substitution profile analysis of these human embryonic stem cell (hESC) derived retinal clones also indicated a reduced point mutation

47 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ frequency, moving from a transversion-predominance precursor cells to migrate and differentiate in a host to a transition-predominance. Stem Cells striatum by using a D3-derived ESC clone that was 2017;35:1189-1196. transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent Armstrong, L., et al. (2010). "Human induced protein (GFP)-labeled construct. This procedure pluripotent stem cell lines show stress defense allowed easy monitoring of all transplanted cells mechanisms and mitochondrial regulation similar to because of the green fluorescent labeling of donor those of human embryonic stem cells." Stem Cells cells. This approach also afforded easy estimation of 28(4): 661-673. cell integration and simultaneous observation of the The generation of induced pluripotent stem cells entire transplanted cell population in relation to (iPSC) has enormous potential for the development of immunocytochemically identified neuronal and glial patient-specific regenerative medicine. Human differentiation. After selection of nestin-positive embryonic stem cells (hESC) are able to defend their neural precursor cells in a synthetic medium, they genomic integrity by maintaining low levels of were implanted into the striatum of male adult Wistar reactive oxygen species (ROS) through a combination rats. Their integration was analyzed on morphological of enhanced removal capacity and limited production studies performed 3 days to 4 weeks of these molecules. Such limited ROS production posttransplantation. CONCLUSIONS: The stems partly from the small number of mitochondria investigators found that after transplantation, a present in hESC; thus, it was important to determine subpopulation of GFP-labeled cells differentiated into that human iPSC (hiPSC) generation is able to various neural morphological types that were positive eliminate the extra mitochondria present in the for the mouse-specific Thy-1 antigen, which is known parental fibroblasts (reminiscent of "bottleneck" be expressed on neurons, as well as being positive for situation after fertilization) and to show that hiPSC the astroglial marker glial fibrillary acidic protein. have antioxidant defenses similar to hESC. We were Moreover, GFP-expressing cells that were negative for able to generate seven hiPSC lines from adult human either of these markers remained close to the injection dermal fibroblasts and have fully characterized two of site, presumably representing other derivatives of the those clones. Both hiPSC clones express pluripotency neural lineage. Together, these findings contribute to markers and are able to differentiate in vitro into cells basic research regarding future transplantation belonging to all three germ layers. One of these clones strategies in neurodegenerative diseases such as PD. is able to produce fully differentiated teratoma, whereas the other hiPSC clone is unable to silence the Arpornmaeklong, P., et al. (2009). "Phenotypic viral expression of OCT4 and c-, produce fully characterization, osteoblastic differentiation, and bone differentiated teratoma, and unable to downregulate regeneration capacity of human embryonic stem cell- the expression of some of the pluripotency genes derived mesenchymal stem cells." Stem Cells Dev during the differentiation process. In spite of these 18(7): 955-968. differences, both clones show ROS stress defense To enhance the understanding of differentiation mechanisms and mitochondrial biogenesis similar to patterns and bone formation capacity of hESCs, we hESC. Together our data suggest that, during the determined (1) the temporal pattern of osteoblastic reprogramming process, certain cellular mechanisms differentiation of human embryonic stem cell-derived are in place to ensure that hiPSC are provided with the mesenchymal stem cells (hESC-MSCs), (2) the same defense mechanisms against accumulation of influence of a three-dimensional matrix on the ROS as the hESC. osteogenic differentiation of hESC-MSCs in long-term culture, and (3) the bone-forming capacity of Arnhold, S., et al. (2000). "Differentiation of osteoblast-like cells derived from hESC-MSCs in green fluorescent protein-labeled embryonic stem cell- calvarial defects. Incubation of hESC-MSCs in derived neural precursor cells into Thy-1-positive osteogenic medium induced osteoblastic neurons and glia after transplantation into adult rat differentiation of hESC-MSCs into mature osteoblasts striatum." J Neurosurg 93(6): 1026-1032. in a similar chronological pattern to human bone OBJECT: The aim of this investigation was to marrow stromal cells and primary osteoblasts. assess new information concerning the capacity of Osteogenic differentiation was enhanced by culturing transplanted embryonic stem cell (ESC)-derived the cells on three-dimensional collagen scaffolds. neuronal cells to migrate into host brain and to Fluorescent-activated cell sorting of alkaline evaluate these cells as a possible source for cell phosphatase expressing cells was used to obtain an replacement therapy in neurodegenerative disorders enriched osteogenic cell population for in vivo such as Parkinson's disease (PD). METHODS: The transplantation. The identification of green authors investigated the ability of ESC-derived neural fluorescence protein and expression of human-specific

48 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ nuclear antigen in osteocytes in newly formed bone MATERIALS AND METHODS: Adult human verified the role of transplanted human cells in the adipose tissue-derived stem cells (ADSCs) were bone regeneration process. The current cell culture harvested from 13 patients after obtaining patients' model and osteogenic cell enrichment method could consent. The specific markers of ESCs included provide large numbers of osteoprogenitor cells for surface proteins CD10, CD13, CD44, CD59, CD105, analysis of differentiation patterns and cell and CD166, and further nucleostemin, (NS) NANOG, transplantation to regenerate skeletal defects. peroxisome proliferator-activated receptor-ggamma, collagen type 1 (Coll1), alkaline phosphate, (ALP) Arpornmaeklong, P., et al. (2010). "Expansion osteocalcin (OC), and 1 (Cbfa1) and characterization of human embryonic stem cell- were analyzed using by reverse transcription- derived osteoblast-like cells." Cell Reprogram 12(4): polymerase chain reaction, (RT-PCR) 377-389. immunofluorescence (IF), and western blot. Human embryonic stem cells (hESCs) have the RESULTS: All the proteins were expressed distinctly, potential to serve as a repository of cells for the except CD13 and OC. CD13 was found individually replacement of damaged or diseased tissues and organs. with different expressions, and OC expression was However, to use hESCs in clinically relevant scenarios, discernable. CONCLUSIONS: Although the ESC with a large number of cells are likely to be required. The its proven self-renewal capacity and pluripotency aim of this study was to demonstrate an alternative cell seems appropriate for clinical use, the recent work on culture method to increase the quantity of osteoblast- ADSCs suggests that these adult stem cells would be a like cells directly derived from hESCs (hESCs-OS). valuable source for future biotechnology, especially Undifferentiated hESCs were directly cultivated and since there is a relative ease of procurement. serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast- Auerbach, W. and T. M. DeChiara (2017). related genes to osteoblast-like cells derived from "Injecting Embryonic Stem Cells into Eight-Cell-Stage mesenchymal stem cells derived from hESCs (hESCs- Mouse Embryos." Cold Spring Harb Protoc 2017(9): MSCs-OS) and human bone marrow stromal cells pdb prot094367. (hBMSCs-OS). In comparison to hESCs-MSCs-OS, In this protocol, eight-cell-stage precompaction the hESCs-OS required a shorter expansion time to embryos from outbred mouse strains are used for the generate a homogenous population of osteoblast-like injection of hybrid or inbred embryonic stem (ES) cells that did not contain contaminating cells. This process often leads to generation of fully undifferentiated hESCs. Identification of human ES cell-derived so-called F0 mice (VelociMice). specific nuclear antigen (HuNu) in the newly formed Postinjection culture of embryos is necessary to bone in calvarial defects verified the role of the achieve the highest ratio of fully ES cell-derived mice transplanted hESCs-OS as active bone forming cells in and high-degree chimeras. Typically, 50 embryos are vivo. Taken together, this study suggests that injected per ES cell clone. osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of Bahena, I., et al. (2014). "Role of Mael in early osteoprogenitors for bone tissue engineering strategies. oogenesis and during germ-cell differentiation from embryonic stem cells in mice in vitro." Zygote 22(4): Arumugam, S. B., et al. (2011). "Detection of 513-520. embryonic stem cell markers in adult human adipose In a previous study, we have identified a set of tissue-derived stem cells." Indian J Pathol Microbiol conserved spermatogenic genes whose expression is 54(3): 501-508. restricted to testis and ovary and that are BACKGROUND: Bone marrow transplantation developmentally regulated. One of these genes, the is already an established therapy, which is now widely Mael, has been reported to play an used in medicine to treat leukemia, lymphoma, and essential role in mouse spermatogenesis. Nevertheless, several inherited blood disorders. The culture of the role of Mael in mouse oogenesis has not been multilineage cells from easily available adipose tissue defined. In order to analyse the role of Mael in mouse is another source of multipotent mesenchymal stem oogenesis, the expression of this gene was blocked cells, and is referred to as adipose tissue-derived stem during early oogenesis in mouse in vitro using RNAi cells (ADSCs). While ADSCs are being used to treat technology. In addition, the role of Mael during various conditions, some lacuna exists regarding the differentiation of embryonic stem cells (ESC) into specific proteins in these. It was therefore decided to germ cells in vitro was analysed. Results show that analyze the specific proteins of embryonic cells in downregulation of Mael by a specific short interfering ADSCs. AIMS: To analyze the specific protein of RNA disrupted fetal oocyte growth and differentiation embryonic stem cells (ESCs) in ADSCs. in fetal ovary explants in culture and the expression of

49 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ several germ-cell markers in ESC during their efficiency to generate embryoid bodies (EBs) in vitro differentiation. These results suggest that there is an and the formation of teratoma in vivo were important role for Mael in early oogenesis and during significantly increased in Bcl-xL-overexpressing germ-cell differentiation from embryonic stem cells in hESCs after single-cell dissociation. Interestingly, the mouse in vitro. number and size of hESC colonies from cluster cultures were not affected by Bcl-xL overexpression. Bahrami, S. B., et al. (2011). "Temporal changes Several genes of extracellular matrix and adhesion in expression accompany endothelial cell molecules were upregulated by Bcl-xL in hESCs differentiation of embryonic stem cells." Cell Adh without single-cell dissociation, suggesting that Bcl- Migr 5(2): 133-141. xL regulates adhesion molecular expression In pluripotent embryonic stem cells (ESCs), independent of cell dissociation. In addition, the gene expression of the Hox master regulatory transcription expressions of FAS and several TNF signaling factors that play essential roles in organogenesis, mediators were downregulated by Bcl-xL. These data angiogenesis, and maintenance of differentiated tissues, support a model in which Bcl-xL promotes cell is globally suppressed. We investigated whether survival and increases cloning efficiency of differentiation of endothelial cells (ECs) from mouse dissociated hESCs without altering hESC self-renewal ESCs was accompanied by activation of distinct Hox by i) attenuation of apoptosis, and ii) upregulation of gene expression profiles. Differentiation was observed adhesion molecules to facilitate cell-cell or cell-matrix within 3 days, as indicated by the appearance of cells interactions. expressing specific endothelial marker genes (Flk-1+ /VE-Cadherin+ ). Expression of HoxA3 and HoxD3, Bai, Q., et al. (2015). "Temporal analysis of which drive adult endothelial cell invasion and genome alterations induced by single-cell passaging in angiogenesis, peaked at day 3 and declined thereafter, human embryonic stem cells." Stem Cells Dev 24(5): whereas expression of HoxA5 and HoxD10, which 653-662. maintain a mature quiescent EC phenotype, was low at Simplified culture conditions are essential for day 3, but increased over time. The temporal and large-scale drug screening and medical applications of reciprocal changes in HoxD3 and HoxA5 expression human pluripotent stem cells (hPSCs). However, were accompanied by corresponding changes in hPSCs [ie, human embryonic stem cells (hESCs), and expression of established downstream target genes human induced pluripotent stem cells (iPSCs) are including integrin beta3 and Thrombospondin-2. Our prone to genomic instability, a phenomenon that is results indicate that differentiation and maturation of highly influenced by the culture conditions. Enzymatic ECs derived from cultured ESCs mimic changes in dissociation, a cornerstone of large-scale hPSC culture Hox gene expression that accompany maturation of systems, has been reported to be deleterious, but the immature angiogenic endothelium into differentiated extent and the timeline of the genomic alterations quiescent endothelium in vivo. induced by this passaging technique are still unclear. We prospectively monitored three hESC lines that Bai, H., et al. (2012). "Bcl-xL enhances single- were initially derived and cultured on human feeders cell survival and expansion of human embryonic stem and passaged mechanically before switching to cells without affecting self-renewal." Stem Cell Res enzymatic single-cell passaging. We show that 8(1): 26-37. karyotype abnormalities and copy number variations Robust expansion and genetic manipulation of are not restricted to long-term culture, but can occur human embryonic stem cells (hESCs) and induced- very rapidly, within five passages after switching pluripotent stem (iPS) cells are limited by poor cell hESCs to enzymatic dissociation. Subchromosomal survival after enzymatic dissociation into single cells. abnormalities preceded or accompanied karyotype Although inhibition of apoptosis is implicated for the abnormalities and were associated with increased single-cell survival of hESCs, the protective role of occurrence of DNA double-strand breaks. Our results attenuation of apoptosis in hESC survival has not been indicate that enzymatic single-cell passaging can be elucidated. Bcl-xL is one of several anti-apoptotic highly deleterious to the hPSC genome, even when proteins, which are members of the Bcl-2 family of used only for a limited period of time. Moreover, proteins. Using an inducible system, we ectopically hPSC culture techniques should be reappraised by expressed Bcl-xL gene in hESCs, and found a complementing the routine karyotype analysis with significant increase of hESC colonies in the single-cell more sensitive techniques, such as microarrays, to suspension cultures. Overexpression of Bcl-xL in detect subchromosomal abnormalities. hESCs decreased apoptotic caspase-3(+) cells, suggesting attenuation of apoptosis in hESCs. Without Bak, X. Y., et al. (2011). "Human embryonic altering the kinetics of pluripotent gene expression, the stem cell-derived mesenchymal stem cells as cellular

50 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ delivery vehicles for prodrug gene therapy of Ballabeni, A., et al. (2011). "Cell cycle glioblastoma." Hum Gene Ther 22(11): 1365-1377. adaptations of embryonic stem cells." Proc Natl Acad Mesenchymal stem cells (MSCs) possess tumor- Sci U S A 108(48): 19252-19257. tropic properties and consequently have been used to ES cells proliferate with very short gap phases deliver therapeutic agents for cancer treatment. Their yet maintain their capacity to differentiate. It had been potential in cancer therapy highlights the need for a thought that the levels of cyclins and other substrates consistent and renewable source for the production of of ubiquitin ligase APC/C remain nearly constant and uniform human MSCs suitable for clinical applications. Cdk activity remains constitutively high in mouse ES In this study, we seek to investigate whether human cells. Here we demonstrate that APC/C (anaphase- embryonic stem cells can be used as a cell source to promoting complex/cyclosome) enzyme is active in fulfill this goal. We generated MSC-like cells from ES cells but attenuated by high levels of the Emi1 two human embryonic stem cell lines, HuES9 and H1, (early mitotic inhibitor-1) protein. Despite the and observed that MSC-like cells derived from human presence of high Cdk activity during the G1 phase, embryonic stem cells were able to migrate into human chromatin can be effectively licensed for DNA glioma intracranial xenografts after being injected into replication and fast entry into the S phase can still the cerebral hemisphere contralateral to the tumor occur. High Cdk activity during S-G2-M phases inoculation site. We engineered these cells with produces high levels of the DNA replication factor baculoviral and lentiviral vectors, respectively, for Cdt1, and this leads to efficient Mcm proteins loading transient and stable expression of the herpes simplex on chromatin after mitotic exit. Although disturbing virus thymidine kinase gene. In tumor-bearing mice the usual balance between Cdk activity and APC/C the engineered MSC-like cells were capable of activity found in somatic cells, a few key adaptations inhibiting tumor growth and prolonging survival in the allow normal progression of a very rapid cell cycle. presence of ganciclovir after they were injected either directly into the xenografts or into the opposite Bandi, S. and R. Akkina (2008). "Human hemisphere. Our findings suggest that human embryonic stem cell (hES) derived dendritic cells are embryonic stem cell-derived MSCs may be a viable functionally normal and are susceptible to HIV-1 and attractive alternative for large-scale derivation of infection." AIDS Res Ther 5: 1. targeting vehicles for cancer therapy. BACKGROUND: Human embryonic stem (hES) cells hold considerable promise for cell replacement Balconi, G., et al. (2000). "Development of and gene therapies. Their remarkable properties of endothelial cell lines from embryonic stem cells: A pluripotency, self-renewal, and tractability for genetic tool for studying genetically manipulated endothelial modification potentially allows for the production of cells in vitro." Arterioscler Thromb Vasc Biol 20(6): sizeable quantities of therapeutic cells of the 1443-1451. hematopoietic lineage. Dendritic cells (DC) arise from Totipotent embryonic stem cells can be induced CD34+ hematopoietic progenitor cells (HPCs) and are to differentiate to endothelium in vitro. This may be a important in many innate and adaptive immune useful tool for obtaining cultures of genetically functions. With respect to HIV-1 infection, DCs play manipulated endothelial cells because embryonic stem an important role in the efficient capture and transfer cells are relatively easy to transfect and are commonly of the virus to susceptible cells. With an aim of used for gene inactivation experiments in mice. generating DCs from a renewable source for HIV-1 However, embryonic stem cell-derived endothelial studies, here we evaluated the capacity of hES cell cells could not be easily separated from embryoid derived CD34+ cells to give rise to DCs which can bodies and maintained in culture. In this study, we support HIV-1 infection. RESULTS: Undifferentiated describe the isolation and characterization of hES cells were cultured on S17 mouse bone marrow immortalized endothelial cell lines obtained from stromal cell layers to derive CD34+ HPCs which were embryonic stem cells differentiated in vitro. The cell subsequently grown in specific cytokine differentiation lines were analyzed for expression of endothelial cell media to promote the development of DCs. The hES markers, including growth factor receptors and derived DCs (hES-DC) were subjected to phenotypic adhesion molecules, and compared with endothelial and functional analyses and compared with DCs cells obtained from the yolk sac, the embryo proper, or derived from fetal liver CD34+ HPC (FL-DC). The the heart microcirculation of the adult. We propose mature hES-DCs displayed typical DC morphology that this approach may be useful for obtaining consisting of veiled stellate cells. The hES-DCs also endothelial cells carrying gene mutations that are displayed characteristic phenotypic surface markers lethal at very early stages of development. CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES- DCs were found to be capable of antigen uptake and stimulating naive allogeneic CD4+ T cells in a mixed

51 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ leukocyte reaction assay. Furthermore, the hES-DCs translation factors without transport activity, as they supported productive HIV-1 viral infection akin to lack transmembrane domains, and one pseudogene. To standard DCs. CONCLUSION: Phenotypically normal understand the roles of ABC genes in pluripotency and and functionally competent DCs that support HIV-1 multipotency, we performed a sensitive qRT-PCR infection can be derived from hES cells. hES-DCs can analysis of their expression in embryonic stem cells now be exploited in applied immunology and HIV-1 (hESCs), bone marrow-derived mesenchymal stem infection studies. Using gene therapy approaches, it is cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). now possible to generate HIV-1 resistant DCs from We confirm that hES-MSCs represent an intermediate anti-HIV gene transduced hES-CD34+ hematopoietic developmental stage between hESCs and hMSCs. We progenitor cells. observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These Banerjee, S. and M. Bacanamwo (2010). "DNA variations are mainly due to plasma membrane methyltransferase inhibition induces mouse embryonic transporters with low but significant gene expression: stem cell differentiation into endothelial cells." Exp 18 are expressed in hESCs compared with 16 in hES- Cell Res 316(2): 172-180. MSCs and 8 in hMSCs, suggesting important roles in Understanding endothelial cell (EC) pluripotency. Several of these ABCs shared similar differentiation is a step forward in tissue engineering, substrates but differ regarding gene regulation. controlling angiogenesis, and endothelial dysfunction. ABCA13 and ABCB4, similarly to ABCB1, could be We hypothesized that epigenetic activation of EC new markers to select primitive hMSCs with specific lineage specification genes is an important mediator of plasma membrane transporter (low) phenotypes. ABC embryonic stem cell (ESC) differentiation into EC. proteins performing basal intracellular functions, Mouse ESC was differentiated by removing leukemia including translation factors and mitochondrial heme inhibitory factor (LIF) from the maintenance media in transporters, showed the highest constant gene the presence or absence of the specific DNA expression among the three populations. Peptide methyltransferase (DNMT) inhibitor 5'-aza-2'- transporters in the endoplasmic reticulum, Golgi and deoxycytidine (aza-dC). Expression of EC lysosome were well expressed in hESCs and slightly specification and marker genes was monitored by upregulated in hMSCs, which play important roles quantitative PCR, western, immunocytochemistry, and during the development of stem cell niches in bone flow cytometry. Functionality of differentiated EC was marrow or meningeal tissue. These results will be assessed by angiogenesis assay. The methylation status useful to study specific cell cycle regulation of in the proximal promoter CpGs of the mediators of EC pluripotent stem cells or ABC dysregulation in differentiation VEGF-A, BMP4, and EPAS-1 as well complex pathologies, such as cancers or neurological as of the mature EC marker VE-cadherin was disorders. determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 Barbuti, A., et al. (2009). "Molecular expression in both the absence and presence of aza-dC composition and functional properties of f-channels in treatment. However, significant increase in murine embryonic stem cell-derived pacemaker cells." angiogenesis and expression of the mediators of EC J Mol Cell Cardiol 46(3): 343-351. differentiation and EC-specific genes was only Mouse embryonic stem cells (mESCs) observed in aza-dC-treated cells. The DNMT differentiate into all cardiac phenotypes, and thus inhibition-mediated increase in EC specification and represent an important potential source for cardiac marker gene expression was not associated with regenerative therapies. Here we characterize the demethylation of these genes. These studies suggest molecular composition and functional properties of that DNMT inhibition is an efficient inducer of EC "funny" (f-) channels in mESC-derived pacemaker differentiation from ESC. cells. Following differentiation, a fraction of mESC- derived myocytes exhibited action potentials Barbet, R., et al. (2012). "Expression of the 49 characterized by a slow diastolic depolarization and human ATP binding cassette (ABC) genes in expressed the I (f) current. I (f) plays an important role pluripotent embryonic stem cells and in early- and in the pacemaking mechanism of these cells since late-stage multipotent mesenchymal stem cells: ivabradine (3 microM), a specific f-channel inhibitor, possible role of ABC plasma membrane transporters in inhibited I (f) by about 50% and slowed rate by about maintaining human stem cell pluripotency." Cell Cycle 25%. Analysis of I (f) kinetics revealed the presence of 11(8): 1611-1620. two populations of cells, one expressing a fast- and The 49-member human ATP binding cassette one a slow-activating I (f); the two components are (ABC) gene family encodes 44 membrane transporters present both at early and late stages of differentiation for lipids, ions, peptides or xenobiotics, four and had also distinct activation curves.

52 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

Immunofluorescence analysis revealed that HCN1 and (GFP) cDNA targeted to the INS locus by homologous HCN4 are the only isoforms of the pacemaker channel recombination (INS (GFP/w)) and an untargeted hESC expressed in these cells. Rhythmic cells responded to line (HES2). INS (GFP/w) allowed efficient beta-adrenergic and muscarinic agonists: isoproterenol identification and purification of GFP-producing (1 microM) accelerated and acetylcholine (0.1 microM) (INS:GFP (+)) cells. Insulin (+) cells were examined slowed spontaneous rate by about 50 and 12%, for key features of adult beta cells using microarray, respectively. The same agonists caused quantitatively quantitative PCR, secretion assays, imaging and different effects on I (f): isoproterenol shifted electrophysiology. RESULTS: Immunofluorescent activation curves by about 5.9 and 2.7 mV and staining showed complete co-localisation of insulin acetylcholine by -4.0 and -2.0 mV in slow and fast I with GFP; however, cells were often multihormonal, (f)-activating cells, respectively. Accordingly, beta1- many with granules containing insulin and glucagon. and beta2-adrenergic, and M2-muscarinic receptors Electrophysiological recordings revealed variable K were detected in mESC-derived myocytes. Our data (ATP) and voltage-gated Ca (2+) channel activity, and show that mESC-derived pacemaker cells functionally reduced glucose-induced cytosolic Ca (2+) uptake. express proteins which underlie generation and This translated into defective glucose-stimulated modulation of heart rhythm, and can therefore insulin secretion but, intriguingly, appropriate represent a potential cell substrate for the generation of glucagon responses. Gene profiling revealed biological pacemakers. differences in global gene expression between INS:GFP (+) cells and adult human islets; however, Barta, T., et al. (2013). "Cell cycle regulation in INS:GFP (+) cells had remarkably similar expression human embryonic stem cells: links to adaptation to of endocrine-lineage transcription factors and genes cell culture." Exp Biol Med (Maywood) 238(3): 271- involved in glucose sensing and exocytosis. 275. CONCLUSIONS/INTERPRETATION: INS:GFP (+) Cell cycle represents not only a tightly cells can be purified from differentiated hESCs, orchestrated mechanism of cell replication and cell providing a superior source of insulin-producing cells. division but it also plays an important role in Genomic analyses revealed that INS:GFP (+) cells regulation of cell fate decision. Particularly in the collectively resemble immature endocrine cells. context of pluripotent stem cells or multipotent However, insulin (+) cells were heterogeneous, a fact progenitor cells, regulation of cell fate decision is of that translated into important functional differences paramount importance. It has been shown that human within this population. The information gained from embryonic stem cells (hESCs) show unique cell cycle this study may now be used to generate new iterations characteristics, such as short doubling time due to of functioning beta cells that can be purified for abbreviated G1 phase; these properties change with the transplant. onset of differentiation. This review summarizes the current understanding of cell cycle regulation in Batista, P. J., et al. (2014). "m (6)A RNA hESCs. We discuss cell cycle properties as well as modification controls cell fate transition in mammalian regulatory machinery governing cell cycle progression embryonic stem cells." Cell Stem Cell 15(6): 707-719. of undifferentiated hESCs. Additionally, we provide N6-methyl-adenosine (m (6)A) is the most evidence that long-term culture of hESCs is abundant modification on messenger RNAs and is accompanied by changes in cell cycle properties as linked to human diseases, but its functions in well as configuration of several cell cycle regulatory mammalian development are poorly understood. Here molecules. we reveal the evolutionary conservation and function of m (6)A by mapping the m (6)A methylome in Basford, C. L., et al. (2012). "The functional and mouse and human embryonic stem cells. Thousands of molecular characterisation of human embryonic stem messenger and long noncoding RNAs show conserved cell-derived insulin-positive cells compared with adult m (6)A modification, including transcripts encoding pancreatic beta cells." Diabetologia 55(2): 358-371. core pluripotency transcription factors. m (6)A is AIMS/HYPOTHESIS: Using a novel directed enriched over 3' untranslated regions at defined differentiation protocol, we recently generated up to sequence motifs and marks unstable transcripts, 25% insulin-producing cells from human embryonic including transcripts turned over upon differentiation. stem cells (hESCs) (insulin (+) cells). At this juncture, Genetic inactivation or depletion of mouse and human it was important to functionally and molecularly Mettl3, one of the m (6)A methylases, led to m (6)A characterise these hESC-derived insulin (+) cells and erasure on select target genes, prolonged Nanog identify key differences and similarities between them expression upon differentiation, and impaired ESC exit and primary beta cells. METHODS: We used a new from self-renewal toward differentiation into several reporter hESC line with green fluorescent protein lineages in vitro and in vivo. Thus, m (6)A is a mark

53 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ of transcriptome flexibility required for stem cells to human ES cells. This early establishment of H4 gene differentiate to specific lineages. regulation, which is independent, is consistent with co-expression of the cognate transcriptional Becker, K. A., et al. (2006). "Self-renewal of regulators HiNF-P and p220(NPAT). Human ES cells human embryonic stem cells is supported by a differ from somatic cells in the expression of members shortened G1 cell cycle phase." J Cell Physiol 209(3): of the E2F family and RB-related pocket proteins 883-893. (p105(RB1), p107(RBL1), and p130(RBL2/RB2)) that Competency for self-renewal of human control expression of genes encoding enzymes for embryonic stem (ES) cells is linked to pluripotency. nucleotide metabolism and DNA synthesis. Human ES However, there is a critical paucity of fundamental cells rapidly and robustly (>200-fold) induce the parameters of human ES cell division. In this study we cyclin dependent kinase (CDK) inhibitor show that human ES cells (H1 and H9; NIH- p21(WAF1/CIP1) upon gamma-irradiation. This DNA designated WA01 and WA09) rapidly proliferate due damage response promptly reduces histone gene to a very short overall cell cycle (15-16 h) compared to expression as well as mRNA levels for HiNF-P and somatic cells (e.g., normal diploid IMR90 fibroblasts p220(NPAT) and causes accumulation of unprocessed and NT-2 teratocarcinoma cells). The human ES cell histone H4 precursor RNAs. Furthermore, while , cycle maintains the four canonical cell cycle stages G1, and p130(RBL2/RB2) are the major E2F and S, G2, and M, but the duration of G1 is dramatically pocket protein mRNAs in actively proliferating ES shortened. Bromodeoxyuridine (BrdU) incorporation cells, expression levels of E2F5, E2F6, and p105(RB1) and FACS analysis demonstrated that 65% of are most strongly elevated during cell cycle arrest in asynchronously growing human ES cells are in S cells responding to DNA damage. Our data suggest phase. Immunofluorescence microscopy studies that the brief G1 phase of ES cells is supported by a detecting BrdU labeled mitotic chromosomes, Ki67 potent p21(WAF1/CIP1) related DNA damage domains, and p220(NPAT) containing Cajal bodies response that functions through several mechanisms to revealed that the durations of the S ( approximately 8 rapidly inhibit cell cycle progression. This response h), G2 ( approximately 4 h), and M phases may alter the E2F/pocket protein combinations that ( approximately 1 h) are similar in ES and somatic control E2F dependent genes and block H4 gene cells. We determined that human ES cells remain expression by inhibiting histone-specific transcription viable after synchronization with either nocodazole or factors and processing of histone gene transcripts, as the anti-tumor drug Paclitaxel (taxol) and have an well as by destabilizing histone mRNAs. abbreviated G1 phase of only 2.5-3 h that is significantly shorter than in somatic cells. Molecular Behroozi, F., et al. (2018). "Smart liposomal drug analyses using quantitative RT-PCR demonstrate that delivery for treatment of oxidative stress model in human ES cells and somatic cells express similar cell human embryonic stem cell-derived retinal pigment cycle markers. However, among cyclins and cyclin- epithelial cells." Int J Pharm 548(1): 62-72. dependent kinases (CDKs), we observed high mRNA Oxidative stress has been implicated in the levels for the G1-related CDK4 and cyclin D2 genes. progression of age-related macular degeneration We conclude that human ES cells exhibit unique G1 (AMD). Treatment with antioxidants seems to delay cell cycle kinetics and use CDK4/cyclin D2 related progression of AMD. In this study, we suggested an mechanisms to attain competency for DNA replication. antioxidant delivery system based on redox-sensitive liposome composed of phospholipids and a diselenide Becker, K. A., et al. (2007). "Establishment of centered alkyl chain. Dynamic light scattering histone gene regulation and cell cycle checkpoint assessment indicated that the liposomes had an control in human embryonic stem cells." J Cell Physiol average size of 140nm with a polydispersity index 210(2): 517-526. below 0.2. The percentage of encapsulation efficiency Rapid self-renewal of human embryonic stem of the liposomes was calculated by high-performance (ES) cells (NIH designation WA01 and WA09) is liquid chromatography. The carriers were loaded with accommodated by an abbreviated cell cycle due to a N-acetyl cysteine as a model antioxidant drug. We reduction in the G1 phase. Thus, molecular demonstrated responsiveness of the nanocarrier and its mechanisms operative in ES cells may expedite the efficiency in drug delivery in an oxidative stress model cellular commitment to progress into S phase to of human embryonic stem cell-derived retinal pigment initiate replication of DNA and biosynthesis of histone epithelial (hESC-RPE) cells. The modeled cells treated proteins to form new chromatin. Here we show that with diselenide containing liposomes loaded with the selective cell cycle regulated expression of 10mM NAC, showed a better therapeutic effect with a individual histone H4 gene copies, which is typical for cell metabolic activity of 90%, which was significantly somatic cell types, is already firmly established in higher compared to insensitive liposomes or NAC

54 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ treated groups (P<0.05). In addition, the expression of UNLABELLED: Congenital human oxidative-sensitive gene markers in diselenide cytomegalovirus (HCMV) infection is a major cause containing liposomes groups were improved. Our of central nervous system structural anomalies and results demonstrated fabricated smart liposomes opens sensory impairments. It is likely that the stage of fetal new opportunity for targeted treatment of retinal development, as well as the state of differentiation of degeneration. susceptible cells at the time of infection, affects the severity of the disease. We used human embryonic Bel, A., et al. (2010). "Composite cell sheets: a stem (ES) cell-derived primitive prerosette neural stem further step toward safe and effective myocardial cells (pNSCs) and neural progenitor cells (NPCs) regeneration by cardiac progenitors derived from maintained in chemically defined conditions to study embryonic stem cells." Circulation 122(11 Suppl): HCMV replication in cells at the early stages of neural S118-123. development. In contrast to what was observed BACKGROUND: The safety and efficacy of previously using fetus-derived NPCs, infection of ES myocardial regeneration using embryonic stem cells cell-derived pNSCs with HCMV was nonprogressive. are limited by the risk of teratoma and the high rate of At a low multiplicity of infection, we observed only a cell death. METHODS AND RESULTS: To address small percentage of cells expressing immediate-early these issues, we developed a composite construct genes (IE) and early genes. IE expression was found to made of a sheet of adipose tissue-derived stroma cells be restricted to cells negative for the anterior marker and embryonic stem cell-derived cardiac progenitors. FORSE-1, and treatment of pNSCs with retinoic acid Ten Rhesus monkeys underwent a transient coronary restored IE expression. Differentiation of pNSCs into artery occlusion followed, 2 weeks later, by the open- NPCs restored IE expression but not the chest delivery of the composite cell sheet over the transactivation of early genes. Virions produced in infarcted area or a sham operation. The sheet was NPCs and pNSCs were exclusively cell associated and made of adipose tissue-derived stroma cells grown were mostly non-neural tropic. Finally, we found that from a biopsy of autologous adipose tissue and viral genomes could persist in pNSC cultures for up to cultured onto temperature-responsive dishes. a month after infection despite the absence of Allogeneic Rhesus embryonic stem cells were detectable IE expression by immunofluorescence, and committed to a cardiac lineage and infectious virus could be produced upon differentiation immunomagnetically sorted to yield SSEA-1(+) of pNSCs to neurons. In conclusion, our results cardiac progenitors, which were then deposited onto highlight the complex array of hurdles that HCMV the cell sheet. Cyclosporine was given for 2 months must overcome in order to infect primitive neural stem until the animals were euthanized. Preimplantation cells and suggest that these cells might act as a studies showed that the SSEA-1(+) progenitors reservoir for the virus. IMPORTANCE: Human expressed cardiac markers and had lost pluripotency. cytomegalovirus (HCMV) is a betaherpesvirus that is After 2 months, there was no teratoma in any of the 5 highly prevalent in the population. HCMV infection is cell-treated monkeys. Analysis of >1500 histological usually asymptomatic but can lead to severe sections showed that the SSEA-1(+) cardiac consequences in immunosuppressed individuals. progenitors had differentiated into cardiomyocytes, as HCMV is also the most important infectious cause of evidenced by immunofluorescence and real-time congenital developmental birth defects. Manifestations polymerase chain reaction. There were also a robust of fetal HCMV disease range from deafness and engraftment of autologous adipose tissue-derived learning disabilities to more severe symptoms such as stroma cells and increased angiogenesis compared microcephaly. In this study, we have used embryonic with the sham animals. CONCLUSIONS: These data stem cells to generate primitive neural stem cells and collected in a clinically relevant nonhuman primate have used these to model HCMV infection of the fetal model show that developmentally restricted SSEA-1(+) central nervous system (CNS) in vitro. Our results cardiac progenitors appear to be safe and highlight the reveal that these cells, which are similar to those benefit of the epicardial delivery of a construct present in the developing neural tube, do not support harboring cells with a cardiomyogenic differentiation viral replication but instead likely constitute a viral potential and cells providing them the necessary reservoir. Future work will define the effect of viral trophic support. persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral Belzile, J. P., et al. (2014). "Human replication in the CNS. cytomegalovirus infection of human embryonic stem cell-derived primitive neural stem cells is restricted at Bencsik, R., et al. (2016). "Improved transgene several steps but leads to the persistence of viral expression in doxycycline-inducible embryonic stem DNA." J Virol 88(8): 4021-4039.

55 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cells by repeated chemical selection or cell sorting." extract: Cell line-dependent differences." J Neurosci Stem Cell Res 17(2): 228-234. Res 85(5): 1057-1064. Transgene-mediated programming is a In the present study, we compare the capacity of preeminent strategy to direct cellular identity. To two different embryonic stem (ES) cell lines to secrete facilitate cell fate switching, lineage regulating genes neurotrophins in response to cerebral tissue extract must be efficiently and uniformly induced. However, derived from healthy or injured rat brains. The gene expression is often heterogeneous in transgenic intrinsic capacity of the embryonic cell lines BAC7 systems. Consistent with this notion, a non-uniform (feeder cell-dependent cultivation) to release brain- reporter gene expression was detected in our derived neurotrophic factor (BDNF) or neurotrophin-3 doxycycline (DOX)-regulated, murine embryonic stem (NT-3) exceeded the release of these factors by CGR8 (ES) cell clones. Interestingly, a significant fraction of cells (feeder cell-free growth) by factors of 10 and 4, cells within each clone failed to produce any reporter respectively. Nerve growth factor (NGF) was secreted signals upon DOX treatment. We found that the only by BAC7 cells. Conditioning of cell lines with majority of these non-responsive cells neither carry cerebral tissue extract derived from healthy or fluid reporter transgene nor geneticin/G418 resistance. This percussion-injured rat brains resulted in a significant observation suggested that our ES cell clones time-dependent increase in BDNF release in both cell contained non-recombined cells that survived the lines. The increase in BDNF release by BAC7 cells G418 selection which was carried out during the was more pronounced when cells were incubated with establishment of these clones. We successfully brain extract derived from injured brain. However, eliminated most of these corrupted cells with repeated differences in neurotrophin release associated with the chemical (G418) selection, however, even after origin of brain extract were at no time statistically prolonged G418 treatments, a few cells remained non- significant. Neutrophin-3 and NGF release was responsive due to epigenetic silencing. We found that inhibited when cell lines were exposed to cerebral cell sorting has been the most efficient approach to tissue extract. The magnitude of the response to select those cells which can uniformly and stably cerebral tissue extract was dependent on the intrinsic induce the integrated transgene in this ES cell based capacity of the cell lines to release neurotrophins. Our platform. Together, our data revealed that post-cloning results clearly demonstrate significant variations in the chemical re-selection or cell sorting strongly facilitate intrinsic capability of different stem cell lines to the production of ES cell lines with a uniform produce neurotrophic factors. Furthermore, a transgene induction capacity. significant modulation of neurotrophic factor release was observed following conditioning of cell lines with Bendall, S. C., et al. (2008). "Human embryonic tissue extract derived from rat brains. A significant stem cells: lessons from stem cell niches in vivo." modulation of neurotrophin release dependent on the Regen Med 3(3): 365-376. source of cerebral tissue extract used was not observed. In vivo the stem cell niche is an essential component in controlling and maintaining the stem Ben-Yehudah, A., et al. (2009). "Evaluating cells' ability to survive and respond to injury. Human protocols for embryonic stem cell differentiation into embryonic stem cells (hESCs) appear to be an insulin-secreting beta-cells using insulin II-GFP as a exception to this rule as they can be removed from specific and noninvasive reporter." Cloning Stem Cells their blastocytic microenvironment and maintained 11(2): 245-257. indefinitely in vitro. However, recent observations Stable and full differentiation of pluripotent stem reveal the existence of an autonomously derived in cells into functional beta-cells offers the potential to vitro hESC niche. This provides a previously treat type I diabetes with a theoretically inexhaustible unappreciated mechanism to control hESC expansion source of replacement cells. In addition to the and differentiation. Recognizing this, it may now be difficulties in directed differentiation, progress toward possible to take aspects of in vivo stem cell niches, an optimized and reliable protocol has been hampered namely extracellular matrices, paracrine signals and by the complication that cultured cells will concentrate accessory cell types, and exploit them in order to gain insulin from the media, thus making it difficult to tell fidelity in directed hESC differentiation. In doing so, which, if any, cells are producing insulin. To address routine customization of hESC lines and their this, we utilized a novel murine embryonic stem cell application in regenerative therapies may be further (mESC) research model, in which the green enhanced using unique hESC niche-based approaches. fluorescent protein (GFP) has been inserted within the C-peptide of the mouse insulinII gene (InsulinII-GFP). Bentz, K., et al. (2007). "Embryonic stem cells Using this method, cells producing insulin are easily produce neurotrophins in response to cerebral tissue identified. We then compared four published protocols for differentiating mESCs into beta-cells to evaluate

56 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ their relative efficiency by assaying intrinsic insulin tKO) mice display cobblestone lissencephaly and are production. Cells differentiated using each protocol perinatally lethal. To circumvent this problem, we were easily distinguished based on culture conditions generated APP triple knockout embryonic stem (ES) and morphology. This comparison is strengthened cells and differentiated these to APP triple knockout because all testing is performed within the same neurons in vitro and in vivo. In comparison with wild- laboratory by the same researchers, thereby removing type (WT) ES cell-derived neurons, APP tKO neurons interlaboratory variability in culture, cells, or analysis. formed equally pure neuronal cultures, had unaltered Differentiated cells were analyzed and sorted based on in vitro migratory capacities, had a similar acquisition GFP fluorescence as compared to wild type cells. Each of polarity, and were capable of extending long differentiation protocol increased GFP fluorescence neurites and forming active excitatory synapses. These but only modestly. None of these protocols yielded data were confirmed in vivo in chimeric mice with more than 3% of cells capable of insulin biosynthesis APP tKO neurons expressing the enhanced green indicating the relative inefficiency of all analyzed fluorescent protein (eGFP) present in a WT protocols. Therefore, improved beta-cells background brain. The results suggest that the loss of differentiation protocols are needed, and these insulin the APP family of proteins has no major effect on II GFP cells may prove to be an important tool to these critical neuronal processes and that the apparent accelerate this process. multitude of functions in which APP has been implicated might be characterized by molecular Berger, C. N., et al. (1995). "The development of redundancy. Our stem cell culture provides an haematopoietic cells is biased in embryonic stem cell excellent tool to circumvent the problem of lack of chimaeras." Dev Biol 170(2): 651-663. viability of APP/APLP triple knockout mice and will The haematopoietic development of embryonic help to explore the function of this intriguing protein stem (ES) cell injection chimaeras was analysed using further in vitro and in vivo. beta-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived Bernreuther, C., et al. (2006). "Neural cell cell population from the host cells. The number of adhesion molecule L1-transfected embryonic stem cells in the different haematopoietic cell cells promote functional recovery after excitotoxic subpopulations was determined by flow cytometry. lesion of the mouse striatum." J Neurosci 26(45): When the proportions of ES-derived cells in the 11532-11539. antigen-positive lineages were compared to the ES cell We have generated a murine embryonic stem cell contribution to all cells in teh organs, we found an line constitutively expressing L1 at all stages of neural unexpected bias in the haematopoietic differentiation differentiation to investigate the effects of L1 of ES-derived cells. ES descendants were overexpression on stem cell proliferation, migration, overrepresented in the bone marrow B lymphoid cell differentiation, cell death, and ability to influence population and the splenic myeloid cells but were drug-induced rotation behavior in an animal model of underrepresented in the CD4-positive T lymphoid cells Huntington's disease. L1-transfected cells showed in the spleen. These results were obtained by decreased cell proliferation in vitro, enhanced neuronal comparison with control female animals that were X differentiation in vitro and in vivo, and decreased chromosome mosaic for beta-galactosidase expression. astrocytic differentiation in vivo without influencing These findings of uneven contribution to cell death compared with nontransfected cells. L1 haematopoietic development by ES cells indicate that overexpression also resulted in an increased yield of the commitment of ES cell descendants may be GABAergic neurons and enhanced migration of different from that of the host cells. embryonic stem cell-derived neural precursor cells into the lesioned striatum. Mice grafted with L1- Bergmans, B. A., et al. (2010). "Neurons transfected cells showed recovery in rotation behavior generated from APP/APLP1/APLP2 triple knockout 1 and 4 weeks, but not 8 weeks, after transplantation embryonic stem cells behave normally in vitro and in compared with mice that had received nontransfected vivo: lack of evidence for a cell autonomous role of cells, thus demonstrating for the first time that a the amyloid precursor protein in neuronal recognition molecule is capable of improving differentiation." Stem Cells 28(3): 399-406. functional recovery during the initial phase in a Alzheimer's disease amyloid precursor protein syngeneic transplantation paradigm. (APP) has been implicated in many neurobiologic processes, but supporting evidence remains indirect. Berrill, A., et al. (2004). "Assessment of stem Studies are confounded by the existence of two cell markers during long-term culture of mouse partially redundant APP homologues, APLP1 and embryonic stem cells." Cytotechnology 44(1-2): 77-91. APLP2. APP/APLP1/APLP2 triple knockout (APP

57 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

Embryonic stem (ES) cells have been in the fore from ES cells. We showed that even a complex front of scientific literature lately as having the cocktail of HGFs (stem cell factor, interleukin 3, IL6, potential for regeneration of many tissue types. Two IL11, granulocyte colony-stimulating factor, and important issues that need to be addressed are the erythropoietin) is unable to induce significant myeloid culture conditions for maintaining ES cells and the differentiation in day 12 embryoid bodies. Cocultures accuracy of ES cell markers in monitoring the of MS-5 stromal cells with ES cells were slightly more undifferentiated state. Leukaemia inhibitory factor productive than HGFs. A strong synergistic effect was (LIF) is routinely used to sustain mouse ES cells (mES) observed on the growth of myeloid colonies and MKs in a pluripotent fashion. In this paper, we assessed when we used a combination of MS-5 cells plus the three markers during long-term maintenance of ES HGF cocktail. Conditioned medium from MS-5 cells cells with various concentrations of LIF to see if also synergized with the HGF cocktail to produce a decreasing concentration would lead to changes in substantial number of mixed colonies containing MKs. marker expressions and growth behavior. Common The addition of fibroblast growth factor-2 (FGF-2) to markers of pluripotency such as alkaline phosphatase the HGF cocktail plus MS-5 nearly doubled the enzyme activity (ALP), surface staining for stage number of myeloid progenitors, including those with specific embryonic antigen 1 (SSEA-1), Oct-4 MKs. Thrombopoietin (TPO) alone or in any transcription factor, cell doubling time, as well as combination with MS-5 or HGFs, did not increase the visual observations of cell morphology were analyzed number of MK-containing colonies. However, when during long-term maintenance of mES cells with LIF TPO was added to the HGF cocktail + FGF-2 + MS-5, concentrations ranging from 0 to 500 pM. The the number of MKs in liquid cultures and mixed morphology of the cells at LIF concentrations of 0 25 colonies increased, and many exhibited a "hairy" pM changed from being tight clusters to more appearance resembling pseudopodial proplatelet flattened shapes while cells in 50-500 pM retained the formation. Having defined the culture conditions of ES clustered shape but growth rates remained essentially cells that allow the production of all the myeloid identical at between 10 and 16 h. ES cells at all lineages including MKs, we conclude that the concentrations of LIF continued expressing ALP, hematopoietic differentiation model of ES cells is SSEA-1 and Oct-4 markers over a period of 6 weeks, especially useful for studying the regulation of which indicate that mES cells are capable of either expression of any gene important in early producing autocrine LIF or are able to proliferate at hematopoiesis. very low levels of LIF. Pluripotency markers such as Oct-4 and SSEA-1 are only moderately reduced after Beyer, T. A., et al. (2013). "Switch enhancers 5-6 weeks. Oct-4 mRNA expression levels were interpret TGF-beta and Hippo signaling to control cell partially diminished in LIF free conditions only at fate in human embryonic stem cells." Cell Rep 5(6): weeks 5 and 6 compared to controls with LIF at 500 1611-1624. pM. Changes in morphology of cells by visual A small toolkit of morphogens is used repeatedly observation seemed to be a faster indication of the to direct development, raising the question of how onset of differentiation in mES cells, although other context dictates interpretation of the same cue. One reliable means also include decreased levels of Oct-4, example is the transforming growth factor beta (TGF- SSEA-1 and ALP markers. It is preferable to maintain beta) pathway that in human embryonic stem cells long-term cultures of mES cells above 50 pM of LIF fulfills two opposite functions: pluripotency to have a more homogenous, stable population of maintenance and mesendoderm (ME) specification. pluripotent cells. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex Berthier, R., et al. (1997). "The MS-5 murine composed of transcriptional effectors of the Hippo stromal cell line and hematopoietic growth factors pathway (TAZ/YAP/TEAD), the TGF-beta pathway synergize to support the megakaryocytic (SMAD2/3), and the pluripotency regulator OCT4 differentiation of embryonic stem cells." Exp Hematol (TSO). TSO collaborates with NuRD repressor 25(6): 481-490. complexes to buffer pluripotency gene expression Murine embryonic stem (ES) cells are able to while suppressing ME genes. Importantly, the SMAD differentiate into erythroid, mast, and DNA binding partner FOXH1, a major specifier of ME, granulomonocytic cells by using appropriate culture is found near TSO elements, and upon fate conditions. Because we were interested in the specification we show that TSO is disrupted with regulation of tissue-specific expression of the platelet subsequent SMAD-FOXH1 induction of ME. These glycoprotein IIb gene, we studied the culture studies define switch-enhancer elements and provide a conditions, aiming at the reproducible production of framework to understand how cellular context dictates myeloid cells that included megakaryocytes (MKs)

58 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ interpretation of the same morphogen signal in Embryonic stem cells (ESCs) are an outstanding development. model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for Bhartiya, D., et al. (2012). "Very small investigating the development of early hematopoietic embryonic-like stem cells with maximum regenerative progenitor cells (HPCs). Gene expression in ESCs can potential get discarded during cord blood banking and be manipulated by several techniques that allow the bone marrow processing for autologous stem cell role for individual molecules in development to be therapy." Stem Cells Dev 21(1): 1-6. determined. One difficulty is that expression of Very small embryonic-like stem cells (VSELs) specific genes often has different phenotypic effects are possibly lost during cord blood banking and bone dependent on their temporal expression. This problem marrow (BM) processing for autologus stem cell can be circumvented by the generation of ESCs that therapy mainly because of their small size. The present inducibly express a gene of interest using technology study was conducted on human umbilical cord blood such as the doxycycline-inducible transgene system. (UCB, n=6) and discarded red blood cells (RBC) However, generation of these inducible cell lines is fraction obtained after separation of mononuclear cells costly and time consuming. Described here is a from human BM (n=6), to test this hypothesis. The method for disaggregating ESC-derived embryoid results show that VSELs, which are pluripotent stem bodies (EBs) into single cell suspensions, retrovirally cells with maximum regenerative potential, settle infecting the cell suspensions, and then reforming the along with the RBCs during Ficoll-Hypaque density EBs by hanging drop. Downstream differentiation is separation. These cells are very small in size (3-5 then evaluated by flow cytometry. Using this protocol, mum), have high nucleo-cytoplasmic ratio, and it was demonstrated that exogenous expression of a express nuclear Oct-4, cell surface protein SSEA-4, microRNA gene at the beginning of ESC and other pluripotent markers such as Nanog, Sox-2, differentiation blocks HPC generation. However, when Rex-1, and Tert as indicated by immunolocalization expressed in EB derived cells after nascent mesoderm and quantitative polymerase chain reaction (Q-PCR) is produced, the microRNA gene enhances studies. Interestingly, a distinct population of slightly hematopoietic differentiation. This method is useful larger, round hematopoietic stem cells (HSCs) with for investigating the role of genes after specific germ cytoplasmic Oct-4 were detected in the "buffy" coat, layer tissue is derived. which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the Boast, S. and C. D. Stern (2013). "Simple umbilical cord tissue (UCT) sections (n=3) showed the methods for generating neural, bone and endodermal presence of nuclear Oct-4-positive VSELs and many cell types from chick embryonic stem cells." Stem Cell fibroblast-like mesenchymal stem cells (MSCs) with Res 10(1): 20-28. cytoplasmic Oct-4. These VSELs with nuclear Oct-4, Most work on embryonic stem cell differentiation detected in UCB, UCT, and discarded RBC fraction uses mammalian cells derived from the blastocyst obtained after BM processing, may persist throughout stage and some of the most widely used protocols to life, maintain tissue homeostasis, and undergo induce differentiation involve growing these cells in asymmetric cell division to self-renew as well as monolayer culture. Equivalent stem cells can be produce larger progenitor stem cells, viz. HSCs or obtained from embryos of non-mammalian vertebrates, MSCs, which follow differentiation trajectories but to date this has only been successful in birds. depending on the somatic niche. Hence, it can be These cells can contribute to all somatic lineages in concluded that the true stem cells in adult body tissues chimaeras and can be induced to differentiate into a are the VSELs, whereas the HSCs and MSCs are variety of cell types in vitro via embryoid body actually progenitor stem cells that arise by asymmetric formation. However to date there are no reliable cell division of VSELs. The results of the present methods for differentiating them into descendants study may help explain low efficacy reported during from each of the germ layers in monolayer culture, adult autologous stem cell trials, wherein unknowingly comparable to the protocols used in mammals. Here progenitor stem cells are injected rather than the we describe three simple and reproducible protocols pluripotent stem cells with maximum regenerative for differentiation of chick embryonic stem cells into potential. mesoderm (bone), endoderm and neuroectoderm (neurons and glia) in monolayer culture. These Bikorimana, E., et al. (2014). "Retroviral methods open the way for more direct comparisons of infection of murine embryonic stem cell derived the properties of mammalian and avian embryonic embryoid body cells for analysis of hematopoietic stem cells that may highlight similarities and differentiation." J Vis Exp (92): e52022. differences.

59 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

Boheler, K. R., et al. (2011). "Embryonic stem Bonig, H., et al. (2008). "Blood types of current cell-derived cardiomyocyte heterogeneity and the embryonic stem cell lines are not conducive to isolation of immature and committed cells for cardiac culturing "universal-donor" red blood cells." remodeling and regeneration." Stem Cells Int 2011: Transfusion 48(5): 1039-1040. 214203. Pluripotent stem cells represent one promising Boras-Granic, K., et al. (2014). "Embryonic cells source for cell replacement therapy in heart, but contribute directly to the quiescent stem cell differentiating embryonic stem cell-derived population in the adult mouse mammary gland." cardiomyocytes (ESC-CMs) are highly heterogeneous Breast Cancer Res 16(6): 487. and show a variety of maturation states. In this study, INTRODUCTION: Studies have identified multi- we employed an ESC clonal line that contains a potent stem cells in the adult mammary gland. More cardiac-restricted ncx1 promoter-driven puromycin recent studies have suggested that the embryonic resistance cassette together with a mass culture system mammary gland may also contain stem/progenitor to isolate ESC-CMs that display traits characteristic of cells that contribute to initial ductal development. We very immature CMs. The cells display properties of were interested in determining whether embryonic proliferation, CM-restricted markers, reduced cells might also directly contribute to long-lived stem mitochondrial mass, and hypoxia-resistance. cells that support homeostasis and development in the Following transplantation into rodent hearts, adult mammary gland. METHODS: We used DNA- bioluminescence imaging revealed that immature cells, label retention to detect long label-retaining cells in but not more mature CMs, survived for at least one the mammary gland. Mouse embryos were labeled month following injection. These data and with 5-ethynl-2'-deoxyuridine (EdU) between comparisons with more mature cells lead us to embryonic day 14.5 and embryonic day 18.5 and were conclude that immature hypoxia resistant ESC-CMs subsequently sacrificed and examined for EdU can be isolated in mass in vitro and, following retention at various intervals after birth. EdU retaining injection into heart, form grafts that may mediate long- cells were co-stained for various lineage markers and term recovery of global and regional myocardial identified after fluorescence activated cell sorting contractile function following infarction. analysis of specific epithelial subsets. EdU-labeled mice were subjected to subsequent 5-bromo-2'- Bonde, S., et al. (2010). "Cell fusion of bone deoxyuridine administration to determine whether marrow cells and somatic cell reprogramming by EdU-labeled cells could re-enter the cell cycle. Finally, embryonic stem cells." FASEB J 24(2): 364-373. EdU-labeled cells were grown under non-adherent Bone marrow transplantation is a curative conditions to assess their ability to form treatment for many diseases, including leukemia, mammospheres. RESULTS: We demonstrate autoimmune diseases, and a number of embryonically-derived, long label-retaining cells immunodeficiencies. Recently, it was claimed that (eLLRCs) in the adult mammary gland. eLLRCs stain bone marrow cells transdifferentiate, a much desired for basal markers and are enriched within the property as bone marrow cells are abundant and mammary stem cell population identified by cell therefore could be used in regenerative medicine to sorting. eLLRCs are restricted to the primary ducts treat incurable chronic diseases. Using a Cre/loxP near the nipple region. Interestingly, long label system, we studied cell fusion after bone marrow retaining cells (labeled during puberty) are found just transplantation. Fused cells were chiefly Gr-1(+), a in front of the eLLRCs, near where the ends of the myeloid cell marker, and found predominantly in the ducts had been at the time of DNA labeling in early bone marrow; in parenchymal tissues. Surprisingly, puberty. A subset of eLLRCs becomes mitotically fused cells were most abundant in the kidney, Peyer's active during periods of mammary growth and in patches, and cardiac tissue. In contrast, after cell response to ovarian hormones. Finally, we show that fusion with embryonic stem cells, bone marrow cells eLLRCs are contained within primary and secondary were reprogrammed into new tetraploid pluripotent mammospheres. CONCLUSIONS: Our findings stem cells that successfully differentiated into beating suggest that a subset of proliferating embryonic cells cardiomyocytes. Together, these data suggest that cell subsequently becomes quiescent and contributes to the fusion is ubiquitous after cellular transplants and that pool of long-lived mammary stem cells in the adult. the subsequent sharing of genetic material between the eLLRCs can re-enter the cell cycle, produce both fusion partners affects cellular survival and function. mammary lineages and self-renew. Thus, our studies Fusion between tumor cells and bone marrow cells have identified a putative stem/progenitor cell could have consequences for tumor malignancy. population of embryonic origin. Further study of these cells will contribute to an understanding of how quiescent stem cells are generated during development

60 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ and how fetal exposures may alter future breast cancer autonomous capacity to thrive when the ERK pathway risk in adults. is inhibited arises late during blastocyst development and is lost after implantation. The frequency of Borghese, L., et al. (2010). "Inhibition of notch deriving clonal ESC lines suggests that all E4.5 signaling in human embryonic stem cell-derived epiblast cells can become ESCs. We further show that neural stem cells delays G1/S phase transition and ICM cells from early blastocysts can progress to ERK accelerates neuronal differentiation in vitro and in independence if provided with a specific laminin vivo." Stem Cells 28(5): 955-964. substrate. These findings suggest that formation of the The controlled in vitro differentiation of human epiblast coincides with competence for ERK- embryonic stem cells (hESCs) and other pluripotent independent self-renewal in vitro and consequent stem cells provides interesting prospects for generating propagation as ESC lines. large numbers of human neurons for a variety of biomedical applications. A major bottleneck Bose, B., et al. (2012). "Human embryonic stem associated with this approach is the long time required cell differentiation into insulin secreting beta-cells for for hESC-derived neural cells to give rise to mature diabetes." Cell Biol Int 36(11): 1013-1020. neuronal progeny. In the developing vertebrate hESC (human embryonic stem cells), when nervous system, Notch signaling represents a key differentiated into pancreatic beta ILC (islet-like regulator of neural stem cell (NSC) maintenance. Here, clusters), have enormous potential for the cell we set out to explore whether this signaling pathway transplantation therapy for Type 1 diabetes. We have can be exploited to modulate the differentiation of developed a five-step protocol in which the EBs hESC-derived NSCs (hESNSCs). We assessed the (embryoid bodies) were first differentiated into expression of Notch pathway components in hESNSCs definitive endoderm and subsequently into pancreatic and demonstrate that Notch signaling is active under lineage followed by formation of functional endocrine self-renewing culture conditions. Inhibition of Notch beta islets, which were finally matured efficiently activity by the gamma-secretase inhibitor N-[N-(3,5- under 3D conditions. The conventional cytokines difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl activin A and RA (retinoic acid) were used initially to ester (DAPT) in hESNSCs affects the expression of obtain definitive endoderm. In the last step, ILC were human homologues of known targets of Notch and of further matured under 3D conditions using amino acid several cell cycle regulators. Furthermore, DAPT- rich media (CMRL media) supplemented with anti- mediated Notch inhibition delays G1/S-phase hyperglycaemic hormone-Glp1 (glucagon-like peptide transition and commits hESNSCs to neurogenesis. 1) analogue Liraglutide with prolonged t ((1/2)) and Combined with growth factor withdrawal, inhibition of Exendin 4. The differentiated islet-like 3D clusters Notch signaling results in a marked acceleration of expressed bonafide mature and functional beta-cell differentiation, thereby shortening the time required markers-PDX1 (pancreatic and duodenal homoeobox- for the generation of electrophysiologically active 1), C-peptide, insulin and MafA. Insulin synthesis de hESNSC-derived neurons. This effect can be exploited novo was confirmed by C-peptide ELISA of culture for neural cell transplantation, where transient Notch supernatant in response to varying concentrations of inhibition before grafting suffices to promote the onset glucose as well as agonist and antagonist of functional of neuronal differentiation of hESNSCs in the host 3D beta islet cells in vitro. Our results indicate the tissue. Thus, interference with Notch signaling presence of almost 65% of insulin producing cells in provides a tool for controlling human NSC 3D clusters. The cells were also found to ameliorate differentiation both in vitro and in vivo. hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non-obese diabetic/severe Boroviak, T., et al. (2014). "The ability of inner- combined immunodeficiency) mouse up to 96 days of cell-mass cells to self-renew as embryonic stem cells transplantation. This protocol provides a basis for 3D is acquired following epiblast specification." Nat Cell in vitro generation of long-term in vivo functionally Biol 16(6): 516-528. viable islets from hESC. The precise relationship of embryonic stem cells (ESCs) to cells in the mouse embryo remains Bottai, D., et al. (2010). "Embryonic stem cells controversial. We present transcriptional and promote motor recovery and affect inflammatory cell functional data to identify the embryonic counterpart infiltration in spinal cord injured mice." Exp Neurol of ESCs. Marker profiling shows that ESCs are 223(2): 452-463. distinct from early inner cell mass (ICM) and closely The purpose of this study was to determine the resemble pre-implantation epiblast. A characteristic fate and the effects of undifferentiated embryonic stem feature of mouse ESCs is propagation without ERK cells (ESCs) in mice after contusive lesion of the signalling. Single-cell culture reveals that cell- spinal cord (SCI). Reproducible traumatic lesion to the

61 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cord was performed at T8 level by means of the Infinite Horizon Device, and was followed by Bourne, S., et al. (2004). "Osteogenic intravenous injection of one million of undifferentiated differentiation of mouse embryonic stem cells: ESCs through the tail vein within 2 h from the lesion. differential gene expression analysis by cDNA The ESCs-treated animals showed a significant microarray and purification of osteoblasts by cadherin- improvement of the recovery of motor function 28 11 magnetically activated cell sorting." Tissue Eng days after lesion, with an average score of 4.61+/-0.13 10(5-6): 796-806. points of the Basso Mouse Scale (n=14), when We have previously shown osteogenic compared to the average score of vehicle treated mice, differentiation of mouse embryonic stem (ES) cells 3.58+/-0.23 (n=10). The number of identified ESCs and temporal enrichment with osteoblastic cells, by found at the lesion site was 0.6% of the injected cells stimulation with serum-containing culture medium at 1 week after transplantation, and further reduced to supplemented with beta-glycerophosphate, ascorbate, 0.04% at 1 month. It is, thus, apparent that the and dexamethasone. In our present study we have used promoted hind-limb recovery cannot be correlated to a similar culture conditions to further investigate substitution of the lost tissue performed by the osteogenic differentiation of mouse ES cells. Using exogenous ESC. The extensive evaluation of reverse transcription-polymerase chain reaction (RT- production of several neuroprotective and PCR) we demonstrated the expression of genes inflammatory cytokines did not reveal any effect by associated with osteoblast differentiation including the ESC-treatment, but unexpectedly the number of bone matrix protein osteocalcin and the transcription invading macrophages and neutrophils was greatly factor Cbfa-1/. Furthermore, results of cDNA reduced. This may explain the improved preservation microarray analysis, and subsequent RT-PCR analysis of lesion site ventral myelin, at both 1 week (29+/- of differentiating ES cells after exposure to osteogenic 11%) and 1 month (106+/-14%) after injury. No stimuli, revealed a combination of upregulation of teratoma formation was observed, although an genes involved in osteoblast differentiation including inappropriate colonization of the sacral cord by osteopontin, HSP-47, and IGF-II coupled with differentiated nestin- and beta-tubulin III-positive downregulation of genes involved in differentiation of ESCs was detected. other phenotypes such as the neuroectoderm factor Stra-13. Finally, we have applied magnetically Boulanger, C. A., et al. (2013). "Embryonic stem activated cell-sorting methods to ES cell cultures cells are redirected to non-tumorigenic epithelial cell treated with osteogenic stimuli and, using an antibody fate by interaction with the mammary to cadherin-11, have purified a subpopulation of cells microenvironment." PLoS One 8(4): e62019. with osteoblastic characteristics. Experiments were conducted to redirect mouse Embryonic Stem (ES) cells from a tumorigenic Boyd, A. S., et al. (2005). "Transplanting stem phenotype to a normal mammary epithelial phenotype cells: potential targets for immune attack. Modulating in vivo. Mixing LacZ-labeled ES cells with normal the immune response against embryonic stem cell mouse mammary epithelial cells at ratios of 1:5 and transplantation." Adv Drug Deliv Rev 57(13): 1944- 1:50 in phosphate buffered saline and immediately 1969. inoculating them into epithelium-divested mammary The curative promise of stem cells and their fat pads of immune-compromised mice accomplished descendants for tissue regeneration and repair is this. Our results indicate that tumorigenesis occurs currently the subject of an intense research effort only when normal mammary ductal growth is not worldwide. If it proves feasible to differentiate stem achieved in the inoculated fat pads. When normal cells into specific tissues reliably and safely, this mammary gland growth occurs, we find ES cells approach will be invaluable in the treatment of (LacZ+) progeny interspersed with normal mammary diseases that lead to organ degeneration or failure, cell progeny in the mammary epithelial structures. We providing an alternative or supplementary source of demonstrate that these progeny, marked by LacZ tissue for transplantation. Embryonic stem (ES) cells expression, differentiate into multiple epithelial are pluripotent cells derived from the inner cell mass subtypes including steroid receptor positive luminal of a pre-implantation blastocyst that can produce all cells and myoepithelial cells indicating that the ES cells and tissues of the foetus. In recent years, several cells are capable of epithelial multipotency in this laboratories have described the directed differentiation context but do not form teratomas. In addition, in of ES cells into multiple mature cell types including: secondary transplants, ES cell progeny proliferate, cardiomyocytes; haemopoietic cells; hepatocytes; contribute apparently normal mammary progeny, neurones; muscle cells and both endocrine and maintain their multipotency and do not produce exocrine cells of the pancreas. How the immune teratomas. system of the host will respond when these ES cell-

62 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ derived mature cells are transplanted is ill defined. show that the derived cells do not form teratomas in This review will focus on the potential mechanisms SCID mice and independently derived lines show that the immune system could use to target ES cell- similar patterns of gene expression by microarray derived transplants and how unwanted responses analysis. When exposed to platelet-derived growth might be prevented. factor-BB, the platelet-derived growth factor receptor beta is activated and hES-MC migrate in response to a Boyd, A. S., et al. (2008). "A comparison of gradient. We also show that in a serum-free medium, protocols used to generate insulin-producing cell transforming growth factor beta1 (TGFbeta1) induces clusters from mouse embryonic stem cells." Stem robust expression of multiple contractile proteins Cells 26(5): 1128-1137. (alpha smooth muscle actin, smooth muscle myosin Embryonic stem cells (ESCs) have the capacity heavy chain, smooth muscle 22alpha, and calponin). to generate a panoply of tissue types and may therefore TGFbeta1 signaling is mediated through the provide an alternative source of tissue in regenerative TGFbetaR1/Alk5 pathway as demonstrated by medicine to treat potentially debilitating conditions inhibition of alpha smooth muscle actin expression by like Type 1 diabetes mellitus. However, the ability of treatment of the Alk5-specific inhibitor SB525334 and mouse ESCs to generate insulin-producing cell stable retroviral expression of the Alk5 dominant clusters (IPCCs) remains highly contentious. In an negative (K232R). Coculture of human umbilical vein attempt to clarify this issue, three protocols for the endothelial cell (HUVEC) with hES-MC maintains ESC-based generation of IPCCs (referred to as network integrity compared to HUVEC alone in three- Blyszczuk, Hori, and Lumelsky protocols) were dimensional collagen I-fibronectin by paracrine modified and evaluated for their ability to express signaling. Using high-resolution laser confocal pancreatic islet genes and proteins and their capacity microscopy, we show that hES-MC also make direct to function. Herein, we show that the Blyszczuk contact with HUVEC. This demonstrates that hESC- protocol reproducibly generated IPCCs with gene- derived mesenchymal cells possess the molecular expression characteristics that were qualitatively and machinery expected in a perivascular progenitor cells quantitatively most reminiscent of those found in and can play a functional role in stabilizing EC pancreatic islets. Furthermore, compared to the Hori networks in in vitro three-dimensional culture. and Lumelsky protocols, Blyszczuk-derived IPCCs exhibited superior expression of c-peptide, a by- Boyd, N. L., et al. (2013). "Dissecting the role of product of de novo insulin synthesis. Functionally, human embryonic stem cell-derived mesenchymal Blyszczuk IPCCs, in contrast to Hori and Lumelsky cells in human umbilical vein endothelial cell network IPCCs, were able to transiently restore normal blood stabilization in three-dimensional environments." glucose levels in diabetic mice (<1 week). Longer Tissue Eng Part A 19(1-2): 211-223. normoglycemic rescue (>2 weeks) was also achieved The microvasculature is principally composed of in a third of diabetic recipients receiving Blyszczuk two cell types: endothelium and mural support cells. IPCCs. Yet Blyszczuk IPCCs were less able to rescue Multiple sources are available for human endothelial experimental diabetes than isolated syngeneic cells (ECs) but sources for human microvascular mural pancreatic islet tissue. Therefore, depending on the cells (MCs) are limited. We derived multipotent mode of differentiation, ESCs can be driven to mesenchymal progenitor cells from human embryonic generate de novo IPCCs that possess limited stem cells (hES-MC) that can function as an MC and functionality. Further modifications to differentiation stabilize human EC networks in three-dimensional (3D) protocols will be essential to improve the generation of collagen-fibronectin culture by paracrine mechanisms. functional IPCCs from mouse ESCs. Here, we have investigated the basis for hES-MC- mediated stabilization and identified the pleiotropic Boyd, N. L., et al. (2011). "Microvascular mural growth factor hepatocyte growth factor/scatter factor cell functionality of human embryonic stem cell- (HGF/SF) as a putative hES-MC-derived regulator of derived mesenchymal cells." Tissue Eng Part A 17(11- EC network stabilization in 3D in vitro culture. 12): 1537-1548. Pharmacological inhibition of the HGF receptor (Met) Microvascular mural or perivascular cells are (1 mum SU11274) inhibits EC network formation in required for the stabilization and maturation of the the presence of hES-MC. hES-MC produce and remodeling vasculature. However, much less is known release HGF while human umbilical vein endothelial about their biology and function compared to large cells (HUVEC) do not. When HUVEC are cultured vessel smooth muscle cells. We have developed lines alone the networks collapse, but in the presence of of multipotent mesenchymal cells from human recombinant human HGF or conditioned media from embryonic stem cells (hES-MC); we hypothesize that human HGF-transduced cells significantly more these can function as perivascular mural cells. Here we networks persist. In addition, HUVEC transduced to

63 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ constitutively express human HGF also form stable networks by autocrine mechanisms. By enzyme-linked Brederlau, A., et al. (2006). "Transplantation of immunosorbent assay, the coculture media were human embryonic stem cell-derived cells to a rat enriched in both angiopoietin-1 (Ang1) and model of Parkinson's disease: effect of in vitro angiopoietin-2 (Ang2), but at significantly different differentiation on graft survival and teratoma levels (Ang1=159+/-15 pg/mL vs. Ang2=30,867+/- formation." Stem Cells 24(6): 1433-1440. 2685 pg/mL) contributed by hES-MC and HUVEC, Human embryonic stem cells (hESCs) have been respectively. Although the coculture cells formed proposed as a source of dopamine (DA) neurons for stabile network architectures, their morphology transplantation in Parkinson's disease (PD). We have suggests the assembly of an immature plexus. When investigated the effect of in vitro predifferentiation on HUVEC and hES-MC were implanted subcutaneously in vivo survival and differentiation of hESCs in immune compromised Rag1 mice, hES-MC implanted into the 6-OHDA (6-hydroxydopamine)- increased their contact with HUVEC along the axis of lesion rat model of PD. The hESCs were cocultured the vessel. This data suggests that HUVEC and hES- with PA6 cells for 16, 20, or 23 days, leading to the in MC form an immature plexus mediated in part by vitro differentiation into DA neurons. Grafted hESC- HGF and angiopoietins that is capable of maturation derived cells survived well and expressed neuronal under the correct environmental conditions (e.g., in markers. However, very few exhibited a DA neuron vivo). Therefore, hES-MC can function as phenotype. Reversal of lesion-induced motor deficits microvascular MCs and may be a useful cell source for was not observed. Rats grafted with hESCs testing EC-MC interactions. predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs Boyd, N. L., et al. (2009). "Human embryonic predifferentiated for 20 and 23 days remained healthy stem cell-derived mesoderm-like epithelium until the end of the experiment. This indicates that transitions to mesenchymal progenitor cells." Tissue prolonged in vitro differentiation of hESCs is essential Eng Part A 15(8): 1897-1907. for preventing formation of teratomas. Human embryonic stem cells (hESC) have the potential to produce all of the cells in the body. They Brehany, J. (2005). "Nontraditional sources of are able to self-renew indefinitely, potentially making pluripotent stem cells: a new chapter in the debate them a source for large-scale production of therapeutic about embryonic stem cell research." Health Care cell lines. Here, we developed a monolayer Ethics USA 13(2): E3. differentiation culture that induces hESC (WA09 and Recent efforts to resolve the political impasse BG01) to form epithelial sheets with mesodermal gene over human embryonic stem cells (ESC) have expression patterns (BMP4, RUNX1, and GATA4). generated proposals for obtaining ESC while avoiding These E-cadherin+ CD90low cells then undergo the destruction of human embryos. This new chapter in apparent epithelial-mesenchymal transition for the the scientific and ethical debate provides an important derivation of mesenchymal progenitor cells (hESC- opportunity to introduce additional ethical derived mesenchymal cells [hES-MC]) that by flow considerations to enhance public discourse. cytometry are negative for hematopoietic (CD34, CD45, and CD133) and endothelial (CD31 and CD146) Brochner, C. B., et al. (2012). "YKL-40 is markers, but positive for markers associated with differentially expressed in human embryonic stem mesenchymal stem cells (CD73, CD90, CD105, and cells and in cell progeny of the three germ layers." J CD166). To determine their functionality, we tested Histochem Cytochem 60(3): 188-204. their capacity to produce the three lineages associated The secreted glycoprotein YKL-40 participates in with mesenchymal stem cells and found they could cell differentiation, inflammation, and cancer form osteogenic and chondrogenic, but not adipogenic progression. High YKL-40 expression is reported lineages. The derived hES-MC were able to remodel during early human development, but its functions are and contract collagen I lattice constructs to an unknown. Six human embryonic stem cell (hESC) equivalent degree as keloid fibroblasts and were lines were cultured in an atmosphere of low or high induced to express alpha-smooth muscle actin when oxygen tension, in culture medium with or without exposed to transforming growth factor (TGF)-beta1, basic fibroblast growth factor, and on feeder layers but not platelet derived growth factor-B (PDGF-B). comprising mouse embryonic fibroblasts or human These data suggest that the derived hES-MC are foreskin fibroblasts to evaluate whether hESCs and multipotent cells with potential uses in tissue their progeny produced YKL-40 and to characterize engineering and regenerative medicine and for YKL-40 expression during differentiation. Secreted providing a highly reproducible cell source for adult- YKL-40 protein and YKL-40 mRNA expression were like progenitor cells. measured by enzyme-linked immunosorbent assay

64 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

(ELISA) and quantitative RT-PCR. Serial-sectioned their capacity to treat type 2 diabetes has not been colonies were stained for YKL-40 protein and for reported. An immunodeficient model of type 2 pluripotent hESC (OCT4, NANOG) and germ layer diabetes was generated by high-fat diet (HFD) feeding (HNF-3beta, PDX1, CD34, p63, nestin, PAX6) in SCID-beige mice. Exposure to HFDs did not impact markers. Double-labeling showed YKL-40 expression the maturation of macroencapsulated pancreatic in OCT4-positive hESCs, PAX6-positive progenitor cells into glucose-responsive insulin- neuroectodermal cells, and HNF-3beta-positive secreting cells following transplantation, and the cell endodermal cells. The differentiating progeny showed therapy improved glucose tolerance in HFD-fed strong YKL-40 expression. Abrupt transition between transplant recipients after 24 weeks. However, since YKL-40 and OCT4-positive hESCs and YKL-40- diet-induced hyperglycemia and obesity were not fully positive ecto- and neuroectodermal lineages was ameliorated by transplantation alone, a second cohort observed within the same epithelial-like layer. YKL- of HFD-fed mice was treated with pancreatic 40-positive cells within deeper layers lacked contact progenitor cells combined with one of three with OCT4-positive cells. YKL-40 may be important antidiabetic drugs. All combination therapies rapidly in initial cell differentiation from hESCs toward improved body weight and co-treatment with either ectoderm and neuroectoderm, with retained epithelial sitagliptin or metformin improved hyperglycemia after morphology, whereas later differentiation into only 12 weeks. Therefore, a stem cell-based therapy endoderm and mesoderm involves a transition into the may be effective for treating type 2 diabetes, deeper layers of the colony. particularly in combination with antidiabetic drugs.

Brolen, G. K., et al. (2005). "Signals from the Brzeszczynska, J., et al. (2014). "Differentiation embryonic mouse pancreas induce differentiation of and molecular profiling of human embryonic stem human embryonic stem cells into insulin-producing cell-derived corneal epithelial cells." Int J Mol Med beta-cell-like cells." Diabetes 54(10): 2867-2874. 33(6): 1597-1606. The recent success in restoring normoglycemia in It has been suggested that the isolation of type 1 diabetes by islet cell transplantation indicates scalable populations of limbal stem cells may lead to that cell replacement therapy of this severe disease is radical changes in ocular therapy. In particular, the achievable. However, the severe lack of donor islets derivation and transplantation of corneal stem cells has increased the demand for alternative sources of from these populations may result in therapies beta-cells, such as adult and embryonic stem cells. providing clinical normality of the diseased or Here, we investigate the potential of human embryonic damaged cornea. Although feasible in theory, the lack stem cells (hESCs) to differentiate into beta-cells. of donor material in sufficient quantity and quality Spontaneous differentiation of hESCs under two- currently limits such a strategy. A potential scalable dimensional growth conditions resulted in source of corneal cells could be derived from differentiation of Pdx1(+)/Foxa2(+) pancreatic pluripotent stem cells (PSCs). We developed an in progenitors and Pdx1(+)/Isl1(+) endocrine progenitors vitro and serum-free corneal differentiation model but no insulin-producing cells. However, which displays significant promise. Our stepwise cotransplantation of differentiated hESCs with the differentiation model was designed with reference to dorsal pancreas, but not with the liver or telencephalon, development and gave rise to cells which displayed from mouse embryos resulted in differentiation of similarities to epithelial progenitor cells which can be beta-cell-like cell clusters. Comparative analysis of the specified to cells displaying a corneal epithelial basic characteristics of hESC-derived insulin (+) cell phenotype. We believe our approach is novel, provides clusters with human adult islets demonstrated that the a robust model of human development and in the insulin (+) cells share important features with normal future, may facilitate the generation of corneal beta-cells, such as synthesis (proinsulin) and epithelial cells that are suitable for clinical use. processing (C-peptide) of insulin and nuclear Additionally, we demonstrate that following continued localization of key beta-cell transcription factors, cell culture, stem cell-derived corneal epithelial cells including Foxa2, Pdx1, and Isl1. undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model Bruin, J. E., et al. (2015). "Treating diet-induced of disease. diabetes and obesity with human embryonic stem cell- derived pancreatic progenitor cells and antidiabetic Burdon, T., et al. (2002). "Signalling, cell cycle drugs." Stem Cell Reports 4(4): 605-620. and pluripotency in embryonic stem cells." Trends Human embryonic stem cell (hESC)-derived Cell Biol 12(9): 432-438. pancreatic progenitor cells effectively reverse Pluripotent mouse embryonic stem (ES) cells can hyperglycemia in rodent models of type 1 diabetes, but be expanded in large numbers in vitro owing to a

65 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ process of symmetrical self-renewal. Self-renewal later time points. CONCLUSIONS: We demonstrate entails proliferation with a concomitant suppression of the utility of NADH fluorescence intensity as a non- differentiation. Here we describe how the cytokine invasive indicator of cell death in stem cell aggregates leukaemia inhibitory factor (LIF) sustains self-renewal when measured using multi-photon excitation. In through activation of the transcription factor STAT3, addition, we show that the degree of stem cell death at and how two other signals - extracellular-signal- early stages of differentiation is predictive for the related kinase (ERK) and phosphatidylinositol-3-OH formation of functional cardiomyocytes. kinase (PI3K) - can influence differentiation and propagation, respectively. We relate these observations Byrne, J. A., et al. (2007). "Producing primate to the unusual cell-cycle properties of ES cells and embryonic stem cells by somatic cell nuclear transfer." speculate on the role of the cell cycle in maintaining Nature 450(7169): 497-502. pluripotency. Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell Busch, C., et al. (2007). "Embryonic stem cells in nuclear transfer (SCNT) holds the potential to cure or human sacrococcygeal teratomas: Isolation and alleviate the symptoms of many degenerative diseases characterization of an embryonic stem cell line." J while circumventing concerns regarding rejection by Stem Cells Regen Med 2(1): 76. the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient Buschke, D. G., et al. (2012). "Cell death, non- reprogramming and poor embryonic development invasively assessed by intrinsic fluorescence intensity characterizes the results obtained in primates. Here, we of NADH, is a predictive indicator of functional used a modified SCNT approach to produce rhesus differentiation of embryonic stem cells." Biol Cell macaque blastocysts from adult skin fibroblasts, and 104(6): 352-364. successfully isolated two ES cell lines from these BACKGROUND INFORMATION: Continued embryos. DNA analysis confirmed that nuclear DNA advances in stem cell biology and stem cell was identical to donor somatic cells and that transplantation rely on non-invasive biomarkers to mitochondrial DNA originated from oocytes. Both cell characterise cells and stem cell aggregates. The non- lines exhibited normal ES cell morphology, expressed invasive quality of such biomarkers is essential key stem-cell markers, were transcriptionally similar because exogenous labels, probes or reporters can to control ES cells and differentiated into multiple cell unintentionally and dramatically alter stem cell state as types in vitro and in vivo. Our results represent can disruption of cell-cell and cell-matrix interactions. successful nuclear reprogramming of adult somatic Here, we investigate the utility of the autofluorescent cells into pluripotent ES cells and demonstrate proof- metabolite, nicotinamide adenine dinucleotide of-concept for therapeutic cloning in primates. (NADH), as a non-invasive, intrinsic biomarker of cell death when detected with multi-photon optical-based Calderon, D., et al. (2012). "Immune response to approaches. To test this possibility, cell death was human embryonic stem cell-derived cardiac induced in murine embryoid bodies (EBs) at an early progenitors and adipose-derived stromal cells." J Cell stage (day 3) of differentiation using staurosporine, an Mol Med 16(7): 1544-1552. ATP-competitive kinase inhibitor of electron transport. Transplantation of allogeneic human embryonic Several hours after staurosporine treatment, EBs were stem cell-derived cardiac progenitors triggers an stained with a single-colour, live/dead probe. A single- immune response. We assessed whether this response cross-sectional plane of each EB was imaged to detect could be modulated by the concomitant use of the fluorescence intensity of the live/dead probe adipose-derived stromal cells (ADSC). Peripheral (extrinsic fluorescence) as well as the fluorescence blood mononuclear cells were collected from 40 intensity of NADH (intrinsic fluorescence). EBs were patients with coronary artery disease (CAD) and nine assessed at subsequent time points (days 6-12) for the healthy controls. Cardiac progenitors (CD15(+) formation of beating areas as an indicator of functional Mesp1(+)) were generated as already reported from differentiation. RESULTS: Statistical comparison the I6 cell line treated with bone morphogenetic indicated a strong positive correlation between protein (BMP)-2. Adipose-derived stromal cells were extrinsic fluorescence intensity of the live/dead stain obtained from abdominal dermolipectomies. We and intrinsic fluorescence of NADH, suggesting that assessed the proliferative response of peripheral the intensity of NADH fluorescence could be used to lymphocytes from patients and controls to cardiac reliably and non-invasively assess death of cells of progenitors cultured on a monolayer of ADSC, to EBs. Furthermore, EBs that had high levels of cell allogeneic lymphocytes in mixed lymphocyte culture death soon after aggregate formation had limited and to the T cell mitogen phytohemaglutin A in ability to give rise to functional cardiomyocytes at presence or absence of ADSC. Cardiac progenitors

66 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cultured on a monolayer of ADSC triggered a identifying possibilities for the modulation of these proliferation of lymphocytes from both patients and molecular pathways should allow the in vitro controls albeit lower than that induced by allogeneic expansion of HSPCs for a multitude of therapeutic lymphocytes. When cultured alone, ADSC did not uses. induce any proliferation of allogeneic lymphocytes. When added to cultures of lymphocytes, ADSC Carr, A. J., et al. (2009). "Molecular significantly inhibited the alloantigen or mitogen- characterization and functional analysis of induced proliferative response. Compared to healthy phagocytosis by human embryonic stem cell-derived controls, lymphocytes from patients presenting CAD RPE cells using a novel human retinal assay." Mol Vis expressed a decreased proliferative capacity, in 15: 283-295. particular to mitogen-induced stimulation. Adipose- PURPOSE: To examine the ability of retinal derived stromal cells express an immunomodulatory pigment epithelial (RPE) cells derived from human effect that limits both alloantigen and mitogen-induced embryonic stem cells (HESC) to phagocytose lymphocyte responses. Furthermore, lymphocytes photoreceptor outer segments, and to determine from patients with CAD are low responders to whether exposure to human retina induces any conventional stimuli, possibly because of their age and morphological changes in these cells. METHODS: disease-associated treatment regimens. We propose HESC-RPE cells were derived from a super-confluent that, in combination, these factors may limit the in preparation of the Shef1 HESC line. Pigmented vivo immunogenicity of cardiac progenitors co- colonies were isolated and expanded into pigmented implanted with ADSC in patients with CAD. monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer Campbell, C., et al. (2015). "Zebrafish embryonic segments isolated from the porcine eye and assessed stromal trunk (ZEST) cells support hematopoietic stem for phagocytic activity at regular intervals. Expression and progenitor cell (HSPC) proliferation, survival, and of molecules associated with RPE phagocytosis was differentiation." Exp Hematol 43(12): 1047-1061. analyzed by RT-PCR, immunocytochemistry, and Forward genetic screens in zebrafish have been western blot. The role of Mer Tyrosine Kinase used to identify genes essential for the generation of (MERTK) in the phagocytosis of outer segments was primitive blood and the emergence of hematopoietic investigated using antibodies directed against MERTK stem cells (HSCs), but have not elucidated the genes to block function. In a novel approach, cells were also essential for hematopoietic stem and progenitor cell exposed to fresh human neural retina tissue then (HSPC) proliferation and differentiation because of the examined by electron microscopy for evidence of lack of methodologies to functionally assess these phagocytosis and changes in cell morphology. processes. We previously described techniques used to RESULTS: HESC-derived RPE cells are capable of test the developmental potential of HSPCs by culturing phagocytosing isolated porcine outer segments and them on zebrafish kidney stromal (ZKS) cells, derived express molecules associated with RPE-specific from the main site of hematopoiesis in the adult teleost. phagocytosis, including MERTK. Pre-incubation with Here we describe an additional primary stromal cell antibodies against MERTK blocked phagocytosis of line we refer to as zebrafish embryonic stromal trunk photoreceptor outer segments, but not polystyrene (ZEST) cells, derived from tissue surrounding the beads. HESC-RPE cells also phagocytosed outer embryonic dorsal aorta, the site of HSC emergence in segments in a novel human retinal explant system. developing fish. ZEST cells encouraged HSPC Furthermore co-culture adjacent to human retina tissue differentiation toward the myeloid, lymphoid, and in this preparation resulted in the appearance of erythroid pathways when assessed by morphologic and features in HESC-derived RPE cells normally quantitative reverse transcription polymerase chain observed only as the RPE matures. CONCLUSIONS: reaction analyses. Additionally, ZEST cells The ingestion of photoreceptor outer segments from an significantly expanded the number of cultured HSPCs isolated population and an artificial ex vivo human in vitro, indicating that these stromal cells are retina system demonstrates HESC-derived RPE cells supportive of both HSPC proliferation and are functional. HESC-derived RPE possess the multilineage differentiation. Examination of ZEST relevant molecules required for phagocytosis, cells indicates that they express numerous cytokines including MERTK, which is essential for the and Notch ligands and possess endothelial phagocytosis of outer segments but not latex beads. characteristics. Further characterization of ZEST cells Furthermore, some changes observed in cell should prove to be invaluable in understanding the morphology after co-culture with human retina may complex signaling cascades instigated by the have implications for understanding the full embryonic hematopoietic niche required to expand and development and differentiation of RPE cells. differentiate HSPCs. Elucidating these processes and

67 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

Caspi, O. and L. Gepstein (2006). "Regenerating reversal in vivo, and by elevated expression of the heart using human embryonic stem cells--from cell pancreatic endocrine makers, beta-cell differentiation to bedside." Isr Med Assoc J 8(3): 208-214. yield, and insulin production in vitro. Our studies The adult human heart has limited regenerative confirm the importance of oxygen modulation as a key capacity and, therefore, functional restoration of the variable to consider in the design of beta-cell damaged heart presents a great challenge. Despite the differentiation protocols and open the door to future progress achieved in the pharmacological and surgical strategies for the transplantation of fully mature beta treatment of degenerative myocardial diseases, they cells. are still considered a major cause of morbidity and mortality in the western world. Repopulation of the Cerdan, C., et al. (2006). "Complement targeting damaged heart with cardiomyocytes represents a novel of nonhuman sialic acid does not mediate cell death of conceptual therapeutic paradigm but is hampered by human embryonic stem cells." Nat Med 12(10): 1113- the lack of sources for human cardiomyocytes. The 1114; author reply 1115. recent derivation of pluripotent human embryonic stem cell lines may provide a solution for this cell Chaddah, R., et al. (2012). "Clonal neural stem sourcing problem. This review will focus on the cells from human embryonic stem cell colonies." J derivation of the hESC lines, their mechanism of self- Neurosci 32(23): 7771-7781. renewal, and their differentiation to cardiomyocytes. Clonal cell culture is crucial for experimental The possible signals and cues involved in the protocols that require growth or selection of pure commitment and early differentiation of populations of cells. High-density derivation of neural cardiomyocytes in this model will be discussed as well progenitors from human embryonic stem cells (hESCs) as the molecular, structural and electrophysiologic can lead to incomplete differentiation, and characteristics of the generated hESC-derived transplantation of resulting heterogeneous cell cardiomyocytes. Finally, the hurdles and challenges mixtures can cause proliferation of tumorigenic toward fully harnessing the potential clinical clusters in vivo. We have identified the neural applications of these unique cells will be described. precursor that resides among normal hESC colonies as a TRA-1-60(-)/SSEA4(-)/SOX1(+) cell and developed Cechin, S., et al. (2014). "Influence of in vitro a method that allows for the clonal expansion of these and in vivo oxygen modulation on beta cell FACS-selected progenitors to neural stem cells (NSCs) differentiation from human embryonic stem cells." in serum-free conditions. Single TRA-1-60(- Stem Cells Transl Med 3(3): 277-289. )/SSEA4(-)/SOX1(+) cells grown in serum-free media The possibility of using human embryonic stem give rise to multipotent NSCs with an efficiency of (hES) cell-derived beta cells as an alternative to 0.7%. The fate of the TRA-1-60(-)/SSEA4(-)/SOX1(+) cadaveric islets for the treatment of type 1 diabetes is neural precursor becomes specified in maintenance now widely acknowledged. However, current conditions by inhibition of BMP signaling. This clonal differentiation methods consistently fail to generate culture method can be scaled up to produce NSCs for meaningful numbers of mature, functional beta cells. differentiation and use in cell therapies. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of Chaerkady, R., et al. (2011). "Quantitative pancreatic progenitor (PP) cells differentiated from temporal proteomic analysis of human embryonic stem hES cells. We have previously determined that cell differentiation into oligodendrocyte progenitor oxygenation is a powerful driver of murine PP cells." Proteomics 11(20): 4007-4020. differentiation along the endocrine lineage of the Oligodendrocytes (OLs) are glial cells of the pancreas. We hypothesized that targeting central nervous system, which produce myelin. physiological oxygen partial pressure (pO2) levels Cultured OLs provide immense therapeutic seen in mature islets would help the differentiation of opportunities for treating a variety of neurological PP cells along the beta-cell lineage. This hypothesis conditions. One of the most promising sources for was tested both in vivo (by exposing PP-transplanted such therapies is human embryonic stem cells (ESCs) immunodeficient mice to a daily hyperbaric oxygen as well as providing a model to study human OL regimen) and in vitro (by allowing PP cells to mature development. For these purposes, an investigation of in a perfluorocarbon-based culture device designed to proteome level changes is critical for understanding carefully adjust pO2 to a desired range). Our results the process of OL differentiation. In this report, an show that oxygen modulation does indeed contribute iTRAQ-based quantitative proteomic approach was to enhanced maturation of PP cells, as evidenced by used to study multiple steps during OL differentiation improved engraftment, segregation of alpha and beta including neural progenitor cells, glial progenitor cells cells, body weight maintenance, and rate of diabetes and oligodendrocyte progenitor cells (OPCs)

68 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ compared to undifferentiated ESCs. Using a 1% false Maturation State of a Population by Quantifying discovery rate cutoff, approximately 3145 proteins Parameters in Individual Cells." Stem Cells Int 2015: were quantitated and several demonstrated progressive 586908. stage-specific expression. Proteins such as transferrin, Quantitative methods were established to neural cell adhesion molecule 1, apolipoprotein E and determine the level of maturation of human embryonic wingless-related MMTV integration site 5A showed stem cell-derived ventricular cardiomyocytes (hESC- increased expression from the neural progenitor cell to vCMs) that were treated with different metabolic the OPC stage. Several proteins that have stimulants (i.e., isoproterenol and oleic acid) during demonstrated evidence or been suspected in OL early differentiation. Cells were double- maturation were also found upregulated in OPCs immunolabeled with alpha-actinin and COX IV including fatty acid-binding protein 4, THBS1, bone antibodies, to label the myofibrils and mitochondria, morphogenetic protein 1, CRYAB, transferrin, respectively, after which images were acquired via tenascin C, COL3A1, TGFBI and EPB41L3. Thus, by confocal microscopy. In order to determine the extent providing the first extensive proteomic profiling of of differentiation, image analysis protocols were then human ESC differentiation into OPCs, this study used to quantify cell shape and area, as well as the provides many novel proteins that are potentially degree of myofibrillar organization and intercalation involved in OL development. of mitochondria between the myofibrils within the cells. We demonstrated that oleic acid or isoproterenol Chambers, C. A., et al. (1994). "Exogenous Mtv- alone, or a combination of the two, induced a more 7 superantigen transgene expression in major elongated hESC-vCM phenotype than the untreated histocompatibility complex class II I-E- mice controls. In addition, cells treated with isoproterenol reconstituted with embryonic stem cell-derived alone exhibited a similar level of myofibrillar hematopoietic stem cells." Proc Natl Acad Sci U S A organization as the controls, but those treated with 91(3): 1138-1142. oleic acid with/without isoproterenol exhibited a more Direct genetic manipulation of hematopoietic organized (parallel) orientation of myofibrils. The cells is limited by the lack of an established combined isoproterenol/oleic acid treatment also hematopoietic stem cell line. It has been demonstrated resulted in enhanced intercalation of mitochondria that embryonic stem (ES) cell<-->tetraploid embryos between the myofibrils. We suggest that these are completely ES cell-derived and that fetal liver (FL) quantitative morphometric methods might serve as cells from these embryos support hematopoiesis in simple and effective tools that can be utilized in the lethally irradiated recipients. In this report, we determination of the level of structural maturation of demonstrate that FL cells from ES cell<-->tetraploid hESC-vCMs. embryos support normal lymphopoiesis and T-cell repertoire development. Moreover, the introduction of Chan, K. M., et al. (2008). "Hematopoiesis and the Mtv-7 superantigen transgene coding for minor immunity of HOXB4-transduced embryonic stem cell- lymphocyte stimulatory antigen 1 into murine derived hematopoietic progenitor cells." Blood 111(6): hematopoietic cells via reconstitution with ES cell<-- 2953-2961. >tetraploid FL cells demonstrates that this method can The ability of embryonic stem (ES) cells to form effectively confer stable genetic changes into the cells and tissues from all 3 germ layers can be hematopoietic tissues without going through the germ exploited to generate cells that can be used to treat line. Long-term and secondary reconstitution with ES diseases. In particular, successful generation of cell<-->tetraploid FL cells expressing the Mtv-7 hematopoietic cells from ES cells could provide safer superantigen transgene clonally deleted minor and less immunogenic cells than bone marrow cells, lymphocyte stimulatory antigen 1-reactive T-cell which require severe host preconditioning when receptor V beta 6+, -8.1+, and -9+ T cells, but not V transplanted across major histocompatibility complex beta 7+ T cells, in H-2b (I-E-) mice. This model barriers. Here, we exploited the self-renewal properties system will be extremely important for analyzing of ectopically expressed HOXB4, a structure-function relationships of molecules involved transcription factor, to generate hematopoietic in proliferation, differentiation, and selection of progenitor cells (HPCs) that successfully induce high- hematopoietic cells in vivo and for examining level mixed chimerism and long-term engraftment in hematopoiesis-specific effects of mutations that are recipient mice. The HPCs partially restored splenic lethal during embryogenesis. architecture in Rag2(-/-)gamma (c) (-/-)- immunodeficient mice. In addition, HPC-derived Chan, H. Y., et al. (2015). "Morphometric newly generated T cells were able to mount a peptide- Analysis of Human Embryonic Stem Cell-Derived specific response to lymphocytic choriomeningitis Ventricular Cardiomyocytes: Determining the virus and specifically secreted interleukin-2 and

69 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ interferon-gamma upon CD3 stimulation. In addition, PRDM14 is an important determinant of the HPC-derived antigen presenting cells in chimeric mice human embryonic stem cell (ESC) identity and works efficiently presented viral antigen to wild-type T cells. in concert with the core ESC regulators to activate These results demonstrate for the first time that pluripotency-associated genes. PRDM14 has been leukocytes derived from ES cells ectopically previously reported to exhibit repressive activity in expressing HOXB4 are immunologically functional, mouse ESCs and primordial germ cells; and while opening up new opportunities for the use of ES cell- PRDM14 has been implicated to suppress derived HPCs in the treatment of hematologic and differentiation genes in human ESCs, the exact immunologic diseases. mechanism of this repressive activity remains unknown. In this study, we provide evidence that Chan, K. M., et al. (2013). "Hepatic stellate cells PRDM14 is a direct repressor of developmental genes promote the differentiation of embryonic stem cell- in human ESCs. PRDM14 binds to silenced genes in derived definitive endodermal cells into hepatic human ESCs and its global binding profile is enriched progenitor cells." Hepatol Res 43(6): 648-657. for the repressive trimethylation of histone H3 lysine AIM: Hepatic non-parenchymal cells are well 27 (H3K27me3) modification. Further investigation known to be capable of providing an important reveals that PRDM14 interacts directly with the microenvironment and growth factors for hepatic chromatin regulator polycomb repressive complex 2 regeneration, but their capacity for directing (PRC2) and PRC2 binding is detected at PRDM14- embryonic stem cells (ESC) toward hepatocytes bound loci in human ESCs. Depletion of PRDM14 remains to be assessed. Thus, this study aims to reduces PRC2 binding at these loci and the investigate the role of hepatic stellate cells (HSC), the concomitant reduction of H3K27me3 modification. major type of hepatic non-parenchymal cells, in the Using reporter assays, we demonstrate that gene loci differentiation of ESC as well as exploring the bound by PRDM14 exhibit repressive activity that is potentiality of ESC in regeneration medicine for cell- dependent on both PRDM14 and PRC2. In based therapy. METHODS: A two-step differentiation reprogramming human fibroblasts into induced procedure that utilized the capability of HSC to pluripotent stem cells (iPSCs), ectopically expressed regulate proliferation and differentiation of PRDM14 can repress these developmental genes in hepatocytes was used to develop an approach for fibroblasts. In addition, we show that PRDM14 directing the differentiation of ESC towards hepatic recruits PRC2 to repress a key mesenchymal gene progenitor cells. Mouse ESC were cultivated in a ZEB1, which enhances mesenchymal-to-epithelial serum-free medium containing Activin A and transition in the initiation event of iPSC fibroblast growth factor to generate definitive reprogramming. In summary, our study reveals a endodermal cells characterized by the CXCR4 cell- repressive role of PRDM14 in the maintenance and surface marker. After 6-8 days in culture, induction of pluripotency and identifies PRDM14 as a approximately 60% of the differentiated cells new regulator of PRC2. expressed CXCR4, and more than 90% of the CXCR4 positive cells could be recovered by cell sorting. The Chandrasekar, M. P., et al. (2010). "The role of purified CXCR4 positive cells were co-cultured with AMP Kinase on the beta-cell differentiation of mouse mouse HSC as feeder cells in basal medium without embryonic stem cells." J Stem Cells Regen Med 6(2): additional hepatocyte growth factors. Differentiation 55. was complete after 10-12 days of co-culture, and hepatic progenitor cell markers such as alpha- Chang, D. J., et al. (2013). "Contralaterally fetoprotein (afp) and albumin (alb) were detected in transplanted human embryonic stem cell-derived the terminally differentiated ESC. CONCLUSION: neural precursor cells (ENStem-A) migrate and These results show that HSC provide an appropriate improve brain functions in stroke-damaged rats." Exp microenvironment and pivotal growth factors for Mol Med 45: e53. generation of hepatic progenitor cells from ESC- The transplantation of neural precursor cells derived definitive endodermal cells, and suggest that (NPCs) is known to be a promising approach to this approach possibly allows for hepatic ameliorating behavioral deficits after stroke in a rodent differentiation of ESC imitating the process of hepatic model of middle cerebral artery occlusion (MCAo). regeneration. Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and Chan, Y. S., et al. (2013). "A PRC2-dependent differentiate into mature neurons to some extent. repressive role of PRDM14 in human embryonic stem However, the spatiotemporal dynamics of NPC cells and induced pluripotent stem cell migration following transplantation into stroke animals reprogramming." Stem Cells 31(4): 682-692. have yet to be elucidated. In this study, we

70 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ investigated the fates of human embryonic stem cell that contained the beta (A)-globin gene. We obtained (hESC)-derived NPCs (ENStem-A) for 8 weeks ES cells in which the beta (S) was corrected to the beta following transplantation into the side contralateral to (A) sequence. Hematopoietic cells differentiated from the infarct region using 7.0T animal magnetic these ES cells produced both hemoglobin A and resonance imaging (MRI). T2- and T2*-weighted MRI hemoglobin S. This approach can be applied to human analyses indicated that the migrating cells were clearly ES cells to correct the sickle mutation as well as beta- detectable at the infarct boundary zone by 1 week, and thalassemia mutations. the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the Chang, T. C., et al. (2010). "Rho kinases regulate signals were slightly increased or unchanged. At 8 the renewal and neural differentiation of embryonic weeks, we performed Prussian blue staining and stem cells in a cell plating density-dependent manner." immunohistochemical staining using human-specific PLoS One 5(2): e9187. markers, and found that high percentages of BACKGROUND: Rho kinases (ROCKs) mediate transplanted cells migrated to the infarct boundary. cell contraction, local adhesion, and cell motility, Most of these cells were CXCR4-positive. We also which are considered to be important in cell observed that the migrating cells expressed markers differentiation. We postulated that ROCKs are for various stages of neural differentiation, including involved in controlling embryonic stem (ES) cell Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, renewal and differentiation. indicating that the transplanted cells may partially METHODOLOGY/PRINCIPAL FINDINGS: CCE, a contribute to the reconstruction of the damaged neural murine ES cell, was treated with Y-27632 for 48 to 96 tissues after stroke. Interestingly, we found that the hours and colony formation was evaluated. Y-27632 extent of gliosis (glial fibrillary acidic protein-positive blocked CCE colony formation and induced CCE to cells) and apoptosis (TUNEL-positive cells) were grow as individual cells, regardless of the initial significantly decreased in the cell-transplanted group, seeding cell density either at 10(4)/cm (2) ("high" suggesting that hESC-NPCs have a positive role in seeding density) or 2x10(3)/cm (2) ("low" density). reducing glia scar formation and cell death after stroke. However, at high seeding density, Y-27632-treated No tumors formed in our study. We also performed cells exhibited reduction of alkaline phosphatase (AP) various behavioral tests, including rotarod, stepping staining and Oct3/4 expression. They expressed SOX- and modified neurological severity score tests, and 1, nestin, and MAP2c, but not betaIII-tubulin or NG-2. found that the transplanted animals exhibited They did not express endoderm or mesoderm lineage significant improvements in sensorimotor functions markers. After removal of Y-27632, the cells failed to during the 8 weeks after transplantation. Taken form colonies or regain undifferentiated state. together, these results strongly suggest that hESC- Silencing of ROCK-1 or ROCK-2 with selective small NPCs have the capacity to migrate to the infarct region, interference RNA induced CCE morphological form neural tissues efficiently and contribute to changes similar to Y-27632. Silencing of ROCK-1 or behavioral recovery in a rodent model of ischemic ROCK-2 individually was sufficient to cause reduction stroke. of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs Chang, J. C., et al. (2006). "Correction of the did not augment the effects exerted by individual sickle cell mutation in embryonic stem cells." Proc ROCK siRNA. Y-27632-treated CCE cells seeded at Natl Acad Sci U S A 103(4): 1036-1040. 2x10(3) or 6.6x10(3) cells/cm (2) did not lose renewal Sickle cell anemia is one of the most common factors or express differentiation markers. Furthermore, genetic diseases worldwide. Patients often suffer from they were able to form AP-positive colonies after anemia, painful crises, infections, strokes, and removal of Y-27632 and reseeding. Similar to ROCK cardiopulmonary complications. Although current inhibition by Y-27632, silencing of ROCK-1 or management has improved the quality of life and ROCK-2 in cells seeded at 2x10(3)/cm (2) did not survival of patients, cure can be achieved only with change renewal factors. bone marrow transplantation when histocompatible CONCLUSIONS/SIGNIFICANCE: We conclude that donors are available. The ES cell technology suggests ROCKs promote ES cell colony formation, maintain that a therapeutic cloning approach may be feasible for them at undifferentiated state, and prevent them from treatment of this disease. Using a transgenic/knockout neural differentiation at high seeding density. ROCK sickle cell anemia mouse model, which harbors 240 kb inhibition represents a new strategy for preparing large of human DNA sequences containing the beta (S)- numbers of neural progenitor cells. globin gene, we prepared ES cells from blastocysts that had the sickle cells anemia genotype and carried Chao, J. R., et al. (2017). "Transplantation of out homologous recombination with DNA constructs Human Embryonic Stem Cell-Derived Retinal Cells

71 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ into the Subretinal Space of a Non-Human Primate." analysis showed that SSCs retained high levels of Transl Vis Sci Technol 6(3): 4. alkaline phosphatase activity and stained strongly for PURPOSE: Previous studies have demonstrated anti-stage-specific embryonic antigen (SSEA)-1, the ability of retinal cells derived from human OCT4 and CD49f. They also expressed the genes embryonic stem cells (hESCs) to survive, integrate OCT4, SOX3 and STRA8 as detected by reverse into the host retina, and mediate light responses in transcription polymerase chain reaction (RT-PCR) murine mouse models. Our aim is to determine analysis. These data clearly illustrate a novel approach whether these cells can also survive and integrate into for the growth of human SSCs using hdFs as feeder the retina of a nonhuman primate, Saimiri sciureus, cells, potentially eliminating xenogeneic contaminants. following transplantation into the subretinal space. This system provides a new opportunity for the study METHODS: hESCs were differentiated toward retinal of the regulatory mechanism of the 'niche' that governs neuronal fates using our previously published SSC self-renewal, and will be a valuable source of technique and cultured for 60 to 70 days. SSCs for potential clinical applications. Differentiated cells were further treated with 20 muM N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S- Chen, B. Z., et al. (2011). "Identification of phenylglycine t-butyl ester (DAPT) for a period of 5 microRNAs expressed highly in pancreatic islet-like days immediately prior to subretinal transplantation. cell clusters differentiated from human embryonic Differentiated cells were labeled with a lentivirus stem cells." Cell Biol Int 35(1): 29-37. expressing GFP. One million cells (10,000 cells/muL) Type 1 diabetes is an autoimmune destruction of were injected into the submacular space into a squirrel pancreatic islet beta cell disease, making it important monkey eye, using an ab externo technique. to find a new alternative source of the islet beta cells to RESULTS: RetCam imaging demonstrated the replace the damaged cells. hES (human embryonic presence and survival of human donor cells 3 months stem) cells possess unlimited self-renewal and after transplantation in the S. sciureus eye. Injected pluripotency and thus have the potential to provide an cells consolidated in the temporal macula. GFP (+) unlimited supply of different cell types for tissue axonal projections were observed to emanate from the replacement. The hES-T3 cells with normal female central consolidation of cells at 1 month, with some karyotype were first differentiated into EBs (embryoid projecting into the optic nerve by 3 months after bodies) and then induced to generate the T3pi transplantation. CONCLUSIONS: Human ES cell- (pancreatic islet-like cell clusters derived from T3 derived retinal neurons injected into the submacular cells), which expressed pancreatic islet cell-specific space of a squirrel monkey survive at least 3 months markers of insulin, glucagon and somatostatin. The postinjection without immunosuppression. Some expression profiles of microRNAs and mRNAs from donor cells appeared to integrate into the host inner the T3pi were analysed and compared with those of retina, and numerous donor axonal projections were undifferentiated hES-T3 cells and differentiated EBs. noted throughout, with some projecting into the optic MicroRNAs negatively regulate the expression of nerve. TRANSLATIONAL RELEVANCE: These data protein-coding mRNAs. The T3pi showed very high illustrate the feasibility of hESC-derived retinal cell expression of microRNAs, miR-186, miR-199a and replacement in the nonhuman primate eye. miR-339, which down-regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, Chen, B., et al. (2009). "Xeno-free culture of PLEKHH1, RBPMS2 and PAK6. Therefore, these human spermatogonial stem cells supported by human microRNAs and their target genes are very likely to embryonic stem cell-derived fibroblast-like cells." play important regulatory roles in the development of Asian J Androl 11(5): 557-565. pancreas and/or differentiation of islet cells, and they Spermatogonial stem cells (SSCs) divide may be manipulated to increase the proportion of beta continuously to support spermatogenesis throughout cells and insulin synthesis in the differentiated T3pi postnatal life and transmit genetic information to the for cell therapy of type I diabetics. next generation. Here, we report the successful establishment of the method for the isolation and Chen, C., et al. (2011). "Characterization of an in identification of human SSCs from testicular tissue, vitro differentiation assay for pancreatic-like cell and to determine the culture conditions required to development from murine embryonic stem cells: expand SSCs on human embryonic stem cell-derived detailed gene expression analysis." Assay Drug Dev fibroblast-like cells (hdFs). Large-scale cultures of Technol 9(4): 403-419. SSCs were maintained on hdF feeder layers and Embryonic stem (ES) cell technology may serve expanded in the presence of a combination of as a platform for the discovery of drugs to treat cytokines and glial cell line-derived neurotrophic diseases such as diabetes. However, because of factor for at least 2 months. Cell surface marker difficulties in establishing reliable ES cell

72 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ differentiation methods and in creating cost-effective embryoid body formation or by adding exogenous plating conditions for the high-throughput format, BMP4. The defect in trophoblast formation was due to screening for molecules that regulate pancreatic beta the lack of GPI-anchored BMP coreceptors, resulting cells and their immediate progenitors has been limited. in the impairment of full BMP4 signaling activation in A relatively simple and inexpensive differentiation the GPI-AP-deficient hES cells. These data reveal that protocol that allows efficient generation of insulin- GPI-AP-enhanced full activation of BMP signaling is expressing cells from murine ES cells was previously required for human trophoblast formation. established in our laboratories. In this report, this system is characterized in greater detail to map Chen, H. F., et al. (2007). "Derivation, developmental cell stages for future screening characterization and differentiation of human experiments. Our results show that sequential embryonic stem cells: comparing serum-containing activation of multiple gene markers for versus serum-free media and evidence of germ cell undifferentiated ES cells, epiblast, definitive endoderm, differentiation." Hum Reprod 22(2): 567-577. foregut, and pancreatic lineages was found to follow BACKGROUND: This study was designed to the sequence of events that mimics pancreatic establish human embryonic stem cell (hESC) lines, to ontogeny. Cells that expressed enhanced green identify the differences when maintained in serum- fluorescent protein, driven by pancreatic and duodenal containing versus serum-free medium and to test their homeobox 1 or insulin 1 promoter, correctly expressed potential of in vitro differentiation. METHODS: known beta cell lineage markers. Overexpression of Procedures including immunosurgery were performed Sox17, an endoderm fate-determining transcription on 11 donated human blastocysts to establish hESC factor, at a very early stage of differentiation (days 2-3) lines. The cell lines were characterized and maintained enhanced pancreatic gene expression. Overexpression using either serum-free or serum-containing media to of neurogenin3, an endocrine progenitor cell marker, compare their morphology, Oct-4 expression, induced glucagon expression at stages when pancreatic apoptosis and growth speed. Differentiation of these and duodenal homeobox 1 message was present (days lines was evaluated by the morphology and the 10-16). Forced expression (between days 16 and 25) of expression of genes belonging to the three embryonic MafA, a pancreatic maturation factor, resulted in germ layers and the germ cell lineage. RESULTS: enhanced expression of insulin genes, glucose Three hESC lines were established, and they grew at transporter 2 and glucokinase, and glucose-responsive similar speed in both media (serum-containing or insulin secretion. Day 20 cells implanted in vivo serum-free), but hESC cultured in serum-containing resulted in pancreatic-like cells. Together, our medium yielded significantly higher percentages of differentiation assay recapitulates the proceedings and morphologically good colonies and cells expressing behaviors of pancreatic development and will be Oct-4. These cell lines differentiated spontaneously in valuable for future screening of beta cell effectors. vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, Chen, G., et al. (2008). "Trophoblast including c-Kit, STELLA, VASA and growth differentiation defect in human embryonic stem cells differentiation factor 9 (GDF9), in directly adherent lacking PIG-A and GPI-anchored cell-surface culture. CONCLUSIONS: Three hESC lines with proteins." Cell Stem Cell 2(4): 345-355. Taiwanese ancestry have been established, and they Pluripotent human embryonic stem (hES) cells retain the in vitro differentiation potential with or can differentiate into various cell types derived from without embryoid body (EB) formation. The data the three embryonic germ layers and extraembryonic support that hESC may be capable of differentiation tissues such as trophoblasts. The mechanisms into germ cells although further confirmation is needed. governing lineage choices of hES cells are largely It is also suggested that strategies such as stepwise unknown. Here, we report that we established two adaptation will be needed before implementing a independent hES cell clones lacking a group of cell serum-free culture condition for hESC lines that have surface molecules, glycosyl-phosphatidyl-inositol- previously been derived in a medium containing serum. anchored proteins (GPI-APs). The GPI-AP deficiency in these two hES clones is due to the deficiency in the Chen, J., et al. (2005). "Cell adhesion molecule gene expression of PIG-A (phosphatidyl-inositol- l1-transfected embryonic stem cells with enhanced glycan class A), which is required for the first step of survival support regrowth of corticospinal tract axons GPI synthesis. GPI-AP-deficient hES cells were in mice after spinal cord injury." J Neurotrauma 22(8): capable of forming embryoid bodies and initiating cell 896-906. differentiation into the three embryonic germ layers. Previous studies have indicated that the cell However, GPI-AP-deficient hES cells failed to form adhesion molecule L1 enhances neuronal survival and trophoblasts after differentiation induction by neurite outgrowth. L1-mediated promotion of neurite

73 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ outgrowth has been shown to occur also in an inhibitory environment not only in vitro, but also in Chen, W., et al. (2012). "Retinoic acid regulates vivo. To further investigate the effects of L1 in spinal germ cell differentiation in mouse embryonic stem cord injury, we transfected embryonic stem cells with cells through a Smad-dependent pathway." Biochem a plasmid encoding the full-length mouse L1 molecule Biophys Res Commun 418(3): 571-577. under the control of PGK promoter. An embryonic Murine embryonic stem cells (ESCs) are stem cell line derived from C57BL/6J transgenic mice pluripotent cells that differentiate into multiple cell that express green fluorescent protein under control of lineages. It was recently observed that all-trans the beta-actin promoter was transfected with L1 and retinoic acid (RA) provides instructive signals for the injected into the lesion site of 3-month-old C57BL/6J commitment of the germ cell lineage from ESCs. female mice 7 days after compression injury. Non- However, little is known about the molecular transfected embryonic stem cells were detectable at the mechanisms by which RA signals lead to germ cell lesion site 3 days after transplantation, but lost their commitment. In this study, we determined if RA cellular integrity 7 days after transplantation and were induced ESC differentiation to the germ lineage barely detectable 1 month after transplantation. In through modulation of the (bone morphogenetic contrast, L1-transfected embryonic stem cells were protein) BMP/Smad pathway activity. In a monolayer detected 1 month after transplantation in numbers culture, RA significantly induced both the expression comparable to those of the injected cells and of the early germ-specific genes, Stra8, Dazl and Mvh, demonstrated extended processes. Further, in contrast and prolonged activation of Smad1/5 (for at least 24h). to the few detectable nontransfected stem cells that Meanwhile, dorsomorphin (a BMP-Smad1/5 specific remained at the injection site 1 month post- inhibitor) significantly reduced the RA-induced germ- transplantation, the L1-transfected embryonic stem specific gene expression and completely blocked the cells had migrated rostrally and caudally from the RA-induced activation of Smad1/5. Moreover, RA- lesion. Anterogradely labeled corticospinal tract axons induced germ-specific gene expression was showed interdigitation with L1-transfected embryonic significantly increased by treatment with the potential stem cells and, in contrast to non-transfected stem cells, activator of Smad1/5, SB431542. Furthermore, the extended into the lesion site 1 month after biochemical manipulation of Smad1/5 expression transplantation and, in some cases, extended beyond it. through shRNA knockdown significantly reduced RA- Our observations encourage the use of L1-transfected mediated up-regulation of germ-specific gene embryonic stem cells that express L1 not only at the expression. Our results clearly demonstrate that the cell surface, but also as a soluble and secreted form. Smad1/5 pathway is specifically required at an early Their use could condition the inhibitory environment stage of germ cell differentiation, corresponding to the for homophilic L1-enhanced axon regrowth not only in RA-dependent commitment of ESCs. spinal cord regeneration, but also in other lesion paradigms. Chen, W., et al. (2018). "Angiogenic and osteogenic regeneration in rats via calcium phosphate Chen, T., et al. (2012). "Cell growth arrest and scaffold and endothelial cell co-culture with human apoptosis induced by Oct4 or Nanog knockdown in bone marrow mesenchymal stem cells (MSCs), human mouse embryonic stem cells: a possible role of umbilical cord MSCs, human induced pluripotent stem Trp53." Mol Biol Rep 39(2): 1855-1861. cell-derived MSCs and human embryonic stem cell- It has been clear that both Oct4 and Nanog play derived MSCs." J Tissue Eng Regen Med 12(1): 191- essential roles in maintaining embryonic stem cells 203. (ESCs) undifferentiation. However, the roles of Oct4 Angiogenesis is a limiting factor in regenerating and Nanog in ESCs growth and apoptosis have been large bone defects. The objective of this study was to much less explored. In this study, we systematically investigate angiogenic and osteogenic effects of co- examined the effects of Oct4 or Nanog knockdown on culture on calcium phosphate cement (CPC) scaffold mouse ESCs (mESCs) growth and apoptosis as well as using human umbilical vein endothelial cells potential mechanisms. Our results show that Oct4 or (hUVECs) and mesenchymal stem cells (MSCs) from Nanog knockdown induces growth arrest and different origins for the first time. hUVECs were co- apoptosis in mESCs, indicating that the two genes also cultured with four types of cell: human umbilical cord play important roles in mESCs survival and growth. MSCs (hUCMSCs), human bone marrow MSCs Moreover, upregulation in Trp53 and its downstream (hBMSCs) and MSCs from induced pluripotent stem genes expression was detected in Oct4 or Nanog cells (hiPSC-MSCs) and embryonic stem cells (hESC- knockdown mESCs, suggesting a possible role of MSCs). Constructs were implanted in 8 mm cranial Trp53 in Oct4 or Nanog knockdown induced mESCs defects of rats for 12 weeks. CPC without cells served growth arrest and apoptosis. as control 1. CPC with hBMSCs served as control 2.

74 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

Microcapillary-like structures were successfully Conclusions: HCEC-like cells could be differentiated formed on CPC in vitro in all four co-cultured groups. from hESC by simply using a two-step, preconditioned, Microcapillary lengths increased with time (p < 0.05). medium-mediated approach, which could significantly Osteogenic and angiogenic gene expressions were minimize the workload to generate HCEC for potential highly elevated and mineralization by co-cultured cells clinical use. This research may provide an ideal cell increased with time (p < 0.05). New bone amount and source for corneal regenerative medicine and clinical blood vessel density of co-cultured groups were much treatment for corneal diseases in the future. greater than controls (p < 0.05) in an animal study. hUVECs co-cultured with hUCMSCs, hiPSC-MSCs Chen, X., et al. (2014). "-overexpressed and hESC-MSCs achieved new bone and vessel human embryonic stem cell-derived mesenchymal density similar to hUVECs co-cultured with hBMSCs stem cells for tendon tissue engineering with knitted (p > 0.1). Therefore, hUCMSCs, hiPSC-MSCs and silk-collagen scaffold." Tissue Eng Part A 20(11-12): hESC-MSCs could serve as alternative cell sources to 1583-1592. hBMSCs, which require an invasive procedure to AIM: Despite our previous study that harvest. In conclusion, this study showed for the first demonstrates that human embryonic stem cells (hESCs) time that co-cultures of hUVECs with hUCMSCs, can be used as seed cells for tendon tissue engineering hiPSC-MSCs, hESC-MSCs and hBMSCs delivered after stepwise induction, suboptimal tendon via CPC scaffold achieved excellent osteogenic and regeneration implies that a new strategy needs to be angiogenic capabilities in vivo. The novel co-culture developed for tendon repair. We investigated whether constructs are promising for bone reconstruction with overexpression of the tendon-specific transcription improved angiogenesis for craniofacial/orthopaedic factor scleraxis (SCX) in hESC-derived mesenchymal applications. Copyright (c) 2017 John Wiley & Sons, stem cells (hESC-MSCs) together with knitted silk- Ltd. collagen sponge scaffold could promote tendon regeneration. METHODS AND RESULTS: hESCs Chen, X., et al. (2018). "Directed Differentiation were initially differentiated into MSCs and then of Human Corneal Endothelial Cells From Human engineered with scleraxis (SCX+hESC-MSCs). Embryonic Stem Cells by Using Cell-Conditioned Engineered tendons were constructed with Culture Media." Invest Ophthalmol Vis Sci 59(7): SCX+hESC-MSCs and a knitted silk-collagen sponge 3028-3036. scaffold and then mechanical stress was applied. SCX Purpose: A shortage of human corneal elevated tendon gene expression in hESC-MSCs and endothelial cells (HCEC) for transplant and current concomitantly attenuated their adipogenic and methods of differentiation induction require chemical chondrogenic potential. Mechanical stress further compounds, which might cast further influences after augmented the expression of tendon-specific genes in differentiation induction. Therefore, we developed a SCX+hESC-MSC-engineered tendon. Moreover, in simple and straightforward approach to endothelial vivo mechanical stimulation promoted the alignment cell differentiation from human embryonic stem cells of cells and increased the diameter of collagen fibers (hESC). Methods: HESC are used to differentiate into after ectopic transplantation. In the in vivo tendon HCEC by employing a two-stage method, which repair model, the SCX+hESC-MSC-engineered tendon involves the application of two different types of enhanced the regeneration process as shown by conditioned culture medium, human corneal stromal histological scores and superior mechanical cell-conditioned medium (HCSC-CM) and lens performance compared with control cells, especially at epithelial cell (LEC) plus HCSC-CM (LEC- early stages. CONCLUSION: Our study offers new CM+HCEC-CM). In brief, hESCs were treated with evidence concerning the roles of SCX in tendon different conditioned media to induce directed differentiation and regeneration. We demonstrated a endothelial differentiation. Results: In the presence of novel strategy of combining hESCs, genetic conditioned culture medium, embryonic stem cells engineering, and tissue-engineering principles for differentiate first under the control of periocular tendon regeneration, which are important for the future mesenchymal precursors (POMPs). Consequently, the application of hESCs and silk scaffolds for tendon expression of several POMP markers was observed. repair. Following this first stage differentiation, POMPs were further directed to differentiate into corneal endothelial Chen, Y., et al. (2018). "Long-Term Engraftment cell (CEC)-like cells in the presence of the second- Promotes Differentiation of Alveolar Epithelial Cells conditioned culture medium. The differentiation of from Human Embryonic Stem Cell Derived Lung POMPs into CEC-like cells is regulated by a TGFbeta- Organoids." Stem Cells Dev 27(19): 1339-1349. 2/FOXC1 signaling pathway that is activated by the Human embryonic stem cell (hESC) derived 3D factors present in the conditioned culture medium. human lung organoids (HLOs) provide a promising

75 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ model to study human lung development and disease. methods of derivation of smooth muscle progenitors HLOs containing proximal or/and immature distal and cells from human ESCs. The primary emphasis is airway epithelial cells have been successfully on the inherent heterogeneity of smooth muscle generated in vitro, such as early staged alveolar type 2 progenitors and cells and the limitations of current in (AT2) cells (SPC (+)/SOX9(+)) and immature alveolar vitro characterisation. Essential transplantation issues type 1 (AT1) cells (HOPX (+)/SOX9(+)). When HLOs such as the type and source of therapeutic cells, mode were transplanted into immunocompromised mice for of cell delivery, measures to enhance cell viability, further differentiation in vivo, only few distal putative mechanisms of benefit and long-term tracking epithelial cells could be observed. In this study, we of cell fate are also discussed. Finally, we highlight the transplanted different stages of HLOs into challenges of clinical compatibility and scaling up for immunocompromised mice to assess whether HLOs medical use in order to eventually realise the goal of could expand and mature in vivo. We found that short- human ESC-based vascular regenerative medicine. term transplanted HLOs contained lung progenitor cells (NKX2.1(+), SOX9(+), and P63(+)), but not SPC Chikhovskaya, J. V., et al. (2012). "Human testis- (+) AT2 cells or AQP5(+) AT1 cells. Meanwhile, derived embryonic stem cell-like cells are not long-term engrafted HLOs could differentiate into pluripotent, but possess potential of mesenchymal lung distal bipotent progenitor cells (PDPN (+)/SPC progenitors." Hum Reprod 27(1): 210-221. (+)/SOX9(+)), AT2 cells (SPC (+), SPB (+)), and BACKGROUND: Spontaneous in vitro transition immature AT1 cells (PDPN (+), AQP5(-)). However, of undifferentiated spermatogonia into the pluripotent HLOs at late in vitro stage turned into mature AT1- cell state has been achieved using neonatal and adult like cells (AQP5(+)/SPB (-)/SOX9(-)) in vivo. mouse testis tissue. In an effort to establish an Immunofluorescence staining and transmission analogous source of human patient-specific pluripotent electron microscopy (TEM) results revealed that stem cells, several research groups have described the transplanted HLOs contained mesenchymal cells derivation of embryonic stem cell-like cells from (collagen I (+)), vasculature (ACTA2(+)), primary cultures of human testis. These cells are neuroendocrine-like cells (PGP9.5(+)), and nerve fiber characterized in all studies as growing in compact structures (myelin sheath structure). Together, these colonies, expressing pluripotency-associated markers data reveal that hESC-derived HLOs would be useful and possessing multilineage differentiation capabilities for human lung development modeling, and in vitro, but only one study claimed their ability to transplanted HLOs could mimic lung organ-like induce teratomas. This controversy initiated a debate structures in vivo by possessing vascular network and about the pluripotent state and origin of human testis- neuronal network. derived ES-like cells (htES-like cells). METHODS: htES-like cell colonies were obtained from primary Cheung, C. and S. Sinha (2011). "Human testicular cultures of three individuals and selectively embryonic stem cell-derived vascular smooth muscle expanded using culture conditions known to support cells in therapeutic neovascularisation." J Mol Cell the propagation of blastocyst-derived human Cardiol 51(5): 651-664. embryonic stem cells (ESCs), mouse epiblast stem Ischemic diseases remain one of the major causes cells and 'naive' human ESCs. The stem cell properties of morbidity and mortality throughout the world. In of htES-like cells were subsequently assessed by recent clinical trials on cell-based therapies, the use of testing the expression of ESC-specific markers, adult stem and progenitor cells only elicited marginal differentiation abilities in vitro and in vivo, and benefits. Therapeutic neovascularisation is the Holy microarray profiling. RESULTS: The expression of Grail for ischemic tissue recovery. There is compelling pluripotency-associated markers in htES-like cells and evidence from animal transplantation studies that the their differentiation abilities differed significantly from inclusion of mural cells in addition to endothelial cells those of ESCs. Gene expression microarray analysis (ECs) can enhance the formation of functional blood revealed that htES-like cells possess a transcriptome vessels. Vascular smooth muscle cells (SMCs) and distinct from human ESCs and fibroblasts, but closely pericytes are essential for the stabilisation of nascent resembling the transcriptome of mesenchymal stem immature endothelial tubes. Despite the intense cells (MSCs). The similarity to MSCs was confirmed interest in the utility of human embryonic stem cells by detection of SSEA4/CD146 expressing cells within (ESCs) for vascular regenerative medicine, ESC- htES-like colonies and efficient in vitro differentiation derived vascular SMCs have received much less toward three mesodermal lineages (adipogenic, attention than ECs. This review begins with osteogenic, chondrogenic). CONCLUSIONS: Taken developmental insights into a range of smooth muscle together, these results indicate that htES-like cells, in progenitors from studies on embryos and ESC contrast to pluripotent stem cells derived from adult differentiation systems. We then summarise the

76 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ mouse testis, are not pluripotent and most likely not of developed a novel approach to convert mESCs to XEN germ cell but of mesenchymal origin. cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription Chinzei, R., et al. (2002). "Embryoid-body cells factor Nanog and the expression of primitive derived from a mouse embryonic stem cell line show endoderm-associated genes Gata6, Gata4, Sox17 and differentiation into functional hepatocytes." Pdgfra are necessary for cXEN cell derivation. This Hepatology 36(1): 22-29. approach highlights an important function for Fgf4 in Embryonic stem (ES) cells have a potential to cXEN cell derivation. Paracrine FGF signalling differentiate into various progenitor cells. Here we compensates for the loss of endogenous Fgf4, which is investigated the differentiation capacity of mouse ES necessary to exit mESC self-renewal, but not for XEN cells into hepatocytes both in vitro and in vivo. During cell maintenance. Our cXEN protocol also reveals that the culture of embryoid bodies (EBs) derived from ES distinct pluripotent stem cells respond uniquely to cells, albumin (ALB) messenger RNA (mRNA) was differentiation promoting signals. cXEN cells can be expressed within 12 days after removal of leukemia derived from mESCs cultured with Erk and Gsk3 inhibitory factor, and alpha-fetoprotein (AFP) mRNA inhibitors (2i), and LIF, similar to conventional was observed within 9 days without additional mESCs. However, we find that epiblast stem cells exogenous growth factors. In ES cells and early EBs, (EpiSCs) derived from the post-implantation embryo by contrast, neither ALB mRNA nor AFP mRNA was are refractory to cXEN cell establishment, consistent observed. ALB protein was first detected at day 15 and with the hypothesis that EpiSCs represent a pluripotent the level increased with the culture period. The state distinct from mESCs. In all, these findings differentiation of EBs facilitated the synthesis of urea suggest that the potential of mESCs includes the with the culture period, whereas early EBs and ES capacity to give rise to both extra-embryonic and cells produced no urea. These results suggest that embryonic lineages. cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from Cho, M., et al. (2006). "An alternative method of EBs were transplanted through portal vein to the liver deriving embryonic stem cell-like clones by after 30% partial hepatectomy of female mice aggregation of diploid cells with tetraploid embryos." pretreated with 2-acetylaminofluorene. Four weeks Fertil Steril 85 Suppl 1: 1103-1110. after transplantation with isolated cells from day-9 OBJECTIVE: To assess whether embryonic stem EBs, ES-derived cells containing Y-chromosome in (ES) cells could be derived from the aggregation of the liver were positive for ALB (0.2% of total liver diploid cells with tetraploid embryos. DESIGN: cells), whereas teratoma was found in mice Randomized, prospective study. SETTING: University transplanted with ES cells or EBs up to day 6. The embryology and gamete biotechnology laboratory. incidence of teratoma was decreased with the culture ANIMAL (S): F1 (C57BL6/DBA2) mice. duration and no teratoma was observed in the liver INTERVENTION (S): Four- to eight-cell F1 tetraploid transplanted with isolated cells from day-9 EBs. In embryos were aggregated with 10 to 15 donor E14 ES conclusion, our in vitro and in vivo experiments cells. MAIN OUTCOME MEASURE (S): revealed that cultured EBs contain functional Embryogenesis and ES cell establishment. RESULT hepatocytes or hepatocyte-like cells. (S): No difference (78% to 89%) in blastocyst formation was detected between the aggregated Cho, L. T., et al. (2012). "Conversion from tetraploid and the control diploid embryos. In a total of mouse embryonic to extra-embryonic endoderm stem 27 transfers, pregnancy was detected in three cells reveals distinct differentiation capacities of tetraploid (23.1%) and five diploid (35.7%) cases, and pluripotent stem cell states." Development 139(16): three live births developed from the aggregated 2866-2877. tetraploid embryos. The tetraploid blastocysts without The inner cell mass of the mouse pre- aggregation were plated, but no ES cell-like colony implantation blastocyst comprises epiblast progenitor was formed. Six of eight aggregated blastocysts and primitive endoderm cells of which cognate derived well-proliferated colonies, which were embryonic (mESCs) or extra-embryonic (XEN) stem positive for anti-stage-specific embryonic antigen cell lines can be derived. Importantly, each stem cell (SSEA)-1 antibody, Oct-4, and alkaline phosphatase. type retains the defining properties and lineage The microsatellite assay confirmed the homogenous restriction of their in vivo tissue of origin. Recently, makeup among the donor E14 cells and live-birth and we demonstrated that XEN-like cells arise within ES-like cells derived from the E14-aggregated, mESC cultures. This raises the possibility that mESCs tetraploid embryo. CONCLUSION (S): The can generate self-renewing XEN cells without the aggregation of pluripotent diploid cells with tetraploid requirement for gene manipulation. We have

77 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ embryos yielded live births and ES-like cells that were SNUhES3 by the addition of betacellulin and homogenous to the donor diploid cells. nicotinamide. We reproduced in vivo pancreatic islet differentiation by directing the cells through stages Cho, M. S., et al. (2012). "Generation of retinal that resembled in vivo pancreatic organogenesis. The pigment epithelial cells from human embryonic stem addition of betacellulin and nicotinamide sustained cell-derived spherical neural masses." Stem Cell Res PDX1 expression and induced beta-cell differentiation. 9(2): 101-109. C-peptide-a genuine marker of de novo insulin Dysfunction and loss of retinal pigment production-was identified in the differentiated cells, epithelium (RPE) are major pathologic changes although the insulin mRNA content was very low. observed in various retinal degenerative diseases such Further studies are necessary to develop more efficient as aged-related macular degeneration. RPE generated and universal protocols for beta-cell differentiation. from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we Choi, H. S., et al. (2008). "Development of a show the differentiation of human embryonic stem decoy immunization strategy to identify cell-surface cells (hESCs) toward RPE with the generation of molecules expressed on undifferentiated human spherical neural masses (SNMs), which are pure embryonic stem cells." Cell Tissue Res 333(2): 197- masses of hESCs-derived neural precursors. During 206. the early passaging of SNMs, cystic structures arising Little is known about the cell-surface molecules from opened neural tube-like structures showed that are related to the undifferentiated and pluripotent pigmented epithelial morphology. These pigmented state of human embryonic stem cells (hESCs). Here, cells were differentiated into functional RPE by we generated a panel of murine monoclonal antibodies neuroectodermal induction and mechanical (MAb) against undifferentiated hESCs by a purification. Most of the differentiated cells showed modification of a previously described decoy typical RPE morphologies, such as a polygonal-shaped immunization strategy. H9 hESCs were differentiated epithelial monolayer, and transmission electron in the presence of retinoic acid and used as a decoy microscopy revealed apical microvilli, pigment immunogen. Twelve Balb/c mice were immunized in granules, and tight junctions. These cells also the right hind footpads with differentiated H9 cells and expressed molecular markers of RPE, including Mitf, in the left hind footpads with undifferentiated H9 cells. ZO-1, RPE65, CRALBP, and bestrophin. The After immunization, the left popliteal lymph node cells generated RPE also showed phagocytosis of isolated were collected and were fused with mouse myeloma bovine photoreceptor outer segment and secreting cells. The fusion resulted in 79 hybridomas secreting pigment epithelium-derived factor and vascular MAbs that bound to the undifferentiated H9 cells as endothelial growth factor. Functional RPE could be shown by flow cytometric analysis. Of these, 70 MAbs generated from SNM in our method. Because SNMs bound to the undifferentiated H9 cells, but only have several advantages, including the capability of weakly or not at all to the differentiated H9 cells. We expansion for long periods without loss of characterized 37 MAbs (32 IgGs, 5 IgMs) recognizing differentiation capability, easy storage and thawing, surface molecules that were down-regulated during and no need for feeder cells, our method for RPE embryoid body cell formation. One of the MAbs, differentiation may be used as an efficient strategy for L125-C2, was confirmed to immunoprecipitate CD9, generating functional RPE cells for retinal previously known as a surface molecule on the regeneration therapy. undifferentiated hESCs. To investigate the relationship between the MAbs and hESC-specific antibodies, two Cho, Y. M., et al. (2008). "Betacellulin and representative MAbs, viz., L125-C2 and 291-D4, were nicotinamide sustain PDX1 expression and induce selected and studied by multi-color flow cytometric pancreatic beta-cell differentiation in human analysis. This showed that more than 60% of L125- embryonic stem cells." Biochem Biophys Res C2- and 291-D4-positive cells were also positive for Commun 366(1): 129-134. the expression of hESC-specific surface molecules The major obstacle in cell therapy of diabetes such as SSEA3, SSEA4, TRA-1-60, and TRA-1-81, mellitus is the limited source of insulin-producing beta indicating the close relationship between the two cells. Very recently, it was shown that a five-stage MAbs and the hESC-specific surface molecules. Our protocol recapitulating in vivo pancreatic results suggest that the decoy immunization strategy is organogenesis induced pancreatic beta cells in vitro; an efficient method for isolating a panel of MAbs however, this protocol is specific to certain cell lines against undifferentiated hESCs, and that the generated and shows much line-to-line variation in MAbs should be useful for studying the surface differentiation efficacy. Here, we modified the five- molecules on hESCs in the pluripotent and stage protocol for the human embryonic stem cell line undifferentiated state.

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Choi, S., et al. (2013). "Regulation of Choi, H. S., et al. (2014). "Antibody approaches Pluripotency-related Genes and Differentiation in to prepare clinically transplantable cells from human Mouse Embryonic Stem Cells by Direct Delivery of embryonic stem cells: identification of human Cell-penetrating Peptide-conjugated CARM1 embryonic stem cell surface markers by monoclonal Recombinant Protein." Dev Reprod 17(1): 9-16. antibodies." Biotechnol J 9(7): 915-920. Coactivator-associated arginine Human embryonic stem cells (hESCs) are unique methyltransferase 1 (CARM1) is included in the cell populations, possessing both unlimited self- protein arginine methyltransferase (PRMT) family, renewal capacity and pluripotency, i.e. the potential to which methylates histone arginine residues through give rise to all kinds of specialized cells in the human posttranslational modification. It has been proposed body. Marker molecules expressed on the surface of that CARM1 may up-regulate the expression of hESCs are important for the identification, pluripotency-related genes through the alteration of the characterization, and clinical application of hESCs. chromatin structure. Mouse embryonic stem cells Compared with conventional genomics- or (mESCs) are pluripotent and have the ability to self- proteomics-based approaches, generating monoclonal renew. The cells are mainly used to study the genetic antibody (mAb) libraries against hESCs using function of novel genes, because the cells facilitate the alternative methodologies expands the repertoire of transmission of the manipulated genes into target mice. mAbs raised against non-protein markers, for example, Since the up-regulated methylation levels of histone glycolipid antigens. Additional information about the arginine residue lead to the maintenance of conformation and post-translational modification of pluripotency in embryos and stem cells, it may be surface molecules can also be obtained. In this article, suggested that CARM1 overexpressing mESCs elevate we review how mAb libraries against hESC surface the expression of pluripotency-related genes in markers have been developed using whole-cell and reconstituted embryos for transgenic mice and may decoy immunization strategies. resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 Choi, J. H., et al. (2011). "Generation of viable and 7X-arginine (R7). As a cell-penetrating peptide embryos and embryonic stem cell-like cells from (CPP), can translocate CARM1 protein into mESCs. cultured primary follicles in mice." Biol Reprod 85(4): CPP-CARM1 protein was detected in the nuclei of the 744-754. mESCs after a treatment of 24 hours. Accordingly, the Primary follicles retrieved from B6CBAF1 expression of pluripotency-related genes was up- prepubertal mice were cultured in a stepwise manner regulated in CPP-CARM1-treated mESCs. In addition, in an alpha-minimum essential medium-based medium CPP-CARM1-treated mESC-derived embryoid bodies to generate viable embryos and embryonic stem cell (EBs) showed an elevated expression of pluripotency- (ESC)-like cells. A significant increase in follicle related genes and delayed spontaneous differentiation. growth and oocyte maturation accompanied by This result suggests that the treatment of recombinant increased secretion of 17beta-estradiol and CPP-CARM1 protein elevates the expression of progesterone was achieved by exposing primary pluripotency-related genes of mESCs by epigenetic follicles to 100 or 200 mIU of follicle-stimulating modification, and this protein-delivery system could hormone (FSH) during culture. More oocytes be used to modify embryonic fate in reconstituted developed into blastocysts following in vitro embryos with mESCs. fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture Choi, Y. J., et al. (2018). "Phthalazinone period than with 100 mIU. Eleven ESC-like cell lines, Pyrazole Enhances the Hepatic Functions of Human consisting of four heterozygotic and seven Embryonic Stem Cell-Derived Hepatocyte-Like Cells homozygotic phenotypes, were established from 25 via Suppression of the Epithelial-Mesenchymal trials of primary follicle culture combined with IVF or Transition." Stem Cell Rev 14(3): 438-450. parthenogenetic activation. In conclusion, primary During liver development, nonpolarized hepatic follicles can potentially yield developmentally progenitor cells differentiate into mature hepatocytes competent oocytes, which produce viable embryos and with distinct polarity. This polarity is essential for ESC-like cell lines following in vitro manipulation. maintaining the intrinsic properties of hepatocytes. We suggest a method to utilize immature follicles, The balance between the epithelial-mesenchymal which are most abundant in ovaries, to improve transition (EMT) and mesenchymal-epithelial reproductive efficiency and for use in regenerative transition (MET) plays a decisive role in medicine. differentiation of polarized hepatocytes. In this study, we found that phthalazinone pyrazole (PP), a selective inhibitor of Aurora-A kinase (Aurora-A), suppressed

79 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ the EMT during the differentiation of hepatocyte-like Growing evidence suggests that physical cells (HLCs) from human embryonic stem cells. The microenvironments and mechanical stresses, in differentiated HLCs treated with PP at the hepatoblast addition to soluble factors, help direct mesenchymal- stage showed enhanced hepatic morphology and stem-cell fate. However, biological responses to a functions, particularly with regard to the expression of local force in embryonic stem cells remain elusive. drug metabolizing enzymes. Moreover, we found that Here we show that a local cyclic stress through focal these effects were mediated though suppression of the adhesions induced spreading in mouse embryonic stem AKT pathway, which is involved in induction of the cells but not in mouse embryonic stem-cell- EMT, and upregulation of hepatocyte nuclear factor differentiated cells, which were ten times stiffer. This 4alpha expression rather than Aurora-A inhibition. In response was dictated by the cell material property conclusion, these findings provided insights into the (cell softness), suggesting that a threshold cell regulatory role of the EMT on in vitro hepatic deformation is the key setpoint for triggering maturation, suggesting that inhibition of the EMT may spreading responses. Traction quantification and drive transformation of hepatoblast cells into mature pharmacological or shRNA intervention revealed that and polarized HLCs. myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress Chowdhury, F., et al. (2010). "Soft substrates led to oct3/4 gene downregulation in mES cells. Our promote homogeneous self-renewal of embryonic stem findings demonstrate that cell softness dictates cellular cells via downregulating cell-matrix tractions." PLoS sensitivity to force, suggesting that local small forces One 5(12): e15655. might have far more important roles in early Maintaining undifferentiated mouse embryonic development of soft embryos than previously stem cell (mESC) culture has been a major challenge appreciated. as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, Christoforou, N., et al. (2010). "Implantation of fluctuating expression of pluripotency genes, and mouse embryonic stem cell-derived cardiac progenitor genes of specialized cells. Here we show that, in sharp cells preserves function of infarcted murine hearts." contrast to the mESCs seeded on the conventional PLoS One 5(7): e11536. rigid substrates, the mESCs cultured on the soft Stem cell transplantation holds great promise for substrates that match the intrinsic stiffness of the the treatment of myocardial infarction injury. We mESCs and in the absence of exogenous LIF for 5 recently described the embryonic stem cell-derived days, surprisingly still generated homogeneous cardiac progenitor cells (CPCs) capable of undifferentiated colonies, maintained high levels of differentiating into cardiomyocytes, vascular Oct3/4, Nanog, and Alkaline Phosphatase (AP) endothelium, and smooth muscle. In this study, we activities, and formed embryoid bodies and teratomas hypothesized that transplanted CPCs will preserve efficiently. A different line of mESCs, cultured on the function of the infarcted heart by participating in both soft substrates without exogenous LIF, maintained the muscle replacement and neovascularization. capacity of generating homogeneous undifferentiated Differentiated CPCs formed functional colonies with relatively high levels of Oct3/4 and AP electromechanical junctions with cardiomyocytes in activities, up to at least 15 passages, suggesting that vitro and conducted action potentials over cm-scale this soft substrate approach applies to long term distances. When transplanted into infarcted mouse culture of different mESC lines. mESC colonies on hearts, CPCs engrafted long-term in the infarct zone these soft substrates without LIF generated low cell- and surrounding myocardium without causing matrix tractions and low stiffness. Both tractions and teratomas or arrhythmias. The grafted cells stiffness of the colonies increased with substrate differentiated into cross-striated cardiomyocytes stiffness, accompanied by downregulation of Oct3/4 forming gap junctions with the host cells, while also expression. Our findings demonstrate that mESC self- contributing to neovascularization. Serial renewal and pluripotency can be maintained echocardiography and pressure-volume catheterization homogeneously on soft substrates via the biophysical demonstrated attenuated ventricular dilatation and mechanism of facilitating generation of low cell- preserved left ventricular fractional shortening, matrix tractions. systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the Chowdhury, F., et al. (2010). "Material properties functional output of the infarcted heart. of the cell dictate stress-induced spreading and differentiation in embryonic stem cells." Nat Mater Chuang, C. Y., et al. (2012). "Meiotic competent 9(1): 82-88. human germ cell-like cells derived from human embryonic stem cells induced by BMP4/WNT3A

80 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ signaling and OCT4/EpCAM (epithelial cell adhesion of the key signaling pathways that have recently been molecule) selection." J Biol Chem 287(18): 14389- identified, which regulate germ cell specification. In 14401. addition, the new cell-based models for differentiating The establishment of an effective germ cell germ cells from both mouse and human embryonic selection/enrichment platform from in vitro stem cells (ESCs) will be summarized. differentiating human embryonic stem cells (hESCs) is crucial for studying the molecular and signaling Clark, A. T. and R. A. Reijo Pera (2006). processes governing human germ cell specification "Modeling human germ cell development with and development. In this study, we developed a germ embryonic stem cells." Regen Med 1(1): 85-93. cell-enriching system that enables us to identify There has previously been no robust cell-based signaling factors involved in germ cell-fate induction model for examining the genetic and epigenetic from differentiating hESCs in vitro. First, we mechanisms of human germ cell formation. Human demonstrated that selection through an OCT4-EGFP embryonic stem cells (hESCs) could potentially fill reporter system can successfully increase the this need, as all cell types analyzed to date (including percentage of meiotic-competent, germ cell-like cells mature germ cells) can be identified by marker from spontaneously differentiating hESCs. analysis during hESC differentiation. Furthermore, Furthermore, we showed that the pluripotency hESCs could also be used to differentiate mature associated surface marker, epithelial cell adhesion female germ cells (oocytes) in culture as an alternate molecule (EpCAM), is also expressed in human fetal reprogramming cell for somatic cell nuclear transfer. gonads and can be used as an effective selection However, to differentiate and isolate a functional germ marker for germ cell enrichment from differentiating cell from hESCs, the mechanisms that regulate germ hESCs. Combining OCT4 and EpCAM selection can cell formation need to be understood. The purpose of further enrich the meiotic-competent germ cell-like this review is to summarize the current understanding cell population. Also, with the percentage of of the earliest events in human germ cell formation OCT4(+)/EpCAM (+) cells as readout, we and to describe some of the known genetic pathways demonstrated the synergistic effect of that regulate germ cell specification and development BMP4/pSMAD1/5/8 and WNT3A/beta-CATENIN in in the mouse. Finally, the current literature on the promoting hESCs toward the germline fate. formation of germ cells from ESCs will be described. Combining BMP4/WNT3A induction and OCT4/EpCAM selection can significantly increase the Cobo, F., et al. (2008). "Electron microscopy putative germ cell population with meiotic reveals the presence of viruses in mouse embryonic competency. Co-transplantation of these cells with fibroblasts but neither in human embryonic fibroblasts dissociated mouse neonatal ovary cells into SCID mice nor in human mesenchymal cells used for hESC resulted in a homogenous germ cell cluster formation maintenance: toward an implementation of in vivo. The stepwise platform established in this study microbiological quality assurance program in stem cell provides a useful tool to elucidate the molecular banks." Cloning Stem Cells 10(1): 65-74. mechanisms of human germ cell development, which Human embryonic stem cells (hESCs) are has implications not only for human fertility research expected to open up new avenues in regenerative but regenerative medicine in general. medicine by allowing the generation of transplantable cells to be used in future cell replacement therapies. Clark, A. T. (2007). "Establishment and Maintenance of hESCs in the presence of xenogenic differentiation of human embryonic stem cell derived compounds is likely to prevent their use in future germ cells." Soc Reprod Fertil Suppl 63: 77-86. therapeutic applications in humans. Recently, it has Germ cells are absolutely essential for fertility. been claimed that human foreskin-derived human Aberrant germ cell development can result in embryonic fibroblast (HEFs) and human adult marrow abnormal gonadal function, incomplete embryogenesis cells have the ability to support prolonged expansion and infertility, or germ cell tumors. Our understanding of hESCs in culture similar to murine feeders. Here, to of the molecular regulation of normal germ cell minimize the use of xenogenic components for hESC development in mammals has progressed significantly maintenance, we performed transmission electron due to the utility of the mouse as a genetic model microscopy-based microbiological studies in an system. However, the molecular regulation of human attempt to implement a microbiological Quality germ cell development is almost completely unknown Assurance Program in Stem Cell Banks by due to the historical lack of a malleable model. The determining the potential presence of viral particles in purpose of this review is to compare the cell-based MEFs compared with human HEFs and bone marrow- events leading up to the specification of the germ cell derived mesenchymal cells. We observed in three out lineage in both mice and humans and to discuss some of nine MEF samples (33.3%) viruses belonging to the

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Retroviridae family. Within the Retroviridae family, and sequencing analysis of antibiotic resistant clones these viruses have a C morphology, which indicates indicated targeted integration of the reporter cassette at they belong to the subfamily Orthoretroviridae. In the 3' of the CRX gene, generating a CRX-GFP fusion. contrast, no viral particles could be observed in either Further analysis of a clone exhibiting homozygote the HEF samples (n = 5) or the human BM-derived integration of the GFP reporter was conducted mesenchymal cells (n = 9) analyzed. Based on these suggesting genomic stability was preserved and no experimental microbiological data, we recommend the other copies of the targeting cassette were inserted implementation of microbiological Quality Assurance elsewhere within the genome. This clone was selected Programs by means of transmission electron for differentiation towards the retinal lineage. microscopy as a routine technique to assess the Immunocytochemistry of sections obtained from potential presence of viral particles in any feeder cell embryoid bodies and quantitative reverse transcriptase used in stem cell banks and support the use of human PCR of GFP positive and negative subpopulations cells rather than murine cells as feeders to maintain purified by fluorescence activated cell sorting during hESC cultures in an undifferentiated state. the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Coll, J. L., et al. (1995). "Targeted disruption of Furthermore, GFP expression was found in vinculin genes in F9 and embryonic stem cells changes photoreceptor precursors emerging during hESC cell morphology, adhesion, and locomotion." Proc differentiation, but not in the retinal pigmented Natl Acad Sci U S A 92(20): 9161-9165. epithelium, retinal ganglion cells, or neurons of the Vinculin, a major constituent of focal adhesions developing inner nuclear layer. Together our data and zonula adherens junctions, is thought to be demonstrate the successful application of ZFN involved in linking the microfilaments to areas of cell- technology to generate CRX-GFP labeled hESC lines, substrate and cell-cell contacts. To test the role of which can be used to study and isolate photoreceptor vinculin in cell adhesion and motility, we used precursors during hESC differentiation. homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones Cong, S., et al. (2014). "Effects of different homozygous for a disrupted vinculin gene. When feeder layers on culture of bovine embryonic stem compared to wild-type cells, vinculin-mutant cells cell-like cells in vitro." Cytotechnology 66(6): 995- displayed a rounder morphology and a reduced ability 1005. to adhere and spread on plastic or fibronectin. To find a suitable feeder layer is important for Decreased adhesion of the mutant cells was associated successful culture conditions of bovine embryonic with a reduction in lamellipodial extensions, as stem cell-like cells. In this study, expression of observed by time-lapse video microscopy. The pluripotency-related genes OCT4, and NANOG locomotive activities of control F9 and the vinculin- in bovine embryonic stem cell-like cells on mouse null cells were compared in two assays. Loss of embryonic fibroblast feeder layers at 1-5 passages vinculin resulted in a 2.4-fold increase in cell motility. were monitored in order to identify the possible reason These results demonstrate an important role for that bovine embryonic stem cell-like cells could not vinculin in determining cell shape, adhesion, surface continue growth and passage. Here, we developed two protrusive activity, and cell locomotion. novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at Collin, J., et al. (2016). "Using different ratios and sources including mouse fibroblast Nuclease Technology to Generate CRX-Reporter cell lines. The bovine embryonic stem cell-like cells Human Embryonic Stem Cells as a Tool to Identify generated in our study displayed typical stem cell and Study the Emergence of Photoreceptors Precursors morphology and expressed specific markers such as During Pluripotent Stem Cell Differentiation." Stem OCT4, stage-specific embryonic antigen 1 and 4, Cells 34(2): 311-321. alkaline phosphatase, SOX2, and NANOG mRNA The purpose of this study was to generate human levels. When feeder layers and cell growth factors embryonic stem cell (hESC) lines harboring the green were removed, the bovine embryonic stem cell-like fluorescent protein (GFP) reporter at the endogenous cells formed embryoid bodies in a suspension culture. loci of the Cone-Rod Homeobox (CRX) gene, a key Furthermore, we compared the expression of the transcription factor in retinal development. Zinc finger pluripotent markers during bovine embryonic stem nucleases (ZFNs) designed to cleave in the 3' UTR of cell-like cell in culture on mixed embryonic fibroblast CRX were transfected into hESCs along with a donor feeder layers, including mouse fibroblast cell lines construct containing homology to the target region, feeder layers and mouse embryonic fibroblast feeder eGFP reporter, and a puromycin selection cassette. layers by real-time quantitative polymerase chain Following selection, polymerase chain reaction (PCR) reaction. Results suggested that mixed embryonic

82 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ fibroblast and sources including mouse fibroblast cell as those derived from spinal cord and offer great lines feeder layers were more suitable for long-term promise as an unlimited source of neural stem cells for culture and growth of bovine embryonic stem cell-like transplantation. We found that embryonic stem cell- cells than mouse embryonic fibroblast feeder layers. derived neural stem cells can differentiate into motor The findings may provide useful experimental data for neurons in vitro and in vivo. In addition, following the establishment of an appropriate culture system for their intrathecal transplantation into spinal muscular bovine embryonic stem cell lines. atrophy mice, the neural stem cells, like those derived from spinal cord, survived and migrated to appropriate Corrales, C. E., et al. (2006). "Engraftment and areas, ameliorated behavioural endpoints and lifespan, differentiation of embryonic stem cell-derived neural and exhibited neuroprotective capability. Neural stem progenitor cells in the cochlear nerve trunk: growth of cells obtained using a drug-selectable embryonic stem processes into the organ of Corti." J Neurobiol 66(13): cell line yielded the greatest improvements. As with 1489-1500. cells originating from primary tissue, the embryonic Hearing loss in mammals is irreversible because stem cell-derived neural stem cells integrated cochlear neurons and hair cells do not regenerate. To appropriately into the parenchyma, expressing neuron- determine whether we could replace neurons lost to and motor neuron-specific markers. Our results primary neuronal degeneration, we injected EYFP- suggest translational potential for the use of expressing embryonic stem cell-derived mouse neural pluripotent cells in neural stem cell-mediated therapies progenitor cells into the cochlear nerve trunk in and highlight potential safety improvements and immunosuppressed animals 1 week after destroying benefits of drug selection for neuroepithelial cells. the cochlear nerve (spiral ganglion) cells while leaving hair cells intact by ouabain application to the round Costa, A. and D. Henrique (2015). window at the base of the cochlea in gerbils. At 3 days "Transcriptome profiling of induced hair cells (iHCs) post transplantation, small grafts were seen that generated by combined expression of Gfi1, Pou4f3 expressed endogenous EYFP and could be and Atoh1 during embryonic stem cell differentiation." immunolabeled for neuron-specific markers. Twelve Genom Data 6: 77-80. days after transplantation, the grafts had neurons that To gain new insights about the genetic networks extended processes from the nerve core toward the controlling hair cell (HC) development, we previously denervated organ of Corti. By 64-98 days, the grafts developed a direct genetic programming strategy to had sent out abundant processes that occupied a generate an inexhaustible supply of HC-like cells significant portion of the space formerly occupied by (induced HCs, iHCs) in vitro, starting from mouse the cochlear nerve. The neurites grew in fasciculating embryonic stem cells (ESC). We found that combined bundles projecting through Rosenthal's canal, the activity of three transcription factors, Gfi1, Pou4f3, former site of spiral ganglion cells, into the osseous and Atoh1, can program ESC-derived progenitors spiral lamina and ultimately into the organ of Corti, towards HC fate with efficiencies of 55%-80%. These where they contacted hair cells. Neuronal counts iHCs express several HC markers and exhibit showed a significant increase in neuronal processes polarized structures that are highly reminiscent of the near the sensory epithelium, compared to animals that mechanosensitive hair bundles, with many microvilli- were denervated without subsequent stem cell like stereocilia. Here, we describe the experimental transplantation. The regeneration of these neurons design, methodology, and data validation for the shows that neurons differentiated from stem cells have microarray analysis used to characterize the the capacity to grow to a specific target in an animal transcriptome profile of iHCs at different stages of model of neuronal degeneration. their differentiation. This approach based on FACS sorting and microarray analysis revealed a highly Corti, S., et al. (2010). "Embryonic stem cell- similar iHC transcriptome to that of endogenous HCs derived neural stem cells improve spinal muscular in vivo. The data obtained in this study is available in atrophy phenotype in mice." Brain 133(Pt 2): 465-481. the Gene Expression Omnibus (GEO) database Spinal muscular atrophy, characterized by (accession number GSE60352). selective loss of lower motor neurons, is an incurable genetic neurological disease leading to infant mortality. Coulombel, L. (2010). "[A big step forward in We previously showed that primary neural stem cells the identification of therapeutic human embryonic derived from spinal cord can ameliorate the spinal stem cells-derived progenitors for cardiac cell muscular atrophy phenotype in mice, but this primary therapy]." Med Sci (Paris) 26(4): 439-441. source has limited translational value. Here, we illustrate that pluripotent stem cells from embryonic Couteaudier, M., et al. (2016). "Keratinocytes stem cells show the same potential therapeutic effects derived from chicken embryonic stem cells support

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Marek's disease virus infection: a highly differentiated lineage by using a combination of stromal induction, cell model to study viral replication and ascorbic acid, BMP4 and chicken serum. During the morphogenesis." Virol J 13: 7. induction period, we observed a downregulation of BACKGROUND: Marek's disease is a virus pluripotency markers and an upregulation of epidermal disease with worldwide distribution that causes major markers. Three homogenous cell populations were losses to poultry production. Vaccines against Marek's derived, which were morphologically similar to disease virus, an oncogenic alphaherpesvirus, reduce chicken primary keratinocytes, displaying intracellular tumour formation but have no effect on virus shedding. lipid droplets in almost every pavimentous cell. These Successful horizontal virus transmission is linked to cells could be serially passaged without alteration of the active viral replication in feather follicle epithelial their morphology and showed gene and protein cells of infected chickens, from which infectious viral expression profiles of epidermal markers similar to particles are shed into the environment. The feather chicken primary keratinocytes. These cells represent follicle epithelium is the sole tissue in which those an alternative to the isolation of chicken primary infectious particles are produced and no in vitro cell- keratinocytes, being less cumbersome to handle and systems can support this highly efficient reducing the number of experimental animals used for morphogenesis. We previously characterized the preparation of primary cells. embryonic stem-cell-derived keratinocytes, showing they display a marker-gene profile similar to skin Crocker, S. J., et al. (2011). "Intravenous keratinocytes, and therefore we tested their administration of human embryonic stem cell-derived susceptibility to Marek's disease virus infection. neural precursor cells attenuates cuprizone-induced FINDINGS: We show herein that keratinocytes central nervous system (CNS) demyelination." derived from chicken embryonic stem-cells are fully Neuropathol Appl Neurobiol 37(6): 643-653. permissive to the replication of either non-pathogenic AIMS: Previous studies have demonstrated the or pathogenic Marek's disease viruses. All viruses therapeutic potential for human embryonic stem cell- replicated on all three keratinocyte lines and kinetics derived neural precursor cells (hES-NPCs) in of viral production as well as viral loads were similar autoimmune and genetic animal models of to those obtained on primary cells. Morphogenesis demyelinating diseases. Herein, we tested whether studies were conducted on infected keratinocytes and intravenous (i.v.) administration of hES-NPCs would on corneocytes, showing that all types of impact central nervous system (CNS) demyelination in capsids/virions were present inside the cells, but a cuprizone model of demyelination. METHODS: extracellular viruses were absent. CONCLUSIONS: C57Bl/6 mice were fed cuprizone (0.2%) for 2 weeks The keratinocyte lines are the first epithelial cell-line and then separated into two groups that either received showing ectodermal specific markers supporting an i.v. injection of hES-NPCs or i.v. administration of Marek's disease virus replication. In this in vitro model media without these cells. After an additional 2 weeks the replication lead to the production of cell-associated of dietary cuprizone treatment, CNS tissues were viral progeny. Further work will be devoted to the analysed for detection of transplanted cells and study of relationship between 3D differentiation of differences in myelination in the region of the corpus keratinocytes and Marek's disease virus replication. callosum (CC). RESULTS: Cuprizone-induced demyelination in the CC was significantly reduced in Couteaudier, M., et al. (2015). "Derivation of mice treated with hES-NPCs compared with keratinocytes from chicken embryonic stem cells: cuprizone-treated controls that did not receive stem establishment and characterization of differentiated cells. hES-NPCs were identified within the brain proliferative cell populations." Stem Cell Res 14(2): tissues of treated mice and revealed migration of 224-237. transplanted cells into the CNS. A limited number of A common challenge in avian cell biology is the human cells were found to express the mature generation of differentiated cell-lines, especially in the oligodendrocyte marker, O1, or the astrocyte marker, keratinocyte lineage. Only a few avian cell-lines are glial fibrillary acidic protein. Reduced apoptosis and available and very few of them show an interesting attenuated microglial and astrocytic responses were differentiation profile. During the last decade, also observed in the CC of hES-NPC-treated mice. mammalian embryonic stem cell-lines were shown to CONCLUSIONS: These findings indicated that differentiate into almost all lineages, including systemically administered hES-NPCs migrated from keratinocytes. Although chicken embryonic stem cells circulation into a demyelinated lesion within the CNS had been obtained in the 1990s, few differentiation and effectively reduced demyelination. Observed studies toward the ectodermal lineage were reported. reductions in astrocyte and microglial responses, and Consequently, we explored the differentiation of the benefit of hES-NPC treatment in this model of chicken embryonic stem cells toward the keratinocyte

84 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ myelin injury was not obviously accountable to tissue 1 among the undifferentiated cells in the ES cell replacement by exogenously administered cells. colonies was evident by immunohistochemistry and immuno-SEM, supporting the flow cytometry findings. Cruz, A. C., et al. (2004). "Tumor necrosis In contrast, most undifferentiated ES cells strongly factor-alpha-converting enzyme controls surface expressed CD9. SSEA-1 was located preferentially on expression of c-Kit and survival of embryonic stem the edge of low protuberances and microvilli and cell-derived mast cells." J Biol Chem 279(7): 5612- formed clusters or linear arrays of 3-20 particles. 5620. PECAM-1 and ICAM-1 were randomly localized on Transmembrane metalloproteinases of the the free cell surfaces, whereas CD9 was preferentially disintegrin and metalloproteinase (ADAM) family localized on the microvilli or protuberances, especially control cell signaling interactions via hydrolysis of in the cell periphery. Both the SSEA-1(+) fraction and protein extracellular domains. Prior work has shown the SSEA-1(-) fraction of magnetic cell sorting that the receptor tyrosine kinase, c-Kit (CD117), is (MACS) formed undifferentiated colonies after plating. essential for mast cell survival and that serum levels of Flow cytometry showed that these populations c-Kit increase in proliferative mast cell disorders, reverted separately again to a culture with a mixed suggesting the existence of c-Kit shedding pathways in phenotype. Differentiation induced by retinoic acid mast cells. In the present work, we report that tumor downregulated the expression of all CAMs. Immuno- necrosis factor alpha-converting enzyme (TACE; SEM showed decreases of SSEA-1 in the ADAM-17) mediates shedding of c-Kit. Stimulation of differentiated ES cells, although some clustering still transfected cells with phorbol 12-myristate 13-acetate remained. Our findings help to elucidate the (PMA) induced metalloproteinase-mediated release of significance of these molecules in ES cell maintenance c-Kit ectodomain, which increased further upon TACE and differentiation and suggest that cell surface overexpression. By contrast, TACE-deficient antigens may be useful for defining the phenotype of fibroblasts did not demonstrate inducible release, thus undifferentiated and differentiated ES cells. identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface Cusulin, C., et al. (2012). "Embryonic stem cell- expression of c-Kit by the human mast cell-1 line derived neural stem cells fuse with microglia and decreased upon phorbol-induced shedding, which mature neurons." Stem Cells 30(12): 2657-2671. involved metalloproteinase activity susceptible to Transplantation of neural stem cells (NSCs) is a inhibition by tissue inhibitor of metalloproteinase novel strategy to restore function in the diseased brain, (TIMP)-3. To further explore the role of TACE in acting through multiple mechanisms, for example, shedding of c-Kit from mast cells, we compared the neuronal replacement, neuroprotection, and behavior of mast cells derived from murine embryonic modulation of inflammation. Whether transplanted stem cells. In these studies, PMA decreased surface c- NSCs can operate by fusing with microglial cells or Kit levels on mast cells expressing wild-type (+/+) mature neurons is largely unknown. Here, we have TACE but not on those expressing an inactive mutant studied the interaction of a mouse embryonic stem (DeltaZn/DeltaZn), confirming the role of TACE in cell-derived neural stem (NS) cell line with rat and PMA-induced c-Kit shedding. Compared with TACE mouse microglia and neurons in vitro and in vivo. We (+/+) cells, TACE (DeltaZn/DeltaZn) mast cells also show that NS cells spontaneously fuse with cocultured demonstrated decreased constitutive shedding and cortical neurons, and that this process requires the increased basal surface expression of c-Kit, with presence of microglia. Our in vitro data indicate that diminished apoptosis in response to c-Kit ligand the NS cells can first fuse with microglia and then with deprivation. These data suggest that TACE controls neurons. The fused NS/microglial cells express mast cell survival by regulating shedding and surface markers and retain genetic and functional expression of c-Kit. characteristics of both parental cell types, being able to respond to microglia-specific stimuli (LPS and IL- Cui, L., et al. (2004). "Spatial distribution and 4/IL-13) and to differentiate to neurons and astrocytes. initial changes of SSEA-1 and other cell adhesion- The NS cells fuse with microglia, at least partly, related molecules on mouse embryonic stem cells through interaction between phosphatidylserine before and during differentiation." J Histochem exposed on the surface of NS cells and CD36 receptor Cytochem 52(11): 1447-1457. on microglia. Transplantation of NS cells into rodent We examined the distribution of cell adhesion- cortex results in fusion with mature pyramidal neurons, related molecules (CAMs) among mouse embryonic which often carry two nuclei, a process probably stem (ES) cells and the spatial distribution on cell mediated by microglia. The fusogenic role of surfaces before and during differentiation. The cell- microglia could be even more important after NSC cell heterogeneity of SSEA-1, PECAM-1, and ICAM- transplantation into brains affected by

85 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ neurodegenerative diseases associated with microglia that the TH-expressing cells did not co-localize with activation. It remains to be elucidated how the GAD. The transplantation of these DA-induced hNSCs occurrence of the fused cells will influence the into the non-human primate MPTP model of PD functional outcome after NSC transplantation in the demonstrated that the cells maintain their DA-induced diseased brain. phenotype, extend neurite outgrowths and express synaptic markers. Czerwinska, A. M., et al. (2016). "Cell cycle regulation of embryonic stem cells and mouse Daadi, M. M. and G. K. Steinberg (2009). embryonic fibroblasts lacking functional Pax7." Cell "Manufacturing neurons from human embryonic stem Cycle 15(21): 2931-2942. cells: biological and regulatory aspects to develop a The transcription factor Pax7 plays a key role safe cellular product for stroke cell therapy." Regen during embryonic myogenesis and in adult organisms Med 4(2): 251-263. in that it sustains the proper function of satellite cells, Demographic trends, particularly those related to which serve as adult skeletal muscle stem cells. longer life expectancy, suggest that the demand for Recently we have shown that lack of Pax7 does not tissue and organ transplants will further increase since prevent the myogenic differentiation of pluripotent many disorders result from degeneration, injury or stem cells. In the current work we show that the organ failure. The most urgent problem in absence of functional Pax7 in differentiating transplantation medicine is the shortage or lack of embryonic stem cells modulates cell cycle facilitating suitable donor organs and tissue, leading to ethical and their proliferation. Surprisingly, deregulation of Pax7 societal problems such as organ trafficking. The function also positively impacts at the proliferation of discovery of stem cells in the inner cell mass of mouse embryonic fibroblasts. Such phenotypes seem developing embryos and in adult tissue has to be executed by modulating the expression of revolutionized the medical field by introducing new positive cell cycle regulators, such as cyclin E. therapeutic dimensions to consider for previously untreatable diseases and injuries. The unlimited self- Daadi, M. M., et al. (2012). "Dopaminergic renewal ability and pluripotent capacity to become any neurons from midbrain-specified human embryonic cell type of the organism make human embryonic stem stem cell-derived neural stem cells engrafted in a cells (hESCs) a compelling source of cells to study monkey model of Parkinson's disease." PLoS One 7(7): tissue histogenesis and to apply in a wide array of e41120. tissue engineering, cell transplantation therapy and The use of human embryonic stem cells (hESCs) drug discovery applications. In this article, we will to repair diseased or injured brain is promising focus on hESCs and address the derivation of technology with significant humanitarian, societal and therapeutic neural stem cell lines from hESCs, as well economic impact. Parkinson's disease (PD) is a as the biological and regulatory aspects to developing neurological disorder characterized by the loss of a safe cellular product for stroke cell therapy. midbrain dopaminergic (DA) neurons. The generation of this cell type will fulfill a currently unmet Dabelsteen, S., et al. (2009). "Epithelial cells therapeutic need. We report on the isolation and derived from human embryonic stem cells display perpetuation of a midbrain-specified self-renewable p16INK4A senescence, hypermotility, and human neural stem cell line (hNSCs) from hESCs. differentiation properties shared by many P63+ These hNSCs grew as a monolayer and uniformly somatic cell types." Stem Cells 27(6): 1388-1399. expressed the neural precursor markers nestin, Human embryonic stem (hES) cells can generate vimentin and a radial glial phenotype. We describe a cells expressing p63, K14, and involucrin, which have process to direct the differentiation of these hNSCs been proposed to be keratinocytes. Although these towards the DA lineage. Glial conditioned media acted hES-derived, keratinocyte-like (hESderK) cells form synergistically with fibroblastic growth factor and epithelioid colonies when cultured in a fibroblast leukemia inhibitory factor to induce the expression of feeder system optimal for normal tissue-derived the DA marker, tyrosine hydroxylase (TH), in the keratinocytes, they have a very short replicative hNSC progeny. The glial-derived neurotrophic factor lifespan unless engineered to express HPV16 E6E7. did not fully mimic the effects of conditioned media. We report here that hESderK cells undergo senescence The hNSCs expressed the midbrain-specific associated with p16(INK4A) expression, unrelated to transcription factors Nurr1 and Pitx3. The inductive telomere status. Transduction to express bmi1, a effects did not modify the level of the glutamic acid repressor of the p16(INK4A)/p14(ARF) locus, decarboxylase (GAD) transcript, a marker for conferred upon hESderK cells and keratinocytes a GABAergic neurons, while the TH transcript increased substantially extended lifespan. When exposed to 10-fold. Immunocytochemical analysis demonstrated transforming growth factor beta or to an incompletely

86 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ processed form of Laminin-332, three lifespan- forces primarily influence nondifferentiated cells. extended or immortalized hESderK lines that we Understanding the effects of forces on stem cell studied became directionally hypermotile, a wound differentiation provides a means of controlling their healing and invasion response previously characterized differentiation for later use in regenerative medicine in keratinocytes. In organotypic culture, hESderK cells applications and sheds light on their involvement in stratified and expressed involucrin and K10, as do embryogenesis. epidermal keratinocytes in vivo. However, their growth requirements were less stringent than Dang, L. T., et al. (2012). "Zfhx1b induces a keratinocytes. We then extended the comparison to definitive neural stem cell fate in mouse embryonic endoderm-derived, p63(+)/K14(+) urothelial and stem cells." Stem Cells Dev 21(15): 2838-2851. tracheobronchial epithelial cells. Primary and Inducing a stable and predictable program of immortalized lines of these cell types had growth neural cell fate in pluripotent cells in vitro is an requirements and hypermotility responses similar to important goal for utilizing these cells for modeling keratinocytes and bmi1 expression facilitated their human disease mechanisms. However, the extent to immortalization by engineering to express the catalytic which in vitro neural specification recapitulates in vivo subunit of telomerase (TERT). In organotypic culture, neural specification remains to be fully established. they stratified and exhibited squamous metaplasia, We previously demonstrated that in the mouse embryo, expressing involucrin and K10. Thus, hESderK cells activation of fibroblast growth factor (FGF) signalling proved to be distinct from all three normal p63(+) cell promotes definitive neural stem cell (NSC) types tested. These results indicate that hESderK cells development through the upregulation of the cannot be identified conclusively as keratinocytes or transcription factor Zfhx1b. Here, we asked whether even as ectodermal cells, but may represent an Zfhx1b is similarly required during neural lineage incomplete form of, or deviation from, normal p63(+) development of embryonic stem (ES) cells. Zfhx1b lineage development. gene expression is rapidly upregulated in mouse ES cells cultured in a permissive neural-inducing Dado-Rosenfeld, D., et al. (2015). "Tensile forces environment, compared to ES cells in a standard applied on a cell-embedded three-dimensional scaffold pluripotency maintenance environment, and is can direct early differentiation of embryonic stem cells potentiated by FGF signalling. However, toward the mesoderm germ layer." Tissue Eng Part A overexpression of Zfhx1b in ES cells in maintenance 21(1-2): 124-133. conditions, containing serum and leukemia inhibitory Mechanical forces play an important role in the factor (LIF), is sufficient to induce Sox1 expression, a initial stages of embryo development; yet, the marker found in neural precursors and to promote influence of forces, particularly of tensile forces, on definitive NSC colony formation. Knockdown of embryonic stem cell differentiation is still unknown. Zfhx1b in ES cells using siRNA did not affect the The effects of tensile forces on mouse embryonic stem initial transition of ES cells to a neural cell fate, but cell (mESC) differentiation within a three-dimensional did diminish the ability of these neural cells to develop (3D) environment were examined using an advanced further into definitive NSCs. Thus, our findings using bioreactor system. Uniaxial static or dynamic stretch ES cells are congruent with evidence from mouse was applied on cell-embedded collagen constructs. embryos and support a model, whereby intercellular Six-day-long cyclic stretching of the seeded constructs FGF signaling induces Zfhx1b, which promotes the led to a fourfold increase in (BRACH-T) development of definitive NSCs subsequent to an expression, associated with the primitive streak phase initial neural specification event that is independent of in gastrulation, confirmed also by this pathway. immunofluorescence staining. Further examination of gene expression characteristic of mESC differentiation Dang, S. M. and P. W. Zandstra (2005). and pluripotency, under the same conditions, revealed "Scalable production of embryonic stem cell-derived changes mostly related to mesodermal processes. cells." Methods Mol Biol 290: 353-364. Additionally, downregulation of genes related to Embryonic stem (ES) cells have the ability to pluripotency and stemness was observed. Cyclic self-renew as well as differentiate into any cell type in stretching of the 3D constructs resulted in actin fiber the body. These traits make ES cells an attractive "raw alignment parallel to the stretching direction. BRACH- material" for a variety of cell-based technologies. T expression decreased under cyclic stretching with However, uncontrolled cell aggregation in ES cell addition of myosin II inhibitor. No significant changes differentiation culture inhibits cell proliferation and in gene expression were observed when mESCs were differentiation and thwarts the use of stirred first differentiated in the form of embryoid bodies and suspension bioreactors. Encapsulation of ES cells in then exposed to cyclic stretching, suggesting that agarose microdrops prevents physical interaction

87 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ between developing embryoid bodies (EBs) that, in transcription-induced pluripotency, 2 techniques that turn, prevents EB agglomeration. This enables use of have been used in attempts to circumvent the need to stirred suspension bioreactors that can generate large derive ES cells by the harvest of embryonic tissue. numbers of ES-derived cells under controlled conditions. David, R., et al. (2005). "Magnetic cell sorting purification of differentiated embryonic stem cells Danova-Alt, R., et al. (2012). "Very small stably expressing truncated human CD4 as surface embryonic-like stem cells purified from umbilical cord marker." Stem Cells 23(4): 477-482. blood lack stem cell characteristics." PLoS One 7(4): Embryonic stem (ES) cells offer great potential e34899. in regenerative medicine and tissue engineering. Very small embryonic-like (VSEL) cells have Clinical applications are still hampered by the lack of been described as putatively pluripotent stem cells protocols for gentle, high-yield isolation of specific present in murine bone marrow and human umbilical cell types for transplantation expressing no cord blood (hUCB) and as such are of high potential immunogenic markers. We describe labeling of stably interest for regenerative medicine. However, there transfected ES cells expressing a human CD4 remain some questions concerning the precise identity molecule lacking its intracellular domain (DeltaCD4) and properties of VSEL cells, particularly those under control of the phosphoglycerate kinase promoter derived from hUCB. For this reason, we have carried for magnetic cell sorting (MACS). To track the labeled out an extensive characterisation of purified ES cells, we fused DeltaCD4 to an intracellular populations of VSEL cells from a large number of enhanced green fluorescent protein domain UCB samples. Consistent with a previous report, we (DeltaCD4EGFP). We showed functionality of the find that VSEL cells are CXCR4(+), have a high membrane-bound fluorescent fusion protein and its density, are indeed significantly smaller than HSC and suitability for MACS leading to purities greater than have an extremely high nuclear/cytoplasmic ratio. 97%. Likewise, expression of DeltaCD4 yielded up to Their nucleoplasm is unstructured and stains strongly 98.5% positive cells independently of their with Hoechst 33342. A comprehensive FACS screen differentiation state. Purities were not limited by the for surface markers characteristic of embryonic, initial percentage of DeltaCD4(+) cells, ranging from mesenchymal, neuronal or hematopoietic stem cells 0.6%-16%. The viability of MACS-selected cells was revealed negligible expression on VSEL cells. These demonstrated by reaggregation and de novo formation cells failed to expand in vitro under a wide range of of embryoid bodies developing all three germ layers. culture conditions known to support embryonic or Thus, expression of DeltaCD4 in differentiated ES adult stem cell types and a microarray analysis cells may enable rapid, high-yield purification of a revealed the transcriptional profile of VSEL cells to be desired cell type for tissue engineering and clearly distinct both from well-defined populations of transplantation studies. pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an David, R., et al. (2008). "Connexin 40 promoter- aneuploid karyotype in the majority of purified VSEL based enrichment of embryonic stem cell-derived cells by fluorescence in situ hybridisation. These data cardiovascular progenitor cells." Cells Tissues Organs support neither an embryonic nor an adult stem cell 188(1-2): 62-69. like phenotype, suggesting rather that hUCB VSEL BACKGROUND: Pluripotent embryonic stem cells are an aberrant and inactive population that is not (ES) cells that can differentiate into functional comparable to murine VSEL cells. cardiomyocytes as well as vascular cells in cell culture may open the door to cardiovascular cell Das, S., et al. (2008). "Generation of embryonic transplantation. However, the percentage of ES cells in stem cells: limitations of and alternatives to inner cell embryoid bodies (EBs) which spontaneously undergo mass harvest." Neurosurg Focus 24(3-4): E4. cardiovascular differentiation is low (<10%), making Embryonic stem (ES) cells are pluripotent cells strategies for their specific labeling and purification derived from the inner cell mass of the early indispensable. METHODS: The human connexin 40 mammalian embryo. Because of their plasticity and (Cx40) promoter was isolated and cloned in the vector potentially unlimited capacity for self-renewal, ES pEGFP. The specificity of the construct was initially cells have generated tremendous interest both as assessed in Xenopus embryos injected with Cx40- models for developmental biology and as possible EGFP plasmid DNA. Stable Cx40-EGFP ES cell tools for regenerative medicine. This excitement has clones were differentiated and fluorescent cells were been attenuated, however, by scientific, political, and enriched manually as well as via fluorescence- ethical considerations. In this article the authors activated cell sorting. Characterization of these cells describe somatic cell nuclear transfer and was performed with respect to spontaneous beating as

88 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ well as via RT-PCRs and immunofluorescent stainings. INTRODUCTION: Human mesenchymal stem RESULTS: Cx40-EGFP reporter plasmid injection led cells (hMSCs) are promising candidates for bone to EGFP fluorescence specifically in the abdominal engineering and regeneration with a considerable aorta of frog tadpoles. After crude manual enrichment number of experimental successes reported over the of highly Cx40-EGFP-positive EBs, the appearance of last years. However, hMSCs show several limitations cardiac and vascular structures was increased for tissue engineering applications, which can be approximately 3-fold. Immunofluorescent stainings overcome by using human embryonic stem cell- showed EGFP expression exclusively in vascular-like derived mesodermal progenitors (hES-MPs). The aim structures simultaneously expressing von Willebrand of this study was to investigate and compare the factor and in formerly beating areas expressing alpha- osteogenic differentiation potential of hMSCs and actinin. Cx40-EGFP-expressing EBs revealed hES-MPs. MATERIALS AND METHODS: The significantly higher numbers of beating osteogenic differentiation and mineralization behavior cardiomyocytes and vascular-like structures. of both cell types were evaluated at passage 5, 10, 15, Semiquantitative RT-PCRs confirmed an enhanced and 20. Expression of COL1A1, RUNX2, OPN, and cardiovascular differentiation as shown for the cardiac OC was evaluated by reverse transcription (RT)- markers Nkx2.5 and MLC2v, as well as the polymerase chain reaction, whereas mineralization endothelial marker vascular endothelial cadherin. was examined by photospectrometry, von Kossa CONCLUSIONS: Our work shows the feasibility of staining, and time-of-flight secondary ion mass specific labeling and purification of cardiovascular spectrometry. The immunoprofile of both cell types progenitor cells from differentiating EBs based on the was investigated by flow cytometry. RESULTS: We Cx40 promoter. We provide proof of principle that the demonstrated that, under proper stimulation, hES-MPs deleted CD4 (DeltaCD4) surface marker-based method undergo osteogenic differentiation and exhibit for magnetic cell sorting developed by our group will significantly increased mineralization ability compared be ideally suitable for transference to this promoter. to hMSCs after protracted expansion. hES-MPs were also found to express lower amount of human de Peppo, G. M. and D. Marolt (2012). "State of leukocyte antigens class II proteins. CONCLUSIONS: the art in stem cell research: human embryonic stem The high osteogenic ability of hES-MPs, together with cells, induced pluripotent stem cells, and low expression of human leukocyte antigens class II, transdifferentiation." J Blood Transfus 2012: 317632. makes these cells an attractive alternative for bulk Stem cells divide by asymmetric division and production of cells for bone engineering applications. display different degrees of potency, or ability to differentiate into various specialized cell types. Owing de Peppo, G. M., et al. (2013). "Human to their unique regenerative capacity, stem cells have embryonic stem cell-derived mesodermal progenitors generated great enthusiasm worldwide and represent display substantially increased tissue formation an invaluable tool with unprecedented potential for compared to human mesenchymal stem cells under biomedical research and therapeutic applications. Stem dynamic culture conditions in a packed bed/column cells play a central role in the understanding of bioreactor." Tissue Eng Part A 19(1-2): 175-187. molecular mechanisms regulating tissue development Bone tissue engineering represents a promising and regeneration in normal and pathological strategy to obviate bone deficiencies, allowing the ex conditions and open large possibilities for the vivo construction of bone substitutes with discovery of innovative pharmaceuticals to treat the unprecedented potential in the clinical practice. most devastating diseases of our time. Not least, their Considering that in the human body cells are intrinsic characteristics allow the engineering of constantly stimulated by chemical and mechanical functional tissues for replacement therapies that stimuli, the use of bioreactor is emerging as an promise to revolutionize the medical practice in the essential factor for providing the proper environment near future. In this paper, the authors present the for the reproducible and large-scale production of the characteristics of pluripotent stem cells and new engineered substitutes. Human mesenchymal stem developments of transdifferentiation technologies and cells (hMSCs) are experimentally relevant cells but, explore some of the biomedical applications that this regardless the encouraging results reported after emerging technology is expected to empower. culture under dynamic conditions in bioreactors, show important limitations for tissue engineering de Peppo, G. M., et al. (2010). "Osteogenic applications, especially considering their limited potential of human mesenchymal stem cells and proliferative potential, loss of functionality following human embryonic stem cell-derived mesodermal protracted expansion, and decline in cellular fitness progenitors: a tissue engineering perspective." Tissue associated with aging. On the other hand, we Eng Part A 16(11): 3413-3426. previously demonstrated that human embryonic stem

89 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cell-derived mesodermal progenitors (hES-MPs) hold capacity to generate all tissues, we induced murine ES great potential to provide a homogenous and unlimited cells to differentiate towards ectodermal and source of cells for bone engineering applications. neuroectodermal cell types and from there investigated Based on prior scientific evidence using different types their commitment towards the hair cell lineage in the of stem cells, in the present study we hypothesized that presence of EGF. Cells were collected at three points dynamic culture of hES-MPs in a packed bed/column along the differentiation pathway and their expression bioreactor had the potential to affect proliferation, profiles were determined using the Soares NMIE expression of genes involved in osteogenic mouse inner ear cDNA library printed in microarray differentiation, and matrix mineralization, therefore format. RESULTS: Three genes up-regulated after resulting in increased bone-like tissue formation. The addition of EGF (serine (or cysteine) proteinase reported findings suggest that hES-MPs constitute a inhibitor, clade H, member 1 (Serpinh1), solute carrier suitable alternative cell source to hMSCs and hold family 2 (facilitated glucose transporter), member 10 great potential for the construction of bone substitutes (Slc2a10) and secreted acidic cysteine-rich for tissue engineering applications in clinical settings. glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, De Repentigny, Y. and R. Kothary (2010). Serpinh1 and Slc2a10 have never been implicated in "Production of mouse chimeras by injection of the hearing process. embryonic stem cells into the perivitelline space of one-cell stage embryos." Transgenic Res 19(6): 1137- De Smedt, A., et al. (2008). "Optimisation of the 1144. cell cultivation methods in the embryonic stem cell Generation of mouse chimeras is useful for the test results in an increased differentiation potential of elucidation of gene function. In the present report, we the cells into strong beating myocard cells." Toxicol In describe a new technique for the production of Vitro 22(7): 1789-1796. chimeras by injection of R1 embryonic stem (ES) cells In order to support drug research in the selection into the perivitelline space of one-cell stage mouse process for non-embryotoxic pharmaceutical embryos. One-cell embryos are injected with 2-6 ES compounds, a screening method for embryotoxicity is cells into the perivitelline space under the zona needed. The murine embryonic stem cell test (EST) is pellucida without laser-assistance. Our embryo culture a validated in vitro test based on two permanent mouse experiments reveal that ES cells injected at the one- cell lines and delivering results in 10-days. cell stage embryo start to be incorporated into the Implementation of this test within our laboratory, blastomeres beginning at the 8-cell stage and form a revealed variability in the differentiation potential of chimeric blastocyst after 4 days. We have used this the embryonic stem cells and, as a consequence, a lot approach to successfully produce a high rate of mouse of assays needed to be rejected due the fact the chimeras in two different mouse genetic backgrounds acceptance criteria were not reached. In order to gain a permitting the establishment of germ line transmitters. better yield of contracting myocardial cells, we used (1) This method allows for the earlier introduction of ES a stringent control of the cell growth during cells into mouse embryos, and should free up the subcultivation and a standardised hanging drop culture possibility of using frozen one-cell embryos for this method and (2) a non-enzymatic cell harvest instead of purpose. a trypsin/EDTA cell harvest. Implementing of these cell culture modifications resulted in a decreased De Silva, M. G., et al. (2006). "Gene expression variability in the size of embryonic bodies, an increase changes during step-wise differentiation of embryonic of the number of acceptable tests and a significant stem cells along the inner ear hair cell pathway." Acta increase of the differentiation potential of embryonic Otolaryngol 126(11): 1148-1157. cells into strong beating myocardium, which made CONCLUSION: Our study outlines an scoring less time consuming. Testing of 6 reference alternative approach for the selection and investigation compounds in the optimized EST showed that the cell of genes involved in inner ear function. OBJECTIVE: culture modifications did not changed the in vitro To gain understanding of the gene pathways involved classification. in the development of the normal cochlea. MATERIALS AND METHODS: Microarray de Waard, H., et al. (2008). "Cell-type-specific technology currently offers the most efficient approach consequences of nucleotide excision repair to investigate gene expression and identify pathways deficiencies: Embryonic stem cells versus fibroblasts." involved in cell differentiation. Epidermal growth DNA Repair (Amst) 7(10): 1659-1669. factor (EGF) induces cultures derived from the organ Pluripotent embryonic stem cells (ES cells) are of Corti to proliferate and produce new hair cells. the precursors of all different cell types comprising the Since pluripotent embryonic stem (ES) cells have the organism. Since persistent DNA damage in this cell

90 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ type might lead to mutations that cause huge embryonic kidney cells. RESULTS: 293T cells can be malformations in the developing organism, genome readily cultured and passaged as spheres in serum-free caretaking is of prime importance. We first compared stem cell promoting culture conditions. Cells cultured the sensitivity of wild type mouse embryonic in vitro as three-dimensional spheres (3D) were shown fibroblasts (MEFs) and ES cells for various genotoxic to contain higher ALDH1 and CD44+/CD24- agents and show that ES cells are more sensitive to population compared to monolayer cells. These cells treatment with UV-light, gamma-rays and mitomycin were also resistant to radiation and upregulate stem C than MEFs. We next investigated the contribution of cell survival signaling including beta-catenin, Notch1 the transcription-coupled (TC-NER) and global and Survivin in response to radiation. Moreover, 3D genome (GG-NER) sub-pathways of nucleotide spheres generated from the 293T cells have increased excision repair (NER) in protection of ES cells, using expression of mesenchymal genes including vimentin, cells from mouse models for the NER disorders n-cadherin, , snail and slug as well as pro- xeroderma pigmentosum (XP) and Cockayne metastatic genes RhoC, Tenascin C and MTA1. In syndrome (CS). TC-NER-deficient Csb (-/-) and GG- addition, microRNAs implicated in self-renewal and NER/TC-NER-defective Xpa (-/-) MEFs are metastases were markedly reduced in 3D spheres. hypersensitive to UV, whereas GG-NER-deficient Xpc CONCLUSIONS: 293T cells exhibit a cancer stem (-/-) MEFs attribute intermediate UV sensitivity. The cell-like phenotype when cultured as 3D spheres and observed UV-hypersensitivity in Csb (-/-) and Xpa (-/-) represent an important research tool for studying the MEFs correlates with increased apoptosis. In contrast, molecular and biological mechanisms of cancer stem Xpa (-/-) and Xpc (-/-) ES cells are highly UV- cells and for testing and developing novel targets for sensitive, while a Csb deficiency only causes a mild cancer therapy. increase in UV-sensitivity. Surprisingly, a UV-induced hyperapoptotic response is mainly observed in Xpa (-/-) Delacroix, L., et al. (2010). "Cell-specific ES cells, suggesting a different mechanism of interaction of retinoic acid receptors with target genes apoptosis induction in ES cells, mainly triggered by in mouse embryonic fibroblasts and embryonic stem damage in the global genome rather than in transcribed cells." Mol Cell Biol 30(1): 231-244. genes (as in MEFs). Moreover, we show a pronounced All-trans retinoic acid (RA) induces transforming S-phase delay in Xpa (-/-) and Xpc (-/-) ES cells, growth factor beta (TGF-beta)-dependent autocrine which might well function as a safeguard mechanism growth of mouse embryonic fibroblasts (MEFs). We for heavily damaged ES cells in case the apoptotic have used chromatin immunoprecipitation to map 354 response fails. Although Xpa (-/-) and Xpc (-/-) ES RA receptor (RAR) binding loci in MEFs, most of cells are totally NER-defective or GG-NER-deficient which were similarly occupied by the RAR alpha and respectively, mutation induction upon UV is similar RAR gamma receptors. Only a subset of the genes compared to wild type ES cells indicating that the associated with these loci are regulated by RA, among observed apoptotic and cell cycle responses are indeed which are several critical components of the TGF-beta sufficient to protect against proliferation of damaged pathway. We also show RAR binding to a novel series cells. In conclusion, we show a double safeguard of target genes involved in cell cycle regulation, mechanism in ES cells against NER-type of damages, transformation, and metastasis, suggesting new which mainly relies on damage detection in the global pathways by which RA may regulate proliferation and genome. cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The Debeb, B. G., et al. (2010). "Characterizing majority comprised either degenerate DRs or no cancer cells with cancer stem cell-like features in 293T identifiable DRs but anomalously spaced half sites. human embryonic kidney cells." Mol Cancer 9: 180. Furthermore, we identify 462 RAR target loci in BACKGROUND: Since the first suggestion of embryonic stem (ES) cells and show that their prospectively identifiable cancer stem cells in solid occupancy is cell type specific. Our results also show tumors, efforts have been made to characterize that differences in the chromatin landscape regulate reported cancer stem cell surrogates in existing cancer the accessibility of a subset of more than 700 cell lines, and cell lines rich with these surrogates have identified loci to RARs, thus modulating the repertoire been used to screen for cancer stem cell targeted of target genes that can be regulated and the biological agents. Although 293T cells were derived from human effects of RA. embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that Deleu, S., et al. (2009). "Human cystic fibrosis self-renew as evidenced by serial xenograft passages embryonic stem cell lines derived on placental through transplantation. Herein we fully characterize mesenchymal stromal cells." Reprod Biomed Online cancer stem cell-like features in 293T human 18(5): 704-716.

91 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

This study describes the production of two new occurs prior to the onset of PAX6 expression, within a human embryonic stem cell (hESC) lines affected by pre-neuroepithelial stage. cystic fibrosis. These cell lines are heterozygous compounds, each a carrier of the DF508 mutations Denker, H. W. (2006). "Potentiality of embryonic associated either with E585X or with 3849+10 kb C-- stem cells: an ethical problem even with alternative >T. The derivation process was performed on stem cell sources." J Med Ethics 32(11): 665-671. irradiated human placental mesenchymal stromal cells The recent discussions about alternative sources and designed to minimize contact with xeno- of human embryonic stem cells (White Paper of the components. This new source of feeder cells is easy to US President's Council on Bioethics, 2005), while obtain and devoid of ethical concerns. The cells have a stirring new interest in the developmental potential of great capacity to proliferate which reduces the need for the various abnormal embryos or constructs proposed continuous preparation of new feeder cell lines. In as such sources, also raise questions about the addition, three normal hESC lines were obtained in the potential of the derived embryonic stem cells. The data same conditions. The five stem cell lines retained on the developmental potential of embryonic stem hESC-specific features, including an unlimited and cells that seem relevant for ethical considerations and undifferentiated proliferation capacity, marker aspects of patentability are discussed. Particular expression and the maintenance of stable karyotype. attention is paid to the meaning of "totipotency, They also demonstrated pluripotency in vitro, forming omnipotency and pluripotency" as illustrated by a cell lineages of the three germ layers, as indicated by comparison of the developmental potential of three- immunolocalization of beta-tubulin, alpha-fetoprotein dimensional clusters of blastomeres (morula), and actin. These new genetic cell lines represent an embryonic stem cells, somatic or (adult) stem cells or important in-vitro tool to study the physiological other somatic (non-stem) cells. This paper focuses on processes underlying this genetic disease, drug embryoid bodies and on direct cloning by tetraploid screening, and tissue engineering. complementation. Usage and patenting of these cells cannot be considered to be ethically sound as long as Denham, M., et al. (2012). "Glycogen synthase totipotency and tetraploid complementability of kinase 3beta and activin/nodal inhibition in human embryonic stem cells are not excluded for the specific embryonic stem cells induces a pre-neuroepithelial cell line in question. Testing this poses an ethical state that is required for specification to a floor plate problem in itself and needs to be discussed in the cell lineage." Stem Cells 30(11): 2400-2411. future. The floor plate is one of the major organizers of the developing nervous system through its secretion of Desai, N., et al. (2013). "Development of a xeno- sonic hedgehog (Shh). Although the floor plate is free non-contact co-culture system for derivation and located within the neural tube, the derivation of the maintenance of embryonic stem cells using a novel floor plate during development is still debatable and human endometrial cell line." J Assist Reprod Genet some studies suggest that floor plate cells are specified 30(5): 609-615. by Shh in a temporarily restricted window different to PURPOSE: Mouse embryonic fibroblast feeder neuroepithelial cells. Using human embryonic stem layers (MEF) have conventionally been used to culture cells (hESC) as a model of neurogenesis, we sought to and maintain the pluripotency of embryonic stem cells determine how floor plate cells may be temporarily (ESC). This study explores the potential of using a specified by SHH signaling during human novel human endometrial cell line to develop a non- embryogenesis. We found that inhibition of both xeno, non-contact co-culture system for ESC GSK3beta and activin/nodal pathways in hESC propagation and derivation. Such xeno-free systems induces a cellular state of SOX2+/PAX6- expression, may prove essential for the establishment of clinical we describe as "pre-neuroepithelial." Exposure of grade human ESC lines suitable for therapeutic SHH during this pre-neuroepithelial period causes the application. METHODS: A novel line of human expression of GLI transcription factors to function as endometrial cells were seeded in a 6-well dish. Filter activators and consequently upregulate expression of inserts containing mouse ESCs were placed on these the floor plate marker, FOXA2, while also supressing wells and passaged 2-3 times per week. Inner cell PAX6 expression to inhibit neuroepithelial fate. masses derived from mouse blastocysts were also FOXA2+ cells were able to efficiently generate cultured on transwells in the presence of the feeder mesencephalic dopaminergic neurons, a floor plate layer. In both cases, staining for SSEA-1, SOX-2, derivative. Overall, this study demonstrates a highly OCT-4 and alkaline phosphatase were used to monitor efficient system for generating floor plate cells from the retention of stem cells. RESULTS: ESC colonies hESC and, most importantly, reveals that specification retained their stem cell morphology and attributes for of floor plate cells is temporally dependent, whereby it over 120 days in culture and 44 passages to date. Inner

92 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cell mass derived ESC cultures were maintained in a procedure. The established lineage-specific pluripotent state for 45 days, through 6 passages with differentiation protocols developed for ESCs are being retention of all stem cell characteristics. The stem cell applied to iPSCs, as they have great potential in colonies expressed stem cell specific markers SSEA-1, regenerative medicine for cell therapy, disease Sox 2, Oct-4 and alkaline phosphatase. Upon removal modeling either for drug development or for of the human feeder layer, there was a distinct change fundamental science, and, last but not least, toxicity in cell morphology within the colonies and evidence of testing. This paper reviews efforts aimed at practical ESC differentiation. CONCLUSIONS: Human feeder development of iPSC differentiation to neural/cardiac layers offer a simple path away from the use of MEF lineages and further the use of these iPSCs-derived feeder cells or MEF conditioned medium for ESC cells for drug development and toxicity testing. culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent Devereaux, M. W. (2007). "Alternative sources contamination of stem cell preparations with feeder of adult stem cells: a possible solution to the cells during passaging. embryonic stem cell debate." Gend Med 4(1): 85; author reply 86. Desai, N., et al. (2011). "Vitrification of mouse embryo-derived ICM cells: a tool for preserving Di Giorgio, F. P., et al. (2008). "Human embryonic stem cell potential?" J Assist Reprod Genet embryonic stem cell-derived motor neurons are 28(2): 93-99. sensitive to the toxic effect of glial cells carrying an PURPOSE: Vitrification technology presents ALS-causing mutation." Cell Stem Cell 3(6): 637-648. new opportunities for preservation of embryo derived It has been proposed that human embryonic stem stem cells without first establishing a viable ESC line. cells could be used to provide an inexhaustible supply This study tests the feasibility of cryopreserving ICM of differentiated cell types for the study of disease cells using vitrification. MATERIALS AND processes. Although methods for differentiating METHODS: ICMs from mouse embryos were isolated embryonic stem cells into specific cell types have and vitrified in HSV straws or on cryoloops. Upon become increasingly sophisticated, the utility of the warming, the vitrified ICMs were cultured and resulting cells for modeling disease has not been observed for attachment and morphology. Colonies determined. We have asked whether specific neuronal were passaged every 3-6 days. ICMs and ICM-derived subtypes produced from human embryonic stem cells ESC colonies were tested for expression of stem cell can be used to investigate the mechanisms leading to specific markers. RESULTS: ICMs vitrified on both neural degeneration in amyotrophic lateral sclerosis the cryoloop and the HSV straw had high survival (ALS). We show that human spinal motor neurons, but rates. ICM derived ESCs remained undifferentiated for not interneurons, are selectively sensitive to the toxic several passages and demonstrated expression of effect of glial cells carrying an ALS-causing mutation typical stem cell markers; SSEA-1, Sox-2, Oct 4 and in the SOD1 gene. Our findings demonstrate the alkaline phosphatase. CONCLUSION: This is the first relevance of these non-cell-autonomous effects to report on successful vitrification of isolated ICMs and human motor neurons and more broadly demonstrate the subsequent derivation of ESC colonies. the utility of human embryonic stem cells for studying Vitrification of isolated ICMs is a novel approach for disease and identifying potential therapeutics. preservation of the "stem cell source" material. Dihazi, H., et al. (2011). "Multipotent adult Deshmukh, R. S., et al. (2012). "Drug discovery germline stem cells and embryonic stem cells models and toxicity testing using embryonic and functional proteomics revealed an important role of induced pluripotent stem-cell-derived cardiac and eukaryotic initiation factor 5A (Eif5a) in stem cell neuronal cells." Stem Cells Int 2012: 379569. differentiation." J Proteome Res 10(4): 1962-1973. Development of induced pluripotent stem cells Multipotent adult germline stem cells (maGSCs) (iPSCs) using forced expression of specific sets of are pluripotent cells that can be differentiated into transcription factors has changed the field of stem cell somatic cells of the three primary germ layers. To research extensively. Two important limitations for highlight the protein profile changes associated with research application of embryonic stem cells (ESCs), stem cell differentiation, retinoic acid (RA) treated namely, ethical and immunological issues, can be mouse stem cells (maGSCs and ESCs) were compared circumvented using iPSCs. Since the development of to nontreated stem cells. 2-DE and DIGE reference first iPSCs, tremendous effort has been directed to the maps were created, and differentially expressed development of methods to increase the efficiency of proteins were further processed for identification. In the process and to reduce the extent of genomic both stem cell types, the RA induced differentiation modifications associated with the reprogramming resulted in an alteration of 36 proteins of which 18

93 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ were down-regulated and might be potential phenotypic conversion of stem cells into inner ear pluripotency associated proteins, whereas the other 18 hair-cell-like cells. Here, we induced the proteins were up-regulated. These might be correlated differentiation of human embryonic stem cells (hESCs) to stem cell differentiation. Surprisingly, eukaryotic into otic epithelial progenitors (OEPs), and further initiation factor 5A (Eif5a), a protein which is essential induced the differentiation of OEPs into hair-cell-like for cell proliferation and differentiation, was cells using different substrates. Our results showed significantly down-regulated under RA treatment. A that OEPs cultured on the chicken utricle stromal cells time-dependent investigation of Eif5a showed that the with the induction medium could differentiate into RA treatment of stem cells resulted in a significant up- hair-cell-like cells with stereociliary bundles. Co- regulation of the Eif5a in the first 48 h followed by a culture with stromal cells, however, may be progressive down-regulation thereafter. This effect problematic for subsequent examination of the induced could be blocked by the hypusination inhibitor hair-cell-like cells. In order to avoid the interference ciclopirox olamine (CPX). The alteration of Eif5a from stromal cells, we cultured OEPs on laminin with hypusination, as confirmed by mass spectrometry, different induction media and examined the effects of exerts an antiproliferative effect on ESCs and maGSCs the induction medium on the differentiation potentials in vitro, but does not affect the cell pluripotency. Our of OEPs into hair-cell-like cells. The results revealed data highlights the important role of Eif5a and its that the culture of OEPs on laminin with the hypusination for stem cell differentiation and conditioned medium from chicken utricle stromal cells proliferation. supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into Dihne, M., et al. (2006). "Embryonic stem cell- epithelial clusters displaying hair-cell-like cells with derived neuronally committed precursor cells with stereociliary bundles. These cells also displayed the reduced teratoma formation after transplantation into expected electrophysiological properties. the lesioned adult mouse brain." Stem Cells 24(6): 1458-1466. Ding, X., et al. (2012). "Polycomb group protein The therapeutic potential of embryonic stem (ES) Bmi1 promotes hematopoietic cell development from cells in neurodegenerative disorders has been widely embryonic stem cells." Stem Cells Dev 21(1): 121-132. recognized, and methods are being developed to Bmi1 is a component of the Polycomb repressive optimize culture conditions for enriching the cells of complexes and essential for maintaining the pool of interest and to improve graft stability and safety after adult stem cells. Polycomb repressive complexes are transplantation. Whereas teratoma formation rarely key regulators for embryonic development by occurs in xenogeneic transplantation paradigms of ES modifying chromatin architecture and maintaining cell-derived neural progeny, more than 70% of mice gene repression. To assess the role of Bmi1 in that received murine ES cell-derived neural precursor pluripotent stem cells and on exit from pluripotency cells develop teratomas, thus posing a major safety during differentiation, we studied forced Bmi1 problem for allogeneic and syngeneic transplantation expression in mouse embryonic stem cells (ESC). We paradigms. Here we introduce a new differentiation found that ESC do not express detectable levels of protocol based on the generation of substrate-adherent Bmi1 RNA and protein and that forced Bmi1 ES cell-derived neural aggregates (SENAs) that expression had no obvious influence on ESC self- consist predominantly of neuronally committed renewal. However, upon ESC differentiation, Bmi1 precursor cells. Purified SENAs that were effectively enhanced development of hematopoietic differentiated into immature but postmitotic neurons cells. Global transcriptional profiling identified a large did not form tumors up to four months after syngeneic array of genes that were differentially regulated during transplantation into the acutely degenerated striatum ESC differentiation by Bmi1. Importantly, we found and showed robust survival. that Bmi1 induced a prominent up-regulation of Gata2, a zinc finger transcription factor, which is essential for Ding, J., et al. (2016). "Induction of primitive hematopoietic cell generation from differentiation of human embryonic stem cells into mesoderm. In addition, Bmi1 caused sustained growth functional hair-cell-like cells in the absence of stromal and a >100-fold expansion of ESC-derived cells." Int J Biochem Cell Biol 81(Pt A): 208-222. hematopoietic stem/progenitor cells within 2-3 weeks Sensorineural hearing loss and vestibular of culture. The enhanced proliferative capacity was dysfunction have become the most common forms of associated with reduced Ink4a/Arf expression in sensory defects. Stem cell-based therapeutic strategies Bmi1-transduced cells. Taken together, our for curing hearing loss are being developed. Several experiments demonstrate distinct activities of Bmi1 in attempts to develop hair cells by using chicken utricle ESC and ESC-derived hematopoietic progenitor cells. stromal cells as feeder cells have resulted in In addition, Bmi1 enhances the propensity of ESC in

94 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ differentiating toward the hematopoietic lineage. Thus, spontaneously active neural networks. Until recently Bmi1 could be a candidate gene for engineered adult very few studies have reported hESC-derived neurons stem cell derivation from ESC. capable of forming such networks, suggesting lack of certain components in culture media to promote Do, E. K., et al. (2014). "Reptin regulates mature synaptogenesis. In this review we discuss the pluripotency of embryonic stem cells and somatic cell various factors that enhance functional synapse reprogramming through Oct4-dependent mechanism." formation in primary and stem cell-derived neuronal Stem Cells 32(12): 3126-3136. cultures. These factors include astrocytes, astrocyte- Oct4 has been implicated in regulation of derived factors, cell adhesion molecules and pluripotency in embryonic stem cells (ESCs) and neurotrophins. We discuss the current literature on reprogramming of somatic cells into induced studies that have used these factors for functional pluripotent stem cells. However, the molecular differentiation of primary neural cultures, and discuss mechanisms involved in Oct4-dependent regulation of its implications for stem cell -derived neural cultures. pluripotency and reprogramming have not been clear. To gain insight into the mechanism of regulation of Domev, H., et al. (2012). "Efficient engineering Oct4-mediated self-renewal of ESCs and of vascularized ectopic bone from human embryonic reprogramming of somatic cells, we attempted to stem cell-derived mesenchymal stem cells." Tissue identify Oct4-binding proteins using affinity Eng Part A 18(21-22): 2290-2302. purification and mass spectrometry. We identified Human mesenchymal stem cells (hMSCs) can be Reptin, a key component of ATP-dependent chromatin derived from various adult and fetal tissues. However, remodeling complexes, as an Oct4-binding protein. the quality of tissues for the isolation of adult and fetal Depletion of endogenous Reptin using lentiviral short hMSCs is donor dependent with a nonreproducible hairpin RNA (shRNA) led to a decrease in the number yield. In addition, tissue engineering and cell therapy and size of alkaline phosphatase-positive colonies of require large-scale production of a pure population of mouse ESCs. In addition, shRNA-mediated silencing lineage-restricted stem cells that can be easily induced of Reptin resulted in decreased expression of to differentiate into a specific cell type. Therefore, pluripotency-specific marker genes, including Oct4, human embryonic stem cells (hESCs) can provide an Sox2, Nanog, and SSEA-1. Results of the Oct4 alternative, plentiful source for generation of reporter assay showed synergism between Oct4 and reproducible hMSCs. We have developed efficient Reptin, and depletion of endogenous Reptin abolished differentiation protocols for derivation of hMSCs from Oct4 transcriptional activity. Results of a chromatin hESCs, including coculture with murine OP9 stromal immunoprecipitation assay showed the overlapping cells and feeder layer-free system. Our protocols have interaction of Reptin and Oct4 to CR4 in the Oct4 resulted in the generation of up to 49% of hMSCs, enhancer in ESCs. Knockdown of Reptin using which expressed CD105, CD90, CD29, and CD44. shRNA suppressed the reprogramming of mouse The hMSCs exhibited high adipogenic, chondrocytic, embryonic fibroblasts to induced pluripotent stem cells, and osteogenic differentiation in vitro. The latter whereas overexpression of Reptin resulted in enhanced correlated with osteocalcin secretion and vascular efficiency of induced pluripotent stem cell generation. endothelial growth factor (VEGF) production by the These results strongly suggest that Reptin plays a key differentiating hMSCs. hMSC-derived osteoblasts role in maintaining the pluripotency of ESCs and in further differentiated and formed ectopic bone in vivo, establishing the pluripotency during reprogramming of and induced the formation of blood vessels in Matrigel somatic cells by regulation of Oct4-mediated gene implants. Our protocol enables generation of a purified regulation. population of hESC-derived MSCs, with the potential of differentiating into several mesodermal lineages, Dodla, M. C., et al. (2010). "Role of astrocytes, and particularly into vasculogenesis-inducing soluble factors, cells adhesion molecules and osteoblasts, which can contribute to the development neurotrophins in functional synapse formation: of bone repair protocols. implications for human embryonic stem cell derived neurons." Curr Stem Cell Res Ther 5(3): 251-260. Dong, W., et al. (2013). "Antitumor effect of Availability of human embryonic stem cells embryonic stem cells in a non-small cell lung cancer (hESCs) and its neural derivatives has opened up wide model: antitumor factors and immune responses." Int J possibilities of using these cells as tools for Med Sci 10(10): 1314-1320. developmental studies, drug screening and cell Research in recent years has revealed that therapies for treating neurodegenerative diseases. embryonic stem cells (ESCs) could generate obvious However, for hESC-derived neurons to fulfill their antitumor effects in both vitro and vivo. In vitro, ESCs potential they need to form functional synapses and could secrete soluble factors that are capable of

95 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ blocking cancer cells proliferation, moreover, smooth muscle cells, epithelial-like cells, neuronal-like embryonic microenvironments could effectively cells, osteoblasts and monocytes. Interestingly, inhibit tumorigenesis and metastasis; while in vivo, development of cardiomyocytes from the BMP2+ cells administration of ESCs in tumor-bearing mice could requires secondary EB formation. CONCLUSION: generate significant antitumor effects by indirectly This is the first study to identify the complete activating the antitumor immune system. In this study, transcriptome of BMP2+ cells and cell phenotypes non-small cell lung cancer cells (Lewis Lung from a mesodermal origin, thus offering an insight into Carcinoma cells, LLCs) and ESCs were co-injected the role of BMP2+ cells during embryonic together into mice, after that subcutaneous tumor developmental processes in vivo. growth was monitored, cellular and humoral immune responses were detected, and different control groups Doss, M. X., et al. (2004). "Embryonic stem cells: were set to compare the results in different conditions. a promising tool for cell replacement therapy." J Cell Our results suggested that compared to be injected Mol Med 8(4): 465-473. alone, ESCs co-injected with cancer cells could inhibit Embryonic stem (ES) cells are revolutionizing cancer cell growth more efficiently in vivo, with more the field of developmental biology as a potential tool CD8+ lymphocytes generated in both peripheral to understand the molecular mechanisms occurring circulation and spleen, and with higher serum during the process of differentiation from the anticancer cytokine level (interleukin (IL)-2 and embryonic stage to the adult phenotype. ES cells interferon (IFN)-gamma). We conclude that the harvested from the inner cell mass (ICM) of the early boosted antitumor effects induced by ESCs and cancer embryo can proliferate indefinitely in vitro while cells co-injection may be both the effects of antitumor retaining the ability to differentiate into all somatic factors secreted by ESCs and immune responses cells. Emerging results from mice models with ES induced by ESCs in vivo. cells are promising and raising tremendous hope among the scientific community for the ES-cell based Doss, M. X., et al. (2007). "Transcriptomic and cell replacement therapy (CRT) of various severe phenotypic analysis of murine embryonic stem cell diseases. ES cells could potentially revolutionize derived BMP2+ lineage cells: an insight into medicine by providing an unlimited renewable source mesodermal patterning." Genome Biol 8(9): R184. of cells capable of replacing or repairing tissues that BACKGROUND: Bone morphogenetic protein have been damaged in almost all degenerative diseases (BMP)2 is a late mesodermal marker expressed during such as diabetes, myocardial infarction and vertebrate development and plays a crucial role in Parkinson's disease. This review updates the progress early embryonic development. The nature of the of ES cell research in CRT, discusses about the BMP2-expressing cells during the early stages of problems encountered in the practical utility of ES embryonic development, their transcriptome and cell cells in CRT and evaluates how far this approach is phenotypes developed from these cells have not yet successful experimentally. been characterized. RESULTS: We generated a transgenic BMP2 embryonic stem (ES) cell lineage Doss, M. X., et al. (2010). "Global transcriptomic expressing both puromycin acetyltransferase and analysis of murine embryonic stem cell-derived enhanced green fluorescent protein (EGFP) driven by brachyury (+) (T) cells." Genes Cells 15(3): 209-228. the BMP2 promoter. Puromycin resistant and EGFP Brachyury (+) mesodermal cell population with positive BMP2+ cells with a purity of over 93% were purity over 79% was obtained from differentiating isolated. Complete transcriptome analysis of BMP2+ brachyury embryonic stem cells (ESC) generated with cells in comparison to the undifferentiated ES cells brachyury promoter driven enhanced green fluorescent and the control population from seven-day-old protein and puromycin-N-acetyltransferase. A embryoid bodies (EBs; intersection of genes comprehensive transcriptomic analysis of brachyury (+) differentially expressed between undifferentiated ES cells enriched with puromycin application from 6-day- cells and BMP2+ EBs as well as differentially old embryoid bodies (EBs), 6-day-old control EBs and expressed between seven-day-old control EBs and undifferentiated ESCs led to identification of 1573 BMP2+ EBs by t-test, p < 0.01, fold change >2) by uniquely up-regulated and 1549 uniquely down- microarray analysis led to identification of 479 regulated transcripts in brachyury (+) cells. specifically upregulated and 193 downregulated Furthermore, transcripts up-regulated in brachyury (+) transcripts. Transcription factors, apoptosis promoting cells have overrepresented the factors and other signaling molecules involved in early annotations (cell differentiation, blood vessel embryonic development are mainly upregulated in morphogenesis, striated muscle development, placenta BMP2+ cells. Long-term differentiation of the BMP2+ development and cell motility) and Kyoto cells resulted in neural crest stem cells (NCSCs), Encyclopedia of Genes and Genomes pathway

96 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ annotations (mitogen-activated protein kinase SRF-dependent SMC differentiation autonomously or signaling and transforming growth factor beta in concert with myocardin. signaling). Transcripts representing Larp2 and Ankrd34b are notably up-regulated in brachyury (+) Duggal, G., et al. (2015). "Exogenous cells. Knockdown of Larp2 resulted in a significantly supplementation of Activin A enhances germ cell down-regulation BMP-2 expression, and knockdown differentiation of human embryonic stem cells." Mol of Ankrd34b resulted in alteration of NF-H, Hum Reprod 21(5): 410-423. PPARgamma and PECAM1 expression. The Human embryonic stem cells (hESCs) derived in elucidation of transcriptomic signatures of ESCs- the presence of Activin A (ActA) demonstrate an derived brachyury (+) cells will contribute toward increased differentiation propensity toward the germ defining the genetic and cellular identities of cell lineage. In addition, mouse epiblast stem cells and presumptive mesodermal cells. Furthermore, there is a mouse epiblast-like cells are poised toward germ cell possible involvement of Larp2 in the regulation of the differentiation and are derived in the presence of ActA. late mesodermal marker BMP-2. Ankrd34b might be a We therefore investigated whether supplementation positive regulator of neurogenesis and a negative with ActA enhances in vitro hESC differentiation regulator of adipogenesis. toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 Du, K. L., et al. (2004). "Megakaryoblastic (developmental pluripotency associated 3) and leukemia factor-1 transduces cytoskeletal signals and tyrosine kinase receptor cKIT in both ActA-derived induces smooth muscle cell differentiation from and standard-derived hESCs indicating its role in undifferentiated embryonic stem cells." J Biol Chem priming hESCs toward the PGC lineage. Indeed, ActA 279(17): 17578-17586. plus bone morphogenic protein 4 (BMP4) strongly The SAP domain transcription factor myocardin increased germ cell differentiation potential of hESCs plays a critical role in the transcriptional program based on the high expression of late PGC markers regulating smooth muscle cell differentiation. In this DAZL (deleted in azoospermia-like) and report, we describe the capacity of myocardin to VASA/DDX4 (DEAD-box polypeptide 4) at mRNA physically associate with megakaryoblastic leukemia and protein level. Hence, the combination of ActA factor-1 (MKL1) and characterize the function of with BMP4 provides an additional boost for hESCs to MKL1 in smooth muscle cells (SMCs). The MKL1 develop into postmigratory germ cells. Together with gene is expressed in most human tissues and increased VASA expression in the presence of ActA myocardin and MKL are co-expressed in SMCs. and BMP4, we also observed up-regulation of MKL1 and myocardin physically associate via endoderm-specific genes GATA4 (GATA binding conserved domains. Overexpression of protein 4) and GATA6. Finally, we were able to MKL1 transactivates (SRF)- further mature these in vitro-derived PGC-like cells dependent SMC-restricted transcriptional regulatory (PGCLCs) by culturing them in in vitro maturation elements including the SM22alpha promoter, smooth (IVM) medium, resulting in the formation of germ muscle myosin heavy chain promoter/enhancer, and cell-like clusters and induction of meiotic gene SM-alpha-actin promoter/enhancer in non-SMCs. expression. In conclusion, we demonstrate for the first Moreover, forced expression of MKL1 and SRF in time a synergism between ActA and BMP4 in undifferentiated SRF (-/-) embryonic stem cells facilitating germ cell-directed differentiation of hESCs, activates multiple endogenous SMC-restricted genes at which is enhanced by extended culture in IVM levels equivalent to, or exceeding, myocardin. Forced medium, as shown by cytoplasmic VASA-expressing expression of a dominant-negative MKL1 mutant PGCLCs. We propose a novel relationship between reduces myocardin-induced activation of the SMC- the endoderm and germ cell lineage during hESC specific SM22alpha promoter. In NIH3T3 fibroblasts differentiation. MKL1 localizes to the cytoplasm and translocates to the nucleus in response to serum stimulation, actin Dukhovny, A., et al. (2012). "Varicella-zoster treadmilling, and RhoA signaling. In contrast, in virus infects human embryonic stem cell-derived SMCs MKL1 is observed exclusively in the nucleus neurons and neurospheres but not pluripotent regardless of serum conditions or RhoA signaling. embryonic stem cells or early progenitors." J Virol However, when actin polymerization is disrupted 86(6): 3211-3218. MKL1 translocates from the nucleus to the cytoplasm Pluripotent human stem cells are a powerful tool in SMCs. Together, these data were consistent with a for the generation of differentiated cells that can be model wherein MKL1 transduces signals from the used for the study of human disease. We recently cytoskeleton to the nucleus in SMCs and regulates demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected

97 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ by the highly host-restricted human alphaherpesvirus cloned embryos were successfully used to derive ntES- varicella-zoster virus (VZV), permitting the interaction like cells. The rate of primary colony formation rate of VZV with neurons to be readily evaluated in culture. was 62.50% +/- 4.62 for fibroblast donor cell derived In the present study, we examine whether pluripotent embryos. The same was 60.60% +/- 4.62 for putative hESC and neural progenitors at intermediate stages of ES donor cell derived embryos and 66.66% +/- 4.62 differentiation are permissive for VZV infection. We for lymphocyte donor cell derived embryos, demonstrate here that VZV infection is blocked in respectively. The putative ntES colonies were naive hESC. A block to VZV replication is also seen positively characterized for alkaline phosphatase, Oct- when a bacterial artificial chromosome (BAC) 4, TRA-1-60, TRA-1-81, Sox-2, and Nanog by containing the VZV genome is transfected into hESC. Immunocytochemistry and Reverse Transcription PCR. In contrast, related alphaherpesviruses herpes simplex To further validate the stem ness, the produced virus 1 (HSV-1) and pseudorabies virus (PrV) putative ntES colonies were differentiated to embryoid productively infect naive hESC in a cell-free manner, bodies. Immunocytochemistry revealed that embryoid and PrV replicates from a BAC transfected into hESC. bodies expressed NESTIN specific for ectodermal Neurons differentiate from hESC via neural progenitor lineage; GATA-4 for endodermal lineage and smooth intermediates, as is the case in the embryo. The first in muscle actin-I, and troponin-I specific for mesodermal vitro stage at which permissiveness of hESC-derived lineage. The study has established an efficient protocol neural precursors to VZV replication is observed is for putative ntES cell derivation from HMC embryos. upon formation of "neurospheres," immediately after It could be of substantial significance as patient detachment from the inductive stromal feeder layer. specific ntES cells have proven therapeutic These findings suggest that hESC may be useful in significance. deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication. Easley, C. A., et al. (2014). "Gamete derivation from embryonic stem cells, induced pluripotent stem Dutta, R., et al. (2011). "A comparative study on cells or somatic cell nuclear transfer-derived efficiency of adult fibroblast, putative embryonic stem embryonic stem cells: state of the art." Reprod Fertil cell and lymphocyte as donor cells for production of Dev 27(1): 89-92. handmade cloned embryos in goat and characterization Generating gametes from pluripotent stem cells of putative ntES cells obtained from these embryos." (PSCs) has many scientific justifications and several Theriogenology 76(5): 851-863. biomedical rationales. Here, we consider several The main purpose of the experiment was to strategies for deriving gametes from PSCs from mice compare the efficiency of three cell types, namely and primates (human and non-human) and their adult fibroblast, putative embryonic stem (ES) cell, anticipated strengths, challenges and limitations. and lymphocyte, as donor cells for somatic cell nuclear Although the 'Weismann barrier', which separates the transfer by handmade cloning in goats. The outcome mortal somatic cell lineages from the potentially clearly shows that putative embryonic stem cells, with immortal germline, has long existed, breakthroughs a cleavage and blastocyst production rate of 74.69% first in mice and now in humans are artificially +/- 3.92 and 39.75% +/- 3.86, respectively, performs creating germ cells from somatic cells. Spermatozoa better in comparison to adult fibroblast cell and with full reproductive viability establishing multiple lymphocyte. Between adult fibroblast cell and generations of seemingly normal offspring have been lymphocyte no statistically significant difference exists reported in mice and, in humans, haploid spermatids at P < 0.05. An overall cleavage and blastocyst with correct parent-of-origin imprints have been formation rate of 67.41% +/- 3.92 and 26.96% +/- 3.86 obtained. Similar progress with making oocytes has was obtained using adult fibroblast donor cells. The been published using mouse PSCs differentiated in study establishes beyond doubt the reprogrammability vitro into primordial germ cells, which are then of lymphocyte by handmade cloning (HMC) protocol cultured after xenografting reconstructed artificial with a cleavage and blastocyst production rate of 56.47% ovaries. Progress in making human oocytes artificially +/- 3.92 and 24.70% +/- 3.86, respectively. PCR is proving challenging. The usefulness of these analysis of highly polymorphic 286 bp fragment of artificial gametes, from assessing environmental MHC II DRB genes of cloned embryos and three exposure toxicity to optimising medical treatments to donor cells were performed to verify the cloned prevent negative off-target effects on fertility, may embryos. The amplified PCR products were subjected prove invaluable, as may basic discoveries on the to SSCP to confirm their genetic identity. The fundamental mechanisms of gametogenesis. karyotyping of the cloned embryos showed normal chromosomal status as expected in goat. Significantly, Eckardt, S., et al. (2008). "In vivo and in vitro in the second stage of the experiment, the produced differentiation of uniparental embryonic stem cells

98 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ into hematopoietic and neural cell types." disease modeling, drug/toxicology screens, and Organogenesis 4(1): 33-41. cellular replacement therapy. The biological role of genomic imprinting in adult tissue is central to the consideration of Egozi, D., et al. (2007). "Regulation of the cell transplanting uniparental embryonic stem (ES) cell- cycle inhibitor p27 and its ubiquitin ligase Skp2 in derived tissues. We have recently shown that both differentiation of human embryonic stem cells." maternal (parthenogenetic/gynogenetic) and paternal FASEB J 21(11): 2807-2817. (androgenetic) uniparental ES cells can differentiate, Embryonic stem cells combine the features of both in vivo in chimeras and in vitro, into adult- robust proliferation with precise differentiation repopulating hematopoietic stem and progenitor cells. capacity. p27 is a cell cycle inhibitor that is involved This suggests that, at least in some tissues, the in the regulation of proliferation and differentiation in presence of two maternal or two paternal genomes many developing tissues. Recent studies in murine does not interfere with stem cell function and tissue embryonic stem cells have suggested that p27 is homeostasis in the adult. Here, we consider involved in the progression of normal differentiation implications of the contribution of uniparental cells to programs in these cells. However, the expression and hematopoiesis and to development of other organ regulation of p27 and its role in the differentiation of systems, notably neural tissue for which consequences human embryonic stem cells (hESc) has not been of genomic imprinting are associated with a known previously explored. Herein we show that p27 bias in development and behavioral disorders. Our expression was low in undifferentiated hESc, but findings so far indicate that there is little or no limit to increased markedly in differentiated cells. The the differentiation potential of uniparental ES cells expression of Skp2, the ubiquitin ligase that targets outside the normal developmental paradigm. As a p27 for degradation, was inversely related to p27 potentially donor MHC-matching source of tissue, expression. Moreover, embryoid bodies (EBs) with uniparental transplants may provide not only a clinical low p27 expression and high Skp2/p27 ratio showed resource but also a unique tool to investigate aspects of poorer differentiation than those with high p27 genomic imprinting in adults. expression. Modulation of Skp2 expression is mainly regulated by its rate of degradation. In contrast to Efthymiou, A. G., et al. (2014). "Self-renewal somatic cells, which have high levels of Skp2 mainly and cell lineage differentiation strategies in human in S and G2/M, in undifferentiated hESc Skp2 levels embryonic stem cells and induced pluripotent stem were also high in G1. These results point to a cells." Expert Opin Biol Ther 14(9): 1333-1344. potentially important role for p27 regulation in hESc. INTRODUCTION: Since the initial discoveries of human embryonic and induced pluripotent stem Eiges, R., et al. (2001). "Establishment of human cells, many strategies have been developed to utilize embryonic stem cell-transfected clones carrying a the potential of these cells for translational research marker for undifferentiated cells." Curr Biol 11(7): and disease modeling. The success of these aims and 514-518. the development of future applications in this area will Human embryonic stem (ES) cells are pluripotent depend on the ability to generate high-quality and cell lines that have been derived from the inner cell large numbers of differentiated cell types that mass (ICM) of blastocyst stage embryos [1--3]. They genetically, epigenetically, and functionally mimic the are characterized by their ability to be propagated cells found in the body. AREAS COVERED: In this indefinitely in culture as undifferentiated cells with a review, we highlight the current strategies used to normal karyotype and can be induced to differentiate maintain stem cell pluripotency (a measure of stem in vitro into various cell types [1, 2, 4-- 6]. Thus, cell quality), as well as provide an overview of the human ES cells promise to serve as an unlimited cell various differentiation strategies being used to source for transplantation. However, these unique cell generate cells from all three germ lineages. We also lines tend to spontaneously differentiate in culture and discuss the particular considerations that must be therefore are difficult to maintain. Furthermore, addressed when utilizing these cells for translational colonies may contain several cell types and may be therapy, and provide an example of a cell type composed of cells other than pluripotent cells [1, 2, 6]. currently used in clinical trials. EXPERT OPINION: In order to overcome these difficulties and establish The major challenge in regenerative medicine and lines of cells with an undifferentiated phenotype, we disease modeling will be in generating functional cells have introduced a reporter gene that is regulated by a of sufficient quality that are physiologically and promoter of an ES cell-enriched gene into the cells. epigenetically similar to the diverse cells that they are For the introduction of DNA into human ES cells, we modeled after. By meeting these criteria, these have established a specific transfection protocol that is differentiated products can be successfully used in different from the one used for murine ES cells.

99 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

Human ES cells were transfected with enhanced green The routine culture and expansion of human fluorescence protein (EGFP), under the control of embryonic stem (hES) cells has been and is still posing murine Rex1 promoter. The transfected cells show a challenge to researchers wishing to take advantage of high levels of GFP expression when in an the cells' unique potential. In contrast to mouse undifferentiated state. As the cells differentiate, this embryonic stem cells, hES cells usually have to be expression is dramatically reduced in monolayer expanded by tedious mechanical microdissection or by cultures as well as in the primitive endoderm of early enzymatic dissociation to cell clusters of a very narrow stage (simple) embryoid bodies (EBs) and in mature size range.It is essential to use a culture system that EBs. The undifferentiated cells expressing GFP can be allows the robust and reproducible enzymatic analyzed and sorted by using a Fluorescence Activated dissociation of viable hES cell cultures to single cells Cell Sorter (FACS). Thus, we have established lines of to allow the scale-up of hES cell cultures as well as the human ES cells in which only undifferentiated cells application of hES cells in various experiments, such are fluorescent, and these cells can be followed and as FACS, electroporation, and clonal selection.By the selected for in culture. We also propose that the development of enzyme-based protocols, which are pluripotent nature of the culture is made evident by the less labor intensive and less time consuming, much ability of the homogeneous cell population to form progress has been made over the recent years with EBs. The ability to efficiently transfect human ES cells regard to improved culture systems for hES cell. We will provide the means to study and manipulate these have developed a culture system that is based on single cells for the purpose of basic and applied research. cell enzymatic dissociation (SCED) in combination with a highly supportive feeder cell layer of human El-Badawy, A. and N. El-Badri (2016). "The cell foreskin fibroblasts (hFFs). The culture system allows cycle as a brake for beta-cell regeneration from defined enzymatic propagation while maintaining the embryonic stem cells." Stem Cell Res Ther 7: 9. hES cell lines in an undifferentiated, pluripotent, and The generation of insulin-producing beta cells normal state.In this chapter, we will show how hES from stem cells in vitro provides a promising source of cells, which have been derived and passaged by cells for cell transplantation therapy in diabetes. traditional mechanical dissection, can be rapidly However, insulin-producing cells generated from adjusted to propagation by enzymatic dissociation to human stem cells show deficiency in many functional single cells. The protocols we describe are widely characteristics compared with pancreatic beta cells. applicable and should therefore be of general use for Recent reports have shown molecular ties between the the reliable mass cultivation of hES cells for various cell cycle and the differentiation mechanism of experiments. embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Ellerstrom, C., et al. (2007). "Facilitated Both beta cells and ES cells possess unique cell cycle expansion of human embryonic stem cells by single- machinery yet with significant contrasts. In this review, cell enzymatic dissociation." Stem Cells 25(7): 1690- we compare the cell cycle control mechanisms in both 1696. ES cells and beta cells, and highlight the fundamental Traditionally, human embryonic stem cells differences between pluripotent cells of embryonic (hESCs) are propagated by mechanical dissection or origin and differentiated beta cells. Through critical enzymatic dissociation into clusters of cells. To analysis of the differences of the cell cycle between facilitate up-scaling and the use of hESC in various these two cell types, we propose that the cell cycle of experimental manipulations, such as fluorescence- ES cells may act as a brake for beta-cell regeneration. activated cell sorting, electroporation, and clonal Based on these differences, we discuss the potential of selection, it is important to develop new, stable culture modulating the cell cycle of ES cells for the large- systems based on single-cell enzymatic propagation. scale generation of functionally mature beta cells in Here, we show that hESCs, which were derived and vitro. Further understanding of the factors that passaged by mechanical dissection, can be rapidly modulate the ES cell cycle will lead to new approaches adjusted to propagation by enzymatic dissociation to to enhance the production of functional mature insulin- single cells. As an indication of the stability of this producing cells, and yield a reliable system to generate culture system, we demonstrate that hESCs can be bona fide beta cells in vitro. maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic Ellerstrom, C., et al. (2010). "Single cell passages. We also demonstrate that a recombinant enzymatic dissociation of human embryonic stem cells: trypsin preparation increases clonal survival compared a straightforward, robust, and standardized culture with porcine trypsin. Finally, we show that human method." Methods Mol Biol 584: 121-134. foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in

100 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ terms of their ability to prevent spontaneous Finally, these results demonstrate an additional differentiation after single-cell passaging. Importantly, application of ROCK inhibition to hESC research. the culture system is widely applicable and should therefore be of general use to facilitate reliable large- Endoh, M., et al. (2017). "PCGF6-PRC1 scale cultivation of hESCs, as well as their use in suppresses premature differentiation of mouse various experimental manipulations. Disclosure of embryonic stem cells by regulating germ cell-related potential conflicts of interest is found at the end of this genes." Elife 6. article. The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 Emre, N., et al. (2010). "The ROCK inhibitor Y- associated factors to form a non-canonical PRC1 27632 improves recovery of human embryonic stem (polycomb repressive complex 1) known as PCGF6- cells after fluorescence-activated cell sorting with PRC1. Here, we demonstrate that PCGF6-PRC1 plays multiple cell surface markers." PLoS One 5(8): e12148. a role in repressing a subset of PRC1 target genes by BACKGROUND: Due to the inherent sensitivity recruiting RING1B and mediating downstream mono- of human embryonic stem cells (hESCs) to ubiquitination of histone H2A. PCGF6-PRC1 bound manipulations, the recovery and survival of hESCs loci are highly enriched for promoters of germ cell- after fluorescence-activated cell sorting (FACS) can be related genes in mouse embryonic stem cells (ESCs). low. Additionally, a well characterized and robust Conditional ablation of Pcgf6 in ESCs leads to robust methodology for performing FACS on hESCs using de-repression of such germ cell-related genes, in turn multiple-cell surface markers has not been described. affecting cell growth and viability. We also find a role The p160-Rho-associated coiled kinase (ROCK) for PCGF6 in pre- and peri-implantation mouse inhibitor, Y-27632, previously has been identified as embryonic development. We further show that a enhancing survival of hESCs upon single-cell heterodimer of the transcription factors MAX and dissociation, as well as enhancing recovery from MGA recruits PCGF6 to target loci. PCGF6 thus links cryopreservation. Here we examined the application of sequence specific target recognition by the Y-27632 to hESCs after FACS to improve survival in MAX/MGA complex to PRC1-dependent both feeder-dependent and feeder-independent growth transcriptional silencing of germ cell-specific genes in conditions. METHODOLOGY/PRINCIPAL pluripotent stem cells. FINDINGS: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated Farifteh, F., et al. (2014). "Histone modification after sorting for 24 hours in either the presence or the of embryonic stem cells produced by somatic cell absence of Y-27632. In both feeder-dependent and nuclear transfer and fertilized blastocysts." Cell J 15(4): feeder-independent conditions, cell survival was 316-323. greater when Y-27632 was applied to the hESCs after OBJECTIVE: Nuclear transfer-embryonic stem sort. Specifically, treatment of cells with Y-27632 cells (NT-ESCs) are genetically identical to the improved post-sort recovery up to four fold. To donor's cells; provide a renewable source of tissue for determine the long-term effects of sorting with and replacement, and therefore, decrease the risk of without the application of Y-27632, hESCs were immune rejection. Trichostatin A (TSA) as a histone further analyzed. Specifically, hESCs sorted with and deacetylase in- hibitor (HDACi) plays an important without the addition of Y-27632 retained normal role in the reorganization of the genome and morphology, expressed hESC-specific markers as epigenetic changes. In this study, we examined measured by immunocytochemistry and flow whether TSA treatment after somatic cell nuclear cytometry, and maintained a stable karyotype. In transfer (SCNT) can improve the developmental rate addition, the hESCs could differentiate into three germ of embryos and establishment rate of NT-ESCs line, as layers in vitro and in vivo in both feeder-dependent well as whether TSA treatment can improve histone and feeder-independent growth conditions. modification in NT-ESCs lines. MATERIALS AND CONCLUSIONS/SIGNIFICANCE: The application of METHODS: In this experimental study, mature Y-27632 to hESCs after cell sorting improves cell oocytes were recovered from BDF1 [C57BL/6xDBA/2) recovery with no observed effect on pluripotency, and F 1 mice] mice and enucleated by micromanipulator. enables the consistent recovery of hESCs by FACS Cumulus cells were injected into enucleated oocytes as using multiple surface markers. This improved donor. Reconstructed embryos were ac- tivated in the methodology for cell sorting of hESCs will aid many presence or absence of TSA and cultured for 5 days. applications such as removal of hESCs from secondary Blastocysts were transferred on inactive mouse cell types, identification and isolation of stem cell embryonic fibroblasts (MEF), so ESCs lines were subpopulations, and generation of single cell clones. estab- lished. ESCs markers were evaluated by reverse transcription-polymerase chain reaction (RT-PCR).

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Histone modifications were analyzed by enzyme better differentiate and maintain the function of linked immunosorbent assay (ELISA). RESULTS: differentiating hESCs, we have hypothesized that Result of this study showed that TSA treatment after hESCs undergo better differentiation into hepatocyte- SCNT can improve devel- opmental rate of embryos like cells (HLCs) when induced on three-dimensional (21.12 +/- 3.56 vs. 8.08 +/- 7.92), as well as nanofibrillar surfaces. We have demonstrated that, establishment rate of NT-ESCs line (25 vs. 12.5). We during stepwise differentiation of induction, the established 6 NT-ESCs in two experimental groups, markers of hepatic lineage expressed and finally lead and three embryonic stem cells (ESCs) lines as control to the generation of functional mature cells. In the group. TSA treatment has no effect in H3K4 presence of an ultraweb nanofiber, HLCs produced acetylation and H3K9 tri-methylation in ESCs. lower AFP, greater urea, glycogen storage, metabolic CONCLUSION: TSA plays a key role in the PROD activity, uptake of LDL and organic anion ICG, developmental rate of embryos, establishment rate of all of which are indicative of the differentiation of ESC lines after SCNT, and regulation of histone HLCs. These results show that topographically treated modification in NT-ESCs, in a man- ner similar to that hESCs at the nano level have a distinct hepatic of ESCs established from normal blastocysts. functionality profile which has implications for cell therapies. Faro-Trindade, I. and P. R. Cook (2006). "A conserved organization of transcription during Fassler, R., et al. (1995). "Lack of beta 1 integrin embryonic stem cell differentiation and in cells with gene in embryonic stem cells affects morphology, high C value." Mol Biol Cell 17(7): 2910-2920. adhesion, and migration but not integration into the Although we have detailed information on the inner cell mass of blastocysts." J Cell Biol 128(5): alterations occurring in steady-state levels of all 979-988. cellular mRNAs during differentiation, we still know A gene trap-type targeting vector was designed to little about more global changes. Therefore, we inactivate the beta 1 integrin gene in embryonic stem investigated the numbers of molecules of RNA (ES) cells. Using this vector more than 50% of the ES polymerase II that are active--and the way those cell clones acquired a disruption in the beta 1 integrin molecules are organized--as two mouse cells gene and a single clone was mutated in both alleles. (aneuploid F9 teratocarcinoma, and euploid and The homozygous mutant did not produce beta 1 totipotent embryonic stem cells) differentiate into integrin mRNA or protein, while alpha 3, alpha 5, and parietal endoderm. Quantitative immunoblotting alpha 6 integrin subunits were transcribed but not shows the number of active molecules roughly halves. detectable on the cell surface. Heterozygous mutants Transcription sites (detected by light and electron showed reduced beta 1 expression and surface microscopy after allowing engaged polymerases to localization of alpha/beta 1 heterodimers. The alpha V extend nascent transcripts in bromouridine- subunit expression was not impaired on any of the triphosphate) are uniformly distributed throughout the mutants. Homozygous ES cell mutants lacked nucleoplasm. The numbers of such sites fall during adhesiveness for laminin and fibronectin but not for differentiation as nuclei become smaller, but site vitronectin and showed a reduced association with a density and diameter remain roughly constant. Similar fibroblast feeder layer. Furthermore, they did not site densities and diameters are found in salamander migrate towards chemoattractants in fibroblast (amphibian) cells with 11-fold larger genomes, and in medium. None of these functions were impaired in aneuploid HeLa cells. We conclude that active heterozygous mutants. Scanning electron microscopy polymerases and their nascent transcripts are revealed that homozygous cells showed fewer cell-cell concentrated in a limited number of discrete junctions and had many microvilli not usually found nucleoplasmic sites or factories, and we speculate that on wild type and heterozygous cells. This profound the organization of transcription is conserved during change in cell shape is not associated with gross both differentiation and evolution to a high C value. alterations in the expression and distribution of cytoskeletal components. Unexpectedly, Farzaneh, Z., et al. (2010). "Enhanced functions microinjection into blastocysts demonstrated full of human embryonic stem cell-derived hepatocyte-like integration of homozygous and heterozygous mutants cells on three-dimensional nanofibrillar surfaces." into the inner cell mass. This will allow studies of the Stem Cell Rev 6(4): 601-610. consequences of beta 1 integrin deficiency in several Human embryonic stem cell (hESC)-derived in vivo situations. hepatocytes provide a promising unlimited resource for the treatment of liver disease. However, current Fathi, A., et al. (2014). "Quantitative proteomics protocols for the generation of mature and functional analysis highlights the role of redox hemostasis and hepatocytes are inefficient. Therefore, in order to

102 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ energy metabolism in human embryonic stem cell including endothelial cells. ESCs have been widely differentiation to neural cells." J Proteomics 101: 1-16. regarded as an unlimited source of cells in UNLABELLED: Neural differentiation of human regeneration medicine and also an ideal in vitro model embryonic stem cells (hESCs) is a unique opportunity to investigate complex developmental processes. Here, for in vitro analyses of neurogenesis in humans. we report a simple and efficient in vitro model to Extrinsic cues through neural plate formation are well derive a nearly pure population of endothelial cells described in the hESCs although intracellular from a murine ESC line. CCE ES cells are exposed to mechanisms underlying neural development are alpha-MEM medium containing 10% FBS for 4 days largely unknown. Proteome analysis of hESC and then cultured in endothelial basal-2 medium differentiation to neural cells will help to further containing vascular endothelial growth factor (VEGF), define molecular mechanisms involved in basic fibroblast growth factor (bFGF), insulin-like neurogenesis in humans. Using a two-dimensional growth factor (IGF), epidermal growth factor (EGF), differential gel electrophoresis (2D-DIGE) system, we and 2% FBS for 42 days. The cells acquired a analyzed the proteome of hESC differentiation to relatively uniform endothelial cell morphology and neurons at three stages, early neural differentiation, were able to propagate and expand in culture. When neural ectoderm and mature neurons. Out of 137 murine ES cell-derived endothelial cells (MESDECs) differentially accumulated protein spots, 118 spots were cultured on Matrigel and incubated for 48 h, were identified using MALDI-TOF/TOF and LC vessel-like tube structures consisting of CD31 MS/MS. We observed that proteins involved in redox (PECAM-1) or BS-1 immunoreactive cells were hemostasis, vitamin and energy metabolism and developed. Immunocytochemistry and RT-PCR ubiquitin dependent proteolysis were more abundant in analyses revealed that MESDECs express endothelial differentiated cells, whereas the abundance of proteins cell-specific marker proteins such as Flk-1, PECAM-1, associated with RNA processing and protein folding Tie-1, and Tie-2, in which the expressions persist for was higher in hESCs. Higher abundance of proteins long periods of time after differentiation. The cells involved in maintaining cellular redox state suggests were also capable of taking up acetylated low-density the importance of redox hemostasis in neural lipoprotein (LDL) in culture. Our data suggest that differentiation. Furthermore, our results support the MESDECs could provide a suitable in vitro model to concept of a coupling mechanism between neuronal study molecular events involved in vascular activity and glucose utilization. The protein network development and open up a new therapeutic strategy in analysis showed that the majority of the interacting regeneration medicine of cardiovascular disorders. proteins were associated with the cell cycle and cellular proliferation. These results enhanced our Feigelman, J., et al. (2016). "Analysis of Cell understanding of the molecular dynamics that underlie Lineage Trees by Exact Bayesian Inference Identifies neural commitment and differentiation. BIOLOGICAL Negative Autoregulation of Nanog in Mouse SIGNIFICANCE: In highlighting the role of redox and Embryonic Stem Cells." Cell Syst 3(5): 480-490 e413. unique metabolic properties of neuronal cells, the Many cellular effectors of pluripotency are present findings add insight to our understanding of dynamically regulated. In principle, regulatory hESC differentiation to neurons. The abundance of mechanisms can be inferred from single-cell fourteen proteins involved in maintaining cellular observations of effector activity across time. However, redox state, including 10 members of peroxiredoxin rigorous inference techniques suitable for noisy, (Prdx) family, mainly increased during differentiation, incomplete, and heterogeneous data are lacking. Here, thus highlighting a link of neural differentiation to we introduce stochastic inference on lineage trees redox. Our results revealed markedly higher (STILT), an algorithm capable of identifying expression of genes encoding enzymes involved in the stochastic models that accurately describe the glycolysis and amino acid synthesis during quantitative behavior of cell fate markers observed differentiation. Protein network analysis predicted a using time-lapse microscopy data collected from number of critical mediators in hESC differentiation. proliferating cell populations. STILT performs exact These proteins included TP53, CTNNB1, SMARCA4, Bayesian parameter inference and stochastic model TNF, TERT, , MYC, RB1, and AR. selection using a particle-filter-based algorithm. We use STILT to investigate the autoregulation of Nanog, Fathi, F., et al. (2008). "Characterizing a heterogeneously expressed core pluripotency factor, endothelial cells derived from the murine embryonic in mouse embryonic stem cells. STILT rejects the stem cell line CCE." Rejuvenation Res 11(2): 371-378. possibility of positive Nanog autoregulation with high Embryonic stem cells (ESC) are defined by two confidence; instead, model predictions indicate weak main properties of self-renewal and their multipotency negative feedback. We use STILT for rational to differentiate into virtually all cell types of the body, experimental design and validate model predictions

103 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ using novel experimental data. STILT is available for the etiology of these defects, the study of embryos can download as an open source framework from be complemented with in vitro assays that use http://www.imsb.ethz.ch/research/claassen/Software/st differentiating embryonic stem (ES) cells. Apoptosis is ilt---stochastic-inference-o n-lineage-trees.html. a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme Filippi, M. D., et al. (2002). "Requirement for cascade. Characteristic cell changes include membrane mitogen-activated protein kinase activation in the blebbing, nuclear shrinking, and DNA fragmentation. response of embryonic stem cell-derived Other forms of PCD do not involve caspase activation hematopoietic cells to thrombopoietin in vitro." Blood and may be the end-result of prolonged autophagy. 99(4): 1174-1182. Regardless of the PCD pathway, dying cells need to be Enforced expression of c-mpl in embryonic stem removed. In adults, the immune cells perform this (ES) cells inactivated for this gene results in protein function, while in embryos, where the immune system expression in all the ES cell progeny, producing cells has not yet developed, removal occurs by an that do not belong to the megakaryocytic lineage and alternative mechanism. This mechanism involves are responsive to PEG-rhuMGDF, a truncated form of neighboring cells (called "non-professional human thrombopoietin (TPO) conjugated to phagocytes") taking on a phagocytic role-they polyethylene glycol. These include a primitive cell recognize the 'eat me' signal on the surface of the called BL-CFC, thought to represent the equivalent of dying cell and engulf it (8-10). After engulfment, the the hemangioblast, and all myeloid progenitor cells. In debris is brought to the lysosome for degradation. this model, PEG-rhuMGDF was able to potentiate the Thus regardless of PCD mechanism, an increase in stimulating effects of other growth factors, including lysosomal activity can be correlated with increased vascular endothelial growth factor, on BL-CFC and a cell death. To study PCD, a simple assay to visualize combination of cytokines on the growth of granulocyte lysosomes in thick tissues and multilayer macrophage-colony-forming units. The importance of differentiating cultures can be useful. LysoTracker dye the C-terminal domain of Mpl and of mitogen- is a highly soluble small molecule that is retained in activated protein kinase (MAPK) activation in TPO- acidic subcellular compartments such as the lysosome dependent megakaryocytic differentiation has been (11-13). The dye is taken up by diffusion and through well studied in vitro. Here, the role of this domain and the circulation. Since penetration is not a hindrance, the involvement of MAPK in upstream and visualization of PCD in thick tissues and multi-layer nonmegakaryocytic cells are examined by using 2 cultures is possible (12,13). In contrast, TUNEL truncated mutants of Mpl (Delta34, deletion of (Terminal deoxynucleotidyl transferase dUTP nick end residues 71 to 121 in the C-terminal domain; and labeling) analysis (14), is limited to small samples, Delta3, deletion of residues 71-94) and specific histological sections, and monolayer cultures because inhibitors of the MAPK pathway. The 2 deleted the procedure requires the entry/permeability of a regions support different functions, mediated by terminal transferase. In contrast to Aniline blue, which different signals. Residues 71 to 121 were required for diffuses and is dissolved by solvents, LysoTracker Red PEG-rhuMGDF-dependent growth of BL-CFC, for DND-99 is fixable, bright, and stable. Staining can be megakaryocytic and other myeloid progenitors, and for visualized with standard fluorescent or confocal megakaryocyte polyploidization. These responses microscopy in whole-mount or section using aqueous were mediated by the ERK1-ERK2 MAPK pathway. or solvent-based mounting media (12,13). Here we In contrast, the only function of the sequence describe protocols using this dye to look at PCD in comprising residues 71 to 94 was to mediate the normal and sonic hedgehog null mouse embryos. In synergistic effects of PEG-rhuMGDF with other addition, we demonstrate analysis of PCD in hematopoietic growth factors. This function is not differentiating ES cell cultures and present a simple mediated by MAPK activation. quantification method. In summary, LysoTracker staining can be a great complement to other methods Fogel, J. L., et al. (2012). "Use of LysoTracker to of detecting PCD. detect programmed cell death in embryos and differentiating embryonic stem cells." J Vis Exp (68). Foja, S., et al. (2013). "Hypoxia supports Programmed cell death (PCD) occurs in adults to reprogramming of mesenchymal stromal cells via maintain normal tissue homeostasis and during induction of embryonic stem cell-specific microRNA- embryological development to shape tissues and 302 cluster and pluripotency-associated genes." Cell organs (1,2,6,7). During development, toxic chemicals Reprogram 15(1): 68-79. or genetic alterations can cause an increase in PCD or Pluripotency is characterized by specific change PCD patterns resulting in developmental transcription factors such as OCT4, NANOG, and abnormalities and birth defects (3-5). To understand SOX2, but also by pluripotency-associated

104 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ microRNAs (miRs). Somatic cells can be passed through the column ('flow-through' fraction) reprogrammed by forced expression of these factors and those retained ('labelled' fraction') were leading to induced pluripotent stem cells (iPSCs) with subsequently analysed using FACS. The maximum characteristics similar to embryonic stem cells (ESCs). efficacy of hESCs retention using MACS was 81.0 +/- However, current reprogramming strategies are 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using commonly based on viral delivery of the pluripotency- FACS, all the undifferentiated hESCs labelled with the associated factors, which affects the integrity of the two cell-surface markers could be removed by genome and impedes the use of such cells in any selective gating. Both hESCs and HepG2 cells in the clinical application. In an effort to establish nonviral, 'flow-through' fraction following MACS separation nonintegrating reprogramming strategies, we were viable in culture whereas by FACS separation examined the influence of hypoxia on the expression only the HepG2 cells were viable. FACS efficiently of pluripotency-associated factors and the ESC- helps to eliminate the undifferentiated hESCs based on specific miR-302 cluster in primary and immortalized their cell-surface antigens expressed. mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) Fujimori, H., et al. (2012). "Induction of treatments led to the induction of OCT4 and NANOG cancerous stem cells during embryonic stem cell in an immortalized cell line L87 and primary MSCs, differentiation." J Biol Chem 287(44): 36777-36791. accompanied with increased doubling rates and Stem cell maintenance depends on their decreased senescence. Most importantly, the surrounding microenvironment, and aberrancies in the endogenous ECS-specific cluster miR-302 was environment have been associated with tumorigenesis. induced upon hypoxic culture and FGF2 However, it remains to be elucidated whether an supplementation. Hypoxia also improved environmental aberrancy can act as a carcinogenic reprogramming of MSCs via episomal expression of stress for cellular transformation of differentiating pluripotency factors. Thus, our data illustrate that stem cells into cancer stem cells. Here, utilizing mouse hypoxia in combination with FGF2 supplementation embryonic stem cells as a model, it was illustrated that efficiently facilitates reprogramming of MSCs. environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing Fong, C. Y., et al. (2009). "Separation of SSEA-4 cancerous characteristics. Analogous to precancerous and TRA-1-60 labelled undifferentiated human stages, DNA lesions were spontaneously accumulated embryonic stem cells from a heterogeneous cell during embryonic stem cell differentiation under population using magnetic-activated cell sorting aberrational environments, which activates barrier (MACS) and fluorescence-activated cell sorting responses such as senescence and apoptosis. However, (FACS)." Stem Cell Rev 5(1): 72-80. overwhelming such barrier responses, piled-up spheres A major concern in human embryonic stem cell were subsequently induced from the previously (hESC)-derived cell replacement therapy is the risk of senescent cells. The sphere cells exhibit aneuploidy tumorigenesis from undifferentiated hESCs residing in and dysfunction of the Arf- module as well as the population of hESC-derived cells. Separation of enhanced tumorigenicity and a strong self-renewal these undifferentiated hESCs from the differentiated capacity, suggesting development of cancerous stem derivatives using cell sorting methods may be a cells. Our current study suggests that stem cells plausible approach in overcoming this problem. We differentiating in an aberrational environment are at therefore explored magnetic activated cell sorting risk of cellular transformation into malignant (MACS) and fluorescence activated cell sorting counterparts. (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and Fujita, A., et al. (2016). "beta-Globin-Expressing hepatocellular carcinoma cells (HepG2) deliberately Definitive Erythroid Progenitor Cells Generated from mixed respectively at different ratios (10:90, 20:80, Embryonic and Induced Pluripotent Stem Cell-Derived 30:70, 40:60 and 50:50) to mimic a standard in vitro Sacs." Stem Cells 34(6): 1541-1552. differentiation protocol, instead of using a hESC- Human embryonic stem (ES) cells and induced differentiated cell population, so that we could be sure pluripotent stem (iPS) cells represent a potential of the actual number of cells separated. HES-3 and alternative source for red blood cell transfusion. HES-4 cells were labelled in separate experiments for However, when using traditional methods with the stem cell markers SSEA-4 and TRA-1-60 using embryoid bodies, ES cell-derived erythroid cells primary antibodies. Anti-PE magnetic microbeads that predominantly express embryonic type varepsilon- recognize the PE-conjugated SSEA-4 labelled hESCs globin, with lesser fetal type gamma-globin and very was added to the heterogeneous cell mixture and little adult type beta-globin. Furthermore, no beta- passed through the MACS column. The cells that globin expression is detected in iPS cell-derived

105 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ erythroid cells. ES cell-derived sacs (ES sacs) have containing a temperature-sensitive SV40 large T been recently used to generate functional platelets. antigen was transfected into the primary fetal liver Due to its unique structure, we hypothesized that ES stromal cells. These immortalized cells, which were sacs serve as hemangioblast-like progenitors capable named as the gp38-positive and Thy1-positive murine to generate definitive erythroid cells that express beta- liver stromal (MLSgt) cells, induced both mouse ESCs globin. With our ES sac-derived erythroid and HPCs to differentiate into mature hepatocyte-like differentiation protocol, we obtained approximately cells using a coculture method. Since MLSgt is not a 120 erythroid cells per single ES cell. Both primitive cloned cell line, one clone, MLSgt20, was selected as (varepsilon-globin expressing) and definitive (gamma- a line with the characteristic to induce hepatic and beta-globin expressing) erythroid cells were differentiation, which was comparable to its parental generated from not only ES cells but also iPS cells. stromal cells. The ESC-derived endoderm cells Primitive erythropoiesis is gradually switched to cocultured with the MLSgt20 cells expressed mature definitive erythropoiesis during prolonged ES sac hepatocyte-specific gene markers, including glucose- maturation, concurrent with the emergence of 6-phosphatase, tyrosine aminotransferase, tryptophan hematopoietic progenitor cells. Primitive and 2,3-dioxgenase, and cytochrome P450 (CYP1a1, definitive erythroid progenitor cells were selected on Cyp1b1, Cyp1a2, and Cyp3a11). In addition, these the basis of glycophorin A or CD34 expression from cells also exhibited hepatic functions, such as cells within the ES sacs before erythroid glycogen storage and ammonia metabolism. differentiation. This selection and differentiation Transmission electron microscopy showed that the strategy represents an important step toward the cocultured ESCs expressed the morphologic features development of in vitro erythroid cell production of mature hepatocytes. In conclusion, a cell line was systems from pluripotent stem cells. Further established that has the characteristic to promote the optimization to improve expansion should be required hepatic maturation of mouse ESCs and HPCs by a for clinical application. Stem Cells 2016;34:1541-1552. coculture method.

Fukuda, H. and J. Takahashi (2005). "Embryonic Fukunaga, N., et al. (2010). "Leukemia inhibitory stem cells as a cell source for treating Parkinson's factor (LIF) enhances germ cell differentiation from disease." Expert Opin Biol Ther 5(10): 1273-1280. primate embryonic stem cells." Cell Reprogram 12(4): Parkinson's disease (PD) is a neurodegenerative 369-376. disease characterised by a loss of midbrain Recently, several research groups have shown dopaminergic (DA) neurons. Transplantation of DA that germ cells can be produced in vitro from neurons represents a promising treatment for PD, and pluripotent embryonic stem cells (ESCs). In the mouse, embryonic stem (ES) cells are a good candidate source live births of offspring using germ cells induced from for DA neurons. However, although recent reports ESCs in vitro have been reported. Furthermore, some have demonstrated that DA neurons can be efficiently efficient methods for inducing the useful number of induced from ES cells and function therapeutically in germ cells from ESCs have also been developed. On an animal model of PD, many problems remain to be the other hand, in primates, despite the appearances of solved in order for ES cells to be used for clinical germ cell-like cells including meiotic cells were applications. This review will describe the current observed by spontaneous differentiation or introducing status of this field and the obstacles yet to be transgenes, it has not been determined whether fully overcome, and will outline future research approaches functional germ cells can be derived from ESCs. To from the clinical perspective. elucidate the property for the germ cells induced from primate ESCs, specification of the promoting factors Fukumitsu, K., et al. (2009). "Establishment of a for the germ cell development and improving the cell line derived from a mouse fetal liver that has the efficiency of germ cell derivation are essential. characteristic to promote the hepatic maturation of Leukemia inhibitory factor (LIF) has been reported as mouse embryonic stem cells by a coculture method." one of the important factors for mouse primordial Tissue Eng Part A 15(12): 3847-3856. germ cell (PGC) survival in vitro. However, the effects Stromal cells residing in murine fetal livers have of LIF on germ cell formation from pluripotent cells of the ability to promote the hepatic maturation of murine primates have not been examined. The aim of this embryonic stem cells (ESCs) and hepatic progenitor study is to determine whether LIF addition can cells (HPCs) 3848 in vitro. These stromal cells were improve in vitro germ cell production from isolated as the CD49f (+/-)CD45(-)Thy1(+)gp38(+) cynomolgus monkey ESCs (cyESCs). After 8 days of cell fraction. The present study established a murine differentiation, LIF added culture induced dome- fetal liver stromal cell line that induced hepatic shaped germ cell colonies as indicated by the intense maturation in mouse ESCs and HPCs. A transgene expression of alkaline phosphatase activity (ALP).

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These cells also demonstrate high-level expression of differentiate predominantly into epiblast cells in a the germ cell-marker VASA, OCT-4, and BLIMP-1, chimeric embryo." Biol Reprod 70(5): 1452-1457. and show SSEA-1 expression that supports their early We examined the expression of cell-surface stage germ cell identity. Finally, we observed that markers on subpopulations of mouse embryonic stem adding LIF to differentiating cultures inhibited meiotic (ES) cells to identify those that were associated with gene expressions and increased the percentage of cells that had the highest pluripotency. Flow cytometry ALP-positive cells, and demonstrate that the addition analysis revealed a wide variation in the expression of of LIF to differentiation media increases platelet endothelial cell adhesion molecule 1 differentiation of early germ cells from the cyESCs. (PECAM-1) and stage-specific embryonic antigen (SSEA)-1 in ES cells. Almost all SSEA-1+ cells Funakoshi, N., et al. (2011). "Comparison of expressed a high level of PECAM- 1, and reversible hepatic-like cell production from human embryonic repopulation was observed between PECAM- stem cells and adult liver progenitor cells: CAR 1+SSEA-1+ and PECAM-1+SSEA-1- cells. The ES transduction activates a battery of detoxification cells carrying the lacZ gene were sorted into three genes." Stem Cell Rev 7(3): 518-531. subpopulations: PECAM- 1-SSEA-1-, PECAM- In vitro production of human hepatocytes is of 1+SSEA-1-, and PECAM-1+SSEA-1+. Quantitative primary importance in basic research, reverse transcription-polymerase chain reaction pharmacotoxicology and biotherapy of liver diseases. revealed a low level of Oct3/4 mRNA expression and We have developed a protocol of differentiation of an elevation in differentiation maker gene expression human embryonic stem cells (ES) towards hepatocyte- in PECAM-1- cells. To compare the pluripotency of like cells (ES-Hep). Using a set of human adult these three subpopulations, a single cell from each was markers including CAAT/enhancer binding protein injected into eight-cell embryo and ES cells identified (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio at later stages by X-gal staining. At the blastocyst (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 stage, PECAM-1+ SSEA-1+/- cells were found to (CYP7A1), CYP3A4 and constitutive androstane have differentiated into epiblast cells in high numbers. receptor (CAR), and fetal markers including alpha- In contrast, PECAM- 1- cell derivatives localized in fetoprotein, CYP3A7 and glutathione S-transferase P1, the primitive endoderm or trophectoderm. At 6.0-7.0 we analyzed the expression of a panel of 41 genes in days post coitum, many PECAM-1+SSEA- 1+ cells ES-Hep comparatively with human adult primary were found in the epiblast, but few beta-gal+ cells hepatocytes, adult and fetal liver. The data revealed were detected in any regions of embryos that were that after 21 days of differentiation, ES-Hep are injected with cells from the other two populations. representative of fetal hepatocytes at less than 20 These results showed that the expression levels of weeks of gestation. The PECAM-1 and SSEA-1 in ES cells correlated closely pathway was functional in ES-Hep. Extending with their pluripotency and/or their ability to protocols of differentiation to 4 weeks did not improve incorporate into the epiblast of chimeric embryos. cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal Gadkari, R., et al. (2014). "Human embryonic epithelial (NPE) cells (NPE-Hep), ES-Hep expressed stem cell derived-mesenchymal stem cells: an several adult and fetal liver makers at much greater alternative mesenchymal stem cell source for levels (at least one order of magnitude), consistent regenerative medicine therapy." Regen Med 9(4): 453- with greater expression of liver-enriched transcription 465. factors Forkhead box A2, C/EBPalpha, HNF4alpha AIM: To enumerate and characterize and HNF6. It therefore seems that ES-Hep reach a mesenchymal stem cells (MSC) derived from human better level of differentiation than NPE-Hep and that embryonic stem cells (hESC) for clinical application. these cells use different lineage pathways towards the MATERIALS & METHODS: hESC were hepatic phenotype. Finally we showed that lentivirus- differentiated into hESC-MSC and characterized by mediated expression of xenoreceptor CAR in ES-Hep the expression of surface markers using flow induced the expression of several detoxification genes cytometry. hESC-MSC were evaluated with respect to including CYP2B6, CYP2C9, CYP3A4, UDP- growth kinetics, colony-forming potential, as well as glycosyltransferase 1A1, solute carriers 21A6, as well osteogenic and adipogenic differentiation capacity. as biotransformation of midazolam, a CYP3A4- Immunosuppressive effects were assessed using specific substrate. peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS: Furusawa, T., et al. (2004). "Embryonic stem hESC-MSC showed similar morphology, and cell cells expressing both platelet endothelial cell adhesion surface markers as adipose (AMSC) and bone marrow- molecule-1 and stage-specific embryonic antigen-1 derived MSC (BMSC). hESC-MSC exhibited a higher

107 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ growth rate during early in vitro expansion and seeding density when optimizing hESC differentiation equivalent adipogenic and osteogenic differentiation protocols. and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive Galat, V., et al. (2010). "[Cell engineering and effects as AMSC and BMSC. CONCLUSION: hESC- genetic approaches to the development of models of MSC were comparable to BMSC and AMSC and human embryonic stem cells for studying genetic hence can be used as an alternative source of MSC for disorders]." Biofizika 55(3): 481-485. clinical applications. A novel approach to the establishment of genetically modified human embryonic stem cell Gage, B. K., et al. (2013). "Initial cell seeding (hESC) lines has been developed, and it has been density influences pancreatic endocrine development shown that mutant hESC may be derived from affected during in vitro differentiation of human embryonic embryos after preimplantation genetic diagnosis stem cells." PLoS One 8(12): e82076. screening for a particular single gene disorder. Here Human embryonic stem cells (hESCs) have the we provide the description of embryo and cell ability to form cells derived from all three germ layers, manipulation procedures, diagnostic lay out, analysis and as such have received significant attention as a of the efficiency of embryo development and hESC possible source for insulin-secreting pancreatic beta- establishment, as well as developments for hESC cells for diabetes treatment. While considerable derivation in animal free conditions. The high advances have been made in generating hESC-derived efficiency of the approach (50%) is especially crucial insulin-producing cells, to date in vitro-derived in the work with rare and unique resources, such as glucose-responsive beta-cells have remained an genetically screened embryos necessary for the elusive goal. With the objective of increasing the in derivation of hESC lines representative of specific vitro formation of pancreatic endocrine cells, we genetic diseases. examined the effect of varying initial cell seeding density from 1.3 x 10(4) cells/cm (2) to 5.3 x 10(4) Gan, Q., et al. (2007). "Concise review: cells/cm (2) followed by a 21-day pancreatic endocrine epigenetic mechanisms contribute to pluripotency and differentiation protocol. Low density-seeded cells cell lineage determination of embryonic stem cells." were found to be biased toward the G2/M phases of Stem Cells 25(1): 2-9. the cell cycle and failed to efficiently differentiate into Epigenetic mechanisms, such as histone SOX17-CXCR4 co-positive definitive endoderm cells modifications and DNA methylation, have been shown leaving increased numbers of OCT4 positive cells in to play a key role in the regulation of gene day 4 cultures. Moderate density cultures effectively transcription. Results of recent studies indicate that a formed definitive endoderm and progressed to express novel "bivalent" chromatin structure marks key PDX1 in approximately 20% of the culture. High developmental genes in embryonic stem cells (ESCs), density cultures contained approximately double the wherein a number of untranscribed lineage-control numbers of PDX1 positive pancreatic progenitor cells genes, such as Sox1, Nkx2-2, Msx1, Irx3, and Pax3, and also showed increased expression of MNX1, are epigenetically modified with a unique combination PTF1a, NGN3, ARX, and PAX4 compared to cultures of activating and repressive histone modifications that seeded at moderate density. The cultures seeded at prime them for potential activation (or repression) high density displayed increased formation of upon cell lineage induction and differentiation. polyhormonal pancreatic endocrine cell populations However, results of these studies also showed that a co-expressing insulin, glucagon and somatostatin. The subset of lineage-control genes, such as Myf5 and maturation process giving rise to these endocrine cell Mash1, were not marked by these histone populations followed the expected cascade of modifications, suggesting that distinct epigenetic pancreatic progenitor marker (PDX1 and MNX1) mechanisms might exist for lineage-control genes in expression, followed by pancreatic endocrine ESCs. In this review article, we summarize evidence specification marker expression (BRN4, PAX4, ARX, regarding possible mechanisms that control these NEUROD1, NKX6.1 and NKX2.2) and then unique histone modifications at lineage-control gene pancreatic hormone expression (insulin, glucagon and loci in ESCs and consider their possible contribution somatostatin). Taken together these data suggest that to ESC pluripotency. In addition, we propose a novel initial cell seeding density plays an important role in "histone modification pulsing" model wherein both germ layer specification and pancreatic individual pluripotent stem cells within the inner cell progenitor commitment, which precedes pancreatic mass of blastocysts undergo transient asynchronous endocrine cell formation. This work highlights the histone modifications at these developmental gene loci, need to examine standard culture variables such as thereby conferring differential responsiveness to environmental cues and morphogenic gradients

108 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ important for cell lineage determination. Finally, we telomerase activity. The expression level was Oct4 consider how these rapid histone modification 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 exchanges become progressively more stable as ESCs 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, undergo differentiation and maturation into specialized which was lower than that in ADSCs, but the purity of cell lineages. DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell Gangemi, R. M., et al. (2004). "Regulatory genes population, and expressed some embryonic stem cell controlling cell fate choice in embryonic and adult markers like ADSCs, which seems to be a good neural stem cells." J Neurochem 89(2): 286-306. candidate source of adult stem cells for the future cell Neural stem cells are the most immature replacement therapy. progenitor cells in the nervous system and are defined by their ability to self-renew by symmetric division as Garcia-Lavandeira, M., et al. (2012). well as to give rise to more mature progenitors of all "Craniopharyngiomas express embryonic stem cell neural lineages by asymmetric division markers (SOX2, OCT4, , and SOX9) as pituitary (multipotentiality). The interest in neural stem cells stem cells but do not coexpress RET/GFRA3 has been growing in the past few years following the receptors." J Clin Endocrinol Metab 97(1): E80-87. demonstration of their presence also in the adult CONTEXT: Adult stem cells maintain some nervous system of several mammals, including markers expressed by embryonic stem cells and humans. This observation implies that the brain, once express other specific markers depending on the organ thought to be entirely post-mitotic, must have at least a where they reside. Recently, stem/progenitor cells in limited capacity for self-renewal. This raises the the rodent and human pituitary have been possibility that the adult nervous system may still have characterized as expressing GFRA2/RET, PROP1, and the necessary plasticity to undergo repair of inborn stem cell markers such as SOX2 and OCT4 (GPS defects and acquired injuries, if ways can be found to cells). OBJECTIVE: Our objective was to detect other exploit the potential of neural stem cells (either specific markers of the pituitary stem cells and to endogenous or derived from other sources) to replace investigate whether craniopharyngiomas (CRF), a damaged or defective cells. A full understanding of the tumor potentially derived from Rathke's pouch molecular mechanisms regulating generation and remnants, express similar markers as normal pituitary maintenance of neural stem cells, their choice between stem cells. DESIGN: We conducted mRNA and different differentiation programmes and their Western blot studies in pituitary extracts, and migration properties is essential if these cells are to be immunohistochemistry and immunofluorescence on used for therapeutic applications. Here, we summarize sections from normal rat and human pituitaries and 20 what is currently known of the genes and the CRF (18 adamantinomatous and two papillary). signalling pathways involved in these mechanisms. RESULTS: Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key Gao, Q., et al. (2012). "Expression pattern of embryonic stem cell markers, SOX2, OCT4, and embryonic stem cell markers in DFAT cells and KLF4, in addition to SOX9 and PROP1 and beta- ADSCs." Mol Biol Rep 39(5): 5791-5804. catenin overexpression. They express the RET Mature adipocytes can revert to a more primitive receptor and its GFRA2 coreceptor but also express phenotype and gain cell proliferative ability under the the coreceptor GFRA3 that could be detected in the condition of ceiling method, named dedifferentiated MZ of paraffin pituitary sections. CRF maintain the fat cells (DFAT cells). These cells exhibit multilineage expression of SOX2, OCT4, KLF4, SOX9, and beta- potential as adipose tissue-derived stromal cells catenin. However, RET and GFRA3 expression was (ADSCs). However, the stem molecular signature of altered in CRF. In 25% (five of 20), both RET and DFAT cells and the difference distinct from ADSCs GFRA3 were detected but not colocalized in the same are still not sure. To study the molecular signature of cells. The other 75% (15 of 20) lose the expression of DFAT cells better, highly purified mature adipocytes RET, GFRA3, or both proteins simultaneously. were obtained from rats and the purity was more than CONCLUSIONS: Human pituitary adult 98%, and about 98.6% were monocytes. These mature stem/progenitor cells (GPS) located in the MZ are adipocytes dedifferentiated into fibroblast-like cells characterized by expression of embryonic stem cell spontaneously by the ceiling culture method, these markers SOX2, OCT4, and KLF4 plus the specific cells proliferated rapidly in vitro, grew in the same pituitary embryonic factor PROP1 and the RET system. direction and formed vertex, and expressed extensively Redundancy in RET coreceptor expression (GFRA2 embryonic stem cell markers such as Oct4, Sox2, c- and GFRA3) suggest an important systematic function Myc, and Nanog, surface antigen SSEA-1, CD105, in their physiological behavior. CRF share the stem and CD31, moreover, these cells possessed ALP and cell markers suggesting a common origin with GPS.

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However, the lack of expression of the RET/GFRA fertilized and cloned blastocysts." Cell Reprogram system could be related to the cell mislocation and 13(3): 263-272. deregulated growth of CRF. Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned Gavrilov, S., et al. (2009). "Non-viable human blastocysts, generated using fibroblasts derived from embryos as a source of viable cells for embryonic stem an adult buffalo (BAF). These nuclear transfer cell derivation." Reprod Biomed Online 18(2): 301- embryonic stem cell-like cells (NT-ES) grew in well- 308. defined and dome-shaped colonies. The expression Human embryonic stem cells (hESC) hold great pattern of pluripotency marker genes was similar in promise for use in regenerative medicine. However, both NT-ES and in vitro fertilization (IVF) embryo- the extraordinary potential of hESC as therapeutic derived embryonic stem cell-like cells (F-ES). Upon tools is tempered by ethical, moral and political issues spontaneous differentiation via embryoid body surrounding their derivation from human embryos. It formation, cells of different morphology were has previously been proposed that ethical criteria observed, among which predominant were applied to essential organ donation could be employed endodermal-like and epithelial-like cell types. The ES for derivation of hESC from irreversibly arrested, and cell-like cells could be passaged only mechanically thus organismically dead, human embryos produced and did not form colonies when plated as single cell during routine IVF procedures. Here, it is shown that suspension at different concentrations. When F-ES arrested embryos do not resume normal development cell-like, NT-ES cell-like, and BAF cells of same during extended culture, yet most of them contain a genotype were used for hand-made cloning (HMC), no substantial number of living cells on embryonic day 6 significant difference (p > 0.05) was observed in (72% have <1 viable cell, 47% have <5 viable cells), cleavage and blastocyst rate. Following transfer of suggesting that this class of non-viable embryos could HMC embryos to synchronized recipients, pregnancies be a rich source of viable cells for derivation of hESC were established only with F-ES cell-like and BAF lines. cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES Geens, M., et al. (2011). "Sertoli cell-conditioned cell-like cells and BAF were used for HMC, no medium induces germ cell differentiation in human significant difference (p > 0.05) was observed between embryonic stem cells." J Assist Reprod Genet 28(5): cleavage and blastocyst rate. In conclusion, here we 471-480. report for the first time the derivation of ES cell-like PURPOSE: To investigate the spontaneous germ cells from an adult buffalo, and its genetic cell differentiation capacity of VUB hESC lines, modification. We also report the birth of a live cloned develop a protocol for the induction of germ cell calf from buffalo ES cell-like cells. differentiation using conditioned medium from Sertoli cells (SCCM) and compare it to existing protocols. Gerecht-Nir, S. and J. Itskovitz-Eldor (2004). METHODS: hESC were allowed to differentiate "Cell therapy using human embryonic stem cells." spontaneously or after the addition of bone Transpl Immunol 12(3-4): 203-209. morphogenetic proteins (BMPs) and/or SCCM. VASA Cell therapy refers to the transplantation of transcripts were measured by relative quantification healthy, functional and propagating cells to restore the real-time RT-PCR to determine the efficiency of germ viability or function of deficient tissues. Stem cells are cell differentiation. RESULTS: VUB hESC lines can characterized by self-renewal and the potential to form differentiate spontaneously towards the germ cell differentiated cells. In early mammalian embryos, at lineage, however, more consistently in an embryoid the blastocyst stage, the inner cell mass is pluripotent. body approach than in monolayer cultures. BMPs and Thus, it has been recognized that human embryonic SCCM significantly improve VASA expression, but stem cells (hESCs), which are derived from such cells do not have a synergistic effect. Direct contact of of blastocysts, may serve as a source of numerous differentiating hESC with Sertoli cells does not types of differentiated cells. The first part of this improve VASA expression. CONCLUSIONS: SCCM review summarizes different techniques for the contains inductive factors for germ cell differentiation derivation and maintenance of undifferentiated hESCs. and could represent an element for in-vitro In the second part, issues concerning the safety and differentiation to germ cells. bulk production, which may enable hESCs use in future clinical applications, are presented. The last part George, A., et al. (2011). "Production of cloned of this review details accumulated data regarding the and transgenic embryos using buffalo (Bubalus bubalis) in vitro differentiation potential of hESCs. embryonic stem cell-like cells isolated from in vitro

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Germano, I. M., et al. (2006). "Apoptosis in cells from hESCs facilitates the process of human human glioblastoma cells produced using embryonic embryonic development through the generation of stem cell-derived astrocytes expressing tumor necrosis neuronal subtypes. We have recently indicated that factor-related apoptosis-inducing ligand." J Neurosurg fibronectin type III domain containing 5 protein 105(1): 88-95. (FNDC5) expression is required for appropriate neural OBJECT: Embryonic stem (ES) cell-derived differentiation of mouse embryonic stem cells astrocytes have several theoretical and practical (mESCs). Bioinformatics analyses have shown the advantages as gene therapy vectors in the treatment of presence of three isoforms for human FNDC5 mRNA. malignant gliomas. The aim of this study was to test To differentiate which isoform of FNDC5 is involved the proapoptotic effects of ES cell-derived astrocytes in the process of human neural differentiation, we have expressing transgenic tumor necrosis factor-related used hESCs as an in vitro model for neural apoptosis-inducing ligand (TRAIL) in human differentiation by retinoic acid (RA) induction. The malignant glioma cells. METHODS: Mouse ES cells hESC line, Royan H5, was differentiated into a neural containing a doxycycline-inducible transgene were lineage in defined adherent culture treated by RA and engineered with human TRAIL (hTRAIL) and then basic fibroblast growth factor (bFGF). We collected all directed to differentiate into astrocytes. The ES cell- cell types that included hESCs, rosette structures, and derived-TRAIL-expressing astrocytes were cocultured neural cells in an attempt to assess the expression of with human malignant glioma cells. Reverse FNDC5 isoforms. There was a contiguous increase in transcriptase polymerase chain reaction, all three FNDC5 isoforms during the neural immunocytochemistry, terminal deoxynucleotidyl differentiation process. Furthermore, the highest level transferase-mediated deoxyuridine triphosphate nick- of expression of the isoforms was significantly end labeling, and flow cytometry were used to observed in neural cells compared to hESCs and the quantify results. In vitro coculture of ES cell-derived rosette structures known as neural precursor cells astrocytes expressing hTRAIL with A172 human (NPCs). High expression levels of FNDC5 in human malignant glioma cells after doxycycline induction fetal brain and spinal cord tissues have suggested the caused a significant decrease in cell viability from 85 involvement of this gene in neural tube development. +/- 2% at baseline to 8 +/- 2% posttreatment (p < Additional research is necessary to determine the 0.001). Labeling with apoptotic markers showed that major function of FDNC5 in this process. cell death occurred by means of apoptosis. A significant increase in apoptotic rate (88 +/- 3%) from Ghodsizadeh, A., et al. (2014). "Galactosylated baseline (4 +/- 2%) was found in A172 cells after collagen matrix enhanced in vitro maturation of human doxycycline induction (p < 0.005). This effect was embryonic stem cell-derived hepatocyte-like cells." superior to the apoptotic rate seen after treatment with Biotechnol Lett 36(5): 1095-1106. recombinant TRAIL (57 +/- 2%). A decrease in cell Due to their important biomedical applications, viability and an increase in the apoptotic rate were not functional human embryonic stem cell-derived found in TRAIL-expressing-ES cell-derived astrocytes hepatocyte-like cells (hESC-HLCs) are an attractive after induction with doxycycline or in A172 cells topic in the field of stem cell differentiation. Here, we exposed to doxycycline alone. CONCLUSIONS: have initially differentiated hESCs into functional Engineering of transgenic hTRAIL by using ES cell- hepatic endoderm (HE) and continued the derived astrocytes induced apoptosis in human differentiation by replating them onto galactosylated malignant glioma cells while sparing nontumor collagen (GC) and collagen matrices. The astrocytes. The apoptotic effects of transgenic hTRAIL differentiation of hESC-HE cells into HLCs on GC are greater than those of recombinant hTRAIL. substrate showed significant up-regulation of hepatic- Analysis of these results suggests that hTRAIL- specific genes such as ALB, HNF4alpha, CYP3A4, expressing-ES cell-derived astrocytes should be G6P, and ASGR1. There was more albumin secretion considered in the development of new in vivo and urea synthesis, as well as more cytochrome p450 strategies to treat malignant human gliomas. activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested Ghahrizjani, F. A., et al. (2015). "Enhanced that GC substrate has the potential to be used for in expression of FNDC5 in human embryonic stem cell- vitro maturation of hESC-HLCs. derived neural cells along with relevant embryonic neural tissues." Gene 557(2): 123-129. Gholamitabar Tabari, M., et al. (2018). Availability of human embryonic stem cells "Evaluation of Novel Mouse-Specific Germ Cell Gene (hESCs) has enhanced the capability of basic and Expression in Embryonic Stem Cell-Derived Germ clinical research in the context of human neural Cell-Like Cells In Vitro with Retinoic Acid differentiation. Derivation of neural progenitor (NP) Treatment." Cell Reprogram 20(4): 245-255.

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We designed a study to induce differentiation of (p=0.90), Scp3 (p=0.61) and Stra8 (p=0.08) were up- Oct4-GFP (expression of Green Fluorescent Protein of regulated without significance differences compared oct4) embryonic stem cells (ESCs) by embryoid body with control group. Flow cytometry analysis showed (EB) culture system into germ cells (GCs) using that the mean number of Mvh-positive cells in the retinoic acid (RA) and evaluated the expression level +BMP4 group was greater when compared with ESCs, of (Fkbp6, Mov10l1, 4930432K21Rik, and Tex13) in -BMP4 and EB groups (p=0.03, p

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Human embryonic stem (ES) cells have an (FISH), we show that this subnuclear organization is expedited cell cycle ( approximately 15 h) due to an compromised in some cancer cell lines. In aneuploid abbreviated G1 phase ( approximately 2.5 h) relative cells, the presence of HLBs correlates with the number to somatic cells. One principal regulatory event during of histone gene loci. More importantly, the in situ co- cell cycle progression is the G1/S phase induction of localization of p220(NPAT) and Lsm10 is disrupted in histone biosynthesis to package newly replicated DNA. HeLa S3 cervical carcinoma cells and MCF7 breast In somatic cells, histone H4 gene expression is adenocarcinoma cells, with most Lsm10 residing in controlled by CDK2 phosphorylation of p220(NPAT) Cajal Bodies. The finding that the subnuclear and localization of HiNF-P/p220(NPAT) complexes integration of transcriptional initiation and 3'end with histone genes at Cajal body related subnuclear processing of histone gene transcripts is deregulated foci. Here we show that this 'S point' pathway is may be causally linked to tumor-related modifications operative in situ in human ES cells (H9 cells; NIH- in molecular pathways controlling histone gene designated WA09). Immunofluorescence microscopy expression during the cell cycle. shows an increase in p220(NPAT) foci in G1 reflecting the assembly of histone gene regulatory Ghule, P. N., et al. (2008). "Staged assembly of complexes in situ. In contrast to somatic cells where histone gene expression machinery at subnuclear foci duplication of p220(NPAT) foci is evident in S phase, in the abbreviated cell cycle of human embryonic stem the increase in the number of p220(NPAT) foci in ES cells." Proc Natl Acad Sci U S A 105(44): 16964- cells appears to precede the onset of DNA synthesis as 16969. measured by BrdU incorporation. Phosphorylation of Human embryonic stem (hES) cells have an p220(NPAT) at CDK dependent epitopes is most abbreviated G (1) phase of the cell cycle. How cells pronounced in S phase when cells exhibit elevated expedite G (1) events that are required for the levels of cyclins E and A. Our data indicate that initiation of S phase has not been resolved. One key subnuclear organization of the HiNF-P/p220(NPAT) regulatory pathway that controls G (1)/S-phase pathway is rapidly established as ES cells emerge from transition is the cyclin E/CDK2-dependent activation mitosis and that p220(NPAT) is subsequently of the coactivator protein nuclear protein, ataxia- phosphorylated in situ. Our findings establish that the telangiectasia locus/histone nuclear factor-P HiNF-P/p220(NPAT) gene regulatory pathway (p220(NPAT)/HiNF-P) complex that induces histone operates in a cell cycle dependent microenvironment gene transcription. In this study, we use the subnuclear that supports expression of DNA replication-linked organization of factors controlling histone gene histone genes and chromatin assembly to expression to define mechanistic differences in the G accommodate human stem cell self-renewal. (1) phase of hES and somatic cells using in situ immunofluorescence microscopy and fluorescence in Ghule, P. N., et al. (2009). "The subnuclear situ hybridization (FISH). We show that histone gene organization of histone gene regulatory proteins and 3' expression is supported by the staged assembly and end processing factors of normal somatic and modification of a unique subnuclear structure that embryonic stem cells is compromised in selected coordinates initiation and processing of transcripts human cancer cell types." J Cell Physiol 220(1): 129- originating from histone gene loci. Our results 135. demonstrate that regulatory complexes that mediate Human histone gene expression is controlled at transcriptional initiation (e.g., p220(NPAT)) and 3'- the level of transcription initiation and subsequent end processing (e.g., Lsm10, Lsm11, and SLBP) of 3'end processing to generate non-polyadenylated stem- histone gene transcripts colocalize at histone gene loci loop containing histone mRNAs. Transcription is in dedicated subnuclear foci (histone locus bodies) that controlled at the G1/S phase transition by the Cyclin are distinct from Cajal bodies. Although appearance of E/CDK2 mediated induction of p220(NPAT)/HiNF-P CDK2-phosphorylated p220(NPAT) in these domains complexes at subnuclear domains designated Histone occurs at the time of S-phase entry, histone locus Locus Bodies (HLBs) that associate with histone gene bodies are formed approximately 1 to 2 h before S clusters. Histone mRNA maturation is mediated by phase in embryonic cells but 6 h before S phase in Lsm10 containing U7snRNP complexes. In normal somatic cells. These temporal differences in the human somatic and embryonic stem cells, the 6p formation of histone locus bodies suggest that the G (1) histone locus, the transcription marker p220(NPAT) phase of the cell cycle in hES cells is abbreviated in and the 3'end processing marker Lsm10 (but not the part by contraction of late G (1). Cajal Body marker coilin) co-localize, reflecting the assembly of an integrated factory for histone gene Gibson, J. D., et al. (2009). "Single-cell transcript expression. Using in situ immuno-fluorescence analysis of human embryonic stem cells." Integr Biol microscopy and fluorescence in situ hybridization (Camb) 1(8-9): 540-551.

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We demonstrate the qualitative and quantitative cells (hESC) would secrete factors that arrest growth power of single-cell transcript analysis to characterize of human epithelial cancer cell lines. MATERIALS transcriptome dynamics in human embryonic stem AND METHODS: Cell proliferation was examined cells (hESC's). Single-cell analysis can systematically using the MTT assay then haemocytometer cell counts. determine unique cellular profiles for use in cell Staining with propidium iodide followed by flow sorting and identification, show the potential to cytometry was used to detect cell cycle stages. Heat augment standing models of cellular differentiation, denaturation and molecular fractionation experiments and elucidate the behavior of stem cells exiting were also performed. RESULTS: We found that hESC pluripotency. Using single-cell analysis of H9 hESC's conditioned medium (hESC CM) inhibited SKOV-3 differentiating under three culture conditions, we and HEY cell proliferation. Similar results were also revealed transient expression of mesendodermal obtained when we used breast and prostate cancer cell markers in all three protocols, followed by lines, whereas little or no inhibitory effect was increasingly stable expression of embryonic endoderm observed when human fibroblasts were tested. and extra-embryonic endoderm markers. Our single- Moreover, a co-culture model confirmed that cell profiles reveal mixed populations of cell types, inhibition of cancer cell proliferation is mediated by with both transcriptional and temporal heterogeneity soluble factors produced by hESCs. We also marking differentiation under all conditions. determined that the proportion of cancer cells in G (1) Interestingly, we also observe extensive and prolonged phase was increased by hESC CM treatment, co-expression of markers regulating both pluripotency accompanied by decrease in cells in S and G (2)/M and lineage differentiation in all culture conditions, phases, suggesting that the factors slow progression of and we find that pluripotency marker transcripts cancer cells by cell cycle inhibition. Heat denaturation remain detectable in the majority of cells for many and molecular fractionation experiments indicated a days. Finally, we show that cells derived from low molecular weight thermostable factor was undifferentiated hESC colonies display consistent gene responsible for these properties. CONCLUSIONS: expression profiles characterized by three cohorts of Our findings provide evidence that the human transcripts: uniform, absent and sporadically detected embryonic microenvironment contains soluble factor messages, and that a striking correlation exists (s) that are capable of inhibiting growth of cancer cells, between genes' membership in these cohorts and their and that exposure to such factors may represent a new hESC promoter chromatin state, with bivalent cancer treatment strategy. promoters dominating the sporadic transcripts. Glaser, D. E., et al. (2011). "Functional Gioviale, M. C., et al. (2013). "Beyond islet characterization of embryonic stem cell-derived transplantation in diabetes cell therapy: from endothelial cells." J Vasc Res 48(5): 415-428. embryonic stem cells to transdifferentiation of adult Endothelial cells (EC) derived from embryonic cells." Transplant Proc 45(5): 2019-2024. stem cells (ESC) require additional functional Exogenous insulin is, at the moment, the therapy characterization before they are used as a cell therapy of choice of diabetes, but does not allow tight in order to enhance their potential for engraftment and regulation of glucose leading to long-term proliferation. We explore several physiologically complications. Recently, pancreatic islet relevant functions of ESC-derived EC (ESC-EC), such transplantation to reconstitute insulin-producing beta as its capacity to produce nitric oxide (NO), regulate cells, has emerged as an alternative promising permeability, activate and express surface molecules therapeutic approach. Unfortunately, the number of for the recruitment of leukocytes in response to donor islets is too low compared with the high number inflammatory stimuli, migrate and grow new blood of patients needing a transplantation leading to a vessels, lay down extracellular matrix, and take up search for renewable sources of high-quality beta-cells. low-density lipoproteins. We also examined the ESC- This review, summarizes more recent promising EC ability to upregulate NO in response to shear stress approaches to the generation of new beta-cells from and downregulate NO in response to pro-inflammatory embryonic stem cells for transdifferentiation of adult TNF-alpha activation. Functional responses of ESC- cells, particularly a critical examination of the seminal EC were compared with those of cultured mouse aortic work by Lumelsky et al. ECs. The ESC-EC exhibit most aspects of functional endothelium, but interesting differences remain. The Giuffrida, D., et al. (2009). "Human embryonic ESC-EC produced less NO on a per cell basis, but the stem cells secrete soluble factors that inhibit cancer same amount of NO if quantified based on the area of cell growth." Cell Prolif 42(6): 788-798. endothelial tissue. They also exhibit increased OBJECTIVES: The aim of this study was to angiogenic sprouting and are more resistant to determine whether normal human embryonic stem inflammatory signals. We further characterized the

114 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ subphenotype of our ESC-EC and observed both transplantation. Among these, only IL-6 reduced venous and arterial markers on individual cells with a neuronal differentiation and promoted glial larger percentage of the cells exhibiting a venous differentiation in vitro. When we added anti-IL-6 phenotype. These data support the hypothesis that the receptor antibodies to NPCs during transplantation, developmental default pathway is toward a venous EC, this single and local blockade of IL-6 signaling and that refinement of methods for differentiation reduced the accumulation of host-derived leukocytes, towards arterial EC is required to maintain a including microglia. Furthermore, it also promoted homogeneous population. neuronal differentiation and reduced glial differentiation from the grafted NPCs to an extent Godfrey, K. J., et al. (2012). "Stem cell-based similar to that with systemic and continuous treatments for Type 1 diabetes mellitus: bone marrow, administration of cyclosporine A. These results embryonic, hepatic, pancreatic and induced pluripotent suggest that local administration of anti-IL-6 receptor stem cells." Diabet Med 29(1): 14-23. antibodies with NPCs may promote neuronal Type 1 diabetes mellitus--characterized by the differentiation during the treatment of neurological permanent destruction of insulin-secreting beta-cells-- diseases with cell replacement therapy. is responsive to cell-based treatments that replace lost beta-cell populations. The current gold standard of Gong, S. P., et al. (2014). "The co-injection of pancreas transplantation provides only temporary somatic cells with embryonic stem cells affects independence from exogenous insulin and is fraught teratoma formation and the properties of teratoma- with complications, including increased mortality. derived stem cell-like cells." PLoS One 9(9): e105975. Stem cells offer a number of theoretical advantages The aim of this study was to assess the biological over current therapies. Our review will focus on the reactions triggered by stem cell transplantation related development of treatments involving tissue stem cells to phenotypic alteration, host-to-cell response, from bone marrow, liver and pancreatic cells, as well chromosomal stability, transcriptional alteration, and as the potential use of embryonic and induced stem cell-like cell re-expansion. B6CBAF1 mouse pluripotent stem cells for Type 1 diabetes therapy. embryonic stem cells (ESCs) were injected While the body of research involving stem cells is at subcutaneously into homologous or heterologous once promising and inconsistent, bone marrow-derived (B6D2F1) recipients, and heterologous injections were mesenchymal stem cell transplantation seems to offer performed with or without co-injection of B6D2F1 the most compelling evidence of efficacy. These cells fetal fibroblasts. All homologous injections resulted in have been demonstrated to increase endogenous teratoma formation, whereas a sharp decrease in insulin production, while partially mitigating the formation was detected after heterologous injection autoimmune destruction of newly formed beta-cells. (100 vs. 14%; p<0.05). The co-injection of somatic However, recently successful experiments involving cells in heterologous injections enhanced teratoma induced pluripotent stem cells could quickly move formation significantly (14 vs. 75%; p<0.05). Next, them into the foreground of therapeutic research. We ESC-like cell colonies with the same genotype as address the limitations encountered by present parental ESCs were formed by culturing teratoma- research and look toward the future of stem cell dissociated cells. Compared with parental ESCs, treatments for Type 1 diabetes. teratoma-derived ESC-like cells exhibited significantly increased aneuploidy, regardless of homologous or Gomi, M., et al. (2011). "Single and local heterologous injections. Repopulation of the parental blockade of interleukin-6 signaling promotes neuronal ESCs was the main factor that induced chromosomal differentiation from transplanted embryonic stem cell- instability, whereas the co-injection of somatic cells derived neural precursor cells." J Neurosci Res 89(9): did not restore chromosomal normality. Different 1388-1399. genes were expressed in the parental ESCs and Safe and efficient transplantation of embryonic teratoma-derived ESC-like cells; the difference was stem (ES) cells to the brain requires that local larger with parental vs. heterologous than parental vs. inflammatory and immune responses to allogeneic homologous co-injections. The co-injection of somatic grafts are inhibited. To investigate cytokines that cells decreased this difference further. In conclusion, affect graft cell survival and differentiation, we used the host-to-cell interactions triggered by ESC stromal cell-derived inducing activity to induce the transplantation could be modulated by co-injection differentiation of neural progenitor cells (NPCs) from with somatic cells. A mouse model using homologous mouse ES cells and transplanted the NPCs into mouse or heterologous transplantation of stem cells could brain. Examination of surrounding brain tissue help monitor cell adaptability and gene expression revealed elevated expression levels of interleukin (IL)- after injection. 1beta, IL-4, and IL-6 in response to NPC

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Gong, S. P., et al. (2010). "Embryonic stem cell- involved in the TGFbeta signaling pathway, which has like cells established by culture of adult ovarian cells a well-established role in hESC maintenance [3], [4], in mice." Fertil Steril 93(8): 2594-2601, 2601 e2591- [5]. The microarray data have been deposited in 2599. NCBI's Gene Expression Omnibus (GEO) and can be OBJECTIVE: To suggest an alternative strategy accessed through GEO Series accession numbers for deriving histocompatible stems cells without GSE62062 and GSE63215. undertaking genetic manipulation. DESIGN: Prospective approach using an animal model. Gonzalez, S., et al. (2011). "Influence of E- SETTING: Stem cell and bioevaluation laboratory, cadherin-mediated cell adhesion on mouse embryonic Seoul National University. ANIMAL (S): F1 (C57BL6 stem cells derivation from isolated blastomeres." Stem X DBA2) and outbred (ICR) mice. INTERVENTION Cell Rev 7(3): 494-505. (S): Ovarian stroma cells of less than 40 mum in Efforts to efficiently derive embryonic stem cells diameter were subcultured with fibroblast monolayer, (ESC) from isolated blastomeres have been done to and colony-forming cells were characterized. MAIN minimize ethical concerns about human embryo OUTCOME MEASURE (S): Stemness, genotype, and destruction. Previous studies in our laboratory imprinted gene methylation. RESULT (S): Two-lines indicated a poor derivation efficiency of mouse ESC of colony-forming cells were established, which lines from isolated blastomeres at the 8-cell stage (1/8 expressed markers specific for embryonic stem cells blastomeres) due, in part, to a low division rate of the (ESC) and formed embryoid bodies and teratomas. single blastomeres in comparison to their counterparts Complete matching of microsatellite markers with the with a higher number of blastomeres (2/8, 3/8 and 4/8 cell donor strain confirmed their establishment from blastomeres). Communication and adhesion between ovarian tissue, and identification of both homozygotic blastomeres from which the derivation process begins and heterozygotic chromosomes raised the possibility could be important aspects to efficiently derive ESC of their derivation from parthenogenetic oocytes. lines. In the present study, an approach consisting in However, the use of cells smaller than mature oocytes the adhesion of a chimeric E-cadherin (E-cad-Fc) to for primary culture, the difference in imprinted gene the blastomere surface was devised to recreate the methylation compared with parthenogenetic ESCs, and signaling produced by native E-cadherin between failure to establish the ESC-like cells by primary neighboring blastomeres inside the embryo. By this follicle culture collectively suggested the irrelevancy approach, the division rate of 1/8 blastomeres to gametes. CONCLUSION (S): Coculture of adult increased from 44.6% to 88.8% and a short exposure ovarian cells with somatic fibroblasts can yield of 24 h to the E-cad-Fc produced an ESC derivation colony-forming cells having ESC-like activity, which efficiency of 33.6%, significantly higher than the 2.2% may provide an alternative for establishing autologous obtained from the control group without E-cad-Fc. By stem cells from adults that can be obtained without contrast, a longer exposure to the same chimeric genetic manipulation. protein resulted in higher proportions of trophoblastic vesicles. Thus, we establish an important role of E- Gonzales, K. A. and H. Liang (2015). cadherin-mediated adherens junctions in promoting "Transcriptomic profiling of human embryonic stem both the division of single 1/8 blastomeres and the cells upon cell cycle manipulation during pluripotent efficiency of the ESC derivation process. state dissolution." Genom Data 6: 118-119. While distinct cell cycle structures have been Gonzalo-Gil, E., et al. (2016). "Human known to correlate with pluripotent or differentiated embryonic stem cell-derived mesenchymal stromal cell states [1], there is no evidence on how the cell cells ameliorate collagen-induced arthritis by inducing cycle machinery directly contributes to human host-derived indoleamine 2,3 dioxygenase." Arthritis embryonic stem cell (hESC) pluripotency. We Res Ther 18: 77. established a determinant role of cell cycle BACKGROUND: The immunosuppressive and machineries on the pluripotent state by demonstrating anti-inflammatory properties of mesenchymal stromal that the specific perturbation of the S and G2 phases cells (MSC) have prompted their therapeutic can prevent pluripotent state dissolution (PSD) [2]. application in several autoimmune diseases, including Active mechanisms in these phases, such as the DNA rheumatoid arthritis. Adult MSC are finite and their damage checkpoint and Cyclin B1, promote the clinical use is restricted by the need for long-term pluripotent state [2]. To understand the mechanisms expansion protocols that can lead to genomic behind the effect on PSD by these pathways in hESCs, instability. Inhibition of Smad2/3 signaling in human we performed comprehensive gene expression analysis pluripotent stem cells (hPSC) provides an infinite by time-course microarray experiments. From these source of MSC that match the phenotype and datasets, we observed expression changes in genes functional properties of adult MSC. Here, we test the

116 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ therapeutic potential of hPSC-MSC of embryonic from the transplantation of the sorted CD34(+) cell origin (embryonic stem cell-derived mesenchymal population was not augmented by transplanting the stromal cells, hESC-MSC) in the experimental model entire cell population from which the CD34(+) cells of collagen-induced arthritis (CIA). METHODS: CIA were isolated. Human DNA and insulin messenger was induced in DBA/1 mice by immunization with RNA were detected in sheep pancreases. An average type II collagen (CII) in Complete Freund's Adjuvant of 1.51 ng/mL human C-peptide was detected in serum (CFA). Mice were treated with either a single dose from eight animals transplanted with differentiated cell (10(6) cells/mouse) of hESC-MSC on the day of populations and assayed up to 55 months immunization (prophylaxis) or with three doses of posttransplantation. Transplantation of as few as hESC-MSC every other day starting on the day of 23,500 cells resulted in long-term sustainable beta- arthritis onset (therapy). Arthritis severity was cell-like activity. Teratomas were absent in the evaluated daily for six weeks and ten days, transplanted animals. CONCLUSION: Our data respectively. Frequency of Treg (FoxP3(+)), Th1 suggest that hESC-derived CD34(+) cells have a (IFNgamma (+)) and Th17 (IL17(+)) CD4(+) T cells potential for long-term in vivo endocrine cellular in inguinal lymph nodes (ILN) was quantified by flow activity that could prove useful in regenerative cytometry. Serum levels of anti-CII antibodies were medicine. Because the same cell population has determined by ELISA. Detection of hESC-MSC and previously been shown to contain hematopoietic quantification of murine and human indoleamine 2,3 potential, it could be used for the induction of dioxygenase (IDO1) expression was performed by immunological tolerance and bone marrow chimerism quantitative real-time PCR. Statistical differences were prior to cellular therapy for diabetes. analyzed by ANOVA and the Mann-Whitney U test. RESULTS: Administration of hESC-MSC to mice Hajizadeh-Saffar, E., et al. (2015). "Inducible with established arthritis reduced disease severity VEGF expression by human embryonic stem cell- compared to control-treated mice. Analysis of CD4 T derived mesenchymal stromal cells reduces the cell populations in treated mice showed an increase in minimal islet mass required to reverse diabetes." Sci FoxP3(+) Treg and IFNgamma (+) Th1 cells but not in Rep 5: 9322. Th17 cells in the ILN. Anti-CII antibody levels were UNLABELLED: Islet transplantation has been not affected by treatment. Migration of hESC-MSC to hampered by loss of function due to poor the ILN in treated mice was associated with the revascularization. We hypothesize that co- induction of murine IDO1. CONCLUSION: Treatment transplantation of islets with human embryonic stem with hESC-MSC ameliorates CIA by inducing cell-derived mesenchymal stromal cells that IFNgamma (+) Th1 cells and IDO1 in the host. Thus, conditionally overexpress VEGF (hESC-MSC:VEGF) hESC-MSC can provide an infinite cellular source for may augment islet revascularization and reduce the treatment of rheumatoid arthritis. minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant Goodrich, A. D., et al. (2010). "In vivo lentiviruses that allowed conditional (Dox-regulated) generation of beta-cell-like cells from CD34(+) cells overexpression of VEGF. HESC-MSC: VEGF were differentiated from human embryonic stem cells." Exp characterized by tube formation assay. After co- Hematol 38(6): 516-525 e514. transplantation of hESC-MSC:VEGF with murine OBJECTIVE: CD34(+) cells, present within the islets in collagen-fibrin hydrogel in the omental pouch bone marrow, have previously been shown to possess of diabetic nude mice, we measured blood glucose, pancreatic endocrine potential. Based on this body weight, glucose tolerance and serum C-peptide. observation, we explored the capacity of CD34(+) As control, islets were transplanted alone or with non- cells derived in culture from the differentiation of transduced hESC-MSCs. Next, we compared human embryonic stem cells (hESC), for their in vivo functional parameters of 400 islets alone versus 200 pancreatic endocrine capacity. MATERIALS AND islets co-transplanted with hESC-MSC:VEGF. As METHODS: Sheep were transplanted with hESC- control, 200 islets were transplanted alone. Metabolic derived CD34(+) cells, as well as nonsorted function of islets transplanted with hESC-MSC:VEGF differentiated cultures. Transplantations were carried significantly improved, accompanied by superior graft out with in utero intraperitoneal injections prior to revascularization, compared with control groups. development of the immune system in the fetus so that Transplantation of 200 islets with hESC-MSC:VEGF tolerance toward foreign antigens was acquired during showed superior function over 400 islets alone. We gestation and persisted in the adult. RESULTS: All conclude that co-transplantation of islets with VEGF- cell populations that were tested demonstrated human expressing hESC-MSCs allowed for at least a 50% cellular activity and long-term presence up to 5 years. reduction in minimal islet mass required to reverse However, the in vivo beta-cell-like activity achieved

117 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ diabetes in mice. This approach may contribute to Han, S., et al. (2018). "Endothelial cells instruct alleviate the need for multiple donor organs per patient. liver specification of embryonic stem cell-derived endoderm through endothelial VEGFR2 signaling and Hall, V. (2008). "Porcine embryonic stem cells: a endoderm epigenetic modifications." Stem Cell Res 30: possible source for cell replacement therapy." Stem 163-170. Cell Rev 4(4): 275-282. Liver organogenesis requires complex cell-cell The development of porcine embryonic stem cell interactions between hepatic endoderm cells and lines (pESC) has received renewed interest given the adjacent cell niches. Endothelial cells are key players advances being made in the production of for endoderm hepatic fate decision. We previously immunocompatible transgenic pigs. However, demonstrated that the endothelial cell niche promotes difficulties are evident in the production of pESCs in- hepatic specification of mouse embryonic stem cell vitro. This may largely be attributable to differences in (ESC)-derived endoderm through dual repression of porcine pre-implantation development compared to the Wnt and Notch pathways in endoderm cells. In the mouse and human. Expression of oct4, nanog and sox2 present study, we dissected further the mechanisms by differs in the zona-enclosed porcine blastocyst which endothelial cells trigger endoderm hepatic compared to its mouse and human counterparts, which specification. Using our previously established in vitro may suggest that other factors may be responsible for mouse ESC system mimicking the early hepatic maintaining porcine pluripotency in the early specification process, endoderm cells were purified blastocyst. In addition, the epiblast forms considerably and co-cultured with endothelial cells to induce later, at days 7 to 8 when the porcine blastocyst begins hepatic specification. The comparison of transcriptome to hatch and is maintained for 4 days before profiles between hepatic endoderm cells isolated from completely differentiating. This review covers an co-cultures and endoderm cells cultured alone revealed outline of the known molecular profile during porcine that VEGF signaling instructs hepatic specification of pre-implantation development and provides a history endoderm cells through endothelial VEGFR2 in the development of putative pESCs to date. Greater activation. Additionally, epigenetic mark inhibition knowledge on the molecular mechanisms that underlie assays upon co-cultures uncovered that histone porcine pluripotency and pre-implantation acetylation and DNA methylation promote hepatic development may aid in improving the development of specification while histone methylation inhibits it. This pESCs. study provides an efficient 2D platform modelling the endothelial cell niche crosstalk with endoderm, and Hall, V. J. (2008). "Embryonic stem cells and reveals mechanisms by which endothelial cells Parkinson's disease: cell transplantation to cell promote hepatic specification of mouse ESC-derived therapy." Ann Acad Med Singapore 37(3): 163-162. endoderm cells through endothelial VEGFR2 activation and endoderm epigenetic modifications. Han, L., et al. (2012). "A chemical small molecule induces mouse embryonic stem cell Han, X., et al. (2017). "Efficient and Fast differentiation into functional vascular endothelial Differentiation of Human Neural Stem Cells from cells via Hmbox1." Stem Cells Dev 21(15): 2762-2769. Human Embryonic Stem Cells for Cell Therapy." Embryonic stem cells (ESCs) can differentiate to Stem Cells Int 2017: 9405204. endothelial progenitor cells and vascular endothelial Stem cell-based therapies have been used for cells (VECs), but the mechanism is largely unknown. repairing damaged brain tissue and helping functional In this study, we synthesized 2 chiral compounds (R- recovery after brain injury. Aberrance neurogenesis is ABO and S-ABO) and identified R-ABO as an related with brain injury, and multipotential neural effective inducer of ESC differentiation into VECs. stem cells from human embryonic stem (hES) cells Furthermore, we found that R-ABO induced ESC provide a great promise for cell replacement therapies. differentiation into VECs via homeobox containing 1 Optimized protocols for neural differentiation are (Hmbox1) that acted upstream of fibroblast growth necessary to produce functional human neural stem factor 2 (FGF-2). The data suggest that R-ABO is a cells (hNSCs) for cell therapy. However, the qualified novel tool for ESC differentiation into VECs, and procedure is scarce and detailed features of hNSCs Hmbox1 is a key regulator in this differentiation originated from hES cells are still unclear. In this process. These findings provide information on a study, we developed a method to obtain hNSCs from novel target and a new platform for further hES cells, by which we could harvest abundant hNSCs investigating the gene control of ESC differentiation to in a relatively short time. Then, we examined the VECs. expression of pluripotent and multipotent marker genes through immunostaining and confirmed differentiation potential of the differentiated hNSCs.

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Furthermore, we analyzed the mitotic activity of these delivery system that is both safe and efficient, but hNSCs. In this report, we provided comprehensive using mRNA as an alternative to DNA can circumvent features of hNSCs and delivered the knowledge about these major roadblocks. In this study, we show that how to obtain more high-quality hNSCs from hES both unmodified and modified mRNA can be cells which may help to accelerate the NSC-based delivered to retinal pigmented epithelial (RPE) cells therapies in brain injury treatment. with a high efficiency compared with conventional plasmid delivery systems. On the other hand, Handschel, J., et al. (2011). "Embryonic stem administration of unmodified mRNA induced a strong cells in scaffold-free three-dimensional cell culture: innate immune response that was almost absent when osteogenic differentiation and bone generation." Head using modified mRNA. Importantly, transfection of Face Med 7: 12. mRNA encoding a key regulator of RPE gene Extracorporeal formation of mineralized bone- expression, microphthalmia-associated transcription like tissue is still an unsolved challenge in tissue factor (MITF), confirmed the functionality of the engineering. Embryonic stem cells may open up new delivered mRNA. Immunostaining showed that therapeutic options for the future and should be an transfection with either type of mRNA led to the interesting model for the analysis of fetal expression of roughly equal levels of MITF, primarily organogenesis. Here we describe a technique for localized in the nucleus. Despite these findings, culturing embryonic stem cells (ESCs) in the absence quantitative RT-PCR analyses showed that the of artificial scaffolds which generated mineralized activation of the expression of MITF target genes was miromasses. Embryonic stem cells were harvested and higher following transfection with modified mRNA osteogenic differentiation was stimulated by the compared with unmodified mRNA. Our findings, addition of dexamethasone, ascorbic acid, and ss- therefore, show that modified mRNA transfection can glycerolphosphate (DAG). After three days of be applied to human embryonic stem cell-derived RPE cultivation microspheres were formed. These spherical cells and that the method is safe, efficient, and three-dimensional cell units showed a peripheral zone functional. consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular Hao, J., et al. (2009). "Human parthenogenetic matrix. Alizarine red staining confirmed evidence of embryonic stem cells: one potential resource for cell mineralization after 10 days of DAG stimulation in the therapy." Sci China C Life Sci 52(7): 599-602. stimulated but not in the control group. Transmission Pluripotent stem cells derived from somatic cells electron microscopy demonstrated scorching through such processes as nuclear transfer or induced crystallites and collagenous fibrils as early indication pluripotent stem (iPS) cells present an important of bone formation. These extracellular structures model for biomedical research and provide potential resembled hydroxyl apatite-like crystals as resources for cell replacement therapies. However, the demonstrated by distinct diffraction patterns using overall efficiency of the conversional nuclear transfer electron diffraction analysis. The micromass culture is very low and the safety issue remains a major technique is an appropriate model to form three- concern for iPS cells. Embryonic stem cells (ESCs) dimensional bone-like micro-units without the need generated from parthenogenetic embryos are one for an underlying scaffold. Further studies will have to attractive alternative as a source of histocompatible show whether the technique is applicable also to cells and tissues for cell therapy. Recent studies on pluripotent stem cells of different origin. human parthenogenetic embryonic stem cells (hPG ESCs) have revealed that these ESCs are very similar Hansson, M. L., et al. (2015). "Efficient delivery to the hESCs derived from IVF or in vivo produced and functional expression of transfected modified blastocysts in gene expression and other characteristics, mRNA in human embryonic stem cell-derived retinal but full differentiation and development potential of pigmented epithelial cells." J Biol Chem 290(9): 5661- these hPG ESCs have to be further investigated before 5672. clinical research and therapeutic interventions. To Gene- and cell-based therapies are promising generate various pluripotent stem cells, diverse strategies for the treatment of degenerative retinal reprogramming techniques and approaches will be diseases such as age-related macular degeneration, developed and integrated. This may help elucidate the Stargardt disease, and retinitis pigmentosa. Cellular fundamental mechanisms underlying reprogramming engineering before transplantation may allow the and stem cell biology, and ultimately benefit cell delivery of cellular factors that can promote functional therapy and regenerative medicine. improvements, such as increased engraftment or survival of transplanted cells. A current challenge in Hao, Q., et al. (2015). "Study of Bone Marrow traditional DNA-based vector transfection is to find a and Embryonic Stem Cell-Derived Human

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Mesenchymal Stem Cells for Treatment of Escherichia those of BM-MSCs, such as for immunomodulation, coli Endotoxin-Induced Acute Lung Injury in Mice." these results highlight the need for a disease-specific Stem Cells Transl Med 4(7): 832-840. potency assay for stem cell-based therapy. UNLABELLED:: Mesenchymal stem cells (MSCs) can be derived from multiple tissue sources. Haraguchi, S., et al. (2012). "Establishment of However, the optimal source of MSCs for cell-based self-renewing porcine embryonic stem cell-like cells therapy for acute lung injury (ALI) is unclear. In the by signal inhibition." J Reprod Dev 58(6): 707-716. present experiments, we studied bone marrow (BM)- Although the establishment of putative porcine derived and embryonic stem cell-derived human MSC embryonic stem cells (ESCs) has been reported, such (ES-MSCs) as a therapeutic agent in Escherichia coli cell lines quickly lose their self-renewal ability, as they endotoxin-induced ALI in mice. We hypothesized that easily differentiate or become extinct after only a ES-MSCs would be more potent than BM-MSCs limited number of passages in culture. ESC-like cells owing to its more primitive source of origin. ALI was exhibiting self-renewal rather than pluripotency are induced by the intratracheal instillation of endotoxin at considered to be a valuable resource in applications 4 mg/kg into 10-12-week-old C57BL/6 mice with or such as drug screening and toxicology testing in without BM-MSCs, ES-MSCs, or normal human lung humans, livestock and veterinary medicine. Here, we fibroblasts as a cellular control. Compared with the report the generation of unique cell lines established endotoxin-injured mice at 48 hours, the administration from the inner cell mass (ICM) of porcine embryos by of ES-MSCs provided results similar to those of BM- using inhibitors of glycogen synthase kinase 3beta and MSCs, significantly reducing the influx of white blood mitogen-activated protein kinase kinase 1. These ICM- cells and neutrophils and decreasing the secretion of derived cell lines were initially cultured and passaged the inflammatory cytokines, macrophage inflammatory in conventional ES medium for human ESCs and protein-2 and tumor necrosis factor-alpha, in the showed porcine ESC-like morphology with alkaline injured alveolus. BM-MSCs also reduced phosphatase (AP) activity. After transfer to culture in extravascular lung water, a measure of pulmonary ES medium containing inhibitors, the morphology of edema, by 60% and the total protein levels, a measure the colonies was dramatically changed, i.e., they were of lung permeability, by 66%. However, surprisingly, closely packed smooth-edged colonies with close cell- ES-MSCs did not have these protective effects, which cell boundaries and showed the expression of was partially explained by the increased secretion of undifferentiated markers including OCT4 (POU5F1) matrix metallopeptidase 9 by ES-MSCs, an enzyme and NANOG. Notably, the self-renewal capacity and known to increase lung protein permeability. In morphology of the cells were LIF-dependent, conclusion, both BM-MSCs and ES-MSCs markedly consistent with the expression of LIF receptors and decreased endotoxin-induced inflammation. However, phosphorylation of signal transducer and activator of ES-MSCs did not show any beneficial effect on transcription 3. To date, our established cell lines have reducing pulmonary edema and lung protein been cultured continuously for over 100 passages permeability compared with BM-MSCs, suggesting without any overt morphological changes. Thus, the that not all MSCs behave in a similar fashion. Our established cell lines reported here provide a new results highlight the need perhaps for a disease- ESC-like cell culture system for use not only in the specific potency assay for MSCs. SIGNIFICANCE: fields of veterinary medicine and livestock but also To determine the optimal source of mesenchymal stem human medical research, since porcine physiology cells (MSCs) for cell-based therapy for acute lung closely resembles that of humans. injury, bone marrow (BM)- and embryonic stem cell- derived human MSC (ES-MSCs) were compared as Haridass, D., et al. (2009). "Repopulation therapeutic agents for Escherichia coli endotoxin- efficiencies of adult hepatocytes, fetal liver progenitor induced lung injury in mice. ES-MSCs behaved cells, and embryonic stem cell-derived hepatic cells in similarly to BM-MSCs by markedly decreasing the albumin-promoter-enhancer urokinase-type inflammatory response induced by endotoxin. plasminogen activator mice." Am J Pathol 175(4): However, unlike BM-MSCs, ES-MSCs provided no 1483-1492. protective effects against increasing lung water and Fetal liver progenitor cell suspensions (FLPC) protein permeability, in part because of an increase in and hepatic precursor cells derived from embryonic expression of matrix metallopeptidase 9 by ES-MSCs. stem cells (ES-HPC) represent a potential source for In patients with acute respiratory distress syndrome, liver cell therapy. However, the relative capacity of impaired alveolar fluid clearance (i.e., no resolution of these cell types to engraft and repopulate a recipient pulmonary edema fluid) has been associated with liver compared with adult hepatocytes (HC) has not higher mortality rates. Although ES-MSCs might been comprehensively assessed. We transplanted ultimately be found to have properties superior to mouse and human HC, FLPC, and ES-HPC into a new

120 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ immunodeficient mouse strain (Alb-uPA (tg (+/- were comparable to other in vitro models previously ))Rag2(-/-)gamma (c) (-/-) mice) and estimated the characterized in the literature. hN2 cells provide a percentages of HC after 3 months. Adult mouse HC model in which to investigate chemical effects on repopulated approximately half of the liver mass (46.6 neurite outgrowth in a non-transformed human-derived +/- 8.0%, 1 x 10(6) transplanted cells), whereas mouse cells and provide an alternative to the use of primary FLPC derived from day 13.5 and 11.5 post conception rodent neural cultures or immortalized clonal cell lines. embryos generated only 12.1 +/- 3.0% and 5.1 +/- 1.1%, respectively, of the recipient liver and smaller Harrison, N. J., et al. (2007). "Culture adaptation cell clusters. Adult human HC and FLPC generated of embryonic stem cells echoes germ cell overall less liver tissue than mouse cells and malignancy." Int J Androl 30(4): 275-281; discussion repopulated 10.0 +/- 3.9% and 2.7 +/- 1.1% of the 281. recipient livers, respectively. Mouse and human ES- Teratocarcinomas are a subset of tumours that HPC did not generate HC clusters in our animal model. result from the neoplastic transformation of primordial We conclude that, in contrast to expectations, adult germ cells. Such germ cell tumours (GCT) are HC of human and mouse origin generate liver tissue histologically heterogeneous, reflecting a capacity for more efficiently than cells derived from fetal tissue or differentiation (pluripotency) of their embryonal embryonic stem cells in a highly immunodeficient carcinoma (EC) stem cells. However, malignant Alb-uPA transgenic mouse model system. These evolution of these tumours may ultimately correlate results have important implications in the context of with a decrease in pluripotency, because this would selecting the optimal strategy for human liver cell tend to increase the propensity of EC cells for self- therapies. renewal. Human embryonic stem (ES) cells, derived from early blastocysts, closely resemble EC cells and, Harrill, J. A., et al. (2010). "Quantitative on prolonged culture in vitro, acquire progressive assessment of neurite outgrowth in human embryonic genetic changes that show striking similarity to those stem cell-derived hN2 cells using automated high- seen in GCT (e.g. gain of material from chromosome content image analysis." Neurotoxicology 31(3): 277- 12). In parallel, these abnormal ES cells show 290. enhanced population growth rates and plating Throughout development neurons undergo a efficiencies, indicative of their adaptation to culture number of morphological changes including neurite conditions. Understanding the mechanisms that drive outgrowth from the cell body. Exposure to neurotoxic such culture adaptation of ES cells may also provide chemicals that interfere with this process may result in insights into the development and progression of GCT. permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and Hartman, B. H., et al. (2018). "Fbxo2(VHC) immortalized human and non-human clonal cell lines mouse and embryonic stem cell reporter lines delineate have been used to investigate the molecular in vitro-generated inner ear sensory epithelia cells and mechanisms controlling neurite outgrowth and enable otic lineage selection and Cre-recombination." examine chemical effects on this process. The present Dev Biol. study characterizes the molecular phenotype of hN2 While the mouse has been a productive model for human embryonic stem cell (hESC)-derived neural inner ear studies, a lack of highly specific genes and cells and uses automated high-content image analysis tools has presented challenges. The absence of to measure neurite outgrowth in vitro. At 24h post- definitive otic lineage markers and tools is limiting in plating hN2 cells express a number of protein markers vitro studies of otic development, where innate cellular indicative of a neuronal phenotype, including: nestin, heterogeneity and disorganization increase the reliance beta (III)-tubulin, microtubule-associated protein 2 on lineage-specific markers. To address this challenge (MAP2) and phosphorylated neurofilaments. Neurite in mice and embryonic stem (ES) cells, we targeted outgrowth in hN2 cells proceeded rapidly, with a the lineage-specific otic gene Fbxo2 with a majority of cells extending one to three neurites by multicistronic reporter cassette (Venus/Hygro/CreER 48h in culture. In addition, concentration-dependent = VHC). In otic organoids derived from ES cells, decreases in neurite outgrowth and ATP-content were Fbxo2(VHC) specifically delineates otic progenitors observed following treatment of hN2 cells with either and inner ear sensory epithelia. In mice, Venus bisindolylmaleimide I, U0126, lithium chloride, expression and CreER activity reveal a cochlear sodium orthovanadate and brefeldin A, all of which developmental gradient, label the prosensory lineage, have previously been shown to inhibit neurite show enrichment in a subset of type I vestibular hair outgrowth in primary rodent neural cultures. Overall, cells, and expose strong expression in adult cerebellar the molecular phenotype, rate of neurite outgrowth and granule cells. We provide a toolbox of multiple sensitivity of hN2 cells to neurite outgrowth inhibitors spectrally distinct reporter combinations for studies

121 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ that require use of fluorescent reporters, hygromycin ESCs, followed by the occurrence of massive selection, and conditional Cre-mediated recombination. apoptosis. Neither p53 nor its target gene was required for G2/M arrest. Instead, p53 and p73 were Hayakawa-Yano, Y., et al. (2017). "An RNA- fully responsible for apoptosis. p53 and p73 were also binding protein, Qki5, regulates embryonic neural required for differentiation-induced apoptosis in stem cells through pre-mRNA processing in cell mouse ESCs. In addition, doxorubicin treatment adhesion signaling." Genes Dev 31(18): 1910-1925. induced the expression of in a Cell type-specific transcriptomes are enabled by p53-dependent manner. Therefore, both p53 and p73 the action of multiple regulators, which are frequently are critical in apoptosis induced by DNA damage and expressed within restricted tissue regions. In the differentiation. present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is He, K., et al. (2016). "Epigenetics changes expressed in early embryonic neural stem cells and caused by the fusion of human embryonic stem cell subsequently down-regulated during neurogenesis. and ovarian cancer cells." Biosci Rep 36(5). mRNA sequencing analysis in neural stem cell culture To observe the effect of gene expression and indicates that Qki proteins play supporting roles in the tumorigenicity in hybrid cells of human embryonic neural stem cell transcriptome and various forms of stem cells (hESCs) and ovarian cancer cells in vitro mRNA processing that may result from regionally and in vivo using a mouse model, and to determine its restricted expression and subcellular localization. Also, feasibility in reprogramming tumour cells growth and our in utero electroporation gain-of-function study apoptosis, for a potential exploration of the role of suggests that the nuclear-type Qki isoform Qki5 hESCs and tumour cells fusion in the management of supports the neural stem cell state. We next performed ovarian cancer. Stable transgenic hESCs (H1) and in vivo transcriptome-wide protein-RNA interaction ovarian cancer cell line OVCAR-3 were established mapping to search for direct targets of Qki5 and before fusion, and cell fusion system was established elucidate how Qki5 regulates neural stem cell function. to analyse the related indicators. PTEN expression in Combined with our transcriptome analysis, this HO-H1 cells was higher than those in the parental mapping analysis yielded a bona fide map of Qki5- stem cells and lower than those in parental tumour RNA interaction at single-nucleotide resolution, the cells; the growth of OV-H1 (RFP+GFP) hybrid cells identification of 892 Qki5 direct target genes, and an with double fluorescence expressions were obviously accurate Qki5-dependent alternative splicing rule in slower than that of human embryonic stem cells and the developing brain. Last, our target gene list OVCAR-3 ovarian cancer cells. The apoptosis signal provides the first compelling evidence that Qki5 is of the OV-H1 hybrid cells was significantly higher associated with specific biological events; namely, than that of the hESCs and OVCAR-3 ovarian cancer cell-cell adhesion. This prediction was confirmed by cells. In vivo results showed that compared with 7 histological analysis of mice in which Qki proteins days, 28 days and 35 days after inoculation of OV-H1 were genetically ablated, which revealed disruption of hybrid cells; also, apoptotic cell detection indicated the apical surface of the lateral wall in the developing that much stronger apoptotic signal was found in OV- brain. These data collectively indicate that Qki5 H1 hybrid cells inoculated mouse. The hESCs can regulates communication between neural stem cells by inhibit the growth of OVCAR-3 cells in vitro by mediating numerous RNA processing events and suppressing p53 and PTEN expression to suppress the suggest new links between splicing regulation and growth of tumour that may be achieved by inducing neural stem cell states. apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and He, H., et al. (2016). "p53 and p73 Regulate hESCs may become a novel direction for treatment of Apoptosis but Not Cell-Cycle Progression in Mouse ovarian cancer. Embryonic Stem Cells upon DNA Damage and Differentiation." Stem Cell Reports 7(6): 1087-1098. Hirata, S., et al. (2005). "Prevention of Embryonic stem cells (ESCs) are fast experimental autoimmune encephalomyelitis by proliferating cells capable of differentiating into all transfer of embryonic stem cell-derived dendritic cells somatic cell types. In somatic cells, it is well expressing myelin oligodendrocyte glycoprotein documented that p53 is rapidly activated upon DNA peptide along with TRAIL or programmed death-1 damage to arrest the cell cycle and induce apoptosis. ligand." J Immunol 174(4): 1888-1897. In mouse ESCs, p53 can also be functionally activated, Experimental autoimmune encephalomyelitis but the precise biological consequences are not well (EAE) is caused by activation of myelin Ag-reactive characterized. Here, we demonstrated that doxorubicin CD4+ T cells. In the current study, we tested a strategy treatment initially led to cell-cycle arrest at G2/M in to prevent EAE by pretreatment of mice with

122 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ genetically modified dendritic cells (DC) presenting to a D3-specific agonist, but this proliferation was not myelin oligodendrocyte glycoprotein (MOG) peptide observed in vivo. Cells cotransduced with D3 and Bcl- in the context of MHC class II molecules and x (L) genes had a phenotype similar to that of simultaneously expressing TRAIL or Programmed lymphoma in vivo, suggesting that the leukemogenesis Death-1 ligand (PD-L1). For genetic modification of induced by retroviral integration required "second hit" DC, we used a recently established method to generate mutations of additional genes. DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected Hiroyama, T., et al. (2008). "Establishment of with an expression vector for TRAIL or PD-L1 and an mouse embryonic stem cell-derived erythroid MHC class II-associated invariant chain-based MOG progenitor cell lines able to produce functional red epitope-presenting vector. Subsequently, double- blood cells." PLoS One 3(2): e1544. transfectant ES cell clones were induced to BACKGROUND: The supply of transfusable red differentiate to ES-DC, which expressed the products blood cells (RBCs) is not sufficient in many countries. of introduced genes. Treatment of mice with either of If erythroid cell lines able to produce transfusable the double-transfectant ES-DC significantly reduced T RBCs in vitro were established, they would be cell response to MOG, cell infiltration into spinal cord, valuable resources. However, such cell lines have not and the severity of MOG peptide-induced EAE. In been established. To evaluate the feasibility of contrast, treatment with ES-DC expressing MOG establishing useful erythroid cell lines, we attempted alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus to establish such cell lines from mouse embryonic PD-L1, or coinjection with ES-DC expressing MOG stem (ES) cells. METHODOLOGY/PRINCIPAL plus ES-DC-expressing TRAIL or PD-L1 had no FINDINGS: We developed a robust method to obtain effect in reducing the disease severity. In contrast, differentiated cell lines following the induction of immune response to irrelevant exogenous Ag (keyhole hematopoietic differentiation of mouse ES cells and limpet hemocyanin) was not impaired by treatment established five independent hematopoietic cell lines with any of the genetically modified ES-DC. The using the method. Three of these lines exhibited double-transfectant ES-DC presenting Ag and characteristics of erythroid cells. Although their simultaneously expressing immune-suppressive precise characteristics varied, each of these lines could molecules may well prove to be an effective therapy differentiate in vitro into more mature erythroid cells, for autoimmune diseases without inhibition of the including enucleated RBCs. Following transplantation immune response to irrelevant Ag. of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently Hirata, Y., et al. (2010). "Transactivation of the differentiated into functional RBCs, and significantly dopamine receptor 3 gene by a single provirus ameliorated the acute anemia. In addition, we did not integration results in development of B-cell lymphoma observe formation of any tumors following in transgenic mice generated from retrovirally transplantation of these cells. transduced embryonic stem cells." Blood 115(19): CONCLUSION/SIGNIFICANCE: To the best of our 3930-3938. knowledge, this is the first report to show the Gene transfer vectors based on retroviruses are feasibility of establishing erythroid cell lines able to commonly used in gene therapy applications because produce mature RBCs. Considering the number of of their unique ability to integrate efficiently into host human ES cell lines that have been established so far, genomes. This ability also forms the basis of a the intensive testing of a number of these lines for transformation event that can be induced in transduced erythroid potential may allow the establishment of cells by transactivation of proto-oncogenes near the human erythroid cell lines similar to the mouse vector integration sites. Here, we report on the erythroid cell lines described here. In addition, our development of lymphoma in mice generated from results strongly suggest the possibility of establishing embryonic stem cells transduced with an enhanced useful cell lines committed to specific lineages other green fluorescent protein. The cells expressed B220, than hematopoietic progenitors from human ES cells. CD5, Mac1, and IgM on their surfaces and expressed transcription factors characteristic of B-cell lymphoma. Hisamatsu-Sakamoto, M., et al. (2008). Importantly, each mouse had a single copy of the "Embryonic stem cells cultured in serum-free medium provirus in its genome; the copy was integrated into acquire bovine apolipoprotein B-100 from feeder cell the second intron of the dopamine receptor 3 (D3) layers and serum replacement medium." Stem Cells gene, and high-level expression of D3 was detected 26(1): 72-78. only in the lymphoma cells. Ectopic expression of D3 Previous studies have demonstrated that cell in murine marrow cells resulted in preferential populations that are cultured with heterologous animal proliferation of cells at the pre-B-cell stage in response products can acquire xenoantigens, potentially limiting

123 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ their clinical utility because of immune responses. BACKGROUND: The potential clinical Embryonic stem cells (ESCs) are an attractive source application of bone marrow or peripheral blood- of multiple potential cellular therapies and are derived progenitor cells for cardiovascular typically derived and routinely cultured on murine regeneration in patients with diabetes mellitus (DM) is embryonic fibroblast (MEF) feeder cell layers in limited by their functional impairment. We sought to commercially available serum replacement (SR) determine the mechanisms of impaired therapeutic medium or fetal calf serum (FCS)-containing medium. efficacy of peripheral blood-derived progenitor cells in Recently, we found that a strong antibody response type 2 DM patients and evaluated the use of cell-free was generated in human subjects after the second conditioned medium obtained from human embryonic infusion of therapeutic cells cultured in FCS- stem cell-derived endothelial-like cells (ESC-ECs) to containing medium. This response was specific for reverse their functional impairment. METHODS: The bovine apolipoprotein B-100 (apoB-100), which is the angiogenic potential of late outgrowth endothelial cells major protein component of low-density lipoproteins (OECs) and cytokine profile of the conditional (LDL) and which targets its binding to abundant low- medium of proangiogenic cells (PACs) derived from density lipoprotein receptors on the cell surface, from peripheral blood-mononuclear cells of healthy control which it is internalized. Here, we have shown that and DM patients and ESC-ECs was compared by in ESCs cultured on MEFs in SR medium acquired vitro tube formation assay and a multiplex bead-based bovine apoB-100 from MEFs and from the SR immunoassay kit, respectively. The in vivo angiogenic medium as well. Our findings also suggest that bovine potential of ESC-ECs derived conditioned medium in LDL are used as critical nutrients for ESC propagation. rescuing the functional impairment of PB-PACs in DM patients was investigated using a hindlimb Ho, H. Y. and M. Li (2006). "Potential ischemia model. RESULTS: Human ESC-ECs had application of embryonic stem cells in Parkinson's similar functional and phenotypic characteristics as disease: drug screening and cell therapy." Regen Med OECs in healthy controls. Cytokine profiling showed 1(2): 175-182. that vascular endothelial growth factor, stromal cell- Embryonic stem (ES) cells are genetically normal, derived factor 1 and placental growth factor were continuous cell lines that can give rise to a variety of down-regulated in PACs from DM patients. Tube somatic cells in culture. These include the midbrain formation assay that revealed functional impairment of dopaminergic neurons, a major cell type lost in OECs from DM patients could be rescued by ESC- Parkinson's disease. With the promising outcome of ECs conditioned medium. Administration of ESC-ECs mesencephalic fetal transplantation in some conditioned medium restored the therapeutic efficacy Parkinson's disease patients, the establishment of of PB-PACs from DM patients in a mouse model of human ES cells has sparked much attention in both the hindlimb ischemia. CONCLUSIONS: Our results scientific and general community regarding their showed that peripheral blood-derived progenitor cells potential as an alternative to aborted fetal tissue for from DM patients have impaired function because of cell replacement therapies. There is also great interest defective secretion of angiogenic cytokines, which in developing the ES cell system as a platform for could be restored by supplementation of ESC-ECs pharmaceutical and toxicological screening. Progress conditioned medium. has been made in developing protocols for dopaminergic neuronal specification in ES cell Husseini, L., et al. (2008). "Functional analysis development. Research to define the criteria for the of embryonic stem cell-derived glial cells after 'right' category of therapeutic dopaminergic cells is integration into hippocampal slice cultures." Stem underway. However, the promise of human ES cells Cells Dev 17(6): 1141-1152. rests largely on our ability to expand stem cells Embryonic stem (ES) cell-derived neural without genetic and epigenetic compromise, and to progenitor cells (ESNPs) generated in vitro are direct stem cell differentiation with absolute multipotent progenitors which can differentiate into phenotypic fidelity. The delivery of these goals will oligodendrocytes, astrocytes, and neurons. Given the require a much better understanding of the control of exciting prospects for ES cell-based treatments of ES cell self-renewal, proliferation and the commitment neurological disorders, several studies investigated the of differentiation. migration, integration, and differentiation of grafted ESNPs into neurons and glial cells. However, little is Ho, J. C., et al. (2012). "Reversal of endothelial known about the functional properties of transplanted progenitor cell dysfunction in patients with type 2 ESNPs on the single cell level. In this study, we diabetes using a conditioned medium of human combined electrophysiology, single cell reverse embryonic stem cell-derived endothelial cells." transcription polymerase chain reaction (RT-PCR) and Diabetes Metab Res Rev 28(5): 462-473. immunochemistry to determine the developmental

124 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ time course of molecular and functional properties of derived spinal GABAergic neurons dramatically ES cell-derived glial precursors (ESGPs) after attenuated the chronic neuropathic pain following SCI, deposition onto hippocampal slice cultures. Based on suggesting that the spinal GABAergic mESC-NPCs functional criteria, donor cells possessed three cultured with low doses of SHH and RA could be different phenotypes. During an observation period of alternative cell sources for treatment of SCI 3 weeks after engraftment, the proportion of donor neuropathic pain by stem cell-based therapies. cells with a passive current pattern (type 3) continuously increased. The majority of these cells Hwang, N. S., et al. (2008). "In vivo commitment expressed astroglial markers. Type 3 host cells and functional tissue regeneration using human underwent similar developmental changes. In contrast, embryonic stem cell-derived mesenchymal cells." Proc donor and host cells expressing time- and voltage- Natl Acad Sci U S A 105(52): 20641-20646. dependent currents (types 1, 2) displayed different Development of clinically relevant regenerative developmental profiles. Importantly, type 2 donor and medicine therapies using human embryonic stem cells host cells also differed in the expression of inwardly (hESCs) requires production of a simple and readily rectifying K (+) channels. This suggests that despite expandable cell population that can be directed to form several similarities in overall current phenotypes and functional 3D tissue in an in vivo environment. We timing of maturation, many donor cells integrated into describe an efficient derivation method and host tissue but did not acquire the full set of ion characterization of mesenchymal stem cells (MSCs) channels present in their native counterparts. These from hESCs (hESCd-MSCs) that have multilineage findings emphasize the need to carefully characterize differentiation potential and are capable of producing ES cell-derived progeny aimed for neural repair and fat, cartilage, and bone in vitro. Furthermore, we cell-mediated gene transfer strategies. highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment Hwang, I., et al. (2016). "Intrathecal comprising chondrocyte-secreted morphogenetic Transplantation of Embryonic Stem Cell-Derived factors and hydrogels. Normal cartilage architecture Spinal GABAergic Neural Precursor Cells Attenuates was established in rat osteochondral defects after Neuropathic Pain in a Spinal Cord Injury Rat Model." treatment with chondrogenically-committed hESCd- Cell Transplant 25(3): 593-607. MSCs. In view of the limited available cell sources for Neuropathic pain following spinal cord injury tissue engineering applications, these embryonic- (SCI) is a devastating disease characterized by derived cells show significant potential in spontaneous pain such as hyperalgesia and allodynia. musculoskeletal tissue regeneration applications. In this study, we investigated the therapeutic potential of ESC-derived spinal GABAergic neurons to treat Hwang, N. S., et al. (2006). "Chondrogenic neuropathic pain in a SCI rat model. Mouse embryonic differentiation of human embryonic stem cell-derived stem cell-derived neural precursor cells (mESC-NPCs) cells in arginine-glycine-aspartate-modified were cultured in media supplemented with sonic hydrogels." Tissue Eng 12(9): 2695-2706. hedgehog (SHH) and retinoic acid (RA) and efficiently Human embryonic stem cells (hESCs) have the differentiated into GABAergic neurons. Interestingly, potential to self-renew and generate multiple cell types, low doses of SHH and RA induced MGE-like producing critical building blocks for tissue progenitors, which expressed low levels of DARPP32 engineering and regenerative medicine applications. and Nkx2.1 and high levels of Irx3 and Pax6. These Here, we describe the efficient derivation and cells subsequently generated the majority of the chondrogenic differentiation of mesenchymal-like DARPP32(-) GABAergic neurons after in vitro cells from hESCs. These cells exhibit mesenchymal differentiation. The spinal mESC-NPCs were stem cell (MSC) surface markers, including CD29, intrathecally transplanted into the lesion area of the CD44, CD105, and platelet-derived growth factor spinal cord around T10-T11 at 21 days after SCI. The receptor-alpha. Under appropriate growth conditions, engrafted spinal GABAergic neurons remarkably the hESC-derived cells proliferated without increased both the paw withdrawal threshold (PWT) phenotypic changes and maintained MSC surface below the level of the lesion and the vocalization markers. The chondrogenic capacity of the cells was threshold (VT) to the level of the lesion (T12, T11, studied in pellet culture and after encapsulation in poly and T10 vertebrae), which indicates attenuation of (ethylene glycol)-diacrylate (PEGDA) hydrogels with chronic neuropathic pain by the spinal GABAergic exogenous extracellular proteins or arginineglycine- neurons. The transplanted cells were positive for aspartate (RGD)-modified PEGDA hydrogels. The GABA antibody staining in the injured region, and hESC-derived cells exhibited growth factor- dependent cells migrated to the injured spinal site and survived matrix production in pellet culture but did not produce for more than 7 weeks in L4-L5. The mESC-NPC- tissue characteristic of cartilage morphology. In

125 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

PEGDA hydrogels containing exogenous hyaluronic since October 2003 and is at passage level 56. Line acid or type I collagen, no significant cell growth or HS306 has been cultured since February 2004, now at matrix production was observed. In contrast, when passage 41. The lines express markers of pluripotent these cells were encapsulated in RGDmodified poly hESCs (Oct-4, SSEA-4, TRA-1-60, TRA-1-81, (ethylene glycol)hydrogels, neocartilage with GCTM-2, and alkaline phosphatase). The pluripotency basophilic extracellular matrix deposition was has been shown in embryoid bodies in vitro, and the observed within 3 weeks of culture, producing pluripotency of line 293 has also been shown in vivo cartilage-specific gene up-regulation and extracellular by teratoma formation in severe combined matrix production. Our results indicate that precursor immunodeficiency/beige mice. The karyotype of cells characteristic of a MSC population can be HS293 is 46,XY, and that of HS306 is 46,XX. Ten cultured from differentiating hESCs through embryoid more early lines have been derived under similar bodies, thus holding great promise for a potentially conditions since September 2004. We conclude that unlimited source of cells for cartilage tissue hESC lines can be successfully derived using SR engineering. medium and postnatal human fibroblasts as feeder cells. This is a step toward xeno-free conditions and Hyka-Nouspikel, N., et al. (2012). "Deficient facilitates the use of these cells in transplantation. DNA damage response and cell cycle checkpoints lead to accumulation of point mutations in human Ishii, S., et al. (2010). "Stromal cell-secreted embryonic stem cells." Stem Cells 30(9): 1901-1910. factors promote the survival of embryonic stem cell- Human embryonic stem cells (hESCs) tend to derived early neural stem/progenitor cells via the lose genomic integrity during long periods of culture activation of MAPK and PI3K-Akt pathways." J in vitro and to acquire a cancer-like phenotype. In this Neurosci Res 88(4): 722-734. study, we aim at understanding the contribution of Neural stem/progenitor cells (NS/PCs) have been point mutations to the adaptation process and at studied extensively with the hope of using them providing a mechanistic explanation for their clinically to repair the damaged central nervous system. accumulation. We observed that, due to the absence of However, little is known about the signals that regulate p21/Waf1/Cip1, cultured hESCs lack proper cell cycle the proliferation, survival, and differentiation of checkpoints and are vulnerable to the kind of DNA NS/PCs in early development. To clarify the damage usually repaired by the highly versatile underlying mechanisms, we took advantage of an in nucleotide excision repair (NER) pathway. In response vitro ES cell differentiation system from which we can to UV-induced DNA damage, the majority of hESCs obtain neurospheres containing NS/PCs with succumb to apoptosis; however, a subpopulation characteristics of the early caudal neural tube, by continues to proliferate, carrying damaged DNA and treating embryoid bodies (EBs) with a low accumulating point mutations with a typical UV- concentration of retinoic acid (RA). We found that induced signature. The UV-resistant cells retain their conditioned medium from the PA6 stromal cell line proliferative capacity and potential for pluripotent (PA6CM) increased the efficiency of neurosphere differentiation and are markedly less apoptotic to formation by suppressing apoptosis and promoting the subsequent UV exposure. These findings demonstrate survival of the NS/PCs. PA6CM also induced the that, due to deficient DNA damage response, the phosphorylation of Erk1/2 and Akt1 in cells derived modest NER activity in hESCs is insufficient to from the EBs. Furthermore, inhibitors of the MAPK prevent increased mutagenesis. This provides for the and PI3K-Akt signaling pathways, U0126 and appearance of genetically aberrant hESCs, paving the LY294002, attenuated the effects of PA6CM, way for further major genetic changes. significantly increasing the number of apoptotic cells and decreasing the number of viable cells among the Inzunza, J., et al. (2005). "Derivation of human ES cell-derived NS/PCs. Thus, PA6CM appears to embryonic stem cell lines in serum replacement contain soluble factors that promote the survival of ES medium using postnatal human fibroblasts as feeder cell-derived early NS/PCs through the activation of the cells." Stem Cells 23(4): 544-549. MAPK and PI3K-Akt pathways. Derivation and culture of human embryonic stem cells (hESCs) without animal-derived material would Ishii, T., et al. (2010). "In vitro hepatic be optimal for cell transplantation. We derived two maturation of human embryonic stem cells by using a new hES (HS293 and HS306) and 10 early cell lines mesenchymal cell line derived from murine fetal using serum replacement (SR) medium instead of livers." Cell Tissue Res 339(3): 505-512. conventional fetal calf serum and human foreskin Hepatocytes derived from human embryonic fibroblasts as feeder cells. Line HS293 has been in stem cells (hESCs) are an attractive cell source for continuous culture, with a passage time of 5-8 days, regenerative medicine. We previously reported the

126 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ differentiation of hESCs into alpha-fetoprotein (AFP)- transplantation. In conclusion, this study demonstrates, producing endodermal cells by using extracellular for the first time, that ESC-derived endodermal cells matrix and growth factors. We also reported the improve the survival rates after transplantation into establishment of the MLSgt20 cell line, which was mice with induced hepatocellular injury. Disclosure of derived from mesenchymal cells residing in murine potential conflicts of interest is found at the end of this fetal livers and accelerated the hepatic maturation of article. both murine hepatic progenitor cells and murine ESCs. In this study, hESC-derived AFP-producing cells were Ishiwata, I., et al. (2003). "Organogenesis of isolated by using a flow cytometer and co-cultured heart-vascular system derived from mouse 2 cell stage with MLSgt20 cells. The co-cultured hESC-derived embryos and from early embryonic stem cells in AFP-producing cells had the immunocytological vitro." Hum Cell 16(1): 15-22. characteristics of hepatocytes, expressed mature Regenerative medical treatment with embryonic hepatocyte markers (as indicated by reverse stem cells (an ES cell) is a goal for organ transcription and the polymerase chain reaction), and transplantation. Structures that are tubular in nature displayed higher hepatocyte functions including (i.e. blood capillaries) were induced from early ammonia removal, cytochrome P450 3A4/7 activity, embryonic stem (EES) cells in vitro using and the ability to produce and store glycogen. embryotrophic factor (ETFs). In addition, cardiac However, the MLSgt20 cells did not directly cause muscle cells could be identified as well. However, undifferentiated hESCs to mature into hepatocyte-like differentiation of EES cells into a complete cells. The co-culture method was thus successfully cardiovascular system was difficult because 3 germ shown to induce the differentiation of hESC-derived layer primordial organs are directed embryologically endodermal cells into functional hepatocyte-like cells. in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after Ishii, T., et al. (2007). "Transplantation of the formation of an embryoid body and were embryonic stem cell-derived endodermal cells into successful in cloning cell clusters that beat, thus mice with induced lethal liver damage." Stem Cells deriving only cardiovascular organs. The application 25(12): 3252-3260. of this to the treatment of various cardiovascular ESCs are a potential cell source for cell therapy. diseases is promising. However, there is no evidence that cell transplantation using ESC-derived hepatocytes is therapeutically Islam, M. S., et al. (2010). "Use of human effective. The main objective of this study was to embryonic stem cells to understand hematopoiesis and assess the therapeutic efficacy of the transplantation of hematopoietic stem cell niche." Curr Stem Cell Res ESC-derived endodermal cells into a liver injury Ther 5(3): 245-250. model. The beta-galactosidase-labeled mouse ESCs Intensive research of hematopoiesis using human were differentiated into alpha-fetoprotein (AFP)- embryonic stem cells (hESC) as a unique starting cell producing endodermal cells. AFP-producing cells or population has enabled differentiation and isolation of ESCs were transplanted into transgenic mice that diverse hematopoietic cell lineages. However, there expressed diphtheria toxin (DT) receptors under the has been only limited success in derivation of control of an albumin enhancer/promoter. Selective hematopoietic stem cells (HSCs) capable of long-term, damage was induced in the recipient hepatocytes by multi-lineage engraftment when transplanted into the administration of DT. Although the transplanted xenogeneic models. Better understanding of the HSC AFP-producing cells had repopulated only 3.4% of the developmental niche, the home for hematopoietic stem total liver mass 7 days after cell transplantation, they and progenitor cells, will aid to advance strategies to replaced 32.8% of the liver by day 35. However, these derive and assay putative HSCs from hESCs. This engrafted cells decreased (18.3% at day 40 and 7.9% review discusses recent status of hematopoietic at day 50) after the cessation of DT administration, development from the hESCs and highlights the and few donor cells were observed by days 60-90. The possibility of developing HSC niche using hESC- survival rate of the AFP-producing cell-transplanted derived niche components. group (66.7%) was significantly higher in comparison with that of the sham-operated group (17.6%). No Israely, E., et al. (2014). "Akt suppression of tumors were detected by day 50 in the AFP-producing TGFbeta signaling contributes to the maintenance of cell-transplanted group; however, splenic teratomas vascular identity in embryonic stem cell-derived did form 60 days or more after transplantation. ESC endothelial cells." Stem Cells 32(1): 177-190. transplantation had no effect on survival rates; The ability to generate and maintain stable in furthermore, there was a high frequency of tumors in vitro cultures of mouse endothelial cells (ECs) has the ESC-transplanted group 35 days after great potential for genetic dissection of the numerous

127 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ pathologies involving vascular dysfunction as well as Cell fate decisions of pluripotent embryonic stem therapeutic applications. However, previous efforts at (ES) cells are dictated by activation and repression of achieving sustained cultures of primary stable murine lineage-specific genes. Numerous signaling and vascular cells have fallen short, and the cellular transcriptional networks progressively narrow and requirements for EC maintenance in vitro remain specify the potential of ES cells. Whether specific undefined. In this study, we have generated vascular microRNAs help refine and limit gene expression and, ECs from mouse embryonic stem (ES) cells and show thereby, could be used to manipulate ES cell that active Akt is essential to their survival and differentiation has largely been unexplored. Here, we propagation as homogeneous monolayers in vitro. show that two serum response factor (SRF)-dependent These cells harbor the phenotypical, biochemical, and muscle-specific microRNAs, miR-1 and miR-133, functional characteristics of ECs and expand promote mesoderm formation from ES cells but have throughout long-term cultures, while maintaining their opposing functions during further differentiation into angiogenic capacity. Moreover, Akt-transduced cardiac muscle progenitors. Furthermore, miR-1 and embryonic ECs form functional perfused vessels in miR-133 were potent repressors of nonmuscle gene vivo that anastomose with host blood vessels. We expression and cell fate during mouse and human ES provide evidence for a novel function of Akt in cell differentiation. miR-1's effects were in part stabilizing EC identity, whereby the activated form of mediated by translational repression of the Notch the protein protects mouse ES cell-derived ECs from ligand Delta-like 1 (Dll-1). Our findings indicate that TGFbeta-mediated transdifferentiation by muscle-specific miRNAs reinforce the silencing of downregulating SMAD3. These findings identify a nonmuscle genes during cell lineage commitment and role for Akt in regulating the developmental potential suggest that miRNAs may have general utility in of ES cell-derived ECs and demonstrate that active regulating cell-fate decisions from pluripotent ES cells. Akt maintains endothelial identity in embryonic ECs by interfering with active TGFbeta-mediated processes Jacobs, V. R., et al. (2005). "[The STEMMAT- that would ordinarily usher these cells to alternate project as part of health initiative BayernAktiv: adult fates. stem cells from umbilical cord and cord blood as alternative to embryonic stem cell research]." Iuchi, S., et al. (2006). "An immortalized drug- Zentralbl Gynakol 127(6): 368-372. resistant cell line established from 12-13-day mouse Adult stem cells from umbilical cord and cord embryos for the propagation of human embryonic stem blood are an interesting alternative to embryonic stem cells." Differentiation 74(4): 160-166. cells because such research is commonly recognized as Human embryonic stem (ES) cells are usually co- ethical undisputed and many aspects are still cultivated with supporting cells consisting of short- insufficiently investigated. In the context of the term cultures of fibroblasts (not an immortalized line) STEMMAT research project (STEM = Stem Cell and in a medium lacking serum. This method has promoted MAT = Material) different aspects of stem cells from important progress in the field, but suffers from certain umbilical cord and cord blood are investigated, to disadvantages. By serial cultivation for 27 consecutive improve basic science understanding and potentially transfers and about 63 cell generations, we have leading someday to a clinical application. evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports Jafary, H., et al. (2008). "Differential effect of the multiplication of H9 cells better than the 3T3 line. activin on mouse embryonic stem cell differentiation It supports the growth of H9 cells as well as do in insulin-secreting cells under nestin-positive available short-term fibroblast cultures, but maintains selection and spontaneous differentiation protocols." more effectively the stem cell character of the H9 cells, Cell Biol Int 32(2): 278-286. judging by their better retention of Oct4. We have Parallel to the importance of the development of made MMM cells resistant to blasticidin and zeocin, cell therapies to treat diabetes is the production of the most efficient antibiotics for selection of stable sufficient numbers of pancreatic endocrine cells that transformants. In the presence of zeocin, the resistant function like primary islets. To increase the efficiency MMM were able to support multiplication and of endocrine pancreatic-like cell differentiation from selection of ES cells transfected with an exogenous mouse embryonic stem cells (ESCs), we applied gene encoding zeocin resistance. activin-B to nestin-positive selection (protocol 1) and spontaneous differentiation (protocol 2) in different Ivey, K. N., et al. (2008). "MicroRNA regulation groups including: [A] activin-B, or [B] basic fibroblast of cell lineages in mouse and human embryonic stem growth factor (bFGF), and/or [C] activin-B+bFGF. cells." Cell Stem Cell 2(3): 219-229. The differentiated cells expressed most pancreatic- related genes. The number of insulin- and C peptide-

128 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ positive cells, as well as dithizone-positive clusters in leveraged for therapeutic applications if production of group A of protocol 1 was higher than in the other secretary molecules were optimized. Pharmacological groups. Significant insulin concentrations in protocol 1 preconditioning using small molecules can increase were produced when glucose was added to the survival of MSCs after transplantation. The aim of this medium, in comparison with protocol 2. Moreover, study was to investigate the effect of secretome of insulin release was increased significantly in group A human embryonic-derived mesenchymal stem cells of protocol 1 even with lower glucose. In conclusion, (hESC-MSCs) preconditioned with MG- Addition of activin-B in a nestin-positive selection 132,Trimetazidine (TMZ) and Diazoxide (DZ) on protocol increased the insulin-secreting cells in immunomodulatory efficiency of these cells in Lipo comparison with the same protocol with bFGF and/or polysaccharide (LPS) challenged mice models. Mice spontaneous differentiation in presence of bFGF were injected intraperitoneally with LPS and groups of and/or activin-B alone. However, improvements of the animals were intraperitoneally given 1 ml 30x current method are required to generate a sufficient secretome 6 h after LPS injection. Serum levels of source of true beta-cells for the treatment of diabetes biochemical parameters were then measured by an mellitus. auto analyser and serum inflammatory cytokine levels were analysed using commercially available RayBio Jagatha, B., et al. (2009). "In vitro differentiation Mouse Inflammation Antibody Array. Ultimately, of retinal ganglion-like cells from embryonic stem cell histopathology and survival studies were conducted. derived neural progenitors." Biochem Biophys Res The results showed that TMZ and DZ-conditioned Commun 380(2): 230-235. medium significantly increasing the survival and ES cells have been reported to serve as an improvement of histopathological score. We found excellent source for obtaining various specialized cell that MG-132-conditioned medium failed to show types and could be used in cell replacement therapy. significant outcomes. This study demonstrated that Here, we demonstrate the potential of ES cells to human MSC secretome has the potential to control differentiate along retinal ganglion cell (RGC) lineage. inflammation. FGF2-induced ES cell derived neural progenitors (ES- NPs) were able to generate RGC-like cells in vitro Jain, A. K., et al. (2012). "p53 regulates cell upon differentiation. These cells expressed RGC cycle and microRNAs to promote differentiation of regulators and markers such as, Ath5, Brn3b, RPF-1, human embryonic stem cells." PLoS Biol 10(2): Thy-1 and Islet-1, confirming their potential to e1001268. differentiate into RGCs. The generation of RGCs from Multiple studies show that tumor suppressor p53 ES-NPs was enhanced with the exposure of FGF2 and is a barrier to dedifferentiation; whether this is strictly Sonic hedgehog (Shh), although Shh treatment alone due to repression of proliferation remains a subject of did not affect RGC differentiation significantly. ES- debate. Here, we show that p53 plays an active role in NPs, after exposure to FGF2, were capable of promoting differentiation of human embryonic stem integrating and differentiating into RGCs in vivo upon cells (hESCs) and opposing self-renewal by regulation transplantation. Thus, our study suggests that ES cells of specific target genes and microRNAs. In contrast to can serve an excellent renewable source for generating mouse embryonic stem cells, p53 in hESCs is RGCs that can be used to treat neurodegenerative maintained at low levels in the nucleus, albeit in a diseases like glaucoma. deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to Jahandideh, S., et al. (2018). "Anti-inflammatory dissociation from E3-ubiquitin ligases HDM2 and effects of human embryonic stem cell-derived TRIM24. Stabilized p53 binds CDKN1A to establish a mesenchymal stem cells secretome preconditioned G (1) phase of cell cycle without activation of cell with diazoxide, trimetazidine and MG-132 on LPS- death pathways. In parallel, p53 activates expression induced systemic inflammation mouse model." Artif of miR-34a and miR-145, which in turn repress stem Cells Nanomed Biotechnol: 1-10. cell factors OCT4, KLF4, LIN28A, and SOX2 and Systemic inflammatory response syndrome is a prevent backsliding to pluripotency. Induction of p53 complex pathophysiologic and immunologic response levels is a key step: RNA-interference-mediated to an insult. Sepsis is a life-threatening condition knockdown of p53 delays differentiation, whereas happening when the body's response to infection depletion of negative regulators of p53 or ectopic causes injury to its own tissues and organs. Stem cell expression of p53 yields spontaneous differentiation of therapy is a new approach to modulate immune hESCs, independently of retinoic acid. Ectopic responses. Mesenchymal stem cells (MSCs) establish a expression of p53R175H, a mutated form of p53 that regenerative niche by secreting secretome and does not bind DNA or regulate transcription, failed to modulating immune responses. MSC secretome can be induce differentiation. These studies underscore the

129 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ importance of a p53-regulated network in determining cognate corepressor. This raises the important question the human stem cell state. of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout Jakobs, P. M., et al. (1999). "Embryonic stem cells, we demonstrate for the first time (to our cells can be used to construct hybrid cell lines knowledge) that mutations that abolish IP4 binding containing a single, selectable murine chromosome." reduce the activity of HDAC1/2 in vivo. Our data Mamm Genome 10(4): 381-384. indicate that HDAC1/2 have essential and pleiotropic Microcell-mediated chromosome transfer is a roles in cellular proliferation and regulate stem cell useful technique for the study of gene function, gene self-renewal by maintaining expression of key regulation, gene mapping, and functional cloning in pluripotent transcription factors. mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have James, D., et al. (2010). "Expansion and been developed. These donor cell lines contain a single maintenance of human embryonic stem cell-derived human chromosome marked with a dominant endothelial cells by TGFbeta inhibition is Id1 selectable gene in a rodent cell background. However, dependent." Nat Biotechnol 28(2): 161-166. a similar panel does not exist for murine chromosomes. Previous efforts to differentiate human To produce mouse monochromosomal donor hybrids, embryonic stem cells (hESCs) into endothelial cells we have utilized embryonic stem (ES) cells with have not achieved sustained expansion and stability of targeted gene disruptions of known chromosomal vascular cells. To define vasculogenic developmental location as starting material. ES cells with mutations in pathways and enhance differentiation, we used an aprt, fyn, and myc were utilized to generate endothelial cell-specific VE-cadherin promoter driving monochromosomal hybrids with neomycin green fluorescent protein (GFP) (hVPr-GFP) to screen phosphotransferase-marked murine Chr 8, 10, or 15 for factors that promote vascular commitment. In respectively in a hamster or rat background. This same phase 1 of our method, inhibition of transforming methodology can be used to generate a complete panel growth factor (TGF)beta at day 7 of differentiation of marked mouse chromosomes for somatic cell increases hVPr-GFP (+) cells by tenfold. In phase 2, genetic experimentaion. TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting Jamaladdin, S., et al. (2014). "Histone in a net 36-fold expansion of endothelial cells in deacetylase (HDAC) 1 and 2 are essential for accurate homogenous monolayers, which exhibited a cell division and the pluripotency of embryonic stem transcriptional profile of Id1(high)VEGFR2(high)VE- cells." Proc Natl Acad Sci U S A 111(27): 9840-9845. cadherin (+) ephrinB2(+). Using an Id1-YFP hESC Histone deacetylases 1 and 2 (HDAC1/2) form reporter line, we showed that TGFbeta inhibition the core catalytic components of corepressor sustains Id1 expression in hESC-derived endothelial complexes that modulate gene expression. In most cell cells and that Id1 is required for increased proliferation types, deletion of both Hdac1 and Hdac2 is required to and preservation of endothelial cell commitment. Our generate a discernible phenotype, suggesting their approach provides a serum-free method for activity is largely redundant. We have therefore differentiation and long-term maintenance of hESC- generated an ES cell line in which Hdac1 and Hdac2 derived endothelial cells at a scale relevant to clinical can be inactivated simultaneously. Loss of HDAC1/2 application. resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon Jaramillo, M. and I. Banerjee (2012). cell cycle progression, because differentiated, "Endothelial cell co-culture mediates maturation of nonproliferating cells retain their viability. human embryonic stem cell to pancreatic insulin Furthermore, we observe increased mitotic defects, producing cells in a directed differentiation approach." chromatin bridges, and micronuclei, suggesting J Vis Exp (61). HDAC1/2 are necessary for accurate chromosome Embryonic stem cells (ESC) have two main segregation. Consistent with a critical role in the characteristics: they can be indefinitely propagated in regulation of gene expression, microarray analysis of vitro in an undifferentiated state and they are Hdac1/2-deleted cells reveals 1,708 differentially pluripotent, thus having the potential to differentiate expressed genes. Significantly for the maintenance of into multiple lineages. Such properties make ESCs stem cell self-renewal, we detected a reduction in the extremely attractive for cell based therapy and expression of the pluripotent transcription factors, regenerative treatment applications. However for its Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is full potential to be realized the cells have to be regulated through binding of an inositol tetraphosphate differentiated into mature and functional phenotypes, molecule (IP4) sandwiched between the HDAC and its which is a daunting task. A promising approach in

130 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ inducing cellular differentiation is to closely mimic the sheet, which aggregates with endothelial cells (ECs) path of organogenesis in the in vitro setting. Pancreatic during the final stages of maturation. Several findings development is known to occur in specific stages, suggest that the interactions with EC play a role in starting with endoderm, which can develop into pancreatic development. In this study, we recapitulated several organs, including liver and pancreas. this phenomenon in an in vitro environment by Endoderm induction can be achieved by modulation of maturing the human ESC (hESC)-derived PP cells in the nodal pathway through addition of Activin A in close contact with ECs. We find that co-culture with combination with several growth factors. Definitive different ECs (but not fibroblast) alone results in endoderm cells then undergo pancreatic commitment pancreatic islet-specific differentiation of hESC- by inhibition of sonic hedgehog inhibition, which can derived PP cells even in the absence of additional be achieved in vitro by addition of cyclopamine. chemical induction. The differentiated cells responded Pancreatic maturation is mediated by several parallel to exogenous glucose levels by enhanced C-peptide events including inhibition of notch signaling; synthesis. The co-culture system aligned well with aggregation of pancreatic progenitors into 3- endocrine development as determined by dimentional clusters; induction of vascularization; to comprehensive analysis of involved signaling name a few. By far the most successful in vitro pathways. By recapitulating cell-cell interaction maturation of ESC derived pancreatic progenitor cells aspects of the developmental niche we achieved a have been achieved through inhibition of notch differentiation model that aligns closely with islet signaling by DAPT supplementation. Although organogenesis. successful, this results in low yield of the mature phenotype with reduced functionality. A less studied Javaherian, A. and A. Kriegstein (2009). "A stem area is the effect of endothelial cell signaling in cell niche for intermediate progenitor cells of the pancreatic maturation, which is increasingly being embryonic cortex." Cereb Cortex 19 Suppl 1: i70-77. appreciated as an important contributing factor in in- The excitatory neurons of the mammalian vivo pancreatic islet maturation. The current study cerebral cortex arise from asymmetric divisions of explores such effect of endothelial cell signaling in radial glial cells in the ventricular zone and symmetric maturation of human ESC derived pancreatic division of intermediate progenitor cells (IPCs) in the progenitor cells into insulin producing islet-like cells. subventricular zone (SVZ) of the embryonic cortex. We report a multi-stage directed differentiation Little is known about the microenvironment in which protocol where the human ESCs are first induced IPCs divide or whether a stem cell niche exists in the towards endoderm by Activin A along with inhibition SVZ of the embryonic cortex. Recent evidence of PI3K pathway. Pancreatic specification of suggests that vasculature may provide a niche for adult endoderm cells is achieved by inhibition of sonic stem cells but its role in development is less clear. We hedgehog signaling by Cyclopamine along with have investigated the vasculature in the embryonic retinoid induction by addition of Retinoic Acid. The cortex during neurogenesis and find that IPCs are final stage of maturation is induced by endothelial cell spatially and temporally associated with blood vessels signaling achieved by a co-culture configuration. during cortical development. Intermediate progenitors While several endothelial cells have been tested in the mimic the pattern of capillaries suggesting patterns of co-culture, herein we present our data with rat heart angiogenesis and neurogenesis are coordinated during microvascular endothelial Cells (RHMVEC), primarily development. More importantly, we find that IPCs for the ease of analysis. divide near blood vessel branch points suggesting that cerebral vasculature establishes a stem cell niche for Jaramillo, M., et al. (2015). "Endothelial cells intermediate progenitors in the SVZ. These data mediate islet-specific maturation of human embryonic provide novel evidence for the presence of a stem cell-derived pancreatic progenitor cells." Tissue neurogenic niche for intermediate progenitors in the Eng Part A 21(1-2): 14-25. embryonic SVZ and suggest blood vessels are It is well recognized that in vitro differentiation important for proper patterning of neurogenesis. of embryonic stem cells (ESC) can be best achieved by closely recapitulating the in vivo developmental niche. Ji, Y., et al. (2017). "Microvesicles released from Thus, implementation of directed differentiation human embryonic stem cell derived-mesenchymal strategies has yielded encouraging results in the area stem cells inhibit proliferation of leukemia cells." of pancreatic islet differentiation. These strategies Oncol Rep 38(2): 1013-1020. have concentrated on direct addition of chemical Human embryonic stem cell derived- signals, however, other aspect of the developmental mesenchymal stem cells (hESCMSCs) are able to niche are yet to be explored. During development, inhibit proliferation of leukemia cells. Microvesicles pancreatic progenitor (PP) cells grow as an epithelial released from human embryonic stem cell derived-

131 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ mesenchymal stem cells (hESCMSCMVs) might play CONCLUSIONS/SIGNIFICANCE: Our data provide an important part in antitumor activity. Microvesicles evidence for the dominance of the tissue-specific were isolated by ultracentrifugation and identified mammary stem cell niche and its role in directing mES under a scanning electron microscopy and cells, undergoing hematopoietic differentiation, to transmission electron microscope separately. After 48- reprogram into mammary epithelial cells and to h cocultured with hESCMSCs and hESCMSCMVs, promote mammary epithelial morphogenesis. These the number of K562 and HL60 was counted and tumor studies should also provide insights into regeneration cell viability was measured by CCK8 assay. The of damaged mammary gland and the role of the expression of proteins Bcl-2 and Bax were estimated mammary microenvironment in reprogramming cell by western blotting. Transmission electron microscope fate. and western blot analysis were adopted to evaluate the autophagy level. Results showed that both hESCMSCs Kaitsuka, T., et al. (2014). "Generation of and hESCMSCMVs inhibited proliferation of functional insulin-producing cells from mouse leukemia cells in a concentration-dependent manner. embryonic stem cells through 804G cell-derived hESCMSCMVs reduced the ratio of Bcl/Bax, extracellular matrix and protein transduction of enhanced the protein level of Beclin-1 and LC3-II transcription factors." Stem Cells Transl Med 3(1): conversion, thus upregulating autophagy and apoptosis. 114-127. In conclusion, microvesicles released from human Embryonic stem (ES) and induced pluripotent embryonic stem cell derived-mesenchymal stem cells stem (iPS) cells have potential applications to inhibited tumor growth and stimulated autophagy and regenerative medicine for diabetes; however, a useful excessive autophagy might induce apoptosis. and safe way to generate pancreatic beta cells has not been developed. In this study, we tried to establish an Jiang, S., et al. (2010). "Reconstitution of effective method of differentiation through the protein mammary epithelial morphogenesis by murine transduction of three transcription factors (Pdx1, embryonic stem cells undergoing hematopoietic stem NeuroD, and MafA) important to pancreatic beta cell cell differentiation." PLoS One 5(3): e9707. development. The method poses no risk of unexpected BACKGROUND: Mammary stem cells are genetic modifications in target cells. Transduction of maintained within specific microenvironments and the three proteins induced the differentiation of mouse recruited throughout lifetime to reconstitute de novo ES and mouse iPS cells into insulin-producing cells. the mammary gland. Mammary stem cells have been Furthermore, a laminin-5-rich extracellular matrix isolated through the identification of specific cell efficiently induced differentiation under feeder-free surface markers and in vivo transplantation into conditions. Cell differentiation was confirmed with the cleared mammary fat pads. Accumulating evidence expression of the insulin 1 gene in addition to marker showed that during the reformation of mammary stem genes in pancreatic beta cells, the differentiated cells cell niches by dispersed epithelial cells in the context secreted glucose-responsive C-peptide, and their of the intact epithelium-free mammary stroma, non- transplantation restored normoglycemia in diabetic mammary epithelial cells may be sequestered and mice. Moreover, Pdx1 protein transduction had reprogrammed to perform mammary epithelial cell facilitative effects on differentiation into pancreatic functions and to adopt mammary epithelial endocrine progenitors from human iPS cells. These characteristics during reconstruction of mammary results suggest the direct delivery of recombinant epithelium in regenerating mammary tissue in vivo. proteins and treatment with laminin-5-rich METHODOLOGY/PRINCIPAL FINDINGS: To extracellular matrix to be useful for the generation of examine whether other types of progenitor cells are insulin-producing cells. able to contribute to mammary branching morphogenesis, we examined the potential of murine Karamali, F., et al. (2018). "Hepatocyte growth embryonic stem (mES) cells, undergoing factor promotes the proliferation of human embryonic hematopoietic differentiation, to support mammary stem cell derived retinal pigment epithelial cells." J reconstitution in vivo. We observed that cells from day Cell Physiol. 14 embryoid bodies (EBs) under hematopoietic Research that pertains to the molecular differentiation condition, but not supernatants derived mechanisms involved in retinal pigment epithelial from these cells, when transplanted into denuded (RPE) development can significantly contribute to cell mammary fat pads, were able to contribute to both the therapy studies. The effects of periocular luminal and myoepithelial lineages in branching ductal mesenchymal cells on the expansion of RPE cells structures resembling the ductal-alveolar architecture remain elusive. We have examined the possible of the mammary tree. No teratomas were observed proliferative role of hepatocyte growth factor (HGF) as when these cells were transplanted in vivo. a mesenchymal cell secretory factor against human

132 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ embryonic stem cell derived RPE (hESC-RPE). We A hallmark of graft-versus-host-disease (GVHD), found that the conditioned medium of human a life-threatening complication after allogeneic mesenchymal stem cells from apical papilla and/or hematopoietic stem cell transplantation, is the exogenous HGF promoted proliferation of the hESC- cytopathic injury of host tissues mediated by persistent RPE cells as single cells and cell sheets, in addition to alloreactive effector T cells (T (E)). However, the rabbit RPE sheets in vitro. Blockage of HGF signaling mechanisms that regulate the persistence of by HGF receptor inhibitor, PHA-665752, inhibited alloreactive T (E) during GVHD remain largely proliferation of hESC-RPE cells. However, unknown. Using mouse GVHD models, we differentiation of hESCs and human-induced demonstrate that alloreactive CD8(+) T (E) rapidly pluripotent stem cells to a rostral fate and eye-field diminished in vivo when adoptively transferred into specification was unaffected by HGF. Our in vivo irradiated secondary congenic recipient mice. In analysis showed HGF expression in periocular contrast, although alloreactive CD8(+) T (E) mesenchymal cells after optic cup formation in underwent massive apoptosis upon chronic exposure chicken embryos. Administration of HGF receptor to alloantigens, they proliferated in vivo in secondary inhibitor at this developmental stage in chicken allogeneic recipients, persisted, and caused severe embryos led to reduced eye size and disorganization of GVHD. Thus, the continuous proliferation of the RPE sheet. These findings suggested that HGF alloreactive CD8(+) T (E), which is mediated by administration could be beneficial for obtaining higher alloantigenic stimuli rather than homeostatic factors, is numbers of hESC-RPE cells in human preclinical and critical to maintaining their persistence. Gene clinical trials. expression profile analysis revealed that although alloreactive CD8(+) T (E) increased the expression of Kasuda, S., et al. (2008). "Establishment of genes associated with cell death, they activated a embryonic stem cells secreting human factor VIII for group of stem cell genes normally expressed in cell-based treatment of hemophilia A." J Thromb embryonic and neural stem cells. Most of these stem Haemost 6(8): 1352-1359. cell genes are associated with cell cycle regulation, BACKGROUND: Hemophilia A is an X- DNA replication, chromatin modification, and chromosome-linked recessive bleeding disorder transcription. One of these genes, Ezh2, which resulting from an F8 gene abnormality. Although encodes a chromatin modifying enzyme, was various gene therapies have been attempted with the abundantly expressed in CD8(+) T (E). Silencing Ezh2 aim of eliminating the need for factor VIII significantly reduced the proliferation of alloantigen- replacement therapy, obstacles to their clinical activated CD8(+) T cells. Thus, these findings identify application remain. OBJECTIVES: We evaluated that a group of stem cell genes could play important whether embryonic stem (ES) cells with a tetracycline- roles in sustaining terminally differentiated inducible system could secrete human FVIII. alloreactive CD8(+) T (E) and may be therapeutic METHODS AND RESULTS: We found that targets for controlling GVHD. embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted Katsman, D., et al. (2012). "Embryonic stem cell- human FVIII after doxycycline induction. Moreover, derived microvesicles induce gene expression changes use of a B-domain variant F8 cDNA (226aa/N6) in Muller cells of the retina." PLoS One 7(11): e50417. dramatically enhanced FVIII secretion. Sorting based Cell-derived microvesicles (MVs), recognized as on green fluorescent protein (GFP)-brachyury (Bry) important components of cell-cell communication, and c-kit revealed that GFP-Bry (+)/c-kit (+) cells contain mRNAs, miRNAs, proteins and lipids and during EB differentiation with serum contain an transfer their bioactive contents from parent cells to endoderm progenitor population. When GFP-Bry cells of other origins. We have studied the effect that (+)/c-kit (+) cells were cultured under the liver cell- MVs released from embryonic stem cells (ESMVs) promoting conditions, these cells secreted FVIII more have on retinal progenitor Muller cells. Cultured efficiently than other populations tested. human Muller cells were exposed to mouse ESMVs CONCLUSION: Our findings suggest the potential for every 48 hours for a total of 9 treatments. future development of an effective ES cell-based Morphological changes were observed by light approach to treating hemophilia A. microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the Kato, K., et al. (2010). "Identification of stem uniform, spindle-like adherent cellular sheets of cell transcriptional programs normally expressed in untreated cells. ESMVs transferred to Muller cells embryonic and neural stem cells in alloreactive CD8+ embryonic stem cell (ESC) mRNAs involved in the T cells mediating graft-versus-host disease." Biol maintenance of pluripotency, including Oct4 and Sox2, Blood Marrow Transplant 16(6): 751-771. and the miRNAs of the 290 cluster, important

133 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ regulators of the ESC-specific cell cycle. Moreover, (infants and young children) and T3GCT (aged men), ESMV exposure induced up-regulation of the basal T2GCT arise from CIS/GCNIS that develops from levels of endogenous human Oct4 mRNA in Muller pre-CIS. Pre-CIS represents undifferentiated, growth- cells. mRNA and miRNA microarrays of ESMV- arrested gonocytes that persist in fetal testes due to treated vs. untreated Muller cells revealed the up- endocrine disruption. However, whether pre-CIS truly regulation of genes and miRNAs involved in the exist, do CIS develop into T2GCT, why no CIS in induction of pluripotency, cellular proliferation, early T1GCT/T3GCT, why germ cell tumors (GCT) also ocular genes and genes important for retinal protection occur along midline at extra-gonadal sites, why and remodeling, as well as the down-regulation of T1GCT show partial erasure and T2GCT show inhibitory and scar-related genes and miRNAs complete erasure of genomic imprints are open involved in differentiation and cell cycle arrest. To questions that are awaiting answers. We propose that further characterize the heterogeneous cell population rather than pre-CIS, pluripotent, very small of ESMV-treated Muller cells, their expression of embryonic-like stem cells (VSELs) get affected by retinal cell markers was compared to that in untreated exposure to endocrine disruption. Since VSELs are control cells by immunocytochemistry. Markers for developmentally equivalent to primordial germ cells amacrine, ganglion and rod photoreceptors were (PGCs), T2GCT cells show complete erasure of present in treated but not in control Muller cells. genomic imprints and CIS represents growth-arrested Together, our findings indicate that ESMs induce de- clonally expanding stem/progenitor cells. differentiation and pluripotency in their target Muller PGCs/VSELs migrate along the midline to various cells, which may turn on an early retinogenic program organs and this explains why GCT occur along the of differentiation. midline, T1GCT show partial erasure of imprints as they develop from migrating PGCs. T3GCT possibly Kattman, S. J., et al. (2007). "Specification of reflects effects of aging due to compromised multipotential cardiovascular progenitor cells during differentiation and expansion of pre-meiotic embryonic stem cell differentiation and embryonic spermatocytes. Absent spermatogenesis in pre-pubertal development." Trends Cardiovasc Med 17(7): 240-246. and aged testes explains absence of CIS in T1GCT and The fully formed heart is composed of diverse T3GCT. Endocrine disruptors possibly alter epigenetic cell lineages including myocytes, endothelial cells, state of VSELs and thus rather than maintaining vascular smooth muscle cells, and fibroblasts that normal tissue homeostasis, VSELs undergo increased derive from distinct subsets of mesoderm during proliferation and compromised differentiation embryonic development. Findings from lineage tracing resulting in reduced sperm count, infertility and TGCT. studies indicate that cardiomyocytes develop from This newly emerging understanding offers alternate cells that express fetal liver kinase-1, suggesting that premise to explain TGCT and warrants further the cardiac lineages may arise from a progenitor cell exploration. with vascular cardiomyocyte potential. Recent studies using the embryonic stem cell model have led to the Kauts, M. L., et al. (2018). "In Vitro identification of a fetal liver kinase-1(+) progenitor Differentiation of Gata2 and Ly6a Reporter cell that displays both vascular and cardiomyocyte Embryonic Stem Cells Corresponds to In Vivo Waves potential. A comparable progenitor was also isolated of Hematopoietic Cell Generation." Stem Cell Reports from the early mouse embryo. Identification and 10(1): 151-165. isolation of these cardiovascular progenitor cells In vivo hematopoietic generation occurs in waves establishes a new model of heart development that will of primitive and definitive cell emergence. provide insights into the mechanisms regulating Differentiation cultures of pluripotent embryonic stem cardiovascular lineage diversification. These cells (ESCs) offer an accessible source of progenitor cells may also represent a novel cell hematopoietic cells for blood-related research and population for models of congenital heart disease and therapeutic strategies. However, despite many cell replacement therapy. approaches, it remains a goal to robustly generate hematopoietic progenitor and stem cells (HP/SCs) in Kaushik, A. and D. Bhartiya (2018). "Pluripotent vitro from ESCs. This is partly due to the inability to Very Small Embryonic-Like Stem Cells in Adult efficiently promote, enrich, and/or molecularly direct Testes - An Alternate Premise to Explain Testicular hematopoietic emergence. Here, we use Gata2Venus Germ Cell Tumors." Stem Cell Rev. (G2V) and Ly6a (SCA1)GFP (LG) reporter ESCs, Developmental exposure to endocrine disruptors derived from well-characterized mouse models of has resulted in the increased incidence of infertility HP/SC emergence, to show that during in vitro and testicular germ cell tumors (T2GCT) in young differentiation they report emergent waves of primitive men residing in developed countries. Unlike T1GCT hematopoietic progenitor cells (HPCs), definitive

134 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

HPCs, and B-lymphoid cell potential. These results, METHODS: Undifferentiated mouse ESCs were facilitated by enrichment of single and double reporter transfected with cDNA by electroporation, cells with HPC properties, demonstrate that in vitro followed by selection with G418. We conducted ESC differentiation approximates the waves of limiting-dilution culture of pax6-transfected cells. We hematopoietic cell generation found in vivo, thus expanded the cloned pax6-transfected cells, which raising possibilities for enrichment of rare ESC- expressed nestin and musashi-1, for further derived HP/SCs. characterization in culture media containing fibronectin. The cells were characterized using RT- Kawazoe, S., et al. (2009). "Extrinsic factors PCR, immunostaining, electron microscopy, renal derived from mouse embryonal carcinoma cell lines subcapsular transplantation assay and Ca imaging. maintain pluripotency of mouse embryonic stem cells RESULTS: We obtained clonally expanding pax6- through a novel signal pathway." Dev Growth Differ transfected cells, all of which were positive for , 51(2): 81-93. sonic hedgehog (shh), math5, brn3, thy1 and Embryonic carcinoma (EC) cells, which are melanopsin, by using several ESCs. When malignant stem cells of teratocarcinoma, have transplanted into a mouse renal capsule, they numerous morphological and biochemical properties differentiated into neurons with elongated axons, in common with pluripotent stem cells such as expressing betaIII tubulin and neurofilament middle embryonic stem (ES) cells. However, three EC cell chain, and were free from teratoma development. lines (F9, P19 and PCC3) show different Electron-microscopic examination showed developmental potential and self-renewal capacity neurotubules and neurofilaments in the axon-like from those of ES cells. All three EC cell lines maintain processes of the cloned pax6-transfected cells. High self-renewal capacity in serum containing medium KCl stimulation increased free Ca influx on Ca2+ without Leukemia Inhibitory factor (LIF) or feeder imaging. CONCLUSIONS: ESCs were applicable for layer, and show limited differentiation capacity into the induction of retinal progenitor cells, including restricted lineage and cell types. To reveal the RGC-like cells, by transfection with the pax6 gene and underlying mechanism of these characteristics, we subsequent limiting-dilution culture. Cloned cell lines took the approach of characterizing extrinsic factors may be useful to analyze the requirements for retinal derived from EC cells on the self-renewal capacity and progenitor cell differentiation, and our study suggests pluripotency of mouse ES cells. Here we demonstrate the clinical application of this cell type. that EC cell lines F9 and P19 produce factor (s) maintaining the undifferentiated state of mouse ES Kayama, M., et al. (2007). "Recent advances in cells via an unidentified signal pathway, while P19 and corneal regeneration and possible application of PCC3 cells produce self-renewal factors of ES cells embryonic stem cell-derived corneal epithelial cells." other than LIF that were able to activate the STAT3 Clin Ophthalmol 1(4): 373-382. signal; however, inhibition of STAT3 activation with The depletion of limbal stem cells due to various Janus kinase inhibitor shows only partial impairment diseases leads to corneal opacification and visual loss. on the maintenance of the undifferentiated state of ES The unequivocal identification and isolation of limbal cells. Thus, these factors present in EC cells-derived stem cells may be a considerable advantage because conditioned medium may be responsible for the self- long-term, functional recovery of corneal epithelium is renewal capacity of EC and ES cells independently of linked to graft constructs that retain viable stem cell LIF signaling. populations. As specific markers of limbal stem cells, the ATP-binding cassette, sub-family G, member2 Kayama, M., et al. (2010). "Transfection with (ABCG2), a member of the multiple drug-resistance pax6 gene of mouse embryonic stem cells and (MDR) family of membrane transporters which leads subsequent cell cloning induced retinal neuron to a side population phenotype, and transcription progenitors, including retinal ganglion cell-like cells, factor p63 were proposed recently. Conventional in vitro." Ophthalmic Res 43(2): 79-91. corneal transplantation is not applicable for patients OBJECTIVE: It is theoretically possible to with limbal stem cells deficiency, because the induce various cell types, including retinal neurons, conventional allograft lacks limbal stem cells. The from embryonic stem cells (ESCs). pax6 regulates introduction of limbal epithelial cell transplantation early events in eye development, including the was a major advance in the therapeutic techniques for generation of retinal ganglion cells (RGCs). We reconstruction of the corneal surface. Limbal epithelial previously reported the successful induction of corneal cell transplantation is clinically conducted when epithelial cells from ESCs transfected with the pax6 cultured allografts as well as autografts are available; gene. Here, we attempted to establish cloned RGC-like however, allografts have a risk of immunologic cells from ESCs transfected with the pax6 gene. rejection and autografts are hardly available for

135 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ patients with bilateral ocular surface disorders. (PGCs) in mice. It has been shown that these BMPs Embryonic stem (ES) cells are characterized by their significantly increase germ cell differentiation from capacity to proliferate indefinitely and to differentiate hESCs in vitro. This protocol describes a method to into any cell type. We induced corneal epithelial cells induce germ cell differentiation from hESCs by the from ES cells by culturing them on type IV collagen or addition of BMPs to hESC differentiation medium. alternatively, by introduction of the pax6 gene into ES The protocol can be used to study the basic mechanism cells. Recent advances in our study supports the of germ cell development in human cells. possibility of their clinical use as a cell source for reconstruction of the damaged corneal surface. This Kelly, O. G., et al. (2011). "Cell-surface markers review summarizes the recent advances in corneal for the isolation of pancreatic cell types derived from regeneration therapies and the possible application of human embryonic stem cells." Nat Biotechnol 29(8): ES cell-derived corneal epithelial cells. 750-756. Using a flow cytometry-based screen of Kee, K., et al. (2006). "Bone morphogenetic commercial antibodies, we have identified cell-surface proteins induce germ cell differentiation from human markers for the separation of pancreatic cell types embryonic stem cells." Stem Cells Dev 15(6): 831-837. derived from human embryonic stem (hES) cells. We The growth factors bone morphogenetic protein- show enrichment of pancreatic endoderm cells using 4 (BMP4), BMP7, and BMP8b are required for CD142 and of endocrine cells using CD200 and specification of primordial germ cells (PGCs) in mice. CD318. After transplantation into mice, enriched Disruption of the genes that encode these factors leads pancreatic endoderm cells give rise to all the to a severe reduction in number, or the complete pancreatic lineages, including functional insulin- absence, of PGCs. In addition, several studies have producing cells, demonstrating that they are pancreatic demonstrated that human BMP4 can promote PGC progenitors. In contrast, implanted, enriched differentiation from mouse embryonic stem (ES) cells polyhormonal endocrine cells principally give rise to and in organ cultures. Here, we sought to determine glucagon cells. These antibodies will aid investigations whether recombinant human BMPs could induce that use pancreatic cells generated from pluripotent differentiation of germ cells from human (h) ES cells. stem cells to study diabetes and pancreas biology. We found that addition of recombinant human BMP4 increased the expression of the germ cell-specific Kerkis, I., et al. (2006). "Isolation and markers VASA and SYCP3 during differentiation of characterization of a population of immature dental hES cells to embryoid bodies (EBs). In addition, pulp stem cells expressing OCT-4 and other BMP7 and BMP8b showed additive effects on germ embryonic stem cell markers." Cells Tissues Organs cell induction when added together with BMP4. 184(3-4): 105-116. Finally, we observed that addition of BMPs to We report the isolation of a population of differentiating ES cells also increased the percentage immature dental pulp stem cells (IDPSC), which of cells that stained positively for VASA. We note that express embryonic stem cell markers Oct-4, Nanog, the effects of recombinant BMPs were modest but SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 as well as reproducible and suggest that addition of BMPs to several other mesenchymal stem cell markers during at differentiation media increases differentiation of least 25 passages while maintaining the normal human germ cells from hES cells. karyotype and the rate of expansion characteristic of stem cells. The expression of these markers was Kee, K. and R. A. Reijo Pera (2008). "Human maintained in subclones obtained from these cells. germ cell lineage differentiation from embryonic stem Moreover, in vitrothese cells can be induced to cells." CSH Protoc 2008: pdb prot5048. undergo uniform differentiation into smooth and INTRODUCTIONBiological and ethical skeletal muscles, neurons, cartilage, and bone under constraints hinder studies of human germ cell chemically defined culture conditions. After in vivo development despite its importance to reproductive transplantation of these cells into health, including fertility and tumorigenesis. Thus, immunocompromised mice, they showed dense most of what we know of human germ cell engraftment in various tissues. The relative ease of development has been extrapolated from studies in recovery and the expression profiles of various model organisms. Human embryonic stem cells markers justify further exploration of IDPSC for (hESCs) may provide an ideal system for probing the clinical therapy. developmental genetics of germ cell formation and differentiation in vitro. The growth factors BMP (bone Kern, I., et al. (2013). "Embryonic stem cell- morphogenetic protein) 4, BMP7, and BMP8b are based screen for small molecules: cluster analysis required for development of primordial germ cells

136 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ reveals four response patterns in developing neural reducing associated costs and efforts. Here, we cells." Curr Med Chem 20(5): 710-723. describe establishment of a C57BL/6 ES cell line Neural differentiation of embryonic stem cells (LK1) and compare its efficacy to a widely used (ESC) is considered a promising model to perform in 129SvJ ES cell line (GSI-1) in generating germline vitro testing for neuroactive and neurotoxic chimeras. In contrast to earlier studies, our data shows compounds. We studied the potential of a dual reporter that highly germline-competent C57BL/6 ES cell lines murine ESC line to identify bioactive and/or toxic can be derived using a simple approach, and thus compounds. This line expressed firefly luciferase support broader use of C57BL/6 ES cell lines for under the control of the neural cell-specific tubulin genetically engineered mouse models. alpha promoter (TUBA1A), and renilla luciferase under the control of the ubiquitous translation Kibschull, M., et al. (2011). "Human embryonic elongation factor 1-alpha-1 (EEF1A1) promoter. fibroblasts support single cell enzymatic expansion of During neural differentiation, TUBA1A activity human embryonic stem cells in xeno-free cultures." increased, while EEF1A1 activity decreased. We first Stem Cell Res 6(1): 70-82. validated our test system using the known neurotoxin The future application of human embryonic stem methyl mercury. This compound altered expression of cells (hESC) for therapeutic approaches requires the both reporter genes, with ESC-derived neural development of xeno-free culture conditions to prevent precursors being affected at markedly lower the potential transmission of animal pathogens or concentrations than undifferentiated ESCs. Analysis of xenobiotic substances to hESC. An important a library of 1040 bioactive compounds picked up 127 component of the majority of hESC culture systems compounds with altered EEF1A1 and/or TUBA1A developed is the requirement for fibroblasts to serve as promoter activity, which were classified in 4 clusters. feeders. For this purpose, several studies have used Cluster 1 (low EEF1A1 and TUBA1A) was the largest human foreskin fibroblasts established under xeno-free cluster, containing many cytostatic drugs, as well as conditions. In this study we report xeno-free known neurodevelopmental toxicants, psychotropic establishment and maintenance of human embryonic drugs and endocrine disruptors. Cluster 2 (high fibroblasts (XHEF) and demonstrate their ability to EEF1A1, stable TUBA1A) was limited to three support long-term self-renewal of hESC under xeno- sulfonamides. Cluster 3 (high EEF1A1 and TUBA1A) free culture conditions, using a commercially available was small, but markedly enriched in neuroactive and complete medium. Importantly, our culture conditions neurotoxic compounds. Cluster 4 (stable EEF1A1, allow enzymatic passaging of hESC. In contrast, hESC high TUBA1A) was heterogeneous, containing cultured on human foreskin fibroblasts (XHFF) under endocrine disruptors, neurotoxic and cytostatic drugs. the same conditions were poorly maintained and The dual reporter gene assay described here might be a rapidly subject to differentiation. Our study clearly useful addition to in vitro drug testing panels. Our shows that the source of human fibroblasts is essential two-dimensional testing strategy provides information for long-term xeno-free hESC maintenance. on complex response patterns, which could not be achieved by a single marker approach. Kidder, B. L., et al. (2008). "Embryonic stem cells contribute to mouse chimeras in the absence of Keskintepe, L., et al. (2007). "Derivation and detectable cell fusion." Cloning Stem Cells 10(2): 231- comparison of C57BL/6 embryonic stem cells to a 248. widely used 129 embryonic stem cell line." Transgenic Embryonic stem (ES) cells are capable of Res 16(6): 751-758. differentiating into all embryonic and adult cell types Typically, embryonic stem (ES) cells derived following mouse chimera production. Although from 129 mouse substrains are used to generate injection of diploid ES cells into tetraploid blastocysts genetically altered mouse models. Resulting chimeric suggests that tetraploid cells have a selective mice were then usually converted to a C57BL/6 disadvantage in the developing embryo, tetraploid background, which takes at least a year, even in the hybrid cells, formed by cell fusion between ES cells case of speed congenics. In recent years, embryonic and somatic cells, have been reported to contribute to stem cells have been derived from various mouse mouse chimeras. In addition, other examples of strains. However, 129 ES cells are still widely used apparent stem cell plasticity have recently been shown partially due to poor germline transmission of ES cells to be the result of cell fusion. Here we investigate derived from other strains. Availability of highly whether ES cells contribute to mouse chimeras germline-competent C57BL/6 ES cells would through a cell fusion mechanism. Fluorescence in situ enormously facilitate generation of genetically altered hybridization (FISH) analysis for X and Y mice in a pure C57BL/6 genetic background by chromosomes was performed on dissociated tissues eliminating backcrossing time, and thus significantly from embryonic, neonatal, and adult wild-type, and

137 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ chimeric mice to follow the ploidy distributions of cellular reprogramming, it remains unknown whether cells from various tissues. FISH analysis showed that microRNAs are absolutely necessary. We endeavored the ploidy distributions in dissociated tissues, notably to answer this fundamental question by attempting to the tetraploid cell number, did not differ between reprogram Dicer-null mouse embryonic fibroblasts chimeric and wild-type tissues. To address the (MEFs) that lack almost all functional microRNAs possibility that early cell fusion events are hidden by using a defined set of transcription factors. subsequent reductive divisions or other changes in cell Transduction of reprogramming factors using either ploidy, we injected Z/EG (lacZ/EGFP) ES cells into lentiviral or piggyBac transposon vector into two, ACTB-cre blastocysts. Recombination can only occur independently derived lines of Dicer-null MEFs failed as the result of cell fusion, and the recombined allele to produce cells resembling embryonic stem cells should persist through any subsequent changes in cell (ESCs). However, expression of human Dicer in the ploidy. We did not detect evidence of fusion in Dicer-null MEFs restored their reprogramming embryonic chimeras either by direct fluorescence potential. Our study demonstrates for the first time that microscopy for GFP or by PCR amplification of the microRNAs are indispensable for dedifferentiation recombined Z/EG locus on genomic DNA from reprogramming. ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which Kim, D. W., et al. (2006). "Stromal cell-derived ES cells contribute to chimeras. inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from Kiechle, M. (2008). "[Human embryonic stem mouse embryonic stem cells." Stem Cells 24(3): 557- cell research in Germany. The scientific reviewing of 567. applications for the import and use of human To induce differentiation of embryonic stem cells embryonic stem cells]." Bundesgesundheitsblatt (ESCs) into specialized cell types for therapeutic Gesundheitsforschung Gesundheitsschutz 51(9): 961- purposes, it may be desirable to combine genetic 964. manipulation and appropriate differentiation signals. From July 2002 to May 2008, 36 applications for We studied the induction of dopaminergic (DA) the import and use of human embryonic stem cells neurons from mouse ESCs by overexpressing the (hES) were reviewed by the German Central Ethics transcription factor Nurr1 and coculturing with PA6 Committee for Stem Cell Research (ZES). A flood of stromal cells. Nurr1-expressing ESCs (N2 and N5) applications anticipated by opponents to human differentiated into a higher number of neurons embryonic stem cell research has not occurred since (approximately twofold) than the naive ESCs (D3). In the enactment of the German Stem Cell Act in 2002. addition, N2/N5-derived cells contained a significantly On the contrary, German hES cell research is below higher proportion (>50%) of tyrosine hydroxylase international average in terms of project numbers. The (TH)+ neurons than D3 (<30%) and an even greater current restrictions for using hES cells in Germany proportion of TH+ neurons (approximately 90%) when might be causative for the opinion that this type of treated with the signaling molecules sonic hedgehog, research is not considered to be very promising. This fibroblast growth factor 8, and ascorbic acid. N2/N5- could hold true especially for research aiming at derived cells express much higher levels of DA clinical applications. Consequently, potential research markers (e.g., TH, dopamine transporter, aromatic goals of premium importance, especially those of amino acid decarboxylase, and G protein-regulated potential clinical relevance, could be seriously inwardly rectifying K+ channel 2) and produce and jeopardized. release a higher level of dopamine, compared with D3- derived cells. Furthermore, the majority of generated Kim, B. M., et al. (2012). "MicroRNAs are neurons exhibited electrophysiological properties indispensable for reprogramming mouse embryonic characteristic of midbrain DA neurons. Finally, fibroblasts into induced stem cell-like cells." PLoS transplantation experiments showed efficient in vivo One 7(6): e39239. integration/generation of TH+ neurons after MicroRNAs play a pivotal role in cellular implantation into mouse striatum. Taken together, our maintenance, proliferation, and differentiation. They results show that the combination of genetic have also been implicated to play a key role in disease manipulation (s) and in vitro cell differentiation pathogenesis, and more recently, cellular conditions offers a reliable and effective induction of reprogramming. Certain microRNA clusters can DA neurons from ESCs and may pave the way for enhance or even directly induce reprogramming, while future cell transplantation therapy in Parkinson's repressing key proteins involved in microRNA disease. processing decreases reprogramming efficiency. Although microRNAs clearly play important roles in

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Kim, E. M., et al. (2014). "Embryonic stem cell- stimulation and induce lethal graft-versus-host disease. derived haematopoietic progenitor cells down-regulate Thymic selection in fetal thymic organ cultures the CD3 xi chain on T cells, abrogating alloreactive T (FTOCs) allowed negative selection and generation of cells." Immunology 142(3): 421-430. T cells tolerant to 'self' and capable of rejecting MHC- Murine embryonic stem (ES) cell-derived mismatched skin allografts. Our data show that ESC- haematopoietic progenitor cells (HPCs) engraft and derived T cells, despite high expression of HoxB4, are populate lymphoid organs. In vivo, HPCs engraft fully immunocompetent. across MHC barriers protecting donor-type allografts from rejection. However, the underlying phenomenon Kim, G. D., et al. (2012). "Honokiol inhibits remains elusive. Here, we sought to determine the vascular vessel formation of mouse embryonic stem mechanism by which ES cell-derived HPCs regulate cell-derived endothelial cells via the suppression of alloreactive T cells. We used the 2C mouse, which PECAM and MAPK/mTOR signaling pathway." Cell expresses a transgenic T-cell receptor against H2-L (d) Physiol Biochem 30(3): 758-770. to determine whether HPCs are deleted by cytotoxic T Embryonic stem cells, which are characterized by lymphocytes (CTLs). Previously, we reported that pluripotency and self-renewal, have recently been HPCs express MHC class I antigens poorly and do not highlighted in drug discovery. In particular, the express class II antigens. In vitro stimulated 2C CTLs potential of ES cells to differentiate into specific-cell failed to lyse H2-L (d) HPCs in a standard 4-hr (51) types make them an extremely useful tool in the chromium release assay. Similarly, when the HPCs evaluation of the biological activity of test compounds. were tested in an ELISPOT assay measuring the Honokiol, a major neolignan derived from the bark of release of interferon-gamma by CTLs, HPCs failed to Magnolia obovata, has been shown an anti-tumor induce CTL degranulation. In addition, mice that were activity. However, the precise mechanism of action in injected with HPCs showed a marked decrease in T- the anti-tumor activity of honokiol is still poorly cell responses to alloantigen and CD3 stimulation, but understood. Here, we evaluated the antiangiogenic showed a normal response to PMA/ionomycin, activity of honokiol using mouse ES cell-derived suggesting that HPCs impaired T-cell signalling embryoid bodies. mES-derived EBs were formed through the T-cell receptor/CD3 complex. Here, we using hanging drop cultures and vascular formation show that HPCs secrete arginase, an enzyme that was induced on gelatincoated plates in EGM-2 scavenges l-arginine, leading to metabolites that down- medium. The growth inhibition of honokiol was found regulate CD3 zeta chain. Indeed an arginase inhibitor to be more sensitive in the differentiated EB-derived partially restored expression of the CD3 zeta chain, endothelial cells compared to the undifferentiated EB- implicating arginase 1 in the down-regulation of T derived cells. Honokiol also inhibited the vascular cells. This previously unrecognized property of ES formation of mES cells on 3-D collagen gel and cell-derived HPCs could positively enhance the decreased the expression of endothelial biomarkers engraftment of ES cell-derived HPCs across MHC VEGFR2 and PECAM in the differentiated EB- barriers by preventing rejection. derived endothelial cells. In addition, honokiol suppressed the MAPK and mTOR signaling pathways Kim, E. M., et al. (2012). "Embryonic stem cell- in the EB-derived endothelial cells. Therefore, the derived T cells induce lethal graft-versus-host disease anti-angiogenic activity of honokiol is associated in and reject allogenic skin grafts upon thymic selection." part with the suppression of PECAM and Am J Transplant 12(3): 600-609. MAPK/mTOR pathways in EB-derived endothelial Efficient differentiation of embryonic stem cells cells. (ESC) into hematopoietic progenitor cells (HPCs) is crucial for the establishment of stem cell-based Kim, G. D., et al. (2009). "Cytotoxicity of 5- therapies targeting the treatment of immunological and fluorouracil: Effect on endothelial differentiation via hematological disorders. However, so far, it has not cell cycle inhibition in mouse embryonic stem cells." been possible to induce long-term survival of murine Toxicol In Vitro 23(4): 719-727. ESC-derived HPCs without the overexpression of Embryonic stem cells (ESCs) are known to HoxB4, a homeobox transcription factor that confers characteristics for pluripotency and self-renewal, but self-renewal properties to hematopoietic cells. Yet it the precise mechanisms of ES-derived cells to specific has not been feasible to generate T cells from HoxB4- toxicants have not been determined. Here, we expressing HPCs, a problem that has been attributed to evaluated the cytotoxicity of 5-fluorouracil (5-FU) and HoxB4. Here, we show that Notch1 signaling in see its effect on cell viability, proliferation, and HoxB4-transduced ESCs leads to efficient derivation differentiation in mouse ESC-derived endothelial of T cells that survive long term. These T cells display differentiation. Mouse ESCs were exposed to 5-FU a normal T-cell Vbeta repertoire, respond to mitogen (10 microM) and combined with probucol (50 microM)

139 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ for 24h, which is an antagonist of 5-FU. Changes in expression in 3(8)21 cells, no expression of CD45, gene expression as a result of 5-FU exposure in mouse CD117, CD62L, CD80, CD86, CD90.1 or ESC-derived endothelial precursor cells (ES-EPCs) CD95L/CD178 was observed. Constitutive and were assessed using an oligonucleotide microarray cytokine-inducible CD95([LOW]) expression was (AB1700). The expression of Oct-4 was decreased detected in STO and 3(8)21 cells, but not in 3(8)21- during the differentiation of mouse ESCs into EGFP cells. MHC (class I (+[LOW])/class II-) and CD endothelial cells; otherwise, the expression of PECAM (CD34+/CD80-/CD86-/CD95L-) expression patterns was increased. Mouse ES-EPCs were shown to have a in STO and STO cell-derived progenitor cells decrease in viability (49.8%) and PECAM expression, resemble patterns reported for human embryonic stem and induce G1/S phase (31.1%/60.6%) when cell lines. Whether these patterns reflect associations compared with/without treatment of 5-FU. Expression with mechanisms that are regulatory of immune of cell cycle-related proteins was increased in privilege or functional tissue-specific plasticity is endothelial precursor cells exposed to 5-FU without unknown. probucol treatment. From theses results suggest that 5- FU inhibit endothelial differentiation as well as Kodama, M., et al. (2008). "Pancreatic endocrine inducing the G1/S phase arrest. We propose that and exocrine cell ontogeny from renal capsule mouse ES-EPCs might be a useful tool for screening transplanted embryonic stem cells in streptozocin- the cytotoxicity of compounds in endothelial cells. injured mice." J Histochem Cytochem 56(1): 33-44. In this study, we describe pancreatic cell Koch, K. S., et al. (2006). "Immune-privileged ontogeny in renal capsule-transplanted embryonic embryonic Swiss mouse STO and STO cell-derived stem cells (ES) after injury by streptozocin (STZ), progenitor cells: major histocompatibility complex and showing pancreatogenesis in situ. Seven-week-old cell differentiation antigen expression patterns female BALB/c nude mice were treated with either a resemble those of human embryonic stem cell lines." single 175- or 200-mg/kg STZ dose, a regimen that Immunology 119(1): 98-115. induces substantial beta-cell damage without overt Embryonic mouse STO (S, SIM; T, 6- hyperglycemia, and transplanted 24 hr later with 1 x thioguanine resistant; O, ouabain resistant) and 3(8)21- 10(5) ES. Immunohistochemistry was performed on enhanced green fluorescent protein (EGFP) cell lines ES tissue at 15, 21, and 28 days after transplantation exhibit long-term survival and hepatic progenitor cell using antibodies against stage- and lineage-specific behaviour after xenogeneic engraftment in non- pancreatic markers. After 21 days, PDX-1+ pancreatic immunosuppressed inbred rats, and were previously foci first appeared in the renal capsule and expressed designated major histocompatibility complex (MHC) both amylase and endocrine hormones (insulin, class I- and class II-negative lines. To determine the glucagon, and somatostatin). These foci increased in molecular basis for undetectable MHC determinants, size by day 28 because of acinar and duct cell the expression and haplotype of H-2K, H-2D, H-2L proliferation, whereas endocrine cells remained non- and I-A proteins were reassessed by reverse dividing, and made up 2-4% of ES tumor volume. transcriptase-polymerase chain reaction (RT-PCR), PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization cDNA sequencing, RNA hybridization, patterns in pancreatic foci were comparable with immunoblotting, quantitative RT-PCR (QPCR), embryonic pancreatogenesis. A prevalence of immunocytochemistry and flow cytometry. To detect multihormonal endocrine cells, a characteristic of adult cell differentiation (CD) surface antigens characteristic beta-cell regeneration, indicated a possible divergence of stem cells, apoptotic regulation or adaptive from embryonic islet cell development. The results immunity that might facilitate progenitor cell status or indicate that beta-cell damage, without overt immune privilege, flow cytometry was also used to hyperglycemia, induces a process of fetal-like screen untreated and cytokine [interferon (IFN)- pancreatogenesis in renal capsule-transplanted ES, gamma]-treated cultures. Despite prior PCR leading to beta-cell neogenesis. genotyping analyses suggestive of H-2q haplotypes in STO, 3(8)21-EGFP and parental 3(8)21 cells, all three Koel, M., et al. (2017). "Optimizing bone lines expressed H-2K cDNA sequences identical to morphogenic protein 4-mediated human embryonic those of d-haplotype BALB/c mice, as well as stem cell differentiation into trophoblast-like cells constitutive and cytokine-inducible H-2K (d) using fibroblast growth factor 2 and transforming determinants. In contrast, apart from H-2L (d [LOW]) growth factor-beta/activin/nodal signalling inhibition." display in 3(8)21 cells, H-2Dd, H-2Ld and I-Ad Reprod Biomed Online 35(3): 253-263. determinants were undetectable. All three lines Several studies have demonstrated that human expressed constitutive and cytokine-inducible CD34; embryonic stem cells (hESC) can be differentiated into however, except for inducible CD117([LOW]) trophoblast-like cells if exposed to bone morphogenic

140 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ protein 4 (BMP4) and/or inhibitors of fibroblast detectable at 1 week and progressed through 4 weeks growth factor 2 (FGF2) and the transforming growth following cell transplantation. The humoral Ab factor beta (TGF-beta)/activin/nodal signalling response was moderate at 2 weeks but frank at 4 pathways. The goal of this study was to investigate weeks. ELISPOT demonstrated a Th1 pathway of how the inhibitors of these pathways improve the donor specific T-lymphocyte response with strong efficiency of hESC differentiation when compared IFN-gamma and Il-2 production (figure A). MHC I with basic BMP4 treatment. RNA sequencing was expression was significant within the graft and used to analyse the effects of all possible inhibitor maximal in the allogeneic groups. CONCLUSIONS: combinations on the differentiation of hESC into An immune response against transplanted ESC was trophoblast-like cells over 12 days. Genes demonstrated and the future use of ESC will likely differentially expressed compared with untreated cells require the use of systemic immunosuppression. were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin Koh, K. P., et al. (2011). "Tet1 and Tet2 regulate (HCG) and its hyperglycosylated form (HCG-H) were 5-hydroxymethylcytosine production and cell lineage determined by immunoassay from cell culture media. specification in mouse embryonic stem cells." Cell We showed that FGF2 inhibition with BMP4 Stem Cell 8(2): 200-213. activation up-regulates syncytiotrophoblast-specific TET family enzymes convert 5-methylcytosine genes (CGA, CGB and LGALS16), induces several (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. molecular pathways involved in embryo implantation Here, we show that Tet1 and Tet2 are Oct4-regulated and triggers HCG-H production. In contrast, inhibition enzymes that together sustain 5hmC in mouse of the TGF-beta/activin/nodal pathway decreases the embryonic stem cells (ESCs) and are induced ability of hESC to form trophoblast-like cells. concomitantly with 5hmC during reprogramming of Information about the conditions needed for hESC fibroblasts to induced pluripotent stem cells. ESCs differentiation toward trophoblast-like cells helps us to depleted of Tet1 by RNAi show diminished expression find an optimal model for studying the early of the Nodal antagonist Lefty1 and display hyperactive development of human trophoblasts in normal and in Nodal signaling and skewed differentiation into the complicated pregnancy. endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture Kofidis, T., et al. (2005). "They are not stealthy conditions, Tet1-depleted ESCs activate the in the heart: embryonic stem cells trigger cell trophoblast stem cell lineage determinant Elf5 and can infiltration, humoral and T-lymphocyte-based host colonize the placenta in midgestation embryo chimeras. immune response." Eur J Cardiothorac Surg 28(3): Consistent with these findings, Tet1-depleted ESCs 461-466. form aggressive hemorrhagic teratomas with increased OBJECTIVE: The in vivo immunogenicity of endoderm, reduced neuroectoderm, and ectopic Embryonic Stem Cells is controversial. At present, appearance of trophoblastic giant cells. Thus, 5hmC is there is only in vitro evidence of MHC I expression by an epigenetic modification associated with the this cell population but vivid speculation about their pluripotent state, and Tet1 functions to regulate the immune-privileged state. The immunology aspect of lineage differentiation potential of ESCs. ESC transplantation deserves thorough investigation. METHODS: We injected mouse ESC (expressing Koifman, G., et al. (2018). "A mutant p53- Green Fluorescent Protein, GFP) into injured dependent embryonic stem cell gene signature is myocardium of syngeneic, allogeneic and SCID associated with augmented tumorigenesis of stem recipients. Furthermore, we monitored host response cells." Cancer Res. for up to 4 weeks post cell transfer. We determined Mutations in the tumor suppressor p53 are the local response (CD 3, CD 11c expression by host most frequent alterations in human cancer. These cells), MHC I expression by donor cells, MHC-II mutations include p53-inactivating mutations as well expression within and around the graft, humoral as oncogenic gain-of-function (GOF) mutations that response of allogeneic hosts using Flow Cytometry endow p53 with capabilities to promote tumor and evaluated the hosts' cytokine response using progression. A primary challenge in cancer therapy is stimulated spleenocytes by means of ELISPOT. Cell targeting stemness features and cancer stem cells (CSC) survival was estimated by morphometry, by that account for tumor initiation, metastasis, and calculating the area of the GFP+ graft over the area of cancer relapse. Here we show that in vitro cultivation infarction at multiple sections of the harvested heart. of tumors derived from mutant p53 murine bone RESULTS: There was significant cellular infiltration marrow (BM) mesenchymal stem cells (MSC) gives into and around the graft consisting of T-lymphocytes rise to aggressive tumor lines (TL). These MSC-TL (CD3+) and dendritic cells (CD 11c). Infiltration was exhibited CSC features as displayed by their

141 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ augmented oncogenicity and high expression of CSC specialized mechanisms of cell-cycle control. In the markers. Comparative analyses between MSC-TL with last few years, several studies have provided evidence their parental mutant p53 MSC allowed for for a direct link between cell-cycle regulation and cell- identification of the molecular events underlying their fate decisions in stem cells. In this review, we discuss tumorigenic properties, including an embryonic stem the peculiarities of embryonic stem cell-cycle control cell (ESC) gene signature specifically expressed in mechanisms, implicate their involvement in cell-fate MSC-TL. Knockout of mutant p53 led to a reduction decisions, and distinguish centrosomes as important in tumor development and tumorigenic cell frequency, players in the self-renewal versus differentiation which was accompanied by reduced expression of roulette. CSC markers and the ESC MSC-TL signature. In human cancer, MSC-TL ESC signature-derived genes Koltsova, A. M., et al. (2015). "[Characteristics correlated with poor patient survival and were highly of New Mesenchymal Stem Cell Line Derived from expressed in human tumors harboring p53 hotspot Human Embryonic Stem Cells]." Tsitologiia 57(11): mutations. These data indicate that the ESC gene 761-770. signature-derived genes may serve as new stemness- New nonimmortalized fibroblast-like cell line based prognostic biomarkers as well as novel cancer SC6-MSC has been obtained from a line of human therapeutic targets. embryonic stem cells (ESC)--SC6. Numerical and structural karyotypic analysis has shown hypodiploidy Kokudo, T., et al. (2008). "Snail is required for karyotypic: 45, X0 in this line. The average cell TGFbeta-induced endothelial-mesenchymal transition population doublings time, for SC6-MSC is 26.0 +/- of embryonic stem cell-derived endothelial cells." J 0.4 h at the 8th passage and 82.0 +/- 9.2 h at the 18th Cell Sci 121(Pt 20): 3317-3324. passage. The growth curves showed active Epithelial-mesenchymal transition (EMT) plays proliferation for 8-10 passages with a consequent important roles in various physiological and gradual decrease of proliferative activity, which ended pathological processes, and is regulated by signaling to 20th passage. To determine the line's status, the pathways mediated by cytokines, including analysis of the surface markers by flow cytometry was transforming growth factor beta (TGFbeta). carried out. We have revealed the expression of Embryonic endothelial cells also undergo surface antigens CD44, CD73, CD90, CD105 and differentiation into mesenchymal cells during heart HLA-ABC characteristic for human MSC, and the valve formation and aortic maturation. However, the absence of CD34 and HLA-DR expression. However, molecular mechanisms that regulate such endothelial- the level of expression of surface markers CD90 and mesenchymal transition (EndMT) remain to be CD105 was significantly lower in comparison with elucidated. Here we show that TGFbeta plays other MSC lines including the line SC5-MSC derived important roles during mural differentiation of mouse from the line human ESC-SC5. Immunofluorescence embryonic stem cell-derived endothelial cells analysis of the expression of the surface markers and (MESECs). TGFbeta2 induced the differentiation of transcription factor Oct-4 characteristic for human MESECs into mural cells, with a decrease in the embryonic stem cells showed the absence of Oct-4 expression of the endothelial marker claudin 5, and an expression and the presence of SSEA-4 and TRA-1-60 increase in expression of the mural markers smooth expression, which is characteristic for a number of muscle alpha-actin, SM22alpha and calponin, whereas MSC lines with normal karyotype. a TGFbeta type I receptor kinase inhibitor inhibited Immunofluorescence analysis has shown the presence EndMT. Among the transcription factors involved in of the markers of early differentiation in the derivates EMT, Snail was induced by TGFbeta2 in MESECs. of three germ layers, characteristic for human ESC, Tetracycline-regulated expression of Snail induced the which in corresponding microenvironments may allow differentiation of MESECs into mural cells, whereas MSC to be useful for reparation of tissue injures. The knockdown of Snail expression abrogated TGFbeta2- directed osteogenic and chondrogenic differentiation induced mural differentiation of MESECs. These of line SC6-MSC has shown. However, no directed results indicate that Snail mediates the actions of adipogenic differentiation of this line has been found. endogenous TGFbeta signals that induce EndMT. The obtained results with high probability may indicate what alteration of chromosomal and, Koledova, Z., et al. (2010). "Cell-cycle regulation accordingly, gene balance, in line SC6-MSC with in embryonic stem cells: centrosomal decisions on karyotype 45, X0 resulted in decrease in differential self-renewal." Stem Cells Dev 19(11): 1663-1678. potential, in expression CD90, associated in particular Embryonic stem cells seem to have the intriguing with the processes of differentiation and aging of cells. capacity to divide indefinitely while retaining their pluripotency. This self-renewal is accomplished by

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Konig, N., et al. (2017). "Murine neural crest measured intensities essentially reflected DNA content. stem cells and embryonic stem cell-derived neuron When quantifying Raman images from single cells in a precursors survive and differentiate after population of methanol-fixed human embryonic stem transplantation in a model of dorsal root avulsion." J cells, the histogram of corrected 783 cm (-1) band Tissue Eng Regen Med 11(1): 129-137. intensities exhibited a profile analogous to that Spinal root avulsion results in paralysis and obtained using flow-cytometry with nuclear stains. sensory loss, and is commonly associated with chronic The two population peaks in the histogram occur at pain. In addition to the failure of avulsed dorsal root Raman intensities corresponding to a 1-fold and 2-fold axons to regenerate into the spinal cord, avulsion diploid DNA complement per cell, consistent with a injury leads to extensive neuroinflammation and distribution of cells with a population peak due to cells degeneration of second-order neurons in the dorsal at the end of G1 phase (1-fold) and a peak due to cells horn. The ultimate objective in the treatment of this entering M phase (2-fold). When treated with EdU to condition is to counteract degeneration of spinal cord label the replicating DNA and block cell division, cells neurons and to achieve functionally useful with higher EdU-related fluorescence generally had regeneration/reconnection of sensory neurons with higher integrated Raman intensities. This provides spinal cord neurons. Here we compare survival and proof-of-principle of an analytical method for label- migration of murine boundary cap neural crest stem free RMS determination in situ of cell cycle phase in cells (bNCSCs) and embryonic stem cells (ESCs)- adherent monolayers or even single adherent cells. derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed Kurtovic, S., et al. (2015). "Leptin enhances dorsal roots L3-L6 and the spinal cord. Both types of endothelial cell differentiation and angiogenesis in cells survived transplantation, but showed distinctly murine embryonic stem cells." Microvasc Res 97: 65- different modes of migration. Thus, bNCSCs migrated 74. into the spinal cord, expressed glial markers and The metabolic regulation of leptin and its formed elongated tubes in the peripheral nervous angiogenic effects have been well characterized in system (PNS) compartment of the avulsed dorsal root adult mammals. However, the role of leptin in the transitional zone (DRTZ) area. In contrast, the ESC differentiation of embryonic stem cells (ESCs) to transplants remained at the site of implantation and endothelial cells (ECs) has not been characterized. We differentiated to motor neurons and interneurons. hypothesized that leptin enhances the generation of These data show that both stem cell types successfully ECs derived from ESCs and, in this way, promotes survived implantation to the acutely injured spinal angiogenesis in embryonic vessels. To address this cord and maintained their differentiation and migration hypothesis, we utilized an in vitro model consisting of potential. These data suggest that, depending on the murine ESCs-derived embryoid bodies (EBs). source of neural stem cells, they can play different Vascular density, EC and angiogenesis markers as beneficial roles for recovery after dorsal root avulsion. well as phosphorylation levels of signal transducer and Copyright (c) 2014 John Wiley & Sons, Ltd. activator of transcription 3 (pSTAT3) were investigated in leptin-treated EBs and in untreated EBs Konorov, S. O., et al. (2013). "Label-free as controls. ESC-derived ECs were isolated by determination of the cell cycle phase in human magnetic sorting based on the expression of platelet embryonic stem cells by Raman microspectroscopy." endothelial cell adhesion molecule (PECAM-1/CD31). Anal Chem 85(19): 8996-9002. Significant upregulation of EC and angiogenic The cell cycle is a series of integrated and markers as well as higher vessel density were found in coordinated physiological events that results in cell leptin-treated EBs compared to controls. CD31 growth and replication. Besides observing the event of positive enriched cells derived from leptin-treated EBs cell division it is not feasible to determine the cell had improved proliferation and survival rate and cycle phase without fatal and/or perturbing invasive showed higher levels of pSTAT3. These results procedures such as cell staining, fixing, and/or suggested that leptin promotes EC differentiation and dissociation. Raman microspectroscopy (RMS) is a angiogenesis in mouse EBs and that janus tyrosine chemical imaging technique that exploits molecular kinase (JAK)/STAT pathway can play a role in this vibrations as a contrast mechanism; it can be applied biological process. Leptin-mediated EC differentiation to single living cells noninvasively to allow and angiogenesis in ESCs can be a useful application unperturbed analysis over time. We used RMS to towards regenerative medicine and tissue engineering. determine the cell cycle phase based on integrating the composite 783 cm (-1) nucleic acid band intensities Lau, Y. T., et al. (2011). "Effects of across individual cell nuclei. After correcting for RNA hyperpolarization-activated cyclic nucleotide-gated contributions using the RNA 811 cm (-1) band, the (HCN) channel blockers on the proliferation and cell

143 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cycle progression of embryonic stem cells." Pflugers fibronectin deposition), matrix metalloproteinase-2 Arch 461(1): 191-202. (MMP-2) activity and percentage of neuronal cells. In Embryonic stem cells (ESCs) can uniquely line with the increased MMP-2 activity levels found, proliferate indefinitely and differentiate into all cell hydrodynamically-cultured neurospheres showed lineages. ESCs may therefore provide an unlimited higher outward migration on laminin. Moreover, when supply of cells for cell-based therapies. Previous study cultured in a 3D fibrin hydrogel, rotary neurospheres reported the presence of hyperpolarization-activated generated an increased percentage of neuronal cells. In inward currents in undifferentiated mouse (m) ESCs, conclusion, the application of a constant orbital speed but the functional role of this hyperpolarization- to single-cell suspensions of ES-NSPCs, besides activated current in mESCs is unknown. In this study, allowing the formation of homogeneously-sized the role of this current in maintaining the proliferative neurospheres, promoted ES-NSPC differentiation and capacity and the cell cycle progression of ESCs was outward migration, possibly by influencing the investigated. In D3 mESCs, this hyperpolarization- expression of cell-cell adhesion molecules and the activated inward current can be blocked by HCN secretion of proteases/extracellular matrix proteins. channel blocker ZD7288. Application of the HCN These findings are important when establishing the channel blockers, cesium (1-10 mM) or ZD7288 (0.1- culture conditions needed to obtain uniformly-sized 30 muM), attenuated cell proliferation in a ES-NSPC aggregates, either for use in regenerative concentration-dependent manner. Both HCN blockers therapies or in in vitro platforms for biomaterial were found to be non-cytotoxic to mESCs as development or pharmacological screening. Copyright determined by cell viability test. Interestingly, ZD7288 (c) 2016 John Wiley & Sons, Ltd. at 10 and 30 muM was found to decrease the proportion of cells in G (0)/G (1) phase and increase Lavial, F., et al. (2009). "Ectopic expression of the proportion of cells in S phase. This suggests that Cvh (Chicken Vasa homologue) mediates the this hyperpolarization-activated current can affect the reprogramming of chicken embryonic stem cells to a cell cycle progression in mESCs. In summary, the germ cell fate." Dev Biol 330(1): 73-82. present investigation suggests that ESC proliferation When they are derived from blastodermal cells of and cell cycle progression can be regulated by this the pre-primitive streak in vitro, the pluripotency of hyperpolarization-activated current. Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These Laundos, T. L., et al. (2017). "Rotary orbital cESC can differentiate into derivatives of the three suspension culture of embryonic stem cell-derived germ layers both in vitro and in vivo, but they only neural stem/progenitor cells: impact of hydrodynamic weakly colonize the gonads of host embryos. By culture on aggregate yield, morphology and cell contrast, non-cultured blastodermal cells and long- phenotype." J Tissue Eng Regen Med 11(8): 2227- term cultured chicken primordial germ cells maintain 2240. full germline competence. This restriction in the Embryonic stem (ES)-derived neural germline potential of the cESC may result from either stem/progenitor cells (ES-NSPCs) constitute a early germline determination in the donor embryos or promising cell source for application in cell therapies it may occur as a result of in vitro culture. We are for the treatment of central nervous system disorders. interested in understanding the genetic determinants of In this study, a rotary orbital hydrodynamic culture germline programming. The RNA binding protein Cvh system was applied to single-cell suspensions of ES- (Chicken Vasa Homologue) is considered as one such NSPCs, to obtain homogeneously-sized ES-NSPC determinant, although its role in germ cell physiology cellular aggregates (neurospheres). Hydrodynamic is still unclear. Here we show that the exogenous culture allowed the formation of ES-NSPC expression of Cvh, combined with appropriate culture neurospheres with a narrower size distribution than conditions, induces cESC reprogramming towards a statically cultured neurospheres, increasing orbital germ cell fate. Indeed, these cells express the Dazl, speeds leading to smaller-sized neurospheres and Tudor and Sycp3 germline markers, and they display higher neurosphere yield. Neurospheres formed under improved germline colonization and adopt a germ cell hydrodynamic conditions (72 h at 55 rpm) showed fate when injected into recipient embryos. Thus, our higher cell compaction and comparable percentages of results demonstrate that Vasa can drive ES cell viable, dead, apoptotic and proliferative cells. Further differentiation towards the germ cell lineage, both in characterization of cellular aggregates provided new vitro and in vivo. insights into the effect of hydrodynamic shear on ES- NSPC behaviour. Rotary neurospheres exhibited Lavial, F. and B. Pain (2010). "Chicken reduced protein levels of N-cadherin and beta-catenin, embryonic stem cells as a non-mammalian embryonic and higher deposition of laminin (without impacting stem cell model." Dev Growth Differ 52(1): 101-114.

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Embryonic stem cells (ESCs) were isolated in the Hand1-Luc EST. We established another cell line to early 1980s from mouse and in the late 1990s from monitor the Hand1 gene expression via a luciferase primate and human. These cells present the unique reporter gene. By mRNA analysis and luciferase assay, property of self-renewal and the ability to generate we examined in detail the luciferase activity during differentiated progeny in all embryonic lineages both cell differentiation, which allowed us to reduce the in vitro and in vivo. The mESCs (mouse embryonic time of measurement from day 6 to day 5 (120 hr). stem cells) can contribute to both somatic and Furthermore, the protocol was improved, with, among germinal lineages once re-injected into a recipient others, the measurement of cytotoxicity and embryo at the blastocyst stage. In avian species, differentiation toxicity taking place in the same 96- chicken embryonic stem cells (cESCs) have been well round bottom plate instead of two different plates. isolated from the in vitro culture of early chicken With the positive control, 5-fluorouracil (5-FU), and 9 blastodermal cells (cBCs) taken from stage X embryo test chemicals, data with high reproducibility and very (EG & K) These cESCs can be maintained under low variation (CV < 50%) in the relevant endpoints specific culture conditions and have been were obtained. This study shows that the Hand1-Luc characterized on the basis of their morphology, EST could provide an accurate and sensitive short- biochemical features, in vitro differentiation term test for prediction of embryotoxicants by potentialities and in vivo morphogenetic properties. measuring cytotoxicity and differentiation toxicity The relationship between these cESCs and some of the from the same sample. chicken germ cells identified and grown under specific culture conditions are still under debate, in particular Leavitt, A. D. and I. Hamlett (2011). with the identification of the Cvh gene as a key factor "Homologous recombination in human embryonic for germ cell determination. Moreover, by cloning the stem cells: a tool for advancing cell therapy and avian homologue of the Oct4 mammalian gene, we understanding and treating human disease." Clin have demonstrated that this gene, as well as the Transl Sci 4(4): 298-305. chicken Nanog gene, was involved in the Human embryonic stem cells (hESCs) hold great characterization and maintenance of the chicken promise for ushering in an era of novel cell therapies pluripotency. These first steps toward the to treat a wide range of rare and common diseases, yet understanding of pluripotency control in a non- they also provide an unprecedented opportunity for mammalian species opens the way for the basic research to yield clinical benefit. HESCs can be development and characterization of putative new cell used to better understand human development, to types such as chicken EpiSC and raises the question of model human diseases, to understand the contribution the existence of reprogramming in avian species. of specific mutations to the pathogenesis of disease, These different points are discussed. and to develop human cell-based screening systems to identify novel therapeutic agents and evaluate Le Coz, F., et al. (2015). "Hand1-Luc embryonic potential toxicity of therapeutic agents under stem cell test (Hand1-Luc EST): a novel rapid and development. Such basic research will benefit greatly highly reproducible in vitro test for embryotoxicity by from efficient methods to perform targeted gene measuring cytotoxicity and differentiation toxicity modification, an area of hESC investigation that is using engineered mouse ES cells." J Toxicol Sci 40(2): currently in its infancy. Moreover, the reality of hESC- 251-261. based cellular therapies will require improved methods The embryonic stem cell test (EST) is a for generating the specific cells of interest, and promising alternative method for evaluating reporter cell lines generated through targeted gene embryotoxicity of test chemicals by measuring modifications are expected to play an important role in cytotoxicity and differentiation toxicity using mouse developing optimal cell-specific differentiation ES cells. Differentiation toxicity is analyzed by protocols. Herein, we review the current status of microscopically counting the beating of embryonic homologous recombination in hESCs, a gene targeting bodies after 10 days of culture. However, technique that is sure to continue to improve, and to improvements are necessary to reduce the laborious play an important role in realizing the maximal human manipulations involved and the time required to obtain benefit from hESCs. results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart Lee, A. S., et al. (2009). "Effects of cell number and neural crest derivatives expressed transcript 1 on teratoma formation by human embryonic stem (Hand1) gene and the establishment of a 96-well cells." Cell Cycle 8(16): 2608-2612. multi-plate-based new EST with luciferase reporter Teratoma formation is a critical obstacle to safe assay 6 days after treatment with test chemicals. Now, clinical translation of human embryonic stem (ES) we propose an even more rapid and easier EST, named cell-based therapies in the future. As current methods

145 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ of isolation are unable to yield 100% pure population expression of the pluripotency-related genes OCT4 of differentiated cells from a pluripotent donor source, and NANOG was similar to the expression in potential development of these tumors is a significant SNUhES3 cells maintained on xenofeeder STO cells. concern. Here we used non-invasive reporter gene To identify the mechanism that enables the cells to be imaging to investigate the relationship between human maintained without exogenous FGF-2, we checked the ES cell number and teratoma formation in a xenogenic secretion of FGF-2 from the mitomycin-C treated model of ES cell transplantation. Human ES cells (H9 autofeeder hESC-MSCs versus xenofeeder STO cells, line) were stably transduced with a double fusion (DF) and confirmed that hESC-MSCs secreted FGF-2 reporter construct containing firefly luciferase and whereas STO cells did not. The level of FGF-2 in the enhanced green fluorescent protein (Fluc-eGFP) media from the autofeeder system without exogenous driven by a human ubiquitin promoter. FGF-2 was comparable to that from the xenofeeder Immunodeficient mice received intramyocardial (n = system with addition of FGF-2. In conclusion, our new 35) or skeletal muscle (n = 35) injection of 1 x 10(2), 1 culture system for hES cells, which employs a feeder x 10(3), 1 x 10(4), 1 x 10(5) or 1 x 10(6) DF positive layer of autologous hESC-MSCs, supplies sufficient ES cells suspended in saline for myocardium and amounts of secreted FGF-2 to eliminate the Matrigel for skeletal muscle. Cell survival and requirement for exogenous FGF-2. proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following Levy, Y. S., et al. (2004). "Embryonic and adult transplantation. Mice negative for Fluc signal after 8 stem cells as a source for cell therapy in Parkinson's weeks were followed out to day 365 to confirm tumor disease." J Mol Neurosci 24(3): 353-386. absence. Significantly, in this study, a minimum of 1 x The rationale behind the use of cells as 10(5) ES cells in the myocardium and 1 x 10(4) cells therapeutic modalities for neurodegenerative diseases in the skeletal muscle was observed to be requisite for in general, and in Parkinson's disease (PD) in teratoma development, suggesting that human ES cell particular, is that they will improve patient's number may be a critical factor in teratoma formation. functioning by replacing the damaged cell population. Engraftment and tumor occurrence were also observed It is reasoned that these cells will survive, grow to be highly dependent on ES cell number. We neurites, establish functional synapses, integrate best anticipate these results should yield useful insights to and durably with the host tissue mainly in the striatum, the safe and reliable application of human ES cell renew the impaired wiring, and lead to meaningful derivatives in the clinic. clinical improvement. To increase the generation of dopamine, researchers have already transplanted non- Lee, E. J., et al. (2012). "New culture system for neuronal cells, without any genetic manipulation or human embryonic stem cells: autologous after introduction of genes such as tyrosine mesenchymal stem cell feeder without exogenous hydroxylase, in animal models of PD. Because these fibroblast growth factor 2." Differentiation 83(1): 92- cells were not of neuronal origin, they developed 100. without control, did not integrate well into the brain Human embryonic stem (hES) cells have been parenchyma, and their survival rates were low. successfully maintained using human-cell feeder Clinical experiments using cell transplantation as a systems or feeder-free systems. However, despite therapy for PD have been conducted since the 1980s. advances in culture techniques, hES cells require Most of these experiments used fetal dopaminergic supplementation with fibroblast growth factor 2 (FGF- cells originating in the ventral mesencephalic tissue 2), an exogenous stemness factor, which is needed to obtained from fetuses. Although it was shown that the sustain the authentic undifferentiated status. We transplanted cells survived and some patients benefited developed a new culture system for hES cells; this from this treatment, others suffered from severe system does not require supplementation with FGF-2 dyskinesia, probably caused by the graft's excessive to obtain hES cells that are suitable for tissue and uncontrolled production and release of dopamine. engineering and regenerative medicine. This culture It is now recognized that cell-replacement strategy will system employed mesenchymal stem cells derived be effective in PD only if the transplanted cells have from hES cells (hESC-MSCs) as autologous human the same abilities, such as dopamine synthesis and feeder cells in the absence of FGF-2. The hES cell line control release, reuptake, and metabolizing dopamine, SNUhES3 cultured in this new autologous feeder as the original dopaminergic neurons. Recent studies culture system maintained the typical morphology of on embryonic and adult stem cells have demonstrated hES cells and expression of pluripotency-related that cells are able to both self-renew and produce proteins, SSEA-4, TRA-1-60, OCT4, and alkaline differentiated tissues, including dopaminergic neurons. phosphatase, without development of abnormal These new methods offer real hope for tissue karyotypes after more than 30 passages. RNA replacement in a wide range of diseases, especially PD.

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In this review we summarize the evidence of were cultured in growth media supplemented with dopaminergic neuron generation from embryonic and various TCHM extracts. The dosage-dependent effects adult stem cells, and discuss their application for cell of TCHM extracts on cell growth, including therapy in PD. proliferation and cytotoxicity, were assessed via EGFP fluorescence measurement. Seven TCHMs were Leydon, C., et al. (2013). "Human embryonic investigated, and among them Panax notoginseng (PN), stem cell-derived epithelial cells in a novel in vitro Rhizoma Atractylodis macrocephalae, Rhizoma model of vocal mucosa." Tissue Eng Part A 19(19-20): chuanxiong, and Ganoderma lucidum spores (GLS) 2233-2241. showed potential to improve mES cell proliferation. A satisfactory in vitro model of vocal fold Eleven mixtures of these four TCHMs were then mucosa does not exist, thus precluding a systematic, studied, and the results showed that the mixture of PN controlled study of vocal fold biology and and GLS had the strongest growth promoting effect, biomechanics. We sought to create a valid, increasing the specific growth rate of mES cells by reproducible three-dimensional (3D) in vitro model of 29.5% at a low dosage of 0.01% (wt/vol) PN/GLS human origin of vocal fold mucosa of human origin. (P<0.01) and 34.2% at 0.1% (wt/vol) PN/GLS (P<0.05) We hypothesized that coculture of human embryonic compared to the control. The growth promoting effect stem cell (hESC)-derived simple epithelial cells with of PN/GLS was further confirmed with ES cells primary vocal fold fibroblasts under appropriate cultured in spinner flasks. A 29.3-fold increase in the conditions would elicit morphogenesis of progenitor total cell number was achieved in the medium cells into vocal fold epithelial-like cells and creation of supplemented with 0.01% PN/GLS after 5 days, while a basement membrane. Using an in vitro prospective the control culture only gave a 16.8-fold increase. This study design, hESCs were differentiated into cells that cell-based screening method thus can provide an coexpressed the simple epithelial cell marker, keratin efficient and high-throughput way to explore potential 18 (K18), and the transcription factor, p63. These stem cell growth promoters from TCHMs. simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. Liew, C. G., et al. (2008). "PAX4 enhances beta- The cells were cultured for 3 weeks in a keratinocyte cell differentiation of human embryonic stem cells." medium at an air-liquid interface. After that time, the PLoS One 3(3): e1783. engineered mucosa demonstrated a stratified, BACKGROUND: Human embryonic stem cells squamous epithelium and a continuous basement (HESC) readily differentiate into an apparently membrane recapitulating the key morphologic and haphazard array of cell types, corresponding to all phenotypic characteristics of native vocal fold mucosa. three germ layers, when their culture conditions are hESC-derived epithelial cells exhibited positive altered, for example by growth in suspension as staining for vocal fold stratified, squamous epithelial aggregates known as embryoid bodies (EBs). However, markers, keratin 13 (K13) and 14 (K14), as well as this diversity of differentiation means that the tight junctions, adherens junctions, gap junctions, and efficiency of producing any one particular cell type is desmosomes. Despite the presence of components inevitably low. Although pancreatic differentiation has critical for epithelial structural integrity, the epithelium been reported from HESC, practicable applications for demonstrated greater permeability than native tissue the use of beta-cells derived from HESC to treat indicating compromised functional integrity. While diabetes will only be possible once techniques are further work is warranted to improve functional barrier developed to promote efficient differentiation along integrity, this study demonstrates that hESC-derived the pancreatic lineages. METHODS AND FINDINGS: epithelial progenitor cells can be engineered to create a Here, we have tested whether the transcription factor, replicable 3D in vitro model of vocal fold mucosa Pax4 can be used to drive the differentiation of HESC featuring a multilayered, terminally differentiated to a beta-cell fate in vitro. We constitutively over- epithelium. expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Li, D., et al. (2013). "Cell-based screening of Ca (2+) microfluorimetry and cell imaging to assess traditional Chinese medicines for proliferation the role of Pax4 in the differentiation and intracellular enhancers of mouse embryonic stem cells." Biotechnol Ca (2+) homeostasis of beta-cells developing in Prog 29(3): 738-744. embryoid bodies produced from such HESC. Cells A high-throughput cell-based method was expressing key beta-cell markers were isolated by developed for screening traditional Chinese herbal fluorescence-activated cell sorting after staining for medicines (TCHMs) for potential stem cell growth high zinc content using the vital dye, Newport Green. promoters. Mouse embryonic stem (mES) cells CONCLUSION: Constitutive expression of Pax4 in expressing enhanced green fluorescent protein (EGFP) HESC substantially enhances their propensity to form

147 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ putative beta-cells. Our findings provide a novel step, a 3D-co-culture system using calcium alginate foundation to study the mechanism of pancreatic beta- encapsulation and testicular somatic cells was applied cells differentiation during early human development to induce differentiation into haploid germ cells, and a and to help evaluate strategies for the generation of culture containing approximately 3% male haploid purified beta-cells for future clinical applications. germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system Lifantseva, N., et al. (2011). "Expression patterns could be used to efficiently induce GSC-like cells in of cancer-testis antigens in human embryonic stem an EB population and to promote the differentiation of cells and their cell derivatives indicate lineage tracks." ESCs into haploid male germ cells. Stem Cells Int 2011: 795239. Pluripotent stem cells can differentiate into Lim, M. N., et al. (2012). "Ex vivo expanded various lineages but undergo genetic and epigenetic SSEA-4+ human limbal stromal cells are multipotent changes during long-term cultivation and, therefore, and do not express other embryonic stem cell require regular monitoring. The expression patterns of markers." Mol Vis 18: 1289-1300. cancer-testis antigens (CTAs) MAGE-A2, -A3, -A4, - PURPOSE: The presence of multipotent human A6, -A8, -B2, and GAGE were examined in limbal stromal cells resembling mesenchymal stromal undifferentiated human embryonic stem (hES) cells, cells (MSC) provides new insights to the characteristic their differentiated derivatives, teratocarcinoma (hEC) of these cells and its therapeutic potential. However, cells, and cancer cell lines of neuroectodermal and little is known about the expression of stage-specific mesodermal origin. Undifferentiated hES cells and embryonic antigen 4 (SSEA-4) and the embryonic embryoid body cells expressed MAGE-A3, -A6, -A4, - stem cell (ESC)-like properties of these cells. We A8, and GAGEs while later differentiated derivatives studied the expression of SSEA-4 surface protein and expressed only MAGE-A8 or MAGE-A4. Likewise, the various ESC and MSC markers in the ex vivo mouse pluripotent stem cells also express CTAs of cultured limbal stromal cells. The phenotypes and Magea but not Mageb family. Despite similarity of the multipotent differentiation potential of these cells were hES and hEC cell expression patterns, MAGE-A2 and also evaluated. METHODS: Limbal stromal cells were MAGE-B2 were detected only in hEC cells but not in derived from corneoscleral rims. The SSEA-4(+) and hES cells. Moreover, our analysis has shown that SSEA-4(-) limbal stromal cells were sorted by CTAs are aberrantly expressed in cancer cell lines and fluorescence-activated cells sorting (FACS). Isolated display low tissue specificity. The identification of cells were expanded and reanalyzed for their CTA expression patterns in pluripotent stem cells and expression of SSEA-4. Expression of MSC and ESC their derivatives may be useful for isolation of markers on these cells were also analyzed by FACS. In abnormally CTA-expressing cells to improve the addition, expression of limbal epithelial and corneal safety of stem-cell based therapy. stromal proteins such as ATP-binding cassette sub- family G member 2 (ABCG2), tumour protein p63 Lim, J. J., et al. (2014). "Three-step method for (p63), paired box 6 (Pax6), cytokeratin 3 (AE5), proliferation and differentiation of human embryonic cytokeratin 10, and keratocan sulfate were evaluated stem cell (hESC)-derived male germ cells." PLoS One either by immunofluorecence staining or reverse 9(4): e90454. transcription polymerase chain reaction. Appropriate The low efficiency of differentiation into male induction medium was used to differentiate these cells germ cell (GC)-like cells and haploid germ cells from into adipocytes, osteocytes, and chondrocytes. human embryonic stem cells (hESCs) reflects the RESULTS: Expanded limbal stromal cells expressed culture method employed in the two-dimensional the majority of mesenchymal markers. These cells (2D)-microenvironment. In this study, we applied a were negative for ABCG2, p63, Pax6, AE-5, and three-step media and calcium alginate-based 3D- keratocan sulfate. After passaged, a subpopulation of culture system for enhancing the differentiation of these cells showed low expression of SSEA-4 but were hESCs into male germ stem cell (GSC)-like cells and negative for other important ESC surface markers such haploid germ cells. In the first step, embryoid bodies as Tra-1-60, Tra-1-81, and transcription factors like (EBs) were derived from hESCs cultured in EB octamer-binding transcription factor 4 (Oct4), SRY medium for 3 days and re-cultured for 4 additional (sex determining region Y)-box 2 (Sox2), and Nanog. days in EB medium with BMP4 and RA to specify Early passaged cells when induced were able to GSC-like cells. In the second step, the resultant cells differentiate into adipocytes, osteocytes and were cultured in GC-proliferation medium for 7 days. chondrocytes. CONCLUSIONS: The expanded limbal The GSC-like cells were then propagated after stromal cells showed features of multipotent MSC. selection using GFR-alpha1 and were further cultured Our study confirmed the expression of SSEA-4 by a in GC-proliferation medium for 3 weeks. In the final subpopulation of cultured limbal stromal cells.

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However, despite the expression of SSEA-4, these by immunohistochemistry (IHC), and the cells did not express any other markers of ESC. content/secretion of insulin was estimated by ELISA Therefore, we conclude that the cells did not show assay. The mice with severe combined properties of ESC. immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma Lim, W. F., et al. (2013). "Hematopoietic cell glucose restoration after + ES implantation. differentiation from embryonic and induced RESULTS: A high efficiency of gene delivery was pluripotent stem cells." Stem Cell Res Ther 4(3): 71. demonstrated when neucleofection was used in the Pluripotent stem cells, both embryonic stem cells present study; approximately 70% cells showed DsRed and induced pluripotent stem cells, are undifferentiated expression 2 d after neucleofection. By selection of cells that can self-renew and potentially differentiate medium-contained G418, the percentage of DsRed into all hematopoietic lineages, such as hematopoietic expressing cells kept high till the end of study. The stem cells (HSCs), hematopoietic progenitor cells and pancreatic differentiation seemed to be accelerated by mature hematopoietic cells in the presence of a pax4 nucleofection. When compared to the group of suitable culture system. Establishment of pluripotent cells with mock control, , mixl1, , higher stem cells provides a comprehensive model to study insulin and somatostatin levels were detected by SQ- early hematopoietic development and has emerged as a PCR 4 d after nucleofection in the group of pax4 powerful research tool to explore regenerative expressing plasmid delivery. Approximately 55% of medicine. Nowadays, HSC transplantation and neucleofected cells showed insulin expression 18 d hematopoietic cell transfusion have successfully cured after neucleofection, and only 18% of cells showed some patients, especially in malignant hematological insulin expression in mock control. The disturbance diseases. Owing to a shortage of donors and a limited was shown by nucleofected pax4 RNAi vector; only 8% number of the cells, hematopoietic cell induction from of cells expressed insulin 18 d after nucleofection. A pluripotent stem cells has been regarded as an higher IPC population was also detected in the insulin alternative source of HSCs and mature hematopoietic content by ELISA assay, and the glucose dependency cells for intended therapeutic purposes. Pluripotent was demonstrated in insulin secretion level. In the stem cells are therefore extensively utilized to animal model, improvement of average plasma facilitate better understanding in hematopoietic glucose concentration was observed in the group of development by recapitulating embryonic pax-4 expressed ES of SCID mice pretreated with STZ, development in vivo, in which efficient strategies can but no significant difference was observed in the group be easily designed and deployed for the generation of of STZ-pretreated SCID mice who were transplanted hematopoietic lineages in vitro. We hereby review the ES with mock plasmid. CONCLUSION: Enhancement current progress of hematopoietic cell induction from of IPC differentiation from EB-dissociated ES cells embryonic stem/induced pluripotent stem cells. can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also Lin, H. T., et al. (2007). "Enhancement of pancreatic differentiation-related genes can be detected insulin-producing cell differentiation from embryonic by SQ-PCR. Expression of relative genes, such as foxa stem cells using pax4-nucleofection method." World J 2, mixl 1, pdx-1, insulin 1 and somatostatin after Gastroenterol 13(11): 1672-1679. nucleofection, suggests that pax4 accelerates the whole AIM: To enhance the differentiation of insulin differentiation progress. The higher insulin production producing cell (IPC) ability from embryonic stem (ES) with glucose dependent modulation suggests that pax4 cells in vitro. METHODS: Four-day embryoid body expression can drive more mature IPCs. Although (EB)-formatted ES cells were dissociated as single further determination of the entire mechanism is cells for the followed plasmid DNA delivery. The use required, the potential of pax-4-nucleofected cells in of Nucleofector electroporator (Amaxa biosystems, medical treatment is promising. Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for Lin, I. Y., et al. (2014). "Suppression of the advanced selection. Neucleofected cells were plated on SOX2 neural effector gene by PRDM1 promotes the top of fibronectin-coated Petri dishes. Addition of human germ cell fate in embryonic stem cells." Stem Ly294002 and raised the glucose in medium at 24 h Cell Reports 2(2): 189-204. before examination. The differentiation status of these The mechanisms of transcriptional regulation cells was monitored by semi-quantitative PCR (SQ- underlying human primordial germ cell (PGC) PCR) detection of the expression of relative genes, differentiation are largely unknown. The such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, transcriptional repressor Prdm1/Blimp-1 is known to glucagons and somatostatin. The percentage of IPC play a critical role in controlling germ cell population on d 18 of the experiment was investigated specification in mice. Here, we show that PRDM1 is

149 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ expressed in developing human gonads and BACKGROUND: Asthma is affecting more than contributes to the determination of germline versus 300 million people worldwide, which represents the neural fate in early development. We show that most common chronic disease among children. We knockdown of PRDM1 in human embryonic stem cells previously found that mesenchymal stem cells (MSCs) (hESCs) impairs germline potential and upregulates derived from induced pluripotent stem cells (iPSCs) neural genes. Conversely, ectopic expression of modulated the immune response on Th2-mediated PRDM1 in hESCs promotes the generation of cells asthma in vivo and in vitro. This study further that exhibit phenotypic and transcriptomic features of evaluated the immunomodulatory effects of MSCs early PGCs. Furthermore, PRDM1 suppresses from human embryonic stem cells (hESCs) on asthma. transcription of SOX2. Overexpression of SOX2 in METHODS: Multipotent hESC-MSCs were obtained hESCs under conditions favoring germline using a feeder-free method. The hESC-MSCs were differentiation skews cell fate from the germline to the analysed for the expression of stem cell surface neural lineage. Collectively, our results demonstrate markers by flow cytometry, their differentiation that PRDM1 serves as a molecular switch to modulate potentials were analysed using in vitro trilineage the divergence of neural or germline fates through differentiation methods hESC-MSCs were repression of SOX2 during human development. transplanted into the murine model with ovalbumin (OVA)-induced airway allergic inflammation. The Lin, J., et al. (2010). "Controlled major expression levels of allergic related genes were histocompatibility complex-T cell receptor signaling measured by the mRNA PCR arrays. RESULTS: The allows efficient generation of functional, antigen- hESC-MSCs expressed classical MSC markers and specific CD8+ T cells from embryonic stem cells and held the capability of differentiation into multiple thymic progenitors." Tissue Eng Part A 16(9): 2709- mesoderm-type cell lineages. hESC-MSCs were able 2720. to suppress allergic inflammation by modulating Th2 Generation of early T cells by coculturing stem cells and eosinophils in the mice, and reversed the cells on notch-ligand-expressing OP9 stromal cells reduction of regulatory T cells. By using PCR array, 5 (OP9-DL1) has been widely reported. However, mRNAs- chemokine (C-C motif) ligand 11 (Ccl11), further differentiation of these cells into mature, Ccl24, interleukin13 (Il13), Il33 and eosinophil- antigen-specific, functional T cells, without retroviral associated, ribonuclease A family, member 11 (Ear11) transduction of T cell receptors (TcRs), is yet to be were identified the most relevant in murine airway achieved. In the thymic niche this differentiation is allergic inflammation and hESC-MSCs treatment. controlled by the interaction of developing TcRs with CONCLUSIONS: The therapeutic effects of hESC- major histocompatibility (MHC) molecules on stromal MSCs were identified in the murine model of airway cells. We hypothesized that by providing exogenous allergic inflammation with key mRNAs involved. This antigen-specific MHC/TcR signals, stem and study will provide a better understanding regarding the progenitor cells could be engineered into functional, mechanisms underlying hESC-MSCs therapeutic effector T cells specific for the same antigen. Here we application in airway allergic inflammation. demonstrate that both thymus-derived immature T cells (double positive [DP]: CD4+CD8+) and mouse Lindgren, A. G., et al. (2015). "ETV2 expression embryonic stem cells can be efficiently differentiated increases the efficiency of primitive endothelial cell into antigen-specific CD8+ T cells using either MHC derivation from human embryonic stem cells." Cell tetramers or peptide-loaded stromal cells. DP cells, Regen (Lond) 4(1): 1. following MHC/TcR signaling, retained elevated BACKGROUND: Endothelial cells line the recombination activating gene-1 levels, suggesting luminal surface of blood vessels and form a barrier continuing TcR gene rearrangement. Both DP and between the blood and other tissues of the body. Ets embryonic stem-cell-derived CD8+ T cells showed variant 2 (ETV2) is transiently expressed in both significant cytotoxic T lymphocytes activity against zebrafish and mice and is necessary and sufficient for antigen-loaded target cells, indicating that these cells vascular endothelial cell specification. Overexpression are functional. Such directed differentiation strategy of this gene in early zebrafish and mouse embryos could provide an efficient method for generating results in ectopic appearance of endothelial cells. functional, antigen-specific T cells from stem cells for Ectopic expression of ETV2 in later development potential use in adoptive T cell therapy. results in only a subset of cells responding to the signal. FINDINGS: We have examined the expression Lin, Y. D., et al. (2018). "The genes involved in pattern of ETV2 in differentiating human embryonic asthma with the treatment of human embryonic stem stem cells (ESCs) to determine when the peak of cell-derived mesenchymal stem cells." Mol Immunol ETV2 expression occurs. We show that 95: 47-55. overexpression of ETV2 in differentiating human ESC

150 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ is able to increase the number of endothelial cells renewal capacities and differentiation potentials. generated when administered during or after the However, the detailed molecular mechanisms endogenous peak of gene expression. controlling the differentiation and renewal programs in CONCLUSIONS: Addition of exogenous ETV2 to ES cells remained unclear. One of the difficulties in human ESCs significantly increased the number of understanding these mechanisms substantially results cells expressing angioblast genes without arterial or from the low efficacies of gene manipulation by venous specification. This may be a viable solution to delivering exogenous gene expression or knockdown generate in vitro endothelial cells for use in research of endogenous gene expression with small interfering and in the clinic. RNA (siRNA) in ES cells. Here we describe an optimized protocol for efficiently transfecting mouse Lindskog, H., et al. (2006). "New insights to ES cells by Effectene, a liposome-based method. The vascular smooth muscle cell and pericyte high transfection efficiency in mouse ES cells is differentiation of mouse embryonic stem cells in demonstrated in this chapter by (1) achieving a vitro." Arterioscler Thromb Vasc Biol 26(7): 1457- percentage of enhanced green fluorescence protein 1464. (EGFP) expression in >98% embryoid bodies after OBJECTIVE: The molecular mechanisms that introducing plasmids encoding the protein; (2) regulate pericyte differentiation are not well decreased SOX-2 and Oct-3/4 expression and understood, partly because of the lack of well- subsequent morphological evidences of cell characterized in vitro systems that model this process. differentiation after introducing siRNA expression for In this article, we develop a mouse embryonic stem suppressing SOX-2 and Oct-3/4, which are known to (ES) cell-based angiogenesis/vasculogenesis assay and be essential for maintenance of stem cell properties in characterize the system for vascular smooth muscle mouse ES cells; and (3) overexpression or attenuated cell (VSMC) and pericyte differentiation. METHODS expression of 14-3-3sigma to regulate cell AND RESULTS: ES cells that were cultured for 5 proliferation of mouse ES cells. days on OP9 stroma cells upregulated their transcription of VSMC and pericyte selective genes. Liour, S. S., et al. (2006). "Further Other SMC marker genes were induced at a later time characterization of embryonic stem cell-derived radial point, which suggests that vascular SMC/pericyte glial cells." Glia 53(1): 43-56. genes are regulated by a separate mechanism. Previously, we showed that radial glia-like (RG) Moreover, sequence analysis failed to identify any cells differentiated from embryonic stem (ES) cells conserved CArG elements in the vascular SMC and after retinoic acid induction (Liour and Yu, 2003: Glia pericyte gene promoters, which indicates that serum 42:109-117). In the present study, we demonstrate that response factor is not involved in their regulation. the production of RG cells from ES cells is Gleevec, a tyrosine kinase inhibitor that blocks independent of the neural differentiation protocol used. platelet-derived growth factor (PDGF) spell-receptor These ES cell-derived RG (ES-RG) cells are similar in signaling, and a neutralizing antibody against morphology to RG cells in vivo and express several transforming growth factor (TGF) beta1, beta2, and characteristic RG cell markers. The processes of these beta3 failed to inhibit the induction of vascular ES-RG cells are organized into radial arrays similar to SMC/pericyte genes. Finally, ES-derived vascular the RG scaffold in developing CNS. Expression of sprouts recruited cocultured MEF cells to pericyte- Pax6, along with other circumstantial data, suggests typical locations. The recruited cells activated that at least some of these ES-RG cells are neural expression of a VSMC- and pericyte-specific reporter progenitors. The progression of neurogenesis into gene. CONCLUSIONS: We conclude that OP9 stroma gliogenesis during the in vitro neural differentiation of cells induce pericyte differentiation of cocultured ES cells recapitulates the in vivo developmental mouse ES cells. The induction of pericyte marker process. The identification of two cell surface markers, genes is temporally separated from the induction of SSEA-1 and GM1, on both the native embryonic RG SMC genes and does not require platelet-derived cells and ES-RG cells, may facilitate purification of growth factor B or TGFbeta1 signaling. radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of Liou, J. Y., et al. (2017). "An Efficient radial glial cells is a common pathway during the Transfection Method for Differentiation and Cell neural differentiation of ES cells. Proliferation of Mouse Embryonic Stem Cells." Methods Mol Biol 1622: 139-147. Liu, H., et al. (2010). "Folic Acid Embryonic stem (ES) cells are an important supplementation stimulates notch signaling and cell source of stem cells in tissue engineering and proliferation in embryonic neural stem cells." J Clin regenerative medicine because of their high self- Biochem Nutr 47(2): 174-180.

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The present study investigated the effect of folic pancreatic beta-cell differentiation from human acid supplementation on the Notch signaling pathway embryonic stem cells." Cell Res 24(10): 1181-1200. and cell proliferation in rat embryonic neural stem The applications of human pluripotent stem cell cells (NSCs). The NSCs were isolated from E14-16 rat (hPSC)-derived cells in regenerative medicine has brain and grown as neurospheres in serum-free encountered a long-standing challenge: how can we suspension culture. Individual cultures were assigned efficiently obtain mature cell types from hPSCs? to one of 3 treatment groups that differed according to Attempts to address this problem are hindered by the the concentration of folic acid in the medium: Control complexity of controlling cell fate commitment and (baseline folic acid concentration of 4 mg/l), low folic the lack of sufficient developmental knowledge for acid supplementation (4 mg/l above baseline, Folate-L) guiding hPSC differentiation. Here, we developed a and high folic acid supplementation (40 mg/l above systematic strategy to study hPSC differentiation by baseline, Folate-H). NSCs were identified by their labeling sequential developmental genes to encompass expression of immunoreactive nestin and proliferating the major developmental stages, using the directed cells by incorporation of 5'bromo-2'deoxyuridine. Cell differentiation of pancreatic beta cells from hPSCs as a proliferation was also assessed by methyl thiazolyl model. We therefore generated a large panel of tetrazolium assay. Notch signaling was analyzed by pancreas-specific mono- and dual-reporter cell lines. real-time PCR and western blot analyses of the With this unique platform, we visualized the kinetics expression of Notch1 and hairy and enhancer of split 5 of the entire differentiation process in real time for the (Hes5). Supplementation of NSCs with folic acid first time by monitoring the expression dynamics of increased the mRNA and protein expression levels of the reporter genes, identified desired cell populations Notch1 and Hes5. Folic acid supplementation also at each differentiation stage and demonstrated the stimulated NSC proliferation dose-dependently. ability to isolate these cell populations for further Embryonic NSCs respond to folic acid characterization. We further revealed the expression supplementation with increased Notch signaling and profiles of isolated NGN3-eGFP (+) cells by RNA cell proliferation. This mechanism may mediate the sequencing and identified sushi domain-containing 2 effects of folic acid supplementation on neurogenesis (SUSD2) as a novel surface protein that enriches for in the embryonic nervous system. pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)- Liu, H., et al. (2016). "High-Efficient derived pancreatic cells and in the developing human Transfection of Human Embryonic Stem Cells by pancreas. Moreover, we captured a series of cell fate Single-Cell Plating and Starvation." Stem Cells Dev transition events in real time, identified multiple cell 25(6): 477-491. subpopulations and unveiled their distinct gene Nowadays, the low efficiency of small interfering expression profiles, among heterogeneous progenitors RNA (siRNA) or plasmid DNA (pDNA) transfection for the first time using our dual reporter hESC lines. is a critical issue in genetic manipulation of human The exploration of this platform and our new findings embryonic stem (hES) cells. Development of an will pave the way to obtain mature beta cells in vitro. efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more Liu, J., et al. (2012). "Role of miRNAs in imperative. In this study, we tried to modify the neuronal differentiation from human embryonic stem traditional transfection protocol by introducing two cell-derived neural stem cells." Stem Cell Rev 8(4): crucial processes, single-cell plating and starvation, to 1129-1137. increase the transfection efficiency in hES cells. microRNAs (miRNAs) are important modulators Furthermore, we comparatively examined the in regulating gene expression at the post- transfection efficiency of some commercially available transcriptional level and are therefore emerging as siRNA or pDNA transfection reagents in hES cells. strong mediators in neural fate determination. Here, by Our results showed that the new developed method use of the model of human embryonic stem cell markedly enhanced the transfection efficiency without (hESC)-derived neurogenesis, miRNAs involved in influencing the proliferation and pluripotency of hES the differentiation from neural stem cells (hNSC) to cells. Lipofectamine RNAiMAX exhibited much neurons were profiled and identified. hNSC were higher siRNA transfection efficiency than the other differentiated into the neural lineage, out of which the reagents, and FuGENE HD was identified as the best neuronal subset was enriched through cell sorting suitable reagent for efficient pDNA transfection of based on select combinatorial biomarkers: CD15- hES cells among the tested reagents. /CD29(Low)/CD24(High). This relatively pure and viable subpopulation expressed the neuronal marker Liu, H., et al. (2014). "Systematically labeling beta III-tubulin. The miRNA array demonstrated that a developmental stage-specific genes for the study of number of miRNAs were simultaneously induced or

152 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ suppressed in neurons, as compared to hNSC. Real- differentiation are mature in both morphology and time PCR further validated the decrease in levels of function with also well organized structures. miR214, but increase in brain-specific miR7 and miR9 in the derived neurons. For functional studies, hNSC Liu, Q., et al. (2007). "Embryonic stem cells as a were stably transduced with lentiviral vectors carrying novel cell source of cell-based biosensors." Biosens specific constructs to downregulate miR214 or to Bioelectron 22(6): 810-815. upregulate miR7. Manipulation of either miR214 or To investigate the use of embryonic stem cells as miR7 did not affect the expression of beta III-tubulin biosensor elements, mouse embryoid bodies were or neurofilament, however miR7 overexpression gave cultured on the surface of the light-addressable rise to enhanced synapsin expression in the derived potentiometric sensor and induce to in vitro neurons. This indicated that miR7 might play an differentiate into cardiomyocytes and neurons. important role in neurite outgrowth and synapse Extracellular potentials of the cells were recorded by formation. In conclusion, our data demonstrate that sensor, to detect stem cells potential applications in miRNAs function as important modulators in neural drugs screening. The experimental results show that lineage determination. These studies shed light on known cardiac stimulants (isoproterenol) and relaxants strategies to optimize in vitro differentiation (carbamylcholine) have characteristic effects on the efficiencies to mature neurons for use in drug cardiomyocytes in terms of the changes of beat discovery studies and potential future clinical frequency, amplitude and duration. Thus, the applications. embryonic stem cells potentially represent a renewable cell source for the cell-based biosensors. Liu, M. L., et al. (2005). "[Directed differentiation of Balb/C mouse embryonic stem cells Liu, S., et al. (2014). "Effect of transplantation of into pancreatic islet-like cell clusters in vitro: human embryonic stem cell-derived neural progenitor observation by atomic force microscope]." Di Yi Jun cells on adult neurogenesis in aged hippocampus." Am Yi Da Xue Xue Bao 25(4): 377-379, 402. J Stem Cells 3(1): 21-26. OBJECTIVE: To observe the morphological Adult neurogenesis occurs within the special changes of Balb/C mouse embryonic stem cells microenvironment in the subgranular zone of the following directed differentiation into pancreatic islet- hippocampus and the subventricular zone of the lateral like cell clusters (PICC) in vitro using atomic force ventricle of the mammalian brain. The special microscope (AFM). METHODS: Balb/C mouse microenvironment is known as neurogenic niches. embryonic stem cells were first cultured into Multiple cell types, including endothelial cells, embryonic bodies (EBs) and allowed to differentiate astroglia, ependymal cells, immature progeny of spontaneously for 4 days. The cells were then neural stem cells, and mature neurons, comprise the transferred to gelatin-coated dishes for the EBs to neurogenic niche. Differentiation of embryonic stem attach and spread on the tissue culture plates, in the cells towards the neural lineage results in the course of which a series of cell growth factors such as generation of different neuronal subtypes and non- basic fibroblast growth factor (bFGF), insulin-like neuronal cells (mainly astrocytes). Therefore, it is growth factor 1 (IGF-1) and nicotinamide were added reasonable to hypothesize that transplantation of into the culture medium at specific time points to human embryonic stem cell-derived neural progenitor induce directed differentiation of the stem cells into cells can be used to modify neurogenic niches for PICC. Immunocytochemistry was employed to detect facilitating adult neurogenesis. Furthermore, if the cells positive for insulin and glucagon, which were generated new neurons are functionally integrated into observed with AFM. RESULTS: The embryonic stem the existing circuits of the aged hippocampus, synaptic cells developed into cell clusters of different sizes, in plasticity in the hippocampus and learning/memory which the cells were tightly arranged. Islet B cells functions in aged mice should be enhanced. In this were numerous in the center of clusters and darkly article, we provide a comprehensive review of the stained, but fewer in the peripherals with lighter stains. concepts in the regulation of adult neurogenesis by Islet A cells expressing glucagon were relatively fewer neurogenic niches and discuss the molecular in the cell clusters, found mainly in the peripherals. mechanisms underlying the effect of stem cell Scanning of the insulin-positive clusters by AFM transplantation on adult neurogenesis in aged revealed large quantity of tissue fibers resembling hippocampus. nerve fibers that formed a reticular structure in disorderly arrangement. Numerous round granules Liu, Y., et al. (2010). "Enhancement of long-term were observed in the cytoplasm of almost identical proliferative capacity of rabbit corneal epithelial cells sizes ranging from 0.5 to 1.0 mum in diameter. by embryonic stem cell conditioned medium." Tissue CONCLUSION: The cell clusters obtained by directed Eng Part C Methods 16(4): 793-802.

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Induction of autologous stem cells for directed addition of cytokines resulted in robust erythroid -like differentiation has become a predominant method to differentiation of hEBs and these hEBs-derived obtain autologous cells for tissue reconstruction. erythroid cells possessed functions similar to mature However, the low inducing efficiency and red blood cells. contamination with other type of cells hinder its clinical utilization. Here we report a novel Lo, I. C., et al. (2016). "TRPV3 Channel phenomenon that the corneal epithelial cells maintain Negatively Regulates Cell Cycle Progression and long-term proliferative capacity and tissue-specific cell Safeguards the Pluripotency of Embryonic Stem phenotype by factors secreted from murine embryonic Cells." J Cell Physiol 231(2): 403-413. stem cells (ESCs). The rabbit corneal epithelial cells Embryonic stem cells (ESCs) have tremendous grew very well in culture medium with addition of 40% potential for research and future therapeutic purposes. ESC conditioned medium (ESC-CM). These corneal However, the calcium handling mechanism in ESCs is epithelial cells have been serially subcultured for more not fully elucidated. Aims of this study are (1) to than 20 passages and maintained high cell purity, investigate if transient receptor potential vanilloid-3 cobble-stone-like morphology, enhanced colony (TRPV3) channels are present in mouse ESCs (mESCs) forming efficiency, normal diploid, and capacity to and their subcellular localization; (2) to investigate the regenerate a functional stratified corneal epithelial role of TRPV3 in maintaining the characteristics of equivalent. More importantly, these cells did not form mESCs. Western blot and immunocytochemistry tumor, and the cells lost their proliferative capacity showed that TRPV3 was present at the endoplasmic after withdrawal of ESC-CM. The long-term reticulum (ER) of mESCs. Calcium imaging showed proliferative capacity of corneal epithelial cells is that, in the absence of extracellular calcium, TRPV3 partly resulted from enhancement of cell survival and activators camphor and 6-tert-butyl-m-cresol increased colony formation, and mediated by ectopic expression the cytosolic calcium. However, depleting the ER store of telomerase. Our findings indicate that this new in advance of activator addition abolished the calcium ESC-CM culture system can generate low- increase, suggesting that TRPV3 released calcium immunogenic autologous cells sufficiently for use in from the ER. To dissect the functional role of TRPV3, regenerative medicine. TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. Liu, Y. X., et al. (2010). "Production of erythriod The results showed that TRPV3 activation led to a cells from human embryonic stem cells by fetal liver decrease in mESC proliferation. Cell cycle analysis cell extract treatment." BMC Dev Biol 10: 85. revealed that TRPV3 activation increased the BACKGROUND: We recently developed a new percentage of cells in G2 /M phase; consistently, method to induce human stem cells (hESCs) Western blot also revealed a concomitant increase in differentiation into hematopoietic progenitors by cell the expression of inactive form of cyclin-dependent extract treatment. Here, we report an efficient strategy kinase 1, suggesting that TRPV3 activation arrested to generate erythroid progenitors from hESCs using mESCs at G2 /M phase. TRPV3 activation did not cell extract from human fetal liver tissue (hFLT) with alter the expression of pluripotency markers Oct-4, cytokines. Human embryoid bodies (hEBs) obtained of Klf4 and c-Myc, suggesting that the pluripotency was human H1 hESCs were treated with cell extract from preserved. Our study is the first study to show the hFLT and co-cultured with human fetal liver stromal presence of TRPV3 at ER. Our study also reveals the cells (hFLSCs) feeder to induce hematopoietic cells. novel role of TRPV3 in controlling the cell cycle and After the 11 days of treatment, hEBs were isolated and preserving the pluripotency of ESCs. transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics Lo Nigro, A., et al. (2017). "PDGFRalpha (+) of the erythroid cells were analyzed by flow cytometry, Cells in Embryonic Stem Cell Cultures Represent the Wright-Giemsa staining, real-time RT-PCR and In Vitro Equivalent of the Pre-implantation Primitive related functional assays. RESULTS: The erythroid Endoderm Precursors." Stem Cell Reports 8(2): 318- cells produced from hEBs could differentiate into 333. enucleated cells and expressed globins in a time- In early mouse pre-implantation development, dependent manner. They expressed not only primitive endoderm (PrE) precursors are platelet- embryonic globins but also the adult-globin with the derived growth factor receptor alpha (PDGFRalpha) maturation of the erythroid cells. In addition, our data positive. Here, we demonstrated that cultured mouse showed that the hEBs-derived erythroid cells were embryonic stem cells (mESCs) express PDGFRalpha able to act as oxygen carriers, indicating that hESCs heterogeneously, fluctuating between a PDGFRalpha+ could generate functional mature erythroid cells. (PrE-primed) and a platelet endothelial cell adhesion CONCLUSION: Cell extract exposure with the molecule 1 (PECAM1)-positive state (epiblast-primed).

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The two surface markers can be co-detected on a third contrast, none of the ES cell clones with more than 50% subpopulation, expressing epiblast and PrE of chromosomally abnormal metaphases transmitted to determinants (double-positive). In vitro, these the germline. Euploid ES cell clones cultured in vitro subpopulations differ in their self-renewal and for more than 20 passages rapidly became severely differentiation capability, transcriptional and aneuploid, and again this correlated closely with the epigenetic states. In vivo, double-positive cells percentage of chimaerism and with the number of ES contributed to epiblast and PrE, while PrE-primed cell-embryo chimaeras obtained per number of cells exclusively contributed to PrE derivatives. The blastocysts injected. At the same time, the ability of transcriptome of PDGFRalpha (+) subpopulations these clones to contribute to the germline was lost differs from previously described subpopulations and when the proportion of euploid cells dropped below shows similarities with early/mid blastocyst cells. The 50%. This study suggests that aneuploidy, rather than heterogeneity did not depend on PDGFRalpha but on 'loss of totipotency', in ES cells, is the major cause of leukemia inhibitory factor and fibroblast growth factor failure in obtaining contributions to all tissues of the signaling and DNA methylation. Thus, PDGFRalpha adult chimaera, including the germline. Because (+) cells represent the in vitro counterpart of in vivo euploidy is predictive of germline transmission, PrE precursors, and their selection from cultured karyotype analysis is crucial and time/cost saving in mESCs yields pure PrE precursors. any gene-targeting experiment.

Lobanok, E. S., et al. (2008). "[Effect of the stem Lorberbaum, D. S. and D. Gottlieb (2011). cell factor on the morphology and functional state of "Regulated expression of transgenes in embryonic mouse embryonic stem cells]." Biofizika 53(4): 646- stem cell-derived neural cells." Genesis 49(2): 66-74. 651. Discovery and characterization of gene The effect of the stem cell factor on the state of promoters, enhancers and repressor binding elements membranes and functional activity of mouse is an important research area in neuroscience. Here, embryonic stem cells cultivated in LIF (Leukemia the suitability of embryonic stem cells and their neural Inhibitory Factor) cytokine-free and LIF-containing derivatives as a model system for this research is media has been studied. It was shown that the stem investigated. Three neural transgenic constructs (from cell factor induces changes in the viscosity of the Mnx1, Fabp7, and tuba1a genes) that have been membrane lipid bilayer and increases the respiration validated in transgenic mice were inserted into rate, the ATP level, and the proliferation activity of embryonic stem cells as stable transgenes. These embryonic stem cells. An intricate character of the transgenic embryonic stem cells were differentiated LIF-dependent modification of biological effects of into neural cultures and the pattern of transgene the stem cell factor was revealed. expression across a series of inducing conditions determined. The pattern of expression matched that Longo, L., et al. (1997). "The chromosome make- predicted from transgenic mouse experiments for each up of mouse embryonic stem cells is predictive of of the three transgenes. The results show that somatic and germ cell chimaerism." Transgenic Res embryonic stem cells and their neural derivatives 6(5): 321-328. comprise a promising model for investigating the Mouse pluripotent embryonic stem (ES) cells, mechanisms that control cell- and temporal-specific once reintroduced into a mouse blastocyst, can neural gene transcription. contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. Lorincz, M. T. (2006). "Optimized neuronal However, the reasons why this contribution often differentiation of murine embryonic stem cells: role of appears erratic are poorly understood. We have tested cell density." Methods Mol Biol 330: 55-69. the notion that the chromosome make-up may be Neuronally differentiated embryonic stem (ES) important in contributing both to somatic cell cells offer a flexible and extremely potent model to chimaerism and to germ line transmission. We found study nervous system development and disease. A that the percentage of chimaerism of ES cell-embryo variety of protocols have been described to facilitate chimaeras, the absolute number of chimaeras and the neuronal differentiation. The density of ES cells used ratio of chimaeras to total pups born all correlate for neuronal differentiation has striking effects on the closely with the percentage of euploid metaphases in proportion and purity of the derived neuronal cells. the ES cell clones injected into the murine blastocyst. Here, the protocols used to optimize ES cell density in The majority of the ES cell clones that we tested, neuronal differentiation with and without an initial which were obtained from different gene targeting aggregation step are described. knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline; in

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Loring, J. F., et al. (2001). "A gene expression critical properties without LIF addition. mESCs profile of embryonic stem cells and embryonic stem cultured in BGC-CM expressed the stem cell markers cell-derived neurons." Restor Neurol Neurosci 18(2-3): Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. 81-88. Moreover, mESCs cultured in BGC-CM gave rise to Embryonic stem (ES) cells have the ability to embryoid bodies and teratomas that differentiated differentiate into a variety of cell lineages. We are effectively to diverse cell populations from endoderm, examining ES cell differentiation in vitro by using mesoderm, and ectoderm. Further, we found that cDNA microarrays to generate a molecular phenotype mESCs cultured in BGC-CM have an increased for each cell type. El4 ES cells induced by retinoic proliferation rate compared with cells grown in the acid after forming embryoid bodies differentiate mESC standard culture medium supplemented with almost exclusively to neurons. We obtained expression LIF. These findings may provide a powerful tool to patterns for about 8500 gene sequences by comparing culture mESCs for long periods of time with high mRNAs from undifferentiated ES cells and their proliferation rate while preserving its basic differentiated derivatives in a competitive characteristics, contributing to the application of these hybridization. Our results indicate that the genes cells to assess potential tissue engineering and cellular expressed by ES cells change dramatically as they therapy applications. differentiate (58 gene sequences up-regulated, 34 down-regulated). Most notably, totipotent ES cells Lotfinia, M., et al. (2016). "Effect of Secreted expressed high levels of a repressor of Hox expression Molecules of Human Embryonic Stem Cell-Derived (the polycomb homolog Mphl) and a co-repressor Mesenchymal Stem Cells on Acute Hepatic Failure (CTBP2). Expression of these genes was undetectable Model." Stem Cells Dev 25(24): 1898-1908. in differentiated cells; the ES cell-derived neurons Adult tissue-derived mesenchymal stem cells expressed a different set of transcriptional regulators, (MSCs) show tremendous promise for a wide array of as weil as markers of neurogenesis. The gene therapeutic applications predominantly through expression profiles indicate that ES cells actively paracrine activity. Recent reports showed that human suppress differentiation by transcriptional repression; embryonic stem cell (ESC)-derived MSCs are an cell-cell contact in embryoid bodies and retinoic acid alternative for regenerative cellular therapy due to treatment may overcome this suppression, allowing manufacturing large quantities of MSCs from a single expression of Hox genes and inducing a suite of donor. However, no study has been reported to neuronal genes. Gene expression profiles will be a uncover the secretome of human ESC-MSCs as useful outcome measure for comparing in vitro treatment of an acute liver failure (ALF) mouse model. treatments of differentiating ES cells and other stem We demonstrated that human ESC-MSCs showed cells. Also, knowing the molecule phenotype of similar morphology and cell surface markers transplantable cells will allow correlation of phenotype compared with bone marrow-derived MSCs. ESC- with the success of the transplant. MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic Losino, N., et al. (2011). "Maintenance of murine differentiation potential. Treatment with ESC-MSC- embryonic stem cells' self-renewal and pluripotency conditioned medium (CM) led to statistically with increase in proliferation rate by a bovine significant enhancement of primary hepatocyte granulosa cell line-conditioned medium." Stem Cells viability and increased immunomodulatory Dev 20(8): 1439-1449. interleukin-10 secretion from lipopolysaccharide- Murine embryonic stem cells (mESCs) are induced human blood mononuclear cells. Analysis of pluripotent cells that can be propagated in an the MSCs secretome by a protein array screen showed undifferentiated state in continuous culture on a feeder an association between higher frequencies of secretory layer or without feeders in the presence of leukemia proteins such as vascular endothelial growth factor inhibitory factor (LIF). Although there has been a (VEGF) and regulation of cell proliferation, cell great advance since their establishment, ESC culture is migration, the development process, immune system still complex and expensive. Therefore, finding culture process, and apoptosis. In this thioacetamide-induced conditions that maintain the self-renewal of ESCs, mouse model of acute liver injury, we observed that preventing their differentiation and promoting their systemic infusion of VEGF led to significant survival. proliferation, is still an area of great interest. In this These data have provided the first experimental work, we studied the effects of the conditioned evidence of the therapeutic potential of human ESC- medium from a bovine granulosa cell line (BGC-CM) MSC-derived molecules. These molecules show on the maintenance of self-renewal and pluripotency trophic support to hepatocytes, which potentially of mESCs. We found that this medium is able to creates new avenues for the treatment of ALF, as an maintain mESCs' self-renewal while preserving its inflammatory condition.

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the zeta-chain gene in murine embryonic stem cells. Lotfinia, M., et al. (2017). "Hypoxia Pre- The mutant alleles are predicted to result either in a Conditioned Embryonic Mesenchymal Stem Cell null phenotype or in the synthesis of a truncated Secretome Reduces IL-10 Production by Peripheral protein capable of supporting T-cell-receptor surface Blood Mononuclear Cells." Iran Biomed J 21(1): 24- expression but deficient in transmembrane signaling. 31. Both of these targeting events were recovered in a BACKGROUND: Mesenchymal stem cells single electroporation experiment with either (MSCs) are important candidates for MSC-based coelectroporation or a combination deletion/truncation cellular therapy. Current paradigm states that MSCs construct. Our results suggest that similar approaches support local progenitor cells in damaged tissue could be used to generate multiple single mutations, through paracrine signaling. Therefore, study of modifications of more than one site within a gene, or paracrine effects and secretome of MSCs could lead to subtle alterations that rely upon coconversion with the the appreciation of mechanisms and molecules selectable marker gene. associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune- Lu, J., et al. (2017). "Interactions of human modulatory effects of MSC secretomes derived from embryonic stem cell-derived cardiovascular progenitor embryonic stem cells (ESCs) and bone marrow cells cells with immobilized extracellular matrix proteins." J after hypoxia and normoxia preconditioning. Biomed Mater Res A 105(4): 1094-1104. METHODS: ESCs differentiated into MSCs and Human embryonic stem cell-derived characterized by flow cytometry and differentiation cardiovascular progenitor cells (hESC-CVPCs) hold into adipocytes and osteoblasts. The experimental great promise for cell-based therapies of heart diseases. groups consisted of individual groups of ESC-MSCs However, little is known about their niche and BM-MSCs (bone marrow-derived mesenchymal microenvironment and in particular the required stromal cells), which were preconditioned with either extracellular matrix (ECM) components. Here we hypoxia or normoxia for 24, 48 and 72 h. After screened combinations of surface-immobilized ECM collecting the cell-free medium from each treatment, proteins to identify substrates that support the secretomes were concentrated by centrifugal filters. attachment and survival of hESC-CVPCs. Covalent Using a peripheral blood mononuclear cell (PBMC) immobilization of ECM proteins laminin (Lm), assay and ELISA, IL-10 concentration in PBMCs was fibronectin (Fn), collagen I (CI), collagen III (CIII), evaluated after their incubation with different and collagen IV (CIV) in multiple combinations and secretomes from preconditioned and non- concentrations was achieved by reductive amination preconditioned MSCs. RESULTS: A significant on transparent acetaldehyde plasma polymer (AAPP) difference was observed between ESC-MSC normoxia interlayer coatings. We identified that CI, CIII, CIV, and ESC-MSC hypoxia in IL-10 concentration, and and Fn and their combinations were important for normoxia secretomes increased IL-10 secretion from hESC-CVPC attachment and survival, while Lm was PBMCs. Moreover, the strongest IL-10 secretion from dispensable. Moreover, for coatings displaying single PBMCs could be detected after the stimulation by ECM proteins, CI and CIII performed better than CIV ESC-MSC conditioned secretomes, but not BM-MSC and Fn, while coatings displaying the combined ECM conditioned medium. CONCLUSIONS: Human proteins CIII + CIV and Fn + CIII + CIV at 100 hypoxia preconditioned ESC-MSC secretome microg/mL were comparable to Matrigel in regard to indicated stronger immune-modulatory effects supporting hESC-CVPC attachment and viability. Our compared to BM-MSC conditioned medium. It could results identify ECM proteins required for hESC- be suggested that induced MSCs confer less immune- CVPCs and demonstrate that coatings displaying modulatory effects but produce more inflammatory multiple immobilized ECM proteins offer a suitable molecules such as tumor necrosis factor alpha, which microenvironment for the attachment and survival of needs further investigation. hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC- Love, P. E., et al. (1992). "Targeting of the T-cell CVPCs and therefore to advances in cardiovascular receptor zeta-chain gene in embryonic stem cells: regeneration. (c) 2017 Wiley Periodicals, Inc. J strategies for generating multiple mutations in a single Biomed Mater Res Part A: 105A: 1094-1104, 2017. gene." Proc Natl Acad Sci U S A 89(20): 9929-9933. The T-cell receptor zeta chain is a member of a Lu, M., et al. (2009). "Enhanced generation of family of related proteins that play a critical role in hematopoietic cells from human hepatocarcinoma cell- coupling cell-surface receptors to intracellular stimulated human embryonic and induced pluripotent signaling pathways. To study the role of zeta chain in stem cells." Exp Hematol 37(8): 924-936. T-cell ontogeny, we generated targeted mutations of

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OBJECTIVE: Human embryonic stem cells pose risks in their therapeutic application. EHT from (hESCs) and human induced pluripotent stem cells NT-ES cell-derived cardiomyocytes was generated (hiPSCs) constitute unique sources of pluripotent cells, through a series of improved techniques in a self-made although the molecular mechanisms involved in their mould to keep the EHTs from contraction and provide differentiation into specific lineages are just beginning static stretch simultaneously. After 7 days of static and to be defined. Here we evaluated the ability of MEDII mechanical stretching, respectively, the EHTs were (medium conditioned by HepG2 cells, a human implanted to the infarcted rat heart. Four weeks after hepatocarcinoma cell line) to selectively enhance transplantation, the suitability of EHT in heart muscle generation of mesodermal derivatives, including repair after myocardial infarction was evaluated by hematopoietic cells, from hESCs and hiPSCs. histological examination, echocardiography and MATERIALS AND METHODS: Test cells were multielectrode array measurement. The results showed exposed to MEDII prior to being placed in conditions that large (thickness/diameter, 2-4 mm/10 mm) that promote embryoid body (EB) formation. spontaneously contracting EHTs was generated Hematopoietic activity was measured by clonogenic successfully. The EHTs, which were derived from NT- assays, flow cytometry, quantitative real-time ES cells, inte grated and electrically coupled to host polymerase chain reaction of specific transcript myocardium and exerted beneficial effects on the left complementary DNAs and the ability of cells to ventricular function of infarcted rat heart. No teratoma repopulate sublethally irradiated nonobese formation was observed in the rat heart implanted with diabetic/severe combined immunodeficient EHTs for 4 weeks. NT-ES cells can be used as a interleukin-2 receptor gamma-chain-null mice for source of seeding cells for cardiac tissue engineering. almost 1 year. RESULTS: Exposure of both hESCs Large contractile EHT grafts can be constructed in and hiPSCs to MEDII induced a rapid and preferential vitro with the ability to survive after implantation and differentiation of hESCs into mesodermal elements. improve myocardial performance of infarcted rat Subsequently produced EBs showed a further hearts. enhanced expression of transcripts characteristic of multiple mesodermal lineages, and a concurrent Lu, S., et al. (2010). "Both the transplantation of decrease in endodermal and ectodermal cell transcripts. somatic cell nuclear transfer- and fertilization-derived Frequency of all types of clonogenic hematopoietic mouse embryonic stem cells with temperature- progenitors in subsequently derived EBs was also responsive chitosan hydrogel improve myocardial increased. In vivo assays of MEDII-treated hESC- performance in infarcted rat hearts." Tissue Eng Part A derived EBs also showed they contained cells able to 16(4): 1303-1315. undertake low-level but longterm multilineage The transplantation of embryonic stem cells repopulation of primary and secondary nonobese could improve cardiac function but was limited by diabetic/severe combined immunodeficient immune rejection as well as low cell retention and interleukin-2 receptor gamma-chain-null mice. survival within the ischemic tissues. The somatic cell CONCLUSIONS: MEDII treatment of hESCs and nuclear transfer (SCNT) is practical to generate hiPSCs alike selectively enhances their differentiation autologous histocompatible stem (nuclear-transferred into mesodermal cells and allows subsequent embryonic stem [NTES]) cells for diseases, but NTES generation of detectable levels of hematopoietic may be arguably unsafe for therapeutic application. progenitors with in vitro and in vivo differentiating The temperature-responsive chitosan hydrogel is a activity. suitable matrix in cell transplantation. As the scaffold, chitosan hydrogel was coinjected with NTES cells into Lu, S., et al. (2010). "Engineered heart tissue the left ventricular wall of rat infarction models. graft derived from somatic cell nuclear transferred Detailed histological analysis and echocardiography embryonic stem cells improve myocardial were performed to determine the structure and performance in infarcted rat heart." J Cell Mol Med functional consequences of transplantation. The 14(12): 2771-2779. myocardial performance in SCNT- and fertilization- The concept of regenerating diseased derived mouse ES cell transplantation with chitosan myocardium by implanting engineered heart tissue hydrogel was also compared. The results showed that (EHT) is intriguing. Yet it was limited by immune both the 24-h cell retention and 4-week graft size were rejection and difficulties to be generated at a size with significantly greater in the NTES + chitosan group contractile properties. Somatic cell nuclear transfer is than that of NTES + phosphate-buffered saline (PBS) proposed as a practical strategy for generating group (p < 0.01). The NTES cells might differentiate autologous histocompatible stem (nuclear transferred into cardiomyocytes in vivo. The heart function embryonic stem [NT-ES]) cells to treat diseases. improved significantly in the chitosan + NTES group Nevertheless, it is controversial as NT-ES cells may (fractional shortening: 28.7% +/- 2.8%) compared with

158 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ that of PBS + NTES group (fractional shortening: 25.2% A 7-day-old hypoxic-ischemic encephalopathy +/- 2.9%) at 4 weeks after transplantation (p < 0.01). (HIE) mouse model was used to study the effect of In addition, the arteriole/venule densities within the transplantation of embryonic stem (ES) cell-derived infarcted area improved significantly in the chitosan + cells on the HIE. After the inducement in vitro, the ES NTES group (280 +/- 17/mm (2)) compared with that cell-derived cells expressed Nestin and MAP-2, rather of PBS + NTES group (234 +/- 16/mm (2)) at 4 weeks than GFAP mRNA. After transplantation, ES cell- after transplantation (p < 0.01). There was no derived cells can survive, migrate into the injury site, difference in the myocardial performance in SCNT- and specifically differentiate into neurons, showing and fertilization-derived mouse ES cell transplantation improvement of the learning ability and memory of the with chitosan hydrogel. The NTES cells with chitosan HIE mouse at 8 months post-transplantation. The non- hydrogel have been proved to possess therapeutic grafted HIE mouse brain showed typical pathological potential to improve the function of infarcted heart. changes in the hippocampus and cerebral cortex, Thus the method of in situ injectable tissue where the number of neurons was reduced, while in engineering is promising clinically. the cell graft group, number of the neurons increased in the same regions. Although further study is Ma, D. K., et al. (2008). "G9a and Jhdm2a necessary to elucidate the precise mechanisms regulate embryonic stem cell fusion-induced responsible for this functional recovery, we believe reprogramming of adult neural stem cells." Stem Cells that ES cells have advantages for use as a donor source 26(8): 2131-2141. in HIE. Somatic nuclei can be reprogrammed to pluripotency through fusion with embryonic stem cells Ma, M., et al. (2010). "Major histocompatibility (ESCs). The underlying mechanism is largely complex-I expression on embryonic stem cell-derived unknown, primarily because of a lack of effective vascular progenitor cells is critical for syngeneic approaches to monitor and quantitatively analyze transplant survival." Stem Cells 28(9): 1465-1475. transient, early reprogramming events. The Donor-recipient cell interactions are essential for transcription factor Oct4 is expressed specifically in functional engraftment after nonautologous cell pluripotent stem cells, and its reactivation from transplantation. During this process, transplant somatic cell genome constitutes a hallmark for engraftment is characterized and defined by effective reprogramming. Here we developed a double interactions between transplanted cells with local and fluorescent reporter system using engineered ESCs recruited inflammatory cells. The outcome of these and adult neural stem cells/progenitors (NSCs) to interactions determines donor cell fate. Here, we simultaneously and independently monitor cell fusion provide evidence that lineage-committed embryonic and reprogramming-induced reactivation of transgenic stem cell (ESC)-derived vascular progenitor cells are Oct4-enhanced green fluorescent protein (EGFP) the target of major histocompatibility complex (MHC) expression. We demonstrate that knockdown of a class I-dependent, natural killer (NK) cell-mediated histone methyltransferase, G9a, or overexpression of a elimination in vitro and in vivo. Treatment with histone demethylase, Jhdm2a, promotes ESC fusion- interferon gamma was found to significantly induced Oct4-EGFP reactivation from adult NSCs. In upregulate MHC class I expression on ESC-derived addition, coexpression of Nanog and Jhdm2a further vascular progenitor cells, rendering them less enhances the ESC-induced Oct4-EGFP reactivation. susceptible to syngeneic NK cell-mediated killing in Interestingly, knockdown of G9a alone in adult NSCs vitro and enhancing their survival and differentiation leads to demethylation of the Oct4 promoter and potential in vivo. Furthermore, in vivo ablation of NK partial reactivation of the endogenous Oct4 expression cells led to enhanced progenitor cell survival after from adult NSCs. Our results suggest that ESC- transplantation into a syngeneic murine ischemic induced reprogramming of somatic cells occurs with hindlimb model, providing additional evidence that coordinated actions between erasure of somatic NK cells mediate ESC-derived progenitor cell epigenome and transcriptional resetting to restore transplant rejection. These data highlight the pluripotency. These mechanistic findings may guide importance of recipient immune-donor cell more efficient reprogramming for future therapeutic interactions, and indicate a functional role for MHC-I applications of stem cells. Disclosure of potential antigen expression during successful ESC-derived conflicts of interest is found at the end of this article. syngeneic transplant engraftment.

Ma, J., et al. (2007). "Treatment of hypoxic- Ma, W., et al. (2008). "Cell-extracellular matrix ischemic encephalopathy in mouse by transplantation interactions regulate neural differentiation of human of embryonic stem cell-derived cells." Neurochem Int embryonic stem cells." BMC Dev Biol 8: 90. 51(1): 57-65.

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BACKGROUND: Interactions of cells with the for regenerative medicine. Taking advantage by AFS extracellular matrix (ECM) are critical for the cells, we studied the ability of AFS cells in supporting establishment and maintenance of stem cell self- undifferentiated propagation and pluripotency of renewal and differentiation. However, the ECM is a Chinese population derived X-01 hESCs. Human AF- complex mixture of matrix molecules; little is known type amniotic fluid stem cells (hAF-AFSCs) about the role of ECM components in human transcribed genes including Activin A, TGF-beta1, embryonic stem cell (hESC) differentiation into neural Noggin and b-FGF, which involved in maintaining progenitors and neurons. RESULTS: A reproducible pluripotency and self-renewal of hESCs. Compared to protocol was used to generate highly homogenous mouse embryonic fibroblasts (MEFs), hAF-AFSCs neural progenitors or a mixed population of neural secreted higher concentration of b-FGF which was progenitors and neurons from hESCs. This defined important in hESCs culture (P < 0.05). The hESCs adherent culture system allowed us to examine the were propagated more than 30 passages on hAF- effect of ECM molecules on neural differentiation of AFSCs layer with exogenous b-FGF supplementation, hESCs. hESC-derived differentiating embryoid bodies keeping undifferentiated status. While exogenous b- were plated on Poly-D-Lysine (PDL), PDL/fibronectin, FGF was obviated, propagation of hESCs with PDL/laminin, type I collagen and Matrigel, and undifferentiated status was dependent on density of cultured in neural differentiation medium. We found hAF-AFSC feeder layer. Lower density of hAF- that the five substrates instructed neural progenitors AFSCs resulted in rapid decline in undifferentiated followed by neuronal differentiation to differing clone number, while higher ones hindered the growth degrees. Glia did not appear until 4 weeks later. Neural of colonies. The most appropriate hAF-AFSCs feeder progenitor and neuronal generation and neurite density to maintain the X-01 hESC line without outgrowth were significantly greater on laminin and exogenous b-FGF was 15-20x10(4)/well. To the best laminin-rich Matrigel substrates than on other 3 of our knowledge, this is the first study demonstrating substrates. Laminin stimulated hESC-derived neural that hAF-AFSCs could support undifferentiated progenitor expansion and neurite outgrowth in a dose- propagation and pluripotency of Chinese population dependent manner. The laminin-induced neural derived hESCs without exogenous b-FGF progenitor expansion was partially blocked by the supplementation. antibody against integrin alpha6 or beta1 subunit. CONCLUSION: We defined laminin as a key ECM Maass, K., et al. (2015). "Isolation and molecule to enhance neural progenitor generation, characterization of embryonic stem cell-derived expansion and differentiation into neurons from cardiac Purkinje cells." Stem Cells 33(4): 1102-1112. hESCs. The cell-laminin interactions involve The cardiac Purkinje fiber network is composed alpha6beta1 integrin receptors implicating a possible of highly specialized cardiomyocytes responsible for role of laminin/alpha6beta1 integrin signaling in the synchronous excitation and contraction of the directed neural differentiation of hESCs. Since laminin ventricles. Computational modeling, experimental acts in concert with other ECM molecules in vivo, animal studies, and intracardiac electrical recordings evaluating cellular responses to the composition of the from patients with heritable and acquired forms of ECM is essential to clarify further the role of cell- heart disease suggest that Purkinje cells (PCs) may matrix interactions in neural derivation of hESCs. also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in Ma, X., et al. (2014). "Human amniotic fluid isolating and studying this rare population of cells, the stem cells support undifferentiated propagation and precise role of PC in arrhythmogenesis and the pluripotency of human embryonic stem cell without b- underlying molecular mechanisms responsible for their FGF in a density dependent manner." Int J Clin Exp proarrhythmic behavior are not fully characterized. Pathol 7(8): 4661-4673. Conceptually, a stem cell-based model system might Human embryonic stem cells (hESCs) are facilitate studies of PC-dependent arrhythmia pluripotent cells which can give rise to almost all adult mechanisms and serve as a platform to test novel cell lineages. Culture system of hESCs is complex, therapeutics. Here, we describe the generation of requiring exogenous b-FGF and feeder cell layer. murine embryonic stem cells (ESC) harboring pan- Human mesenchymal stem cells (MSCs) not only cardiomyocyte and PC-specific reporter genes. We synthesize soluble cytokines or factors such as b-FGF, demonstrate that the dual reporter gene strategy may but also provide other mechanism which might play be used to identify and isolate the rare ESC-derived positive role on sustaining hESCs propagation and PC (ESC-PC) from a mixed population of cardiogenic pluripotency. Human amniotic fluid stem (AFS) cells, cells. ESC-PC display transcriptional signatures and which share characteristics of both embryonic and functional properties, including action potentials, adult stem cells, have been regarded as promising cells intracellular calcium cycling, and chronotropic

160 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ behavior comparable to endogenous PC. Our results microenvironment in the MB facilitates non-neuronal suggest that stem-cell derived PC are a feasible new differentiation, we differentiated mESCs for 8 days by platform for studies of developmental biology, disease using chemically defined basal medium. In this case, pathogenesis, and screening for novel antiarrhythmic differentiated cell morphology differed markedly therapies. between the MB and 6WP cultures: epithelial sheet- like morphology in the MB, whereas rosette Maeda, I., et al. (2013). "Max is a repressor of morphology in the 6WP. Expression of markers from germ cell-related gene expression in mouse embryonic the three germ layers indicated lower neuronal but stem cells." Nat Commun 4: 1754. higher meso- and endo-dermal differentiation of Embryonic stem cells and primordial germ cells mESCs in the MB than the 6WP culture. Moreover, (PGCs) express many pluripotency-associated genes, among various cell-secreted soluble factors, BMP4 but embryonic stem cells do not normally undergo expression was remarkably upregulated in the MB conversion into primordial germ cells. Thus, we culture. Inhibition of BMP4 signaling demonstrated predicted that there is a mechanism that represses that enhanced effect of upregulated BMP4 was primordial germ cell-related gene expression in responsible for the prominent meso- and endo-dermal embryonic stem cells. Here we identify genes involved differentiation in the MB. However, in the 6WP, in this putative mechanism, by using an embryonic downregulated BMP4 had a minimal influence on the stem cell line with a Vasa reporter in an RNA differentiation behavior. Our study demonstrated interference screen of transcription factor genes utilization of a microbioreactor to modulate the effect expressed in embryonic stem cells. We identify five of cell-secreted soluble factors by autoregulation and genes that result in the expression of Vasa when thereby inducing alternative self-capability of mESCs. silenced. Of these, Max is the most striking. Understanding and implementation of autoregulation Transcriptome analysis reveals that Max knockdown of soluble factors similar to this study will lead to the in embryonic stem cells results in selective, global development of robust culture systems to control ESC derepression of germ cell-specific genes. Max interacts behavior. with histone H3K9 methyltransferases and associates with the germ cell-specific genes in embryonic stem Mahmood, A., et al. (2011). "In vitro cells. In addition, Max knockdown results in a differentiation and maturation of human embryonic decrease in histone H3K9 dimethylation at their stem cell into multipotent cells." Stem Cells Int 2011: promoter regions. We propose that Max is part of 735420. protein complex that acts as a repressor of germ cell- Human embryonic stem cells (hESCs), which related genes in embryonic stem cells. have the potential to generate virtually any differentiated progeny, are an attractive cell source for Mahfuz Chowdhury, M., et al. (2012). "Induction transplantation therapy, regenerative medicine, and of alternative fate other than default neuronal fate of tissue engineering. To realize this potential, it is embryonic stem cells in a membrane-based two- essential to be able to control ESC differentiation and chambered microbioreactor by cell-secreted BMP4." to direct the development of these cells along specific Biomicrofluidics 6(1): 14117-1411713. pathways. Basic science in the field of embryonic Cell-secreted soluble factor signaling in a development, stem cell differentiation, and tissue diffusion dominant microenvironment plays an engineering has offered important insights into key important role on early stage differentiation of pathways and scaffolds that regulate hESC pluripotent stem cells invivo. In this study, we utilized differentiation, which have produced advances in a membrane-based two-chambered microbioreactor modeling gastrulation in culture and in the efficient (MB) to differentiate mouse embryonic stem cells induction of endoderm, mesoderm, ectoderm, and (mESCs) in a diffusion dominant microenvironment of many of their downstream derivatives. These findings the top chamber while providing enough nutrient have lead to identification of several pathways through the bottom chamber. Speculating that controlling the differentiation of hESCs into accumulated FGF4 in the small top chamber will mesodermal derivatives such as myoblasts, augment neuronal differentiation in the MB culture, mesenchymal cells, osteoblasts, chondrocytes, we first differentiated mESCs for 8 days by using a adipocytes, as well as hemangioblastic derivatives. chemically optimized culture medium for neuronal The next challenge will be to demonstrate the induction. However, comparison of cellular functional utility of these cells, both in vitro and in morphology and expression of neuronal markers in the preclinical models of bone and vascular diseases. MB with that in the 6-well plate (6WP) indicated relatively lower neuronal differentiation in the MB Maj, M., et al. (2015). "The cell cycle- and culture. Therefore, to investigate whether insulin-signaling-inhibiting miRNA expression pattern

161 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ of very small embryonic-like stem cells contributes to Moreover, the expression of Mvh, alpha6 and beta1 their quiescent state." Exp Biol Med (Maywood) integrins, Stra8 and Piwil2 genes was evaluated in co- 240(8): 1107-1111. culture groups. The molecular results of co-culture Murine Oct4(+), very small embryonic-like stem groups showed higher-but insignificant-Piwil2 and cells (VSELs), are a quiescent stem cell population significant alpha6 integrin expressions in BMP4 that requires a supportive co-culture layer to treated co-culture systems. These results confirmed proliferate and/or to differentiate in vitro. Gene that the EB system and the presence of BMP4 in a expression studies have revealed that the quiescence of STO co-culture system improve the differentiation of these cells is due to changes in expression of ESCs to germ cell. parentally imprinted genes, including genes involved in cell cycle regulation and insulin and insulin-like Makoolati, Z., et al. (2016). "Proliferation in growth factor signaling (IIS). To investigate the role of culture of primordial germ cells derived from microRNAs (miRNAs) in VSEL quiescence, we embryonic stem cell: induction by retinoic acid." performed miRNA studies in highly purified VSELs Biosci Rep 36(6). and observed a unique miRNA expression pattern in An in vitro system that supports primordial germ these cells. Specifically, we observed significant cells (PGCs) survival and proliferation is useful for differences in the expression of certain miRNA species enhancement of these cells and efficient (relative to a reference cell population), including (i) transplantation in infertility disorders. One approach is miRNA-25_1 and miRNA-19 b, whose cultivation of PGCs under proper conditions that allow downregulation has the effect of upregulating cell self-renewal and proliferation of PGCs. For this cycle checkpoint genes and (ii) miRNA-675-3 p and purpose, we compared the effects of different miRNA-675-5 p, miRNA-292-5 p, miRNA-184, and concentrations of retinoic acid (RA), and the effect of miRNA-125 b, whose upregulation attenuates IIS. PGCs co-culture (Co-C) with SIM mouse embryo- These observations are important for understanding derived thioguanine- and ouabain-resistant (STO) cells the biology of these cells and for developing efficient on the proliferation of embryonic stem cells (ESCs)- ex vivo expansion strategies for VSELs isolated from derived PGCs. One-day-old embryoid body (EB) was adult tissues. cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 Makoolati, Z., et al. (2016). "In vitro germ cell (BMP4) (SCB group) for PGC induction. For PGC differentiation from embryonic stem cells of mice: enrichment, ESCs-derived germ cells were cultured for induction control by BMP4 signalling." Biosci Rep 7 days in the presence of different doses (0-5 muM) of 36(6). RA, both in the simple and STO Co-C systems. At the The present study aims to confirm and analyse end of the culture period, viability and proliferation germ cell-related patterns and specific gene rates were assessed and expression of mouse vasa expressions at a very early stage of cell commitment. homologue (Mvh), alpha6 integrin, beta1 integrin, Following the XY cytogenetic confirmation of the stimulated by retinoic acid 8 (Stra8) and piwi CCE mouse embryonic stem cells (mESCs) line, cells (Drosophila)-like 2 (Piwil2) was evaluated using were cultured to form embryoid bodies (EBs). quantitative PCR. Also, the inductive effects were Expression pattern assessment of the mouse vasa investigated immunocytochemically with Mvh and homologue (Mvh), Stra8, alpha6 and beta1 integrin cadherin1 (CDH1) on the selected groups. genes in ESC and 1-3-day-old EB showed that all Immunocytochemistry/PCR results showed higher genes except alpha6 integrin were expressed in the expression of Mvh, the PGC-specific marker, in 3 ESC. The mean calibration of Mvh, Stra8 and alpha6 muM RA concentrations on the top of the STO feeder integrin expression significantly increased upon EB layer. Meanwhile, assessment of the Stra8 mRNA and formation compared with the ESCs. During mouse CDH1 protein, the specific makers for spermatogonia, embryogenesis, the signalling of bone morphogenetic showed no significant differences between groups. protein (BMP) is essential for germ-line formation. To Based on the results, it seems that in the presence of 3 investigate its role in germ-line induction in vitro, muM RA on top of the STO feeder layer cells, the mESCs were cultured as 1-day-old EB aggregates with majority of the cells transdifferentiated into germ cells BMP4 for 4 days in STO co-culture systems, in the were PGCs. presence and absence of 5 ng/ml BMP4. At the end of the culture period, colony assay (number and diameter) Makoolati, Z., et al. (2017). "Embryonic stem was performed and the viability percentage and cell derived germ cells induce spermatogenesis after proliferation rate was determined. There were no transplantation into the testes of an adult mouse significant statistical differences in the azoospermia model." Clin Sci (Lond) 131(18): 2381- abovementioned criteria between these two groups. 2395.

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The present study aimed to: (i) identify the sinusnode, atrium or ventricle of the heart. The cardiac exogenous factors that allow in vitro differentiation of cells of early differentiated stage expressed mouse spermatogonial stem cells (SSCs) from pacemaker-like action potentials similar to those embryonic stem cells (ESCs); (ii) evaluate the effects described for embryonic cardiomyocytes. The action of Sertoli cells in SSC enrichment; and (iii) assess the potentials of terminally differentiated cells revealed success of transplantation using in vitro differentiated shapes, pharmacological characteristics and hormonal SSCs in a mouse busulfan-treated azoospermia model. regulation inherent to adult sinusnodal, atrial or A 1-day-old embryoid body (EB) received 5 ng/ml of ventricular cells. In cardiomyocytes of intermediate bone morphogenetic protein 4 (BMP4) for 4 days, 3 differentiation state, action potentials of very long microM retinoic acid (RA) in a SIM mouse embryo- duration (0.3-1 s) were found, which may represent derived thioguanine and ouabain resistant (STO) co- developmentally controlled transitions between culture system for 7 days, and was subsequently co- different types of action potentials. Therefore, the cultured for 2 days with Sertoli cells in the presence or presented ES cell differentiation system permits the absence of a leukaemia inhibitory factor (LIF), basic investigation of commitment and differentiation of fibroblast growth factor (bFGF) and RA composition, embryonic cells into the cardiomyogenic lineage in and in the presence of these factors in simple culture vitro. medium. Higher viability, proliferation and germ cell gene expression were seen in the presence of the LIF, Manabe, K., et al. (2004). "Developmental bFGF and RA composition, on top of Sertoli cells. changes of Ni (2+) sensitivity and automaticity in Immunocytochemistry results showed higher CDH1 Nkx2.5-positive cardiac precursor cells from murine expression in this group. Sertoli co-culture had no embryonic stem cell." Circ J 68(7): 724-726. effects on SSC proliferation. Eight weeks after BACKGROUND: It is controversial which transplantation, injected cells were observed at the subtypes of T type Ca (2+) channels are implicated in base of the seminiferous tubules and in the recipient automaticity of cardiac cells during the embryonic testes. The number of spermatogonia and the mass of period. METHOD AND RESULTS: The effect of Ni the testes were higher in transplanted testes relative to (2+) on the automaticity of Nkx2.5-positive cardiac the control group. It seems that transplantation of these precursor cells sorted from embryonic stem cells cells can be useful in infertility treatment. during their differentiation was examined using patch clamp techniques. Although 40 micromol/L Ni (2+), Maltsev, V. A., et al. (1993). "Embryonic stem which is enough to block Ni (2+)sensitive T type-Ca cells differentiate in vitro into cardiomyocytes (2+) channels, decreased the spontaneous beating rate representing sinusnodal, atrial and ventricular cell in all cells in the early and intermediate stage, Ni (2+) types." Mech Dev 44(1): 41-50. did not show any effects on the automaticity of 50% of Pluripotent embryonic stem cells (ESC, ES cells) the cells in the late stage. CONCLUSION: These of line D3 were differentiated in vitro and via embryo- results indicate that Ni (2+)-sensitive T-type Ca (2+) like aggregates (embryoid bodies) of defined cell channels expressed in the Nkx2.5-positive cardiac number into spontaneously beating cardiomyocytes. precursor cells are developmentally regulated. By using RT-PCR technique, alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be Manceur, A., et al. (2007). "Flow cytometric expressed in embryoid bodies of early to terminal screening of cell-penetrating peptides for their uptake differentiation stages. The exclusive expression of the into embryonic and adult stem cells." Anal Biochem beta-cardiac MHC gene detected in very early 364(1): 51-59. differentiated embryoid bodies proved to be dependent There is an increasing appreciation of the on the number of ES cells developing in the embryoid potential of cell-penetrating peptides (CPPs) as vectors body. Cardiomyocytes enzymatically isolated from to deliver peptides, proteins, and DNA into cells. embryoid body outgrowths at different stages of However, the absolute and relative efficacy of various development were further characterized by CPPs for applications targeting stem cells and primary immunocytological and electrophysiological cells is unclear. In this study, we have developed a techniques. All cardiomyocytes appeared to be two-step loading method and a flow cytometric assay positive in immunofluorescence assays with to systematically compare the cellular uptake of five monoclonal antibodies against cardiac-specific alpha- CPPs into embryonic stem cells, neurospheres (NSs), cardiac MHC, as well as muscle-specific sarcomeric primary bone marrow hematopoietic progenitor (Sca- myosin heavy chain and desmin. The patch-clamp 1(+)Lin (-)) cells, and hematopoietic cell lines (TF-1, technique allowed a more detailed characterization of K562, and FDCP Mix). The series of CPPs tested the in vitro differentiated cardiomyocytes which were included three arginine-rich peptides; one was derived found to represent phenotypes corresponding to from HIV transactivator of transcription (TAT), one

163 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ was derived from Antennapedia (Antp), and the third derived hepatic cells, in conjunction with new and was a synthetic peptide known as protein transduction improved test methods, for evaluating drug domain 4 (PTD4). Two hydrophobic peptides were metabolism and hepatotoxicity. Recent progress on the also tested; one was derived from Kaposi fibroblast directed differentiation of human embryonic stem cells growth factor (K-FGF), and one was derived from to the functional hepatic phenotype is reported, as well PreS2 surface antigen of hepatitis B virus (PreS2- as the development and adaptation of bioreactors and TLM). Our results indicate, for the first time, that toxicity assay technologies for the testing of hepatic arginine-rich CPPs can internalize into primary NSs cells. The aim of achieving a testing platform for and bone marrow Sca-1(+)Lin (-) cells. In addition, in metabolism and hepatotoxicity assessment, based on all cell types examined, the uptake of arginine-rich hESC-derived hepatic cells, has advanced markedly in CPPs is significantly greater than that of hydrophobic the last 2-3 years. However, great challenges still peptides. remain, before such new test systems could be routinely used by the industry. In particular, we give Mandal, A., et al. (2016). "Long-term culture and an overview of results from the Vitrocellomics project cryopreservation does not affect the stability and (EU Framework 6) and discuss these in relation to the functionality of human embryonic stem cell-derived current state-of-the-art and the remaining difficulties, hepatocyte-like cells." In Vitro Cell Dev Biol Anim with suggestions on how to proceed before such in 52(2): 243-251. vitro systems can be implemented in industrial Human embryonic stem cells (hESCs) are discovery and development settings and in regulatory predicted to be an unlimited source of hepatocytes acceptance. which can pave the way for applications such as cell replacement therapies or as a model of human Mani, V., et al. (2008). "Serial in vivo positive development or even to predict the hepatotoxicity of contrast MRI of iron oxide-labeled embryonic stem drug compounds. We have optimized a 23-d cell-derived cardiac precursor cells in a mouse model differentiation protocol to generate hepatocyte-like of myocardial infarction." Magn Reson Med 60(1): 73- cells (HLCs) from hESCs, obtaining a relatively pure 81. population which expresses the major hepatic markers Myocardial regeneration with stem-cell and is functional and mature. The stability of the transplantation is a possible treatment option to reverse HLCs in terms of hepato-specific marker expression deleterious effects that occur after myocardial and functionality was found to be intact even after an infarction. Since little is known about stem cell extended period of in vitro culture and survival after transplantation, developing techniques cryopreservation. The hESC-derived HLCs have for "tracking" cells would be desirable. Iron-oxide- shown the capability to display sensitivity and an labeled stem cells have been used for in vivo tracking alteration in the level of CYP enzyme upon drug using MRI but produce negative contrast images that induction. This illustrates the potential of such assays are difficult to interpret. The aim of the current study in predicting the hepatotoxicity of a drug compound was to test a positive contrast MR technique using leading to advancement of pharmacology. reduced z-gradient rephasing (GRASP) to aid in dynamically tracking stem cells in an in vivo model of Mandenius, C. F., et al. (2011). "Toward mouse myocardial infraction. Ferumoxides and preclinical predictive drug testing for metabolism and protamine sulfate were complexed and used to hepatotoxicity by using in vitro models derived from magnetically label embryonic stem cell-derived human embryonic stem cells and human cell lines - a cardiac-precursor-cells (ES-CPCs). A total of 500,000 report on the Vitrocellomics EU-project." Altern Lab ES-CPCs were injected in the border zone of infarcted Anim 39(2): 147-171. mice and MR imaging was performed on a 9.4T Drug-induced liver injury is a common reason for scanner using T (2)*-GRE sequences (negative drug attrition in late clinical phases, and even for post- contrast) and positive contrast GRASP technique launch withdrawals. As a consequence, there is a before, 24 hours, and 1 week after ES-CPC broad consensus in the pharmaceutical industry, and implantation. Following imaging, mice were sacrificed within regulatory authorities, that a significant for histology and Perl's staining was used to confirm improvement of the current in vitro test methodologies iron within myocardium. Good correlation was for accurate assessment and prediction of such adverse observed between signal loss seen on conventional T effects is needed. For this purpose, appropriate in (2)* images, bright areas on GRASP, and the presence vivo-like hepatic in vitro models are necessary, in of iron on histology. This demonstrated the feasibility addition to novel sources of human hepatocytes. In this of in vivo stem cell imaging with positive contrast report, we describe recent and ongoing research MRI. toward the use of human embryonic stem cell (hESC)-

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Manley, N. C., et al. (2017). "Human Embryonic chimeric VN:IGF-I protein. Here we demonstrate that Stem Cell-Derived Oligodendrocyte Progenitor Cells: hES cells can be serially propagated and retain their Preclinical Efficacy and Safety in Cervical Spinal undifferentiated state in vitro for up to 35 passages in Cord Injury." Stem Cells Transl Med 6(10): 1917- our feeder-cell-free, serum-free, chemically defined 1929. media. We have utilized real-time polymerase chain Cervical spinal cord injury (SCI) remains an reaction (PCR), immunofluorescence, and important research focus for regenerative medicine fluorescence-activated cell sorter (FACS) analysis to given the potential for severe functional deficits and show that the hES cells have maintained an the current lack of treatment options to augment undifferentiated phenotype. In vitro differentiation neurological recovery. We recently reported the assays demonstrated that the hES cells retain their preclinical safety data of a human embryonic cell- pluripotent potential and the karyotype of the hES derived oligodendrocyte progenitor cell (OPC) therapy cells remains unchanged. This study demonstrates that that supported initiation of a phase I clinical trial for the novel, fully defined, synthetic VN:IGF-I chimera- patients with sensorimotor complete thoracic SCI. To containing medium described herein is a viable support the clinical use of this OPC therapy for alternative to media containing serum, and that in cervical injuries, we conducted preclinical efficacy and conjunction with laminin-coated plates facilitates safety testing of the OPCs in a nude rat model of feeder-cell-free and serum-free growth of hES. cervical SCI. Using the automated TreadScan system to track motor behavioral recovery, we found that Mantsoki, A., et al. (2016). "Gene expression OPCs significantly improved locomotor performance variability in mammalian embryonic stem cells using when administered directly into the cervical spinal single cell RNA-seq data." Comput Biol Chem 63: 52- cord 1 week after injury, and that this functional 61. improvement was associated with reduced BACKGROUND: Gene expression heterogeneity parenchymal cavitation and increased sparing of contributes to development as well as disease myelinated axons within the injury site. Based on large progression. Due to technological limitations, most scale biodistribution and toxicology studies, we show studies to date have focused on differences in mean that OPC migration is limited to the spinal cord and expression across experimental conditions, rather than brainstem and did not cause any adverse clinical differences in gene expression variance. The advent of observations, toxicities, allodynia, or tumors. In single cell RNA sequencing has now made it feasible combination with previously published efficacy and to study gene expression heterogeneity and to safety data, the results presented here supported characterise genes based on their coefficient of initiation of a phase I/IIa clinical trial in the U.S. for variation. METHODS: We collected single cell gene patients with sensorimotor complete cervical SCI. expression profiles for 32 human and 39 mouse Stem Cells Translational Medicine 2017;6:1917-1929. embryonic stem cells and studied correlation between diverse characteristics such as network connectivity Manton, K. J., et al. (2010). "A chimeric and coefficient of variation (CV) across single cells. vitronectin: IGF-I protein supports feeder-cell-free and We further systematically characterised properties serum-free culture of human embryonic stem cells." unique to High CV genes. RESULTS: Highly Stem Cells Dev 19(9): 1297-1305. expressed genes tended to have a low CV and were The therapeutic use of human embryonic stem enriched for cell cycle genes. In contrast, High CV (hES) cells is severely limited by safety concerns genes were co-expressed with other High CV genes, regarding their culture in media containing animal- were enriched for bivalent (H3K4me3 and H3K27me3) derived or nondefined factors and on animal-derived marked promoters and showed enrichment for feeder cells. Thus, there is a pressing need to develop response to DNA damage and DNA repair. culture techniques that are xeno-free, fully defined, CONCLUSIONS: Taken together, this analysis and synthetic. Our laboratory has discovered that demonstrates the divergent characteristics of genes insulin-like growth factor (IGF) and vitronectin (VN) based on their CV. High CV genes tend to form co- bind to each other resulting in synergistic short-term expression clusters and they explain bivalency at least functional effects in several cell types, including in part. keratinocytes and breast epithelial cells. We have further refined this complex into a single chimeric Marchetto, M. C., et al. (2008). "Non-cell- VN:IGF-I protein that functionally mimics the effects autonomous effect of human SOD1 G37R astrocytes obtained upon binding of IGF-I to VN. The aim of the on motor neurons derived from human embryonic current study was to determine whether hES cells can stem cells." Cell Stem Cell 3(6): 649-657. be serially propagated in feeder-cell-free and serum- Amyotrophic lateral sclerosis (ALS) is a free conditions using medium containing our novel neurodegenerative disease characterized by motor

165 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ neuron death. ALS can be induced by mutations in the tyrosinase, bestrophin1, Mertk, and displayed superoxide dismutase 1 gene (SOD1). Evidence for the phagocytic activity. CONCLUSIONS: This study non-cell-autonomous nature of ALS emerged from the confirms the retinal differentiation potential of observation that wild-type glial cells extended the BJNhem20 cells and describes an optimized protocol survival of SOD1 mutant motor neurons in chimeric to generate enriched populations of neuro-retinal and mice. To uncover the contribution of astrocytes to RPE cells. human motor neuron degeneration, we cocultured hESC-derived motor neurons with human primary Maric, D., et al. (2003). "Prospective cell sorting astrocytes expressing mutated SOD1. We detected a of embryonic rat neural stem cells and neuronal and selective motor neuron toxicity that was correlated glial progenitors reveals selective effects of basic with increased inflammatory response in SOD1- fibroblast growth factor and epidermal growth factor mutated astrocytes. Furthermore, we present evidence on self-renewal and differentiation." J Neurosci 23(1): that astrocytes can activate NOX2 to produce 240-251. superoxide and that effect can be reversed by We directly isolated neural stem cells and antioxidants. We show that NOX2 inhibitor, apocynin, lineage-restricted neuronal and glial progenitors from can prevent the loss of motor neurons caused by the embryonic rat telencephalon using a novel strategy SOD1-mutated astrocytes. These results provide an of surface labeling and fluorescence-activated cell assay for drug screening using a human ALS in vitro sorting. Neural stem cells, which did not express astrocyte-based cell model. surface epitopes characteristic of differentiation or apoptosis, were sorted by negative selection. These Mariappan, I., et al. (2015). "Enriched Cultures cells predominantly expressed fibroblast growth factor of Retinal Cells From BJNhem20 Human Embryonic receptor type 1 (FGFR-1), and a minority exhibited Stem Cell Line of Indian Origin." Invest Ophthalmol basic fibroblast growth factor (bFGF), whereas few Vis Sci 56(11): 6714-6723. expressed epidermal growth factor receptor (EGFR) or PURPOSE: To test the retinal differentiation EGF. Clonal analyses revealed that these cells potential and to establish an optimized protocol for primarily self-renewed without differentiating in enriching retinal cells from an Indian origin, human bFGF-containing medium, whereas few survived or embryonic stem cell (hESC) line, BJNhem20. expanded in EGF-containing medium. Culturing of METHODS: The BJNhem20 cells were cultured and neural stem cells in bFGF- and EGF-containing expanded under feeder-free culture conditions. medium permitted both self-renewal and Differentiation was initiated by embryoid body (EB) differentiation into neuronal, astroglial, and formation and were cultured on Matrigel in neural oligodendroglial phenotypes. In contrast, lineage- induction medium (NIM) for 1 week and further restricted progenitors were directly sorted by positive maintained in retinal differentiation medium (RDM). selection using a combination of surface epitopes After 1 month, the neuro-retinal progenitor clusters identifying neuronal or glial phenotypes or both. These located at the center of pigmented retinal patches were cells were also primarily FGFR-1(+), with few EGFR picked and cultured as suspended neurospheres in (+), and most expanded and progressed along their RDM for 3 days and subsequently on Matrigel in expected lineages in bFGF-containing medium but not neuro-retinal medium. The mildly pigmented, in EGF-containing medium. Ca (2+) imaging of self- immature retinal pigmented epithelial (RPE) cells renewing neural stem cells cultured in bFGF- were picked separately and cultured on Matrigel in containing medium revealed that bFGF, but not EGF, RPE medium (RPEM). After 1 week, the confluent induced cytosolic Ca (2+) (Ca (2+)c) responses in neuro-retinal and RPE cultures were maintained in these cells, whereas in bFGF- and EGF-containing RDM for 2 to 3 months and characterized by medium, both bFGF and EGF evoked Ca (2+)c signals immunofluorescence and RT-PCR. RESULTS: The only in differentiating progeny of these cells. The BJNhem20 cells efficiently differentiated into both results demonstrate that bFGF, but not EGF, sustains a neuro-retinal and RPE cells. The early retinal calcium-dependent self-renewal of neural stem cells progenitors expressed Nestin, GFAP, Pax6, Rx, MitfA, and early expansion of lineage-restricted progenitors, Chx10, and Otx2. Neuro-retinal cells expressed the whereas together the two growth factors permit the neural markers, Map2, beta-III tubulin, acetylated initial commitment of neural stem cells into neuronal tubulin and photoreceptor-specific markers, Crx, and glial phenotypes. rhodopsin, recoverin, calbindin, PKC, NeuroD1, RLBP1, rhodopsin kinase, PDE6A, and PDE6C. Marion, R. M., et al. (2009). "Telomeres acquire Mature RPE cells developed intense pigmentation embryonic stem cell characteristics in induced within 3 months and showed ZO-1 and Phalloidin pluripotent stem cells." Cell Stem Cell 4(2): 141-154. staining at cell-cell junctions and expressed RPE65,

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Telomere shortening is associated with organismal aging. iPS cells have been recently derived Markoullis, K., et al. (2009). "Mycoplasma from old patients; however, it is not known whether contamination of murine embryonic stem cells affects telomere chromatin acquires the same characteristics cell parameters, germline transmission and chimeric as in ES cells. We show here that telomeres are progeny." Transgenic Res 18(1): 71-87. elongated in iPS cells compared to the parental Murine embryonic stem cells (mESCs) differentiated cells both when using four (Oct3/4, inoculated at passage P13 with the mycoplasma Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) species M. hominis, M. fermentans and M. orale and reprogramming factors and both from young and aged cultured over 20 passages showed reduced growth rate individuals. We demonstrate genetically that, during and viability (P < 0.0001) compared to control mESCs. reprogramming, telomere elongation is usually Spectral karyotypic analysis of mycoplasma-infected mediated by telomerase and that iPS telomeres acquire mESCs showed a number of non-clonal chromosomal the epigenetic marks of ES cells, including a low aberrations which increased with the duration of density of trimethylated histones H3K9 and H4K20 infection. The differentiation status of the infected and increased abundance of telomere transcripts. mESCs was most affected at passage P13+6 where the Finally, reprogramming efficiency of cells derived infection was strongest and 46.3% of the mESCs from increasing generations of telomerase-deficient expressed both POU5F1 and SSEA-1 markers whereas mice shows a dramatic decrease in iPS cell efficiency, 84.8% of control mESCs expressed both markers. The a defect that is restored by telomerase reintroduction. percentage of germline chimeras from mycoplasma- Together, these results highlight the importance of infected mESCs was examined after blastocyst telomere biology for iPS cell generation and injection and embryo transfer to suitable recipients at functionality. different passages and, compared to the respective control group, was most affected at passage P13+5 (50% Markoulaki, S., et al. (2008). "Somatic cell vs. 90%; P < 0.07). Further reductions were obtained nuclear transfer and derivation of embryonic stem cells at the same passage in the percentage of litters born in the mouse." Methods 45(2): 101-114. (50% vs. 100%; P < 0.07) and in the percentage of Addressing the fundamental questions of nuclear pups born (22% vs. 45%; P < 0.001). Thirty three equivalence in somatic cells has fascinated scientists chimeras (39.8%) obtained from blastocyst injection for decades and has resulted in the development of with mycoplasma-infected mESCs showed reduced somatic cell nuclear transfer (SCNT) or animal cloning. body weight (P < 0.0001), nasal discharge, SCNT involves the transfer of the nucleus of a somatic osteoarthropathia, and cachexia. Flow cytometric cell into the cytoplasm of an egg whose own analysis of plasma from chimeras produced with chromosomes have been removed. In the mouse, mycoplasma-infected mESCs revealed statistically SCNT has not only been successfully used to address significant differences in the proportions of T-cells and the issue of nuclear equivalence, but has been used as increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) a model system to test the hypothesis that embryonic and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) stem cells (ESCs) derived from NT blastocysts have and rheumatoid factor (P < 0.01). The present data the potential to correct--through genetic indicate that mycoplasma contamination of mESCs manipulations--degenerative diseases. This paper aims affects various cell parameters, germline transmission, to provide a comprehensive description of SCNT in and postnatal development of the resulting chimeras. the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very Martin, C. H., et al. (2008). "Differences in challenging and inefficient procedure because it is lymphocyte developmental potential between human technically complex, it bypasses the normal events of embryonic stem cell and umbilical cord blood-derived gamete interactions and egg activation, and it depends hematopoietic progenitor cells." Blood 112(7): 2730- on adequate reprogramming of the somatic cell 2737. nucleus in vivo. Improvements in any or all those Hematopoietic progenitor cells derived from aspects may enhance the efficiency and applicability human embryonic stem cells (hESCs) develop into of SCNT. ESC derivation from SCNT blastocysts, on diverse mature hematopoietic lineages, including the other hand, requires the survival of only a few lymphocytes. Whereas functional natural killer (NK) successfully reprogrammed cells, which have the cells can be efficiently generated in vitro from hESC- capacity to proliferate indefinitely in vitro, maintain derived CD34(+) cells, studies of T- and B-cell correct genetic and epigenetic status, and differentiate development from hESCs have been much more into any cell type in the body--characteristics that are limited. Here, we demonstrate that despite expressing essential for transplantation therapy or any other in functional Notch-1, CD34(+) cells from hESCs did not vivo application. derive T cells when cocultured with OP9 cells

167 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ expressing Delta-like 1, or in fetal thymus organ screening of drugs, small molecules or genes that may culture. hESC-derived CD34(+) cells also did not have potential to influence beta-cell function. produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells Mathieu, J., et al. (2011). "HIF induces human when cultured in the same conditions. Notably, both embryonic stem cell markers in cancer cells." Cancer undifferentiated hESCs, and sorted hESC-derived Res 71(13): 4640-4652. populations with hematopoietic developmental Low oxygen levels have been shown to promote potential exhibited constitutive expression of ID self-renewal in many stem cells. In tumors, hypoxia is family genes and of transcriptional targets of stem cell associated with aggressive disease course and poor factor-induced signaling. These pathways both inhibit clinical outcomes. Furthermore, many aggressive T-cell development and promote NK-cell development. tumors have been shown to display gene expression Together, these results demonstrate fundamental signatures characteristic of human embryonic stem differences between hESC-derived hematopoietic cells (hESC). We now tested whether hypoxia might progenitors and analogous primary human cells. be responsible for the hESC signature observed in Therefore, hESCs can be more readily supported to aggressive tumors. We show that hypoxia, through differentiate into certain cell types than others, hypoxia-inducible factor (HIF), can induce an hESC- findings that have important implications for like transcriptional program, including the induced derivation of defined lineage-committed populations pluripotent stem cell (iPSC) inducers, OCT4, NANOG, from hESCs. SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, Massumi, M., et al. (2016). "An Abbreviated lung, colon, liver, and breast tumors). Furthermore, Protocol for In Vitro Generation of Functional Human nondegradable forms of HIFalpha, combined with the Embryonic Stem Cell-Derived Beta-Like Cells." PLoS traditional iPSC inducers, are highly efficient in One 11(10): e0164457. generating A549 iPSC-like colonies that have high The ability to yield glucose-responsive pancreatic tumorigenic capacity. To test potential correlation beta-cells from human pluripotent stem cells in vitro between iPSC inducers and HIF expression in primary will facilitate the development of the cell replacement tumors, we analyzed primary prostate tumors and therapies for the treatment of Type 1 Diabetes. Here, found a significant correlation between NANOG-, through the sequential in vitro targeting of selected OCT4-, and HIF1alpha-positive regions. Furthermore, signaling pathways, we have developed an abbreviated NANOG and OCT4 expressions positively correlated five-stage protocol (25-30 days) to generate human with increased prostate tumor Gleason score. In Embryonic Stem Cell-Derived Beta-like Cells (ES- primary glioma-derived CD133 negative cells, DBCs). We showed that Geltrex, as an extracellular hypoxia was able to induce neurospheres and hESC matrix, could support the generation of ES-DBCs markers. Together, these findings suggest that HIF more efficiently than that of the previously described targets may act as key inducers of a dynamic state of culture systems. The activation of FGF and Retinoic stemness in pathologic conditions. Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% Matsumoto, M., et al. (2008). "Potential of NKX6.1+/NGN3+ Endocrine Progenitors. The embryonic stem cell-derived neurons for synapse inhibition of Notch and tyrosine kinase receptor AXL, formation with auditory hair cells." J Neurosci Res and the treatment with Exendin-4 and T3 in the final 86(14): 3075-3085. stage resulted in 35% mono-hormonal insulin positive Recent studies have indicated that embryonic cells, 1% insulin and glucagon positive cells and 30% stem cells (ESCs) can be a source for the replacement insulin and NKX6.1 co-expressing cells. Functionally, of spiral ganglion neurons (SGNs), auditory primary ES-DBCs were responsive to high glucose in static neurons, and neurite projections from ESC-derived incubation and perifusion studies, and could secrete neurons to auditory sensory epithelia. However, the insulin in response to successive glucose stimulations. potential of ESC-derived neurons for synapse Mitochondrial metabolic flux analyses using Seahorse formation with auditory hair cells (HCs) has not been demonstrated that the ES-DBCs could efficiently elucidated. The present study therefore aimed to metabolize glucose and generate intracellular signals examine the ability of ESC-derived neurons to form to trigger insulin secretion. In conclusion, targeting synaptic connections with HCs in vitro. Mouse ESC- selected signaling pathways for 25-30 days was derived neural progenitors expressing enhanced green sufficient to generate ES-DBCs in vitro. The ability of fluorescence protein (EGFP) were cocultured with ES-DBCs to secrete insulin in response to glucose explants of cochlea sensory epithelia obtained from renders them a promising model for the in vitro postnatal day 3 mice. After a 7-day culture, neurites of ESC-derived neurons predominantly elongated toward

168 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ inner hair cells (IHCs), which play a crucial role in Matsuoka, H., et al. (2007). "Semi-quantitative sound transmission to SGNs. Immunohistochemical expression and knockdown of a target gene in single- analyses revealed the expression of synapsin 1 and cell mouse embryonic stem cells by high performance synaptophysin in the nerve endings of ESC-derived microinjection." Biotechnol Lett 29(3): 341-350. neurons adjacent to IHCs, indicating the formation of Interactions of multiple genes and associated synaptic connections. Transmission electron factors are involved in the differentiation and de- microscopy demonstrated synaptic contacts between differentiation of embryonic stem (ES) cells. nerve endings of ESC-derived neurons and IHCs. The Quantitative analysis of these genes and factors is present findings show that ESC-derived neurons can essential for the elucidation of their mechanism. To make synaptic connections with IHCs. meet this requirement, we have investigated various experimental conditions for high performance Matsunaga, Y., et al. (2008). "Activation of microinjection into mouse ES cells. A speedy and antigen-specific cytotoxic T lymphocytes by beta 2- rhythmic operation was found to be important and was microglobulin or TAP1 gene disruption and the accomplished robotically by using a single-cell introduction of recipient-matched MHC class I gene in manipulation technique and XY-address registrable allogeneic embryonic stem cell-derived dendritic culture dishes. Among many experimental parameters, cells." J Immunol 181(9): 6635-6643. the tip size of an injection capillary, the pressure A method for the genetic modification of condition, and the DNA concentration in the injection dendritic cells (DC) was previously established based capillary were of critical significance. Their optimum on the in vitro differentiation of embryonic stem (ES) values were 0.5-0.8 microm, 0.7 kgf/cm (2) for 30 ms, cells to DC (ES-DC). The unavailability of human ES and 1-100 ng/microl, respectively. Under these cells genetically identical to the patients will be a conditions, semi-quantitative control of the EGFP gene problem in the future clinical application of this expression in mouse ES cells and its knockdown was technology. This study attempted to establish a successfully demonstrated. strategy to overcome this issue. The TAP1 or beta (2)- microglobulin (beta (2)m) gene was disrupted in 129 Matsuyoshi, H., et al. (2004). "Enhanced priming (H-2(b))-derived ES cells and then expression vectors of antigen-specific CTLs in vivo by embryonic stem for the H-2K (d) or beta (2)m-linked form of K (d) cell-derived dendritic cells expressing chemokine (beta2m-K (d)) were introduced, thus resulting in two along with antigenic protein: application to antitumor types of genetically engineered ES-DC, TAP1(-/-)/K vaccination." J Immunol 172(2): 776-786. (d) ES-DC and beta (2)m (-/-)/beta (2)m-K (d) ES-DC. Dendritic cell (DC)-based immunotherapy is As intended, both of the transfectant ES-DC expressed regarded as a promising means for anti-cancer therapy. K (d) but not the intrinsic H-2(b) haplotype-derived The efficiency of T cell-priming in vivo by transferred MHC class I. Beta (2)m (-/-)/beta (2)m-K (d) and DCs should depend on their encounter with T cells. In TAP1(-/-)/K (d) ES-DC were not recognized by pre- the present study, we attempted to improve the activated H-2(b)-reactive CTL and did not prime H- capacity of DCs to prime T cells in vivo by genetic 2(b) reactive CTL in vitro or in vivo. Beta (2)m (-/- modification to express chemokine with a T cell- )/beta (2)m-K (d) ES-DC and TAP1(-/-)/K (d) ES-DC attracting property. For genetic modification of DCs, had a survival advantage in comparison to beta (2)m we used a recently established method to generate DCs (+/-)/beta (2)m-K (d) ES-DC and TAP1(+/+)/K (d) from mouse embryonic stem cells. We generated ES-DC, when transferred into BALB/c mice. K (d)- double-transfectant DCs expressing a chemokine along restricted RSV-M2-derived peptide-loaded ES-DC with a model Ag (OVA) by sequential transfection of could prime the epitope-specific CTL upon injection embryonic stem cells, and then induced differentiation into the BALB/c mice, irrespective of the cell surface to DCs. We comparatively evaluated the effect of three expression of intrinsic H-2(b) haplotype-encoded kinds of chemokines; secondary lymphoid tissue MHC class I. Beta (2)m (-/-)/beta (2)m-K (d) ES-DC chemokine (SLC), monokine induced by IFN-gamma were significantly more efficient in eliciting immunity (Mig), and lymphotactin (Lptn). All three types of against RSV M2 protein-expressing tumor cells than double transfectant DCs primed OVA-specific CTLs beta (2)m (+/-)/beta (2)m-K (d) ES-DC. The in vivo more efficiently than did DCs expressing only modification of the beta (2)m or TAP gene may OVA, and the coexpression of SLC or Lptn was more therefore be an effective strategy to resolve the effective than that of Mig. Immunization with DCs problem of HLA class I allele mismatch between expressing OVA plus SLC or Mig provided protection human ES or induced pluripotent stem cells and the from OVA-expressing tumor cells more potently than recipients to be treated. did immunization with OVA alone, and SLC was more effective than Mig. In contrast, coexpression of Lptn gave no additive effect on protection from the tumor.

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Collectively, among the three chemokines, expression support neuronal differentiation in the adult rat brain." of SLC was the most effective in enhancing antitumor Stem Cells 33(2): 491-502. immunity by transferred DCs in vivo. The findings A neurogenic niche can be identified by the provide useful information for the development of a proliferation and differentiation of its naturally potent DC-based cellular immunotherapy. residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable Mattei, C., et al. (2018). "Generation of Neural for neural differentiation, other than the areas of active Organoids from Human Embryonic Stem Cells Using neurogenesis, exist in the adult brain. Embryoid body the Rotary Cell Culture System: Effects of (EB) cells derived from embryonic stem cells (ESCs) Microgravity on Neural Progenitor Cell Fate." Stem are endowed with a high potential to respond to Cells Dev 27(12): 848-857. specification and neuralization signals of the embryo. Progress in aeronautics and spaceflight Hence, to identify microenvironments in the postnatal technologies requires in parallel further research on and adult rat brain with the capacity to support how microgravity may affect human tissue. To date, neuronal differentiation, we transplanted dissociated little is known about the effects of microgravity on EB cells to conventional neurogenic and non- human development. In this study we used the rotary neurogenic regions. Our results show a neuronal cell culture system to investigate whether microgravity differentiation pattern of EB cells that was dependent supports the generation and maintenance of neural on the host region. Efficient neuronal differentiation of organoids derived from human embryonic stem cells EB cells occurred within an adjacent region to the (hESCs) as a model of human brain development. Our rostral migratory stream. EB cell differentiation was results show that although neural organoids could be initially patchy and progressed toward an even generated and maintained in microgravity conditions, distribution along the graft by 15-21 days post- there were changes in expression of rostral-caudal transplantation, giving rise mostly to GABAergic neural patterning genes and cortical markers compared neurons. EB cells in the striatum displayed a lower to organoids generated in standard conditions. This level of neuronal differentiation and derived into a phenomenon was also observed in hESC-derived significant number of astrocytes. Remarkably, when cortical organoids exposed to microgravity for EB cells were transplanted to the striatum of adult rats relatively shorter periods. These results are one of the after a local ischemic stroke, increased number of first for analyzing human neurogenesis in a neuroblasts and neurons were observed. Unexpectedly, microgravity environment. we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a Matveeva, N. M., et al. (2015). "Generation of robust neurogenic environment. Therefore, neurally mouse chimeras with high contribution of tetraploid uncommitted cells derived from ESCs can detect embryonic stem cells and embryonic stem cell- regions that support neuronal differentiation within the fibroblast hybrid cells." Methods Mol Biol 1313: 61- adult brain, a fundamental step for the development of 71. stem cell-based replacement therapies. The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation Mazzilli, J. L., et al. (2017). "Derivation and embryos offers the unique possibility of combining ES characterization of the human embryonic stem cell line cells with pre-implantation embryos to generate CR-4: Differentiation to human retinal pigment chimeras, thus facilitating the creation of a bridge epithelial cells." Stem Cell Res 18: 37-40. between in vitro and in vivo investigations. Genomic The CR-4 human embryonic stem cell line was manipulation using ES cells and homologous derived from the inner cell mass of a developing recombination is one of the most outstanding scientific blastocyst. This cell line has been adapted to grow in achievements, resulting in the generation of animals feeder-free conditions and is especially well-suited for with desirable genome modifications. As such, the differentiation to retinal pigment epithelium. The line generation of ES cells with different ploidy via cell demonstrates a normal human 46,XX female fusion also deserves much attention because this karyotype. Pluripotency was assessed through qRT- approach allows for the production of chimeras that PCR for expression of NANOG, OCT-4, and SOX-2. contain somatic cells with various ploidy. Therefore, A teratoma assay was performed and results were this is a powerful tool that can be used to study the positive for all three germ layers. Testing for role of polyploidy in the normal development of Mycoplasma was negative. mammals. McCabe, K. L., et al. (2015). "Efficient Maya-Espinosa, G., et al. (2015). "Mouse Generation of Human Embryonic Stem Cell-Derived embryonic stem cell-derived cells reveal niches that

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Corneal Endothelial Cells by Directed could develop patent lumens. We have shown that Differentiation." PLoS One 10(12): e0145266. endothelial cells derived, purified and expanded in AIM: To generate human embryonic stem cell vitro from ES cells sustain an important endothelial derived corneal endothelial cells (hESC-CECs) for cell function, the ability to undergo vasculogenesis in transplantation in patients with corneal endothelial collagen gels, indicating that endothelial products dystrophies. MATERIALS AND METHODS: Feeder- derived in vitro from stem cells could be useful in free hESC-CECs were generated by a directed regenerative medicine applications. differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal McCloskey, K. E., et al. (2006). "Embryonic endothelial markers, and microarray analysis of gene stem cell-derived endothelial cells may lack complete expression. RESULTS: hESC-CECs were nearly functional maturation in vitro." J Vasc Res 43(5): 411- identical morphologically to primary human corneal 421. endothelial cells, expressed Zona Occludens 1 (ZO-1) Stem cell therapies will only become clinically and Na+/K+ATPasealpha1 (ATPA1) on the apical relevant if the stem cells differentiated in vitro surface in monolayer culture, and produced the key function as their in vivo counterparts. Here, we proteins of Descemet's membrane, Collagen employed our previously developed techniques for VIIIalpha1 and VIIIalpha2 (COL8A1 and 8A2). deriving endothelial cells (>96% purity) from mouse Quantitative PCR analysis revealed expression of all embryonic stem cells (ESC) and compared these with corneal endothelial pump transcripts. hESC-CECs mouse aortic endothelial cells (MAEC) obtained from were 96% similar to primary human adult CECs by thoracic aortas. Immunocytochemical analysis of ESC- microarray analysis. CONCLUSION: hESC-CECs are derived endothelial cells (EC) demonstrates that both morphologically similar, express corneal endothelial cell types are positive for the EC markers endothelial cell markers and express a nearly identical nitric oxide synthase (eNOS), Flk-1, Flt-1, vascular complement of genes compared to human adult endothelial cadherin (VEcad), platelet-endothelial cell corneal endothelial cells. hESC-CECs may be a adhesion molecule-1 (PECAM-1), and CD34. suitable alternative to donor-derived corneal However, ESC-derived EC express slightly lower endothelium. levels of PECAM-1 and VE-cadherin, and significantly lower levels of acetylated low-density McCloskey, K. E., et al. (2005). "Use of lipoprotein (LDL) uptake and von Willebrand factor. embryonic stem cell-derived endothelial cells as a cell Although ESC-derived EC do express VE-cadherin, source to generate vessel structures in vitro." Tissue the VE-cadherin in the ESC-derived EC did not Eng 11(3-4): 497-505. localize as well at the cell-cell junctions as in the Embryonic stem (ES) cells could potentially MAEC. Interestingly, ESC-derived EC express much serve as an excellent cell source for various greater levels of the endothelial and hematopoietic applications in regenerative medicine and tissue stem cell marker CD34 and vasculogenic and engineering. Our laboratory is particularly interested in angiogenic sprouting than MAEC. These results generating a reproducible endothelial cell source for indicate that ESC-derived EC share some key the development of prevascularized materials for characteristics of 'mature' EC, while retaining markers tissue/organ reconstruction. After developing methods of alternate phenotypes including immature to isolate highly purified (>96%) proliferating endothelium. populations of endothelial cells from mouse embryonic stem cells, we tested their ability to form McElroy, S. L. and R. A. Reijo Pera (2008). three-dimensional (3-D) vascular structures in vitro. "Preparation of mouse embryonic fibroblast feeder The ES cell-derived endothelial cells were embedded cells for human embryonic stem cell culture." CSH in 3-D collagen gel constructs with rat tail collagen Protoc 2008: pdb prot5041. type I (2 mg/mL) at a concentration of 10(6) cells/mL INTRODUCTIONEmbryonic stem cells (ESCs) of gel. The gels were observed daily with a phase- are derived from the inner cell mass of day 5-6 contrast microscope to analyze the time course for blastocysts. ESCs are pluripotent, meaning that they endothelial cell assembly. The first vessels were are able to differentiate into all derivatives of the three observed between days 3 and 5 after gel construct primary germ layers (ectoderm, endoderm, and formation. The number and complexity of structures mesoderm). In order to maintain the undifferentiated steadily increased, reaching a maximum before status of human ESCs (hESCs), feeder cells are used beginning to regress. By 2 weeks, all vessel-like to provide both a suitable attachment substrate and structures had regressed back to single cells. Histology critical soluble factors. Since the first hESC lines were and fluorescent images of the vessel-like structures established on mouse embryonic fibroblasts (MEFs), verified that tube structures were multicellular and mitotically inactivated MEFs have commonly been

171 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ used for supporting the culture of undifferentiated contribute to the germline have not been demonstrated hESCs. Some previous studies suggest that MEFs may in any species but mouse. Among mouse strains, support hESC growth better than the human feeder genetic background strongly affects the efficiency of cells typically isolated from post-natal tissues. This ES isolation, and almost all ES lines in use are derived protocol describes a method for isolation and from strain 129 (refs 1,4,5) or, less commonly, irradiation of MEFs for use in hESC culture. C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of McHugh, P. C., et al. (2008). "Proteomic analysis CBA-derived ES lines. Hence, CBA mice may provide of embryonic stem cell-derived neural cells exposed to a convenient model of ES isolation in other species. In the antidepressant paroxetine." J Neurosci Res 86(2): ES derivation it is critical that the primary explant be 306-316. cultured for a sufficient time to allow multiplication of Antidepressant drugs can have significant effects ES cell progenitors, yet without allowing extensive on the mood of a patient suffering from major differentiation. Thus, differences in ES derivation depression or other disorders. The pharmacological between mouse strains may reflect differences in the actions of these drugs generally affect the uptake or control of ES progenitor cells by other lineages within metabolism of the neurotransmitters serotonin, the embryo. Here we describe a strategy to noradrenalin, and, to a lesser extent, dopamine. continuously remove differentiated cells by drug However, many aspects of antidepressant action are selection, which generates germline competent ES not understood. We conducted a proteomic analysis in lines from genotypes that are non-permissive in the a neuronal cell culture model in an attempt to identify absence of selection. molecules important to the operation of pathways functionally relevant to antidepressant action. The Meamar, R., et al. (2010). "Toxicity of ecstasy model involved generating cultures containing mixed (MDMA) towards embryonic stem cell-derived neural and glial cells by controlled differentiation of cardiac and neural cells." Toxicol In Vitro 24(4): mouse embryonic stem cells, followed by exposure to 1133-1138. 1 microM paroxetine for 14 days. After antidepressant "Ecstasy" or methylenedioxymethamphetamine exposure, we observed increased expression or (MDMA) is primarily a recreational drug commonly modification of sepiapterin reductase (SPR), heat used during the child bearing period, thus, there is a shock protein 9A, RAS and EF-hand domain major concern regarding the embryonic and fetal containing, and protein disulfide isomerase associated toxicity of this drug. Here, we report the cardio- and 3 and decreased expression or modification of creatine neuro-toxic effects of MDMA on beating embryoid kinase, actin, prohibitin, a T-cell receptor alpha chain, bodies (EBs) and neural cell-containing EBs derived defensin-related cryptdin 5, and the intermediate from mouse embryonic stem cell (ESCs). Based on our filament proteins glial fibrillary acidic protein and linear discriminate function, MDMA is considered to vimentin. SPR, the most strongly up-regulated protein be a moderate or weak teratogen. Moreover, the observed, controls production of tetrahydrobiopterin, generation of EBs with neural cell morphology and the an essential cofactor for the synthesis of many expression of MAP2, a mature neuron marker, neurotransmitters including serotonin, making it a decrease more when MDMA is administered during plausible and intriguing candidate protein for the EB formation stage rather than post-plated EBs. In involvement in mood control and antidepressant drug addition, the ID50 (inhibition of differentiation) of action. SPR and the other proteins identified may EBs with neural cell morphology is less than cardiac represent links to molecular processes of importance to cells. In conclusion, MDMA causes a marked mood dysregulation and control, and their respective reduction in beating cardiomyocytes and neurons in genes may be novel candidates for the study of ESC cultures, and this drug has a more potent toxicity antidepressant pharmacogenetics. on neural rather than cardiac cell differentiation.

McWhir, J., et al. (1996). "Selective ablation of Medine, C. N., et al. (2008). Robust generation of differentiated cells permits isolation of embryonic hepatocyte-like cells from human embryonic stem cell stem cell lines from murine embryos with a non- populations. StemBook. Cambridge (MA). permissive genetic background." Nat Genet 14(2): Despite progress in modelling human drug 223-226. toxicity, many compounds fail during clinical trials Embryonic stem (ES) cells enable the due to unpredicted side effects. The cost of clinical engineering of precise modifications to the mouse studies are substantial, therefore it is essential that genome by gene targeting. Although there are reports more predictive toxicology screens are developed and of cultured cell contributions to chimaeras in golden deployed early on in drug development (Greenhough hamster, rat and pig, definitive ES cell lines which et al 2010). Human hepatocytes represent the current

172 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ gold standard model for evaluating drug toxicity, but and induced pluripotent stem cells." Methods Mol Biol are a limited resource that exhibit variable function. 1154: 563-578. Therefore, the use of immortalised cell lines and Although 10-15 % of couples are infertile, little animal tissue models are routinely employed due to is known of the diverse, underlying pathologies in men their abundance. While both sources are informative, and women with poor germ cell production; they are limited by poor function, species variability furthermore, for those with few or no high-quality and/or instability in culture (Dalgetty et al 2009). germ cells, there are few options available for Pluripotent stem cells (PSCs) are an attractive treatment. Thus, over the last decade, concerted efforts alternative source of human hepatocyte like cells have been aimed at developing a biological system to (HLCs) (Medine et al 2010). PSCs are capable of self probe the fundamentals of human egg and sperm renewal and differentiation to all somatic cell types production via pluripotent stem cell cells with the found in the adult and thereby represent a potentially hopes of informing clinical decisions and ultimately inexhaustible source of differentiated cells. We have providing alternative methods for therapy which may developed a procedure that is simple, highly efficient, include developing a source of germ cells ultimately amenable to automation and yields functional human for reproductive purposes. HLCs (Hay et al 2008; Fletcher et al 2008; Hannoun et al 2010; Payne et al 2011 and Hay et al 2011). We Mehat, M. S., et al. (2018). "Transplantation of believe our technology will lead to the scalable Human Embryonic Stem Cell-Derived Retinal production of HLCs for drug discovery, disease Pigment Epithelial Cells in Macular Degeneration." modeling, the construction of extra-corporeal devices Ophthalmology. and possibly cell based transplantation therapies. PURPOSE: Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial Medine, C. N., et al. (2011). "Robust generation (RPE) cells offers the potential for benefit in macular of hepatocyte-like cells from human embryonic stem degeneration. Previous trials have reported improved cell populations." J Vis Exp (56): e2969. visual acuity (VA), but lacked detailed analysis of Despite progress in modelling human drug retinal structure and function in the treated area. toxicity, many compounds fail during clinical trials DESIGN: Phase 1/2 open-label dose-escalation trial to due to unpredicted side effects. The cost of clinical evaluate safety and potential efficacy studies are substantial, therefore it is essential that (clinicaltrials.gov identifier, NCT01469832). more predictive toxicology screens are developed and PARTICIPANTS: Twelve participants with advanced deployed early on in drug development (Greenhough Stargardt disease (STGD1), the most common cause of et al 2010). Human hepatocytes represent the current macular degeneration in children and young adults. gold standard model for evaluating drug toxicity, but METHODS: Subretinal transplantation of up to 200 are a limited resource that exhibit variable function. 000 hESC-derived RPE cells with systemic Therefore, the use of immortalised cell lines and immunosuppressive therapy for 13 weeks. MAIN animal tissue models are routinely employed due to OUTCOME MEASURES: The primary end points their abundance. While both sources are informative, were the safety and tolerability of hESC-derived RPE they are limited by poor function, species variability cell administration. We also investigated evidence of and/or instability in culture (Dalgetty et al 2009). the survival of transplanted cells and measured retinal Pluripotent stem cells (PSCs) are an attractive structure and function using microperimetry and alternative source of human hepatocyte like cells spectral-domain OCT. RESULTS: Focal areas of (HLCs) (Medine et al 2010). PSCs are capable of self subretinal hyperpigmentation developed in all renewal and differentiation to all somatic cell types participants in a dose-dependent manner in the found in the adult and thereby represent a potentially recipient retina and persisted after withdrawal of inexhaustible source of differentiated cells. We have systemic immunosuppression. We found no evidence developed a procedure that is simple, highly efficient, of uncontrolled proliferation or inflammatory amenable to automation and yields functional human responses. Borderline improvements in best-corrected HLCs (Hay et al 2008; Fletcher et al 2008; Hannoun et VA in 4 participants either were unsustained or were al 2010; Payne et al 2011 and Hay et al 2011). We matched by a similar improvement in the untreated believe our technology will lead to the scalable contralateral eye. Microperimetry demonstrated no production of HLCs for drug discovery, disease evidence of benefit at 12 months in the 12 participants. modeling, the construction of extra-corporeal devices In one instance at the highest dose, localized retinal and possibly cell based transplantation therapies. thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Medrano, J. V., et al. (2014). "Human germ cell Participant-reported quality of life using the 25-item differentiation from pluripotent embryonic stem cells National Eye Institute Visual Function Questionnaire

173 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ indicated no significant change. CONCLUSIONS: development and disease, and perform drug-screening Subretinal hyperpigmentation is consistent with the and toxicity studies. The realization of these potentials, survival of viable transplanted hESC-derived RPE however, depends on expanding our knowledge about cells, but may reflect released pigment in their absence. the cellular and molecular mechanisms that regulate The findings demonstrate the value of detailed analysis self-renewal and lineage specification. Here, we of spatial correlation of retinal structure and function briefly highlight the potential applications of hESC in determining with appropriate sensitivity the impact and review how flow cytometry has contributed to the of cell transplantation and suggest that intervention in initial characterization of both undifferentiated hESC early stage of disease should be approached with cultures and hematopoietic development arising from caution. Given the slow rate of progressive hESC. We envision that a combination of state-of-the- degeneration at this advanced stage of disease, any art technologies, including cytomics, proteomics and protection against further deterioration may be evident genomics, will be instrumental in moving the field only after a more extended period of observation. forward, ultimately lending invaluable knowledge to research areas such as human embryology, oncology Menchon, C., et al. (2011). "The cell cycle and immunology. inhibitor p27Kip (1) controls self-renewal and pluripotency of human embryonic stem cells by Menendez, P., et al. (2004). "Retroviral regulating the cell cycle, Brachyury and Twist." Cell transduction of hematopoietic cells differentiated from Cycle 10(9): 1435-1447. human embryonic stem cell-derived CD45(neg)PFV The continued turn over of human embryonic hemogenic precursors." Mol Ther 10(6): 1109-1120. stem cells (hESC) while maintaining an Human embryonic stem cells (hESCs) provide a undifferentiated state is dependent on the regulation of unique opportunity to study molecular mechanisms the cell cycle. Here we asked the question if a single that regulate specification of the hematopoietic lineage cell cycle gene could regulate the self-renewal or in the human. Exploitation of this model using pluripotency properties of hESC. We identified that transgenic strategies depends on the ability to target the protein expression of the p27(Kip) (1) cell cycle cells of the hematopoietic lineage effectively and inhibitor is low in hESC cells and increased with establish stable transgene expression. Here, a recently differentiation. By adopting a gain and loss of function defined subpopulation of endothelial-like precursors strategy we forced or reduced its expression in derived from hESCs that is exclusively responsible for undifferentiating conditions to define its functional hematopoietic cell fate (CD45(neg)PFV) is shown to role in self-renewal and pluripotency. Using express GALVR-1 receptor and be efficiently undifferentiation conditions, overexpression of transduced with GALV-pseudotyped retrovirus. p27(Kip) (1) in hESC lead to a G (1)phase arrest with Retroviral transduction, measured by enhanced green an enlarged and flattened hESC morphology and fluorescent protein, of hESC-derived CD45(+) cells consequent loss of self-renewal ability. Loss of differentiated from isolated CD45(neg)PFV precursors p27(Kip) (1) caused an elongated/scatter cell-like was 26.5 +/- 13% with 5.6 +/- 4% of these cells phenotype involving up-regulation of Brachyury and coexpressing CD34. An average of 17.5% of Twist gene expression. We demonstrate the novel clonogenic hematopoietic progenitors derived from finding that p27(Kip) (1) protein occupies the Twist1 CD45(neg)PFV precursors expressed the retroviral gene promoter and manipulation of p27(Kip) (1) by transgene. Addition of serum to cultures after gain and loss of function is associated with Twist gene retroviral exposure supported transgene expression in expression changes. These results define p27(Kip) (1) resulting hematopoietic cells derived from hemogenic expression levels as critical for self-renewal and CD45(neg)PFV precursors. Our study represents the pluripotency in hESC and suggest a role for p27(Kip) first report to demonstrate that retroviral transduction (1) in controlling an epithelial to mesenchymal systems, similar to those used currently in clinical transition (EMT) in hESC. gene therapy protocols, are capable of efficient transduction of hematopoietic progenitors derived Menendez, P., et al. (2006). "Human embryonic from hESCs. stem cells: A journey beyond cell replacement therapies." Cytotherapy 8(6): 530-541. Meng, G., et al. (2008). "A novel method for Success in the derivation of human embryonic generating xeno-free human feeder cells for human stem cell (hESC) lines has opened up a new area of embryonic stem cell culture." Stem Cells Dev 17(3): research in biomedicine. Human ESC not only raise 413-422. hope for cell replacement therapies but also provide a Long-term cultures of human embryonic stem potential novel system to better understand early (hES) cells require a feeder layer for maintaining cells human normal development, model human abnormal in an undifferentiated state and increasing karyotype

174 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ stability. In routine hES cell culture, mouse embryonic Vascular derivatives of human embryonic stem fibroblast (MEF) feeders and animal component- cells (hESC) are being developed as sources of tissue- containing media (FBS or serum replacement) are specific cells for organ regeneration. However, commonly used. However, the use of animal materials identity of developmental pathways that modulate the increases the risk of transmitting pathogens to hES specification of endothelial cells is not known yet. We cells and therefore is not optimal for use in cultures studied phosphatidylinositol 3-kinase (PI3K)-Forkhead intended for human transplantation. There are other box O transcription factor 1A (FOXO1A) pathways limitations with conventional feeder cells, such as during differentiation of hESC toward endothelial MEFs, which have a short lifespan and can only be lineage and on proliferation, maturation, and cell death propagated five to six passages before senescing. of hESC-derived endothelial cells (hESC-EC). During Several groups have investigated maintaining existing differentiation of hESC, expression of FOXO1A hES cell lines and deriving new hES cell lines on transcription factor was linked to the expression of a human feeder layers. However, almost all of these cluster of angiogenesis- and vascular remodeling- human source feeder cells employed in previous related genes. PI3K inhibitor LY294002 activated studies were derived and cultured in animal FOXO1A and induced formation of CD31(+) hESC- component conditions. Even though one group EC. In contrast, differentiating hESC with silenced previously reported the derivation and culture of FOXO1A by small interfering RNA (siRNA) showed human foreskin fibroblasts (HFFs) in human serum- lower mRNA levels of CD31 and angiopoietin2. containing medium, this medium is not optimal LY294002 decreased proliferative activity of purified because HFFs routinely undergo senescence after 10 hESC-EC, while FOXO1A siRNA increased their passages when cultured in human serum. In this study proliferation. LY294002 inhibits migration and tube we have developed a completely animal-free method formation of hESC-EC; in contrast, FOXO1A siRNA to derive HFFs from primary tissues. We demonstrate increased in vitro tube formation activity of hESC-EC. that animal-free (AF) HFFs do not enter senescence After in vivo conditioning of cells in athymic nude rats, within 55 passages when cultured in animal-free cells retain their low FOXO1A expression levels. conditions. This methodology offers alternative and PI3K/FOXO1A pathway is important for function and completely animal-free conditions for hES cell culture, survival of hESC-EC and in the regulation of thus maintaining hES cell morphology, pluripotency, endothelial cell fate. Understanding these properties of karyotype stability, and expression of pluripotency hESC-EC may help in future applications for treatment markers. Moreover, no difference in hES cell of injured organs. maintenance was observed when they were cultured on AF-HFFs of different passage number or independent Merle, N., et al. (2012). "ATAD3B is a human derivations. embryonic stem cell specific mitochondrial protein, re- expressed in cancer cells, that functions as dominant Mengarelli, I., et al. (2016). "Use of Multicolor negative for the ubiquitous ATAD3A." Mitochondrion Flow Cytometry for Isolation of Specific Cell 12(4): 441-448. Populations Deriving from Differentiated Human Here we report on the identification of a human Embryonic Stem Cells." Methods Mol Biol 1307: 191- pluripotent embryonic stem cell (hESC) specific 203. mitochondrial protein that is re-expressed in cancer Flow Cytometry-Sorting (FCM-Sorting) is a cells, ATAD3B. ATAD3B belongs to the AAA+ technique commonly used to identify and isolate ATPase ATAD3 protein family of mitochondrial specific types of cells from a heterogeneous population proteins specific to multicellular eukaryotes. Using of live cells. Here we describe a multicolor flow loss- and gain-of-function approaches, we show that cytometry technique that uses five distinct cell surface ATAD3B associates with the ubiquitous ATAD3A antigens to isolate four live populations with different species, negatively regulates the interaction of surface antigen profiles. These profiles were used to ATAD3A with matrix nucleoid complexes and help distinguishing between neural and nonneural (the contributes to a mitochondria fragmentation phenotype. lens) ectoderm derivatives within a highly We conclude that ATAD3B is a negative regulator of heterogenous population of differentiating human ATAD3A and may function as an adaptor of embryonic stem cells (hESC). mitochondrial homeostasis and metabolism in hESCs and cancer cells. Merkely, B., et al. (2015). "Signaling via PI3K/FOXO1A pathway modulates formation and Motomura, Y., et al. (2006). "Embryonic stem survival of human embryonic stem cell-derived cell-derived dendritic cells expressing glypican-3, a endothelial cells." Stem Cells Dev 24(7): 869-878. recently identified oncofetal antigen, induce protective

175 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ immunity against highly metastatic mouse melanoma, approach, termed stem cell selection, may have B16-F10." Cancer Res 66(4): 2414-2422. widespread applicability to the derivation and We have recently established a method to propagation of stem cells. generate dendritic cells from mouse embryonic stem cells. By introducing exogenous genes into embryonic Mousa, S. A., et al. (2010). "Stress resistant stem cells and subsequently inducing differentiation to human embryonic stem cells as a potential source for dendritic cells (ES-DC), we can now readily generate the identification of novel cancer stem cell markers." transfectant ES-DC expressing the transgenes. A Cancer Lett 289(2): 208-216. previous study revealed that the transfer of genetically Cancer stem cells are known for their inherent modified ES-DC expressing a model antigen, resistance to therapy. Here we investigated whether ovalbumin, protected the recipient mice from a normal stem cells with acquired resistance to stress challenge with an ovalbumin-expressing tumor. In the can be used to identify novel markers of cancer stem present study, we examined the capacity of ES-DC cells. For this, we generated a human embryonic stem expressing mouse homologue of human glypican-3, a cell line resistant to Trichostatin A and analyzed recently identified oncofetal antigen expressed in changes in its gene expression. The resistant cells human melanoma and hepatocellular carcinoma, to over-expressed various genes associated with tumor elicit protective immunity against glypican-3- aggressiveness, many of which are also expressed in expressing mouse tumors. CTLs specific to multiple the CD133+ glioma cancer stem cells. These findings glypican-3 epitopes were primed by the in vivo suggest that stress-resistant stem cells generated in transfer of glypican-3-transfectant ES-DC (ES-DC- vitro may be useful for the discovery of novel markers GPC3). The transfer of ES-DC-GPC3 protected the of cancer stem cells. recipient mice from subsequent challenge with B16- F10 melanoma, naturally expressing glypican-3, and Mueller, D., et al. (2005). "Transplanted human with glypican-3-transfectant MCA205 sarcoma. The embryonic germ cell-derived neural stem cells replace treatment with ES-DC-GPC3 was also highly effective neurons and oligodendrocytes in the forebrain of against i.v. injected B16-F10. No harmful side effects, neonatal mice with excitotoxic brain damage." J such as autoimmunity, were observed for these Neurosci Res 82(5): 592-608. treatments. The depletion experiments and Stem cell therapy is a hope for the treatment of immunohistochemical analyses suggest that both some childhood neurological disorders. We examined CD8+ and CD4+ T cells contributed to the observed whether human neural stem cells (hNSCs) replace lost antitumor effect. In conclusion, the usefulness of cells in a newborn mouse model of brain damage. glypican-3 as a target antigen for antimelanoma Excitotoxic lesions were made in neonatal mouse immunotherapy was thus shown in the mouse model forebrain with the N-methyl-D-aspartate (NMDA) using the ES-DC system. Human dendritic cells receptor agonist quinolinic acid (QA). QA induced expressing glypican-3 would be a promising means for apoptosis in neocortex, hippocampus, striatum, white therapy of melanoma and hepatocellular carcinoma. matter, and subventricular zone. This degeneration was associated with production of cleaved caspase-3. Mountford, P., et al. (1998). "Maintenance of Cells immunopositive for inducible nitric oxide pluripotential embryonic stem cells by stem cell synthase were present in damaged white matter and selection." Reprod Fertil Dev 10(7-8): 527-533. subventricular zone. Three days after injury, mice As gastrulation proceeds, pluripotential stem received brain parenchymal or intraventricular cells with the capacity to contribute to all primary injections of hNSCs derived from embryonic germ germ layers disappear from the mammalian embryo. (EG) cells. Human cells were prelabeled in vitro with The extinction of pluripotency also occurs during the DiD for in vivo tracking. The locations of hNSCs formation of embryoid bodies from embryonic stem within the mouse brain were determined through DiD (ES) cells. In this report we show that if the initial fluorescence and immunodetection of human-specific differentiated progeny are removed from ES cell nestin and nuclear antigen 7 days after transplantation. aggregates, further differentiation does not proceed hNSCs survived transplantation into the lesioned and the stem cell population persists and expands. mouse brain, as evidenced by human cell markers and Significantly, the presence of even minor populations DiD fluorescence. The cells migrated away from the of differentiated cells lead to the complete loss of stem injection site and were found at sites of injury within cells from the cultures. This finding implies that the the striatum, hippocampus, thalamus, and white matter normal elimination of pluripotent cells is dictated by tracts and at remote locations in the brain. Subsets of inductive signals provided by differentiated progeny. grafted cells expressed neuronal and glial cell markers. We have exploited this observation to develop a hNSCs restored partially the complement of striatal strategy for the isolation of pluripotential cells. This neurons in brain-damaged mice. We conclude that

176 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ human EG cell-derived NSCs can engraft successfully and mouse cells are primarily in the effects of heparin into injured newborn brain, where they can survive on the FGF-induced response. and disseminate into the lesioned areas, differentiate into neuronal and glial cells, and replace lost neurons. Munsie, M. J., et al. (2000). "Isolation of (c) 2005 Wiley-Liss, Inc. pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei." Curr Biol 10(16): Mummery, C. L. (2002). "[Human embryonic 989-992. stem cells: possibilities for future cell transplantation Pluripotent human stem cells isolated from early therapy]." Tijdschr Diergeneeskd 127(6): 189-191. embryos represent a potentially unlimited source of Human embryonic stem cells are of great many different cell types for cell-based gene and tissue importance, and Parkinson's disease is given as an therapies [1-3]. Nevertheless, if the full potential of example of a condition that could benefit from the cell lines derived from donor embryos is to be realised, development of stem cell-based transplantation the problem of donor-recipient tissue matching needs therapies. The reason for this is fairly obvious: the to be overcome. One approach, which avoids the disease is caused by the loss of only one cell type from problem of transplant rejection, would be to establish the brain that has one major function, namely the stem cell lines from the patient's own cells through production of dopamine. Replacement of these cells therapeutic cloning [3,4]. Recent studies have shown should in principle cure the disease. But what are stem that it is possible to transfer the nucleus from an adult cells and how far is scientific research from being able somatic cell to an unfertilised oocyte that is devoid of to offer stem cell-based therapy in the clinic to patients maternal chromosomes, and achieve embryonic suffering from Parkinson's disease, and other chronic development under the control of the transferred diseases? These questions are addressed here together nucleus [5-7]. Stem cells isolated from such a cloned with a critical evaluation of short and long-term embryo would be genetically identical to the patient clinical perspectives, and a discussion of possible and pose no risk of immune rejection. Here, we report alternatives such as adult stem cells. the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos Mummery, C. L., et al. (1993). "Fibroblast were generated by direct injection of mechanically growth factor-mediated growth regulation and receptor isolated cumulus cell nuclei into mature oocytes. expression in embryonal carcinoma and embryonic Embryonic stem (ES) cells isolated from cumulus-cell- stem cells and human germ cell tumours." Biochem derived blastocysts displayed the characteristic Biophys Res Commun 191(1): 188-195. morphology and marker expression of conventional FGFs have been implicated in the induction of ES cells and underwent extensive differentiation into mesoderm in amphibian development and are present all three embryonic germ layers (endoderm, mesoderm in the mouse embryo at stages that would be and ectoderm) in tumours and in chimaeric foetuses appropriate for a similar function in mammals. and pups. The ES cells were also shown to Primitive ectoderm would then be the target tissue. We differentiate readily into neurons and muscle in culture. have now changes in the expression of receptors for This study shows that pluripotent stem cells can be FGFs during the differentiation of embryonal derived from nuclei of terminally differentiated adult carcinoma (EC) and embryonic stem (ES) cells from somatic cells and offers a model system for the the mouse. These cells resemble those of the inner cell development of therapies that rely on autologous, mass and later primitive ectoderm. On Northern blots human pluripotent stem cells. of mRNA from undifferentiated cells, transcripts for FGF R1, R2 and R3 are expressed. All are upregulated Murakami, K., et al. (2011). "Choice of random during differentiation of ES cells and are upregulated rather than imprinted X inactivation in female or remain constant as EC cells differentiate. FGF R4 is embryonic stem cell-derived extra-embryonic cells." only expressed after differentiation to derivatives Development 138(2): 197-202. resembling parietal endoderm. By contrast in human In female mammals, one of two X chromosomes EC cells, FGF R2 is downregulated during is epigenetically inactivated for gene dosage differentiation, FGF R1 and FGF R3 are unchanged compensation, known as X inactivation (Xi). and FGF R4 is expressed before and after Inactivation occurs randomly in either the paternal or differentiation. In both human and mouse EC cells maternal X chromosome in all embryonic cell lineages, three members of the FGF family (a FGF, b FGF and k designated as random Xi. By contrast, in extra- FGF, also known as FGFs 1,2 and 4) are mitogenic in embryonic cell lineages, which are segregated from serum-free medium and one (KGF or FGF 7) appears somatic cell lineages in pre-implantation development, to have no effect on growth although cellular the paternal X chromosome is selectively inactivated, morphology is altered. Differences between human known as imprinted Xi. Although it is speculated that

177 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ erasure of the imprinted mark on either the maternal or paternal X chromosome in somatic cell lineages might Rohwedel, J., et al. (1994). "Muscle cell change the mode of Xi from imprinted to random, it is differentiation of embryonic stem cells reflects not known when this event is completed in myogenesis in vivo: developmentally regulated development. Here, we tested the mode of Xi during expression of myogenic determination genes and the differentiation of female mouse embryonic stem functional expression of ionic currents." Dev Biol (ES) cells derived from the inner cell mass (ICM) of 164(1): 87-101. blastocyst-stage embryos toward trophectoderm (TE) The mouse blastocyst-derived embryonic stem and primitive endoderm (PrE) lineages induced by cell (ES cell) line BLC6 efficiently differentiates into artificial activation of transcription factor genes Cdx2 myosin heavy chain-, desmin- and -positive and Gata6, respectively. We found that random Xi skeletal muscle cells when cultivated in embryo-like occurs in both TE and PrE cells. Moreover, cloned aggregates (embryoid bodies). Here, we show that the embryos generated by the transfer of nuclei from the muscle-specific determination genes , myogenin, female ES cells showed random Xi in TE, suggesting myoD, and are expressed in these embryoid the complete erasure of all X imprints for imprinted Xi bodies in a characteristic temporal pattern which in ICM-derived ES cells. precisely reflects the sequence observed during mouse development in vivo. Myf5 is the first gene to be Rodriguez-Gomez, J. A., et al. (2012). "T-type expressed followed by myogenin, myoD, and myf6, in Ca2+ channels in mouse embryonic stem cells: this order. In situ hybridization demonstrates modulation during cell cycle and contribution to self- transcripts for myogenin and myoD accumulating in renewal." Am J Physiol Cell Physiol 302(3): C494-504. mono- and multinucleated myogenic cells, while myf5 Ion channels participate in cell homeostasis and mRNA is already found in mononucleated myoblasts. are involved in the regulation of proliferation and The myocytes also express functional nicotinic differentiation in several cell types; however, their cholinoceptors and exhibit T-type Ca2+ currents and presence and function in embryonic stem (ES) cells later L-type Ca2+ currents, demonstrating are poorly studied. We have investigated the existence physiological properties of skeletal muscle cells. of voltage-dependent inward currents in mouse ES During myocyte differentiation the density of L-type cells and their ability to modulate proliferation and Ca2+ channels significantly increases while the self-renewal. Patch-clamped ES cells had inactivating density of T-type Ca2+ channels decreases. The effect tetrodotoxin (TTX)-sensitive Na (+) currents as well as of external signals on myogenic differentiation of transient Ca (2+) currents abolished by the external BLC6 cells was demonstrated by cocultivation with application of Ni (2+). Biophysical and visceral endodermal END-2 cells and the activin A- pharmacological data indicated that the Ca (2+) secreting WEHI-3 cells. END-2 cells essentially current is predominantly mediated by T-type (Ca prevent skeletal muscle differentiation, whereas basic (v)3.2) channels. The number of cells expressing T- fibroblast growth factor, transforming growth factor- type channels and Ca (v)3.2 mRNA levels increased at beta, and WEHI-3 cells have no or an attenuating the G1/S transition of the cell cycle. TTX had no effect effect, respectively. Our results suggest that ES cells on ES cell proliferation. However, blockade of T-type recapitulate closely the early steps of muscle Ca (2+) currents with Ni (2+) induced a decrease in development in vivo and may serve as an excellent in proliferation and alkaline phosphatase positive vitro system to study this process. colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Rohwedel, J., et al. (1996). "Primordial germ Decreased alkaline phosphatase and Oct3/4 expression cell-derived mouse embryonic germ (EG) cells in vitro were also observed in cells subjected to small resemble undifferentiated stem cells with respect to interfering RNA-induced knockdown for T-type (Ca differentiation capacity and cell cycle distribution." (v)3.2) Ca (2+) channels, thus partially recapitulating Cell Biol Int 20(8): 579-587. the pharmacological effects on self-renewal. These Embryonic germ (EG) cells of line EG-1 derived results indicate that Ca (v)3.2 channel expression in from mouse primordial germ cells were investigated ES cells is modulated along the cell cycle being for their in vitro differentiation capacity. By induced at late G1 phase. They also suggest that these cultivation as embryo-like aggregates EG-1 cells channels are involved in the maintenance of the differentiated into cardiac, skeletal muscle and undifferentiated state of mouse ES cells. We propose neuronal cells accompanied by the expression of that Ca (2+) entry mediated by Ca (v)3.2 channels tissue-specific genes and proteins as shown by RT- might be one of the intracellular signals that PCR analysis and indirect immunofluorescence. In participate in the complex network responsible for ES comparison to embryonic stem (ES) cells of line D3 cell self-renewal. the efficiency of differentiation into cardiac and

178 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ muscle cells was comparatively low, whereas Primary cultures or established cell lines of spontaneous neuronal differentiation was more vertebrates are commonly used to analyse the efficient than in D3 cells. Furthermore, the distribution cytotoxic potential of chemical factors, drugs and of cell cycle phases as a parameter for the xenobiotics in vitro. An alternative approach will be differentiation state was analysed in undifferentiated provided by permanent lines of pluripotent embryonic EG cells and ES cells and compared to data obtained stem (ES) cells, which are able to differentiate into for embryonic carcinoma (EC) cells of line P19 and specialised somatic cell types in vitro. Here, we differentiated, epithelioid EPI-7 cells. Flow cytometric demonstrate the capacity of ES cells to generate analysis revealed similar cell cycle phase distributions functional cardiac, neuronal and pancreatic cells. We in EG, EC and ES cells. In contrast, the somatic show that during ES cell differentiation, tissue-specific differentiated EPI-7 cells showed a longer G1-phase genes, proteins as well as functional properties are and shorter S- and G2/M-phases. Together, our results expressed in a developmentally regulated manner demonstrate that the differentiation state and capacity recapitulating processes of early embryonic of EG cells in vitro resemble that of totipotent ES cells. development. We present data that show the use of ES-derived cardiomyocytes and dopaminergic neurons Rojas-Mayorquin, A. E., et al. (2008). in toxicological studies and the potential of ES-derived "Microarray analysis of striatal embryonic stem cells pancreatic beta-like cells in future in vitro assays. The induced to differentiate by ensheathing cell application of these differentiation systems to human conditioned media." Dev Dyn 237(4): 979-994. ES cells opens up new perspectives in basic and The mammalian central nervous system contains applied toxicology. well-defined regions of plasticity in which cells of the aldynoglia phenotype promote neuronal growth and Romorini, L., et al. (2013). "Effect of antibiotics regeneration. Only now are the factors that regulate the against Mycoplasma sp. on human embryonic stem production of new cells from multipotential neural cells undifferentiated status, pluripotency, cell viability precursors (MNP) starting to be identified. We are and growth." PLoS One 8(7): e70267. interested in understanding how differentiation Human embryonic stem cells (hESCs) are self- towards the aldynoglia phenotype is controlled, and to renewing pluripotent cells that can differentiate into study these events we have induced the differentiation specialized cells and hold great promise as models for of embryonic MNP towards this phenotype in vitro. human development and disease studies, cell- Accordingly, we have used microarrays to analyze replacement therapies, drug discovery and in vitro gene expression in three different cell populations: cytotoxicity tests. The culture and differentiation of olfactory bulb ensheathing cells (EC), a prototypic these cells are both complex and expensive, so it is aldynoglia cell type; undifferentiated MNP; and MNP essential to extreme aseptic conditions. hESCs are differentiated in vitro for 24 hr in EC-conditioned susceptible to Mycoplasma sp. infection, which is hard media. The expression profiles identified support the to detect and alters stem cell-associated properties. idea that the EC are more closely related to Schwann The purpose of this work was to evaluate the efficacy cells and astrocytes than to oligodendrocytes. and cytotoxic effect of Plasmocin (TM) and Following MNP differentiation, more strongly ciprofloxacin (specific antibiotics used for expressed genes define a neuroglial cell phenotype. Mycoplasma sp. eradication) on hESCs. Mycoplasma RT-PCR confirms that S100a6, Mtmr2, and Col5a sp. infected HUES-5 884 (H5 884, stable hESCs H5- were highly expressed by EC, whereas Pou3f3 were brachyury promoter-GFP line) cells were effectively more strongly expressed in MNP than in EC, and cured with a 14 days Plasmocin (TM) 25 microg/ml SafB1 and Mash1 expression were induced in MNP by treatment (curative treatment) while maintaining EC-conditioned media. The profile of gene expression stemness characteristic features. Furthermore, cured after differentiation suggests that Wnt signaling may H5 884 cells exhibit the same karyotype as the be inactivated during this process, while activation of parental H5 line and expressed GFP, through up- the BMP pathway may be elicited through the regulation of brachyury promoter, at day 4 of BMPr1A. These results provide us with a starting differentiation onset. Moreover, H5 cells treated with point to study the genes involved in the induction of ciprofloxacin 10 microg/ml for 14 days (mimic of aldynoglia differentiation from MNP. curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin (TM) 5 microg/ml Rolletschek, A., et al. (2004). "Embryonic stem (prophylactic treatment) for 5 passages retained hESCs cell-derived cardiac, neuronal and pancreatic cells as features, as judged by the expression of stemness- model systems to study toxicological effects." Toxicol related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Lett 149(1-3): 361-369. Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers

179 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

(brachyury, Nkx2.5 and cTnT: mesoderm; AFP: periodontal granulation tissue." Cell Biol Int 38(2): endoderm; nestin and Pax-6: ectoderm) was verified in 179-186. in vitro differentiated antibiotic-treated hESCs. In The aim of this study was to identify if cells conclusion, we found that Plasmocin (TM) and obtained from periodontal granulation tissue possess ciprofloxacin do not affect hESCs stemness and embryonic stem cell properties and osteogenic pluripotency nor cell viability. However, curative capacities in vitro. Periodontal granulation tissue was treatments slightly diminished cell growth rate. This removed from one furcation and one infrabony defect cytotoxic effect was reversible as cells regained (FGTC/IGTC-furcation/infrabony defect derived normal growth rate upon antibiotic withdrawal. granulation tissue cells) of six patients. The extracted tissues were treated with collagenase/dispase solution, Ronaghi, M., et al. (2010). "Challenges of stem cultured and passaged twice, while a fraction of them cell therapy for spinal cord injury: human embryonic was bacteriologically analyzed. Upon reaching stem cells, endogenous neural stem cells, or induced confluence, total RNA was extracted, followed by pluripotent stem cells?" Stem Cells 28(1): 93-99. cDNA synthesis and real-time PCR analysis. Gene Spinal cord injury (SCI) causes myelopathy, expression levels of collagen type I, alkaline damage to white matter, and myelinated fiber tracts phosphatase (ALP), and the embryonic stem cell that carry sensation and motor signals to and from the markers Nanog, Oct-4, Rex-1 and Sox-2 were brain. The gray matter damage causes segmental measured, calibrated against the housekeeping gene losses of interneurons and motoneurons and restricts GAPDH. Further, osteogenic differentiation was therapeutic options. Recent advances in stem cell induced. Mineralized matrix formation was confirmed biology, neural injury, and repair, and the progress by von Kossa staining, and ALP activity was measured toward development of neuroprotective and colorimetrically. The total bacterial load amounted to regenerative interventions are the basis for increased 9.4 +/- 14.6 x 10(6) counts/mg of tissue for IGTC, and optimism. This review summarizes the 11.1 +/- 6.1 x 10(6) counts/of tissue for FGTC. Among pathophysiological mechanisms following SCI and the embryonic stem cell markers (FGTC/IGTC), compares human embryonic, adult neural, and the Nanog was most highly expressed (3.48 +/- 1.2/5.85 induced pluripotent stem cell-based therapeutic +/- 5.7), followed by Oct-4 (1.79 +/- 0.69/2.85 +/- 2.5), strategies for SCI. Sox-2 (0.66 +/- 0.3/1.26 +/- 1.4) and Rex-1 (0.06 +/- 0.0/0.04 +/- 0.0). The osteogenic differentiation Ronaghi, M., et al. (2014). "Inner ear hair cell- process was positive in both FGTC and IGTC, judged like cells from human embryonic stem cells." Stem by increased von Kossa staining, and elevated ALP Cells Dev 23(11): 1275-1284. activity and gene expression. This study provides In mammals, the permanence of many forms of evidence that infected periodontal granulation tissue hearing loss is the result of the inner ear's inability to harbors cells expressing embryonic stem cell markers, replace lost sensory hair cells. Here, we apply a and exhibiting osteogenic capacities when in culture in differentiation strategy to guide human embryonic vitro. stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The Ronay, V., et al. (2013). "Infected periodontal generation of human otic progenitor cells was granulation tissue contains cells expressing embryonic dependent on fibroblast growth factor (FGF) signaling, stem cell markers. A pilot study." Schweiz Monatsschr and protracted culture led to the upregulation of Zahnmed 123(1): 12-16. markers indicative of differentiated inner ear sensory The commonly practiced removal of granulation epithelia. Using a transgenic ESC reporter line based tissue during periodontal surgery, aiming to eliminate on a murine Atoh1 enhancer, we show that infection and optimize healing conditions, may also differentiated hair cell-like cells express multiple hair remove progenitor stem cells that could otherwise cell markers simultaneously. Hair cell-like cells support periodontal regeneration. The present study displayed protrusions reminiscent of stereociliary aimed to investigate if cells with embryonic stem cell bundles, but failed to fully mature into cells with properties are present in periodontal granulation tissue. typical hair cell cytoarchitecture. We conclude that During the course of flap surgery inflammatory optimized defined conditions can be used in vitro to granulation tissue was obtained from four patients and attain otic progenitor specification and sensory cell five periodontal defects. Tissues were processed in a differentiation. collagenase/dispase solution to release the cells. Part of the resulting suspension was processed for Ronay, V., et al. (2014). "Expression of bacteriological analysis (IAI PadoTest 4.5), whereas embryonic stem cell markers and osteogenic the remaining cell suspension was cultured and differentiation potential in cells derived from passaged once. Upon reaching confluence, total RNA

180 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ was extracted, followed by cDNA synthesis. PCR was much greater signal in undifferentiated ESCs than in then performed (SYBR Green-based protocols) to their differentiated derivatives, among them the measure gene expression levels of Collagen type I, and putative stemness gene encoding junctional adhesion embryonic stem cell markers Nanog, Oct4, Rex-1 and molecule B (Jam-B gene). However, in spite of the Sox2. Results are expressed as 2(-)Delta (Ct) values of specific expression in undifferentiated ESCs, Jam-B the target gene, calibrated against a house-keeping mutant ESCs had normal morphology and gene (GAPDH). A high total bacterial load up to 20.6 pluripotency. Furthermore, Jam-B homozygous mutant +/- 11.0x10(6) counts/mg of tissue was found. mice are fertile and have no overt developmental Collagen type I was strongly expressed, confirming defects. Moreover, we found that neural and the predominance of mesenchymal/fibroblastic cells. hematopoietic stem cells recovered from Jam-B Among the studied embryonic stem cells markers, mutant mice are not impaired in their ability to self- Nanog was most highly expressed (2.3 +/- 1.2), renew and differentiate. These results demonstrate that followed by Oct4 (1.1 +/- 0.5), Rex-1 (0.6 +/- 0.2) and Jam-B is dispensable for normal mouse development Sox2 (0.3 +/- 0.2). This is the first study that and stem cell identity in embryonic, neural, and demonstrates the presence of cells expressing hematopoietic stem cells. embryonic stem cell markers among infected granulation tissue. This knowledge needs to be Salguero-Aranda, C., et al. (2016). considered when devising future strategies to improve "Differentiation of Mouse Embryonic Stem Cells periodontal wound healing and regeneration. toward Functional Pancreatic beta-Cell Surrogates through Epigenetic Regulation of Pdx1 by Nitric Saito, M. and H. Matsuoka (2010). "Semi- Oxide." Cell Transplant 25(10): 1879-1892. quantitative analysis of transient single-cell gene Pancreatic and duodenal homeobox 1 (Pdx1) is a expression in embryonic stem cells by femtoinjection." transcription factor that regulates the embryonic Methods Mol Biol 650: 155-170. development of the pancreas and the differentiation Single-cell manipulation supporting robot toward beta cells. Previously, we have shown that (SMSR) has enabled femtoinjection, a high-throughput exposure of mouse embryonic stem cells (mESCs) to and semi-quantitative microinjection in the range of high concentrations of diethylenetriamine nitric oxide femtogram (fg) DNA and other molecules. An adduct (DETA-NO) triggers differentiation events and enhanced green fluorescent protein (EGFP) gene promotes the expression of Pdx1. Here we report expression vector can be introduced directly into evidence that Pdx1 expression is associated with mouse embryonic stem cells at 100 cells/h with a 10% release of polycomb repressive complex 2 (PRC2) and success rate. The intensity of EGFP fluorescence in P300 from its promoter region. These events are single ES cells or single colonies of ES cells increases accompanied by epigenetic changes in bivalent as the concentration of an EGFP gene expression markers of histones trimethylated histone H3 lysine 27 vector in the injection capillary increases from 1 to 50 (H3K27me3) and H3K4me3, site-specific changes in ng/muL. On the other hand, the knockdown of EGFP DNA methylation, and no change in H3 acetylation. gene expression can be demonstrated by On the basis of these findings, we developed a femtoinjection of siRNA against EGFP or an shRNA protocol to differentiate mESCs toward insulin- expression vector using an EGFP expressing ES cell producing cells consisting of sequential exposure to line. Femotoinjection can provide a useful method for DETA-NO, valproic acid, and P300 inhibitor (C646) quantitative analysis of transient gene expression in to enhance Pdx1 expression and a final maturation step single cells using RNAi. of culture in suspension to form cell aggregates. This small molecule-based protocol succeeds in obtaining Sakaguchi, T., et al. (2006). "Putative "stemness" cells that express pancreatic beta-cell markers such as gene jam-B is not required for maintenance of stem PDX1, INS1, GCK, and GLUT2 and respond in vitro cell state in embryonic, neural, or hematopoietic stem to high glucose and KCl. cells." Mol Cell Biol 26(17): 6557-6570. Many genes have been identified that are Santolaya-Forgas, J., et al. (2007). "A study to specifically expressed in multiple types of stem cells determine if human umbilical cord hematopoietic stem in their undifferentiated state. It is generally assumed cells can survive in baboon extra-embryonic celomic that at least some of these putative "stemness" genes fluid: a prerequisite for determining the feasibility of are involved in maintaining properties that are in-utero stem cell xeno-transplantation via common to all stem cells. We compared gene celocentesis." Fetal Diagn Ther 22(2): 131-135. expression profiles between undifferentiated and OBJECTIVES: To determine if: (1) human differentiated embryonic stem cells (ESCs) using umbilical cord stem cells could survive for 20 h in DNA microarrays. We identified several genes with extra-embryonic celomic fluid obtained at 40 days of

181 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ development from baboon pregnancies by ultrasound- the InsP3 receptor, as well as vasopressin and guided celocentesis and, (2) human hematopoietic bradykinin, which act via the InsP3 pathway, increased stem cell survival could be enhanced by adding the frequency of [Ca2+]i spikes. In the prescence of increasing concentrations of hematopoietic stem cell brefeldin A (50 microM) or monensin (20 microM), culture medium to the celomic fluid. METHODS: which both inhibit endo/exocytotic vesicle pathways, CD34+ cells were isolated from umbilical cord blood an immediate transient increase in spiking activity was using a magnetic activated cell sorter and flow followed by a decline within 1 to 2 h. In the presence cytometry. Cells were then cultured in five platforms of brefeldin A or thapsigargin or in the absence of containing different combinations of baboon extra- extracellular Ca2+, endocytotic vesicles were absent, embryonic celomic fluid and hematopoietic stem cell suggesting that oscillating [Ca2+]i transients are culture medium to determine cell survival kinetics involved in the exo/endocytotic vesicle shuttle. over a 20-hour period in each of the conditions. RESULTS: Human umbilical cord stem cells can Schumacher, A., et al. (2003). "Staurosporine is a survive for at least 20 h in baboon's celomic fluid. Chi- potent activator of neuronal, glial, and "CNS stem cell- square for linear trends demonstrated that the number like" neurosphere differentiation in murine embryonic of viable cells was significantly enhanced by adding stem cells." Mol Cell Neurosci 23(4): 669-680. the increasing concentrations of culture medium to the Staurosporine (STS), a broad spectrum protein celomic fluid (29% cell survival in pure celomic fluid, kinase inhibitor, was previously shown to induce 48% at 1:8, 50% at 1:2, 54.5% at 1:1, 61% in pure neurite outgrowth in several neuroblastoma cell lines. culture medium; p < 0.001). CONCLUSIONS: Human However, data on the neurotrophic potential of this umbilical cord stem cells survive in baboon celomic alkaloid in embryonic stem cell systems were not fluid and cell survival improves when the celomic available. Therefore, three mouse ES cell lines, IB10, fluid is mixed with greater concentrations of RW4, and Bruce 4, were induced to enter neurogenesis hematopoietic stem cell culture medium. Based on in culture at low concentrations of STS. These cells these findings, future in-vivo experiments in the differentiated into epidermal growth factor-responsive pregnant baboon animal model can be directed at neural precursor cells, formed neurospheres, and determining whether in-utero injection of human further developed to neurons and astrocytes. The hematopoietic stem cells prior to 10 weeks of clonally derived neurospheres consisted of multipotent pregnancy can lead to permanent chimeras. cells which exhibited some of the classical characteristics of early CNS stem cells and could be Sauer, H., et al. (1998). "Spontaneous calcium propagated in vitro. STS was antagonistic in several oscillations in embryonic stem cell-derived primitive ways to retinoic acid (RA), a vitamin A metabolite, endodermal cells." Exp Cell Res 238(1): 13-22. which promotes neuritogenesis. Results from RT-PCR In vitro differentiation of mouse embryonic stem experiments and inhibition studies with RA provided cells within three-dimensional cell aggregates called evidence that staurosporine exerted its neurotrophic embryoid bodies parallels the development of effects through the induction of very late levels of the postimplantation embryos at the egg cylinder stage, nerve growth factor and protein kinase C neurogenesis where visceral and parietal endoderm diverge from the pathways. primitive endoderm. We have investigated spontaneous [Ca2+]i oscillations by means of confocal Scott, G. J., et al. (2018). "Trans-inner Cell Mass laser-scanning microscopy in primitive endodermal Injection of Embryonic Stem Cells Leads to Higher cell layers of embryoid bodies during their Chimerism Rates." J Vis Exp (135). differentiation to parietal and visceral endoderm. The In an effort to increase efficiency in the creation frequency of [Ca2+]i oscillations increased from day 4 of genetically modified mice via ES Cell to day 19 of development, whereas their duration methodologies, we present an adaptation to the current decreased from day 3 to days 16-17. Oscillations blastocyst injection protocol. Here we report that a depended on both extracellular Ca2+ and Ca2+ release simple rotation of the embryo, and injection through from intracellular stores as they were abolished in Ca Trans-Inner cell mass (TICM) increased the (2+)-free solution and in the prescence of Ni2+ and percentage of chimeric mice from 31% to 50%, with thapsigargin. Signal transduction operated via the no additional equipment or further specialized training. phospholipase C (PLC)-mediated inositol 1,4,5- 26 different inbred clones, and 35 total clones were triphosphate (InsP3) pathway with a negative feedback injected over a period of 9 months. There was no loop via protein kinase C (PKC) as U73,122, a blocker significant difference in either pregnancy rate or of PLC; bisindolylmaleimide 1, staurosporine, and H-7, recovery rate of embryos between traditional injection blockers of PKC; and 10 mM caffeine totally inhibited techniques and TICM. Therefore, without any major [Ca2+]i spiking. Thimerosal, which hypersensitizes alteration in the injection process and a simple

182 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ positioning of the blastocyst and injecting through the (ES) cells to differentiate in culture to test ICM, releasing the ES cells into the blastocoel cavity embryotoxicity in vitro. The EST represents a can potentially improve the quantity of chimeric scientifically validated in vitro system for the production and subsequent germline transmission. classification of compounds according to their teratogenic potential based on the morphological Seiler, A., et al. (2002). "[Improving the analysis of beating cardiomyocytes in embryoid body embryonic stem cell test (EST) by establishing outgrowths compared to cytotoxic effects on murine molecular endpoints of tissue specific development ES cells and differentiated 3T3 fibroblasts. Through a using murine embryonic stem cells (D3 cells)]." number of prevalidation and validation studies, the ALTEX 19 Suppl 1: 55-63. EST has been demonstrated to be a reliable alternative Blastocyst-derived pluripotent embryonic stem method for embryotoxicity testing based on the most (ES) cells of the mouse can be induced to differentiate important mechanisms in embryotoxicity-cytotoxicity in culture into a variety of cell types, including cardiac and differentiation--as well as on differences in muscle cells. In the embryonic stem cell test (EST) the sensitivity between differentiated and embryonic capacity of ES cells of the mouse cell line D3 to tissues. Improvements of the EST protocol using flow differentiate into contracting cardiomyocytes is used to cytometry analysis showed that differential expression assess the embryotoxic potential of test compounds of sarcomeric myosin heavy chain and alpha-actinin and in addition, the effects on the viability of ES cells proteins quantified under the influence of a test and differentiated mouse fibroblasts (cell line 3T3) are compound is a useful marker for detecting potential compared. The three endpoints are used to classify the teratogenicity. The in vitro embryotoxicity test embryotoxic potential of chemicals after 10 days of described in this chapter is rapid, simple, and sensitive exposure: (i) the inhibition of differentiation of ES and can be usefully employed as a component of the cells into cardiomyocytes (ID50) and (ii) the decrease risk/hazard assessment process. of viability of 3T3 cells (IC503T3) and (iii) ES cells (IC50D3) in a MTT cytotoxicity test. Applying linear Semrau, S., et al. (2017). "Dynamics of lineage analysis of discriminance, a biostatistical prediction commitment revealed by single-cell transcriptomics of model (PM) was developed to assign test chemicals to differentiating embryonic stem cells." Nat Commun three classes of embryotoxicity. In an international 8(1): 1096. validation study funded by ECVAM it could be Gene expression heterogeneity in the pluripotent demonstrated that the EST can predict the embryotoxic state of mouse embryonic stem cells (mESCs) has potential of a test compound as good as frequently been increasingly well-characterized. In contrast, exit used mammalian systems based on pregnant animals. from pluripotency and lineage commitment have not In a joint project with major German pharmaceutical been studied systematically at the single-cell level. companies we are attempting to improve the EST by Here we measure the gene expression dynamics of establishing molecular endpoints of differentiation (e.g. retinoic acid driven mESC differentiation from cardiac, neuronal, chondrogenic) in cultured ES cells. pluripotency to lineage commitment, using an We have studied the expression of tissue specific unbiased single-cell transcriptomics approach. We find proteins in ES cell cultures in the presence of that the exit from pluripotency marks the start of a embryotoxic chemicals by immunofluorescent lineage transition as well as a transient phase of antibody techniques, e.g. FACS analysis. The other increased susceptibility to lineage specifying signals. groups are focusing on endogenous gene expression in Our study reveals several transcriptional signatures of early development by RT-PCR methods or the DNA this phase, including a sharp increase of gene microarray technique. The results obtained recently expression variability and sequential expression of two using molecular markers specific for cardiac classes of transcriptional regulators. In summary, we differentiation and employing intracellular flow provide a comprehensive analysis of the exit from cytometry for quantification will be presented. pluripotency and lineage commitment at the single cell Molecular endpoints will allow improvement of the level, a potential stepping stone to improved lineage EST by measuring gene expression patterns in a small manipulation through timing of differentiation cues. number of murine ES cells. Senju, S., et al. (2007). "Genetically manipulated Seiler, A. E., et al. (2006). "Use of murine human embryonic stem cell-derived dendritic cells embryonic stem cells in embryotoxicity assays: the with immune regulatory function." Stem Cells 25(11): embryonic stem cell test." Methods Mol Biol 329: 2720-2729. 371-395. Genetically manipulated dendritic cells (DC) are The embryonic stem cell test (EST) takes considered to be a promising means for antigen- advantage of the potential of murine embryonic stem specific immune therapy. This study reports the

183 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ generation, characterization, and genetic modification robust, sequence-conserving DNA damage repair of DC derived from human embryonic stem (ES) cells. mechanisms such as homologous recombination (HR). The human ES cell-derived DC (ES-DC) expressed We used fluorescence microscopy and 3-dimensional surface molecules typically expressed by DC and had image analysis to compare mESC and differentiated the capacities to stimulate allogeneic T lymphocytes cells, with regard to HR-mediated repair of and to process and present protein antigen in the spontaneous and X-ray-induced double-strand breaks context of histocompatibility leukocyte antigen (HLA) (DSBs). Microscopic analysis of repair foci, flow class II molecule. Genetic modification of human ES- cytometry, and functional assays of the major DSB DC can be accomplished without the use of viral repair pathways indicate that HR is greater in mESC vectors, by the introduction of expression vector compared to fibroblasts. Strikingly, HR appears to be plasmids into undifferentiated ES cells by the predominant pathway choice to repair induced or electroporation and subsequent induction of spontaneous DNA damage throughout the ESC cycle differentiation of the transfectant ES cell clones to ES- in contrast to fibroblasts, where it is restricted to DC. replicated chromatin. This suggests that alternative templates, such as homologous chromosomes, are Serfozo, P., et al. (2006). "Selective migration of more frequently used to repair DSB in ESC. Relatively neuralized embryonic stem cells to stem cell factor and frequent HR utilizing homolog chromosome sequences media conditioned by glioma cell lines." Cancer Cell preserves genome integrity in ESC and has distinctive Int 6: 1. and important genetic consequences to subsequent BACKGROUND: Pluripotent mouse embryonic somatic and germ cell lineages. stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural Shah, S. M., et al. (2015). "RETRACTED: Bone progenitors migrate toward areas of damage and morphogenetic protein 4 (BMP4) induces buffalo inflammation in the CNS. We tested whether (Bubalus bubalis) embryonic stem cell differentiation undifferentiated and neuralized mouse ES cells into germ cells." Biochimie 119: 113-124. migrate toward media conditioned by glioma cell lines This article has been retracted: please see (C6, U87 & N1321) or Stem Cell Factor (SCF). Elsevier Policy on Article Withdrawal RESULTS: Cell migration assays revealed selective (http://www.elsevier.com/locate/withdrawalpolicy). migration by neuralized ES cells to conditioned media This article has been retracted at the request of the as well as to synthetic SCF. Migration of Editor-in-Chief. Problems related to images published undifferentiated ES cells was extensive, but not in this paper in Figure 12 were brought to the authors' significantly different from that of controls attention. Unfortunately this figure contains duplicate (Unconditioned Medium). RT-PCR analysis revealed images for ESC controls for VASA, GDF9, and ZP4, that all the three tumor cell lines tested synthesized which display identical patterns superimposed on SCF and that both undifferentiated and neuralized ES varying intensities of background. Therefore, the cells expressed c-kit, the receptor for SCF. authors retract the paper with the agreement of the CONCLUSION: Our results demonstrate that editors and deeply regret this situation and apologize undifferentiated ES cells are highly mobile and that for any inconvenience to the editors and readers of neural progenitors derived from ES cells are Biochimie. selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize Shah, S. M., et al. (2017). "Cumulus cell- SCF, SCF may be one of several factors that conditioned medium supports embryonic stem cell contribute to the selective migration observed. differentiation to germ cell-like cells." Reprod Fertil Dev 29(4): 679-693. Serrano, L., et al. (2011). "Homologous Cumulus cells provide cellular interactions and recombination conserves DNA sequence integrity growth factors required for oogenesis. In vitro studies throughout the cell cycle in embryonic stem cells." of oogenesis are limited primarily because of the Stem Cells Dev 20(2): 363-374. paucity of their source, first trimester fetal gonads, and The maintenance of genomic integrity is crucial the small number of germ lineage precursor cells to embryonic stem cells (ESC) considering the present within these tissues. In order to understand this potential for propagating undesirable mutations to the obscure but vitally important process, the present resulting somatic and germ cell lineages. Indeed, study was designed to direct differentiation of mouse ESC (mESC) exhibit a significantly lower embryonic stem (ES) cells into germ lineage cells. For mutation frequency compared to differentiated cells. this purpose, buffalo ES cells were differentiated, as This could be due to more effective elimination of embryoid bodies (EBs) and monolayer adherent genetically damaged cells via apoptosis, or especially cultures, in the presence of different concentrations of

184 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ cumulus-conditioned medium (CCM; 10%, 20% and identify the optimum dose-and-time period. We 40%) for different periods of culture (4, 8 and 14 days) observed that 40% TCM dose induces highest to identify the optimum differentiation-inducing expression of primordial germ cell-specific (DAZL, concentration and time. Quantitative polymerase chain VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, reaction analysis revealed that 20%-40% CCM and PRM2), spermatocyte-specific (BOULE and induced the highest expression of primordial germ TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) cell-specific (deleted in Azoospermia- like (Dazl), for a culture period of 14 days under both floating and dead (Asp-Glu-Ala-Asp) box polypeptide 4 (Vasa also adherent differentiation. Immunocytochemical known as DDX4) and promyelocytic leukemia zinc analysis of EBs and adherent cultures revealed finger protein (Plzf)); meiotic (synaptonemal complex presence of primordial germ cell markers (c-KIT, protein 3 (Sycp3), mutl homolog I (Mlh1), transition DAZL, and VASA), meiotic markers (SYCP3, MLH1 protein 1/2 (Tnp1/2) and protamine 2 (Prm2); and PROTAMINE1), spermatocyte markers spermatocyte-specific boule-like RNA binding protein (ACROSIN and HAPRIN), and oocyte markers (Boule) and tektin 1 (Tekt1)) and oocyte-specific (GDF9 and ZP4), indicating progression into post- growth differentiation factor 9 (Gdf9) and zona meiotic gametogenesis. pellucida 2 /3 (Zp2/3)) genes over 8-14 days in culture. Immunocytochemical analysis revealed expression of Shao, J., et al. (2017). "Experimental Study of the primordial germ cell (c-KIT, DAZL and VASA), Biological Properties of Human Embryonic Stem Cell- meiotic (SYCP3, MLH1 and PROTAMINE 1), Derived Retinal Progenitor Cells." Sci Rep 7: 42363. spermatocyte (ACROSIN and HAPRIN) and oocyte Retinal degenerative diseases are among the (GDF9 and ZP4) markers in both EBs and monolayer leading causes of blindness worldwide, and cell differentiation cultures. Western blotting revealed replacement is considered as a promising therapeutic. germ lineage-specific protein expression in Day 14 However, the resources of seed cells are scarce. To EBs. The significantly lower (P<0.05) concentration of further explore this type of therapy, we adopted a 5-methyl-2-deoxycytidine in differentiated EBs culture system that could harvest a substantial quantity compared to undifferentiated EBs suggests that of retinal progenitor cells (RPCs) from human methylation erasure may have occurred. Oocyte-like embryonic stem cells (hESCs) within a relatively short structures obtained in monolayer differentiation period of time. Furthermore, we transplanted these stained positive for ZONA PELLUCIDA protein 4 and RPCs into the subretinal spaces of Royal College of progressed through various embryo-like Surgeons (RCS) rats. We quantified the thickness of developmental stages in extended cultures. the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography Shah, S. M., et al. (2016). "Testicular cell- (ERG). It was found that the differentiated cells conditioned medium supports embryonic stem cell expressed RPC markers and photoreceptor progenitor differentiation toward germ lineage and to markers. The transplanted RPCs survived for at least spermatocyte- and oocyte-like cells." Theriogenology 12 weeks, resulting in beneficial effects on the 86(3): 715-729. morphology of the host retina, and led to a significant Testicular cells are believed to secrete various improvement in the visual function of the treated growth factors that activate signaling pathways finally animals. These therapeutic effects suggest that the leading to gametogenesis. In vitro gametogenesis is an hESCs-derived RPCs could delay degeneration of the obscure but paramountly important task primarily retina and partially restore visual function. because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these Shapira-Schweitzer, K., et al. (2009). "A limitations for development of in vitro gametes, the photopolymerizable hydrogel for 3-D culture of present study was designed to induce differentiation of human embryonic stem cell-derived cardiomyocytes buffalo embryonic stem (ES) cells into germ lineage and rat neonatal cardiac cells." J Mol Cell Cardiol cells on stimulation by testicular cell-conditioned 46(2): 213-224. medium (TCM), on the basis of the assumption that The purpose of this study was to assess the in ES cells have the intrinsic property to differentiate into vitro ability of two types of cardiomyocytes any cell type and TCM would provide the necessary (cardiomyocytes derived from human embryonic stem growth factors for differentiation toward germ cell cells (hESC-CM) and rat neonatal cardiomyocytes lineage. For this purpose, buffalo ES cells were (rN-CM)) to survive and generate a functional cardiac differentiated as embryoid bodies (EB) in floating syncytium in a three-dimensional in situ polymerizable cultures and as monolayer adherent cultures in hydrogel environment. Each cell type was cultured in a different doses (10%, 20%, and 40%) of TCM for PEGylated fibrinogen (PF) hydrogel for up to two different culture intervals (4, 8, and 14 days), to weeks while maturation and cardiac function were

185 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ documented in terms of spontaneous contractile (P<0.05). It increased plating efficiency of single-cell behavior and biomolecular organization. Quantitative suspensions of ES cells (P<0.05). Following contractile parameters including contraction amplitude vitrification, the presence of Y-27632 in the and synchronization were measured by non-invasive vitrification solution or thawing medium or both did image analysis. The rN-CM demonstrated the fastest not improve ES cell colony survival. However, maturation and the most significant spontaneous following seeding of clumps of ES cells transfected contraction. The hESC-CM maturation occurred with pAcGFP1N1 carrying green fluorescent protein between 10-14 days in culture, and exhibited less (GFP), Y-27632 increased colony formation rate contraction amplitude and synchronization in (P<0.01). ES cell colonies that formed in all Y-27632- comparison to the rN-CMs. The maturation of both supplemented groups were confirmed for expression cell types within the hydrogels was confirmed by of pluripotency markers alkaline phosphatase, SSEA-4 cardiac-specific biomolecular markers, including and TRA-1-60, and for their ability to generate alpha-sarcomeric actin, actinin, and connexin-43. embryoid bodies containing cells that expressed Cellular responsiveness to isoproterenol, markers of ectoderm, mesoderm and endoderm. In carbamylcholine and heptanol provided further conclusion, Y-27632 improves survival of buffalo ES evidence of the cardiac maturation in the 3-D PF cells under unfavourable conditions such as enzymatic hydrogel as well as identified a potential to use this dissociation to single cells or antibiotic-assisted system for in vitro drug screening. These findings selection after transfection, without compromising indicate that the PF hydrogel biomaterial can be used their pluripotency. as an in situ polymerizable biomaterial for stem cells and their cardiomyocyte derivatives. Sharma, R., et al. (2012). "Growth factor expression pattern of homologous feeder layer for Sharma, A. D., et al. (2008). "Murine embryonic culturing buffalo embryonic stem cell-like cells." stem cell-derived hepatic progenitor cells engraft in Reprod Fertil Dev 24(8): 1098-1104. recipient livers with limited capacity of liver tissue The present study examined the expression formation." Cell Transplant 17(3): 313-323. profile of buffalo fetal fibroblasts (BFF) used as a Directed endodermal differentiation of murine feeder layer for embryonic stem (ES) cell-like cells. embryonic stem (ES) cells gives rise to a subset of The expression of important growth factors was cells with a hepatic phenotype. Such ES cell-derived detected in cells at different passages. Mitomycin-C hepatic progenitor cells (ES-HPC) can acquire features inactivation increased relative expression levels of of hepatocytes in vitro, but fail to form substantial ACTIVIN-A, TGF-beta1, BMP-4 and GREMLIN but hepatocyte clusters in vivo. In this study, we not of fibroblast growth factor-2 (FGF-2). The investigated whether this is due to inefficient expression level of ACTIVIN-A, transforming growth engraftment or an immature phenotype of ES-HPC. ES factor-beta1 (TGF-beta1), bone morphogenetic cells engrafted into recipient livers of NOD/SCID protein-4 (BMP-4) and FGF-2 was similar in buffalo mice with a similar efficacy as adult hepatocytes after fetal fibroblast (BFF) cultured in stem cell medium 28 days. Because transplanted unpurified ES-HPC (SCM), SCM+1000IU mL (-1) leukemia inhibitory formed teratomas in the spleen and liver, we applied factor (LIF), SCM+5 ngmL (-1) FGF-2 or an albumin promoter/enhancer-driven reporter system SCM+LIF+FGF-2 for 24 h whereas GREMLIN to purify ES-HPC by cell sorting. expression was higher in FGF-2-supplemented groups. In spent medium, the concentration of ACTIVIN-A Sharma, R., et al. (2013). "ROCK inhibitor Y- was higher in FGF-2-supplemented groups whereas 27632 enhances the survivability of dissociated that of TGF-beta1 was similar in SCM and LIF+FGF- buffalo (Bubalus bubalis) embryonic stem cell-like 2, which was higher than when either LIF or FGF-2 cells." Reprod Fertil Dev 25(2): 446-455. was used alone. This study investigated the effects of supplementation of culture medium with 10 muM Y- Sharma, R., et al. (2011). "Optimization of 27632, a specific inhibitor of Rho kinase activity, for 6 culture conditions to support long-term self-renewal of days on self-renewal of buffalo embryonic stem (ES) buffalo (Bubalus bubalis) embryonic stem cell-like cell-like cells at Passage 50-80. Y-27632 increased cells." Cell Reprogram 13(6): 539-549. mean colony area (P<0.05) although it did not improve A culture system capable of sustaining self- their survival. It decreased OCT4 expression (P<0.05), renewal of buffalo embryonic stem (ES) cell-like cells increased NANOG expression (P<0.05), but had no in an undifferentiated state over a long period of time effect on SOX2 expression. It also increased was developed. Inner cell masses were seeded on KO- expression of anti-apoptotic gene BCL-2 (P<0.05) and DMEM+15% KO-serum replacer on buffalo fetal decreased that of pro-apoptotic genes BAX and BID fibroblast feeder layer. Supplementation of culture

186 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF mature neural cells, including neurotransmitter- gave the highest (p<0.05) rate of primary colony secreting neurons and glial cells. Investigating the formation. The ES cell-like cells' colony survival rate susceptibility of these hESCs-derived neural cells to and increase in colony size were highest (p<0.05) neurotrophic viruses, such as Japanese encephalitis following supplementation with FGF-2 and LIF virus (JEV), provides insight into the viral cell tropism compared to other groups examined. FGF-2 in the infected human brain. We demonstrate that supplementation affected the quantitative expression hESC-derived NPCs are highly vulnerable to JEV of NANOG, SOX-2, ACTIVIN A, BMP 4, and infection at a low multiplicity of infection (MOI). In TGFbeta1, but not OCT4 and GREMLIN. addition, glial fibrillary acid protein (GFAP)- Supplementation with SU5402, an FGFR inhibitor expressing glial cells are also susceptible to JEV (>/=20 muM) increased (p<0.05) the percentage of infection. In contrast, only a few mature neurons were colonies that differentiated. FGFR1-3 and ERK1, K- infected at MOI 10 or higher on the third day post- RAS, E-RAS, and SHP-2, key signaling intermediates infection. In addition, functional neurotransmitter- of FGF signaling, were detected in ES cell-like cells. secreting neurons are also resistant to JEV infection at Under culture conditions described, three ES cell lines high MOI. Moreover, we discover that vimentin were derived that, to date, have been maintained for intermediate filament, reported as a putative 135, 95, and 85 passages for over 27, 19, and 17 neurovirulent JEV receptor, is highly expressed in months, respectively, whereas under other conditions NPCs and glial cells, but not mature neurons. These examined, ES cell-like cells did not survive beyond results indicate that the expression of vimentin in passage 10. The ES cell-like cells were regularly neural cells correlates to the cell tropism of JEV. monitored for expression of pluripotency markers and Finally, we further demonstrate that membranous their potency to form embryoid bodies. vimentin is necessary for the susceptibility of hESC- derived NPCs to JEV infection. Shaykhiev, R., et al. (2013). "Airway basal cells of healthy smokers express an embryonic stem cell Shetty, D. K. and M. S. Inamdar (2016). signature relevant to lung cancer." Stem Cells 31(9): "Generation of a transgenic human embryonic stem 1992-2002. cell line ectopically expressing the endosomal protein Activation of the human embryonic stem cell Asrij that regulates pluripotency in mouse embryonic (hESC) signature genes has been observed in various stem cells: BJNhem20-Asrij." Stem Cell Res 16(2): epithelial cancers. In this study, we found that the 331-333. hESC signature is selectively induced in the airway Asrij is an endocytic protein expressed in mouse basal stem/progenitor cell population of healthy embryonic stem cells (mESCs) and is essential for smokers (BC-S), with a pattern similar to that maintenance of stemness of mESCs (Mukhopadhyay activated in all major types of human lung cancer. We et al., 2003; Sinha et al., 2013). Its human ortholog further identified a subset of 6 BC-S hESC genes, named Ovarian Carcinoma Immuno-reactive Antigen whose coherent overexpression in lung domain containing protein 1 (OCIAD1) is 85% adenocarcinoma (AdCa) was associated with reduced identical. We ectopically expressed Asrij in epiblast lung function, poorer differentiation grade, more stage equivalent-human embryonic stem cells (hESCs) advanced tumor stage, remarkably shorter survival, to test for induction of naive pluripotency in primed and higher frequency of TP53 mutations. BC-S shared pluripotent cells. The construct pCAG-Asrij was with hESC and a considerable subset of lung introduced into hESCs by microporation. Ectopic carcinomas a common TP53 inactivation molecular expression of Asrij in BJNhem20 hESC line was pattern which strongly correlated with the BC-S hESC performed by selecting for plasmid transfection, gene expression. These data provide transcriptome- followed by stable cell line generation. based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might Shi, C., et al. (2011). "Derivation and represent a common early molecular event in the characterization of Chinese human embryonic stem development of aggressive lung carcinomas in humans. cell line with high potential to differentiate into pancreatic and hepatic cells." Chin Med J (Engl) Shen, S. C., et al. (2014). "Susceptibility of 124(7): 1037-1043. human embryonic stem cell-derived neural cells to BACKGROUND: Human embryonic stem cells Japanese encephalitis virus infection." PLoS One have prospective uses in regenerative medicine and 9(12): e114990. drug screening. Every human embryonic stem cell line Pluripotent human embryonic stem cells (hESCs) has its own genetic background, which determines its can be efficiently directed to become immature specific ability for differentiation as well as neuroepithelial precursor cells (NPCs) and functional susceptibility to drugs. It is necessary to compile many

187 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ human embryonic stem cell lines with various state by stromal cell co-culture. Forty-hours after the backgrounds for future clinical use, especially in induction of HIE, animals were grafted with ES-NPCs China due to its large population. This study targeting the deep layer of the motor cortex in the contributes to isolating new Chinese human embryonic ischemic brain. Motor function was evaluated 3 weeks stem cell lines with clarified directly differentiation after transplantation. ability. METHODS: Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer Shirasawa, S., et al. (2011). "Pancreatic exocrine (IVF-ET) center were collected to establish human enzyme-producing cell differentiation via embryoid embryonic stem cells lines with informed consent. The bodies from human embryonic stem cells." Biochem classic growth factors of basic fibroblast growth factor Biophys Res Commun 410(3): 608-613. (bFGF) and recombinant human leukaemia inhibitory Mouse embryonic stem cells (ESCs) can be factor (hLIF) for culturing embryonic stem cells were induced to form pancreatic exocrine enzyme- used to capture the stem cells from the plated embryos. producing cells in vitro in a stepwise fashion that Mechanical and enzymetic methods were used to recapitulates the development in vivo. However, there propagate the newly established human embryonic is no protocol for the differentiation of pancreatic-like stem cells line. The new cell line was checked for cells from human ESCs (hESCs). Based upon the pluripotent characteristics with detecting the mouse ESC model, we have induced the in vitro expression of stemness genes and observing formation of pancreatic exocrine enzyme-producing spontaneous differentiation both in vitro and in vivo. cells from hESCs. The protocol took place in four Finally similar step-wise protocols from definitive stages. In Stage 1, embryoid bodies (EBs) were endoderm to target specific cells were used to check formed from dissociated hESCs and then treated with the cell line's ability to directly differentiate into the growth factor activin A, which promoted the pancreatic and hepatic cells. RESULTS: We generated expression of Foxa2 and Sox17 mRNAs, markers of a new Chinese human embryonic stem cells line, CH1. definitive endoderm. In Stage 2, the cells were treated This cell line showed the same characteristics as other with all-trans retinoic acid which promoted the reported Chinese human embryonic stem cells lines: transition to cells that expressed gut tube endoderm normal morphology, karyotype and pluripotency in mRNA marker HNF1b. In Stage 3, the cells were vitro and in vivo. The CH1 cells could be directly treated with fibroblast growth factor 7 (FGF7), which differentiated towards pancreatic and hepatic cells induced expression of Pdx1 typical of pancreatic with equal efficiency compared to the H1 cell line. progenitor cells. In Stage 4, treatment with FGF7, CONCLUSIONS: This newly established Chinese cell glucagon-like peptide 1, and nicotinamide induced the line, CH1, which is pluripotent and has high potential expression amylase (AMY) mRNA, a marker for to differentiate into pancreatic and hepatic cells, will mature pancreatic exocrine cells. provide a useful tool for embryo development research, Immunohistochemical staining showed the expression along with clinical treatments for diabetes and some of AMY protein at the edges of cell clusters. These hepatic diseases. cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, Shinoyama, M., et al. (2013). "Cortical region- and pancreatic lipase in culture. Production of these specific engraftment of embryonic stem cell-derived hESC-derived pancreatic enzyme-producing cells neural progenitor cells restores axonal sprouting to a represents a critical step in the study of pancreatic subcortical target and achieves motor functional organogenesis and in the development of a renewable recovery in a mouse model of neonatal hypoxic- source of human pancreatic-like exocrine cells. ischemic brain injury." Front Cell Neurosci 7: 128. Hypoxic-ischemic encephalopathy (HIE) at birth Simonstein, F. (2008). "Embryonic stem cells: could cause cerebral palsy (CP), mental retardation, the disagreement debate and embryonic stem cell and epilepsy, which last throughout the individual's research in Israel." J Med Ethics 34(10): 732-734. lifetime. However, few restorative treatments for While some people claim that the present ischemic tissue are currently available. Cell disagreement over embryonic stem (ES) cell research replacement therapy offers the potential to rescue brain cannot be resolved, others argue that developing damage caused by HI and to restore motor function. In transparency and trust are key elements that could the present study, we evaluated the ability of resolve the existing disagreements over such research. embryonic stem cell-derived neural progenitor cells This paper reveals that transparency is not necessarily (ES-NPCs) to become cortical deep layer neurons, to a requirement for advancing ES cell research, since in restore the neural network, and to repair brain damage Israel, for instance, there is (almost) no transparency, in an HIE mouse model. ES cells stably expressing the and research nevertheless flourishes. Moreover, trust is reporter gene GFP are induced to a neural precursor not independent of cultural values and religious beliefs.

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Because of these beliefs, the environment in Israel for approximately 60%. Mechanistically, our data suggest ES cell research has been pragmatic and liberal. The that E-cadherin plays a role in hESC aggregation and Israeli case illustrates the key role that culture and that dissociation and re-aggregation upon passaging religion can play in biomedical research; it also functions as a purification step towards a pluripotency suggests that as far as cultural values or religious markers-enriched population. Mass expansion of beliefs of people in Western countries strongly oppose hESC was readily achieved by up-scaling 2 ml research on embryonic tissue, it would be very cultures to serial passaging in 50 ml spinner flasks. A difficult, if not impossible, to overcome the media comparison revealed that mTeSR was superior disagreements. to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency Simpson, D. L., et al. (2012). "Use of human markers was achieved for two lines (hES2, hES3) that embryonic stem cell derived-mesenchymal cells for were used at higher passages (>86). In contrast, rapid cardiac repair." Biotechnol Bioeng 109(1): 274-283. down regulation of Oct4, Tra-1-60, and SSEA4 was Human mesenchymal stem cells (hMSC) have observed for ESI049, a clinically compliant line, used proven beneficial in the repair and preservation of at passages 20-36. The up-scaling strategy has infarcted myocardium. Unfortunately, MSCs represent significant potential to provide pluripotent cells on a a small portion of the bone marrow and require ex clinical scale. Nevertheless, our data also highlights a vivo expansion. To further advance the clinical significant line-to-line variability and the need for a usefulness of cellular cardiomyoplasty, derivation of critical assessment of novel methods with numerous "MSC-like" cells that can be made available "off-the- relevant cell lines. shelf" are desirable. Recently, human embryonic stem cell-derived mesenchymal cells (hESC-MC) were Singh, N., et al. (2012). "Cloning and described. We investigated the efficacy of hESC-MC characterization of buffalo NANOG gene: alternative for cardiac repair after myocardial infarction (MI) transcription start sites, splicing, and polyadenylation compared to hMSC. Because of increased efficacy of in embryonic stem cell-like cells." DNA Cell Biol cell delivery, cells were embedded into collagen 31(5): 721-731. patches and delivered to infarcted myocardium. NANOG is a critical homeodomain transcription Culture of hMSC and hESC-MCs in collagen patches factor responsible for maintaining embryonic stem cell did not induce differentiation or significant loss in (ESC) self-renewal and pluripotency. In the present viability. Transplantation of hMSC and hES-MC study, we isolated, sequenced, and characterized the patches onto infarcted myocardium of athymic nude NANOG gene in buffalo ESC-like cells. Here, we rats prevented adverse changes in infarct wall demonstrated that NANOG mRNA is expressed as thickness and fractional area change compared to a multiple isoforms and uses four alternative non-viable patch control. Hemodynamic assessment transcriptional start sites (TSSs) and five different showed that hMSCs and hES-MC patch application polyadenylation sites. The TSSs identified by 5'-RNA improved end diastolic pressure equivalently. There ligase-mediated rapid amplification of cDNA ends were no changes in systolic function. hES-MC and (RLM-5'-RACE) were positioned at 182, 95, 35, and hMSC construct application enhanced neovessel 17 nucleotides upstream relative to the translation formation compared to a non-viable control, and each initiation codon. 3'-RACE experiment revealed the cell type had similar efficacy in stimulating endothelial presence of tandem polyadenylation signals, which cell growth in vitro. In summary, the use of hES-MC leads to the expression of at least five different 3'- provides similar efficacy for cellular cardiomyoplasty untranslated regions (269, 314, 560, 566, and 829 as compared to hMSC and may be considered a nucleotides). Expression analysis showed that these suitable alternative for cell therapy. alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open Singh, H., et al. (2010). "Up-scaling single cell- reading frame of buffalo NANOG codes for a 300- inoculated suspension culture of human embryonic amino-acid-long protein. Further, results showed that stem cells." Stem Cell Res 4(3): 165-179. alternative splicing leads to the expression of two We have systematically developed single cell- types of transcript variants encoded by four and five inoculated suspension cultures of human embryonic exons. In silico analysis of cloned 5'-flanking region stem cells (hESC) in defined media. Cell survival was (3366 nucleotides upstream of translation start codon) dependent on hESC re-aggregation. In the presence of identified several putative transcription factors binding the Rho kinase inhibitor Y-27632 (Ri) only sites in addition to a TATA box and CAAT box at -30 approximately 44% of the seeded cells were rescued, and -139 bp (upstream to the distal most TSS), but an optimized heat shock treatment combined with respectively, in the buffalo NANOG promoter. Ri significantly increased cell survival to

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Singh, S. A., et al. (2014). "p53-Independent cell injection of media alone. Histochemistry and cycle and erythroid differentiation defects in murine immunofluorescence were used to track the embryonic stem cells haploinsufficient for Diamond transplanted ES cells and identify the resulting cell Blackfan anemia-proteins: RPS19 versus RPL5." types. Echocardiography assessed the cardiac size and PLoS One 9(2): e89098. function in a blinded fashion. Two weeks post-MI, Diamond Blackfan anemia (DBA) is a rare engraftment of the transplanted ES cells was inherited bone marrow failure syndrome caused by demonstrated by eGFP or beta-galactosidase-positive ribosomal protein haploinsufficiency. DBA exhibits cells in the infarct region without evidence for tumor marked phenotypic variability, commonly presenting formation. Co-immunolabeling demonstrated that the with erythroid hypoplasia, less consistently with non- transplanted ES cells had become cardiomyocytes, erythroid features. The p53 pathway, activated by vascular smooth muscle, and endothelial cells. abortive ribosome assembly, is hypothesized to Echocardiographic analysis showed that ES cell contribute to the erythroid failure of DBA. We studied transplantation resulted in reduced post-MI remodeling murine embryonic stem (ES) cell lines harboring a of the heart and improved cardiac function. In gene trap mutation in a ribosomal protein gene, either conclusion, transplanted mouse ES cells can Rps19 or Rpl5. Both mutants exhibited ribosomal regenerate infarcted myocardium in part by becoming protein haploinsufficiency and polysome defects. cardiomyocytes, vascular smooth muscle, and Rps19 mutant ES cells showed significant increase in endothelial cells that result in an improvement in p53 protein expression, however, there was no similar cardiac structure and function. Therefore, ES cells increase in the Rpl5 mutant cells. Embryoid body hold promise for myocardial cellular therapy. formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When Tirotta, E., et al. (2012). "IFN-gamma-induced embryoid bodies were further differentiated to apoptosis of human embryonic stem cell derived primitive erythroid colonies, both mutants exhibited a oligodendrocyte progenitor cells is restricted by marked reduction in colony formation, which was CXCR2 signaling." Stem Cell Res 9(3): 208-217. again nonspecifically rescued by p53 inhibition. Cell Engraftment of human embryonic stem cell cycle analyses were normal in Rps19 mutant ES cells, (hESC)-derived OPCs in animal models of but there was a significant delay in the G2/M phase in demyelination results in remyelination and clinical the Rpl5 mutant cells, which was unaffected by p53 recovery, supporting the feasibility of cell replacement knockdown. Concordantly, Rpl5 mutant ES cells had a therapies in promoting repair of damaged neural tissue. more pronounced growth defect in liquid culture A critical gap in our understanding of the mechanisms compared to the Rps19 mutant cells. We conclude that associated with repair revolves around the effects of the defects in our RPS19 and RPL5 haploinsufficient the local microenvironment on transplanted cell mouse ES cells are not adequately explained by p53 survival. We have determined that treatment of human stabilization, as p53 knockdown appears to increase ESC-derived OPCs with the pleiotropic cytokine IFN- the growth and differentiation potential of both gamma promotes apoptosis that is associated with parental and mutant cells. Our studies demonstrate that mitochondrial cytochrome c released into the cytosol gene trap mouse ES cells are useful tools to study the with subsequent caspase 3 activation. IFN-gamma- pathogenesis of DBA. induced apoptosis is mediated, in part, by secretion of the CXC chemokine ligand 10 (CXCL10) from IFN- Singla, D. K., et al. (2006). "Transplantation of gamma-treated cells. Signaling through the chemokine embryonic stem cells into the infarcted mouse heart: receptor CXCR2 by the ligand CXCL1 functions in a formation of multiple cell types." J Mol Cell Cardiol tonic manner by muting apoptosis and this is 40(1): 195-200. associated with reduced levels of cytosolic cytochrome Initial studies have suggested that transplantation c and impaired cleavage of caspase 3. These findings of embryonic stem (ES) cells following myocardial support a role for both IFN-gamma and CXCL10 in infarction (MI) in animal models is beneficial; contributing to neuropathology by promoting OPC however, the mechanism of benefit is largely unknown. apoptosis. In addition, these data suggest that hOPCs The present study investigated the fate of mouse ES used for therapeutic treatment for human neurologic cells transplanted post-MI to determine if the ES cells disease/damage are susceptible to death through give rise to the range of major cell types present in the exposure to local inflammatory cytokines present native myocardium. MI was produced by coronary within the inflammatory milieu. artery ligation in C57BL/6 mice. Two different mouse ES cell lines, expressing eGFP and beta-galactosidase, Toh, W. S., et al. (2010). "Cartilage repair using respectively, were tested. Post-MI intramyocardial hyaluronan hydrogel-encapsulated human embryonic injection of 3 x 10(4) ES cells was compared to

190 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ stem cell-derived chondrogenic cells." Biomaterials signaling factors derived from differentiated hBMSCs. 31(27): 6968-6980. For 28 days, hESCs were grown in a transwell co- Human embryonic stem cells (hESCs) have the culture system with hBMSCs that had been previously potential to offer a virtually unlimited source of differentiated in growth medium containing defined chondrogenic cells for use in cartilage repair and osteogenic supplements for 7-24 days. As a control. regeneration. We have recently shown that expandable hESCs were co-cultured with undifferentiated chondrogenic cells can be derived from hESCs under hBMSCs and alone. Von Kossa and Alizarin Red selective growth factor-responsive conditions. In this staining as well as immunohistochemistry confirmed study, we explore the potential of these hESC-derived that the hESCs co-cultured with differentiated chondrogenic cells to produce an extracellular matrix hBMSCs formed mineralized bone nodules and (ECM)-enriched cartilaginous tissue construct when secreted extracellular matrix protein osteocalcin cultured in hyaluronic acid (HA)-based hydrogel, and (OCN). Quantitative Alizarin Red assays showed further investigated the long-term reparative ability of increased mineralization as compared to the control the resulting hESC-derived chondrogenic cell- with undifferentiated hBMSCs. RT-PCR revealed the engineered cartilage (HCCEC) in an osteochondral loss of pluripotent hESC markers with the concomitant defect model. We hypothesized that HCCEC can gain of osteoblastic markers such as collagen type I, provide a functional template capable of undergoing runx2, and osterix. We demonstrate that osteogenic orderly remodeling during the repair of critical-sized growth factors derived from differentiated hBMSCs osteochondral defects (1.5 mm in diameter, 1 mm within the local microenvironment may help to depth into the subchondral bone) in a rat model. In the promote hESC osteogenic differentiation. process of repair, we observed an orderly spatial- temporal remodeling of HCCEC over 12 weeks into Toya, S. P., et al. (2011). "Interaction of a osteochondral tissue, with characteristic architectural specific population of human embryonic stem cell- features including a hyaline-like neocartilage layer derived progenitor cells with CD11b+ cells with good surface regularity and complete integration ameliorates sepsis-induced lung inflammatory injury." with the adjacent host cartilage and a regenerated Am J Pathol 178(1): 313-324. subchondral bone. By 12 weeks, the HCCEC- Human embryonic stem cells differentiated under regenerated osteochondral tissue resembled closely mesoderm-inducing conditions have important that of age-matched unoperated native control, while therapeutic properties in sepsis-induced lung injury in only fibrous tissue filled in the control defects which mice. Single cell suspensions obtained from day 7 left empty or treated with hydrogel alone. Here we human embryoid bodies (d7EBs) injected i.v. 1 hour demonstrate that transplanted hESC-derived after cecal ligation and puncture significantly reduced chondrogenic cells maintain long-term viability with lung inflammation and edema as well as production of no evidence of tumorigenicity, providing a safe, tumor necrosis factor-alpha and interferon-gamma in highly-efficient and practical strategy of applying lungs compared with controls, whereas interleukin-10 hESCs for cartilage tissue engineering. production remained elevated. d7EB cell transplantation also reduced mortality to 50% from 90% Tong, W., et al. (2007). "Human Embryonic in the control group. The protection was ascribed to Stem Cells Undergo Osteogenic Differentiation in d7EB cell interaction with lung resident CD11b+ cells, Human Bone Marrow Stromal Cell and was correlated with the ability of d7EB cells to Microenvironments." J Stem Cells 2(3): 139-147. reduce it also reduced production of proinflammatory Human embryonic stem cells (hESCs) may offer cytokines by CD11+ cells, and to endothelial NO an unlimited supply of cells that can be directed to synthase-derived NO by d7EB cells, leading to differentiate into all cell types within the body and inhibition of inducible macrophage-type NO synthase used in regenerative medicine for tissue and cell activation in CD11b+ cells. The protective progenitor replacement therapies. Previous work has shown that cells were positive for the endothelial and exposing hESCs to exogenous factors such as hematopoietic lineage marker angiotensin converting dexamethasone, ascorbic acid and beta- enzyme (ACE). Only the ACE+ fraction modulated glycerophosphate can induce osteogenesis. The the proinflammatory profile of CD11b+ cells and specific factors that induce osteogenic differentiation reduced mortality in septic mice. In contrast to the of hESCs have not been identified yet, however, it is nonprotective ACE-cell fraction, the ACE+ cell possible that differentiated human bone marrow fraction also produced NO. These findings suggest that stromal cells (hMBSCs) may secrete factors within the an ACE+ subset of human embryonic stem cell- local microenvironment that promote osteogenesis. derived progenitor cells has a highly specialized anti- Here we report that the lineage progression of hESCs inflammatory function that ameliorates sepsis-induced to osteoblasts is achieved in the presence of soluble lung inflammation and reduces mortality.

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transcriptionally active beta-catenin is associated with Tropepe, V., et al. (2001). "Direct neural fate both neural and primitive streak commitment. These specification from embryonic stem cells: a primitive observations confirm and extend previous suggestions mammalian neural stem cell stage acquired through a that pluripotency genes influence lineage commitment default mechanism." Neuron 30(1): 65-78. and demonstrate how their dynamic expression affects Little is known about how neural stem cells are the direction of lineage commitment, whilst illustrating formed initially during development. We investigated two ways in which the Wnt signalling pathway acts on whether a default mechanism of neural specification this network during cell fate assignment. could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells Trounson, A. (2002). "Human embryonic stem cultured in defined, low-density conditions readily cells: mother of all cell and tissue types." Reprod acquire a neural identity. We characterize a novel Biomed Online 4 Suppl 1: 58-63. primitive neural stem cell as a component of neural Pluripotential embryonic stem (ES) cells have lineage specification that is negatively regulated by been derived very efficiently from spare human TGFbeta-related signaling. Primitive neural stem cells embryos produced by IVF and grown in culture to the have distinct growth factor requirements, express nascent blastocyst stage. The inner cell mass (ICM) is neural precursor markers, generate neurons and glia in isolated by immunosurgery and grown on selected vitro, and have neural and non-neural lineage potential embryonic fibroblast monolayer cultures. ICM cells in vivo. These results are consistent with a default lose their memory for axis during formation of ES cell mechanism for neural fate specification and support a colonies and are then unable to integrate tissue model whereby definitive neural stem cell formation is formation with a body plan. ES cells form teratomas in preceded by a primitive neural stem cell stage during vivo with cells and tissues representative of the three neural lineage commitment. major embryonic lineages (ectoderm, mesoderm, endoderm). The ES cells are continuously renewable Trott, J. and A. Martinez Arias (2013). "Single and can be directed to differentiate into early cell lineage analysis of mouse embryonic stem cells at progenitors of neural stem cells (Noggin cells) and the exit from pluripotency." Biol Open 2(10): 1049- from there into mature neurons and glia (astrocytes 1056. and oligodendrocytes). The neural stem cells formed Understanding how interactions between from human ES cells repopulate the brains of newborn extracellular signalling pathways and transcription mice when injected into the lateral cerebral ventricles, factor networks influence cellular decision making forming astrocytes dominantly in the parenchyma. The will be crucial for understanding mammalian human neural cells can be observed migrating from the embryogenesis and for generating specialised cell subventricular areas along the rostral migratory stream. types in vitro. To this end, pluripotent mouse Human neurons can be found in the olfactory bulb. Embryonic Stem (mES) cells have proven to be a Human ES cells can also be directed into useful model system. However, understanding how cardiomyocytes when co-cultured with visceral transcription factors and signalling pathways affect endoderm-like cells (END-2). These observations decisions made by individual cells is confounded by provide further scope to explore stem cell therapies, the fact that measurements are generally made on gene therapies and drug discovery. For compatible groups of cells, whilst individual mES cells transplantation, ES may need to be derived with a differentiate at different rates and towards different range of HLA types or by nuclear transplantation or lineages, even in conditions that favour a particular stem cell fusion. lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/beta-catenin Troy, T. C. and K. Turksen (2005). signalling activity to investigate their effects on "Commitment of embryonic stem cells to an epidermal lineage commitment decisions made by individual cell fate and differentiation in vitro." Dev Dyn 232(2): cells. We find that pluripotent mES cells exhibit 293-300. differing degrees of heterogeneity in their expression The epidermis develops from a stem cell of important regulators from pluripotency, depending population in the surface ectoderm that feeds a single on the signalling environment to which they are vertical terminal differentiation pathway. To date, exposed. As mES cells differentiate, downregulation however, the limited capacity for the isolation or of Nanog and Oct4 primes cells for neural purification of epidermal stem or precursor cells has commitment, whilst loss of Sox2 expression primes hampered studies on early commitment and cells for primitive streak commitment. Furthermore, differentiation events. We have developed a two-step we find that Wnt signalling acts through Nanog to culture scheme in which pluripotent mouse embryonic direct cells towards a primitive streak fate, but that stem (ES) cells are induced first to a surface ectoderm

192 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ phenotype and then are positively selected for putative been previously determined. In this investigation, 206 epidermal stem cells. We show that the earliest stages genes were found to be targets of the four hES cell- of epidermal development follow an ordered sequence specific miRNAs of miR-302d, miR-372, miR-200c, that is similar to that observed in vivo (expression of and/or miR-367 by using two-fold differential keratin 8, keratin 19, keratin 17, and keratin 14), expression and inverse expression levels (highly suggesting that ES cell-derived surface ectoderm-like negative correlations) of miRNAs to their target cells can be induced to follow the epidermal mRNAs. That YWHAZ (14-3-3 zeta) is a target of developmental pathway. At a low frequency, keratin miR-302d and miR-372 was further confirmed by 14-positive early epidermal cells progressed to keratin proteomic comparison between T3ES cells and their 1-positive and terminally differentiated cells producing differentiated T3DF fibroblasts. According to a cornified envelope. This culturing protocol provides GeneOntology analyses, almost 50% of these 206 an invaluable system in which to study both the target proteins are nuclear and are involved in gene mechanisms that direct stem cells along the epidermal transcription. Identifying the target mRNAs of hES pathway as well as those that influence their cell-specific miRNAs will provide a better subsequent epidermal differentiation. understanding of the complex regulatory networks in hES cells. Furthermore, these miRNA-targeted Tsai, M., et al. (2002). "Mast cells derived from proteins play important roles in differentiation of hES embryonic stem cells: a model system for studying the cells and during embryo development. effects of genetic manipulations on mast cell development, phenotype, and function in vitro and in Tsang, K. M., et al. (2017). "Embryonic Stem vivo." Int J Hematol 75(4): 345-349. Cell Differentiation to Functional Arterial Endothelial Large quantities of highly enriched populations Cells through Sequential Activation of ETV2 and of mast cells can be generated from mouse embryonic NOTCH1 Signaling by HIF1alpha." Stem Cell Reports stem (ES) cells using an in vitro differentiation system. 9(3): 796-806. These embryonic stem cell-derived mast cells (ESMCs) The generation of functional arterial endothelial exhibit many similarities to mouse bone marrow- cells (aECs) from embryonic stem cells (ESCs) holds derived cultured mast cells (BMCMCs), including the great promise for vascular tissue engineering. abilities to survive and to orchestrate immunologically However, the mechanisms underlying their generation specific immunoglobulin E (IgE)-dependent reactions and the potential of aECs in revascularizing ischemic in vivo after transplantation into genetically mast cell- tissue are not fully understood. Here, we observed that deficient KitW/KitW-v mice. Coupled with the current hypoxia exposure of mouse ESCs induced an initial spectrum of techniques for genetically manipulating phase of HIF1alpha-mediated upregulation of the ES cells, ESMCs represent a unique model system to transcription factor Etv2, which in turn induced the analyze the effects of specific alterations in gene commitment to the EC fate. However, sustained structure, expression, or function, including embryonic activation of HIF1alpha in these EC progenitors lethal mutations, on mast cell development, phenotype, thereafter induced NOTCH1 signaling that promoted and function in vitro and in vivo. the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the Tsai, Z. Y., et al. (2011). "Proteomic comparison mouse hindlimb ischemia model. Furthermore, of human embryonic stem cells with their transplantation of aECs in mice showed engraftment in differentiated fibroblasts: Identification of 206 genes ischemic myocardium and restored cardiac function in targeted by hES cell-specific microRNAs." Kaohsiung contrast to ECs derived under normoxia. Thus, J Med Sci 27(8): 299-306. HIF1alpha activation of Etv2 in ESCs followed by Human embryonic stem (hES)-T3 (T3ES) cells NOTCH1 signaling is required for the generation aECs were spontaneously differentiated into autogeneic that are capable of arteriogenesis and revascularization fibroblast-like T3DF cells, as feeder cells with the of ischemic tissue. capacity to support the growth of undifferentiated hES cells. The proteomes of undifferentiated T3ES cells Wakao, H., et al. (2008). "In vitro induction of and their differentiated T3DF fibroblasts were natural killer T cells from embryonic stem cells quantitatively compared. Several heterogeneous prepared using somatic cell nuclear transfer." FASEB nuclear ribonucleoproteins and glycolytic enzymes, J 22(7): 2223-2231. including l-lactate dehydrogenase A (M), were found The ectopic expression of the Notch receptor to be abundantly and differentially expressed in T3ES ligand delta-like 1 on stromal cells allows the cells and T3DF fibroblasts, respectively. Both miRNA induction of T cells from embryonic stem cells (ESCs). and mRNA profiles from the undifferentiated T3ES However, these in vitro-generated T cells are not cells and their differentiated T3DF fibroblasts had transplantable because they are too immature to mount

193 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ an immune response in an immunocompromised Recent advances in mouse cloning are reported to animal. We efficiently generated a subset of T cells illustrate its strengths and promise in the study of called invariant natural killer T (iNKT) cells from mammalian biology and biomedicine. ESCs derived from peripheral iNKT cells using somatic cell nuclear transfer (ntESCs). These iNKT Wakayama, T. (2006). "Establishment of nuclear cells matured autonomously in vivo and exhibited an transfer embryonic stem cell lines from adult somatic adjuvant effect accompanying the production of cells by nuclear transfer and its application." Ernst interferon-gamma in an antigen-specific manner. This Schering Res Found Workshop (60): 111-123. adjuvant effect culminated in the inhibition of Nuclear transfer can be used to generate inoculated tumor cell growth. Our results indicate that embryonic stem cell (ntESC) lines from a patient's ntESC-derived iNKT cells are transplantable own somatic cells. We have shown that ntESCs can be lymphocytes that will be beneficial for the induction of generated relatively easily from a variety of mouse immune tolerance and the treatment of autoimmune genotypes and cell types of both sexes, even though it diseases, tumors, and infections. may be more difficult to generate clones directly. Several reports have already demonstrated that ntESCs Wakayama, S., et al. (2005). "Propagation of an can be used in regenerative medicine in order to rescue infertile hermaphrodite mouse lacking germ cells by immunodeficient or infertile phenotypes. However, it using nuclear transfer and embryonic stem cell is unclear whether ntES cells are identical to fertilized technology." Proc Natl Acad Sci U S A 102(1): 29-33. embryonic stem cells (ESCs). This review seeks to Animals generated by systematic mutagenesis describe the phenotype and possible abnormalities of and routine breeding are often infertile because they ntESC lines. lack germ cells, and maintenance of such lines of animals has been impossible. We found a Wakayama, T., et al. (2001). "Differentiation of hermaphrodite infertile mouse in our colony, a genetic embryonic stem cell lines generated from adult male with an abnormal Y chromosome lacking somatic cells by nuclear transfer." Science 292(5517): developing germ cells. We tried to clone this mouse by 740-743. conventional nuclear transfer but without success. ES Embryonic stem (ES) cells are fully pluripotent cells produced from blastocysts, which had been in that they can differentiate into all cell types, cloned by using somatic cell nuclear transfer (ntES including gametes. We have derived 35 ES cell lines cells) from this mouse, were also unable to produce via nuclear transfer (ntES cell lines) from adult mouse offspring when injected into enucleated oocytes. somatic cells of inbred, hybrid, and mutant strains. Although we were able to produce two chimeric ntES cells contributed to an extensive variety of cell offspring using these ntES cells by tetraploid types, including dopaminergic and serotonergic complementation, they were infertile, because they neurons in vitro and germ cells in vivo. Cloning by also lacked developing germ cells. However, when transfer of ntES cell nuclei could result in normal such ntES cells were injected into normal diploid development of fertile adults. These studies blastocysts, many chimeric offspring were produced. demonstrate the full pluripotency of ntES cells. One such male offspring transmitted hermaphrodite mouse genes to fertile daughters via X chromosome- Walker, L. M., et al. (2018). "The Validated bearing sperm. Thus, ntES cells were used to Embryonic Stem Cell Test with Murine Embryonic propagate offspring from infertile mice lacking germ Stem Cells." Methods Mol Biol 1797: 97-124. cells. Birth defects are the leading cause of infant mortality in the USA, yet the causes of most of these Wakayama, T. (2003). "Cloned mice and conditions are unknown. While a combination of embryonic stem cell lines generated from adult genetic and environmental factors are suspected in somatic cells by nuclear transfer." Oncol Res 13(6-10): most cases, little information exists about the health 309-314. risks that prenatal exposure to many common Mice can now be cloned from cultured and chemicals poses for the fetus. Thus, development and noncultured adult-, fetus-, male-, or female-derived refinement of procedures that can accurately predict cells. Using the mouse as a model, research is moving embryotoxicity of compounds is important for towards a comprehensive description of clones curtailing the number of infants born with birth defects. generated by somatic cell nuclear transfer. In addition, The embryonic stem cell test (EST) is a procedure that embryonic stem (ES) cell lines can be generated from utilizes comparison of cytotoxicity in embryonic and adult somatic cells via nuclear transfer (ntES cells). adult cells and inhibition of differentiation to predict ntES cells contribute to an extensive variety of cell embryotoxicity of compounds tested. Because of its types including neurons in vitro and germ cells in vivo. use of existing cell lines, the EST dramatically reduces

194 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ the need for animal test subjects in toxicity testing. In attach to the bottoms of culturing disks and grow. OA addition, because of its use of inhibition of was added into the medium to induce the EBs to differentiation as an endpoint, the EST is extremely differentiate. Retinoic acid (RA) was used as the versatile in the range of complications it can test for. positive drug. After 72 h, total RNA was extracted, In this chapter, procedures for use of the validated cDNA was synthesized, and real-time fluorescence embryonic stem cell test with the traditional quantitative PCR was performed to measure the cardiomyocyte differentiation endpoint are explained. transcriptional expression profiles of 11 reproduction- The protocol includes discussion of routine stem cell related genes affected by OA and RA, respectively. culture, the cardiomyocyte differentiation procedure, When the data were compared, it was found that OA and methods for utilization of molecular endpoints for up-regulated the transcriptional levels of Oct-4, GDF-9, assessing embryotoxicity of compounds. Stra8, Mvh, ZP2, ZP3, Itga6, and TP2, and down- regulated transcriptional levels of SCP3, ZP1, and Walter, J. and M. Dihne (2012). "Species- Itgb1; RA up-regulated the transcriptional levels of dependent differences of embryonic stem cell-derived GDF-9, Stra8, Mvh, ZP2, ZP3, Itga6, Itgb1, and TP2, neural stem cells after Interferon gamma treatment." and down-regulated the transcriptional levels of Oct-4, Front Cell Neurosci 6: 52. SCP3, and ZP1. The data showed that OA and RA had Pluripotent stem cell (pSC)-derived, neural stem similar effects in inducing differentiation of MESC cells (NSCs) are actually extensively explored in the towards GC. field of neuroregeneration and to clarify disease mechanisms or model neurological diseases in vitro. Wan, Q., et al. (2014). "Retinoic acid can induce Regarding the latter, proliferation and differentiation mouse embryonic stem cell R1/E to differentiate of pSC-derived NSCs are investigated under the toward female germ cells while oleanolic acid can influence of a variety of different substances among induce R1/E to differentiate toward both types of germ them key players of inflammation. However, results cells." Cell Biol Int 38(12): 1423-1429. generated on a murine genetic background are not Retinoic acid (RA) and oleanolic acid (OA) were always representative for the human situation which studied about their potential to induce mouse increasingly leads to the application of human cell embryonic stem cell R1/E (MESC-R1/E) to culture systems derived from human pSCs. We differentiate toward germ cells. Embryoid bodies (EBs) investigated here, if the recently described interferon first formed from MESC-R1/E and EBs were allowed gamma (IFNgamma)-induced dysregulated neural to attach to the bottoms of normal cell-culturing plate phenotype characterized by simultaneous expression and grow. Then, different compounds including RA, of glial and neuronal markers on murine NSCs (Walter OA and so on were respectively added to induce et al., 2011, 2012) can also be found on a human MESC-R1/E to differentiate. After 72 h, microscopy genetic background. For this purpose, we performed images were taken for all interventions, then total experiments with human embryonic stem cell-derived RNAs were extracted, cDNAs were synthesized and NSCs. We could show that the IFNgamma-induced real-time fluorescence quantitative PCR (qPCR) was dysregulated neural phenotype cannot be induced in performed to detect the transcriptional expression human NSCs. This difference occurs, although typical patterns of 11 reproductive-differentiation-related genes like signal transducers and activators of genes for different compounds respectively. During transcription 1 (Stat 1) or interferon regulatory factor 9 the data analysis, it was found RA significantly up- (IRF-9) are similarly regulated by IFNgamma in both, regulated the expression levels of GDF-9, Stra8, SCP3, murine and human populations. These results illustrate Mvh, ZP1, ZP2, and ZP3, while significantly down- that fundamental differences between murine and regulated the levels of Itag6 and Itgb1, and the level of human neural populations exist in vitro, independent Oct-4 was down-regulated insignificantly, while the of anatomical system-related properties. level of TP2 was up-regulated insignificantly; OA significantly up-regulated the expression levels of Wan, Q., et al. (2014). "Oleanolic acid has Stra8, SCP3, Mvh, ZP1, ZP2, Itgb1, and TP2, and the similar effects as retinoic acid in inducing mouse levels of Oct-4, GDF-9, ZP3, and Itga6 were up- embryonic stem cell 1B10 to differentiate towards regulated insignificantly. The data showed that RA can germ cells." Hum Cell 27(1): 5-11. induce MESC-R1/E to differentiate toward female This study investigated the effects of the germ cells while OA can induce MESC-R1/E to compound oleanolic acid (OA) in inducing mouse differentiate toward male and female germ cells. embryonic stem cells (MESC) to differentiate towards germ cells (GC). MESC 1B10 was used as the model Wang, C., et al. (2017). "Basic fibroblast growth cell. 1B10 was cultured, and embryoid bodies (EBs) factor is critical to reprogramming buffalo (Bubalus were produced from 1B10. The EBs were allowed to

195 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ bubalis) primordial germ cells into embryonic germ Wang, H., et al. (2016). "Germ Cell Nuclear stem cell-like cells." Theriogenology 91: 112-120. Factor (GCNF) Represses Oct4 Expression and Primordial germ cells (PGCs) are destined to Globally Modulates Gene Expression in Human form gametes in vivo, and they can be reprogrammed Embryonic Stem (hES) Cells." J Biol Chem 291(16): into pluripotent embryonic germ (EG) cells in vitro. 8644-8652. Buffalo PGC have been reported to be reprogrammed Oct4 is considered a key transcription factor for into EG-like cells, but the identities of the major pluripotent stem cell self-renewal. It binds to specific signaling pathways and culture media involved in this regions within target genes to regulate their expression derivation remain unclear. Here, the effects of basic and is downregulated upon induction of differentiation fibroblast growth factor (bFGF) and downstream of pluripotent stem cells; however, the mechanisms signaling pathways on the reprogramming of buffalo that regulate the levels of human Oct4 expression PGCs into EG-like cells were investigated. Results remain poorly understood. Here we show that showed bFGF to be critical to buffalo PGCs to expression of human Oct4 is directly repressed by dedifferentiate into EG-like cells (20 ng/mL is optimal) (GCNF), an orphan nuclear with many characteristics of pluripotent stem cells, receptor, in hES cells. Knockdown of GCNF by including alkaline phosphatase (AP) activity, siRNA resulted in maintenance of Oct4 expression expression of pluripotency marker genes such as during RA-induced hES cell differentiation. While OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1- overexpression of GCNF promoted repression of Oct4 81, and the capacity to differentiate into all three expression in both undifferentiated and differentiated embryonic germ layers. After chemically inhibiting hES cells. The level of Oct4 repression was dependent pathways or components downstream of bFGF, data on the level of GCNF expression in a dose-dependent showed that inhibition of the PI3K/AKT pathway led manner. mRNA microarray analysis demonstrated that to significantly lower EG cell derivation, while overexpression of GCNF globally regulates gene inhibition of P53 activity resulted in an efficiency of expression in undifferentiated and differentiated hES EG cell derivation comparable to that in the presence cells. Within the group of altered genes, GCNF down- of bFGF. These results suggest that the role of bFGF regulated 36% of the genes, and up-regulated 64% in in PGC-derived EG-like cell generation is mainly due undifferentiated hES cells. In addition, GCNF also to the activation of the PI3K/AKT/P53 pathway, in showed a regulatory gene pattern that is different from particular, the inhibition of P53 function. RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms Wang, D., et al. (2010). "Transplantation of that maintain hES cell pluripotency and regulate gene human embryonic stem cell-derived alveolar epithelial expression during the differentiation process. type II cells abrogates acute lung injury in mice." Mol Ther 18(3): 625-634. Wang, L. (2006). "Endothelial and hematopoietic Respiratory diseases are a major cause of cell fate of human embryonic stem cells." Trends mortality and morbidity worldwide. Current treatments Cardiovasc Med 16(3): 89-94. offer no prospect of cure or disease reversal. The endothelial cells, lining the inside of blood Transplantation of pulmonary progenitor cells derived vessels, and the blood-forming hematopoietic cells from human embryonic stem cells (hESCs) may play crucial roles in vasculogenesis. The establishment provide a novel approach to regenerate endogenous of human embryonic stem cells (hESCs) provides a lung cells destroyed by injury and disease. Here, we unique tool to study the early development of examine the therapeutic potential of alveolar type II endothelial and hematopoietic cells, opening new epithelial cells derived from hESCs (hES-ATIICs) in a avenues of research to explore organ vascularization mouse model of acute lung injury. When transplanted and regeneration. The current study demonstrates that into lungs of mice subjected to bleomycin (BLM)- a population of intermediate-stage precursors, which induced acute lung injury, hES-ATIICs behaved as possesses primitive endothelial properties during normal primary ATIICs, differentiating into cells hESC differentiation, is capable of giving rise to expressing phenotypic markers of alveolar type I endothelial and hematopoietic cells. Single cell epithelial cells. Without experiencing tumorigenic side analysis reveals that these primitive endothelial-like effects, lung injury was abrogated in mice transplanted precursors contain rare bipotent cells with with hES-ATIICs, demonstrated by recovery of body hemangioblast properties, responsible for both weight and arterial blood oxygen saturation, decreased endothelial and hematopoietic cell fates. These collagen deposition, and increased survival. Therefore, findings will facilitate the further study of cellular transplantation of hES-ATIICs shows promise as an commitment, lineage restriction, and terminal effective therapeutic to treat acute lung injury. differentiation of endothelial and hematopoietic

196 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ compartments and may lead to novel regenerative for dissecting the molecular pathways in early germ therapies. cell formation during embryogenesis.

Wang, L., et al. (2004). "Endothelial and Wang, R. and Y. L. Guo (2012). "Transient hematopoietic cell fate of human embryonic stem cells inhibition of cell proliferation does not compromise originates from primitive endothelium with self-renewal of mouse embryonic stem cells." Exp Cell hemangioblastic properties." Immunity 21(1): 31-41. Res 318(16): 2094-2104. The cellular organization and relationships Embryonic stem cells (ESCs) have unlimited among precursors that initiate embryonic angiogenesis capacity for self-renewal and can differentiate into and hematopoiesis in the human have yet to be various cell types when induced. They also have an characterized. Here, we identify a subpopulation of unusual cell cycle control mechanism driven by primitive endothelial-like cells derived from human constitutively active cyclin dependent kinases (Cdks). embryonic stem cells (hESCs) that express PECAM-1, In mouse ESCs (mESCs). It is proposed that the rapid Flk-1, and VE-cadherin, but not CD45 (CD45negPFV cell proliferation could be a necessary part of cells), and that are uniquely responsible for endothelial mechanisms that maintain mESC self-renewal and and hematopoietic development. Molecular profiling pluripotency, but this hypothesis is not in line with the of CD45negPFV cells is consistent with endothelial finding in human ESCs (hESCs) that the length of the and hematopoietic competency. Clonal isolation cell cycle is similar to differentiated cells. Therefore, demonstrates that the CD45negPFV population whether rapid cell proliferation is essential for the includes bipotent cells with endothelial and maintenance of mESC state remains unclear. We hematopoietic capacity. We suggest that human provide insight into this uncertainty through chemical hematopoiesis and endothelial maturation originate intervention of mESC cell cycle. We report here that exclusively from a subset of embryonic endothelium inhibition of Cdks with olomoucine II can dramatically that possesses hemangioblastic properties and offers a slow down cell proliferation of mESCs with model system to study these lineage relationships in concurrent down-regulation of cyclin A, B and E, and the human. the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II Wang, Q., et al. (2013). "GASZ promotes germ and are able to resume normal cell proliferation cell derivation from embryonic stem cells." Stem Cell without losing self-renewal and pluripotency, as Res 11(2): 845-860. demonstrated by the expression of ESC markers, Primordial germ cells (PGCs) are the first germ- colony formation, embryoid body formation, and line population that forms from the proximal epiblast induced differentiation. We provide a mechanistic of the developing embryo. Despite their biological explanation for these observations by demonstrating importance, the regulatory networks whereby PGCs that Oct4 and Nanog, two major transcription factors arise, migrate, and differentiate into gametes during that play critical roles in the maintenance of ESC embryonic development remains elusive, largely due properties, are up-regulated via de novo protein to the limited number of germ cells in the early synthesis when the cells are exposed to olomoucine II. embryo. To elucidate the molecular mechanisms that Together, our data suggest that short-term inhibition of govern early germ cell development, we utilized an in cell proliferation does not compromise the basic vitro differentiation model of embryonic stem cells properties of mESCs. (ESCs) and screened a series of candidate genes with specific expression in the adult reproductive organs. Wang, Y. and R. Blelloch (2009). "Cell cycle We discovered that gain of function of Gasz, a gene regulation by MicroRNAs in embryonic stem cells." previously reported to participate in meiosis of Cancer Res 69(10): 4093-4096. postnatal spermatocytes, led to the most robust The cell cycle is tightly orchestrated during upregulation of PGC formation from both human and normal development. Embryonic stem (ES) cells have murine ESCs. In contrast, Gasz deficiency resulted in a unique cell cycle structure, in which the G1/S pronounced reduction of germ cells during ESC restriction is largely absent, enabling cells to rapidly differentiation and decreased expression of MVH and move through the G1 phase and enter the S phase. This DAZL in genital ridges during early embryonic hastened cell cycle allows the early embryo to rapidly development. Further analyses demonstrated that grow. Recent experiments suggest that small GASZ interacted with DAZL, a key germ cell noncoding RNAs, the microRNAs (miRNAs), play a regulator, to synergistically promote germ cell central role in achieving this unique cell cycle derivation from ESCs. Thus, our data reveal a structure. The responsible miRNAs function by potential role of GASZ during embryonic germ cell suppressing multiple inhibitors of the G1/S transition. development and provide a powerful in vitro system Expression of these miRNAs drops dramatically as the

197 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ

ES cells differentiate and as the G1 phase extends. computational method that measures the responses of Some of the same miRNAs are overexpressed in biochemical pathways to the panel of treatments and cancers, in which they can promote tumor growth, showed that these responses were discriminative for suggesting common mechanisms of miRNA-regulated the three toxicity classes and linked to carcinogenesis cell cycle control in ES cells and cancers. This review through p53, mitogen-activated protein kinases, and discusses these recent findings in the context of apoptosis pathway modules. It could further be shown broader knowledge of cell cycle control in normal and that the discrimination of toxicity classes was abnormal development. improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, Wang, Y., et al. (2005). "Embryonic stem cell- for the first time, the potential of human embryonic derived hematopoietic stem cells." Proc Natl Acad Sci stem cell--derived hepatic cells as an in vitro model for U S A 102(52): 19081-19086. hazard assessment of chemical carcinogenesis, Despite two decades of studies documenting the although it should be noted that more compounds are in vitro blood-forming potential of murine embryonic needed to test the robustness of the assay. stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells Yin, X., et al. (2006). "Proteomic analysis reveals in irradiated mice has proven difficult. We have higher demand for antioxidant protection in embryonic exploited the -Hox pathway, a genetic program stem cell-derived smooth muscle cells." Proteomics important for blood development, to enhance the 6(24): 6437-6446. differentiation of ESCs along the hematopoietic Embryonic stem (ES) cells can differentiate into lineage. Using an embryonic stem cell line engineered vascular smooth muscle cells (SMCs), but differences with tetracycline-inducible Cdx4, we demonstrate that in protein composition, function and behaviour ectopic Cdx4 expression promotes hematopoietic between stem cell-derived and mature SMCs remain to mesoderm specification, increases hematopoietic be characterized. Using differential in gel progenitor formation, and, together with HoxB4, electrophoresis (DIGE) and MS, we identified 146 enhances multilineage hematopoietic engraftment of proteins that differed between ES cell-derived SMCs lethally irradiated adult mice. Clonal analysis of (esSMCs) and aortic SMCs, including proteins retroviral integration sites confirms a common stem involved in DNA maintenance (higher in esSMCs), cell origin of lymphoid and myeloid populations in cytoskeletal proteins and calcium-binding proteins engrafted primary and secondary mice. These data (higher in aortic SMCs). Notably, esSMCs showed document the cardinal stem cell features of self- decreased expression of mitochondrial, but a renewal and multilineage differentiation of ESC- compensatory increase of cytosolic antioxidants. derived hematopoietic stem cells. Subsequent experiments revealed that mitochondrial- derived reactive oxygen species (ROS) were markedly Yildirimman, R., et al. (2011). "Human increased in esSMCs. Despite a three-fold rise in embryonic stem cell derived hepatocyte-like cells as a glutathione (GSH) reductase activity, esSMCs had tool for in vitro hazard assessment of chemical lower levels of reduced GSH, and depletion of GSH carcinogenicity." Toxicol Sci 124(2): 278-290. by diethyl maleate or inhibition of GSH reductase by Hepatocyte-like cells derived from the carmustine (BCNU) resulted in more pronounced cell differentiation of human embryonic stem cells (hES- death compared to aortic SMCs, while addition of Hep) have potential to provide a human relevant in antioxidants improved the viability of esSMCs. We vitro test system in which to evaluate the carcinogenic present the first proteomic analysis of esSMCs hazard of chemicals. In this study, we have demonstrating that stem cell-derived SMCs are more investigated this potential using a panel of 15 sensitive to oxidative stress due to increased chemicals classified as noncarcinogens, genotoxic generation of mitochondrial-derived ROS and require carcinogens, and nongenotoxic carcinogens and additional antioxidant protection for survival. measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA Yla-Outinen, L., et al. (2014). "Three- model that identified 592 genes highly discriminative dimensional growth matrix for human embryonic stem for the panel of chemicals. Supervised classification cell-derived neuronal cells." J Tissue Eng Regen Med with these genes achieved a cross-validation accuracy 8(3): 186-194. of > 95%. Moreover, the expression of the response The future of tissue engineering applications for genes in hES-Hep was strongly correlated with that in neuronal cells will require a supportive 3D matrix. human primary hepatocytes cultured in vitro. In order This particular matrix should be soft, elastic and to infer mechanistic information on the consequences supportive for cell growth. In this study, we of chemical exposure in hES-Hep, we developed a characterized the suitability of a 3D synthetic hydrogel

198 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ matrix, PuraMatrix, as a growth platform for human cell proliferation of neural stem/progenitor cells embryonic stem cell (hESC)-derived neural cells. The derived from embryonic hippocampus." J Pharmacol viability of the cells grown on top of, inside and under Sci 115(2): 182-195. the hydrogel was monitored. The maturation and Nitric oxide (NO) activates the cyclic GMP electrical activity of the neuronal networks inside the (cGMP) / protein kinase G (PKG) pathway during hydrogel were further characterized. We showed that physiological processes in numerous types of cells. cells stayed viable on the top of the PuraMatrix surface Here, we evaluated whether this NO/cGMP/PKG and growth of the neural cells and neural processes pathway is involved in the proliferation of neural was good. Further, hESC-derived neurons, astrocytes stem/progenitor cells (NPCs) derived from the and oligodendrocytes all grew, matured and migrated hippocampus of embryonic mice. In culture, the when cultured inside the hydrogel. Importantly, exposure to the NO synthase inhibitor N (omega)- neuronal cells were able to form electrically active nitro-L-arginine methyl ester (L-NAME) significantly connections that were verified using microelectrode decreased the number of viable cells and 5-bromo-2'- array. Thus, PuraMatrix is a good supportive growth deoxyuridine (BrdU) incorporation into the cells, as matrix for human neural cells and may serve as a well as the levels of intracellular reactive oxygen matrix for neuronal scaffolds in neural tissue species, extracellular NO (2), and intracellular cGMP. engineering. Like L-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) Yoffe, Y., et al. (2016). "Cap-independent and PKG inhibitor KT5823 also decreased cell translation by DAP5 controls cell fate decisions in viability and BrdU incorporation. The membrane- human embryonic stem cells." Genes Dev 30(17): permeable cGMP analogue 8-bromo-cGMP partially 1991-2004. abolished the L-NAME-induced decrease in the BrdU Multiple transcriptional and epigenetic changes incorporation. BrdU incorporation was decreased by drive differentiation of embryonic stem cells (ESCs). Ca (2+)-channel blockers, including dantrolene, MK- This study unveils an additional level of gene 801, ifenprodil, and nifedipine. Interestingly, the NO expression regulation involving noncanonical, cap- (2) level was decreased by dantrolene, but not by the independent translation of a select group of mRNAs. other 3 blockers. L-NAME and ODQ attenuated This is driven by death-associated protein 5 phosphorylation of Akt, but not that of extracellular (DAP5/eIF4G2/NAT1), a translation initiation factor signal-regulated kinases or epidermal growth factor mediating IRES-dependent translation. We found that receptors. Our data suggest that endogenous NO the DAP5 knockdown from human ESCs (hESCs) generation linked to dantrolene-sensitive ryanodine resulted in persistence of pluripotent gene expression, receptors activates the cGMP/PKG signaling pathway delayed induction of differentiation-associated genes for positive regulation of proliferation of hippocampal in different cell lineages, and defective embryoid body NPCs derived from embryonic mice. formation. The latter involved improper cellular organization, lack of cavitation, and enhanced Yoo, H., et al. (2016). "Ultrastructural mislocalized apoptosis. RNA sequencing of polysome- comparison of porcine putative embryonic stem cells associated mRNAs identified candidates with reduced derived by in vitro fertilization and somatic cell translation efficiency in DAP5-depleted hESCs. These nuclear transfer." J Reprod Dev 62(2): 177-185. were enriched in mitochondrial proteins involved in The ultrastructure of porcine putative embryonic oxidative respiration, a pathway essential for stem cells and porcine fetal fibroblasts (PFFs) was differentiation, the significance of which was analyzed by transmission electron microscopy. The confirmed by the aberrant mitochondrial morphology aim of this study was to compare the features of and decreased oxidative respiratory activity in DAP5 organelles in in vitro fertilization (IVF) derived knockdown cells. Further analysis identified the porcine embryonic stem cells (IVF-pESCs) and chromatin modifier HMGN3 as a cap-independent somatic cell nuclear transfer (SCNT) derived pESCs DAP5 translation target whose knockdown resulted in (SCNT-pESCs). Also, the features of organelles in defective differentiation. Thus, DAP5-mediated high-passage IVF-pESCs were compared with those in translation of a specific set of proteins is critical for low-passage cells. The ultrastructure of PFFs showed the transition from pluripotency to differentiation, rare microvilli on the cell surfaces, polygonal or highlighting the importance of cap-independent irregular nuclei with one to two reticular-shaped translation in stem cell fate decisions. nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic Yoneyama, M., et al. (2011). "Endogenous nitric reticulum, elongated mitochondria, rich lysosomes and oxide generation linked to ryanodine receptors rich phagocytic vacuoles. IVF-pESCs showed rare activates cyclic GMP / protein kinase G pathway for microvilli on the cell surfaces, round or irregular

199 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ nuclei with one to two reticular-shaped nucleoli and doxycycline in a nonviral engineering approach. euchromatin, low cytoplasm-to-nucleus ratios, rich Regulatable expression in a prospective nonleaky Tet- ribosomes, long stacks of rough endoplasmic off system could hold promise for therapy, based on reticulum, elongated mitochondria, rare lysosomes and the multifunctional roles of L1, including neuronal rare autophagic vacuoles. By contrast, SCNT-pESCs migration and survival, neuritogenesis, myelination, showed rich microvilli with various lengths and and synaptic plasticity. frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, Yoo, S. J., et al. (2005). "Efficient culture system high cytoplasm-to-nucleus ratios, rare ribosomes, rare for human embryonic stem cells using autologous rough endoplasmic reticulum, round mitochondria, human embryonic stem cell-derived feeder cells." Exp rich lysosomes and rich phagocytic vacuoles with clear Mol Med 37(5): 399-407. intercellular junctions. Furthermore, high-passage Human embryonic stem cells (hESCs) need IVF-pESCs showed irregularly shaped colonies, feeder cells for their maintenance in an pyknosis and numerous lysosomes associated with undifferentiated state. In conventional culture systems, autophagic vacuoles showing signs of apoptosis. In mouse embryonic fibroblasts (MEFs) serve as feeder conclusion, this study confirms that the ultrastructural cells to maintain hESCs. However, the use of MEFs characteristics of pESCs differ depending on their elevates the risk of transmitting mouse pathogens and origin. These ultrastructural characteristics might be thus limits the potential of hESCs in cell replacement useful in biomedical research using pESCs, leading to therapy. Consequently, the use of human feeder cells new insights regarding regenerative medicine and would be an important step forward in this in vitro tissue repair. technology. To address this issue, we used fibroblast- like cells differentiated from the Miz-hES6 hESC line Yoo, M., et al. (2014). "Analysis of human (DiffMiz-hES6) as feeder cells to support the in vitro embryonic stem cells with regulatable expression of growth of three hESC lines. Immunofluorescence the cell adhesion molecule l1 in regeneration after microscopy and reverse transcription-PCR assessing spinal cord injury." J Neurotrauma 31(6): 553-564. the expression of undifferentiated hESC markers Cell replacement therapy is one potential avenue revealed all three hESC lines were maintained in an for central nervous system (CNS) repair. However, undifferentiated state. In vitro proliferation proceeded transplanted stem cells may not contribute to long- as efficiently as when the hESCs were cultured on term recovery of the damaged CNS unless they are MEFS. Moreover, karyotype analysis revealed the engineered for functional advantage. To fine tune chromosomal normality of the hESC lines and the regenerative capabilities, we developed a human DiffMiz-hES6 feeders themselves after even 50 neural cell line expressing L1, a regeneration- passages. Furthermore, the hESC lines maintained conducive adhesion molecule, under the control of a their pluripotency since they remained capable of doxycycline regulatable Tet-off promoter. Controlled forming embryoid bodies (EBs) in vitro. Thus, hESC- expression of L1 is desired because overexpression derived fibroblast-like cells successfully support in after regenerative events may lead to adverse vitro hESC propagation. consequences. The regulated system was tested in several cell lines, where doxycycline completely Yoon, S. W., et al. (2014). "Rad51 regulates cell eliminated green fluorescent protein or L1 expression cycle progression by preserving G2/M transition in by 3-5 days in vitro. Increased colony formation as mouse embryonic stem cells." Stem Cells Dev 23(22): well as decreased proliferation were observed in 2700-2711. H9NSCs without doxycycline (hL1-on). To test the Homologous recombination (HR) maintains role of L1 in vivo after acute compression spinal cord genomic integrity against DNA replication stress and injury of immunosuppressed mice, quantum dot deleterious lesions, such as double-strand breaks labeled hL1-on or hL1-off cells were injected at three (DSBs). Rad51 recombinase is critical for HR events sites: lesion; proximal; and caudal. Mice transplanted that mediate the exchange of genetic information with hL1-on cells showed a better Basso Mouse Scale between parental chromosomes in eukaryotes. score, when compared to those with hL1-off cells. As Additionally, Rad51 and HR accessory factors may compared to the hL1-off versus hL1-on cell facilitate replication fork progression by preventing transplanted mice 6 weeks post-transplantation, replication fork collapse and repair DSBs that expression levels of L1, migration of transplanted cells, spontaneously arise during the normal cell cycle. In and immunoreactivity for tyrosine hydroxylase were this study, we demonstrated a novel role for Rad51 higher, whereas expression of chondroitin sulfate during the cell cycle in mouse embryonic stem cells proteoglycans was lower. Results indicate that L1 (mESCs). In mESCs, Rad51 was constitutively expression is regulatable in human stem cells by expressed throughout the cell cycle, and the formation

200 Stem Cell 2019;10(4) http://www.sciencepub.net/stem SCJ of Rad51 foci increased as the cells entered S phase. embryonic carcinoma NCCIT cells showing gene Suppression of Rad51 expression caused cells to expression profiles similar to embryonic stem cells. accumulate at G2/M phase and activated the DNA Down-regulation of the gene by either small damage checkpoint, but it did not affect the self- interfering RNAs targeting Nanog or histone renewal or differentiation capacity of mESCs. Even deacetylase inhibitor apicidin causes reversion of though Rad51 suppression significantly inhibited the expression pattern of embryonic stem cell signature proliferation rate of mESCs, Rad51 suppression did including Oct4, Sox2, and their target genes, leading to not affect the replication fork progression and speed, cell cycle arrest, inhibition of colony formation in soft indicating that Rad51 repaired DNA damage and agar, and induction of differentiation into all three promoted DNA replication in S phase through an germ layers. These effects are antagonized by independent mechanism. In conclusion, Rad51 may reintroduction of Nanog. Interestingly, embryonic contribute to G2/M transition in mESCs, while carcinoma cells (NCCIT, NTERA2, and P19) exhibit a preserving genomic integrity in global organization of higher sensitivity to apicidin in down-regulation of DNA replication fork. Nanog compared with embryonic stem cells. Furthermore, the down-regulation of Nanog Yoshie, S., et al. (2012). "Establishment of novel expression by apicidin is mediated by a coordinated detection system for embryonic stem cell-derived change in recruitment of epigenetic modulators and hepatocyte-like cells based on nongenetic transcription factors to the promoter region. These manipulation with indocyanine green." Tissue Eng findings indicate that overexpression of stemness gene Part C Methods 18(1): 12-20. Nanog in NCCIT cells is associated with maintaining Hepatocytes derived from embryonic stem cells stem cell-like phenotype and suggest that targeting (ESCs) are expected to be useful for basic research and Nanog might be an approach for improved therapy of clinical applications. However, in several studies, poorly differentiated tumors. genetic methods used to detect and obtain them are difficult and pose major safety problems. Therefore, in Youngblood, B. A., et al. (2014). "CstF-64 this study, we established a novel detection system for supports pluripotency and regulates cell cycle hepatocytes by using indocyanine green (ICG), which progression in embryonic stem cells through histone 3' is selectively taken up by hepatocytes, based on end processing." Nucleic Acids Res 42(13): 8330- nongenetic manipulation. ICG has maximum light 8342. absorption near 780 nm, and it fluoresces between 800 Embryonic stem cells (ESCs) exhibit a unique and 900 nm. Making use of these properties, we cell cycle with a shortened G1 phase that supports developed flow cytometry equipped with an excitation their pluripotency, while apparently buffering them lazer of 785 nm and specific bandpass filters and against pro-differentiation stimuli. In ESCs, expression successfully detected ESC-derived ICG-positive cells of replication-dependent histones is a main component that were periodic acid-Schiff positive and expressed of this abbreviated G1 phase, although the details of hepatocyte phenotypic mRNAs. These results this mechanism are not well understood. Similarly, the demonstrate that this detection system based on role of 3' end processing in regulation of ESC nongenetic manipulation with ICG will lead to isolate pluripotency and cell cycle is poorly understood. To hepatocytes generated from ESCs and provide the better understand these processes, we examined mouse appropriate levels of stability, quality, and safety ESCs that lack the 3' end-processing factor CstF-64. required for cell source for cell-based therapy and These ESCs display slower growth, loss of pharmaceutical studies such as toxicology. pluripotency and a lengthened G1 phase, correlating with increased polyadenylation of histone mRNAs. You, J. S., et al. (2009). "Depletion of embryonic Interestingly, these ESCs also express the tauCstF-64 stem cell signature by histone deacetylase inhibitor in paralog of CstF-64. However, tauCstF-64 only NCCIT cells: involvement of Nanog suppression." partially compensates for lost CstF-64 function, Cancer Res 69(14): 5716-5725. despite being recruited to the histone mRNA 3' end- The embryonic stem cell-like gene expression processing complex. Reduction of tauCstF-64 in CstF- signature has been shown to be associated with poorly 64-deficient ESCs results in even greater levels of differentiated aggressive human tumors and has histone mRNA polyadenylation, suggesting that both attracted great attention as a potential target for future CstF-64 and tauCstF-64 function to inhibit cancer therapies. Here, we investigate the potential of polyadenylation of histone mRNAs. These results the embryonic stem cell signature as molecular target suggest that CstF-64 plays a key role in modulating the for the therapy and the strategy to suppress the cell cycle in ESCs while simultaneously controlling embryonic stem cell signature. The core stemness gene histone mRNA 3' end processing. Nanog is abnormally overexpressed in human

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Yu, G. and Q. Wen (2018). "Expression of that the closest in vivo equivalent of an ES cell is an embryonic liver fodrin (ELF) and stem cell markers in early germ cell. CD13 liver cancer stem cells." Eur Rev Med Pharmacol Sci 22(6): 1653-1657. Zweigerdt, R., et al. (2003). "Generation of OBJECTIVE: Investigating the molecular confluent cardiomyocyte monolayers derived from signaling pathways using CD13 as a marker for embryonic stem cells in suspension: a cell source for Cancer Stem Cells (CSCs) in human liver cancer. new therapies and screening strategies." Cytotherapy PATIENTS AND METHODS: In the present study, 5(5): 399-413. liver carcinoma biopsies were obtained from the liver BACKGROUND: Cellular cardiomyoplasty is cancer patients, as well as healthy controls. evolving as a new strategy to treat cardiac diseases. A Immunohistochemistry and Immunoblotting prerequisite is a reliable source of pure experiments were performed accordingly to conclude cardiomyocytes, which could also help in the the data. RESULTS: Immunohistochemistry along exploitation of recent advances in genomics and drug with immunoblot data showed the expression of Oct-4, screening. Our goal was to establish a robust lab-scale STAT3 and interestingly Embryonic Liver Fodrin process for the generation of embryonic stem (ES)- (ELF) in the CD13 positive liver cancer stem cells. cell-derived cardiomyocytes in suspension. Though embryonic liver fodrin (ELF) is a pro- METHODS: A 71 ES cell clone carrying a construct differentiation protein, it was expressed in the CD13 consisting of the alpha-cardiac myosin heavy chain positive liver cancer stem cells along with stem cell (alphaMHC) promoter driving the neomycin resistance markers such as Oct-4 and STAT3. CONCLUSIONS: gene was used for antibiotic-driven cardiomyocyte Our finding concludes that an association of ELF enrichment. Rotating suspension culture was expression was noted in liver cancer patients. Hence, established to initiate embryoid body (EB) formation. ELF may have value as prognostic indicators and may To track growth and differentiation kinetics, cell count facilitate the development of novel therapeutics for and flow cytometry for SSEA-I, E-cadherin (stem-cell liver cancer. marker)and sarcomeric myosin (cardiomyocytes marker) was performed. Oct4 expression was Zwaka, T. P. and J. A. Thomson (2005). measured via real time (RT)-PCR. RESULTS: "Differentiation of human embryonic stem cells occurs Cultures comprising 2.5-8 x 10(6) differentiating FS through symmetric cell division." Stem Cells 23(2): cells/mL were obtained after 9 days in rotating 146-149. suspension. Upon G418 addition,vigorous contracting Embryonic (ES) stem cells can be expanded spheres, termed cardiac bodies (CB), developed. These indefinitely, yet retain the ability to form all cell types cultures consisted of about 2.1 x 10(5) enriched of the body. Here we report that human ES cells cardiomyocytes/mL after 6- 10 days of selection. differentiate exclusively by symmetric cell division in Suspensions comprising 90- 95%viable single cells each of four distinct differentiation conditions were generated using an improved dissociation method. examined. This suggests that, in some respects, ES Seeding of cardiomyocytes with 7 x 10(4) cell/cm (2) cells more closely resemble precursor or transit resulted in a homogeneous monolayer of amplifying cells rather than adult stem cells. synchronously contracting cells. Myocyte specific immunohistochemistry indicated purity of > 99%. Zwaka, T. P. and J. A. Thomson (2005). "A germ DISCUSSION: We have established a reliable lab- cell origin of embryonic stem cells?" Development scale protocol to generate cultures of highly enriched 132(2): 227-233. cardiomyocytes in suspension. This will facilitate Because embryonic stem (ES) cells are generally development of larger-scale processes for stem-cell derived by the culture of inner cell mass (ICM) cells, based cardiomyocyte supply. An improved method is they are often assumed to be the equivalent of ICM provided to derive vital suspensions of cardiomyocytes, cells. However, various evidence indicates that ICM which could be utilized for transplantation as well as cells transition to a different cell type during ES-cell for drug screening purposes. derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm The above contents are the collected information cells derived directly from the ICM. However, from Internet and public resources to offer to the differences between ES cells and primitive ectoderm people for the convenient reading and information cells have caused developmental biologists to question disseminating and sharing. whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue References culture environment. Here, we review recent evidence 1. Abboud, N., et al. (2017). "Culture conditions have an impact on the maturation of traceable, transplantable mouse

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