© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs203018. doi:10.1242/jcs.203018

REVIEW ARTICLE SERIES: STEM CELLS Hematopoietic stem cells from induced pluripotent stem cells – considering the role of microRNA as a cell differentiation regulator Aline F. Ferreira1, George A. Calin2, Virgıniá Picanço-Castro3, Simone Kashima3, Dimas T. Covas3,4 and Fabiola A. de Castro1,*

ABSTRACT microRNA (miRNA) expression and epigenetic markers are Although (HSC) therapy for hematological distinct, indicating that the stem cell differentiation process needs diseases can lead to a good outcome from the clinical point of view, the to be further investigated. limited number of ideal donors, the comorbidity of patients and the Stem cells have proven to be a powerful tool in studies aimed at in vitro increasing number of elderly patients may limit the application of this understanding cell differentiation, and advances in therapy. HSCs can be generated from induced pluripotent stem cells developing cell reprogramming protocols have meant that these (iPSCs), which requires the understanding of the bone marrow and cells can now be induced to differentiate into a number of different in vitro liver niches components and function in vivo. iPSCs have been tissues (Takayama et al., 2010; Teng et al., 2014; Menon extensively applied in several studies involving disease models, drug et al., 2016). iPSC generation has now been achieved by several screening and cellular replacement therapies. However, the somatic methods, including through integration of viruses and episomal reprogramming by transcription factors is a low-efficiency process. plasmids (Meng et al., 2012; Slamecka et al., 2016). Key aspects of Moreover, the reprogramming process is also regulated by microRNAs iPSC production, such as the target tissue, reprogramming factors, (miRNAs), which modulate the expression of the transcription factors method of cell delivery, culture conditions and the biological assays OCT-4 (also known as POU5F1), SOX-2, KLF-4 and MYC, leading to confirm the resulting cell pluripotency potential, are all time- somatic cells to a pluripotent state. In this Review, we present an consuming, arduous and expensive (Maherali and Hochedlinger, overview of the challenges of cell reprogramming protocols with regard 2008). In fact, the technology used to integrate viruses for the to HSC generation from iPSCs, and highlight the potential role of generation of iPSCs represents the main bottleneck for the miRNAs in cell reprogramming and in the differentiation of induced therapeutic application of iPSCs owing to the possibility of viral pluripotent stem cells. vectors being integrated into the genome, which can result in tumorigenesis (Maherali and Hochedlinger, 2008). By contrast, KEY WORDS: HSC, iPSC, miRNA, Cell reprogramming episomal plasmids do not integrate in the genome and typically disappear from iPSCs after 10 to 14 passages (Chou et al., 2011; INTRODUCTION Meng et al., 2012). Stem cells are undifferentiated cells that present an indeterminate Somatic cell reprogramming and iPSC differentiation have the expansion potential to produce progeny through self-renewal or potential to be used in a wide range of therapeutic applications in vitro, differentiation (Sakaki-Yumoto et al., 2013). Furthermore, stem including disease modelling, drug screening and cellular replacement cells have a low-turnover profile, in contrast to their differentiated therapies (Maherali and Hochedlinger, 2008; Giani et al., 2016; progeny (Eckfeldt et al., 2005; Cheng et al., 2000). We focus here Tiyaboonchai et al., 2014); however, the culture conditions of stem on the role of miRNAs in cell reprogramming and induced cells and the particular protocols applied have a great influence on pluripotent stem cell (iPSC) differentiation. The first study to whether the desirable final results are obtained (Fig. 1). demonstrate the formation of iPSCs upon viral-mediated During differentiation, it is expected that stem cells lose the transduction of murine embryonic and adult fibroblasts with expression of the pluripotency-related genes OCT4 and NANOG octamer-binding transcription factor 4 (OCT-4; also known as and begin to express markers associated with differentiation, such as POU5F1), sex-determining region Y-box 2 (SOX-2), v-myc avian GATA4 and GATA6 (Miyamoto et al., 2015). An important issue myelocytomatosis viral oncogene homolog (MYC) and Kruppel- regarding epigenetic markers in stem cells is that the current like factor 4 (KLF-4) was published in 2006 (Takahashi and methods of iPSC cultivation allow the maintenance of the Yamanaka, 2006). Although iPSCs are similar in morphology and epigenetic profile over a long period (Philonenko et al., 2017). pluripotent potential to embryonic stem cells (ESCs), these types of Such epigenetic control may be also mediated by miRNAs (Fig. 1). stem cells are not identical and their gene expression profile, miRNAs are endogenous small non-coding RNA (ncRNAs) consisting of 20 to 22 nucleotides that impair protein expression by binding to mRNAs and interfering with their translation (Ambros, 1Department of Clinical Analysis, Toxicology and Food Science, School of 2004). In this way, miRNAs are involved in fundamental biological Pharmaceutical Sciences, University of São Paulo (USP), Ribeirão Preto, São Paulo 14040-903, Brazil. 2Department of Experimental Therapeutics, MD Anderson processes, including tissue development, cell differentiation, Cancer Center, Houston, TX 77054, USA. 3Center of Cell Therapy, Regional proliferation and apoptosis. Besides all this knowledge with regard Center of Ribeirão Preto, Ribeirão Preto, São Paulo 14051-140, Brazil. 4Department to miRNA function, their role in stem cells differentiation is not that of Internal Medicine, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo 14049-900, Brazil. well understood (Kim et al., 2017). Here, we aim to highlight miRNAs as a relevant regulator of iPSC generation and reprogramming. *Author for correspondence ([email protected]) Protocols for cell differentiation in vitro aim to generate specific A.F.F., 0000-0001-6874-184X; G.A.C., 0000-0002-7427-0578; S.K., 0000-0002- cell types from undifferentiated stem cells by using embryoid bodies

1487-0141; D.T.C., 0000-0002-7364-2595; F.A.d., 0000-0003-3347-5873 as an initial step (Brickman and Serup, 2017). Embryonic bodies are Journal of Cell Science

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Addition of OCT-4, SOX-2, KLF-4, MYC

5Ј 3Ј 3Ј 5Ј miRNA Fibroblasts regulation

iPSC 5Ј 3Ј 3Ј 5Ј Blood cells 3Ј 5Ј 3Ј 5Ј 3Ј 5Ј Somatic cells 3Ј Ј 5 5Ј 3Ј PatientPa en Addition of 5Ј 3Ј miRNAs

Fig. 1. General diagram of a somatic cell reprogramming protocol. Somatic cells from patients (fibroblasts or peripheral blood cells), can be reprogrammed by using the transcription factors OCT-4, SOX-2, KLF-4 and MYC, together with a cocktail of miRNAs, or by the addition of miRNAs alone. The resulting iPSCs that have been generated in vitro through somatic cell reprogramming have the potential to be used in a wide range of therapeutic applications and as a research tool (e.g. in vitro disease model). a three-dimensional, multicellular aggregates consisting of the three In cases of autologous HSC transplantation, patients are not germ-layers – endoderm, mesoderm and ectoderm – and are affected by severe GVHD nor by risk of rejection. However, the obtained from spontaneous differentiation of iPSCs when they are disease relapse index is higher in comparison with allogeneic HSC cultured under low-oxygen conditions (Hawkins et al., 2013). transplantation, as no GVHD and graft-versus-leukemia effect Besides the ability to obtain embryonic bodies from iPSCs, (GVL) will occur (i.e. immune response against neoplastic cells) another advantage in using iPSCs for in vitro cell differentiation is and any remaining leukemic cells could then induce the disease that technical manipulations, such as selection of clones or cell relapse. Taken together, all these disadvantages of bone marrow sorting, do not interfere with their ability to differentiate into HSC transplantation highlight the requirement of an alternative hematopoietic tissue (Philonenko et al., 2017). Even if iPSCs require source of HSCs, which aims to reduce the rate of HSC rejection, genetic manipulations (e.g. the introduction of reprogramming disease relapse and bone marrow failure syndromes (graft failure or factors to induce cell pluripotency and self-renewal, such as poor graft function), as well as increasing the possibility of OCT-4, SOX-2 and KLF-4) their properties as iPSCs are obtaining HSCs more easily (van Bekkum and Mikkers, 2012; maintained due to their high genetic and epigenetic stability Masouridi-Levrat et al., 2016). In this regard, iPSCs cells represent a (Philonenko et al., 2017). suitable source that may be used to generate sufficient amounts of Although numerous studies have linked iPSCs or iPSC-differentiated HSCs in vitro with limited immunogenicity (Araki et al., 2013). cells with cell-based therapy, there are, however, still challenges and Despite the success in obtaining in vitro hematopoietic cells from methodology limitations to be overcome. We consider the main iPSCs, the strategy used is laborious and expensive (Yang et al., challenges in generating iPSCs to be: choosing the ideal somatic cell 2017). Furthermore, there is a the lack of a specific surface marker source for iPSC generation, the reprogramming method used, and the for the human hemangioblast, a single mesodermal cell that gives control and the optimization of iPSC differentiation (Box 1). rise to blood cells and endothelium, which hinders the derivation of Here, we discuss the processes and protocols currently employed HSCs from iPSCs in practice (Lacaud and Kouskoff, 2017). to obtain hematopoietic stem cells (HSCs) in vitro from iPSCs, and A particular advantage for the use of iPSC-derived HSCs is that also highlight the potential roles of miRNAs in cell reprogramming they do not induce GVHD because they are autologous. However, and differentiation. these cells nevertheless exhibit some bias, such as their inability to self-renew in culture and a failure to engraft and survive long-term iPSCs as a potential tool for HSC generation in vitro after the transplant (Shepard and Talib, 2014). Another advantage of The current therapies for hematological neoplasms are iPSCs as a source of HSCs is their ability to differentiate into primitive chemotherapy, immunotherapy, tyrosine kinase inhibitors and cells, such as erythrocytes, which express fetal-type hemoglobin, and transplantation of hematopoietic stem cells (HSCs) obtained from definitive cells including lymphocytes (Seiler et al., 2011). bone marrow, peripheral blood or umbilical cord (Jaramillo et al., iPSCs might be also useful as disease models, as the maintenance 2017; Ye et al., 2017; Baron and Nagler, 2017). of human primary cells in culture over long periods is difficult, and Patients who receive allogeneic transplants of bone marrow or the use of animal models often involves interspecies variabilities umbilical cord may exhibit a rejection of the bone marrow (De Lázaro et al., 2014). Moreover, numerous reports have transplanted cells due to graft-versus-host disease (GVHD), demonstrated the therapeutic potential of iPSCs in diverse toxicity owing to the conditioning regimen used, disease relapse hematological conditions, such as myelodysplastic syndrome or infections (van Bekkum and Mikkers, 2012). In addition, patients (MDS), sickle cell anemia and hemophilia (Kotini et al., 2015; receiving HSCs by allogeneic bone marrow transplantation should Hanna et al., 2007; Park et al., 2014). iPSCs derived from a variety be under 55 years old and should have a compatible human of monogenic diseases can accurately recapitulate disease leukocyte antigen (HLA) donor, which restricts the applicability of phenotypes in vitro when differentiated into disease-relevant cell the procedure. types (Hamazaki et al., 2017). Journal of Cell Science

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Box 1. Potential challenges in iPSC generation Choice of the somatic cell source Induced pluripotent stem cells can be generated from different sources of somatic cells (fibroblasts, blood cells, keratinocytes, melanocytes, liver, gastric, epithelial, neural, stomach and pancreatic-β cells) by using ectopic expression of reprogramming factors (Aoi et al. 2008; Stadtfeld et al., 2008; Polo et al., 2010; Bar-Nur et al., 2011). Fibroblasts are widely used in the generation of iPSCs, but the procedure for collecting them is considered invasive. Blood is an easily accessible tissue and some groups have already demonstrated the successful generation of iPSCs from peripheral blood cells (Loh et al., 2010; Dowey et al., 2012). However, the cell type and its source influence the molecular and functional properties of iPSCs (Polo et al., 2010; Araki et al., 2013). Thus, although iPSCs acquire markers of pluripotent cells and differentiate into the three embryonic germ layers, they also maintain the transcriptional and epigenetic memory of their cell of origin (Kim et al., 2010; Polo et al., 2010; Bar-Nur et al., 2011), which can influence their phenotype and differentiation potential. Thus, the desired iPSC characteristics should be considered when choosing the ideal somatic cell source and application. Reprogramming method Somatic cell reprogramming changes the cell fate, thus allowing iPSCs to differentiate into tissues distinct from their tissue of origin, and can be achieved by the ectopic expression of defined factors, such as OCT4, SOX-2, KLF4 and MYC (reviewed by Patel and Yang, 2010). Some studies suggest that the transcription factors should be chosen depending on the future application (i.e. in regenerative medicine or as a tool to study disease) (Saha and Jaenisch, 2009; reviewed by Patel and Yang, 2010). Here, we also highlight the potential of miRNAs to boost reprogramming efficiency, particularly in iPSCs from peripheral blood cells, which may be used for HSC production. In order to improve iPSC reprogramming efficiency, it is mandatory to establish a robust and reproducible reprogramming process. The most common method uses integration of viral vectors into the cell genome. However, viral vectors are associated with mutagenesis and oncogenesis potential in iPSCs (Maherali and Hochedlinger, 2008; Zhang et al., 2012; Nishimori et al., 2014). Nonintegrative methods can be also used, including use of episomal DNA (Schlaeger et al., 2015, Goh et al., 2013). Furthermore, the factor delivery technique and electroporation parameters are also crucial; these include electrical parameters, DNA amount and purity, cell density and temperature (Yildirim et al., 2016). Culture optimization The somatic cell culture conditions and reagents used during iPSC generation also need to be optimized to avoid any adverse reactions with recipient tissues and to generate the ideal target cell. Feeder cells can be replaced by human extracellular matrix proteins, and culture media containing serum can be replaced with a serum-free medium with recombinant molecules (Nakagawa et al., 2014, Warren and Wang et al., 2013). In addition, variation between batches is another important issue to consider (Warren and Wang, 2013; Yang et al., 2011; Liang et al., 2013; Kim et al., 2017). Other factors that currently limit the wider use of iPSC technology include the cost of developing cell lines and the time needed to fully characterize them. Differentiation control The controlled differentiation of iPSCs into any desired cell type is the greatest technical challenge in iPSC-based approaches. After iPSC generation, their differentiation potential needs to be evaluated in both animal models and pharmaceutical screens (Teng et al., 2014; Choi et al., 2017) to ensure the resulting iPSC is functional and capable of differentiating into the target cell. The generation of blood cells from iPSCs has been particularly challenging, and the generation of HSCs with long-term self-renewal capability, as well as their differentiation into the different blood cell types capable of effective oxygen transport and still remains a significant challenge (Rowe et al., 2016).

Based on these properties, iPSCs should be considered as a Another approach that has been reported for the generation of potential and relevant source for HSCs in vitro. Below, we will hematopoietic cells in vitro is the co-culture of ESCs with pre- discuss the current protocols used for hematopoietic differentiation adipocytic stromal cells, favoring conditions conducive for of pluripotent stem cells, both ESCs and iPSCs. We will also hematopoietic differentiation without the need for to generate emphasize the role of miRNAs as reprogramming and embryonic bodies (reviewed by Seiler et al., 2011). differentiation regulators. However, the differentiation of iPSCs in vitro might be impaired by the transgenes that are used to reprogram somatic cells, such as Current means of obtaining HSCs in vitro SOX-2, OCT-4 and KLF-4, as these have been shown to reduce the The derivation of blood cells from iPSCs has been reported by ability of mesodermal-like cells to differentiate in hematopoietic several research groups, including ours (reviewed by Zhang, 2013; progenitors (Ebina and Rossi, 2015). In fact, SOX-2 Smith et al., 2013; Sweeney et al., 2016). downregulation is important in hematopoietic development, The embryoid bodies formed from iPSCs, as well as the culture of because its expression appears to be inversely related to the iPSCs in a differentiation medium supplemented with human bone hematogenic potency of a cell (Seiler et al., 2011). morphogenetic protein 4 (hBMP-4), human vascular endothelial In addition to the need of establishing a standardized protocol for growth factor (hVEGF) and human WNT3A Wnt family member the generation of HSCs in vitro, there is also a great demand for 3A (hWnt3a), have been used to differentiate iPSCs into the mature blood cells, such as erythrocytes and , that have been hematopoietic lineage in vitro (Sweeney et al., 2016; Smith et al., generated from hematopoietic progenitor cells, obtained either from 2013). Dissociation of embryonic bodies and culture of their cells the bone marrow, peripheral blood or cord blood. Pluripotent HSCs with OP9 cells (a marrow stromal cell line) have been successfully and early multipotent progenitors (MPPs) all originate from used to obtain myeloid precursors, , and erythroblasts, which generate erythrocytes (Palis, 2008). (Sweeney et al., 2016). These authors also reported that Mouse ESCs have been successfully used for in vitro the potential for colony formation in iPSCs is associated with the co- differentiation into erythroid cells by using two strategies expression of the hematopoietic markers CD34 and CD45 (also (Nakano, 1996; Carotta et al., 2004; Kitajima et al., 2003). The known as PTPRC) during iPSC differentiation; however, these first one utilizes disaggregated embryonic bodies that have been iPSCs were not able to promote long-term hematopoietic engrafting cultured with erythropoietin (EPO) and Kit ligand (KL-1) to in mice (Sweeney et al., 2016). In order to generate hematopoietic stimulate the growth and differentiation of erythroid progenitors cells from iPSCs, growth factors, such as prostaglandin-E2 (PGE2) (Carotta et al., 2004). EPO stimulates cell growth by binding to and StemRegenin 1 (SR1), have been used to increase the iPSC EPO-R in burst-forming unit-erythrocytes (BFU-Es) and colony- differentiation efficiency and to give rise to a long-term HSC forming unit-erythrocyte (CFU-Es) (Elliott et al., 2008; Metcalf, phenotype (Zhang, 2013). 2008). KL-1, in synergy with other cytokines, stimulates growth of Journal of Cell Science

3 REVIEW Journal of Cell Science (2018) 131, jcs203018. doi:10.1242/jcs.203018 hematopoietic progenitors in vitro and increases blood cell miRNAs in cell reprogramming and differentiation production in vivo in animals (Broxmeyer et al., 1991). The As cell reprogramming and stem cell differentiation may be affected protocol involving EPO and KL-1 requires 10 days of culture to by miRNAs, we discuss here how this epigenetic regulator could obtain erythroid colonies (Carotta et al., 2004). Using only EPO interfere with these processes (Wang et al., 2015; Ong et al., 2016). without KL-1 results in primitive erythroid colonies, which are miRNAs have been reported to contribute to somatic cell characterized by their nucleated morphology and expression of reprogramming by upregulating the expression of the pluripotent embryonic globin (Carotta et al., 2004). By contrast, definitive reprogramming, factors OCT-4, SOX-2, KLF-4 and MYC, thus erythroid colonies are composed of cells that express adult globin, promoting reprogramming (Vitaloni et al., 2014; Wang et al., 2015; which is an important feature when considering the use of these cells Hu et al., 2013). In addition, miRNAs can also induce the for hemotherapy (Carotta et al., 2004). pluripotent state of somatic cells in the absence of exogenous The second protocol uses OP9 cells, which are able to create an factors, as has been reported for miR-302, which inhibits nuclear environment that can induce hematopoietic differentiation when co- receptor subfamily 2 group F member 2 (NR2F2) and promotes cultured with ESCs. After 5 days of co-culturing, the presence of pluripotency by upregulating OCT-4 (Vitaloni et al., 2014; Wang colonies formed of hematopoietic tissue can be noticed (Kitajima et al., 2015; Hu et al., 2013). et al., 2003). In this setup, the addition of cytokines, such as EPO and Furthermore, Miyoshi et al. have described the possibility of KL-1, also increases the potential of the culture to produce erythroid reprogramming mature cells by inducing the ectopic expression of colonies (Kitajima et al., 2003). Indeed, after 14 days, definitive miR-200c, miR-302 and miR-369 in human adipose stromal cells erythroid colonies are obtained, which can be separated from the and human dermal fibroblasts (Miyoshi et al., 2011). These primitive cells by simply removing the nonadherent cell population miRNAs were able to promote iPSCs pluripotency and self- representing the primitive erythroid progenitors (Carotta et al., 2004). renewal by inducing the overexpression of OCT-3, stage-specific In addition to the above protocols for generating mice erythroid embryonic antigen 3 and 4 (SSEA-3 and -4), SOX-2 and NANOG cells in vitro, human mature erythroid cells had already been obtained transcription factors, which resulted in the establishment of a stem- through human ESC differentiation in vitro nearly a decade ago (Lu cell-like state (Miyoshi et al., 2011; reviewed by Yao, 2016). et al., 2008). Although enucleation, the final step in the development Moreover, in mice, miR-93 and miR-106 have been shown to also of mature erythrocytes remains poorly understood, these authors were give rise to a pluripotent state of somatic cells by increasing the able to generate oxygen-carrying erythrocytes on a large scale. mRNA levels of OCT-4, SOX-2, KLF-4 and MYC (denoted OSKM) Furthermore, the obtained erythroid cells showed the capacity to through targeting their regulator, the CDKN1A gene (encoding express the adult β-globin chain upon further maturation in vitro, p21), which promotes the formation of iPSC colonies (Li et al., indicating their potential functionality (Lu et al., 2008). Blood cells 2011). miR-302 appears to maintain the renewal capacity and that have been generated in vitro could serve as a disease model and, pluripotency of human ESCs, as this miRNA was found to be importantly, also pave the way for the large-scale manufacture of red associated with the inhibition of premature zygotic cell blood cells, which is a global challenge in order to provide a safe differentiation during embryonic development (Miyoshi et al., supply of transfusable erythrocytes (Timmins et al., 2011). 2011). Finally, the miR-302/367 cluster is a direct target of OCT-4 Although all the methods mentioned above yield functional and SOX-2 in ESCs and iPSCs (Card et al., 2008) and has been hematopoietic cells, they also have limitations including the shown to be able to directly reprogram mouse and human somatic presence of byproducts from murine feeder cells, the need for cells without the need for any pluripotent stem cell transcription long culturing periods for embryonic body formation, and the factors (Anokye-Danso et al., 2011). presence of animal-derived culture products, such as bovine serum; It has been suggested that miRNA expression may be all of these will need to be overcome for any future clinical antagonized by pluripotent factors such as MYC (Chang et al., applications (Kim et al., 2017). 2008). Indeed, Yang et al. transduced mouse embryonic fibroblasts Although the literature shows that it is feasible to generate blood (MEFs) with OCT4, SOX2 and KLF4 (denoted OSK) in the cells in vitro, the underlying molecular mechanisms, such as how presence or absence of MYC and observed that expression of miR- the Wnt signaling pathway, iron homeostasis and hypoxia affect the let-7a, miR-16, miR-21, miR-29a and miR-143 decreased during expression of transcriptional factors that contribute to cell fate, are reprogramming, confirming that MYC is involved in the regulation still unclear (Tsiftsoglou et al., 2009a,b; Undi et al., 2016). of miRNA expression in MEFs and is able to sustain MEFs The current view with regard to hematopoietic differentiation in reprogramming. Moreover, when the authors tested the efficiency of vitro demonstrates that ESCs and iPSCs are useful sources for blood the process in the presence of a miR-21 inhibitor, they observed a cells production. However, it would be desirable if blood cell larger number of iPSC colonies, confirming that miRNA inhibition expansion protocols were feeder free, as this would greatly simplify enhances cell reprogramming (Yang et al., 2011). the commercial scalability, as well as reduce the cost of blood cell Similarly, downregulation of miR-29a improves reprogramming production. in mouse fibroblasts, whereas its overexpression reduced it (Hysolli Besides this knowledge and the considerable advances in et al., 2016). Thus, this result suggests that depletion of miR-29a protocols that drive derivation of hematopoietic stem cells (HSCs) alters the DNA methylation profile in somatic cells and may affect from iPSCs, the generation of robust transplantable HSCs and the expression of the genes responsible for cell reprogramming mature blood cells production from iPSCs remains elusive. (Hysolli et al., 2016). These findings demonstrate the capacity of Researchers should discovery strategies to overcome the miRNAs to regulate DNA methylation and/or demethylation and challenges and obstacles to produce a functional HSC in vitro. It highlight the need for further studies in order to better characterize is also important to optimize methodologies to control or stabilize the iPSC methylome. cell epigenetic states and understand the pathways that are linked to In order to evaluate the importance of Dicer, which is involved in functional HSC generation and differentiation. Here, we point out miRNA biogenesis, for cell reprogramming, Kim et al. investigated the role of miRNAs in somatic cell reprogramming and HSC the reprogramming of Dicer-null MEFs, which do not have a differentiation. functional miRNA biogenesis pathway (Kim et al., 2012). Use of Journal of Cell Science

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OCT-4, SOX-2, KLF-4, MYC and LIN-28 to reprogram Dicer-null CD34+ cells from cord blood results in the impaired proliferation MEFs was unsuccessful, but reprogramming could be achieved and accelerated differentiation of erythroid cells, coupled with when the human Dicer homolog was introduced in Dicer-null MEFs down-modulation of Kit protein. miR-221 and miR-222 exert this before their differentiation, raising the hypothesis that miRNAs are effect by modulating the expression of the Kit receptor, an important essential for iPSC generation (Kim et al., 2012; Judson et al., 2009; protein involved in HSC maintenance, upregulation Pfaff et al., 2017). and erythroleukemic cell expansion (Felli et al., 2005; An et al., Altogether, the role of miR-125a, miR-125b, miR-155, miR-181, 2016) (Fig. 2). miR-221, miR-222, miR-223 in HSC self-renewal and differentiation The differentiation of lymphoid cells might also be regulated by has been extensively explored in in vitro experiments (Chen, 2004; miR-181, which is highly expressed in thymus (Li et al., 2007; Fazi et al., 2005; Felli et al., 2005; Shaham et al., 2012; Kozakowska, Henao-Mejia et al., 2013). Li et al. show that increasing miR-181a et al., 2014, Yao, 2016; Wojtowicz et al., 2016) (Fig. 2). For instance, expression in mature T cells augments their sensitivity to peptide the ectopic expression of miR-125a in human multipotent progenitors antigens, whereas inhibiting miR-181a expression in the immature T (MPPs) increased their self-renewal, and these cells were also able to cells reduces sensitivity and impairs both positive and negative repopulate transplanted mice with a robust long-term multi-lineage selection (Li et al., 2007). Another study has also reported the engraftment (Wojtowicz et al., 2016). Conversely, downregulation relevance of miR-181 in natural killer T cell (NKT cell) ontogenesis of miR-125a decreased HSC self-renewal and so impaired the and lymphocyte T development (Henao-Mejia et al., 2013). The production of blood cells (Wojtowicz et al., 2014, 2016) (Fig. 2). authors described a severe defect in lymphoid development and T Furthermore, overexpression of miR-125b has been shown to cell homeostasis in miR-181-deficient mice, which was linked to promote the generation of blood cells such as in deregulation of the phosphoinositide 3-kinase pathway. Similarly, vitro and therefore constitutes a potential therapeutic target in miR-223 has been identified as a hematopoietic-specific miRNA blood disorders (Shaham et al., 2012). However, it is worth noting and has crucial functions in myeloid and lymphoid lineage that the expression of high levels of miR-125b alone in mice development and their cell fate due to its location in the bone causes a very aggressive form of transplantable myeloid leukemia. marrow, which contains HSCs and hematopoietic cells at various miR-125b exerts this effect by upregulating the number of stages of maturation (Chen, 2004). common myeloid progenitors, while inhibiting the development Thus, the abovementioned miRNAs might all be involved in of pre-B cells. In this context, miR-125b targets Lin28A, whose regulating differentiation of myeloid and lymphoid hematopoietic cells. downregulation can mimic the preleukemic state in mice The modulation of miRNAs could also be exploited for the (Chaudhuri et al., 2012). treatment of solid cancer and leukemias (Sun et al., 2017; Fan et al., In addition, miR-155, miR-221 and miR-222 also regulate 2017; Lu et al., 2016). Indeed, miR-223 modulates the differentiation megakaryocytic (Georgantas et al., 2007) and erythroid of human myeloid progenitor cells during granulocytic differentiation differentiation (Felli et al., 2005). miR-155 impaired both of acute promyelocytic leukemia (APL) cells in response to their processes when it was transduced into K562 cells, a model cell treatment with retinoic acid, as shown both in vitro and in APL line for human hematopoiesis (Georgantas et al., 2007). Here, miR- patients (Fazi et al., 2005). , the generation of myeloid 155 was shown to interfere with the generation of colonies from leukocytes, which includes , monocytopoiesis and hematopoietic progenitor cells (CD34+) that have been transduced megakariocytopoiesis, is controlled by a unique combination of with miR-155, thereby giving rise to only a few myeloid and transcription factors, such as nuclear factor 1 A-type (NFI-A), erythroid colonies, demonstrating its role as a negative regulator of CCAAT-enhancer-binding proteins (C/EBPα), core-binding factor-β normal myelopoiesis and erythropoiesis (Georgantas et al., 2007). (CBF-β) and retinoic acid receptor-α (RAR-α), which cooperatively Furthermore, the ectopic expression of miR-221 and miR-222 in regulate promoters and enhancers present on myeloid-specific-genes.

HSC Fig. 2. Role of miRNAs in HSC self-renewal and differentiation. The expression of miR-125a increases the self-renewal and miR-125a pluripotency capability of hematopoietic stem cells (HSCs) (bright blue). The targeting of LIN-28 by miR-125b overexpression induces m uncontrolled proliferation of myeloid progenitor cells (MPCs), leading miR-223iR Self-renewal -181 -181 to myeloid leukemia (green). miR-155 targets the ETS1 and MEIS transcription factor genes that are responsible for miR miR-125b miR-223 (MK) proliferation and differentiation (dark purple). Thus, enforced miR-155 expression of miR-155 impairs MK development. In addition, miR- LIN-28 MPC LPC 155 may also target additional genes, including CEBPB (encoding β C/EBPβ; MEIS C/EBP ), MEIS, CREB1, JUN, SPI1, AGTR1, AGTR2 and FOS, Generation of CREB1; JUN which regulate the differentiation of HSCs into myeloid progenitor uncontrolled SPI1; AGTR1 cells (MPCs) and lymphoid progenitor cells (LPCs), and thereby AGTR2 FOS MPC ; stimulates the formation of MPCs and erythroid colonies (EC) (orange). miR-181 and miR-223 regulate HCS differentiation into Myeloid leukemia Increase MPC and MPCs and LPCs (dark blue), whereas miR-221 and miR-222 inhibit erythroid colonies erythropoiesis by modulating KIT protein modulation (pink). miR-155 MK EC miR-221 ETS1; miR-222 MEIS1

MK proliferation KIT Inhibition of erythropoiesis

and differentiation Journal of Cell Science

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Fazi et al. showed that granulocytic differentiation is controlled by a Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T. regulatory circuitry involving miR-223 and the NFI-A and C/EBPα and Yamanaka, S. (2008). Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 321, 699-702. transcription factors (Fazi et al., 2005). NFI-A maintains miR-223 at Araki, R., Uda, M., Hoki, Y., Sunayama, M., Nakamura, M., Ando, S., Sugiura, M., low levels, whereas, following retinoic acid (RA)-induced Ideno, H., Shimada, A., Nifuji, A. et al. (2013). Negligible immunogenicity of differentiation, it is replaced by C/EBPα, which in turn upregulates terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature 494, 100-104. miR-223 expression (Fazi et al., 2005). In acute promyelocytic Bar-Nur, O., Russ, H. A., Efrat, S. and Benvenisty, N. (2011). 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