US 20140.038291A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0038291 A1 Ahlfors et al. (43) Pub. Date: Feb. 6, 2014
(54) METHODS FOR REPROGRAMMING CELLS Publication Classification AND USES THEREOF (51) Int. Cl. (71) Applicant: New World Laboratories Inc., Laval CI2N 15/85 (2006.01) (CA) (52) U.S. Cl. CPC ...... CI2N 15/85 (2013.01) (72) Inventors: Jan-Eric Ahlfors, Laval, CA (US); USPC ...... 435/.441; 435/455; 435/325 Rouwayda Elayoubi, Laval, CA (US) (73) Assignee: New World Laboratories Inc., Laval (57) ABSTRACT (CA) (21) Appl. No.: 13/843,713 Described herein are reprogrammed cells, and methods for cell dedifferentiation, transformation and eukaryotic cell (22) Filed: Mar 15, 2013 reprogramming. Also descried are cells, cell lines, and tissues that can be transplanted in a patient after steps of in vitro Related U.S. Application Data dedifferentiation and in vitro reprogramming. In particular (63) Continuation-in-part of application No. 13/464,987, embodiments the cells are Stem-Like Cell s (SLCs), including filed on May 5, 2012, which is a continuation-in-part Neural Stem-Like Cells (NSLCs), Cardiac Stem-Like Cells of applicationy - No. 13/504,988,s filed on Apr. 30, 2012, (CSLC), Hematopoietic Stem-Like Cells (HSLC), Pancreatic filed as application No. PCT/CA10/01727 on Nov. 1, Progenitor-Like Cells, and Mesendoderm-like Cells. Also 2010 described are methods for generating these cells from human Somatic cells and other types of cells. Also provided are (60) Provisional application No. 61/256,967, filed on Oct. compositions and methods of using of the cells so generated 31, 2009. in human therapy and in other areas. Patent Application Publication Feb. 6, 2014 Sheet 1 of 28 US 2014/0038291 A1
Figure 1
NCAM Day 6
firslf in Patent Application Publication Feb. 6, 2014 Sheet 2 of 28 US 2014/0038291 A1
Figure 3 Patent Application Publication Feb. 6, 2014 Sheet 3 of 28 US 2014/0038291 A1
(A) ( B) Hoechst fill-tubulin Musashi
NPC
NSLC
T Hoechs O4 NGFrec
NPC
NSC
Hoechst Co133 Neun
NPC
NSC
Hoechst Nestin
NoC
NSLC
Figure 4 Patent Application Publication Feb. 6, 2014 Sheet 4 of 28 US 2014/0038291 A1
Utransfected x GFAP Fibroretin Fibroblasparker fibroblastices
Figure 5 Patent Application Publication Feb. 6, 2014 Sheet 5 of 28 US 2014/0038291 A1
Hoechst (no Sox2) Sox2 3-tubulin
Week
Week 2
Week
Figure 6 Patent Application Publication Feb. 6, 2014 Sheet 6 of 28 US 2014/0038291 A1
Hoechst Sox2 GFA
HFF untransfected
Nestin Sox2 GFAP
HFF transfected
Keratinocytes
CD34
Figure 7 Patent Application Publication Feb. 6, 2014 Sheet 7 of 28 US 2014/0038291 A1
Figure 8
Transfected HFF with Msi1/Ngm2
Figure 9 Patent Application Publication Feb. 6, 2014 Sheet 8 of 28 US 2014/0038291 A1
Ngn2
Msl1/Ngn2
Figure 10
Figure 11 Patent Application Publication Feb. 6, 2014 Sheet 9 of 28 US 2014/0038291 A1
Os all: No Positive
Castul: No Positive (Psti: No Positive
Figure 12 Patent Application Publication Feb. 6, 2014 Sheet 10 of 28 US 2014/0038291 A1
3.3
3.
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2
--control DMso ses s sonic -o-controlcdosporine
3 is so is is 7 is 23 s 2 2s 3 33 3s 37 as 4 3 days post innurnization Figure 13
NAblation 25 Rotarod 20RPM ONo Treatment Treatment with Aiginate Treatment with Aiginate NPC Treatment with Matrix +NSCs 20 OTreatment with Aiginate - NSLCs Treatment with Matrix Only Treatment with Fibrin Girl
5
O
O ^8...... (sssssssss Figure 14 Patent Application Publication Feb. 6, 2014 Sheet 11 of 28 US 2014/0038291 A1
Walking beam
No Aation --Notreatment
--Treatment with Aginate O-Treatment with Aginate + NPC Treatment with Matra 4 NSLs OTreatment with Aginate 4 NSLCs Treatment with Matru Only Treatment with fibringe
4.
a-a-a-a-a-a-a-a-a-a-a- Figure 15 Patent Application Publication Feb. 6, 2014 Sheet 12 of 28 US 2014/0038291 A1
Octa AP
Figure 16
Untransfected ADSCs Untransfected ADSCs
ADSC transfected with Rexl/Oct4 ADSC transfected with Rexl/Klf4
Figure 17 Patent Application Publication Feb. 6, 2014 Sheet 13 of 28 US 2014/0038291 A1
Figure 18 Patent Application Publication Feb. 6, 2014 Sheet 14 of 28 US 2014/0038291 A1
+ve control Transfected HFFINo inhibitornON3N6 Me2 hES cells
-ye control UntransfectedHFF
Figure 19 Patent Application Publication Feb. 6, 2014 Sheet 15 of 28 US 2014/0038291 A1
Brightfield TRA-1-81 SSEA-4s
BgQ1NS, day15
NSLC, day 15
Figure 20 Patent Application Publication Feb. 6, 2014 Sheet 16 of 28 US 2014/0038291 A1
Figure 21
GFAP 8IIl-tubulin oechst B-tubulin
Figure 22 Patent Application Publication Feb. 6, 2014 Sheet 17 of 28 US 2014/0038291 A1
Set 1: NSLCWS, HFF Set 2: NSLC vs. hNPC
E. L -E E ES us E SE E R EL S CE E E E C E. E.R
of arge if 2 iFaldichargeafculateds fogiaea Figure 23 Patent Application Publication Feb. 6, 2014 Sheet 18 of 28 US 2014/0038291 A1
Adiposeserved Sten Cell Neural Stem-like Ce
Figure 24
Hoechst Hoechst, TRA1-60 Hoechst, RA1.81 Hoechst
s Day 15
Eoechst, set- nanog Hoechst, Stx2 E-cadherin
Day 15
echst, it inning Hoechst, Sox2 E-cadherin
Day 32
Figure 25 Patent Application Publication Feb. 6, 2014 Sheet 19 of 28 US 2014/0038291 A1
Day 28
Ectodermal
Mesodermal
2 Endodernal M
Figure 26 Patent Application Publication Feb. 6, 2014 Sheet 20 of 28 US 2014/0038291 A1
° S SC L es5' O. O.6' v S NSCL
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FF
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NTC 300 200 Figure 27a c N c N SS. SS && &8
NSCL :
NP FF is
Nt2
NTC : Figure 27b. Patent Application Publication Feb. 6, 2014 Sheet 21 of 28 US 2014/0038291 A1
NSCL
NP
FF
Nt2
NTC
NSCL
NP
FF
NTC Figure 27d Patent Application Publication Feb. 6, 2014 Sheet 22 of 28 US 2014/0038291 A1
850 50 NSCL S00 400 850 NP 50 400 850 50 FF 400 850 50
850 50 NTC : 400
ATCB Figure 27g Patent Application Publication Feb. 6, 2014 Sheet 23 of 28 US 2014/0038291 A1
red filter red + green filters
green filter red + phase filters
phase filter red + green + phase filters
Figure 28 Patent Application Publication Feb. 6, 2014 Sheet 24 of 28 US 2014/0038291 A1
MAP2/Synaptotagmin/Hoechst Figure 29 Patent Application Publication Feb. 6, 2014 Sheet 25 of 28 US 2014/0038291 A1
Figure 30 Patent Application Publication Feb. 6, 2014 Sheet 26 of 28 US 2014/0038291 A1
Figure 31 is a panel illustration of Nestin second intron CpG methylation in HFF, Keratinocytes, CD34+ cells before and after reprogramming into Neural Stem-Like Cells (NSLC). NPC serve as a positive control. Open circles indicate unmethylated CpG dinucleotides).
FF: FF-S F: OXXXXX XXX s OCCCXXCXXCXC (XXXXX XXXC OOOOOOOXCO OXCXXCXCXCXXC) OCXCOOXOXOXOXO RS CCXXXXXXX) (CXXXCXCXCXCXCXC CO3: O34"-NSLC: OOXXXXXXX) OCCCXCXCXXCXC (CCCCXCXCXCXCXC CCCXCCXCXCXCXC OOOOOOOOO OOCOXOXOXCXC) featinocyte: Keratinocyte-iSLC: Ooooooooo CCXXCOXOXOXOXO. CCCCCXCXCXCXC
ser {OOOOOOXCXC) E (COXXXXXXX)
Figure 32 is a panel of photomicrographs showing all the chromosomesas of NSLC derived from CD34" cells (left) and HFF cells (right) showing that NSLC have a normal Karyotype. (2n=46, XY metaphase spread).
Patent Application Publication Feb. 6, 2014 Sheet 27 of 28 US 2014/0038291 A1
Figure 33 is a panel of photomicrographs of Cardiac Stem-Like Cells (CSLC) taken with a Cellomics' ArrayScan V' HCS System at 10x showing Gata4, Nkx2.5, CXCR4, Brachyury, and Troponin1 positive cells at Day 15. Hoescht (blue) was used to stain all cell nuclei. The pCMV6-XL4-NkX-2.5, pCMV6-XL5-Mesp1, pCMV6-XL5-brachyury (Figure 3 b) transfected cells showed better expression of all markers than pCMV6-XL4 Tbx5, pCMV6-XL5-Mesp1, pCMV6-XL5-brachyury (Figure 3 a) transfected cells, as well as cells grown on the Laminin-211 Coated plates than on 0.1% gelatin-coated plated.
Figure 33a): GAA4 Nix's CXCR4 rathyury Triporin
Transfied Celsink.
TESC. (LAN21)
Figure 33b): GAA. Nexts XCR4 Brachyury Troponin
Track Gokstin.
Tad. LAN21) Patent Application Publication Feb. 6, 2014 Sheet 28 of 28 US 2014/0038291 A1
Figure 34 is a panel of photomicrographs of CSLC-cardiospheres taken with a light microscope at 20X. CSLC were completely dissociated into single cell suspensions and the single CSLC were monitored over time. The single cells formed individual cardiospheres that continuously grew in size and volume (followed for 55 days). SS
.
Figure 35 is a panel of photomicrographs taken with a Cellomics ArrayScan WHCS System at 10X showing early Cardiomyocyte-Like Cells differentiated from CSLC over 20 days and staining positive for Brachyury, NKX2.5, Troponin T, Troponin I, and Connexin 43. Hoescht (blue) was used to stain all cell nuclei.
Art N Tripnin Fix
US 2014/0038291 A1 Feb. 6, 2014
METHODS FOR REPROGRAMMING CELLS 0007 (iii) For a stem cell to efficiently permanently AND USES THEREOF graft (in a functional but not competitive manner) into the patient's tissue, the stem cell generally has to be CROSS REFERENCE TO RELATED autologous (the patient’s own). The main exception to APPLICATIONS this is hematopoietic stem cell grafts (bone marrow transplants) due to the changes in the immune and other 0001. This application is a continuation-in-part of U.S. systems in the body caused by these specific Somatic application Ser. No. 13/464,987, filed May 5, 2012, which is stem cells. a continuation-in-part of U.S. application Ser. No. 13/504, 988, which is the U.S. National Phase Application of Inter 0008 Thus there is a need for an efficient method to create national Appl. No.: PCT/CA2010/001727, filed Nov. 1, 2010, safe and efficacious autologous stem cells for the specific which claims the benefit of U.S. Provisional Appl. No. tissue, organ or condition to be treated, as well as new types of 61/256,967, filed Oct. 31, 2009. The content of the aforesaid stem cells with new or unique features such as enhanced applications are relied upon and are incorporated by reference potency and/or safety. herein in their entirety. 0009. There are several current “non-reprogramming mechanisms for obtaining a certain cell of interest. According FIELD OF THE INVENTION to a first mechanism, a stem cell can naturally divide or differentiate into another stem cell, progenitor, precursor, or 0002 The present invention relates to the field of eukary Somatic cell. According to a second mechanism, a Somatic otic cell reprogramming, and particularly to cell dedifferen cell can sometimes transiently change its phenotype or tiation. The invention is also concerned with methods of express certain markers when placed in certain conditions, generating stable Reprogrammed Cells and Stem-like Cells and then revert back when placed back into the original con (SLCs), including Neural, Stem-Like Cells (NSLCs) from ditions. According to a third mechanism, the phenotype of human somatic cells (and other cells) and the use of the cells many cells can be changed through forced expression of So generated in human therapy. certain genes (for example, stably transfecting the c-myc gene into fibroblasts turns them into immortal cells having BACKGROUND OF THE INVENTION neuroprogenitor characteristics), however once this forced gene expression is removed, the cells slowly revert back to Cell Reprogramming their original state. Therefore, none of the three above mecha nisms should be considered true reprogramming: the first is 0003. There is a desire in the medical, scientific, and diag considered natural differentiation which is part of a cell pro nostic fields to reprogram an easily obtainable cell into a cell gram that is already in place (going from a more undifferen that is generally harder to obtain, or to reprogram a cell to tiated to a more differentiated State), the second is a transient have new or different functionalities, without fusing or phenotypical change, and the third is a constantly forced cell exchanging material with an oocyte or another stem cell. type. A true stem cell: (i) self-renews almost indefinitely (for 0004 Stem cells currently provide a promising venue to significantly longer thanasomatic cell), (ii) is not a cancerous treat diseases or modulate/enhance certain functionalities. cell, (iii) is not artificially maintained by forced gene expres However, not all stem cells are the same and many challenges sion or similar means (must also be able to be maintained in need to be overcome, for example: standard stem cell media), (iv) can differentiate to progenitor, 0005 (i) Embryonic and other pluripotent stem cells precursor, somatic or other more differentiated cell type (of (ex. iPS cells) can turn into any type of cell which makes the same lineage), and (v) has all the characteristics of a stem them very difficult to control and generally results in the cell and not just certain markers or gene expression or mor wrong type of cell to grow in the wrong place, teratoma phological appearance. formation (a mass of cells containing different types of 0010 Despite the numerous scientific and patent publica tissues) and tumors. Even when their differentiation and tions claiming Successful reprogramming or dedifferentia growth can be controlled, pluripotent stem cells will tion, generally into a stem cell, almost all of these publica more likely grow a new competing tissue within the tions do not disclose true reprogramming because they fall existing tissue rather than integrate into the existing under one of the mechanisms mentioned above. For instance, damaged tissue. Bhasin (WO2010/088735), Cifarelli et al., (US2010/ 0006 (ii) Multipotent stem cells (somatic stem cells) 0003223), Kremer et al. (US2004/0009595), and Winnier et are generally lineage-restricted stem cells that regener al. (US2010/0047908) all refer to reprogramming, dediffer ate and grow existing tissues of their specific lineage (for entiation, and/or obtained stem cells (or progenitors) as phe example, neural stem cells regenerate and grow the ner notypical cell changes based only on a change in cell Surface Vous system, skin stem cells regenerate and grow skin, markers after culture in different media with supplements, and hematopoietic stem cells regenerate and grow the with no evidence of true reprogramming oran actual stem cell hematopoietic tissue (blood cells). Somatic stem cells (non-cancerous self-renewal with stem cells markers and no are the most effective when the right type of somatic differentiation markers). The same is true for Benneti stem cells is used for its coresposing tissue to be treated (WO2009/079007) who used increased expression of Oct4 (for example, a hematopoietic or mesenchymal stem cell and Sox2. Others, such as Akamatsu et al. (WO2010/052904) will have limited regenerative and growth abilities if and You et al (WO2007/097494, US2009/0246870), refer to implanted into the central nervous system (CNS), while having made stem cells, but these came about through con neural stem cells are the types of somatic stem cells that stant artificial gene induction delivered by retrovirus (similar have been evolved for this specific purpose and have to cMyc) with no evidence of true stem cells that are not significant regenerative and growth abilities in the immortal/tumorigenic, and stable instead of transient. Others, CNS). such as Chen et al. (US2005/0176707) and You at al. US 2014/0038291 A1 Feb. 6, 2014
(US200910227023), have made “multipotent cells', but not the hippocampus and the Subventricular Zone that replenishes stem cells. In addition these alleged multipotent cells were olfactory bulb neurons (Singec I et al., 2007; Zielton R, not stable (in the case of You et al. the cells could not even 2008). The availability of precursor cells is a key prerequisite proliferate) and both used constant media Supplements and for a transplant-based repair of defects in the mature nervous conditions to force the phenotypical change. Yet others, such system. Thus, donor cells for neural transplants are largely as Oliveri et al. (WO2009/018832) and Zahner et al. derived from the fetal brain. This creates enormous ethical (US2002/0136709), have claimed the making of pluripotent, problems, in addition to immuno-rejection, and it is question totipotent, multipotent, and/or unipotent cells automatically able whether such an approach can be used for the treatment through genome-wide DNA demethylation and histone of a large number of patients since neural stem cells can lose acetylation, but with no evidence of a stable, non-cancerous, some of their potency with each cell division. true cell line. 0016 Neural stem cells provide promising therapeutic 0011 True reprogramming appears to have been achieved potential for cell-replacement therapies in neurodegenerative with induced pluripotent stem cells (iPS cells) created inde disease (Mimeault at al., 2007). To date, numerous therapeu pendently by Yamanaka's group (Takahashi et al., 2007) and tic transplantations have been performed exploiting various Thomson's group (Yu et al., 2007), and potentially by others types of human fetal tissue as the source of donor material. before them, and although many of these cells were later However, ethical and practical considerations and their inac found to be cancerous, Some of them were not. These cells can cessibility limit the availability as a cell source for transplan be induced by true reprogramming since it was later shown tation therapies (Ninomiy Met al., 2006). that they can also be induced by non-gene integrating tran 0017. To overcome barriers and limitations to the deriva sient transfection (Soldner at al., 2009; Wolten et al., 2009; tion of patient specific cells, one approach has been to use skin Yu et al., 2009) as well as by RNA (Warren et al., 2010) or cells and inducing the trans-differentiation to neural stem protein (Kim et al., 2009; Zhou et al., 2009) alone or by small cells and/or to neurons (Levesque at al., 2000). Transdiffer molecules (Lyssiotis at al., 2009), and by similar methods. entiation has been receiving increasing attention during the However, these cells are essentially identical to embryonic past years, and trans-differentiation of mammalian cells has stem cells and have the same problems of uncontrolled been achieved in co-culture or by manipulation of cell culture growth, teratoma formation, and potential tumor formation. conditions. Alteration of cell fate can be induced artificially in In addition they appear to be less potent and less safe than vitro by treatment of cell cultures with microfilament inhibi embryonic stem cells, including the differentiated cells (in tors (Shea of al., 1990), hormones (Yeomans at al., 1976), and cluding multipotent/somatic stem cells) derived using this Calcium-ionophores (Shea, 1990; Sato at al., 1991). Mam iPS method. malian epithelial cells can be induced to acquire muscle-like 0012. A more desirable option is to have multipotent stem shape and function (Paterson and Rudland, 1985), pancreatic cells or pluripotent-like cells whose lineage and differentia exocrine duct cells can acquire an insulin-secreting endocrine tion potential is more restricted so that they do not readily phenotype (Bouwens, 1998a, b), and bone marrow stem cells form teratomas and uncontrolled growth. There is thus a need can be differentiated into liver cells (Theise et al., 2000) and for methods of creating multipotent stem cells, multipotent into neuronal cells (Woodbury et al., 2000). Other such as stem-like cells, and stem-like cells and method of reprogram Page et al. (US 2003/0059939) have transdifferentiated ming or transforming easily obtainable cells to highly desir Somatic cells to neuronal cells by culturing somatic cells in able multipotent stem cells, multipotent stem-like cells, and the presence of cytoskeletal, acetylation, and methylation stem-like cells. inhibitors, but after withdrawal of the priming agent, neuron morphology and established synapses last for not much than Neural Stem-Like Cells (NSLC) a few weeks in vitro, and complete conversion to a fully 0013 Repairing the central nervous system (CNS) is one functional and stable type of neuron has never been demon of the frontiers that medical Science has yet to conquer. Con strated. These are thus transient cell phenotypes. Complete ditions such as Alzheimer's disease, Parkinson's disease, and conversion to a fully functional and stable type of neuropro stroke can have devastating consequences for those who are genitor or neural stem cell has also never been demonstrated. afflicted. A central hope for these conditions is to develop cell Acquisition of a stable phenotype following transdifferentia populations that can reconstitute the neural network, and tion has been one of the major challenges facing the field. bring the functions of the nervous system back in line. For this 0018 Thus, there is a need in the biomedical field for reason, there is a great deal of evolving interest in neural stem stable, potent, and preferably autologous neural stem cells, and progenitor cells. Up until the present time, it was gener neural progenitor cells, as well as neurons and glial cells for ally thought that multipotent neural progenitor cells commit use in the treatment of various neurological disorders and early in the differentiation pathway to either neural restricted diseases. The same is true for many other types of cells. cells or glia restricted cells. Recently, evidence have been obtained that genes of the basic 0014 Neural stem cells have promise for tissue regenera Helix-Loop-Helix (bHLH) class are important regulators of tion from disease or injury; however, such therapies will several steps in neural lineage development, and over-expres require precise control over cell function to create the neces sion of several neurogenic bHLH factors results in conversion sary cell types. There is not yet a complete understanding of of non-determined ectoderm into neuronal tissue. Proneural the mechanisms that regulate cell proliferation and differen bHLH proteins control the differentiation into progenitor tiation, and it is thus difficult to fully explore the plasticity of cells and their progression through the neurogenic program neural stem cell population derived from any given region of throughout the nervous system (Bertrand et al., 2002). the brain or developing fetus. MASH1, NeuroD, NeuroD2, MATH1-3, and Neurogenin 1-3 0015 The CNS, traditionally believed to have limited are bHLH transcription factors expressed during mammalian regenerative capabilities, retains a limited number of neural neuronal determination and differentiation (Johnson et al., stem cells in adulthood, particularly in the dentate gyrus of 1990; Takebyashi at al., 1997; McCormicket al., 1996; Aka US 2014/0038291 A1 Feb. 6, 2014
Zawa at al., 1995). Targeted disruptions of MASH1, Ngn1. 0025. One particular aspect relates to the development of a Ngn2 or NeuroD in mice lead to the loss of specific subsets of technology to reprogram a somatic cell or non-neuronal cell neurons (Guillemot at al., 1993; Fode of al., 1998; Miyata at to a cell having one or more morphological physiological, al., 1999). and/or immunological features of a neural stem cell and 0019 U.S. Pat. No. 6,087,168 (Levesque et al.) describes which possess the capacity to differentiate along neuronal a method for converting or transdifferentiating epidermal and glial lineages. According to Some embodiments, the basal cells into viable neurons. In one example, this method invention is more particularly concerned with methods of comprises the transfection of the epidermal cells with one or generating stable Neural Stem-Like Cells (NSLCs) from more expression vector(s) containing at least one cDNA human Somatic cells, human progenitor cells and/or of human encoding for a neurogenic transcription factor responsible for stem cells, as well as cells, cell lines and tissues obtained by neural differentiation. Suitable cDNAs include: basic-helix using such methods. loop-helix activators, such as NeuroD1, NeuroD2, ASH1, and 0026. The invention further relates to compositions and zinc-finger type activators, such as Zic3, and MyT1. The methods to induce de-differentiation of human somatic cells transfection step was followed by adding at least one anti into Neural Stem-Like Cells that express neural stem cell sense oligonucleotide known to Suppress neuronal differen specific markers According to the present invention it is pos tiation to the growth medium, Such as the human MSX1 gene sible to effect the conversion of cells to various types of and/or the human HES1 gene (or non-human, homologous differentiated neuronal cells that can be created from a single counterparts). Finally, the transfected cells were grown in the cell type taken from an individual donor and then repro presence of a retinoid and a least one neurotrophin or cytok grammed and transplanted into the same individual. Upon ine, such as brain derived neurotrophic factor (BDNF), nerve induction cells according to the invention express neural growth factor (NGF), neurotrophin 3 (NT-3), or neurotrophin stem-cell specific markers and become Neural Stem-Like 4 (NT-4). This technology yields 26% of neuronal cells; how cells. ever, neither functionality nor stability of these cells was 0027. Other particular aspects relate to the development of established. In addition, neural stem cells or neuroprogenitor technologies to reprogram a Somatic cell to a cell having one cells are not produced according to this method. or more morphological physiological, and/or immunological 0020. A later process (Levesque et al., 2005: U.S. Pat. No. features of a cardiac stem cell, hematopoietic stem cell, pan 6,949.380) mentions the conversion of the epidermal basal creatic progenitor cell, myogenic (muscle) stem cell, pluri cell into a neural progenitor, neuronal, or glial cell by expos potent stem cell, ectodermal derived cell and/or mesendoder ing the epidermal basal cell to an antagonist of bone morpho mal derived cell and which possess the capacity to genetic protein (BMP) and growing the cell in the presence of differentiate along their respective lineages. According to at least one antisense oligonucleotide comprising a segment Some embodiments, the invention is more particularly con of a MSX 1 gene and/or HES1 gene. However, there is no cerned with methods of generating stable Cardiac Stem-Like evidence or examples that any neural progenitors orglial cells Cells (CSLCs), Hematopoietic Stem-Like Cells (HSLCs), were produced according to this method, let alone any details Pancreatic Progenitor-Like Cells, Myogenic (muscle) Stem or evidence that morphological, physiological or immuno Like Cells, Pluripotent-Like Cells, Ectoderm-Like Cells and logical features of neuronal cells was achieved. In addition, Mesendoderm-Like Cells from human somatic cells, human since there is also no information on functionality, stability, progenitor cells and/or of human stem cells, as well as cells, expansion, and yield about the cells which may or may not cell lines and tissues obtained by using such methods have been produced, it is possible that these cells actually are 0028. The invention further relates to compositions and skin-derived precursor cells (Fernandes et al., 2004) that have methods to induce de-differentiation of human somatic cells been differentiated into neuronal cells. into Cardiac Stem-Like Cells (CSLCs), Hematopoietic Stem 0021. In view of the above, there is thus a need for stable, Like Cells (HSLCs), Pancreatic Progenitor-Like Cells, Myo potent, and preferably autologous neural stem cells, neural genic (muscle) Stem-Like Cells, Pluripotent-Like Cells, progenitor cells, neurons and glial cells, as well as other types Ectoderm-Like Cells and Mesendoderm-Like Cells that of cells, stem cells and progenitor cells. There is also a need express cardiac stem cell, hematopoietic stem cell, pancreatic for methods that could result in true cell dedifferentiation and progenitor cell, myogenic (muscle) stem cell, pluripotent cell reprogramming. stem cell, ectoderm cell and mesendoderm cell specific mark 0022. The present invention addresses these needs and ers, respectively. According to the present invention it is pos provides various types of stem-like and progenitor-like cells sible to effect the conversion of cells to various types of and cells derived or differentiated from these stem-like or differentiated cardiac, hematopoietic (blood), pancreatic, progenitor-like cells, as well as methods that can result in true myogenic (muscle), pluripotent derived, ectoderm derived cell dedifferentiation and cell reprogramming. and/or mesendoderm derived cells that can be created from a 0023. Additional features of the invention will be apparent single cell type taken from an individual donor and then from a review of the disclosure and description of the inven reprogrammed and transplanted into the same individual. tion herein. 0029. According to one particular aspect, the invention relates to a method of transforming a cell of a first type to a SUMMARY OF THE INVENTION desired cell of a different type. This comprises i) obtaining a 0024. The present invention relates to stem-like and pro cell of a first type; ii) transiently increasing in the cell of a first genitor-like cells and cells derived or differentiated from type intracellular levels of at least one reprogramming agent, these stem-like or progenitor-like cells. The invention further whereby the transient increase induces direct or indirect relates to methods for cell dedifferentiation and cell repro endogenous expression of at least one gene regulator; iii) gramming. The invention further features compositions and placing the cell in conditions for Supporting the growth and/or methods that are useful for reprogramming cells and related the transformation of the desired cell and maintaining intra therapeutic compositions and methods. cellular levels of the at least one reprogramming agent for a US 2014/0038291 A1 Feb. 6, 2014 sufficient period of time to, allow stable expression of the at genes is the result of the stable expression of the at least least one gene regulator in the absence of the reprogramming one gene regulator, and wherein: (i) stable expression of agent; and iv) maintaining the cell in culture conditions Sup the plurality of secondary genes is characteristic of phe porting the growth and/or the transformation of the desired notypical and/or functional properties of the desired cell. Such conditions are maintained for a sufficient period of cell, (ii) stable expression of at least one of said second time to allow a stable expression of a plurality of secondary ary genes is not characteristic of phenotypical and func genes. According to the invention the expression of one or tional properties of an embryonic stem cell, and wherein more of the secondary genes is characteristic of phenotypical (i) and (ii) are indicative of successful reprogramming of and functional properties of the desired cell while being not the cell of the first type to the desired cell of the different characteristic of phenotypical and functional properties of an type. embryonic stem cell. Therefore, at the end of the period of 0037. In particular embodiments, the at least one repro time, the desired cell of a different type is obtained. gramming agent is a polypeptide listed in Table A. In addi 0030. According to another particular aspect, the inven tional embodiments the at least one gene regulator may be one tion relates to a method of transforming a cell of a first type to or more of the genes listed in Table A for the desired cell type a cell of a second different type. The method comprises con (desired cell of a second different type). tacting the cell of a first type with one or more agents capable 0038. In particular embodiments, the at least one repro of increasing within said cell levels of at least one reprogram gramming agent in the process is a Msil polypeptide, or a ming agent and directly or indirectly remodeling the chroma Ngn2 polypeptide together with a MDB2 polypeptide. In tin and/or DNA of the cell. The at least one reprogramming particular embodiments, the at least one gene regulator is agentis selected for inducing directly or indirectly the expres Sox2 Msil, or both. In additional embodiments the at least sion of morphological and functional characteristics of a one gene regulator may is one or more of the genes listed in desired cell of a different type or different cell lineage. Table A for Neural Stem-Like Cells. 0031. According to another aspect, the invention relates to 0039. According to another aspect, the invention relates to a method of transforming a cell of a first type to a cell of a a method of obtaining a Stem-Like Cell (SLC), comprising: second different type. The method comprises contacting the 0040 i) providing a cell of a first type: chromatin and/or DNA of a cell of a first type with an agent 0041 ii) transiently increasing in the cell intracellular capable of remodeling chromatin and/or DNA of said cell; levels of at least one reprogramming agent, whereby the and increasing intracellular levels of at least one reprogram transient increase induces direct or indirect endogenous ming agent. The at least one reprogramming agent is selected expression of at least one gene regulator; for inducing directly or indirectly the expression of morpho 0.042 iii) placing the cell in conditions for supporting logical and functional characteristics of a desired cell of a the transformation into the stem-like cell and maintain different type or cell lineage. ing intracellular levels of the at least one reprogramming 0032. A further aspect of the invention relates to a method agent for a sufficient period of time to allow stable of transforming a cell of a first type to a cell of a desired cell expression of the at least one gene regulator in absence of a different type, comprising increasing intracellular levels of the reprogramming agent; of at least one reprogramming agent, wherein the at least one 0.043 iv) maintaining the cell in culture conditions for reprogramming agentis selected for inducing directly or indi Supporting the transformation into the stem-like cell for rectly the expression of morphological and functional char a sufficient period of time to allow stable expression of a acteristics of a desired second cell type; and maintaining the plurality of secondary genes whose expression is char cell of a first type in culture conditions for Supporting the acteristic of phenotypical and/or functional properties of transformation of the desired cell for a sufficient period of the stem-like cell, wherein at least one of the secondary time to allow stable, expression of a plurality of secondary genes is not characteristic of phenotypical and func genes whose expression is characteristic of phenotypical and tional properties of an embryonic stem cell. At the end of functional properties of the desired cell, wherein at least one said period of time a stem-like cell is obtained. of the secondary genes is not characteristic of phenotypical 0044) A Stem-Like Cell (SLC) can be any type of stem and functional properties of an embryonic stem cell. At the like cell such as a Neural Stem-Like Cell, Cardiac Stem-Like end of the period of time the desired cell of a different type is Cell (CSLCs), Hematopoietic Stem-Like Cell (HSLCs), Pan obtained and the obtained cell is further characterized by a creatic Progenitor-Like Cell, Myogenic (muscle) Stem-Like stable repression of a plurality of genes expressed in the first Cell, Pluripotent-Like Cell, Ectoderm-Like Cell or Mesendo cell type. derm-Like Cell. 0033. A further aspect of the invention concerns a process 0045. According to another aspect, the invention relates to wherein a cell of a first type is reprogrammed to a desired cell a method of obtaining a Stem-Like Cell. The method com of a different type, the process comprising: prises increasing intracellular levels of at least one polypep 0034 a transient increase of intracellular levels of at tide specific to the desired stem cell type that is able to drive least one reprogramming agent, wherein the at least one directly or indirectly transformation of the cell of the first type reprogramming agent induces a director indirect endog into the Stem-Like Cell. For increasing the yield or type of enous expression of at least one gene regulator, and Stem-Like Cell, the method may further comprises contact wherein the endogenous expression of the said at least ing chromatin and/or DNA of a cell of a first type with a one gene regulator is necessary for the existence of the histone acetylator, an inhibitor of histone deacetylation, a desired cell of a different type: DNA demethylator, and/or an inhibitor of DNA methylation; 0035 a stable expression of said at least one gene regu and/or increasing intracellular levels of at least one other lator; polypeptide specific to the desired stem cell type that is able 0036 stable expression of a plurality of secondary to drive directly or indirectly transformation of the cell of the genes, wherein the stable expression of the secondary first type into a Stem-Like Cell. US 2014/0038291 A1 Feb. 6, 2014
0046 According to another aspect, the invention relates to otides encoding Brachyury (T) and Mesoderm Posterior 1 a method of obtaining a Stem-Like Cell comprising the use of (MESP1), along with NK2 Homebox 5 (Nkx2.5) and/or one or more compounds that increase intracellular levels of at T-box 5 (TBX5), thereby reprogramming the mesenchymal least one polypeptide specific to the desired stem cell type that stem cell into a CSLC. is able to drive directly or indirectly transformation of the cell 0050. Another aspect of the invention concerns a method of the first type into the Stem-Like Cell. For increasing the of obtaining a Hematopoietic Stem-Like Cell (HSLC). In one yield or type of Stem-Like Cell, the method may further embodiment the method comprises transfecting an adipo comprises contacting chromatin and/or DNA of a cell of a cyte-derived stem cell with a combination of polynucleotides first type with a histone acetylator, an inhibitor of histone encoding Brachyury (T), Caudal Type Homeobox 4 (CDX4), deacetylation, a DNA demethylator, and/or an inhibitor of Homeobox 84 (HOXB4), GATA Binding Factor 1 (GATA1), DNA methylation; and/or increasing intracellular levels of at GATA Binding Factor 2 (GATA2), and/or Kruppel-like Fac least one other polypeptide specific to the desired stem cell tor 1 (KLF1), thereby reprogramming the cell into a HSLC. In type that is able to drive directly or indirectly transformation another embodiment the method comprises exposing an adi of the cell of the first type into a Stem-Like Cell; and/or pocyte-derived stem cell to compounds that result in histone disrupting the cell cytoskeleton or cell integrity with at least acetylation and/or DNA demethylation, and further transfect one cytoskeleton disruptor. ing the cell (either simultaneously, before, or afterwards) with 0047 According to another aspect, the invention relates to a combination of polynucleotides encoding Brachyury (T), a method of obtaining a Neural Stem-Like Cell (NSLC). The Caudal Type Homeobox 4 (CDX4), Homeobox B4 method comprises increasing intracellular levels of at least (HOXB4), GATA Binding Factor 1 (GATA1), GATA Binding one neural stem cell specific polypeptide that is able to drive Factor 2 (GATA2), and/or Kruppel-like Factor 1 (KLF1), directly or indirectly transformation of the cell of the first type thereby reprogramming the cell into a HSLC. into a NSLC. For increasing the yield or type of NSLC, the 0051. Another aspect of the invention concerns a method method further comprises contacting chromatin and/or DNA of obtaining a Pancreatic Progenitor-Like Cell. In one of a cell of a first type with a histone acetylator, an inhibitor of embodiment the method comprises transfecting an adipo histone deacetylation, a DNA demethylator, and/or an inhibi cyte-derived stem cell with a polynucleotide encoding SRY tor of DNA methylation; and/or increasing intracellular levels (Sex determining RegionY)-box 17 (SOX17), Pancreatic and of at least one other neural stem cell specific polypeptide that Duodenal Homebox 1 (PDX1) and Neurogenin 3 (NGN3) is able to drive directly or indirectly transformation of the cell optionally along with Octamer-binding Transcription Factor of the first type into a NSLC. 4 (OCT4), thereby reprogramming the cell into a Pancreatic 0048. Another aspect of the invention concerns a method Progenitor-Like Cell. In another embodiment the method of obtaining a Neural Stem-Like Cell (NSLC). In one comprises exposing an adipocyte-derived stem cell to com embodiment the method comprises transfecting a skin cell pounds that result in histone acetylation and/or DNA dem with a polynucleotide encoding Musashil, Musashil and ethylation; and further transfecting the cell (either simulta Neurogenin 2, Musashil and Methyl-CpG Binding Domain neously, before, or afterwards) with polynucleotides Protein 2 (MBD2), or Neurogenin 2 and Methyl-CpG Bind encoding SRY (Sex determining RegionY)-box 17 (SOX17), ing Domain Protein 2, thereby reprogramming the skin cell Pancreatic and Duodenal Homebox 1 (PDX1) and Neuroge into a NSLC. In another embodiment the method comprises nin 3 (NGN3) optionally along with Octamer-binding Tran exposing a skin cell to: (I) an inhibitor of histone deacetyla scription Factor 4 (OCT4), thereby reprogramming the cell tion, (ii) an inhibitor of DNA methylation, (iii) a histone into a Pancreatic Progenitor-Like Cell. acetylator, and/or (iv) a DNA demethylator such as a MBD2 0.052 Another aspect of the invention concerns a method polypeptide and/or transfecting with a polynucleotide encod of obtaining a Mesendoderm-Like Cell. In one embodiment ing a MBD2 polypeptide; and further transfecting the cell the method comprises transfecting an adipocyte-derived stem (either simultaneously, before, or afterwards) with a poly cell with a polynucleotide encoding Forkhead Box D3 nucleotide encoding MUSASHI1 and/or with a polynucle (FOXD3), MIX1 Homeobox-Like Protein 1 (MIXL1) and otide encoding NGN2, thereby reprogramming the skin cell Neurogenin 3 (NGN3) along with Methyl-CpG Binding into a NSLC. Some other cells, such as keratinocytes and Domain Protein 2 (MBD2), thereby reprogramming the cell CD34" cells, can also be used and reprogrammed. Addition into a Mesendoderm-Like Cell. In another embodiment the ally introducing into the cells one or more of the following method comprises exposing an adipocyte-derived stem cell to polypeptides (ex. by transfection of their corresponding poly compounds that result in histone acetylation and/or DNA nucleotides) reprograms the cells to Ectoderm-Like Cells: demethylation; and further transfecting the cell (either simul Zinc Finger Protein 42 (REX1), Octamer-binding Transcrip taneously, before, or afterwards) with polynucleotides encod tion Factor 4 (OCT4), Kruppel-like Factor 4 (KLF4), Sal-like ing Forkhead Box D3 (FOXD3), MIX1 Homeobox-Like Pro 4 (SALL4), and Nanog Homeobox (NANOG). tein 1 (MIXL1) and Neurogenin 3 (NGN3), thereby 0049. Another aspect of the invention concerns a method reprogramming the cell into a Mesendoderm-Like Cell. of obtaining a Cardiac Stem-Like Cell (CSLC). In one Another aspect of the invention concerns a method of obtain embodiment the method comprises transfecting a mesenchy ing a Pluripotent-Like Cell. In one embodiment the method mal stem cell with a polynucleotide encoding Brachyury (T) comprises transfecting an adipocyte-derived stem cell with a and Mesoderm Posterior 1 (MESP1), along with NK2 Home polynucleotide encoding a) Zinc Finger Protein 42 (REX1), box 5 (NRX2.5) and/or T-box 5 (TBX5), thereby reprogram Octamer-binding Transcription Factor 4 (OCT4) and Krup ming the mesenchymal stem cell into a CSLC. In another pel-like Factor 4 (KLF4), or b) Sal-like 4 (SALL4), Octamer embodiment the method comprises exposing a mesenchymal binding Transcription Factor 4 (OCT4), Kruppel-like Factor stem cell to compounds that result in histone acetylation 4 (KLF4) and Nanog Homeobox (NANOG), thereby repro and/or DNA demethylation, and further transfecting the cell gramming the cell into a Pluripotent-Like Cell. In another (either simultaneously, before, or afterwards) with polynucle embodiment the method comprises exposing an adipocyte US 2014/0038291 A1 Feb. 6, 2014 derived stem cell to compounds that result in histone acety tissue or organ, thereby ameliorating the Subjects condition. lation and/or DNA demethylation; and further transfecting In another embodiment the method comprises the adminis the cell (either simultaneously, before, or afterwards) with tration of a reprogrammed cell that has been genetically polynucleotides encoding a) Zinc Finger Protein 42 (REX1), modified (for therapeutic or other purposes) or genetically Octamer-binding Transcription Factor 4 (OCT4) and Krup corrected (in cases of a genetic disease or similar instance) to pel-like Factor 4 (KLF4), or b) Sal-like 4 (SALL4), Octamer a subject in need thereof, wherein the administration provides binding Transcription Factor 4 (OCT4), Kruppel-like Factor a dose of reprogrammed genetically-modified/-corrected 4 (KLF4) and Nanog Homeobox (NANOG), thereby repro cells sufficient to increase, Support, replace or correct a bio gramming the cell into a Pluripotent-Like Cell. logical function of a given cell, tissue or organ, thereby ame 0053. In one particular embodiment, the method of obtain liorating or correcting the Subjects condition or allowing for ing a Neural Stem-Like Cell (NSLC), comprises: a desired condition. 0054 providing a cell of a first type: 0068. The benefits of the present invention are significant 0055 introducing into the cell one or more polynucle and include more potent and/or safer and/or lower cost of cell otide capable of transient expression of one or more the therapy by eliminating the need of immuno-Suppressive following polypeptides: Musashil (Msil); a Musashil agents, no need for embryos or fetal tissue, thus eliminating (Msil) and a Neurogenin 2 (Ngn2); a Musashil (Msil) ethical and time constraints, lower cost of production, and no and methyl-CpG binding domain protein 2 (MBD2); health risks due to possible transmission of viruses or other and Neurogenin 2 (Ngn2) and methyl-CpG binding disease, and the availability of more potent and/or safer stem domain protein 2 (MBD2); and like cells and progenitor-like cells. In addition, since the cells 0056 placing the cell in culture conditions supporting are created fresh, they tend to be more potent than cells that the transformation into a NSLC for a sufficient period of have been passaged multiple times. time to allow a stable expression of a plurality of genes 0069. Additional aspects, advantages and features of the whose expression is characteristic of phenotypical and present invention will become more apparent upon reading of functional properties of a NSLC. the following non-restrictive description of preferred 0057. At the end of the period of time a NSLC is obtained embodiments and Examples which are exemplary and should and the obtained NSLC is further characterized by a stable not be interpreted as limiting the scope of the invention. repression of a plurality of genes expressed in the first cell type. DETAILED DESCRIPTION OF THE INVENTION 0058 According to another embodiment, the method of obtaining a Neural Stem-Like Cell (NSLC), comprises: 0070 The present invention relates to reprogrammed cells, methods for cell dedifferentiation and cell reprogram 0059 providing a cell of a first type which is not a ming, and use of these cells. A significant aspect of the present NSLC; invention is that it permits the use of a patients own cells to 0060 increasing intracellular levels of at least one neu develop different types of cells either in situ (in the patient) or ral stem cell specific polypeptide, wherein the polypep that can be transplanted after steps of in vitro dedifferentia tide is capable of driving directly or indirectly transfor tion and in vitro reprogramming. Thus, this technology elimi mation of the cell of the first type into a NSLC; and nates the problems associated with a) lack of the patients 0061 contacting the chromatin and/or DNA of the cell own desired cells; b) lack of potency or other desired func of a first type with a histone acetylator, an inhibitor of tional features of the patients own desired cells; and/or c) histone deacetylation, a DNA demethylator, and/or a problems associated with transplantation of non-host cells, chemical inhibitor of DNA methylation. Such as, immunological rejection and the risk of transmitting 0062 According to another embodiment, the method of disease, and no permanent functional grafting. In addition, obtaining a Neural Stem-Like Cell (NSLC), comprises: since the cells are “newly created, they have the potential to 0063 obtaining a non-NSLC: be more potent than alternative sources of natural cells that 0064 co-transfecting the non-NSLC with a first poly have already divided multiple times. nucleotide encoding a MBD2 polypeptide and with at least one second polynucleotide encoding a DEFINITIONS MUSASHI1 polypeptide and/or encoding a NGN2 polypeptide; 0071. As used herein and in the appended claims, the 0065 placing the co-transfected cell in culture condi singular forms “a,” “an', and “the’, include plural referents tions for supporting the transformation of NSLC until unless the context clearly indicates otherwise. Thus, for said NSLC is obtained. example, reference to “a cell includes one or more of such, 0066 Certain aspects of the invention concerns isolated cells or a cell line derived from such a cell, reference to “an cells, cell lines, compositions, 3D assembly of cells, and agent includes one or more of Such agent, and reference to tissues comprising cells obtained using the methods “the method’ includes reference to equivalent steps and described herein. Additional aspects concerns the use of Such methods known to those of ordinary skill in the art that could isolated cells, cell lines, compositions, 3D assembly of cells, be modified or substituted for the methods described herein. and tissues of medical treatment and methods of regenerating 0072. As used herein, the term “polynucleotide' refers to a mammalian tissue or organ. any DNA or RNA sequence or molecule, comprising encod 0067. Yet, a further aspect concerns a method for repairing ing nucleotide sequences. The term is intended to encompass or regenerating a tissue in a Subject. In one embodiment the all polynucleotides whether occurring naturally or non-natu method comprises the administration of a reprogrammed cell rally in a particular cell, tissue or organism. This includes as defined herein to a subject in need thereof, wherein the DNA and fragments thereof, RNA and fragments thereof, administration provides a dose of reprogrammed cells Suffi cDNAS and fragments thereof, expressed sequence tags, arti cient to increase or Support a biological function of a given ficial sequences including randomized artificial sequences. US 2014/0038291 A1 Feb. 6, 2014
0073. As used herein, the term “polypeptide' refers to any Cell” or “NSLC refers to any cell-derived multipotent stem amino acid sequence having a desired functional biological cell having, as a physiological feature, a capacity to form activity (e.g. DNA demethylation). The term is intended to other neural stem-like cells and neuroprogenitor-like cells encompass complete proteins, fragments thereof, fusion pro and under physiological conditions that favor differentiation teins and the like, including carbohydrate or lipid chains or to form neuron-like cells and glial-like cells. compositions. I0083) “Neurosphere' refers to a cellular aggregate of neu 0074 “Trans-differentiation refers to a direct switch of ral stem cells and neuroprogenitor cells that form a floating an already differentiated cell to another type of differentiated sphere formed as a result of proliferation of the neural stem cell. cells and neuroprogenitor cells in appropriate proliferation 0075 “De-differentiation” refers to the loss of phenotypic conditions. NSLCs also form neurospheres consisting of characteristics of a differentiated cell by activating or deacti aggregates of NSLCs and neuroprogenitor-like cells. Vating genes or metabolic pathways. I0084 “Cardiosphere refers to a cellular aggregate of car 0076 “Marker” refers to a gene, polypeptide, or biological daic stem cells and cardiac progenitor cells that form a float function that is characteristic of a particular cell type or cel ing sphere formed as a result of proliferation of the cardaic lular phenotype. stem cells and cardiac progenitor cells in appropriate prolif 0077 “Genetically-engineered DNA sequence” is meanta eration conditions. CSLCs also form cardiospheres consist DNA sequence wherein the component sequence elements of ing of aggregates of CSLCs and cardiac progenitor-like cells. DNA sequence are organized within the DNA sequence in a “Reprogrammed cell refers to a cell that has undergone manner not found in nature. stable trans-differentiation, de-differentiation, or transforma 0078 “Signal sequence” refers to a nucleic acid sequence tion. Some reprogrammed cells can be subsequently induced which, when incorporated into a nucleic acid sequence to re-differentiate. The reprogrammed cell stably expresses a encoding a polypeptide, directs secretion of the translated cell-specific marker or set of markers, morphology, and/or polypeptide from cells which express said polypeptide, or biological function that was not characteristic of the original allows the polypeptide to readily cross the cell membrane into cell. “Reprogrammed somatic cell refers to a process that a cell. The signal sequence is preferably located at the 5' end alters or reverses the differentiation status of a somatic cell, of the nucleic acid sequence encoding the polypeptide. Such which can be either complete or partial conversion of the that the polypeptide sequence encoded by the signal sequence differentiated state to an either less differentiated state or a is located at the N-terminus of the translated polypeptide. By new differentiated state. 'signal peptide' is meant the peptide sequence resulting from I0085 “Regeneration” refers to the capability of contribut translation of a signal sequence. ing to the repair or do novo construction of a cell, tissue or 0079) “Ubiquitous promoter” refers to a promoter that Organ. drives expression of a polypeptide or peptides encoded by I0086) “Differentiation” refers to the developmental pro nucleic acid sequences to which promoter is operably linked. cess of lineage commitment of a cell. Differentiation can be Preferred ubiquitous promoters include human cytomega assayed by measuring an increase in one or more cell-differ lovirus immediate early (CMV); simian virus 40 early entiation specific markers relative to the expression of the promter (SV40); Rous sarcoma virus (RSV); or adenovirus undifferentiated cell markers. major late promoter. I0087) “Lineage” refers to a pathway of cellular develop 0080 “Gene expression profiling' means an assay that ment, in which a more undifferentiated cell undergoes pro measures the activity of multiple genes at once, creating a gressive physiological changes to become a more differenti global picture of cellular function. For example, these profiles ated cell type having a characteristic function (e.g., neurons can distinguish between human neural stem cells and Somatic and glia are of a neuroprogenitor linage, which is of an ecto cells that are actively dividing or differentiating. derm lineage which formed from blastocysts and embryonic 0081 “Transfection” refers to a method of gene delivery stem (ES) cells). that introduces a foreign nucleotide sequences (e.g. DNA I0088 “Tissue' refers to an ensemble of cells (identical or molecules) into a cell preferably by a non-viral method. In not) and an extracellular matrix (ECM) that together carry out preferred embodiments according to the present invention a specific function or set of functions. foreign DNA is introduced to a cell by transient transfection I0089. “CDM is meanta living tissue equivalent or matrix, of an expression vector encoding a polypeptide of interest, a living scaffold, or cell-derived matrix. whereby the foreign DNA is introduced but eliminated over time by the cell and during mitosis. By “transient transfec Cell Transformation tion' is meant a method where the introduced expression 0090 Some aspects of the invention concerns methods vectors and the polypeptide encoded by the vector, are not and cells to transform or reprogram a given somatic cell into permanently integrated into the genome of the host cell, or a pluripotent, multipotent and/or unipotent cell. Some aspects anywhere in the cell, and therefore may be eliminated from of the invention relates to methods for conditioning a Somatic the host cell or its progeny over time. Proteins, polypeptides, cell to reprogramming into a pluripotent, multipotent or uni or other compounds can also be delivered into a cell using potent cell. transfection methods. (0091. The terms “transform” or “reprogram” are used 0082 “Neuroprogenitor Cell refers to an immature cell interchangeably to refer to the phenomenon in which a cell is of the nervous system, which can differentiate into neurons dedifferentiated or transdifferentiated to become pluripotent, and glia (oligodendrocytes and astrocytes). “Neural Stem multipotent and/or unipotent. The dedifferentiated cell could Cell' is an ectoderm germ layer derived multipotent stem cell subsequently be redifferentiated into a different type of cell. having, as a physiological feature, a capacity to form neuro Cells can be reprogrammed or converted to varying degrees. progenitor cells and under physiological conditions that favor For example, it is possible that only a small portion of cells are differentiation to form neurons and glia. “Neural Stem-Like converted or that an individual cell is reprogrammed to be US 2014/0038291 A1 Feb. 6, 2014 multipotent but not necessarily pluripotent. Thus, the terms lymphocyte (B and T lymphocyte), granulocyte, macroph “transforming” or “reprogramming methods can refer to age, monocyte, mononuclear cell, pancreatic islet cell, Sertoli methods wherein it is possible to reprogram a cell Such that cell, neuron, glial cell, cardiac muscle cell, and other muscle the “new” cell shows morphological and functional charac cell. teristics of a new or different specific cell lineage (e.g. the 0096. As used herein, examples of cells of a second type transformation of fibroblast cells into neuronal cells). include, but are not limited to germ cells, embryonic stem 0092. As used herein, the term “somatic cell refers to any cells and derivations thereof, adult stem cells and derivations differentiated cell forming the body of an organism, apart thereof, progenitor cells and derivations thereof, cells derived from stem cells, progenitor cells, and germline cells (i.e. from mesoderm, endoderm or ectoderm, and a cell of meso oVogonies and spermatogonies) and the cells derived there derm, endoderm or ectoderm lineage Such as an adipose from (e.g. oocyte, spermatozoa). For instance, internal derived stem cell, mesenchymal stem cell, hematopoietic organs, skin, bones, blood, and connective tissue are all made stem cell, skin derived precursor cell, hair follicle cell, fibro up of Somatic cells. Somatic cells according to the invention blast, keratinocyte, epidermal cell, endothelial cell, epithelial can be differentiated cells isolated from adult or can be fetal cell, granulosa epithelial cell, melanocyte, adipocyte, chon Somatic cells. Somatic cells are obtained from animals, pref drocyte, hepatocyte, lymphocyte (B and T lymphocyte), erably human Subjects, and cultured according to standard granulocyte, macrophage, monocyte, mononuclear cell, pan cell culture protocols available to those of ordinary skill in the creatic islet cell, Sertolicell, neuron, glial cell, cardiac muscle art cell, and other muscle cell. In addition, each of the above 0093. As used herein, “Stem cell refers to those cells “-like” cell (a cell that has similar but not completely identical which retain the ability to renew themselves through mitotic characteristics of the known natural type of the cell) is also cell division and which can differentiate into a diverse range included in the examples of cells of a second type. of specialized cell types. It includes both embryonic stem 0097. According to one particular aspect, the method of cells that are found in blastocysts, and adult stem cells that are transforming a cell of a first type into a cell of a second found in adult tissues. “Totipotent cells' refers to cells that different type comprises the steps of: have the ability to develop into cells derived from all three 0.098 i) providing a cell of a first type: embryonic germ layers (mesoderm, endodermand ectoderm) 0099 ii) transiently increasing in the cell of a first type and an entire organism (e.g., human being if placed in a intracellular levels of at least one reprogramming agent, womans, uterus in the case of humans). Totipotent cells may whereby the transient increase induces director indirect give rise to an embryo, the extra embryonic membranes and endogenous expression of at least one gene regulator, all post-embryonic tissues and organs. The term “pluripotent 0.100 iii) placing the cell in conditions for supporting as used herein is intended to mean the ability of a cell to give the transformation of the desired cell and maintaining rise to differentiated cells of all three embryonic germ layers. intracellular levels of the at least one reprogramming “Multipotent cells' refers to cells that can produce only cells agent for a sufficient period of time to allow stable of a closely related family of cells (e.g. hematopoietic stem expression of the at least one gene regulator in absence cells differentiate into red blood cells, white blood cells, of the reprogramming agent; and platelets, etc.). “Unipotent cells' refers to cells that have the 0101 iv) maintaining the cell in culture conditions sup capacity to develop? differentiate into only one type of tissue/ porting the transformation of the desired cell for a suf cell type (e.g. skin cells). ficient period of time to allow a stable expression of a 0094. The present invention allows the reprogramming of plurality of secondary genes whose expression is char any cell to a different type of cell. Although the present acteristic of phenotypical and functional properties of application focuses primarily on the preparation of Stem the desired cell. At the end of said period of time the cell Like cells, especially, Neural Stem-Like Cells (NSLCs), the of the first type has been transformed into the desired cell invention is not so restricted because many different types of of a different type. Preferably, the cell of a different type cells can be generated according to the principles described obtained after the transformation is further characterized herein. Similarly, while the Examples section describes by a stable repression of a plurality of genes expressed in embodiments where fibroblasts, keratinocytes, CD34" cells, the first cell type. adipose-derived stem cells (ADSCs), neural stem cells (in 0102) According to one particular aspect, the method of cluding NSLCs), and cells within a Cell-Derived Matrix transforming a cell of a first type into a cell of a second (CDM) are reprogrammed, the invention is not limited such different type comprises the steps of: cells. The invention may be employed for the reprogramming 0.103 i) providing a cell of a first type: of virtually any cell of interest. 0.104 ii) transiently increasing in the cell of a first type 0095 Accordingly, a general aspect of the invention intracellular levels of at least one reprogramming agent, relates to a method of transforming a cell of a first type to a whereby the transient increase induces director indirect cell of a second different type. As used herein, examples of endogenous expression of at least one gene regulator, cells of a first type include, but are not limited to germ cells, 0105 iii) placing the cell in conditions for supporting embryonic stem cells and derivations thereof, adult stem cells the transformation of the desired cell and maintaining and derivations thereof, progenitor cells and derivations intracellular levels of the at least one reprogramming thereof, cells derived from mesoderm, endoderm or ecto agent for a sufficient period of time to allow stable derm, and a cell of mesoderm, endoderm or ectoderm lineage expression of the at least one gene regulator in absence Such as an adipose-derived stem cell (ADSC), mesenchymal of the reprogramming agent; and stem cell, hematopoietic stem cell (CD34" cell), skin derived 0106 iv) maintaining the cell in culture conditions sup precursor cell, hair follicle cell, fibroblast, keratinocyte, epi porting the transformation of the desired cell for a suf dermal cell, endothelial cell, epithelial cell, granulosa epithe ficient period of time to allow a stable expression of a lial cell, melanocyte, adipocyte, chondrocyte, hepatocyte, plurality of secondary genes whose expression is char US 2014/0038291 A1 Feb. 6, 2014
acteristic of phenotypical and functional properties of wherein (i) and (ii) are indicative of successful repro the desired cell. At least one of the stably expressed gramming of the cell of the first type to the desired cell secondary genes is not characteristic of phenotypical of the different type. and functional properties of an embryonic stem cell. At 0112. As used herein, “transiently increasing refers to an the end of said period of time the cell of the first type has increase that is not necessarily permanent and therefore, been transformed into the desired cell of a different type. which may decrease or disappear over time. For instance, Preferably, the cell of a different type obtained after the when referring to transiently increasing intracellular levels of at least one reprogramming agent in a cell, it means that the transformation is further characterized by a stable increase in present for a sufficient period of time for causing repression of a plurality of genes expressed in the first particular cellular events to occur (e.g. inducing stable endog cell type. enous expression of a gene regulator). Typically a transient 0107 According to various embodiments, step iii) may be increase is not permanent and is not associated for instance to carried out, consecutively to step ii), simultaneously with step genome integration of an expression vector. ii), or before step ii). 0113. As used herein the term “reprogramming agent' 0108. According to a related aspect, the invention relates refers to a compound that is capable of inducing directly or to a process wherein a cell of a first type is reprogrammed to indirectly the expression of morphological and/or functional a desired cell of a different type, the process comprising: characteristics of the desired cell of a different type. Preferred compounds include those capable of driving directly or indi 0.109 a transient increase of intracellular levels of at rectly transformation of the cell of the first type into the least one reprogramming agent, wherein the at least one desired cell of a different type. In preferred embodiment, the reprogramming agent induces a director indirect endog reprogramming agent is selected for inducing a direct or enous expression of at least one gene regulator, wherein indirect endogenous expression of at least one gene regulator the endogenous expression of the at least one gene regu as defined herein. There are many compounds that may be lator is necessary for the existence of the desired cell of helpful in reprogramming a cell according to the invention a different type: and these compounds can be used alone or in combinations. 0110 a stable expression of said at least one gene regu For example, the compound may be a molecule that induces lator; epigenetic changes (chromatin remodeling, ex. histone acety 0111 stable expression of a plurality of secondary lation and/or DNA demethylation) or a cytoskeleton disruptor genes, wherein the stable expression of the plurality of that is helpful in reprogramming a cell according to the inven secondary genes is the result of the stable expression of tion (or alternatively the culture conditions can include one or the at least one gene regulator, and wherein: (i) stable more compounds, materials, environmental (physical or expression of the plurality of secondary genes is char chemical) effects or conditions that induce epigenetic acteristic of phenotypical and/or functional properties of changes and/or cytoskeleton disruptors that Support the trans the desired cell, (ii) stable expression of at least one of formation to the desired cell). In various embodiments, the the secondary genes is not characteristic of phenotypical reprogramming agent is a polynucleotide or polypeptide and functional properties of an embryonic stem cell, and selected according to TABLE A: TABLE A Reprogramming agent RefSeq, UniProt TM, UniGene TM Examples of GenBank TM (NCBI) Swiss-Port Accession Desired Cell Type Name Accession No. Accession No. No. Pluripotent-like AGR2 NM OO6408.3 O95994 HSS30009 Cells AGR3 NM 176813.3 Q8TD06 HS1 OO686 Markers: BRDX1 NM 018321.3 Q8TDN6 HS.718510 OCT4 CRABP2 NM OO1878.2 P29373 HS.405662 Nanog DNMT3B, NM OO6892.3 Q9UBC3 HS.713611 SSEA-4 isoform 1 TRA1-60 DNMT3B, NM 175848.1 Q9UBC3 HS.713611 TRA1-81 isoform 2 AP DNMT3B, NM 175849.1 Q9UBC3 HS.713611 isoform 3 DNMT3B, NM 175850.1 Q9UBC3 HS.713611 isoform 6 DPPA2 NM 138815.3 Q7Z7J5 HS.351113 DPPA3 NM 199286.2 Q6WOC5 HS131358 (STELLA) DPPA4 NM 018189.3 Q7L190 HS.317659 DPPA5 NM 001025290.1 A6NC42 HS.125331 (ESG1) FOXD3 NM 012183.2 Q9UJUS HSS46573 FOXH1 NM OO3923.2 O75593 HS.70836S GABRB3, NM 000814.5 P28472 HS.302352 isoform 1 GABRB3, NM 021912.4 P28472 HS.302352 isoform 2 GABRB3, NM OO1191320.1 P28472 HS.302352 isoform 3
US 2014/0038291 A1 Feb. 6, 2014 14
TABLE A-continued Reprogramming agent
RefSeq, UniProt TM, UniGene TM Examples of GenBank TM (NCBI) Swiss-Port Accession Desired Cell Type Name Accession No. Accession No. No. PAX7, NM O13945.2 P23759 HS1132S3 isoform 2 PAX7, NM OO1135254.1 P23759 HS1132S3 isoform 3
0114. In some embodiments, the reprogramming agent is period, the number of transformed desired cells is substan a polypeptide which shares at least 75%, 80%, 85%, 90%, tially equivalent or even higher than an amount of cells a first 95%, 97%, 99% or more of the functionality or sequence type provided at the beginning. identity of any one of the reprogramming agents in the table 0116. The present invention encompasses various types of hereinbefore. compounds that are Suitable for increasing in a cell of a first 0115 Identifying the “sufficient period of time' to allow type the intracellular levels of at least one reprogramming stable expression of the at least one gene regulator in absence agent, Preferably, the compound should also be able to of the reprogramming agent and the “sufficient period of directly or indirectly remodel the chromatin and/or DNA of time' in which the cell is to be maintained in culture condi the cell, thus resulting directly or indirectly in the expression tions Supporting the transformation of the desired cell is of morphological and functional characteristics of the desired within the skill of those in the art. The sufficient or propertime cell of a different type. Preferred compounds are reprogram period will vary according to various factors, including but ming agents as defined herein or any other compound having not limited to, the particular type and epigenetic status of cells a similar activity and having the ability to activate or enhance (e.g. the cell of the first type and the desired cell), the amount the expression of the endogenous version of genes listed in of starting material (e.g. the number of cells to be trans the table of reprogramming agents hereinbefore and which formed), the amount and type of reprogramming agent(s), the are capable of driving directly or indirectly transformation of gene regulator(s), the culture conditions, presence of com the cell of the first type into the desired cell of a different type. pounds that speed up reprogramming (ex, compounds that 0117. As will be explained hereinafter, the increase in increase cell cycle turnover, modify the epigenetic status, intracellular levels of the at least one reprogramming agent and/or enhance cell viability), etc. In various embodiments can be achieved by different means. In preferred embodi the sufficient period of time to allow a stable expression of the ments the reprogramming agent is a polypeptide and increas at least one gene regulator in absence of the reprogramming ing intracellular levels of such polypeptide include transfec agent is about 1 day, about 2-4 days, about 4-7 days, about 1-2 tion (or co-transferction) of an expression vector having a weeks, about 2-3 weeks or about 3-4 weeks. In various polynucleotide (ex. DNA or RNA) encoding the polypeptide embodiments the sufficient period of time in which the cells (S), or by an intracellular delivery of polypeptide(s). Accord are to be maintained in culture conditions Supporting the ing to the invention, transient expression is generally prefer transformation of the desired cell and allow a stable expres able. Additional Suitable compounds may include sion of a plurality of secondary genes is about 1 day, about 2-4 compounds capable of increasing the expression of the days, about 4-7 days, or about 1-2 weeks, about 2-3 weeks, endogenous version of genes listed in the table of reprogram about 3-4 weeks, about 4-6 weeks or about 6-8 weeks. In ming agents and gene regulators including, but not limited to, preferred embodiments, at the end of the transformation reprogramming factors listed in Table B. TABLE B Desired cell type Reprogramming Factor Pluripotent-like Cells Y27632, Butyric acid, Hydrocortisone, Sodium Selenite, Insulin, TGFB1 (R&D), NODAL, BMP4, Wnt/B-Catenin, Pluripotin, Glucogen synthetase kinase-3 inhibitor, CHIR99021, PDO325901, 6 Bromoindirubib-3-oxime (BIO), KSOR, vitamin A, histone deacetylase inhibitor (HDAC), RG 108, R(+)BayK8644, SB431542, thiazovivin, BIXO1294, Fibroblast growth factor 2 (FGF2), Activin A, Wnt3a, L-Ascorbic Acid, Cyclic Pifithrin-C, Tranylcypromine hydrochloride, Kenpaullone, 5-AZacytidine, Valproic Acid (VPA), Theanine. Ectoderm-like Cells a retinoid compound (ex. ATRA), L-AScorbic acid, sonic hedgehog (SHH), Wnt3a, a neurotrophic factor, FGF2, Epidermal growth factor (EGF), Transforming growth factor (TGF) alpha, Estrogen, Noggin, 5 Azacytidine, VPA, BIXO1294, R(+)BayK8644, RG108, Butyric acid, Lithium, Cytosine arabinoside, Chordin, b-Catenin, CHIR99021, SB431542, rapamycin (mTOR). US 2014/0038291 A1 Feb. 6, 2014 15
TABLE B-continued Desired cell type Reprogramming Factor Mesendoderm-like Cells BIO, Dorsomorphin, vitamin E, VPA, HAS, Bone morphogenetic protein 4 (BMP4), EGF, FGF2, CHIR99021, Activin A, Insulin-like growth factor 1 (IGF-1), SB431542, PDO325901, Butyric acid, Epidermal growth factor-CriptOfFRL-1/Cryptic (EGF CFC) and the TGFBs, Nodal, SHH, Vg1/GDF1 (growth and differentiation factor-1). Neural Stem-like cells a retinoid compound (ex. ATRA), a neurotrophic factor, L-AScorbic acid, Estrogen, 5-AZacytidine, VPA, BIXO1294, R(+)BayK8644, RG108, Butyric acid, Lithium, Activin A, Noggin, EGF, FGF2, Wnt3a, TGF, cAMP, Follistatin, SHH, BDNF, IGF-1, CNTF, PDFG, SDNSF/MCFD2, Neuropeptide Y, Forskolin. Cardiac Stem-like cells Wnt11, Cardiogenol C, BMP4, FGF2, Activin A, VEGF, DKK1 (dickkopf homologue 1), Insulin-like growth factor 1 (IGF-1), Oxytocin, Cardiotropin, Hepatocyte growth factor (HGF), 5-AZacytidine, L 3,3',5-triiodothyronine, Valproic Acid, BDXO1294, R(+)BayK8644, RG108, Cardiogenol C hydrochloride, Butyric acid, Stem Cell factor. Pancreatic Progenitor-like Cells Activin A, GLP-1, FGF2, Reg1, nicotinamide, Betacellulin, SHH, (-)-Indolactam V, a retinoid compound, Cyclopamine, IDE-1 and 2,5-AZacytidine, Valproic Acid, BIXO1294, R(+)BayK8644, RG108, Butyric acid. Hematopoietic Stem-like Cells SCGM, Thrombopoietin, Interleukin-3, Stem Cell actor (SCF), GM-CSF, Interleukin-6, SHH, Wnt5a, insulin-like Growth factor, Angiopoietin factor 2 or 3, butyric acid, 5-azacytidine, estrogen. Myogenic Stem-like Cells HGF, FGF2, IGF, BMP-2, TGFB, TGFB3, Wnt3a, Wnt11, 5-AZacytidine, Valproic Acid, BDXO1294, R(+)BayK8644, RG108, Trichostatin A (TSA), retinoic acid, Lithium, Butyric acid.
0118. According to the principles of the invention, it is within their skill to select suitable conditions (e.g. culture increasing intracellular levels of at least one reprogramming conditions) favoring growth or transformation of desired cell agent should induce a direct or indirect endogenous expres types. sion of at least one gene regulator. As used herein, “gene I0121 The terms “phenotypical and functional properties”. regulator” refers to a polynucleotide or polypeptide whose when, referring to a desired cell or to an embryonic stem cell, expression is associated with a series of intracellular events means the biological, biochemical, physiological and visual leading to the transformation of a given cell of a first type into characteristics of a cell, including expression of certain genes a pluripotent, multipotent and/or unipotent cell Typically and cell Surface markers, which can be measured or assessed expression of a gene regulator directly or indirectly activates for confirming its identity or function(s). genes necessary for the phenotypical and functional charac 0.122 An example of a Suitable reprogramming agent teristic of pluripotent, multipotent and/or unipotent cells, according to preferred embodiments of the invention is while repressing genes of the cell of a first type. The gene MUSASHI1. In some embodiments this polypeptide is pre regulator may be the same or be different than the reprogram ferred for driving a first cell, such as a fibroblast, into a Neural ming agent. Examples of gene regulators according to the Stem-Like Cell (NSLC). In other embodiments, the at least invention include, but are not limited to, the polynucleotides one reprogramming agent which said intracellular levels is and polypeptides listed herein before in TABLE A. increased is (are) either Musashil (Msil) alone; Musashil 0119. In some embodiments, the gene regulator is a (Msil) and Neurogenin 2 (Ngn2); Musashil (Msil)) and polypeptide which shares at least 75%, 80% 85%, 90%. 95%, methyl-CpG binding domain protein 2 (MBD2); or Neuro 97%, 99% or more of the functionality or sequence identity of genin 2 (Ngn2) and methyl-CpG binding domain protein 2 any one of the gene regulators provided in the Table Ahere (MBD2). Adequate intracellular levels of these polypeptides inbefore. are preferred since they tend to be expressed throughout an 0120. As used herein, “conditions supporting growth' or entire cell lineage, from as early as embryonic stem cells (or “conditions Supporting the transformation' when referring to even earlier) to pre-Somatic cells (or even later). a desired cell refers to various suitable, culture conditions (0123 MBD2 is a member of a family of methyl-CpG (temperature, pH, O, tension, osmolarity, cell media, factors, binding proteins that has been reported to be both a transcrip compounds, growth Substrate (ex. laminin, collagen, tional repressor and a DNA demethylase (dMTase). As used fibronectin, MatrigelTM, low-bind surface, nanostructured or herein, the term “MBD2 generally refers to the human charged surface, etc.), 3D environment, etc.) favoring growth methyl-CpG binding domain protein 2. The GeneBankTM of the desired cell type and/or favoring transformation (NCBI) accession number of human MBD2 is NM 003927. towards such desired cell type. Those skilled in the art know 3/AF072242, the UniProtTM accession number is that growth or transformation of particular cell types is stimu NP-003918/Q9UBB5 and the UniGeneTM accession number lated under specific conditions, while inhibited by others, and is HS.25674. US 2014/0038291 A1 Feb. 6, 2014
0.124. As used herein, the term "Msil” generally refers to tion of DNA, while closing up of the chromatin structure the human musashi homolog 1. The GeneBankTM (NCBI) refers more, specifically to deacetylation of histones, and accession number of human Msil is NM 002442.2/ methylation of DNA. AB012851, the UniProtTM accession number is NP-002433/ 0140. As used herein, “compound” refers to a compound 043347 and the UniGeneTM accession number is HS. 158311. capable of effecting a desired biological function. The term 0.125. As used herein, the term “Ngn2generally refers to includes, but is not limited to, DNA, RNA, protein, polypep the human neurogenin 2. The GeneBankTM (NCBI) accession tides, and other compounds including growth factors, cytok number of human Ngn2 is NM 024019.2/BC036847, the ines, hormones or Small molecules. As used herein, com UniProtTM accession number is NP-076924/Q9H2A3 and the pounds capable of remodeling chromatin and/or DNA UniGeneTM accession number is HS.567563. include, but are not limited to, histone acetylators, inhibitors 0126. According to additional aspects, the method of of histone deacetylation, DNA demethylators, inhibitors of transforming a cell of a first type to a desired cellofa different DNA methylation and combination thereof. type comprises the steps of either: 0.141. “Inhibitor of DNA methylation” refers to an agent I0127 1) contacting the cell of a first type with one or that can inhibit DNA methylation. DNA methylation inhibi more compounds capable of increasing intracellular lev tors have demonstrated the ability to restore Suppressed gene els of at least one reprogramming agent within the cell expression. Suitable agents for inhibiting DNA methylation and directly or indirectly remodeling the chromatin and/ include, but are not limited to 5-azacytidine, 5-aza-2-deoxy or DNA of the cell; or cytidine, 1-3-D-arabinofuranosil-5-azacytosine, and dihy I0128 2) contacting the chromatin and/or DNA of a cell dro-5-azacytidine, and Zebularine (ZEB), BIX (histone lysine of a first type with an agent capable of remodeling the methyltransferase inhibitor), and RG108. chromatin and/or DNA of the cell; and increasing intra 0.142 “Inhibitor of histone deacetylation” refers to an cellular levels of at least one reprogramming agent. agent that prevents the removal of the acetyl groups from the 0129. According to various embodiments, step 2) may be lysine residues of histones that would otherwise lead to the carried out consecutively to step 1), simultaneously with step formation of a condensed and transcriptionally silenced chro 1), or before step 1). matin. Histone deacetylase inhibitors fall into several groups, 0130. According to a particular aspect, the invention including: (1) hydroxamic acids such as trichostatin (A), (2) relates to a method for obtaining a particular stem-like cell, cyclic tetrapeptides, (3) benzamides, (4) electrophilic comprising: ketones, and (5) aliphatic acid group of compounds Such as I0131 providing a cell of a first type: phenylbutyrate and Valporic acid. Suitable agents to inhibit I0132) contacting chromatin and/or DNA of a cell of a histone deacetylation include, but are not limited to, Valporic first type with a histone acetylator, an inhibitor of histone acid (VPA), phenylbutyrate Trichostatin A (TSA), Na-bu deacetylation, a DNA demethylator, and/or a chemical tyrate, and benzamides. VPA promotes neuronal fate and inhibitor of DNA methylation; and inhibits glial fate simultaneously through the induction of 0.133 increasing intracellular levels of at least one gene neurogenic transcription factors including NeuroD. regulator for that particular stem-like cells, wherein the 0.143 “Histone Acetylator refers to an agent that inserts gene regulator is capable of driving directly or indirectly acetyl groups to the lysine residues of histones that opens up transformation of the cell of the first type into the par the chromatin and turns it into a transcriptionally active state. ticular stem-like cell. Suitable Histone Acetylator agents include, but are not lim 0134. According to a particular aspect, the invention ited to, Polyamine, CREB (cAMP element binding protein), relates to a method for obtaining a Neural Stem-Like Cell and BniP3. (NSLC), comprising: 0144) “DNA demethylator refers to an agent that 0.135 providing a cell of a first type which is not a removes the methyl groups from DNA and possesses the NSLC; ability to inhibit hypermethylation and restore suppressed 0.136 increasing intracellular levels of at least one neu gene expression. A demethylase is expected to activate genes ral stem cell specific polypeptide, by removing the repressive methyl residues. Suitable DNA 0.137 wherein the polypeptide is capable of driving demethylators include, but are not limited to, MBD2 and directly or indirectly transformation of the cell of the Gadd45b. first type into a NSLC; and 0145. In some embodiments, the reprogramming agent 0.138 contacting chromatin and/or DNA of a cell of a has one or more of the following functions: it decrease the first type with a histone acetylator, an inhibitor of histone expression of one or more markers of cells of the first type (ex. deacetylation, a DNA demethylator, and/or a chemical see Table C), and/or increase the expression of one or more inhibitor of DNA methylation. markers of the desired cell of the different type (ex. see Table 0.139. With respect to the second step, the term “remodel A). Cells that exhibit a selectable marker for the desired cell ling the chromatin and/or DNA refers to dynamic structural of a different type are then selected and assessed for charac changes to the chromatin. These changes can range from local teristics of the desired cell of a different type. changes necessary for transcriptional regulation, to global 0146 According to the invention, transformation into the changes necessary for opening up the chromatin structure or desired cell results in stable expression of a plurality of sec chromosome segregation to allow transcription of the new set ondary genes whose expression is characteristic of pheno of genes characteristic of the desired cellofa different type, to typical and/or functional properties of the desired cell. Genes closing up of the chromatin structure or chromosome segre whose expression is characteristic of phenotypical and/or gation to prevent transcription of certain genes that are not functional properties of the desired cell include, but is not characteristic of the desired cell of a different type. In some limited to, those listed in Table A. embodiments, opening up of the chromatin structure refers 0.147. In some embodiments, expression of secondary more specifically to acetylation of histones, and demethyla genes whose expression is characteristic of phenotypical and US 2014/0038291 A1 Feb. 6, 2014
functional properties of the desired cell results in the expres- -continued sion of markers defined according to Table A or the following table: Desired cell type Markers Mesendoderm-like cells Sox17, FoxA2, CXCR4, GATA4, MixI1, Eomesodermin Pluripotent-like cells Octa, SSEA4, TRA-1-60, TRA-1-81, AP Desired cell type Markers 0.148. In some embodiments, transformation of a cell of a Neural stem-like cells Nestin, Sox2, GFAP, Msi1 first type into the desired cell results in a stable repression of Neural-like cells BIII-tubulin, Map2b, Synapsin, ACHE a plurality of genes typically expressed in the cell of the first Ectoderm-like cells Sox2, Sox1, Zic1, Nestin, Notch 1, Fox.J3, type but not in the desired cell of the different type. Examples Otx2, Criptol, Vimentin of such Suppressed genes include, but are not limited to, those defined in Table C: TABLE C Examples of Suppressed genes Cell-type specific genes typically repressed during Reprogramming RefSeq, UniProt TMF GenBankTM Swiss-Prot UniGene TM (NCBI) Accession Accession Accession Cell Type Name No No No Markers Keratinocytes TP63, NM 003722.4 Q9H3D4 Hs.137569 Keratin 14 isoform 1 TP63, NM 001114978.1 Q9H3D4 Hs.137569 Basonuclin isoform 2 TP63, NM 001114979.1 Q9H3D4 Hs.137569 P63 isoform 3 TP63, NM 001114980.1 Q9H3D4 Hs.137569 isoform 4 TP63, NM 001114981.1 Q9H3D4 Hs.137569 isoform 5 TP63, NM 001114982.1 Q9H3D4 Hs.137569 isoform 6 BNC1 NM OO1717.3 Q01954 HS.459153 BCN2 NM O17637.5 Q6ZN30 HS.656581 KRT14 NM 000526.4 PO2533 HS.65438O nvolucrin NM 005547.2 PO7476 HS.516439 Fibroblasts THY1 NM OO6288.3 PO4216 HS.724411 CD90 FBN2 NM OO1999.3 P35556 Hs.519294 Fibrillin 2 FN1 NM 212482.1 PO2751 Hs.203717 Fibronectin VIM NM OO3380.3 P08670 HS.455.493 Vimentin COLSA2 NM OOO393.3 PO5997 Hs.445827 Collagen 5a2 DNMT1, NM 001130823.1 P26358 HS2O2672 FSP1 isoform a DNMT1, NM 001379.2 P26358 HS2O2672 isoform b Mesenchymal FN1 NM 212482.1 PO2751 HS2O3717 Fibronectin stem cells MCAM NM OO6500.2 P43121 HS.599.039 STRO-1 THY1 NM OO6288.3 PO4216 HS.724411 CD90 VIM NM OO3380.3 P08670 HS.455.493 Vimentin CD34+ sl1 NM OO2202.2 P61371 HSSOS VEGFR HOXA9 NM 152739.3 P31269 Hs.659350 Cytokeratin HOXB4 NM OO24015.4 P17483 HS.6647O6 CD34 KIk-1 NM OO2257.2 PO6870 HS.123107 Bry NM OO3181.2 O15178 HS.389457 Adipose- ALCAM NM OO1627.2 Q13740 HS.S91293 ALBO derived stem VCAM-1 NM 001078.2 P1932O Hs.10922S Adiponectin cells (ADSC) VCAM-1, NM 080682.1 P1932O Hs.109225 Leptin isoform b PROM1, NM OO6017.2 O43490 HS.614734 isoform 1 PROM1, NM 001145847.1 O43490 HS.614734 isoform 2 NM 001145848.1 PROM1, NM 001145852.1 O43490 HS.614734 isoform 4 PROM1, NM 001145851.1 O43490 HS.614734 isoform 5 PROM1, NM 001145850.1 O43490 HS.614734 isoform 6 US 2014/0038291 A1 Feb. 6, 2014
TABLE C-continued Examples of suppressed genes Cell-type specific genes typically repressed during Reprogramming RefSeq, UniProt TMF GenBank TM Swiss-Prot UniGene TM (NCBI) Accession Accession Accession Cell Type Name No No No Markers PROM1, NM 001145849.1 O43490 HS.614734 isoform 7 FUT4 NM 002033.3 P22083 HS.39042O
0149. In preferred embodiments, stable repression of any but are not limited to, the cytochalasin family of actin cytosk one or more of the genes listed in Table C being expressed in eleton inhibitors, such as Cytochalasin B or D. microtubule the first cell type is also characterized by a disappearance of inhibitors such as colchicine, and myosin inhibitors such as the corresponding markers (see Table C). 2,3-butanedione monoxime; physical effects include freez 0150. Those skilled in the art will understand that there ing, low osmolarity of the medium resulting in osmotic build exist many alternative steps for facilitating cell reprogram up within the cell and bursting and disruption (or removal) of ming. Additional steps for facilitating cell reprogramming cell membrane and cell cytoskeleton, and other forms of include destabilizing the cell's cytoskeletal structure (for removal of the cells physical memory. Such pretreatment example, by exposing the cell to cytochalasin B), loosening may boost reprogramming. In a preferred embodiment, the the chromatin structure of the cell (for example, by using cell is cultured in the presence of at least one cytoskeleton agents such as 5azacytidine (5-Aza) and Valproic acid (VPA) inhibitor one day before, during, or after introducing a neu or DNA demethylator agents such as MBD2), transfecting the rogenic transcription factor(s). cell with one or more expression vector(s) containing at least 0152 Placing the cell in conditions for supporting the one cDNA encoding a neurogenic transcription factor (for transformation of the desired cell, and/or maintaining the cell example, Msil or Ngn2), using an appropriate medium for the in culture conditions supporting the transformation of the desired cell of a different type and an appropriate differentia desired cell may comprise culturing the cell in a media com tion medium to induce differentiation commitment of the prising one or more factors appropriate for inducing the desired cell of a different type, inhibiting repressive pathways expression of the morphological and functional characteris that negatively affects induction into commitment into the tics of the desired cell of a different type. In some embodi desired cell of a different type, growing the cells on an appro ments the one or more factors are reprogramming factors priate substrate for the desired cell of a different type (for helpful in reprogramming a cell and these reprogramming example, laminin for NSLCs or a low-bind surface for cul factors can be used alone or in combinations. turing floating neurospheres), and growing the cells in an 0153. In other embodiments, the step of culturing the cell environment that the desired cell of a different type (or “-like' in a media comprising one or more factors appropriate for cell) would be normally exposed to in viva such as the proper inducing the expression of the morphological and functional temperature, pH and low oxygen environment (for example characteristics of the desired cell of a different type is carried about 2-5% O2). In various embodiments, the invention out Subsequently or simultaneously to steps iii) or iv), or encompasses these and other related methods and techniques Subsequently or simultaneously to steps 1) or 2), as defined for facilitating cell reprogramming. hereinbefore. 0151. Accordingly, the method of transforming a cell of a 0154 Those skilled in the art know many different types of first type into a cell of a second different type may comprise media and many reprogramming factors that may be helpful additional facultative steps. In one embodiment, the method in reprogramming a cell and these reprogramming factors can of transforming a cell further comprises the step of pretreat be used alone or in combinations. In various embodiments, ing the cell of a first type with a cytoskeleton disruptor. As the reprogramming factor is selected according to TABLE B. used herein “cytoskeleton” refers to the filamentous network 0.155. In some embodiments, reprogramming factors have of F-actin, Myosin light and heavy chain, microtubules, and one or more of the following functions: decrease the expres intermediate filaments (IFs) composed of one of three chemi sion of one or more markers of the first type of cell and/or cally distinct subunits, actin, tubulin, or one of several classes increase the expression of one or more markers of the desired of IF protein, as well as the cell's cytoskeletal structure cell. Cells that exhibit a selectable marker for the desired cell including the cell membrane and portions of the cell cyto are then selected and assessed for unipotency, multipotency, plasm and integrated organelles and components. This also pluripotency, or similar characteristics (as appropriate). refers to other structures and molecules that are directly or 0156. In particular embodiments, the cells are cultured in indirectly part of or affected by the cytoskeleton. Accord serum-free medium before, during or after any one of stepsi) ingly, the term “cytoskeleton disruptor” refers to any mol to iv) as defined hereinbefore, or during or after steps 1) or 2), ecule, compound or process that can inhibit, disrupt, remove as defined hereinbefore. or affect the cell cytoskeleton to destabilize the cell and con 0157. In some embodiments, Mesendoderm-like cells can sequently remove or change the feedback mechanisms be created from cells such as ADSCs by up-regulating the between the cells shape and cellular and nuclear function expression of FoxD3, MixI1, Ngn3 and MBD2. Additionally (thus removing or changing the cell's memory). Suitable up-regulating the expression of Oct4, Sox17, Brachyury, and/ cytoskeleton disruptors according to the invention include, or FoxA2 can be used to create Mesendoderm-like cells. In US 2014/0038291 A1 Feb. 6, 2014
addition, media compositions detailed in Table 37 can be used a neural stem-like cell for CNS applications; a cardiac stem to create mesendoderm-like cells from adipocyte-derived like cell for cardiac applications, a pancreatic progenitor-like stem cells (ADSCs) and similar cells, along with the use of cells or Bislet-like cells for diabetes, an ectoderm-like cell, a proper cell substrates formesendoderm cells known in the art mesoderm-like cells, an endoderm-like cell, etc.). In addition (e.g., gelatin-coated plates). derived cells of the invention can be a relatively homogeneous 0158. In some embodiments, cells such as ADSCs can be population of autologous cells of a particular phenotype of reprogrammed into pancreatic progenitor-like cells and B interest. islet-like cells by up-regulating the expression of Sox 17, 0162 The methods of the present invention allow the abil Pdx1, Ngn3, and Oct4 (e.g., Oct4+Sox17+Pdx1 +Ngn3; ity to create the native types of stem cells for particular Sox17+Pdx1 +Ngn3; Oct4+Pdx1 +Ngn3). Additionally applications (e.g., neural stem-like cells for CNS applica up-regulating the expression of FoxA2 and MBD2 can be tions; cardiac stem-like cells for cardiac applications, pancre used to create these cells. In addition, media compositions atic progenitor-like cells or 3-like cells for diabetes, etc.) as detailed in Table 39 can be used to create pancreatic progeni well as creating autologous (from the patients own cells) tor-like cells and B islet-like cells from adipocyte-derived versions of these stem cells that allow them to graft more stem cells (ADSCs) and similar cells, along with the use of appropriately when delivered to the patient as a treatment or proper cell Substrates for growing pancreatic progenitor cells for augmenting the health or functionality of a particular and Bislet-like cells known in the art (e.g., fibronectin-coated tissue or organ, or for diagnostic purposes. The cells can also collagen gels). be used for modeling (e.g., disease modeling, or testing the 0159. In some embodiments, cells such as ADSCs or mes effects of particular compounds or other molecules (e.g., for enchymal stem cells (MSCs) can be reprogrammed into car personalized medicine purposes)). The cells can also easily diac stem-like cells and mesoderm-like cells by up-regulating be enhanced by specific genes of interest or a defective gene the expression of a combination of Mesp1, FoxD3, Tbx5, repaired/replaced before delivery to the patient for treatment Brachyury (T), Nkx2.5, Sox 17 and/or Gata4 (e.g., Foxd3+ of a genetic disorder or for enhanced therapeutic value or Sox17+Mesp1+Tbx5; Foxd3+Sox17+Mesp1+NkX2.5; augmenting the health or functionality of a particular cell, Foxd3+T+Mesp1+Gata4, T+Mesp1+NkX2.5, T+Mesp1+ tissue, organ, System, or organism. Tbx5), which increase the expression of mesoderm and car diac stem cell markers. Additionally up-regulating the Obtaining Neural Stem-Like Cells (NSLCs) expression of Gatao and Bafö0c can be used to create these 0163 According to preferred embodiments for creating cells. In addition, media compositions detailed in Table 44 Neural Stem-Like Cells (NSLCs), the methods of the inven can be used to create cardiac stem-like cells from adipocyte tion are carried out such that cells are treated with selected derived stem cells (ADSCs) or mesenchymal stem cells agents, compounds and factors to promote the reprogram (MSCs) and similar cells, along with the use of proper cell ming and/or dedifferentiation towards Stem-Like Cells Substrates for growing cardiac stem cells known in the art (SLCs). Such reprogrammed somatic cells can then be further (e.g., matrigel, gelatin, or laminin). In some embodiments, treated with agents and/or cultured under conditions Suitable cells such as ADSCs can be reprogrammed into pluripotent for promoting reprogramming towards Neural Stem-Like tike stem cells up-regulating the expression of Rex 1, Oct4 and Cells (NSLCs), and expansion of the NSLCs for the long Klf4, or SalI4, Oct4, Klf4 and Nanog. Reprogramming by term. NSLCs according to the invention have the potential to transient transfection of a combination of the above genes can differentiate to neuronal-like and/or glial-like cells, as well as be achieved efficiently. These cells can be differentiated into neuronal and/or glial cells, for potential treatment of neuro ectoderm-like cells, endoderm-like cells, or mesendoderm logical diseases and injuries such as Parkinson's disease and like cells by methods known in the art for differentiating spinal cord injury. The methods described herein are also pluripotent stem cells into these lineages. In addition, these useful for producing histocompatible cells for cell therapy. pluripotent-like stem cells have protective and/or therapeutic/ 0164. Accordingly, some aspects of the present invention regenerative effect on other cells (e.g., hepatocytes). relates to generating neurons from an individual patient, thus 0160 The NSLCs that is a subject of this invention have making autologous transplantations possible as a treatment benefits over native human neural stem/progenitor cells as modality for many neurological conditions including neu well as embryonic stem cells. The NSLCs do not readily form rotrauma, stroke, neurodegenerative diseases such as Mul tumors or teratomas (as tested in NOD-SCID mice), can be tiple Sclerosis, Parkinson's disease, Huntington disease, created from easily obtainable Somatic cells (e.g., fibroblasts, Alzheimer's diseases. Thus, the invention provides for neu CD34+ blood cells, keratinocytes) and differentiated towards rological therapies to treat the disease or trauma of interest. any specific neuronal lineage, express neurotrophic growth 0.165. Therefore, another aspect of the invention concerns factors (e.g., BDNF and GDNF), express some neuronal dif a method of obtaining a Neural Stem-Like Cell (NSLC), ferentiation genes while maintaining a stem cell like state comprising either: allowing a higher proportion of neuronal differentiation when 0166 1) contacting the cell of a first type with one or placed in differentiation conditions (compared to native more neural stem cell regulating polypeptide capable of human neural stem/progenitor cells), form functional gap increasing intracellular levels of neural stem cell specific junctions and readily form synapses, and are capable of polypeptides within said cell and directly or indirectly attaching and Surviving on 3D scaffolds and environments. remodeling the chromatin and/or DNA of the cell and 0161 The stem-like cells of the present invention have driving directly or indirectly transformation of the cell of numerous advantages over the prior art Such as efficient the first type into a NSLC; or reprogramming without gene integration or constant artificial 0.167 2) contacting the chromatin and/or DNA of a cell forced gene expression, greater potency and safety over of a first type with a histone acetylator, an inhibitor of native cells (esp. stem/progenitor cells), and having the capa histone deacetylation, a DNA demethylator, and/or an bility to be the cell of interest for aparticular application (e.g., inhibitor of DNA methylation; and increasing intracel US 2014/0038291 A1 Feb. 6, 2014 20
lular levels of at least one neural stem cell specific neered DNA sequence into human Somatic cells via transient polypeptide driving directly or indirectly transformation transfection. Since the DNA introduced in the transfection of the cell of the first type into a NSLC. process is not inserted, into the nuclear genome, the foreign 0.168. In preferred embodiments, the step 1) comprises DNA decreases over time and when the cells undergo mitosis. increasing intracellular levels of a MUSASHI1 polypeptide. Nonviral vectors remain in a non-replicative form, have low As it will be explained hereinafter this can be achieved by immunogenicity, and are easy and safe to prepare and to use. different means including, but not limited to, transient expres Furthermore, plasmids may accommodate large fragments of sion of the MUSASHI1 polypeptide, preferably by transfect DNA ing an expression vector encoding the polypeptide. 0.175. In one particular embodiment, the method starts 0169. In preferred embodiments, the step 2) comprises with obtaining cells from the individual, and reprogramming increasing intracellular levels of a MBD2 polypeptide or the cells in vitro to generate NSLCs. The significant aspect of treating the cells with VPA and 5-AZA. As it will be explained the present invention is the stable reprogramming of a hereinafter this can be achieved by different means including, somatic cell or non-neuronal cell into a NSLC that can give but not limited to, transient expression of the MBD2 polypep rise to different types of neuronal or glial cells (including tide, preferably by transfecting an expression vector encoding neuronal-like or glial-like cells), as well as neural crest the polypeptide(s), and/or pre-treating and/or treating the derived cells such as cardiomyocytes. These can then be cells with VPA and 5-AZA. implanted back into the same patient from which the cells 0170 In one particular embodiment, reprogramming a were obtained, thus making an autologous treatment modal cell of a first type to another type of cell that exhibits at least ity for many neurological conditions including neurotrauma, two selectable markers for neural stem cells requires trans stroke, and neurodegenerative disease possible, as well as fecting the cell of a first type with one vector containing a cardiac conditions such as myocardial infarction. These can cDNA encoding for a neurogenic transcription factor and one also be implanted into a different individual from which the DNA demethylator. To enhance the de-differentiation the cells were obtained. Accordingly, the cells and methods of the cells are exposed or pre-exposed to an agent(s) that inhibits present invention may be helpful to treat, prevent, or to sta DNA methylation, inhibits histone deacetylation, and/or dis bilize a neurological disease Such as Alzheimer's disease, rupts the cell cytoskeleton. For example, the dedifferentiation Parkinson's disease, multiple Sclerosis, or spinal cord injury, can be enhanced by pre treating the cells with an agent that or a cardiac disease such as myocardial infarction. This tech disrupts the cell cytoskeleton followed by transfecting the nology provides an ample source of neural stem cells, neuro cells with one or more vector(s) containing two neurogenic progenitor cells, neurons and glia, as well as neural crest transcription factors in the presence of a DNA demethylator derived cells, for clinical, treatment, which can be performed and/or inhibitor of DNA methylation and histone deacetyla by implantation of NSLCs in vivo or inducing the differen tion. The histone deacetylator, inhibitor of histone deacetyla tiation in vitro and implantation of neuro-progenitor cells or tion, DNA demethylator, and/or an inhibitor of DNA methy specific neurons or glia in vivo. lation are as defined previously. 0176). In another embodiment, the method comprises iso 0171 As defined previously, the method may further com lating somatic or non-neuronal cells and exposing the cells to prise a preliminary step of pre-treating the cell of a first type one or more agents that alter cell morphology and chromatin with a cytoskeleton disruptor, as defined previously, and/or structure, and transfecting the cells with one or more genes culturing the cell in a media comprising one or more repro containing at least one cDNA encoding for a neurogenic gramming factors appropriate for appearance and mainte transcription factor. The gene transfection step may be nance of the morphological and functional characteristics of replaced with alternative agents that induce the expression of NSLCs as defined previously (e.g. a retinoid compound, a the neurogenic transcription factor(s) in the cell. Inducing neurotrophic factor, bFGF, EGF, SHH, Wnt3a, neuropeptide epigenetic modifications to DNA and histones (especially Y. Estrogen). In some embodiment the method further com DNA demethylation and an open chromatic structure) facili prises inhibiting cellular BMP signaling pathways (e.g. by tate true reprogramming of the cells. In another embodiment, NOGGIN, fetuin, or follistatin). the cells are incubated in a low oxygen environment, for 0172. In preferred embodiments, generation of a NSLC example 5% O2, thereby helping in reprogramming the cells. from a first cell comprises the use of one or more reprogram 0177. This methodology allows the reprogramming of a ming agents. Suitable agents include, but are not limited to, cell into a NSLC. The further course of development and the Musashi-1 (Msil) and Neurogenin 2 (Ngn2). Other potential expansion of the reprogrammed cell depend on the in situ agents are listed in Table A and B. environment cues to which it is exposed. The embodiments of 0173 The present invention is also directed to the use of the invention further include growing the reprogrammed cell DNA expression vectors encoding a protein or transcript in an appropriate proliferation medium to expand the gener which upregulates the expression of neurogenesis. The ated NSLC, for example Neural Progenitor proliferation genetically-engineered DNA sequence, encoding a defined Medium (StemCell Technologies) with the presence of epi reprogramming agent such as Msil and Ngn2, can be intro dermal growth factor (EGF) and basic fibroblast growth fac duced into cells by using a mono-, bi-, or poly-cistronic tor (bFGF), to promote the neural stem cell to proliferate. vectors. The expression of an endogenous multipotency gene 0.178 The NSLCs obtained according to the invention can indicates that the cDNA encodes a protein whose expression be differentiated into neuronal, astrocyte, and/or oligoden in the cell result directly or indirectly in the de-differentiation drocyte lineages in appropriate differentiation medium, for of the cell. The newly de-differentiated mammalian cells are example NS-A differentiation medium (StemCell, Technolo capable of re-differentiating to neuronal lineages to regener gies) or NbActive medium (BrainBitsTM) including a retinoid ate said mammalian cells, tissues, and organs. compound, such as all-trans-retinoic acid or vitamin A, and 0.174. The present invention is further directed to a method BDNF, to induce the differentiation of NSLCs towards neu for generating NSLCs by introducing a genetically-engi ronal and/or glial cells. Neuronal cells include cells that dis US 2014/0038291 A1 Feb. 6, 2014
play one or more neural-specific morphological, physiologi compound is to be used in Vivo, either a constitutive or an cal, functional and/or immunological features associated inducible promoter or compound is used, depending on the with a neuronal cell type. Useful criteria features includes: particular use of the protein. In addition, a weak promoter can morphological features (e.g., long processes or neurites), be used, such as HSVTK or a promoter having similar activ physiological and/or immunological features such as expres ity. The promoter typically can also include elements that are sion of a set of neuronal-specific markers orantigens, synthe responsive to transactivation, e.g., hypoxia response ele sis of neurotransmitter(s) such as dopamine or gamma ami ments, Ga14 response elements, lac repressor response ele nobutyric acid (GABA), and functional features such as ion ment, and Small molecule control systems such as tet-regu channels or action potentials characteristic of neurons. lated systems and the RU-486 system. 0179. In accordance with the method, reprogrammed cells 0183 In addition to a promoter, an expression vector typi can be selected based on differential adherence properties as cally contains a transcription unit or expression cassette that compared to untransfected cells; for example, reprogrammed contains additional elements required for the expression of cells can form floating neurospheres or grow well on laminin the nucleic acid in host cells, either prokaryotic or eukaryotic. while untransfected fibroblasts attach and grow well on regu A typical expression cassette thus contains a promoter oper lar cell culture treated plates. Reprogrammed cells include ably linked, e.g., to the nucleic acid sequence, and signals cells that exhibit one or more neural stem specific markers required, e.g., for efficient polyadenylation of the transcript, and morphology and the loss of Some or all of the specific transcriptional termination, ribosome binding, and/or trans markers related to the original cells. Furthermore, some of the lation termination. Additional elements of the cassette may functionality of the neural-like cells (NLCs) can be assessed include, e.g., enhancers, and heterologous spliced intronic at different time points by, for example, patch-clamping, signals. immunostaining for synaptophysin and MAP2b, and by 0.184 Expression vectors containing regulatory elements immunochemical means such as by enzyme-linked immun from eukaryotic viruses are often used in eukaryotic expres osorbent assay (ELISA). sion vectors, e.g., SV40 vectors, papilloma virus vectors, and 0180. In certain embodiments, the present invention pro vectors derived from Epstein-Barr virus. Other exemplary vides NSLCs that are able to initiate and direct central ner eukaryotic vectors include pMSG, paV009/A+, pMTO10/ Vous system regenerationata site of tissue damage and can be A+, pMAMneo-5, baculovirus plSVE, and any other vector customized for individual patients using their own cells as the allowing expression of proteins under the direction of the donor or starting cell. The present invention can be used to SV40 early promoter, SV40 late promoter, metallothionein generate cells from an individual patient, thus making autolo promoter, murine mammary tumor virus promoter, Roussar gous transplantations possible as a treatment modality for coma virus promoter, polyhedrin promoter, or other promot many neurological conditions. Thus, this technology elimi ers shown effective for expression in eukaryotic cells. nates the problems associated with transplantations of non 0185. Standard transfection methods can be used to pro host cells, such as, immunological rejection and the risk of duce bacterial, mammalian, yeast, insect, or other cell lines transmitted disease. The great advantage of the present inven that express large quantities of dedifferentiation proteins, tion is that it provides an essentially limitless Supply for which can be purified, if desired, using standard techniques. autologous grafts suitable for transplantation. Therefore, it Transformation of eukaryotic and prokaryotic cells is per will obviate Some significant problems associated with cur formed according to standard techniques. rent Source of materials and methods of transplantation. 0186. Any procedure for introducing foreign nucleotide sequences into host cells can be used. These include, but are Delivery of Polynucleotides not limited to, the use of calcium phosphate transfection, 0181. In certain embodiments, the invention concerns the DEAE-dextran-mediated transfection, polybrene, protoplast use of polynucleotides, e.g. a polynucleotide encoding a fusion, electroporation, lipid-mediated delivery (e.g., lipo MBD2 polypeptide, a MUSASHI1 polypeptide and/or a Somes), microinjection, particle bombardment, introduction Ngn2 polypeptide. Means for introducing polynucleotides of naked DNA, plasmid vectors, viral vectors (both episomal into a cell are well known in the art. Transfection methods of and integrative) and any of the other well known methods for a cell Such as nucleofection and/or lipofection, or other types introducing cloned genomic DNA, cDNA, synthetic DNA or of transfection methods may be used. For instance a poly other foreign genetic material into a host cell (see, e.g., Sam nucleotide encoding a desired polypeptide can be cloned into brook et al. Supra). It is only necessary that the particular intermediate vectors for transfection in eukaryotic cells for genetic engineering procedure used be capable of Success replication and/or expression. Intermediate vectors for Stor fully introducing at least one gene into the host cell capable of age or manipulation of the nucleic acid or production of expressing the protein of choice. protein can be prokaryotic vectors, (e.g., plasmids), shuttle 0187 Conventional viral and non-viral based gene trans vectors, insect vectors, or viral vectors for example. A desired fermethods can be used to introduce nucleic acids into mam polypeptide can also be encoded by a fusion nucleic acid. malian cells or target tissues. Such methods can be used to 0182 To obtain expression of a cloned nucleic acid, it is administer nucleic acids encoding reprogramming polypep typically Subcloned into an expression vector that contains a tides to cells in vitro. Preferably, nucleic acids are adminis promoter to direct transcription. Suitable bacterial and tered for in vivo or ex vivo gene therapy uses, Non-viral eukaryotic promoters are well known in the art and described, vector delivery systems include DNA plasmids, naked e.g., in Sambrook and Russell (Molecular Cloning: a labora nucleic acid, and nucleic acid complexed with a delivery tory manual, Cold Spring Harbor Laboratory Press). The vehicle such as a liposome. Viral vector delivery systems promoter used to direct expression of a nucleic acid of choice include DNA and RNA viruses, which have either episomal depends on the particular application. For example, a strong or integrated genomes after delivery to the cell. constitutive promoter is typically used for expression and 0188 Methods of non-viral delivery of nucleic acids purification. In contrast, when a dedifferentiation protein or include lipofection, microinjection, ballistics, Virosomes, US 2014/0038291 A1 Feb. 6, 2014 22 liposomes, immunoliposomes, polycation or lipid-nucleic a particular composition, a particular route can often provide acid conjugates, naked DNA, artificial virions, and agent a more immediate and more effective reaction than another enhanced uptake of DNA. Lipofection reagents are sold com rOute. mercially (e.g., TransfectamTM and LipofectinTM). Cationic 0194 Pharmaceutically acceptable carriers are deter and neutral lipids suitable for efficient receptor-recognition mined in part by the particular composition being adminis lipofection of polynucleotides are known. Nucleic acid can be tered, as well as by the particular method used to administer delivered to cells (ex vivo administration) or to target tissues the composition. Accordingly, there is a wide variety of Suit (in vivo administration). The preparation of lipid:nucleic acid able formulations of pharmaceutical compositions of the complexes, including targeted liposomes Such as immuno present invention. lipid complexes, is well known to those of skill in the art. (0189 The use of RNA or DNA virus-based systems for the Delivery of Polypeptides delivery of nucleic acids take advantage of highly evolved 0.195. In most, if not all the methods described herein, an processes for targeting a virus to specific cells in the body and alternative possibility consists of bypassing the use of a poly trafficking the viral payload to the nucleus. Viral vectors can nucleotide and contacting a cell of a first type cell directly be administered directly to patients (in vivo) or they can be with a compound (e.g. a polypeptide) for which an increased used to treat cells in vitro, wherein the modified cells are intracellular level is desired. In other embodiments, for administered to patients (ex vivo). Conventional viral based example in certain in vitro situations, the cells are cultured in systems for the delivery include retroviral, lentiviral, pox a medium containing one or more functional polypeptides. yiral, adenoviral, adeno-associated viral, Vesicular stomatitis 0196. An important factor in the administration of viral and herpesviral vectors, althoughntegration in the host polypeptides is ensuring that the polypeptide has the ability to genome is possible with certain viral vectors, including the traverse the plasma membrane of a cell, or the membrane of retrovirus, lentivirus, and adeno-associated virus gene trans an intra-cellular compartment such as the nucleus. Cellular fer methods, often resulting in long term expression of the membranes are composed of lipid-protein bilayers that are inserted transgene. Additionally, high transduction efficien freely permeable to Small, nonionic lipophilic compounds cies have been observed in many different cell types and and are inherently impermeable to polar compounds, macro target tissues. molecules, and therapeutic or diagnostic agents. (0190 plASN and MFG-S are examples of retroviral vec 0.197 However, proteins, lipids and other compounds, tors that have been used in clinical trials. In applications for which have the ability to translocate polypeptides across a which transient expression is preferred, adenoviral-based cell membrane, have been described. For example, “mem systems are useful. Adenoviral based vectors are capable of brane translocation polypeptides have amphiphilic or hydro very high transduction efficiency in many cell types and are phobic amino acid Subsequences that have the ability to act as capable of infecting, and hence delivering nucleic acid to, membrane-translocating carriers. Polypeptides for which an both dividing and non-dividing cells. With such vectors, high increased intracellular level is desired according to the inven titers and levels of expression have been obtained. Adenovi tion can be linked to Suitable peptide sequences for facilitat rus vectors can be produced in large quantities in a relatively ing their uptake into cells. Other suitable chemical moieties simple system. that provide enhanced cellular uptake can also be linked, 0191 Gene therapy vectors can be delivered in vivo by either covalently or non-covalently, to the polypeptides. administration to an individual patient, typically by Systemic Other suitable carriers having the ability to transport polypep administration (e.g., intravenous, intraperitoneal, intramus tides across cell membranes may also be used. cular, Subdermal, or intracranial infusion) or topical applica 0198 Adesired polypeptide can also be introduced into an tion. Alternatively, vectors can be delivered to cells ex vivo, animal cell, preferably a mammalian cell, via liposomes and Such as cells explanted from an individual patient (e.g., lym liposome derivatives such as immunoliposomes. The term phocytes, bone marrow aspirates, tissue biopsy) or universal “liposome' refers to vesicles comprised of one or more con donor hematopoietic stem cells, followed by reimplantation centrically ordered lipid bilayers, which encapsulate an aque of the cells into a patient, usually after selection for cells ous phase. The aqueous phase typically contains the com which have been reprogrammed. pound to be delivered to the cell. 0.192 Ex vivo cell transfection for diagnostics, research, 0199. In certain embodiments, it may be desirable to target or for gene therapy (e.g., via re-infusion of the transfected a liposome using targeting moieties that are specific to a cells into the host organism) is well known to those of skill in particular cell type, tissue, and the like. Targeting of lipo the art. In a preferred embodiment, cells are isolated from the Somes using a variety of targeting moieties (e.g., ligands, Subject organism, transfected with a nucleic acid (gene or receptors, and monoclonal antibodies) has been previously cDNA), and re-infused back into the Subject organism (e.g., described. patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art. Cells and Cell Lines 0193 Vectors (e.g., retroviruses, adenoviruses, lipo 0200. The invention encompasses the cells, cell lines, stem Somes, etc.) containing therapeutic nucleic acids can be also cells and purified cell preparations derived from any of the administered directly to the organism for transfection of cells methods described herein. In some embodiments, the cells, in vivo. Alternatively, naked DNA can be administered. cells lines, stem cells and purified cells preparations of the Administration is by any of the routes normally used for invention are of mammalian origins, including but not limited introducing a molecule into ultimate contact with blood or to human, primates, rodent, dog, cat, horse, cow, or sheep. In tissue cells. Suitable methods of administering such nucleic preferred embodiments, they originate from a human. acids are available and well known to those of skill in the art, 0201 Accordingly, another aspect of the invention relates and, although more than one route can be used to administer to modified cells, cell lines, pluripotent, multipotent or uni US 2014/0038291 A1 Feb. 6, 2014 potent cells and purified cell preparations, wherein any of 0222. In preferred embodiments, a NSLC according to the these cells comprise an exogenous polynucleotide encoding inventions possesses all of the following characteristics: Musashil (Msil); Msil and Ngn2; Msil and MBD2; and 0223 ability to self-renew for significantly longer than Ngn2 and MBD2: Msi1, Ngn2 and MBD2: Msi1, Ngn2, a Somatic cell; Nestin and MBD2; and other potential combinations from 0224 is not a cancerous cell; Table A preferably including Msil and Ngn2 and MBD2. In 0225 is stable and not artificially maintained by forced preferred embodiments the cell according to the invention is gene expression or by similar means and may be main a stem-like cell, more preferably a Neural Stem-Like Cell tained in standard neural stem cell media; (NSLC), the cell possessing one or more of the following 0226 can differentiate to a progenitor, precursor, somatic cellor to another more differentiated cell type of characteristics: the same lineage; 0202 expression of one or more neural stem cell marker 0227 has the characteristics of a stem cell and not just Selected from the group consisting of Sox2, Nestin, certain markers or gene expression or morphological GFAP, Msi1, and Ngn2: appearance; and 0203 decreased expression of one or more genes spe 0228) does not exhibit uncontrolled growth, teratoma cific to the cell that the NSLC was obtained from (e.g. formation, and tumor formation in vivo. see Table C); 0229. In one particular embodiment, the reprogrammed 0204 forms neurospheres in the neurosphere colony cells (NSLCs) according to the invention are capable of pro formation assay; liferating for several months without losing their neural stem 0205 capable of being cultured in suspension or as an cell markers and their ability to differentiate towards neuron adherent culture; like, astrocyte-like, and oligodendrocyte-like cells. The gen 0206 capable of proliferating without the presence of eration of the neural lineages is characterized based on mor an exogenous reprogramming agent for over 1 month, phology, phenotypic changes and functionality. preferably over 2 months, over 3 months, over 5 months 0230. In preferred embodiments the cell according to the and even for more than a year, invention is a stem-like cell, the stem-like cell possessing one 0207 capable of dividing every 36 hours at low pas or more of the following characteristics: Sage, 0231 expression of one or more stem cell marker 0208 positive for telomerase activity; selected from Table A: 0209 capable of differentiation into a neuronal-like 0232 decreased expression of one or more genes spe cell, astrocyte-like cell, oligodendrocyte-like cell and cific to the cell that the stem-like cell was obtained from: combinations thereof; 0233 capable of being cultured in suspension (as spheres) or as an adherent culture; 0210 decreased expression of telomerase and one or 0234 capable of proliferating without the presence of more neural stem cell markers after differentiation; an exogenous reprogramming agent for over 1 month, 0211 having one or more morphological neurite-like preferably over 2 months, over 3 months, over 5 months processes (axons and/or dendrites) greater than one cell or for more than a year; diameter in length after differentiation into a neuronal 0235 positive for telomerase activity; like cell; 0236 capable of differentiation into cells according to 0212 expression of at least one neural-specific antigen the lineage of that stem-like cell; Selected from the group consisting of neural-specific 0237 decreased expression of telomerase and one or tubulin, microtubule associated protein 2, NCAM, and more stem cell markers after differentiation; marker for a neurotransmitter after differentiation into a 0238 having one or more morphological features of the neuronal-like cell; stem cells that the stem-like cell is like: 0213 expression of one or more functional neural 0239 expression of one or more antigen expressed spe markers (e.g. synapsin) after differentiation into a neu cifically in the stem cells that the one or more is like: ronal-like cell; 0240 expression of one or more functional markers of 0214 capable of releasing one or more neurotrophic lineage specific differentiated cells after differentiation factors (e.g. BDNF) after differentiation into a neuronal of the stem-like cell; like cell; 0241 negative in a tumor colony forming assay; 0215 negative in a tumor colony forming assay; 0242 negative for tumor growth in SCID mice; 0216 negative for tumor growth in SCID mice; 0243 negative for teratoma growth in SCID mice; 0244 capable of significantly improving one or more 0217 negative for teratoma growth in SCID mice; functional measures after placement of an adequate 0218 capable of significantly improving one or more number of stem-like cells in a model assessing the regen functional measures after placement of an adequate erative potential of those types of stem cells. number of NSLCs into the Void in a brain ablation 0245. In some embodiments, the cells of the invention may model; have one or more of the following characteristics and prop 0219 capable of significantly improving or maintain erties: self-renewal, multilineage differentiation in vitro and ing one or more functional measures after injecting an in vivo, clonogenicity, a normal karyotype, extensive prolif adequate number of NSLCs into an EAE model; and eration in vitro under well defined culture conditions, and the 0220 capable of improving one or more functional ability to be frozen and thawed, as well as any of the com measures more significantly than hNPCs in CNS injury monly known and/or desired properties or characteristics or neurodegenerative models. typical of stem cells. The cells of the invention may further 0221 Examples of all of the above items can be found in express molecular markers of multipotent or pluripotent cells the Examples section of this application. (i.e. gene and Surface markers as defined previously). US 2014/0038291 A1 Feb. 6, 2014 24
0246. Another aspect of the invention relates to the pro appropriate number of these hematopoietic stem-like cells duction of tissue specific autologous (self) stem and/or pro into the patient (according to methods known in the art) to genitor cells. These stem and/or progenitor cells may be used allow for proper grafting of these cells into the patient’s bone in cell therapy applications to treat diseases of cellular degen marrow. Since the HIV cannot enter into the CCR5 (nega eration. Diseases of cellular degeneration include, for tive) hematopoietic stem-like cells and their progeny (differ example, neurodegenerative diseases such as stroke, Alzhe entiated lineage cells), these cells will over time allow the imer's disease, Parkinson's disease, multiple Sclerosis, patient’s blood system to function normally and potentially Amyotrophic lateral Sclerosis, macular degeneration, the HIV may even be eradicated. It should be noted that the osteolytic diseases such as osteoporosis, osteoarthritis, bone genetic engineering/modification can take place before, dur fractures, bone breaks, diabetes, liver injury, degenerative ing or after the creation of the stem-like cell of interest for all diseases, myocardial infarct, burns and cancer. These stem the above methods and processes in this paragraph. It should and/or progenitor cells may also be used in cell therapy appli also be noted that an allogeneic cell could also be used for all cations to treat injury Such as spinal cord injury, traumatic the above methods and processes in this paragraph. brain injury, burns, and muscle injury as well as other diseases 0248. Hematopoietic stem-like cells of the present inven or other aspects of a disease Such as cancer, hepatitis, HIV/ tion can also be used to repopulate the blood system in AIDS, leukemia and other blood disorders. It is envisioned patients after certain types of radiotherapy (ex. for cancer). that cells according to the invention may be implanted or Other appropriate stem-like cells (or cells differentiated from transplanted into a host. An advantage of the invention is that these stem-like cells) can be used to repopulate tissues, organs large numbers of autologous stem cells can be produced for or a system in a patient after certain types of therapy, Surgical implantation without the risk of immune system mediated intervention or injury has left the patient devoid of enough of rejection. Those cells can lead to production of tissue suitable certain types of cells that can be provided by the cells, meth for transplant into the individual. Since the tissue is derived ods and/or uses of the present invention. from the transplant recipient, it should not stimulate an immune response, as would tissue from an unrelated donor. 0249. Another aspect of the invention relates to a method Such transplants can constitute tissues (e.g. vein, artery, skin, to produce ex vivo engineered tissues for Subsequent implan muscle), Solid organ transplants (e.g., heart, liver, kidney), tation or transplantation into a host, wherein the cellular neuronal cell transplants, or bone marrow transplants such as components of those engineered tissues comprise cells are used in the treatment of various malignancies such as for according to the invention, or cells derived therefrom. For example, leukemias and lymphomas. Neural stem cell, neu example, expanded cultures of the cells of the invention may roprogenitor, or neuronal cell (as well as NSLCs and deriva be differentiated by in vitro treatment with growth factors tions thereof) transplants can also be used in the treatment of and/or morphogens. Populations of differentiated cells are for example, neurological disorders, stroke, spinal cord then implanted into the recipient host near the site of injury or injury, Parkinson's disease, and the like, as well as potentially damage, or cultured in vitro to generate engineered tissues, as Some non-neurological disorders such as a cardiac infarct. described. 0247 Another aspect of the invention relates to a method (0250. The methods and cells of the invention described of genetically engineering a cell created according to the hereincan be used to immortalize (or near-immortalize) cells, present invention, and using said cell for treating a disease or for example to generate a cell line. Using the methods dis enhancing and/or modulating and/or changing the function closed herein, a Somatic cell can be transformed into one ality or characteristics of a cell, tissue, organ or system in a possessing a dedifferentiated phenotype, thereby facilitating patient. An example includes genetically correcting the dys the generation of cell lines from a variety of tissues. There trophin or LAMA gene in a cell obtained from a patient fore, the invention encompasses such immortalized (or near Suffering from muscular dystrophy and creating muscle stem immortalize) cells. like cells according to the methods of the present invention 0251. In addition, the methods of deriving the cells that are then administered to the patient according to appro according to the invention, may be helpful in Scientific and priate methods in the art; these muscle stem-like cells can therapeutic applications including, but not limited to, (a) Sci then help to regenerate the muscles of the patient as well as entific discovery and research involving cellular development potentially gene correcting existing muscle cells through and genetic research (e.g. uses in lieu of human stem cells as fusion of the stem-like cells with the patient’s existing muscle a model cell line to study the differentiation, dedifferentia cells whereby the corrected gene in the stem-like cells tion, or reprogramming of human cells), (b) drug develop replaces the defective gene in the muscle cell. The same ment and discovery (e.g., Screening for efficacy and toxicity process above can be used for treating other genetic diseases of certain drug candidates and chemicals, Screening for pro whereby the appropriate type of stem-like cell is created spective drugs or agents which mediate the differentiation, according to methods of the present invention to regenerate dedifferentiation, or reprogramming of cells), (c) gene and/or correct cells, tissues and/or organs in the patient therapy (e.g., as a delivery device for gene therapy), and (d) affected by the disease. The same method can also be used for treatment of injuries, trauma, diseases and disorders includ enhancing and/or modulating and/or changing the function ing, but not limited to, Parkinson’s, Alzheimer's, Hunting ality or characteristics of a cell, tissue, organ or system in a tons, Tay-Sachs, Gauchers, spinal cord injury, stroke, burns patient. For example, a patient suffering from HIV/AIDS and other skin damage, heart disease, diabetes, Lupus, could be treated by obtaining an appropriate cell that has not osteoarthritis, liver diseases, hormone disorders, kidney dis been infected by HIV, knocking out the CCR5 gene of the cell ease, leukemia, lymphoma, infectious diseases, HIV/AIDS, so that the CCR5 receptor cannot be expressed (if, for multiple sclerosis, rheumatoid arthritis, Duchenne's Musclar example, the cell was a hematopoletic stem cell or any dif Dystrophy, Ontogenesis Imperfecto, birth defects, infertility, ferentiated cell of this lineage), reprogramming this cell into pregnancy loss, and other cancers, degenerative and other a hematopoletic stem-like cells, and then administering an diseases and disorders. US 2014/0038291 A1 Feb. 6, 2014
0252) Additional aspects concern therapeutic methods, Neural Stem-Like Cells according to the invention readily methods of treatment and methods of regenerating a tissue or differentiate into neuronal-like cells, astrocyte-like cells, and organ in a mammal (e.g. a human Subject). One particular oligodendrocyte-like cells within the CDM. It is thus possible method concerns a method of regenerating a mammalian to use CDM and reprogramming methods of the invention to tissue or organ which comprises contacting the tissue or organ reprogram the cells within the CDM to form 3D Neuronal to be regenerated with a SLC, NSLC, or other desired cell or Like multilayer tissue (up to >30 cell layers). Such 3D tissue artificial tissue construct as defined herein. The SLC, NSLC, comprises neurons (or specifically, neuron-like cells), astro desired cell or artificial tissue construct may be placed in cytes (or specifically, astrocyte-like cells), and oligodendro proximity to the tissue or organ to be regenerated by admin cytes (or specifically, oligodendrocyte-like cells) and it can be istering to the Subject using any suitable route (e.g. injecting made completely autologously, can be manually handled and the cell intrathecally, directly into the tissue or organ, or into implanted with relative ease, or can used as an in vitro CNS the blood stream). tissue model. 0253) Another method for repairing or regenerating a tis 0260 One particular aspect concerns an artificial tissue Sue or organ in a Subject in need thereof comprises adminis construct which comprises a 3D assembly of in vitro cultured tering to the Subject a compound inducing a director indirect cells and extracellular matrix produced by these cells. The endogenous expression of at least one gene regulator in cells cells may be desired cells, SLC and/or a plurality of Neural of the tissue or organ and/or a compound inducing a director Stem-Like Cell (NSLC) obtained using any one of the meth indirect endogenous expression of at least one gene regulator ods described herein. in cells capable of transformation or dedifferentiation in vivo in the Subject. Accordingly, the expression of the at least one gene regulator reprograms the cells into desired cells of a Screening Methods different type (e.g. neural stem-like cells), and these cells of a 0261) Another aspect of the invention relates to methods different type are effective in repairing or regenerating said for identifying new compounds (e.g. Small molecules, drugs, tissue or organ. etc) capable of transforming a cell of a first type to a desired 0254 Another method comprises obtaining cells or tissue cell of a different type, as well as identifying the molecular from a patient (e.g. hematopoietic stem cells, fibroblasts, or and cellular pathways for reprogramming. These new com keratinocytes), reprogramming a plurality of such cells or the pounds may be useful for research purposes or as medica tissue, and reintroducing the reprogrammed cells or tissue ments for use in repairing or regenerating cells, tissues or into the patient. A related aspect concerns pharmaceutical organs in a subject; while these pathways may be useful for compositions comprising a plurality of a desired cell, SLC research purposes to develop research tools, diagnostics, and and/or Neural Stem-Like Cell (NSLC) or reprogrammed tis medicaments for use in repairing or regenerating cells, tissues Sue as defined herein. or organs in a Subject and for other uses. Those skilled in the 0255. The therapeutic methods of the invention may be art will understand that it is conceivable to screen for com applicable to the regeneration or repair of various tissues and pounds that will induce transformation of a cell of a first type organs including, but not limited to, the brain, the spinal cord, to a stem-like cell by replacing the “induction” or “biological the heart, the eye, the retina, the cochlea, the skin, muscles, activity” provided by the transient increase of the reprogram intestines, pancreas (including beta cells), kidney, liver, ming agent or gene regulator in the cell by a candidate com lungs, bone, bone marrow, blood system, cartilage, cartilage pound to be tested (e.g. a library of Small molecules or com discs, hair follicles, teeth, blood vessels, glands (including pounds) and measuring activity or efficacy of the candidate endocrine and exocrine glands), ovaries, reproductive organs, compound in generating the stem-like cell; individual or a mammary and breast tissue. mixture of active compounds would be selected if they have 0256 A related aspect concerns pharmaceutical composi the same activity and/or if they can provide the same or tions comprising a plurality of desired cell, SLC and/or Neu similar effects as these polypeptides or compounds (e.g. cell ral Stem-Like Cell (NSLC) as defined herein. transformation and/or appearance of any desirable markers or desirable characteristics as defined hereinbefore). Another Tissues aspect of the invention relates to methods for identifying the 0257 Another aspect of the invention relates to a tissue molecular and cellular pathways for reprogramming. containing reprogrammed cells as defined herein that can be 0262 The Examples section provides principles, methods implanted into a Subject in need thereof. and techniques useful for screening and identifying Such 0258. In some embodiments the present invention pro desirable active compounds. For instance, those skilled in the vides for the reprogramming of cells within a tissue, for art will understand that it is conceivable to screen for com example an in vitro produced 3D tissue construct comprising pounds that will induce transformation of a cell of a first type cells and extracellular matrix produced by these cells. In to a NSLC by replacing the “induction” or “biological activ addition, transfected cells can be seeded on top of these 3D ity’ provided by the transient increase of Musashi1, NGN2 or tissue constructs that can be made completely autologously, MBD2 in the cell by a candidate compound to be tested (e.g. thus preventing host rejection, making it completely immu a library of Small molecules or compounds) and measuring nocompatible and as carrier for reprogrammed cells to be activity or efficacy of the candidate compound in generating transplanted in vivo. Advantageously, these newly created the NSLC. Individual or mixture of active compounds would cells can be used in their undifferentiated and/or differenti be selected if they have the same activity and/or if they can ated State within these tissues for in vitro diagnostic purposes provide the same or similar effects as these polypeptides (e.g. ortransplanted into a patient in need of such a construct in cell cell transformation and/or appearance of any desirable mark therapy/tissue replacement approaches. ers or desirable characteristics, as defined hereinbefore). For 0259. The invention further encompasses 3D Neuronal example, a compound or mixture of compounds capable of Like multilayer tissue. Cells within CDM reprogrammed to transforming a fibroblast into a NSLC could be identified by: US 2014/0038291 A1 Feb. 6, 2014 26
0263 (i) Setting up, culturing and transforming the tional regenerative responses in neurons such as increased fibroblasts into NSLC as in Example 1: neurotransmitter turnover and/or axonal sprouting (neurore 0264 (ii) Screening a library of compounds by replac generation). Up to date, several therapeutic strategies to ing Msil, Ngn2 and/or MBD2 with each candidate com deliver neurotrophic-factors in animal models have been pound in a different well; explored, but so far testing of the effects of growth factors on 0265 (iii) Identify a compound hit when the candidate the brain and nervous system have been limited to direct compound is able to transform the fibroblasts into peripheral injection of large doses of these factors, which NSLCs approximately as well as the replaced Msil, carries a significant risk of side effects. Accordingly, a related Ngn2 and/or MBD2: aspect of the invention relates to overcoming these problems 0266 (iv) If compound from part (iii) did not replace all by using NSLC cells and cell lines according to the invention of Msi1, Ngn2 and MBD2, and is not able to transform which can stably express and secrete growth factors of poten the fibroblasts into NSLCs by itself, then by including tial interest after transplantation. the compound from (iii) in each well, screening a library 0275 To summarize, the present invention provides a of compounds by replacing the Msil, Ngn2 and/or plentiful source of Stem-Like Cells and their progeny (differ MBD2 that was not removed in part (ii) with each can entiated cells) for potential clinical treatments which require didate compound in a different well; transplantation of specific stem cells to 1) compensate for a 0267 (v) Identify a compound hit when the candidate loss of host cells or 2) as vehicles to deliver genetically-based compound is able to transform, along with the com drugs. For example, Neural Stem-Like Cells, Neuron-Like pound from part (iii), the fibroblasts into NSLCs Cells, Astrocyte-Like Cells or Oligodendrocyte-Like Cells approximately as well as the replaced Msil, Ngn2 and/ can be used for potential clinical treatments which require or MBD2: transplantation of neural stem cells, neurons, astrocytes or 0268 (vi) if compound from part (V) did not replace all oligodendrocytes 1) to compensate for a loss of CNS host of Msil, Ngn2 and/or MBD2, and is notable to trans cells (ex. neurons) or 2) as vehicles to deliver genetically form the fibroblasts into NSLCs together with the com based drugs (ex. BDNF). Further, the invention provides a pound from part (iii), then by including the compound novel neurological tool for use in basic research and drug from (iii) and (V) in each well, screening a library of Screening. compounds by replacing the Msil, Ngn2 or MBD2 that 0276 Those skilled in the art will recognize, or be able to was not removed in part (ii) and (iv) with each candidate ascertain using no more than routine experimentation, numer compound in a different well; ous equivalents to the specific procedures, embodiments, 0269 (vii) Identify a compound hit when the candi claims, and examples described herein. Such equivalents are date compound is able to transform, along with the com considered to be within the scope of this invention and cov pound from part (iii) and (v), the fibroblasts into NSLCs ered by the claims appended hereto. The invention is further approximately as well as the replaced Msil, Ngn2 or illustrated by the following examples, which should not be MBD2: construed as further limiting. 0270 (viii) A combination of the compounds from part (iii), (v) and (vii) will be able to transform the fibroblasts EXAMPLES into NSLC: modifications to these compounds can be made and further screened to identify more effective or 0277. The examples set forth herein below provide exem safe versions of these compounds. plary methods for obtaining Reprogrammed and Dedifferen 0271 The same principles are applicable for other desired tiated cells, including Neural Stem-Like Cells (NSLCs). Also types of stem-like cells including pluripotent-like cells, provided are exemplary protocols, molecular tools, probes, mesendoderm-like cells, pancreatic progenitor-like cells, etc. primers and techniques. Tables A and B. and the Examples section provides, for each of these types of cells, a list of potential genes and/or com Example I pounds to be considered in Such screening methods. 0272 Accordingly, the present invention encompasses Preparation of Human Fibroblast Cells these and any equivalent Screening methods where candidate (0278 Human Foreskin fibroblast (HFF) cells were pur compounds are tested for their efficacy in transforming a cell chased from American Type Culture Collection (ATCC, of a first type to a desired cell of a different type when Manassas, Va.) and expanded in cell culture flasks with Dul compared to the efficacy of the reprogramming agent, repro becco's Modified Eagle's Medium (DMEM, Invitrogen), gramming factor and/or gene regulator as defined herein. supplemented with 10% heat-inactivated fetal calf serum (FCS, Hyclone Laboratories), 0.1 mM non-essential amino Delivery of Neurotrophic Factors acids, and 1.0 mM sodium pyruvate (invitrogen) at 37°C., 5% 0273 A related aspect of the invention relates to local CO. The medium was changed twice per week. Cells were delivery of trophic factors (ex. growth factors) to a tissue or trypsinized using Trypsin 0.25% for 4 minutes at 37° C. organ of interest by transplanting stem-like cells and cell lines followed by adding trypsin inhibitor solution, pelleting the according to the invention which can stably express and cells by centrifugation, washing the cells once with PBS, and secrete growth factors of potential interest after transplanta plating the cells at a ratio of 1:2 onto tissue culture flasks until tion to said tissue or organ. a suitable number of cells was reached. 0274 Local delivery of neurotrophic factors has been sug (0279 Cells were then trypsinized and plated (8x10 cells/ gested as a method to treat several neurological conditions. well) in cell culture plates pre-coated with Laminin (10 Strategies using neurotrophic molecules focus on preventing ug/ml. Sigma) in two different composition of CDM medium: the progressive loss of neurons, maintaining neuronal con CDMI Medium consisting of a 3:1 ratio of Dulbecco's modi nections and function (neuroprotection), and inducing addi fied Eagle medium (DMEM, high glucose (4.5 g/L) with US 2014/0038291 A1 Feb. 6, 2014 27
L-glutamine and sodium pyruvate) and Ham's F-12 medium manufacturers instructions. DNase I treatment was per supplemented with the following components: EGF (4.2x10 formed on the RNeasyTM Column to further remove the trans IoM), bFGF (2.8x10" M), ITS (8.6x10 M), dexametha fected plasmid DNA using Rnase-Free DNase Set (Qiagen). sone (1.0x107M), L-3,3',5-triiodothyronine (2.0x10'M), RNA was eluted in 35ul of RNase-free water. Before cDNA ethanolamine (10 M), GlutaMAXTM (4x10M), and glu synthesis, all RNA samples were quantified using the Nano tathione (3.3x10M), but without the presence of L-ascorbic Drop 1000TM (ThermoScientific). cDNA was prepared using acid. the High Capacity cDNA archive kit (Applied Biosystems) as 0280 CDM II Medium consisting of a 3:1 ratio of Dul per the manufacturers instructions. 400ng of RNA was used becco's modified Eagle medium (DMEM, high glucose (4.5 in each 50 ul RT reaction. The resulting cDNA samples were g/L) with L-glutamine and Sodium pyruvate) and Ham's F-12 used immediately for TLDA analysis. For each card of the medium supplemented with the following components: EGF TaqmanTM low-density array (TLDA), there are eight sepa (2.5 ng/ml), bFGF (10 ng/ml), ethanolamine (2.03 mg/ml), rate loading ports that feed into 48 separate wells for a total of insulin (10 mg/ml), Selenious acid (2.5 ug/ml), dexametha 384 wells per card. Each 2 ul well contains specific, user sone (19.7 g/ml), L-3,3',5-triiodothyronine (675 ng/ml), defined primers and probes, capable of detecting a single GlutaMAXTM (4x10M), and glutathione (3.3x10M). gene. In this study, a customized Neuronal Markers 2 TLDA was configured into eight identical 48-gene sets, i.e. 1 loading Transient Transfection of HFF by Lipofectamine Using port for each 48-gene set. Genes were chosen based on litera Constructed Vectors ture. Each set of 48 genes also contains three housekeeping genes: ACTIN, GAPDH, and PPIA. 0281. After two days in culture, cells were transfected with pCMV6-XL5-MBD2 (24 (a DNA demethylator) using 0285) A sample-specific master mix was made for each lipofectamine reagent (Invitrogen) as per the manufacturers sample by mixing cDNA (160 ng for each loading port), 2x protocol. The DNA-lipid complex was added to cells and TaqmanTM Gene Expression Master Mix (Applied Biosys incubated for 24 h at 37° C., 5% CO. After 24 hours of tems) and nuclease-free water (USB) for a total of 100 ul per transfection with the DNA demethylator, the medium was loading port. Aftergentle mixing and centrifugation, the mix changed and cells were transfected by pCMV6-XL5 ture was then transferred into a loading port on a TLDA card. Musashil (2 Lig, Origene) or pCMV6-XL4-Ngn2 (2 ug, Ori The array was centrifuged twice for 1 minute each at 1200 gene) for 24 h. After 24 hours, the medium was changed to rpm to distribute the samples from the loading port into each Neural Progenitor Basal Medium (NPBM, Lonza) supple well. The card was then sealed and PCR amplification was mented with Noggin (20 ng/ml. Peprotech), EGF (20 ng/ml. performed using Applied Biosystems 7900HTTM Fast Real Peprotech), and bFGF (20 ng/ml, Peprotech) and cultured in time PCR system. Thermal cyclerconditions were as follows: this Proliferation Medium. Cells were retransfected after 2 minutes at 50° C., 10 minutes at 94.5°C., and 30 seconds at three days and incubated at 37° C., 5% CO, and 5% O. After 97° C., 1 minute at 59.7° C. for 40 cycles. 1 TLDA's was 7 days in proliferation conditions, 50% of the Proliferation prepared for 8 samples. Medium was changed to Differentiation Medium (Nb Active, 0286 Relative Expression values were calculated using BrainBitsTM) supplemented with Forskolin (10 uM, Tocris), the Comparative C. method. Briefly, this technique uses the all-trans-Retinoic Acid (ATRA, 5uM, Spectrum), bFGF (20 formula 2^* to calculate the expression of target genes ng/ml, Peprotech), NGF (20 ng/ml, Peprotech), and BDNF normalized to a calibrator. The threshold cycle (C) indicates (20 ng/ml. Peprotech); medium was changed every day by the cycle number at which the amount of amplified target increasing the percentage of Differentiation Medium over reaches a fixed threshold. C values range from 0 to 40 (the Proliferation Medium, and the cells were cultured for 20 latter representing the default upper limit PCR cycle number days. that defines failure to detect a signal). AC values AC, C 0282 Visual observation of reprogrammed cells was per (target gene)-C (Average of 3 Housekeeping genes) were formed by light microscopic observation every day following calculated for HFF Ctrl, and subsequently used as the cali transfection using bright field at 10x magnification. Samples brator for the respective samples. All gene expression values were collected at different time points (6, 12, and 20 days) to were assigned a relative value of 1.00 for the calibrator, which analyze neuronal gene expression and protein levels by gene is used to determine comparative gene expression Such that array and immunohistochemistry. Following transfection, AAC, AC (Treated)-AC (HFF Ctrl). Relative Expression reprogramming cells displayed a rapid change in cellular is calculated using the formula 2^^. morphology within 3 days post-transfection. The cells were 0287 Quantitative comparison of astrocyte, neuron, and more rounded and the cell's cytoplasm retracted towards the oligodendrocyte gene expression allowed identification of nucleus forming contracted cell bodies with extended cyto the majority of the genes that are differentially expressed in plasmic extensions and exhibiting neuronal perikaryal reprogrammed cells. Data in Table 1 were analyzed by using appearance at day 6 and 12, which was maintained until day a significance analysis algorithm to identify genes that were 20. However, this morphology was not observed in untrans reproducibly found to be enriched in reprogrammed cells fected cells at day 6 and 12. compared to untransfected cells. After the transfection with Msil or Ngn2 in the presence of MBD2, the expression of Gene Array Analysis oligodendrocytes progenitors such as NKX2.2, olig2, and MAG and two markers for astrocytes (GFAP and AQP4) were 0283 Characterization of the newly engineering cells highly increased. Also, several markers of early neuronal after transfection was performed using a neuronal gene-array cells were enhanced after the transfection of HFF.TDLA data containing 48 partial cDNAS coding for these genes and con revealed a remarkable increase in specific markers for inter trols. neurons, such as Somatostatin and calbindin1. The induction 0284 RNA was isolated from samples using QIAshred of Doublecortin (DCX), which is expressed by migrating derTM (Qiagen) and RNeasyTM Plus mini Kit (Qiagen) as per immature cells during development, and acetylcholine US 2014/0038291 A1 Feb. 6, 2014 28
(ACHE) mRNA, an early marker of neuronal cells, were cells. The expression of VEGF and GDNF genes were up highly expressed in the reprogrammed cells (Table 1). Trans regulated to almost five fold and seven fold by Msil and fection increased the expression of dihydropyrimidinase-like Ngn2, respectively, However, the expression of BDNF, EGF, 3 (DPYSL3), an early marker of newborn neurons, to fivefold and bFGF were not activated and even down-regulated as with Msil and seven fold with Ngn2. Expression, of Micro compared to untransfected cells. The expression of growth tubule-Associated Protein 2 (MAP2), an essential marker for associated protein (GAP-43), a growth- and regeneration development and maintenance of early neuronal morphology associated marker of neurons, and expression ofnetrin, impli and neuronal cell adhesion molecule, were highly expressed cated in neuronal development and guidance, were highly with Msil and Ngn2 (Table 1). The expression of enolase-2, enriched in reprogrammed cells. Expression of receptors for a marker of mature neurons, was 20-fold enhanced by Msil growth and neurotrophin factors was increased, such as type and Ngn2. Member of the NeuroD family NeuroD1 was III receptor tyrosine kinase, neurotrophic tyrosine kinase, and highly expressed after transfection with Msil to 84 fold and to neurotrophic tyrosine kinase receptor. Vimentin and 34 by Ngn2. fibronectin, markers for fibroblasts, were down-regulated in 0288 Gene expression of growth factors such as IGF-1, reprogrammed cells compared to the untransfected control IGF2, NPY and CSF-3 was also enhanced in reprogrammed fibroblast cultures. TABLE 1 Gene array of transfected human fibroblast cells by Msi1/MBD2 and Ngn2/MBD2. Gene array was performed on samples after two weeks of differentiation. Expression values are given relative to untransfected fibroblasts. Relative Relative Company expression to expression Symbol Common name and description Gene ID Msi1 to Ngn2 Astrocytes and oligodendrocytes markers NKX2-2 Markers for oligodendrocyte NMOO2509.2 very high very high progenitors OLIG2 Oligodendrocyte lineage NMOO58.06.2 47.511 8.38 transcription factor 2 MAG Myelin-associated glycoprotein NM-080600.1 212.61 4.51 GFAP Glial fibrillary acidic protein NM 002055.4 very high very high AQP4 Aquaporin 4 NM OO1650.4 83.77 56.86 NC markers SST Somatostatin, specific marker for NM OO1048.3 32.73 35.34 interneurons CALB1 Calbindin 1, interneuron marker NM 004929.2 1821 1322 Tubulin 1A Are necessary for axonal growth NM OO60.09.2 7.45 9.32 NES Precursor neurons (nestin) NM OO6617.1 1.61 1.54 DCX An early neuronal marker NM 178151.1 very high very high (Doublecortin) ACHE Acetylcholinesterase, marker of NM O15831.2 9.02 1322 early neuronal development ENO2 A marker for neurons cells, NM OO1975.2 22.62 20.68 enolase NEUROD1 Neural marker; expression NMOO2500.2 84.22 34.27 gradually increased from neural precursor to fully differentiated (Oil DPYSL3 Dihydropyrimidinase-like3, NM OO1387.2 5.33 7.02 marker of immature neurons MAP2 Microtubule-associated protein 2, NM 002374.3 86.38 89.67 essential for development of early neuronal morphology and maintenance, of adult neuronal morphology NCAM Neural cell adhesion molecule 1 NM 18135.3 very high very high CEND1 Cell cycle exit & neuronal NM O16564.3 4.8O 5.57 differentiation, early marker of proliferating precursor cells that will differentiate to neurons Neuroregeneration and Survival genes FGF2 Fibroblast growth factor- NMOO2006.4 O.O6 O.11 EGF Epidermal growth factor, HsOO153181 m1 O.99 O.S6 IGF-1 Insulin growth factor-1, NM 000618.2 S8.92 21.21 IGF-2 Insulin growth factor-2 NMOOOO612.3 very high very high CSF3 Granulocyte colony-stimulating NM 2219.1 very high 42.60 BDNF factor-Brain derived growth NM-1992.31.1 O.OS O.O3 factor, neurogenesis US 2014/0038291 A1 Feb. 6, 2014 29
TABLE 1-continued Gene array of transfected human fibroblast cells by Msi1/MBD2 and Ngn2/MBD2. Gene array was performed on samples after two weeks of differentiation. Expression values are given relative to untransfected fibroblasts. Relative Relative Company expression to expression Symbol Common name and description Gene ID Msi1 to Ngn2 GDNF Glial derived neurotrophic factor NM-OOO614.2 4.77 6.89 CNTF Ciliary neurotrophic factor NM 001025366.1 186 1.09 VEGF Vascular endothelial growth NM 130850.1 6.67 7.32 factor BMP-4 Bone morphogenetic protein 4 NM OO2253.1 S.96 8.57 KDR Type III receptor tyrosine kinase) NM 006180.3 31.78 6.83 NTRK2 Neurotrophic tyrosine kinase NM OO905.2 10.31 13.37 receptor (TrkB) NPY Neuropeptide factors NM 002649.2 very high very high PIK3CG phosphoinositide-3-kinase, NM 213662.1 very high very high STAT3 Signal transduction transcription 3 NM 002045.2 2.14 3.65 Gap43 Growth associated protein 43 NM OO6180.3 very high very high NTN1 Netrin1, implicated in neuronal NM 024003.1 26.84 2.98 development and guidance NTRK2 Neurotrophic tyrosine kinase, NM OO3061.1 10.31 13.37 receptor, type 2 Sit Axonal guidance molecules HsOO185584 very high very high Vimentin Radial glia and fibroblast marker NM 212474.1 O.11 O.13 Fibronectin fibronectin is a marker for O.15 O.23 fibroblasts
Immunohistochemical Analysis Example II 0289 Cells were fixed with a 4% formaldehyde/PBS solu Comparison of Reprogramming Efficiency of Three tion for 10 min at room temperature and subsequently per Different Neurogenic Genes meabilized for 5 min with 0.1% Triton X-100TM in 4% form aldehyde/PBS. After two brief washes with PBS, unspecific 0292 HFF cells were cultured as described in Example I antibody binding was blocked by a 30 min incubation with and plated in CDM I medium. Cells were transfected using 5% normal goat serum in PBS. Then primary antibodies were the Amaxa NucleofectorTM Device (Lanza). The HFFs were added in 5% normal goat serum/PBS as follows: Mouse anti harvested with TrypLETM (Gibco), resuspended in CDM Nestin (1:100, BD) as an intermediate microfilament present Medium and centrifuged for 10 min at 90xg (1x10 cells/ in neural stem cells and mouse anti-NCAM (1:100, Neurom tube). The Supernatant was discarded and gently resuspended ics) as neuronal adhesion molecule. After a 2h incubation the in 100 ul of Basic NucleofectarTM Solution (basic Nucleofec cells were washed 4 times for 5 min each with 0.1% TweenTM/ torTM kit for primary mammalian fibroblasts, Lanza). Each PBS. Appropriate fluorescence-tagged secondary antibody 100 ul of cell suspension was combined with a different mix was used for visualization; Goat anti-mouse 546 (1:200, of plasmid DNA (for example, Sample 1 was mixed with 2 Lig invitrogen) prepared in 5% normal goat serum/PBS was used. of pCMV6-XL5-Pax6 and 2 ug pCMV6-XL5-MBD2). Cell After incubation for one hour, cells were washed in 0.1% Suspension was transferred into an Amaxa certified cuvette Tween TM/PBS three times for 5 min each. The DNA stain and transfected with the appropriate program (UO23). The Hoechst33342 (Invitrogen) was used as a marker of nuclei sample was transferred without any further resuspension into (dilution 1:5000 in PBS, 10 min incubation). Fluorescence a coated culture plate with LAS-Lysine/Alanine (Brain images were taken with a CellomicsTM ArrayScan HCS BitsTM, 50 g/ml) and the cells were incubated at 37° C., 5% Reader microscopy system. To determine an estimate of the CO. These steps were repeated for each sample that was percentage of cells adopting neuronal or glial phenotypes, transfected. After 24 hours, the medium was changed to Pro random fields were selected and for each field the total num liferation Medium. After two days, cells were retransfected ber of cells (as determined by counting Hoechst stained using lipofectamine as described in Example I and incubated nuclei) and the total number of cells positive for neuronal or at 37°C.,5% CO, and 5% O. After 6 days, differentiation was glial markers were determined. induced with Differentiation Medium that gradually replaced 0290. To confirm that these cells exhibited markers of the Proliferation Medium over several days. Cells were col neuronal lineages, cells were immunostained for nestin and lected at day 14 for RT-PCR and immunohistochemistry NCAM. This analysis revealed that reprogrammed cells analysis. expressed both proteins. NCAM was present in cells during the 6 days post-transfection and increase at day 12 and 20 Gene Expression Analysis following differentiation, while the inverse pattern was 0293 RNA isolation and quantification was performed as observed for the nestin staining. previously described in Example I. cDNA was prepared using 0291. This study showed the ability to reprogram HFF the High Capacity cDNART kit (Applied Biosystems) as per cells using one neurogenic transcription factor with the pres the manufacturer's instructions with a final cDNA concentra ence of a DNA demethylator towards cells that expressed tion of 2 ng/ul. Real-time PCR was then performed for each neuronal genes and proteins specific to neural stem cells and gene of interest using the FAST PCR master mix (Applied neuronal cells. These reprogrammed cells were stable in cul Biosystems) and the TaqmanTM(R) Gene Expression Assays ture for at least 2 weeks. (Applied Biosystems) listed below: US 2014/0038291 A1 Feb. 6, 2014 30
transfected with Msil or Ngn2 in the presence of MBD2 expressed neural stem cell markers (Nestin and Sox2), how Gene Name Assay ID ever the expression of Sox2 was much more highly expressed ACHE HsOO241307 m1 than nestin following transfection with Ngn2 or Msil. Neu NES HsOO707120 s1 ronal and astrocyte specific genes (BIII-Tubulin, MAP2b, IRB Hist, GFAP, and ACHE) was increased as well. mRNAs level of the PAX6 HsOO240871S m1Il tripotent-associated genes BIII-tubulin, MAP2b, acetycho MSI1 HsO1045894 m1 line, and GFAP were undetectable in Pax6 transfected cells, NGN2 HsOO702774 s1 indicating that Pax6 alone was not implicated in the repro gramming process toward neuronal lineage. TABLE 2 Relative expression of gene expression of different neuronal lineage performed by RT-PCR following the transfection of HFF by Msi1, Ngn2, or Pax6 in the presence of MBD2 and cultured for 14 days.
MSI1 NGN2 PAX6 NES TUBB3
Rel. Std. Std. Std. Rel. Std. Rel. Std. Exp. Dev Rel. Exp Dew. Rel. Exp Dev. Exp. Dev. Exp. Dev
#1 Control 1.OO O.O7 1.08 0.57 1.11 O.67 100 OO2 1.OO O.O1 Untransfect #2 4077.82 248.02 1.18 O.66 487.09 69.58 8.62 O.OO 6.58 0.11 MSI1 MBD2 #3 1416 O.63 478O3.26 192.78 624.31 91.27 8.62 O.O2 8.33 O.O2 NGN2 MBD2 i4 1.70 O.36 0.27 O.O1 2956443 357.89 0.46 O.OO 0.49 O.O2 PAX6MBD2
ACHE GFAP MAP2 SOX2
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dew.
#1 Control 1.02 0.29 1.OO 0.06 1.OO O.O1 1.00 O.09 Untransfect #2 6.58 0.64 215.71 20.65 S.SO 0.46 3499.53 18485 MSI1 MBD2 #3 8.33 O.97 365.60 5.11 S.42 O.OO 4039.03 8.6S NGN2 MBD2 i4 1.98 0.48 1.15 0.13 O.S.S. O.04 1.00 O.O3 PAX6 MBD2
-continued Immunohistochemical Analysis Gene Name Assay ID 0297 Fluorescent immunohistochemical staining was performed as previously described in Example I. In agree MAP2 HsOO258900 m1 ment with the RT-PCR data, immunohistochemical analysis GAPDH (housekeeping gene) Hs99999905 m1 of these cultures revealed that reprogrammed cells (with Msil PPLA (housekeeping gene) Hs99999904 m1 or Ngn2) generated morphologically complex neurons that were positive for MAP2b, indicating the differentiation of 0294 The FAST 96-well reaction was performed with 8 NSLCs to neuron-like cells (NLCs). However, the positive ng cDNA per well in a 10ul reaction with 40 cycles. Thermal staining for these markers was undetectable after transfection cycler conditions were as follows: 20 seconds at 95°C., and with Pax6/MBD2. Moreover, the newly formed neurons 1 second at 95°C., 20 seconds at 60° C. for 40 cycles. expressed the markers for and developed long neurites with 0295 Relative Expression values were calculated as pre growth cones at their ends, expressed neural specific genes, viously described in Example I, except the Average of 2 and ceased to proliferate when they were exposed to differ Housekeeping genes (GAPDH & PPIA) was used for normal entiation conditions. ization instead of the Average of 3 Housekeeping genes. Identification of neuronal lineage genes was investigated fol Example III lowing the transfection with three independent vectors con taining Msil, Ngn2, and Pax6. Transfection of HFF by Various Combinations of Vectors and 0296. As shown in Table 2, after 14 days following trans Disruption of Cell Cytoskeleton fection, relative expression of mRNA of neuronal lineage was 0298 Various combinations of neurogenic regulators and undetectable in untransfected cells (HFF), while the cells cytokines for epigenetic modifications were tested to ascer US 2014/0038291 A1 Feb. 6, 2014 31 tain their effect on reprogramming efficiency. Starting one in Example I. RT-PCR analysis showed that the relative day before transfection, cells were treated with or without expression of Sox2, nestin and GFAP was enhanced after cytochalasin B (Calbiochem), with the concentration transfecting the cells with the neurogenic transcription fac decreased every day over five days during media changes tors. As shown in Table 3, transfecting the cells with one (starting with 10 ug/ml Cytochalasin B on day 1 to 7.5 g/ml. transcription factor Msil in the presence of Gadd45b was 5 g/ml, 2.5 ug/ml, and 0 ug/ml over the Subsequent four associated with up-regulation of relative expression of Sox2 days) in order to investigate the effect of disrupting the cell (22.3+5.26) and GFAP (10.14+0.15) and the expression of cytoskeleton on the process of reprogramming. Cells were the these genes was highly increase when transfecting the transiently transfected as described in Example H with one or cells with Ngn2 by 20 fold and 10 fold respectively. Combin two vectors containing one neurogenic transcription factors ing the two neurogenic factors (Msil and Ngn2) with by nucleofection. Cells were co-transfected with either of two Gadd45b enhanced further the expression of Sox2 and GFAP, DNA demethylators, MBD2 or GAdd45B, (e.g. 2x10 cells Transfecting the cells with one transcription factor (Msil or were transfected with pCMV6-XL5Msi1 (2pg) and pCMV6 Ngn2) in the presence of MBD2 was associated with up XL5-MBD2 (2 ug)). After 24 hours, the medium was changed regulation of relative expression of Sox2, Nestin, and GFAP to Neural Proliferation Medium (NeuroCultTM proliferation and down-regulation of Col5A2, while co-transfection with Kit, StemCell Technologies) consisting of DMEM/F12 (1:1), Gadd45b did not increased the expression of nestin and the glucose (0.6%), sodium bicarbonate (0.1%), glutamine (20 expression of Col5A2 was not regulated. The enhancement of mM), HEPES (5 mM), insulin (230 g/ml), transferrin (100 neural stem cells relative expression was observed when ug/ml), progesterone (200 nM), putrescine (90 g/ml), and transfecting the cells with two neurogenic genes in combina sodium selenite (300 nM) and supplemented with Noggin (20 tion with MBD2; a small increase in the expression was ng/ml, Peprotech), recombinant hFGF(20 ng/ml, Peprotech), noticed in the presence of cytochalasin B under certain con and recombinant hEGF (20 ng/ml, Peprotech) and cells were ditions. An increase in the relative expression of the neural cultured for two weeks at 37° C., 5% CO, and 5% O. Cells stem-specific markers (Sox2, Nestin, GFAP) and a decrease were then analyzed for neural stem cell markers. in the fibroblast-specific gene (COL5A2) was observed after transfection with Msi1/Ngn2/MBD2, Msi1/Ngn2/Gadd45b, Gene Expression Analysis Msi1/MBD2 or Ngn2/MBD2 (Table 3). This study demon 0299 Gene expression analysis was performed for neural strated that MBD2 increased more reprogramming efficiency stem-specific markers (Sox2, Nestin, GFAP) and a fibroblast then GDA45b and showed that cytochalasin B had no effect specific marker (Col5A2) by RT-PCR as previously described of its own in the control cultures. TABLE 3 RT-PCR analysis of relative expression of neuronal precursor cell markers such as nestin, Sox2, and GFAP after transfection of fibroblast cells with different combinations with or without the co-treatment with cytochalasin B. Relative expression of Sox2, nestin, and GFAP in NSLCs was increased after transfection with both transcription factors (Ngn2 and Msi1) with MBD2 as the DNA demethyaltor. As demonstrated, this upregulation of neural stem cell gene expression was associated with a decrease of CoL5A2, a specific gene for fibroblast cells.
COLSA2 FBN2 NES MAP2 TUBB3 SOX2 ACHE GFAP
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dew Exp. Dew Exp. Dew Exp. Dew. #1, +CytoB, 1.OO O.O7 OO O.O1 100 OO4 1.OO O.OS 1.00 O.OS 1.00 O.OS 1.OO O.10 1.00 O.11 Control #2, -CytoB, 1.OO O.O3 OO O.O8 100 OOO 100 0.09 1.00 O.09 1.15 O.80 1.01 O.18 1.00 O.O1 Control #3, +CytoB, O.85 0.04 0.75 O.O2 0.60 OO1 O.29 O.O1 O44 O.OO 22.39 S.26 O.81 O.19 10.14 O.15 Msi1, GAD45b #4, -CytoB, O.87 O.O3 81 (0.09 1.84 O.O4 2.31 O.OO 2.09 O.O3 2O.28 5.33 1.99 0.74 6.03 O.OS Msi1, GAD45b #5, +CytoB, O.84 O.O4 O.77 O.O3 O.44 OOO 0.24 O.OO O.36 O.O1 470.84 13.43 O.63 O.OS 103.22 O.80 Ngn2, GAD45b #6, -CytoB, 0.75 0.07 97 (O.O2 1.83 OOO 440 O16 2.02 O.10 789.33 60.3S 1.7O O.13 110.48 4.90 Ngn2, GAD45b #7, +CytoB, O.74 O. 12 O8 O.OO O.89 O.O1 O.S1 O.OO O.63 O.04 1.64 O.98 0.86 O.12 2.49 O.21 Pax6, GAD45b #8, -CytoB, O.66 0.04 241 O.O9 2.7O O.O3 4.96 O.30 3.48 O.O7 O46 O.33 2.97 1.04 O.43 O.09 Pax6, GAD45b #9, +CytoB, O.14 O.O1 O.28 OO1 1.30 O.O3 4.07 O.11 O.84 O.OO S4768.27 6709.S6 O.81 O.24 3391.96 64.63 Msi1, Ngn2, GAD45b. #10, -CytoB, O.12 O.OO O.73 O.O3 S.28 O.21 SO.84 1.23 4.93 O.28 17400.66 822.88 3.58 0.10 1255.76 5.27 Msi1, Ngn2 GAD45b. #11, +CytoB, O.10 O.OO O.26 OO1 1.11 OO1 3.69 O.09 O.76 O.OO SSS88.41 1331.20 O.S.S O.14 2849.96 261.51 Msi1, Ngn2 MBD2 #12, -CytoB, O44 O.O1 1.47 OO6 5.49 0.14 47.3O O.11 5.50 O.31 14587.46 789.19 3.90 O.13 1424.04 39.29 Msi1, Ngn2 MBD2 US 2014/0038291 A1 Feb. 6, 2014 32
TABLE 3-continued RT-PCR analysis of relative expression of neuronal precursor cell markers such as nestin, Sox2, and GFAP after transfection of fibroblast cells with different combinations with or without the co-treatment with cytochalasin B. Relative expression of Sox2, nestin, and GFAP in NSLCs was increased after transfection with both transcription factors (Ngn2 and Msi1) with MBD2 as the DNA demethyaltor. As demonstrated, this upregulation of neural stem cell gene expression was associated with a decrease of CoL5A2, a specific gene for fibroblast cells.
COLSA2 FBN2 NES MAP2 TUBB3 SOX2 ACHE GFAP
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dew. Exp. Dew Exp. Dew. Exp. Dew. #13, +CytoB, 1.11 O.04 1.09 OO6 O.92 0.08 O.68 OO1 O.82 0.03 63.93 2.81 119 O.17 17.43 1.86 GAD45b. #14, -CytoB, O.94 OO1 2.22 OOO 2.82 0.02 6.49 O3O 4.01 O.OS 6.12 O.61 2.34 0.17 1.42 O.10 GAD45b. #15, +CytoB, O.83 O.OO O.83 O.OS O36 0.01 O.16 OO1 O.36 OOO 3.42 3.74 0.63 0.37 2.18 O.12 MBD2 #16, -CytoB, O.68 O.O2 1.SS O.O4 1.57 0.05 147 O.O1 2.OO O.OO O.S2 O.29 1.45 O.15 0.55 O.04 MBD2 #17, +CytoB, 1.10 OO1 1.16 O.O3 1.37 O.O1 1.12 OO6 (O.86 OO6 5.59 148 1.07 0.27 1.70 O46 Msi1, Ngn2 #18, -CytoB, O.93 O.04 2.52 0.10 3.48 0.01 9.01 O.O2 4.SS O.18 1.78 146 3.83 O42 O.S9 O.O1 Msi1, Ngn2 #19, +CytoB, O.2O O.O3 O.36 OO1 1.25 O.OS 6.68 0.31 O.72 O.O2 66592.29 3481.89 2.57 O.O3 4450.08 131.85 Msi1, MBD2 #20, -CytoB, O.12 OOO O.64 O.O3 4.7O O.22 77.51. O.11 4.12 O.11 19128.03 1542.OO 8.14 O.13 999.22 24.75 Msi1, MBD2 #21, +CytoB, O.17 O.O1 O.28 OOO 116 0.04 5.73 0.06 0.62 O.OO 67945.51 3OOO.74 2.15 O.O4 4736.83 11.92 Ngn2, MBD2 #22, -CytoB, O.17 O.OO O.78 O.O3 4.32 O.O8 68.89 5.26 4.01 O.04 16570.91 92.96 7.04 O.S3 1427.13 13.19 Ngn2, MBD2 #23, +CytoB, O.71 O.OS O.79 0.06 O.87 O.O1 O.63 0.06 0.67 O.04 2.86 O.7O 1.08 O.O8 2.08 O.11 Msi1 #24, -CytoB, O66 0.04 1.92 O.17 2.03 O.O2 2.77 O.O2 2.68 0.02 O.32 O.12 1.85 O.65 O.S8 O.04 Msi1
Immunohistochemical Analysis cytochalasin B. A slight increase of Sox2 positive cells (10. 0300 Fluorescent immunohistochemical staining was 42+10.27%) and nestin positive cells (4.85+1.10%) was performed as previously described in Example I. Table 4 detected following transfection with one transcription factor shows the percentage of Nestin and Sox2 in each condition, Msil and MBD2. Same tendency of nestin and Sox2 positive with the highest percentage of Sox2 (38.18+1.75%) and nes cells was observed following transfection with Ngn2 and tin (28.18+2.77%) positive cells observed after transfecting MBD2. Disrupting the cell cytoskeleton with Cytochalasin B the cells simultaneously with both neurogenic transcription significantly enhanced reprogramming, but had no repro factors and in the presence of a DNA demethylator and gramming effect on its own (Table 4). TABLE 4 Percentage of positive cells for Sox2 and nestin after transfection of fibroblast cells with different expression vectors with or without the presence of cytochalasin B. After transfection the cells were cultured in proliferation medium (StemCell Technologies) Supplemented by EGF (20 ng/ml, Peprotech) and FGF (20 ng/ml, Peprotech) for two weeks at 37° C./5% CO2/5% O2. The percentage of immunopositive cells was determined by Cellomics TM and represented as meant SD (n = 3-5). % of Sox2 positive cells % of Nestin positive cells +CytoB -Cyto B +CytoB -CytoB
Untransfected cells O.O2 OO1 O.O1 O.OO O.14 - 0.04 O. 11 O.O9 Ngn2 O.35 - O.36 O.15 0.05 2.34 - 0.99 1.04 O.21 Msi1 O.23 O.15 O.120.09 1950.11 1.11 - 0.18 Gadd45b. O.30 O.17 O.290.11 4.94 - 0.25 2.33 O42 MBD2 O.22 O.13 O.220.11 2.8 0.11 1530.6 Msil/Ngn2 O.19 O.13 O32- 0.05 1.91 - 0.56 2.59 - 1.28 Msi1 MBD2 10.42 10.27 8.84 - 11.63 4.85 - 1.10 2.06 O.O8 Msi1 Gadd45b O.O6 OO1 O.14 - 0.17 O55, O.O6 O.24 O.11 Ngn2/MBD2 11.17 O.O8 9.07 - 11.31 5.7 0.10 2.18 O.23 Ngn2/GAdd45b O.29 O.11 O.95 - 0.17 1170.54 O.98 O.25 Msi1/Ngn2/MBD2 38.18 1.75 22.03 - 1.90 28.18, 2.77 14.54 - 0.45 Msi1/Ngn2/Gadd45b 22.65 SO3 1854 - 940 1872 - 6.26 8.7O 451 US 2014/0038291 A1 Feb. 6, 2014 33
0301 Various DNA demethylators were tested as well for presence of proliferation medium supplemented with EGF their effect on reprogramming efficiency. Cells were co-trans (20 ng/ml), FGF (20 ng/ml), and Noggin (20 ng/ml) with and fected with one vector (MSI1/NGN2) containing two neuro without VPA (1 mM) treatment for 12 days at 37°C., 5% CO genic pCMV6-Msil-Ngn2 factors with various DNA dem and 5% O. ethylators. Simultaneously another neurogenic factor was 0303 Gene expression analysis and immunohistochemis tested for its effect on cells de-differentiation towards NSCs, try was performed to analyse neural specific gene and protein pCMV-XL-Nestin individually or in combination with expression (BIII-tubulin, GFAP, Sox2, Nestin) as described in pCMV-Msi1-Ngn2, pCMV-XL5-Msi1, or pCMV-XL4 Example II. Transfecting cells with Msil and Ngn2 in the Ngn2 in the presence of MBD2 as previously described in presence of various DNA demethylators revealed and confirm Example II. previous data showing that the among various DNA demethy 0302 Cells were co-transfected pCMV-Msi1-Ngn2 with lators used in this study, MBD2 promotes the expression of different DNA demethylators (MBD1, MBD2, MBD3, neural stem genes (Sox2, GFAP, Nestin) as shown in Table 5. MBD4, MeCP2. AICDA). Another assay was performed to Furthermore, transfecting cells with nestin with and without assess the effect of nestin on the reprogramming efficiency; the presence of one neurogenic factor had no effect on the therefore cells were transfected with nestin individually or in reprogramming efficiency into neural stem-like cells. How combination with one vector containing one neurogenic fac ever co-transfection with nestin and Msi1/Ngn2/MBD2 tor (Msil or Ngn2) or both neurogenic factors in the presence enhanced the expression of neural stem cells genes and this of MBD2. Cells were cultured following transfection in the increase was more pronounced in the presence of VPA. TABLE 5 RT-PCR analysis of relative expression of neuronal precursor cell markers such as nestin, Sox2, BIII-tubulin, and GFAP after transfection of fibroblast cells with various combinations of pCMV-Msi1-Ngn2 (MSI1/NGN2), pCMV-XL5-Msi1, pCMV-XL4-Ngn2, pCMV-XL-Nestin with different combinations of DNA demethylators, with and without the co-treatment with VPA.
TUBB3 GFAP SOX2 NES Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dew. Exp. Dew. Exp. Dew. Exp. Dew. Day 12, Untransfected 1.00 O.11 1.00 O.OS 1.01 O16 1.OO O.13 (-VPA) Day 12, Untransfected 1.00 O.O3 1.00 O.O6 1.OO O.OO 1.OO O.O2 (+VPA) Day 12, MSI1/NGN2/MBD1 O.96 O.O6 2.69 O.13 1.15 O49 0.46 O.O2 (-VPA) Day 12, MSI1/NGN2/MBD1 1.10 O.O6 2.22 O.O6 O.80 O.O1 O.84 O.O2 (+VPA) Day 12, MSI1/NGN2/MBD2 123.52 O.06 1638.53 99.86 61467.29 1487.21 31.77 O.17 (-VPA) Day 12, MSI1/NGN2/MBD2 232.00 O.O8 1889.3O 42.39 72022.15 7894.41 42.69 O.14 (+VPA) Day 12, MSI1/NGN2/MBD3 O.92 O.O7 3.98 O.S9 28.05 4.67 O.S6 O.O1 (-VPA) Day 12, MSI1/NGN2/MBD3 1.23 O.OS 1.66 O.18 11.31 2.35 O.87 O.O2 (+VPA) Day 12, MSI1/NGN2/MBD4 O.85 O.O1 4.8O O.23 S.42 S.2O O.62 O.OO (-VPA) Day 12, MSI1/NGN2/MBD4 O.9S O.O1 1.57 O16 2.27 O.O4 O.79 O.O3 (+VPA) Day 12, 1.11 O.O6 3.80 O.38 6.54 6.45 O.69 O.O1 MSI1/NGN2/MeCP2 (-VPA) Day 12, 1.37 O.09 1.63 O.45 10.53 10.49 1.07 O.O1 MSI1 NGN2 MeCP2 (+VPA) Day 12, 1.07 O.04 4.59 O.O2 O.65 O.O1 O.74 O.O2 MSI1/NGN2/AICDA (-VPA) Day 12, 1.10 O.O1 2.37 O.29 1.21 O.16 0.91 O.04 MSI1/NGN2/AICDA (+VPA) Day 12, Msi1/MBD2 (-VPA) 1.31 O.17 3.78 O.49 O.70 O.O2 0.78 O.OO Day 12, Msi1/MBD2 1.36 O.O7 1.75 O.31 1.26 O.O3 1.15 O.O3 (+VPA) Day 12, Ngn2/MBD2 (-VPA) O.85 O.O6 2.93 O.S1 0.79 O.OS O.S8 O.O2 Day 12, Ngn2/MBD2 1.41 O.OS 1.60 O.11 2.30 O.O6 1.03 O.O3 (+VPA) Day 12, Nes/Msi1 (-VPA) O.84 O.O3 3.21 0.72 O.76 O.O1 O.S1 O.O1 Day 12, Nes/Msi1 (+VPA) O.86 O.09 1.82 O.30 2.14 1.02 0.94 O.O1 Day 12, Nes/Ngn2 (-VPA) O.69 O.OS 2.88. O.32 O.99 O.10 O.S7 O.O2 Day 12, Nes/Ngn2 (+VPA) O.88 O.O1 1.53 O.19 2.71 O.O2 0.83 O.O3 Day 12, Nes/MSI1/NGN2/MBD2 111.58 O.04 1423.56 82.87 72069.27 624.51 S1.52 O.12 (-VPA) Day 12, Nes/MSI1/NGN2/MBD2 321.00 O.04 2600.14 1.90 88932.OO 708.72 82.74 O.18 (+VPA) US 2014/0038291 A1 Feb. 6, 2014 34
TABLE 5-continued RT-PCR analysis of relative expression of neuronal precursor cell markers such as nestin, Sox2, BIII-tubulin, and GFAP after transfection of fibroblast cells with various combinations of pCMV-Msi1-Ngn2 (MSI1/NGN2), pCMV-XL5-Msi1, pCMV-XL4-Ngn2, pCMV-XL-Nestin with different combinations of DNA demethylators, with and without the co-treatment with VPA.
TUBB3 GFAP SOX2 NES Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dew. Exp. Dew. Exp. Dew. Exp. Dew. Day 12, Nes/MSI1/NGN2 (-VPA) 0.74 0.11 2.60 (0.28 1.98 O.97 O.S.S. O.O1 Day 12, Nes/MSI1/NGN2 O.86 O.OO 1.70 0.49 1.70 O.O4 O.88 O.OS (+VPA) Day 12, Nes/MBD2 (-VPA) O.76 O. 12 3.15 0.17 O.87 O.O3 O.44 OOO Day 12, Nes/MBD2 (+VPA) O.87 0.03 2.0S O.O7 2.66 1.64 O.91 O.OO Day 12, Nes/Msi1/MBD2 (-VPA) O.81 O.OS 3.41 O.66 1.11 O.O1 O.S8 O.O1 Day 12, Nes/Msi1/MBD2 1.01 O.13 2.43 O.O7 3.27 O.26 O.93 O.O2 (+VPA) Day 12, Nes/Ngn2/MBD2 (-VPA) 1.19 0.07 5.71 1.30 4.11 O.O7 O.91 O.04 Day 12, Nes/Ngn2/MBD2 1.29 O.O3 2.98 0.66 21.2O O42 1.65 0.02 (+VPA)
0304 Immunohistochemistry analysis performed in par 0305 Another study was designed to test the effect of allel with RT-PCR data indicated that positive Sox2 cells were various neurogenic genes on the reprogramming efficiency undetectable when transfecting the cells with Msi1/Ngn2 in towards neural stem-like cells. HFF cells were cultured as the presence of MBD1, MBD3, MBD4, MeCP1, or AICADA described in Example I, and transfected using the Nucleofec (Table 6) and that among the different types of DNA dem torTMR 96-well Shuttle R Device (Lonza) following proce ethylator genes tested only MBD2 plays a significant positive dure described in Example IV, except for the untreated HFF role in the reprogramming efficiency of HFF towards NSLCs control and the untransfected HFF control (for determining when using the above neurogenic genes. Immunohistochem the effect of the complete media & compound treatments on istry analysis revealed a small increase of immunopositive the cells). The cells that had been pre-treated with VPA and Sox2 cells (89.49+3.18) after co-transfecting the cells with 5-Aza and the untreated cells were transfected with the mixes nestin and Msi1/Ngn2 in the presence of MBD2 (Table 6). of DNA as described in Table 7. The cells were plated on Laminin-coated plates and incubated at 37° C., 5% CO. TABLE 6 Media was changed daily according to Table 7. Cells were analysed at day 3, 7, 12 by immunohistochemistry analysis Percentage of positive cells for Sox2 after transfection of fibroblast and at Day 9 by gene array for multipotent and pluripotent cells with different expression vectors with or without the presence gene expression. the various DNA demethylators. After transfection the cells were cultured in proliferation medium (StemCell Technologies) Supplemented by EGF (20 ng/ml, Peprotech) and FGF (20 ng/ml, Gene Array Analysis Peprotech) for two weeks at 37° C.75% CO/5% O. The percentage of immunopositive cells was determined 0306 An additional batch of cells treated according to Oa by Cellomics TM and represented as meant SD (n = 3-5). and 1a in Table 7 was analyzed at Day 9, along with HFFs, hNPCs, and passage 5 NSLCs (frozen from previous experi % Sox2 positive it stav ments from Example III) by the Pluripotency Gene Array HFF untransfected O.13 - 0.12 (ABI) (Tables 8a and b) and a set of genes (Table 8c) to Msi-Ngn2+MBD1 O.92 0.13 determine the gene expression profile of select pluripotency, Msi-Ngn2+MBD2 79.449.86 ectoderm, endoderm, mesoderm, and neural lineage genes in Msi-Ngn2+MBD3 1220.82 passage 1 and, passage 5 NSLCs compared to HFFs (from Msi-Ngn2+MBD4 O59 0.03 which they were created) and normal human neuroprogenitor Msi-Ngn2+ MeCP2 1.10 - 0.25 cells (hNPCs). The results in Table 8 indicate that all the genes Msi-Ngn2+ AICDA O.690.28 Msi+MBD2 O.79 0.28 related to neural stem cells (some of the significantly Ngn2+ MBD2 1.74 - 1.01 expressed pluripotency markers and mesendoderm markers Nestin + Msi O.91 O.O1 are also expressed in neural stem cells) and the neural lineage Nestin + Ngn2 2.16+ 1.44 were significantly expressed in NSLCS as opposed to HFFs, Nestin + MSI1 NGN2 + MBD2 89.493.18 and the expression pattern was a bit different from hNPCs Nestin + MSI1 NGN2 10.20 - 0.21 indicating that NSLCS are similar to, but not identical, to the Nestin MBD2 OOOOOO hNPCs tested. Passage 5 NSLCs 5 had a higher expression of Nestin + Msi + MBD2 8.45 O.O8 stemness genes than Passage 1 NSLCs. hNPCs had a higher Nestin + Ngn2+MBD2 5.71 - 0.66 expression of neuronally committed genes than NSLCS, indicting their neuroprogenitor status versus the greater stem ness status of NSLCs. US 2014/0038291 A1 Feb. 6, 2014 35
(2) 1 Untreated pCMV6-XL5-Sox2 + Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ pCMV6-XL4-Ngn2+ Fgf-2 Egf+ Fgf-2 Fgf-2 CMV6-XLS-MBD2 2 Untreated pCMV6-XL5-Nanog+ Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ pCMV6-XL4-Ngn2+ Fgf-2 Egf+ Fgf-2 Fgf-2 CMV6-XLS-MBD2 3 Untreated pCMV6-XL4-Octa + Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ pCMV6-XL4-Ngn2+ Fgf-2 Egf+ Fgf-2 Fgf-2 CMV6-XLS-MBD2 4 WPA- Msi1/Ngn2 Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza Fgf-2 Egf+ Fgf-2 Fgf-2 re-reate S VPA- bCMV6-XL5-Musashi Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza Fgf-2 + VPA + 5-Aza Egf+ Fgf-2 Fgf-2 re-reate 6 WPA- bCMV6-XL5-Musashi Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf--Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin re-reate 7 WPA- pCMV6-XL5-Musashi + Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza CMV6-XLS-MBD2 Fgf-2 + Noggin + VPA + 5-Aza Egf-- Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin re-reate 8 WPA- pCMV6-XL4-Ngn2 Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf-- Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin re-reate 9 WPA- CMV6-XLS-MBD2 Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf-- Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin re-reate 2O WPA- Ngn2+ pCMV6-XL5- Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza MBD2 Fgf-2 +Noggin + VPA + 5-Aza Egf-- Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin re-reate 21 WPA- No plasmid Neural proliferation medium + Egf+ Neural proliferation medium + Neural proliferation medium + Egf+ 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf-- Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin re-reate
* Immunohistochemistry analysis performed in parallel with RT-PCR data indicated among all the combinations in this experiment where no cytochalasin B was used, positive Sox2 cells were detectable only in cells transfected with Msi1 Ngn2 with and without MBD2. (2) indicates text missing or illegible when filed
TABLE 8a Results for Human Stem Cell Pluripotency Array (n = 4 for each sample) - Embryonic Stem Cell Markers, Germ. Cell Markers and Trophoblast Markers. For Relative Expression calculations, each sample was normalized to the average Ct of the 6 housekeeping genes (ACTB, 18S, CTNNB1, EEF1A1, GAPD, RAF1), and calibrated to the Untreated HFF (Passage 8) control. Relative Expression values with asterisk (*) indicate values with significant up or down-regulation (>2-fold or <0.5-fold). For these samples, for Ctvalues <35 is considered that the expression of the gene is adequate for quantification. For the Relative Expression values that are >2-fold or <0.5-fold but without asterisk, the values could have significant error due to the low expression of the gene (Ct c 35), and thus the up or down-regulation could be merely a result of the high standard deviation of the high Ctvalues of the genes, or fluctuations in the housekeeping genes. As for the Relative Expression values that are between 0.5-fold and 2-fold, it indicates no significant change in the expression of the gene for these samples. MSI1 NGN2 MBD2 transfected HFF Neural stem-like cells, Untreated HFF Untransfected HFF hNPC neurospheres (Day 9) NSLC Passage 8 Day 9 Passage 4 NSLC. Passage 1 Passage 5 Gene Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Embryonic Stem cell markers
BRIX O3 O.30 O.47 O.10 O.78 O.22 O.78 O.25 O.83 O.10 CD9 O1 O.18 2.46* O.62 186 O.29 2.24: O.19 1.OO O.39 COMMD3 O8 O.S3 O.94 O.36 O.94 O.40 O.98 O.40 1.OS O.S9 DNMT3B O7 O.SO 0.34% O.14 2.96* O.84 1.90 O41 O.35 O.34 EBAF/LEFTY2 OO O.OO 2.10 O.OO 7.95 4.6O 7.79 4.88 70.56* 26.12 FGF4 OO O.OO 2.10 O.OO 1.44 O.OO 1.54 O.OO 1.37 O.OO FOXD3 OO O.OO 2.10 O.OO 1.44 O.OO 7.13 11.18 222.41: 63.43 GABRB3 O6 O.38 4.22* O.71 66.65* 12.52 40.018 4.54 1.62 O.98 GAL OO O.04 9.73% O.32 0.03% O.O1 4.25% O46 2.89* O.83 GBX2 OO O.09 O.04 O.OS 45.28:8 4.59 90.92* 12.14 55.22* 2.36 GDF3 OO O.OO 2.10 O.OO 1.44 O.OO 1.54 O.OO 1.37 O.OO GRB7 O2 O.24 0.30% O16 0.05% O.04 0.29* O.08 0.06% O.O8 IFITM1 O1 O.17 63.96: 6.04 0.04% O.O1 21.80% 4.31 3.35: O.63 IFITM2 OO O.12 3.84% O.89 0.02% O.OO O.65 O.11 0.43% O.09 IL6ST O1 O.21 2.19% O.39 O.8S O.14 1.59 O.26 0.75 O.O6 IMP2 .11 O.66 1.65 O.92 1.06 O.48 O.78 O.26 1.96 0.97 US 2014/0038291 A1 Feb. 6, 2014 36
TABLE 8a-continued Results for Human Stem Cell Pluripotency Array (n = 4 for each sample) - Embryonic Stem Cell Markers, Germ. Cell Markers and Trophoblast Markers. For Relative Expression calculations, each sample was normalized to the average Ct of the 6 housekeeping genes (ACTB, 18S, CTNNB1, EEF1A1, GAPD, RAF1), and calibrated to the Untreated HFF (Passage 8) control. Relative Expression values with asterisk (*) indicate values with significant up or down-regulation (>2-fold or <0.5-fold). For these samples, for Ctvalues <35 is considered that the expression of the gene is adequate for quantification. For the Relative Expression values that are >2-fold or <0.5-fold but without asterisk, the values could have significant error due to the low expression of the gene (Ct c 35), and thus the up or down-regulation could be merely a result of the high standard deviation of the high Ctvalues of the genes, or fluctuations in the housekeeping genes. As for the Relative Expression values that are between 0.5-fold and 2-fold, it indicates no significant change in the expression of the gene for these samples. MSI1 NGN2 MBD2 transfected HFF Neural stem-like cells, Untreated HFF Untransfected HFF hNPC neurospheres (Day 9) NSLC Passage 8 Day 9 Passage 4 NSLC. Passage 1 Passage 5 Gene Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. KIT O2 O.26 1.15 O.30 O.O2: O.OO 0.31% O.09 O.OO: O.OO LEFTB .6 1.15 12.28:8 7.84 5.45 3.15 5.58 2.65 8.96: 4.12 LIFR 2.29 3.57 13.51 16...SS 6.31 7.24 1298 9.81 2.85 4.31 LIN28 4.69 8.62 5.25 8.88 28.38: 19.25 26.97% 8.68 32.13: 14.32 NANOG 7 1.97 18.61 16.43 64.94* 28.32 70.87% 9.88 5.87 3.52 NOG O3 0.27 0.18: O.08 0.18: O.O6 O.22* O.O6 O.O2: O.OO NRSA2 2.04 2.05 6.85 8.8O O38 O.OO 3.89 4.36 O.36 O.OO NR6A1 .1 O.66 1.37 O.31 5.08% 0.37 2.71% O.63 2.04: O.17 PODXL OO O.O7 0.018 O.O1 O8O O.11 2.09: O.04 6.49* O.64 POUSF1 O O.13 0.27% O.17 O89 O.09 O.71 O.09 0.19% O.O6 PTEN OO O.O2 2.68: O.29 O.87 O.04 1.07 O.12 O.8O O.14 RESET O O.12 1.53 O.17 O.94 O.18 1.04 O.21 1.10 O.24 SEMA3A OO O.11 1.99 O.19 O66 O.OS 1.OS O.11 O.90 O16 SFRP2 .1 O.S6 122.57: 14.57 3480.98* 702.37 1500.84% 272.46 2.75 2.85 SOX2 OO O.OO 2.45 O.70 127594.46* 11326.91 88615.76* 150O3.70 137424.37* 26622.02 TDGF1 .4 1.28 2.92 O.68 6.13 1.52 5.46 1.9S 2.2O 1.51 TERT OO O.OO 2.10 O.OO 10.81 18.75 10.74 18.41 6506.88% 893.84 TFCP2L1 OO O.OO 2.10 O.OO 7.84 12.8O 32.49 1O.O1 1.37 O.OO UTF1 OO O.OO 8.21 12.23 27.86 19.24 1.54 O.OO 30.68 25.94 XIST OO O.OO 2.10 O.OO 2460946* 4337.83 22637.95: 3988.10 1.37 O.OO ZFP42 .24 1.06 12.38 12.58 1.41 O.78 2.01 1.85 1.76 O.93 Germ cell markers
DDX4 OO O.OO 2.10 O.OO 1.44 O.OO 5.84 8.60 1911 20.49 SYCP3 S8 1.9S 11.97 8.01 11.12 3.46 1546 11.65 2.25 2.85 Trophoblast markers
CDX2 OO O.OO 2.10 O.OO 1.44 O.OO 1.54 O.OO 1.37 O.OO CGB O2 O.24 2.08: O.74 O.15% O16 0.57 O41 0.09: O.17 EOMES S1 1.14 O.33 O.OO O.71 0.97 O.24 O.OO O.77 1.12 GCM1 2.61 2.80 O42 O.OO 3.25 S.92 S.68 1.44 1.47 2.38 KRT1 OO O.OO 2.10 O.OO 1.44 O.OO 1.54 O.OO 1.37 O.OO
TABLE 8b. Results for Human Stem Cell Pluripotency Array (n = 4 for each sample) - Ectoderm, Endoderm and Mesoderm Markers. For Relative Expression calculations, each sample was normalized to the average Ct of the 6 housekeeping genes (ACTB, 18S, CTNNB1, EEF1A1, GAPD, RAF1), and calibrated to the Untreated HFF (Passage 8) control. Relative Expression values with asterisk (*) indicate values with significant up or down-regulation (>2-fold or <0.5-fold). For these samples, for Ctvalues <35 is considered that the expression of the gene is adequate for quantification. For the Relative Expression values that are >2-fold or <0.5-fold but without asterisk, the values could have significant error due to the low expression of the gene (Ct c 35), and thus the up or down-regulation could be merely a result of the high standard deviation of the high Ctvalues of the genes, or fluctuations in the housekeeping genes. As for the Relative Expression values that are between 0.5-fold and 2-fold, it indicates no significant change in the expression of the gene for these samples. MSI1 NGN2 MBD2 transfected HFF Neural stem-like Untreated HFF Untransfected hNPC neurospheres (Day 9) cells, NSLC Passage 8 HFF (Day 9 Passage 4 NSLC. Passage 1 Passage 5 Gene Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Ectoderm markers
CRABP2 1.04 O.35 26.14 4.28 0.018 O.O1 21.11: 2.80 0.21% O.OS FGF5 1.01 O.15 O.21% O.O7 O.OO* O.OO 0.10: O.O2 O.OO* O.OO GFAP 122 O.84 9.89* 5.46 798.04% 162.37 487.99% 79.84 12052.09: 2984.71 ISL1 1.01 O.12 2.19% 0.27 O.O2: O.O2 0.42* O.08 O.OO* O.OO
US 2014/0038291 A1 Feb. 6, 2014 38
TABLE 8c Results for relative expression of Embryonic Stem Cell, Ectoderm, Endoderm mesoderm, and neuronal markers in untransfected and transfected HFF with Msi1/Ngn2/MBD2 calibrated to untreated HFF (passage 8). Genes with asterisk (*) indicate that the Ct values of the test samples are within the quantifiable range (Ct s35), Suggesting the expression of the gene in the test sample is adequate for quantification. For genes without asterisk, the values may be inaccurate due to the low expression of the gene (Ct c 35) and thus the up or down regulation is merely a result of the high standard deviation of the high Ctvalues of the genes, or fluctuation of the housekeeping genes; the trend for these samples may be correct, but the absolute relative expression values may not. Expression of NGN3 and LIN28 were also tested but these two genes were not expressed in any of the test samples (data not shown). RT-PCR revealed a significant increase of ectodern and neuronal markers. MSI1 NGN2 MBD2- Neural stem-like cells, Untreated HFF Untransfected hNPC neurospheres transfected HFF (Day 9) NSLC Passage 8 HFF (Day 9 Passage 4 NSLC. Passage 1 Passage 5 Gene Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Embryonic Stem Cell Markers
OCT4: .04 O.38 7.27 O.81 6.26 O.OS 6.63 O.S1 3.15 O.S8 OCT4 (5'UTR) .04 O41 O.08 O.OO 2.07 O.11 1.82 O.S3 0.55 O.S9 NANOG (5'UTR) O2 O.32 1929 2.23 11.27 O.89 16.73 6.86 9.94 6.32 FBXO15* OS O46 2.58 O45 3.57 O.23 5.89 122 1.13 O.39 ALPL: O3 O.33 0.57 0.73 652.2O 46.60 19423 10.82 13.04 4.04 SALL4 O2 O.25 9.20 1.35 9.76 O.62 15.84 O.92 2.35 0.55 NROB1 (DAX1)* O1 O.19 1862 4.70 2.64 O.11 11.59 3.17 O.O6 O.OO Ectoderm Markers
ZIC1* O1 O.24 2.01 O.25 1889.80 93.48 1158.21 80.43 15640 12.64 SOX1* OO O.O1 2.05 O.O6 1776.83 128.63 1052.75 243.07 47.98 2.12 CDH1 (E-cadherin)* OO O.O1 2.05 O.O6 264.59 6.22 59.14 7.57 1820 3.73 p63 OO O.O1 68.37 72.49 18.01 5.33 39.72 12.76 37.83 6.76 MSX1 OO O.OS 4.19 O.S6 O.10 O.O1 1.53 O.35 O.09 O.OO NOTCH1* OO O.O7 1.26 O.08 7.38 1.2O 4.51 O.S4 4.75 O.26 SOX2: OO O.O1 2.50 0.57 34O909.59 5659.15, 194495.82 17929.15 219269.76 31399.68 SOX2 (3'UTR)* OO O.O1 7.74 8.11 864191.09 6O2O4.44 452684.8O 26457.7O 61824S.O1 7107.48 Mesoderm Endoderm Markers
CXCR4* OS O46 1245 5.64 SO48.23 17214 2763.82 30.29 3773.11 78.89 Neuronal markers MAP2: O1 O.17 2.98 O.2O 155.33 9.08 88.82 6.48 27.38 O.13 TUBB3: OO O.04 O.38 O.O2 1.15 O.OS O.89 O.OS O.98 O.09 ASCL1 (MASH1)* 29 1.16 1119 O.22 4261846 68.52 23554.16 1588.45 31358.79 23.01.26 NGN28 OO O.O1 2.05 O.O6 1945 6.64 247883.48 164O9.80 968.11 191.73 NGN2 (3'UTR) .83 2.17 1.17 O.76 13.39 S.10 8.45 1.75 539.02 59.72 MSI1 * OO O.O1 263.87 70.10 10O376.36 81.45 479098.OS 2281.62 116105.29 2745.03 MSI1 (3'UTR)* O1 O.20 13.61 2.OO 36O1.96 345.79 21 63.87 59.84 3698.14 160.78 ACHE* OO O.OO 2.00 O.26 2S.OO 3.71 12.84 O.84 21.30 O.30 Glia markers
CNP* O1 O.18 1.43 O.10 3.48 O.S8 2.69 O.12 1.93 O.O7 SOX9* OO O.04 3.54 O.O6 88.25 9.71 41.11 2.70 26.96 O.S3 Note that custom primers (5'UTR) for detecting endogenous gene expression are generally not as sensitive and or effective as standard primers (from the supplier's (Origene) catalog) that dtect overall gene expression (both endogenous and exogenous) of a particular gene,
0307. In another part of the experiment, another batch of Technologies), Nb Active4 (Brain Bits), or CDM II medium; cells that were transfected with Msi1/Ngn2 pCMV6-XL5 these first two were Supplemented with the same cytokines as MBD2 were plated on Poly-Ornithine (30 min at RT) and previously described but with the addition of Fgf-2 (20 Laminin (1 h at RT) coated plates in CDM II medium in 5 ng/ml). On day 12, Fgf-2 was removed from the first two different wells. On day 1, medium in two of the wells was differentiation media while cells in the CDMII medium were switched to the same medium as in condition 1a (Table 7) switched to the NS-A Differentiation Medium (StemCell until day 12. Medium was changed daily until day 12, at Technologies) Supplemented with cytokines without Fgf-2. which point it was switched to either NS-A Differentiation Between day 12 and day 17, media was changed every two to Medium (StemCell Technologies) or NbActive4 (Brain BitsTM) medium that were both supplemented with BDNF (20 three days. During the first 12 days of culture, cells in all 3 ng/ml), NT-3 (20 ng/ml), NGF (20 ng/ml), Retinoic acid (5 media developed into a mix of more spindle shaped cells uM), Noggin (20 ng/ml) and Forskolin (10 uM). These cells compared to untransfected fibroblasts and some into cells showed a typical neural stem-like cell morphology by day 7, with a NSLC morphology; upon removal of Fgf-2 cell mor and proliferated until day 12. During the exposure to either of phology turned into a very pronounced neuronal shape as well the two differentiation media, these NSLC changed to a more as glial cells with a network established between cells as neuronal and glial phenotype as shown in the bright field shown in the bright field pictures that expressed GFAP and pictures, but only expressed GFAP by Day 17. Bill-tubulin by Day 17. 0308 For the other three wells, on day 1 medium was 0309 An additional study was designed to assess the switched to either NS-A Differentiation Medium (StemCell effect of Msil, Ngn2 and MBD2 on their endogenous pro US 2014/0038291 A1 Feb. 6, 2014 39 teins levels in reprogrammed cells. Cells were transfected Day 5 with a large jump in gene expression at Day 7 analysis with the MSI1/NGN2 vector and MBD2 as previously timepoint and stayed at this expression level for the rest of the described and cultured in proliferation condition at 37° C. study period. Expression of the neural stem cell marker Nes 5% CO, and 5% O. Samples were collected at various time tin also started to slowly increase from Day 5 onwards. points from Day 2-10 and analyzed by RT-PCR to investigate Expression of the neuronal genes BIII-tubulin (TUBB3) and the expression of endogenous genes and the expression of Map2b were slightly elevated already from Day 2 onwards, neural stem cell and neuronal genes at different time points. but significantly increased on Day 5 onwards. Expression of RT-PCR revealed a gradual loss of total Msil Ngn2 and a marker for acetylcholine receptors (found in neurons), ace MBD2 gene expression starting from Day 2 to Day 10, with tylcholine esterase (ACHE), was also slightly elevated from the increase in MBD2 expression relative to control having Day 2 onwards, but did not significantly increased until Day been almost completely lost by Day 5. This decrease was 7 onwards. It should be noted that among the neural stem cell associated with a significant activation of endogenous Msil markers that were analyzed, the relative expression of Sox2 and Ngn2 on Day 5, with another jump in endogenous gene was highly and early expressed which could then be directly expression at Day 9 (Table 9). A significant increase in Sox2 or indirectly interact with the exogenous Msi/Ngn2 and/or expression was detected at Day 4, and the expression of this other genes in the activation of Nestin, GFAP, and endog ectoderm/neural stem cell I neuronal gene continued to enous Msil and Ngn2 and other genes that promote the repro increase with each subsequent timepoint (Table 10). GFAP (a gramming and cell fate change, as well as the activation of neural stem cell and astrocyte marker) was slightly elevated neuronal genes like 3111-tubulin (TUBB3), Map2b, and already from Day 2 onwards, but significantly increased on ACHE TABLE 9 RT-PCR analysis of exogenous and endogenous relative expression of Msi1, Ngn2 and MBD2 from Day 2-10 after transfection of fibroblast cells with pCMV-Msi1-Ngn2(Msi1/Ngn2) and MBD2 and cultured for 10 days in proliferation medium. Cells were collected at different time point to analyse endogenous gene expression. Endogenous Endogenous Endogenous MSI1 MSI1 NGN2 NGN2 MBD2 MBD2
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dew. Exp. Dew. Exp. Dew. Exp. Dev. Exp. Dev. Exp. Dev. #1 Day12 1.01 O.18 1.04 O.38 1.01 O.15 1.01 O.15 1.01 O.21 1.OO O.14 Untransfected HFF #2 Day12 HFF 1102.17 91.80 62O.S6 1949 2208.36 375.09 S109 14.69 1.09 OOO 0.83 0.06 Msi1/Ngn2+ MBD2 #3 Day18 HFF 1470.36 164.3S 9SO.Of 152.50 71.57 S2.59 122.66 39.63 1.21 O.O2 0.73 0.08 Msi1/Ngn2+ MBD2 #4. Untransfecte 1.49 NFA 1.01 NFA 1.00 NAA 1.00 NFA 1.02 NFA 1.00 NAA Keratinocytes #5 Day 12 4142.78 872.87 364.2O 60.90 4656.42 232.63 102.01 3.18 O.40 O14 O.74 O.30 Keratinocytes Msi1/Ngn2+ MBD2 #6 Day 18 4830.2O 291.17 486.38 1959 SO.O1 6.99 43.08 13.78 O.4O O.O1 0.67 0.01 Keratinocytes Msi1/Ngn2+ MBD2 #7 Untransfected CD34+ 1.01 O.19 1.00 O.O1 1.01 O16 1.17 O.87 100 0.02 1.OO O.O7 #8 Day 18 CD34+ 3969.52 286.36 147.99 7.08 2.03 0.55 3.72 1.23 O.43 O.06 0.90 O.18 Msi1/Ngn2+ MBD2 hNPC (14-Oct.-09, 7574.57 234.74 1141.14 49.15 8.18 5.64 6.27 S.19 O.S8 O.OO 2.35 0.03 EXPOO67)
TABLE 10 RT-PCR analysis of relative expression of Nestin, Map2b, TUBB3, ACHE, GFAP, and Sox2 from Day 2-10 after transfection of fibroblast cells with pCMV-Msi1-Ngn2 (Msi1/Ngn2) and MBD2 and cultured for 10 days in proliferation medium. Cells were collected at different time point to analyse endogenous gene expression.
NES MAP2 TUBB3 ACHE GFAP SOX2
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Std. Exp. Dev Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dew. Rel. Exp. Dew. #1 Untransfected Day2 1.OO O.04 1.00 O.O1 1.OO O.O3 1.00 OO8 1.01 O.23 1.17 O.87 #2 Msi1/Ngn2+ MBD2+Noggin O.88 OO1 8.59 O.18 138 0.03 S.71 106 4.56 O.08 1.26 O.82 Day2 #3 Untransfected Day3 1.38 O.O7 O.66 O.O3 O.4O O.O2 1.36 OO6 1.9S O.38 2.34 2.29 #4. Msi1-Ngn2+MBD2/+Noggin 1.39 O.O8 431 O.24 O.79 0.09 6.03 O.60 4.66 O.O2 O.96 O.10 Day3 #5 Untransfected Day4 2.43 O.23 1.78 O. 11 O.44 OO1 2.7O O.O2 3.76 O.86 O.93 O.O1 #6 Msi1/Ngn2+ MBD2+Noggin 1.91 OO6 2.81 O.20 O.64 O.O2 6.76 O.64 8.67 1.06 5.37 6.06 Day4 US 2014/0038291 A1 Feb. 6, 2014 40
TABLE 10-continued RT-PCR analysis of relative expression of Nestin, Map2b, TUBB3, ACHE, GFAP, and Sox2 from Day 2-10 after transfection of fibroblast cells with pCMV-Msi1-Ngn2 (Msi1/Ngn2) and MBD2 and cultured for 10 days in proliferation medium. Cells were collected at different time point to analyse endogenous gene expression.
NES MAP2 TUBB3 ACHE GFAP SOX2
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dew. Rel. Exp. Dew. #7 Untransfected Day5 1.40 O.OS 1.13 O.O4 O41 O.O3 1.17 O.37 5.44 O.O2 15.03 8.77 #8 Msi1-Ngn2+MBD2/+Noggin 4.31 O.O8 71.6O 6.43 34 OO1 7.60 0.18 42.28 2.94 66377.25 4089.77 Day5 #9 Untransfected Day7 2.24 OOO 4.02 0.15 22 O.OS 1.10 O-48 7.61 1.24 1.34 O.O2 #10 Msi1/Ngn2+MBD2/+Noggin 3.07 O.11 48.10 2.85 2.7O O.OS 13.11 1.30 3271.10 149.81 442S5.59 2004.08 Day 7 #11 Untransfected Day9 4.37 0.23 14.SS O.96 .75 0.14 3.35 0.36 15.95 O.23 429.09 119.98 #12 Msi1-Ngn2+ MBD2/+Noggin 7.97 0.16 123.SS 3.27 2.79 0.12 16.59 O.O3 3152.25 3.31. 114149.7O 3372.2O Day9 #13 Untransfected Day10 3.48 O44 10.03 0.37 63 0.21 3.20 O.81 5.64 1.92 14.66 S.O3 #14 Msi1/Ngn2+MBD2/+Noggin 7.48 0.22 100.25 6.66 2.87 O.O3 1749 135 3374.O3 22.47 101105.49 3996.44 Day 10
Example IV medium and 25% Proliferation Medium which was supple 0310 Comparison of the NucleofectorTMR. II Device and mented with EGF (20 ng/ml), FGF-2 (20 ng/ml), Noggin (20 the NucleofectorTM.R. 98-Well Shuttle R Device in the Repro ng/ml) and Cytochalasin B (10 ug/ml) and the cells were gramming of HFF into NSLC in Adherent and Floating Con incubated at 37° C., 5% CO, and 5% O. The medium was ditions changed daily with an increased proportion of Neural prolif 0311. HFF cells were cultured as described in Example 1, eration medium up to 100% by Day 4 and a decreased pro and transfected using the NucleofectorTM(R) II Device (Lonza) portion of Cytochalasin B that was completely omitted by as previously described in Example II or the Nucleofec Day 5. Forskolin (10M) was added to the medium from Day torTMR 96-well Shuttle R Device (Lonza). The HFFs were 4 onwards. The cells in floating conditions were pelleted by harvested with TrypILETM (Gibco), and 1x10° cells/transfec centrifugation and their medium changed daily as described tion with the NucleofectorTMR. II Device for 10 min at 90 g for the adherent condition. Cells were collected at Day 3, 7, and 6x10 cells transfection with the NucleofectorTMR and 12 for immunohistochemistry analysis. 96-well Shuttle R Device for 5 min at 80xg. After centrifuga 0312 Fluorescence images were taken with a Cellom tion, the cell pellet was gently resuspended in either 100 ul of icsTM ArrayScan HCS ReaderTM microscopy system to deter Basic NucleofectorTM Solution for the NucleofectorTM(R) II or mine an estimate of the percentage of cells positive for SoX2, 20 ul of SE Solution (Cell line kit. SE, Lonza) for the Nucleo a neural stem cell marker. This analysis revealed that in fectorTMR) 96-well Shuttle(R). For the NucleofectorTMR) II untransfected controls and at 3 days after transfection, no Device, each 100 ul of cell suspension was combined with 2 nuclear Sox2 staining was detectable. However, at Day 7 and different mixes of plasmid DNA (sample 1 was mixed with 2 Day 12 the percentage of Sox2 positive cells increased pro ug of pCMV6-XL5-Msi1 and 2 ugpCMV6-XL5-MBD2, and gressively under all transfection conditions except the sample 2 with 2 ug of Msi1/Ngn2 and 2 ug pCMV6-XL5 pCMV6-XL5-Musashi and pCMV6-XL5-MBD2 Nucleo MBD2). Each cell suspension was transferred into an Amaxa fectorTM(R) II condition. The highest percentage at Day 1.2 certified cuvette and transfected with the appropriate program was obtained with Msi1/Ngn2 and pCMV6-XL5-MBD2 (U-023). Right after transfection, 900 ul of warm CDM1 transfected with the NucleofectorTM(R) 96-well Shuttle(R) medium was added to each cuvette and the sample was trans Device (~80%). The same combination transfected with the ferred into a culture plate coated with Laminin (Stemgent, 10 NucleofectorTM(R) II yielded only ~35% positive cells. The ug/ml) at a cell density of 1x10 to 1.5x10 cells per cm or pCMV6-XL5-Musashi and pCMV6-XL5-MBD2 with the into non-cell culture treated Petri dishes for neurosphere for ShuttleR) produced ~20% positive cells, while generally none mation. The cells were incubated at 37° C., 5% CO over were observed with the NucleofectorTMR. II. The percentage night. However for the NucleofectorTMR 96-well Shuttle(R) of positive cells varied strongly between wells. The staining Device, the steps described before were similar with the fol indicated that the cell population was not homogenous, since lowing exceptions: the cell Suspension was mixed with 0.6 ug fields of densely arranged Sox2 positive cells and complete of each DNA of the same 2 DNA mixes, the cell suspension fields with only negative cells could be found in all cases. In was transferred to a well of a 96-well NucleoplateTM (Lonza) general the ShuttleR) was initially more toxic to cells than the and transfected with the program FF-130TM. After transfec NucleofectorTM(R) however at least in the case of Msi1/Ngn2 tion, 80 ul of warm CDM1 medium was added to each well and pCMV6-XL5-MBD2 shuttle, the Sox2 positive popula and the samples were left for 10 min in the incubator prior to tion rapidly expanded from Day 7 to Day 12 to have twice as being transferred into a laminin coated plate or non-cell cul many Sox2 positive cells as compared to the Nucleofec ture treated Petri dishes at the same cell density as previously torTMR. II. The cells in floating conditions did not form mentioned. For both devices, these steps were repeated for spheres during the 12 day experiment in any of the conditions, each sample that was transfected. Prior to transfection cells Suggesting that the formation of neurospheres requires either were cultured in CDM1 as described in Example I. After 24 the generation of neural stem-like cells inadherent conditions hours, the medium was switched to a mix of 75% CDM first or more time. US 2014/0038291 A1 Feb. 6, 2014
0313 Table 11 shows the percentage of Sox2 positive cells by centrifugation at 150xg for 3 min at room temperature with a typical neural stem cell morphology using both the (RT). The pellet was then resuspended in fresh medium and NucleofectorTM(R) II Device and the NucleofectorTM(R) plated into new uncoated, low-bind cell culture dishes. Cul 96-well Shuttle R Device. The latter had the advantages of tures were incubated at 37°C., 5% CO, 5% O, and were fed requiring a smaller starting material (less cells and less DNA daily for at least two months. required) and in addition gave rise to a higher number of Sox2 0316 To investigate whether a single cell from human positive cells. Moreover a very small population of Sox2 neural precursor cells (hNPCs) and human NSLCs was able positive cells was observed with the Shuttle R Device only to generate a neurosphere (a standard test for proving that a upon transfection with only one neurogenic transcription fac cell is a neural stem cell), neurospheres were dissociated into tor (Msi) in the presence of the DNA demethylator MBD2. single cells and these single cells were isolated and cultured in TABLE 11 Percentage of positive cells for Sox2 after transfection of fibroblast cells with different expression vectors. After transfection the cells were cultured in proliferation medium (StemCell Technologies) supplemented by EGF (20 ng/ml, Peprotech) and FGF (20 ng/ml, Peprotech) for two weeks at 37° C.f5% CO2/5% O2. The percentage of immunopositive cells was determined by Cellonics' and represented as meant SD (n = 3-5). Day3 Day 7 Day 12
Total Total Total Cell Cell Cell Sox2 count Sox2 count Sox2 count
Shuttle MSI1, 1.34 - 0.10 6430566 31 - 20 8.03 10683 - 1112 78.173.10 29341 2S27 NGN2 - MBD2 Msi+ MBD2 1.08 O.61 82S3399 3.19. 3.57 89.53672 1905 - 1788 110822999 NucleofectorTM MSI1, O.87 O3O 21870 4476 14.30 - 1.83 37321 6877 35.937.10 33009 1567 NGN2 - MBD2 Msi+ MBD2 O.64 O.O7 46793 - 8808 O.35016 34854 - 2186 O51 - 0.25 320953236
Example V proliferation medium in Suspension, and neurosphere forma tion was monitored by taking bright field images using light Neurosphere Formation Assay and Cell Differentiation microscope (Nikon, 10x) and by CellomicsTM. These cells Analysis started to proliferate and grew as spheres starting day 6 to day 0314 Based on previous studies showing that greater pro 10. Immunohistochemistry analysis of these spheres on Day portional reprogramming is achieved: by transfecting two 20, revealed immunopositive staining for the neural stem neurogenic genes, this study was designed to evaluate the cells markers Sox2, Musashi, CD133, Nestin, and GFAP. number of reprogramming cells by using, the vector Msil/ Cells also stained positive for BIII-tubulin (a marker for neu Ngn2, containing two neurogenic transcription factors (Msil rons), O4 (a marker for oligodendrocytes), and GFAP (a and Ngn2) and the role of DNA demethylator or DNA methy marker for astrocytes), indicating the tri-potent differentia lation inhibitor (5-azacytidine) and histone deacetylation tion potential of both sets of cells (NSLC and hNPC), and inhibitor (VPA) in the reprogramming process. negative for NGFrec and NeuN (markers for differentiated 0315| HFFs were cultured and treated with cytochalasin B neurons) indicating that the cells were not terminally differ as described in Example 111, and treated simultaneously with entiated. VPA (1 mM) and 5-AZacytidine (0.5uM). After two days of treatment, cells were transfected by Nucleofection as TABLE 12 described in Example II with the constructed vector Msil/ Percentage of positive cells for neural stem cells, and neuronal, astrocyte Ngn2. After preparing the cells, they were mixed with 2 g of and oligodendrocyte lineage markers in neurospheres formed from single total DNA (Msi1/Ngn2) and cells that had not been treated NSLCs and hNPCs cultured in proliferation medium (StemCell with chemical inhibitors (VPA and 5-Aza) were co-transfect Technologies) supplemented by EGF (20 ng/ml, Peprotech) and FGF with MBD2 (2 ug), using the appropriate program (UO23). (20 ng/ml, Peprotech) for 20 days at 37° C./5% CO2/5% O. The samples were transferred into a coated culture plate with The percentage of positive cells was determined by Cellomics TM and Laminin (10 ug/ml, Sigma) and incubated in a humidified 37° represented as meant SD. C./5% O/5% CO, incubator. The medium was changed to % of positive cells NSLCS NPCs the proliferation basal media, Neural Proliferation Medium Musashi 91.8 6.8 88.6 7.9 (NeuroCultTM proliferation Kit, StemCell Technologies), Nestin 78.65.7 754 - 12.O with the presence of Noggin (20 ng/ml, Peprotech), recom GFAP 69.27.4 786 - 8.4 binant hFGF (20 ng/ml, Peprotech), and recombinant hEGF BIII-tubulin 85.6 6.4 76.6 8.4 P75 O O (20 ng/ml, Peprotech). Following 6 days of transfection, cells Neun O O were harvested using AccutaseTM (Millipore), centrifuged O4 65.4 6.6 71.4 7.5 (300xg, 5min, RT) and plated in uncoated cell culture dishes CD133 O O in NeuroCultTMNSC Proliferation medium to investigate the capacity to grow cells in Suspension as neurospheres or on Laminin coated-plates for adherent culture. To prevent loss of 0317| HFF cells were cultured as described in Example 1, floating spheres during media changes, cells were sedimented and transfected using the NucleofectorTM II device (Lanza) as
US 2014/0038291 A1 Feb. 6, 2014 43
Sample Information methylation inhibitor, indicating that down-regulation of dif ferentiated markers of fibroblast cells requires DNA dem 0318 ethylation. The expression of ectoderm genes Such as Msil, Sox2, and Nestin was remarkably increased following trans fection in conjunction with DNA demethylation. The expres sion of neuronal markers, such as synaptoga mini (a synaptic Sample ID Sample Name TLDA Port vesicle protein) and NeuroD1 was up-regulated in transfected HFF Ctrl cells with Msi1/Ngn2/MBD2, and slightly increased in trans ReNcell Undifferentiated Ctrl Msi1-Ngn2/MBD2 fected cells with Msi1/Ngn2/VPA and 5-AZA. The selected Msi1-Ngn2/MBD2 three markers of oligodendrocytes were detected in the trans Msi1-Ngn2/VPA + AZA fected cells with a strong increase of Olig2. Two markers for Msi1-Ngn2 astrocytes, GFAP and ALDH1L1, were enhanced following Msi1-Ngn2/MBD2, neurospheres transfection. The results Support the idea that neurospheres Msi1-Ngn2/MBD2, neurospheres are composed of heterogeneous progenitor Subtypes. 0320 Among the neurotrophic factors, expression of 0319. As shown in Table 14, fibroblast-specific genes CNTF was slightly increased in the reprogrammed cells. The (Col3A1., Lox, S100A4) were down-regulated in repro expression of GAP-43 and neuropeptide Y (NPY) were the grammed cells, indicating the loss offibroblast-specific genes most annotated genes. GAP-43 has long been acknowledged following transfection (note that not all cells got transfected to play a pivotal role in axonal plasticity and is used as a and reprogrammed, so the presence of fibroblast-specific marker of regenerating neurite outgrowth and synaptogen gene expression in the cultures is mostly from the un-pro esis, both in embryonic development and in neuronal regen grammed fibroblasts left in the culture). The expression of eration in injured brain and spinal cord. Expression of recep these genes is observed to increase when HFFs were trans tors for growth and neurotrophic factors was increased. Such fected in the absence of DNA demethylator or the DNA as neurotrophic receptor tyrosine kinase expression. TABLE 1.4 Gene array analysis was performed after one month of transfection of human fibroblast cells with Msi1/Ngn2, in the presence MBD2 or VPA and 5-Aza. Cells were cultured on coated culture plates as adherent cells or on untreated culture plates as neurospheres in proliferation medium (StemCell Technologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml). Untransfected cells were considered as negative control and ReNeel (Millipore) as positive control. Relative expression to #1 HFF Ctrl
#3 hia #7 #8 #2 Msi1- Msi1- #5 #6 Msi1-Ngn2. Msi1-Ngn2, ReNcell Ngn2/ Ngn2, Msi1-Ngn2. Msi1- MBD2, VPA + AZA, Symbol Common name and description Undiff MBD2 MBD2 VPA + AZA Ngn2 neurospheres neurospheres Fibroblast ECM component COL3A1 Collagen, type III, alpha 1, fibroblast O.OO O.O3 O.O2 O.O2 11.92 O.OO O.OO marker LOX Lysyl oxidase, ECM component O.O1 O.O3 O.O1 O.O1 2.38 O.OO O.OO FSP1 Fibroblast transcription site-1, O.04 O.04 O.O6 O.OS 3.22 O.OS O.OS enzyme for ECM remodeling Neuron markers
SYT1 Synaptotagmin1, a synaptic vesicle 106.49 108.40 78.66 26.72 22.42 37.61 16.80 protein in neurons SNAP2S SNAP25, mature neuron marker 4.72 6.10 7.89 3.11 3.19 6.47 4.OO NEUROD1* Neurogenic differentiation 1, neuron 2.32 93.35 100.84 2.02 3.11 271.11 10.23 marker Oligoden drocyte markers
MBP* Myelin Basic Protein, mature 2.32 48.53 18.11 6.94 667.56 16.67 1.67 oligodendrocyte marker NKX2-2* NK2 homeobox 2, remyelination 2.32 75.31 54.65 1.66 3.11 1.67 1.74 OLIG2* Oligodendrocyte lineage transcription 2856.4 15594 67369 38733 3.11 9242O 101733 factor 2, oligodendrocyte progenitor Astrocyte markers
ALDH1L1 * Aldehyde dehydrogenase 1 family 6.20 3.77 4.65 1.66 O.O2 5.87 9.59 member L1, astrocyte DIO2: Deiodinase iodothyronine type II, 23.2O O.OO O.OO O.OO O.S1 O.OO O.OO astrocyte marker US 2014/0038291 A1 Feb. 6, 2014 44
TABLE 14-continued Gene array analysis was performed after one month of transfection of human fibroblast cells with Msi1/Ngn2, in the presence MBD2 or VPA and 5-AZa. Cells were cultured on coated culture plates as adherent cells or on untreated culture plates as neurospheres in proliferation medium (StemCell Technologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml). Untransfected cells were considered as negative control and ReNeel (Millipore) as positive control. Relative expression to #1 HFF Ctrl
#3 i4 #7 #8 #2 Msi1- Msi1- #5 #6 Msi1-Ngn2. Msi1-Ngn2, ReNcell Ngn2, Ngn2. Msi1-Ngn2. Msil- MBD2, VPA + AZA, Symbol Common name and description Undiff MBD2 MBD2 VPA + AZA Ngn2 neurospheres neurospheres GFAP Glial fibrillary acidic protein, 3342.1 6899.O 6291.0 48009 1.27 3.118.7 3222.0 astrocyte marker NSCS markers
NCAM1 NCAM1, neuroblast marker 23.21 43.90 24.45 12.72 1.13 31.93 36.70 PDGFRA Plate-derived growth factor receptor O.OS O.O1 O.O1 O.OO 4.42 O.OO O.O1 alpha, oligodendrocyte progenitor cells NES Nestin, neural progenitor 5.76 1984 19.56 3.46 4.23 16.57 8.36 MSI1 * * * Musashi I, neuroblast marker S120.3 5985.2 5262.7 5.645.1 204:34 3179.6 4113.6 SOX1* SOX1, neural progenitor 679.21 223.59 373.14 361.67 3.11 287.82 323.23 SOX2: Sox2, NSCs 1924084 2265299 1889166 1014816 3.11 1313765 1103212 Neuro trophic? Growth Factor
GDNF: Glial cell derived neurotrophic factor O.O1 O.O2 O.O2 O.OO 1.69 O.OO O.OO NGF: Nerve growth factor O.OO O.OO O.OO O.OO 148 O.OO O.OO BDNF Brain derived neurotrophic factor O.O3 O.O9 O.09 O.OS O.82 O.O2 O.O1 CNTF8 Ciliary neurotrophic factor 9.25 4.32 3.11 2.90 64.05 2.31 3.39 GAP43 Growth associated protein 43, neural 917.52 3506.5 1530.8 452.75 S84.00 746.25 578.52 regeneration NRG1: Neuregulin 1, neural regeneration O.O1 O.OO O.OO O.OO O40 O.OO O.OO NPY: Neuropeptide Y, interneuron 2.32 675.69 465.04 153.54 3.11 1244.O 130.38 CSF3% Colony stimulating factor 3, neural O.SO O.O3 O.O2 O.S8 18.62 O.O2 O.O2 regeneration BMP4 Bone morphogenetic protein 4, O.83 O.26 O.74 O.45 11.03 O.09 O.O7 remyelination marker TGFB1 Transforming growth factor, beta 1 O.85 2.39 O.92 O.83 O.65 O.45 O.S8 Angio genesis
VEGFA Vascular endothelial growth factor 2.77 14.93 1S.O1 2.67 3.82 2.80 3.21 Neuro trophin? Growth Factor Receptors
NGFR/P75 NGFR, neurotrophin receptor 5.35 3.29 5.78 9.10 7.53 7.26 17.51 EGFR Epidermal growth factor receptor O.89 O.77 O.86 0.79 1.63 1.44 1.25 KDR: Kinase insert domain receptor, 210.87 259.42 263.45 51.85 O.O7 11.23 17.50 growth factor receptor
0321) Further analysis and quantification of the adherent TABLE 1.5 population of NSLCs showed that cells were positively The percentage of cells stained positive for neural stem cell markers and stained for Sox2 (93.43+1.9%), nestin (60.76+5.7%), and fibroblast markers in untransfected cells and transfected cells with GABA (37.48+4.9), while these markers were undetectable pMsi1/Ngn2/MBD2. Transfected cells (NSLCs) possess a high percentage of neural stem markers but a very low percentage of fibroblast markers as in untransfected cells. Furthermore, these cells stained posi compared to untransfected cells. The percentage of immunopositive cells tive for p75NTR (31.15+1.6), BIII-tubulin (37.55+0.6%) and was determined by Cellomics TM and represented as GFAP (16.47+0.9). However, untransfected HFFs only meant SD (n = 5). stained positive for HFF markers, such as fibronectin and Untransfected Transfected fibroblast fibroblast fibroblast protein marker, while these markers were undetect cells (% of average cells (% of average able in reprogrammed cells, demonstrating that the repro Marker protein positive cells it stclv) positive cells it stclv) grammed cells lost markers of the original cells and adopted Sox2 93.43 - 1.9 1.90 - O.S morphology and markers of neural stem cells and a neuronal Nestin 60.765.7 O.84 O2 lineage. US 2014/0038291 A1 Feb. 6, 2014 45
TABLE 15-continued TABLE 16-continued The percentage of cells stained positive for neural stem cell markers and Doubling time of NSLCs over serial passages. NSLCs were maintained in fibroblast markers in untransfected cells and transfected cells with proliferation conditions for 35 passages in a 37° C., 5% CO2 and 5% pMsi1/Ngn2/MBD2. Transfected cells (NSLCs) possess a high percentage O2 incubator. The time required for the cell population to double (g) was of neural stem markers but a very low percentage of fibroblast markers as calculated for each passage, and was defined as g = (In2)/k, where k compared to untransfected cells. The percentage of immunopositive cells was the number of generations that occured per unit time (t) defined as, was determined by Cellomics TM and represented as k = (In N- In No)/t, where N, was the final cell meant SD (n = 5). number and No the initial seeded cell number. The average generation time was 25.4 h over 35 passages. Untransfected Transfected fibroblast fibroblast Passage cells (% of average cells (% of average number Time (h) LNNo LNN, k (h) g(h) Marker protein positive cells it stolv) positive cells it stolv) 34 96 13.122 16.077 O.O31 15.519 p75NTR 31.15 1.6 3.95 1.7 35 96 13.122 16.077 O.O31 15.519 NCAM 26.84 3.8 O.87 O2 S100 41.80 O.6 1.60 O.3 GFAP 16.47 0.9 3.84 O.9 BIII-Tubulin 37.SS O.6 1.90 O.9 Gene Expression Microarray GABA 37.48 4.9 2.54 - O.S Fibronectin 1.050.7 94.190.9 0323 Microarray expression analysis was performed to Fibroblast marker protein 4.81 - 1.0 S.O.30 - 7.8 get a global overview to compare the gene expression profile of passage 7 NSLC to both HFF (the cells that the NSLC were 0322. This study showed as well that NSLCs have the created from) and hNPCs. NSLC (n=3), HFF (n=2), and capacity to proliferate in culture and exhibit stable morphol hNPC (n-3) were resuspended in RNAlaterTM (Qiagen) and ogy, gene and protein expression that were maintained for the shipped to Genotypics (India) where the samples were pro entire study period, which was for over five month in culture cessed and the Gene Expression Microarray was performed. 0324. In brief, Genotypics extracted RNA from the (Table 16). samples and performed Quality Control using an Agilent BioanalyzerTM. Labelling was done using Agilent’s Quick TABLE 16 AmpTM kit (cDNA synthesis and in vitro transcription), fol Doubling time of NSLCs over serial passages. NSLCs were maintained in lowed by Labelling QC. Hybridization was then performed proliferation conditions for 35 passages in a 37° C., 5% CO, and 5% using the 8x60K array, and scanning was done using high O2 incubator. The time required for the cell population to double (g) was calculated for each passage, and was defined as g = (In2)/k, where k throughput Agilent scanner with SureScanTM technology. The was the number of generations that occured per unit time (t) defined as, Agilent Feature Extraction software was used for automated k = (In N- In No)/t, where N, was the final cell feature extraction, followed by Raw Data QC and Image QC. number and No the initial seeded cell number. The average generation Advanced Data Analysis was then performed, including Path time was 25.4 h over 35 passages. way and Gene Ontology analyisis using Agilent's Gene Passage Spring GXTM v10.0 and Genotypic's Biointerpreter Soft number Time (h) LNNo LNN, k(h) g(h) ware. The NSLC samples were compared to the HFF samples 2 68 S13 5.577 O.O24 38.6SS (Set 1) and hNPC samples (Set 2) The NSLC samples had a 3 216 S13 6.195 O.O22 31.977 global gene expression pattern that was much closer to the 4 92 S13 8.258 O.O3S 39.730 hNPCs than the HFFs from which the NSLCs were created. 5 44 S13 6.258 O.O33 21.036 Pearson correlation analysis revealed that NSLCs are closely 6 44 S13 6.258 O.O33 21.036 7 44 S13 5.702 O.O29 33.824 related to hNPCs, including in terms of neuronal lineage 8 68 S13 5.870 O.O26 26.729 markers, regenerative genes and migration genes. These data 9 2O S13 6.811 O.O31 32.548 confirm that NSLCs are similar, but not identical, to hNPCs. 10 44 S13 S415 O.O27 35.58O 11 2O 3.122 5.895 O.O23 30 0325 Microarray analysis revealed an up-regulation of 12 2O S13 5.747 O.O3S 19.645 neural precursor genes in the NSLC samples as compared to 13 68 S13 5.870 O.O26 26.729 the HFF samples. ACTL6A and PHF10, which both belong to 14 68 2.429 5.870 O.O2O 23.847 the neural progenitors-specific chromatin remodelling com 15 68 S13 5.520 O.O24 29.059 plex (npbaf complex) and are required for the proliferation of 16 92 S13 6.167 O.O24 28S96 17 44 S13 S.239 O.O26 36.791 neural progenitors, were up-regulated by 2.9-fold and 2.3 fold 18 68 S13 5.790 O.O2S 37.229 respectively. MSI2, which plays a role in the proliferation and 19 2O 3.122 5.870 O.O23 30.276 maintenance of stem cells in the central nervous system, was 2O 44 3.122 6.249 O.O22 31.922 21 96 3.122 5.761 O.O27 25.214 up-regulated by 6-fold (Table X1). Glia genes were up-regu 22 2O 3.122 5.870 O.O23 30.276 lated in the NSLC samples as compared to the HFF samples. 23 2O 3.122 5.761 O.O22 31.518 GFAP, is a neural stem cell- and astrocyte-specific marker 24 96 3.122 5.687 O.O27 25.943 that, during the development of the central nervous system, 25 96 3.122 6.O13 O.O3O 23.022 distinguishes astrocytes from other glial cells, is highly up 26 96 3.122 6.067 O.O31 22.599 27 96 3.122 6.300 O.O33 20.938 regulated in the NSLC sample as compared to HFF (690 28 2O 3.122 6.482 O.O28 24.752 fold). OLIG1, which promotes formation and maturation of 29 96 3.122 6.380 O.O34 2O424 oligodendrocytes, especially within the brain, is also highly 30 96 3.122 6.300 O.O33 19938 31 2O 3.122 6.483 O.O28 22.752 up-regulated in NSLC sample as compared to HFF (370-fold) 32 96 3.122 6.062 O.O31 20.640 (Table X2). 33 96 3.122 6.300 O.O33 20.938 0326 Table X3 lists a subset of regenerative genes that are up-regulated in the NSLC samples as compared to the HFF US 2014/0038291 A1 Feb. 6, 2014 46 samples. SOX2, a gene critical for early embryogenesis and TABLE X1-continued for embryonic stem cell pluripotency as well as neural stem cells, is highly up-regulated in the NSLC samples as com Up-regulated Neural Precursor genes (NSLC vs. HFF pared to the HFF samples (5000-fold). CCND2, which is Fold change of NSLC essential for the control of the cell cycle at the G1/S (start) GeneSymbol Accession Number compared to HFF p-value transition, is also up-regulated in NSLC samples (70-fold as compared to HFF samples). As shown in Table X4, numerous LIMK1 NM 002314 2.44 O.OO2 fibroblast genes were down-regulated in the NSLC samples MAPK8IP1 NM 005456 5.88 O.OO1 MCHR1 NM 005297 68.19 O.OO1 as compared to the HFF samples. This shows that the NSLC MEF2C NM 002397 2.91 O.OOO lose the expression of numerous fibroblast genes as it gets MSI2 NM 170721 6.76 O.OOO reprogrammed from HFF to NSLC. NMB NM 021077 3.65 O.OOO 0327 Table X5 show that neural precursor genes were also NOS2A NMOOO625 279.45 O.OOO up-regulated in the NSLC samples as compared to the hNPC NOTCH1 NM O17617 6.75 O.OOO samples. BDNF, which promotes the survival and differen NPAS3 NM 022123 187.85 O.OOO PHF10 NM 018288 2.28 O.OO1 tiation of selected neuronal populations of the peripheral and PHLPP NM 194449 8.84 O.OOO central nervous systems during development, is even more SMAD1 NMOO5900 4.74 O.OOO highly expressed in NSLC samples than in hNPC samples SNTG1 AL161971 34.05 O.OOO (34-fold up-regulation). Table X6 shows that a subset of Glia SP8 NM 198956 1392.67 O.OOO genes are also up-regulated in the NSLC samples as com STAU2 AKOO2152 3.35 O.OOO pared to the hNPC samples. GFAP, a neural stem cell- and STIL NMOO3035 4.94 O.OO3 astrocyte-specific marker that, during the development of the Fold change represents the up-regulation of the gene in the NSLC samples as compared to central nervous system, distinguishes astrocytes from other the HFF samples, (n = 2 for HFF samples, n = 3 for NSLC samples). glial cells, is more highly expressed in NSLC samples than hNPC samples (13-fold). PLP1, the major myelin protein of the central nervous system which plays an important role in TABLE X2 the formation or maintenance of the multilamellar structure Up-regulated Glia genes (NSLC vs. HFF of myelin, is also more highly expressed in NSLC samples Fold change of than in hNPC samples (20-fold). NSLC compared to 0328 Regenerative genes were also up-regulated in the GeneSymbol Accession Number HFF p-value NSLC samples as compared to the hNPCsamples (TableX7). ASTN1 NM 004319 51.44 O.OOO BMP2, a neural crest marker, but which induces growth espe ATP1B2 NM OO1678 186.64 O.OOO cially of cartilage and bone formation and BMP4, which in B3GAT1 NM 018644 1784.49 O.OOO turn induces cartilage and bone formation and acts in meso BCL2 NM 000633 2.65 O.OO2 derm induction, tooth development, limb formation and frac BMP7 NM OO1719 41.35 O.OOO CA14 NM 012113 43.44 O.OOO ture repair, but also in neural stem cells, were both more CLCN2 NM 004366 4.18 O.OOO highly expressed in NSLC samples than in hNPC samples CNDP1 NM O32649 4.39 O.O10 (18-fold and 20-fold respectively). GAP43, which is a major CP NMOOOO96 93.08 O.OO2 component of the motile growth cones that form the tips of CXCR4 NM OO10O8540 4124.29 O.OOO elongating axons was more highly expressed in NSLC ERBB4 NMOO5235 S3.22 O.OO6 FABP7 NM OO1446 18702.36 O.OOO samples than hNPC samples (4-fold). This suggests the GAB NM 207123 2.44 O.OO1 regenerative potential of NSLC. HOXB4, a transcription fac GFAP NM 002055 696.51 O.OOO tor that is involved in development and also in the expansion GJB2 NM 004.004 13.89 O.OO1 of neural stem cells as well as hematopoietic stem and pro ITGB8 NMOO2214 8.48 O.OOS KCNJ 10 NMOO2241 263.42 O.OOO genitor cells in vivo and in vitro making it a potential candi LMO3 NM 018640 194.32 O.OOO date for therapeutic stem cell expansion, was also more MAP6D1 NM O24871 3.99 O.OOO highly expressed in NSLCs than in hNPCs. This data indi MAPT NM O16835 2.38 O.OO1 cates that NSLCs are more stem-like or have more stem NDE NM O17668 2.21 O.OO2 NEFL NM OO6158 10.30 O.OO1 ness than hNPCs. NKX6-2 NM 177400 10.83 O.O26 NOVA2 NMOO2516 7.51 O.OOO TABLE X1 NTN NMOO4822 S.29 O.O15 NTRK3 NM 00101.2338 15.32 O.OOO Up-regulated Neural Precursor genes (NSLC vs. HFF OLIG1 NM 138983 372.11 O.OOO OLIG2 NMOO5806 1632O O.OOO Fold change of NSLC PARD6A NM 016948 4.12 O.OO1 GeneSymbol Accession Number compared to HFF p-value PASK NM O15148 3.89 O.OO1 PAX6 NM OO1604 28.53 O.OO1 ACTL6A NM 178042 2.90 O.OOO PDCD11 ENSTOOOOO369797 2.23 O.OO1 ADAM9 NM 001005845 2.64 O.004 PDE6B NM 000283 5.55 O.OO1 AIFM1 NMOO4208 2.45 O.OOO PER1 NM 002616 2.43 O.OO1 BCAT1 NMOO5504 3.23 O.OOO PLP1 MS4927 351.09 O.OOO BMP2 NM OO1200 17.49 O.OOO PTK2 NM 153831 4.22 O.OOO DLL1 NMOO5618 40.32 O.OOO QKI NM 206855 8.75 O.OO3 EDNRB NMOO3991 933.03 O.OOO S1 OOB NM OO6272 456.OO O.OOO ERBB4 NMOO5235 S3.22 O.OO6 SLC1A3 NMOO4172 49.49 O.OOO GMNN NM O15895 4.42 O.OOO SORL1 NMOO3105 27.61 O.OOO HESS BCO87840 102.33 O.OOO SOX9 NMOOO346 27.82 O.OOO KIF1B NM O15074 9.45 O.OO2 SPRY2 NM 005842 15.83 O.OOO
US 2014/0038291 A1 Feb. 6, 2014 49
TABLE X6-continued bit antibody S100B (1:100, Abcam), and Chicken antibody CNPase (1:50, Abcam). Secondary antibodies are added in Up-regulated Glia genes (NSLC vs. hNPC 5% normal goat serum/PBS as follows: Goat anti mouse Fold change of NSLC Alexa546TM (1:200, Invitrogen), Goat anti rabbit Alexa488TM GeneSymbol Accession Number compared to hNPC p-value (1:200, Invitrogen), and Goat anti-chicken cy5 (1:100, Jack PDCD11 NM O14976 2.48 O.OOO son ImmunoResearch Labs). PLP1 NM 000533 20.64 O.OOO 0330 Immunohistochemistry analysis showed that TGFB1 NMOOO660 6.66 O.OOO TSPAN12 NM 012338 2.58 O.OO6 Nb Active medium promoted the differentiation equally to neuronal (48.66+14.07%, BIII-tubulin) and potential early oligodendrocyte lineages (50.01 +4.04%, CNPase) and to a TABLE XT lower percentage of astrocyte cells (2.68+1.13%, S100B), while NS-A differentiation medium induced the differentia Up-regulated Regenerative genes (NSLC vs. hNPC tion mainly to neurons (64.89+4.11%, BIII-tubulin) and Fold change of NSLC astrocytes (35.94+4.04%, S100beta), and a low percentage of GeneSymbol Accession Number compared to hNPC p-value potential early oligodendrocytes cells (8.68+2.71%, ATR NM 001184 2.57 O.OOO CNPase). The NSC-A medium was selected over NbActive BMP2 NM OO1200 17.71 O.OOO for further differentiation studies. Differentiation of cells in BMP4 NM 001202 20.55 O.OO1 CAV3 NM OO1234 26.23 O.OOO NS-A differentiation medium promote the differentiation of CCND1 NM O53056 10.34 O.OOO hNPC and NSLC similarly as shown in Table 17 by the CDKN2A NM 058197 5.57 O.OOO CEBPB NM 005194 2.58 O.OOO decrease of the percentage of SOX2, musashi and nestin posi GAL NM O15973 12.21 O.OOO tive cells. NSLCs were differentiated to neuronal (74.3+0.1, GAP43 NM 002045 4.27 O.OOO GABA), astrocyte lineage (65.6+0.0, S100beta) and to a HOXB4 NM 024015 133.37 O.OOO SMAD3 NMOO5902 2.27 O.OOO lower percentage of oligodendrocyte cells (5.2+0.6, CNPase). The same pattern of tripotent lineage differentia tion was observed with hNPCs (Table 17). TABLE 17
The percentage of cells stained positive for neural stem cell and neuronal lineage markers in transfected and untransfected cells. NSLCs and hNPCs were cultured in NS-A-differentiation medium supplemented with BDNF (20 ng/ml) and FGF (40 ng/ml), cultures were incubated at 37°C., 5% CO2, 5% O2 for three weeks. The percentage of immunopositive cells was determined by Cellomics TM and represented as meant SD (n = 5).
Sox2 Nestin Musashi S1 OO O4 GABA
Tripotent hNPC 73.8 OS 46.15.2 22.1 7.O 20.8 - 13 6.4-2.9 68.5 - 1.6 medium NSLC 68.6 3.9 41.05.4 26.7 SO 65.6 O.O 8.20.6 743 O.1
0329. In order to investigate the differentiation potential of 0331. Several additional antibodies to neuronal antigens NSLCs to neuronal lineages (Neurons, astrocytes, and oligo were used to characterize, in more detail, the nature of differ dendocytes), neurospheres were dissociated and plated in entiated cells. Antibodies against microtubule-associated laminin/poly-D-Lysine (10 ug/ml, Sigma) in differentiation protein (MAP2b), NCAM, and synaptophysin were used as medium for two weeks. The differentiation towards neuronal recommended by the antibody manufacturer. After three lineage was performed using two different mediums: NbAc weeks in differentiation medium, there was a differentiation tive medium (Brain BitsTM) supplemented with Brain Derived induced reduction in markers of precursors cells and an Neurotrophin Factor (BDNF, 20 ng/ml, Peprotech), all-trans increase in mature neuronal markers. The percentage of neu retinoic acid (ATRA, 5uM, Spectrum), and bFGF (40 ng/ml, ral precursor markers such as Sox2 were decreased during Peprotech) or NeuroCultTM differentiation medium (NeuroC differentiation, while p75NTR, BIII-tubulin and GABA were ultTM Differentiation kit, StemCell Technologies), supple increased with lengthening differentiation time; however, O4 mented with BDNF (20 ng/ml, Peprotech) and bFGF (40 positive cells were very low after 3 weeks of differentiation of ng/ml, Peprotech). After two weeks in culture, the cells were hNPCs (6.4+2.9) and NSLCs (8.2+0.6). Synaptophysin, an stained with the neuronal marker BIII-tubulin, astrocyte antibody used to identify functional neuronal cells, was markers GFAP and 51 0013, andoligodendrocyte marker increased following 2 and 3 weeks of differentiation, indicat CNPase. The cells were fixed with 4% formaldehyde and the ing maturity of the neuronal cells. GABA and acetycholine primary antibodies were added in 5% normal goat serum/PBS markers were increased following 2 weeks of differentiation as follows: Mouse antibody BIII-tubulin (1:200, Abcam), rab and decreased at week 3. US 2014/0038291 A1 Feb. 6, 2014 50
0332 The morphological changes and expression of a a neuronal cell type. Useful criteria include morphological number of neuronal antigens and genes show that the above features (long processes or neurites), physiological, and/or method results in normal and viable neuronal cells. Addition immunological features Such as expression of a set of neu ally, the newly formed neuronal cells have the morphological ronal-specific markers or antigens. Furthermore, NSLCs criteria of neurons. In addition to the above markers, the readily turn into a tripotent-like precursor cell with differen differentiated cells were evaluated by characterizing morpho tiation potential to a high percentage of neuronal, astrocytes logical markers of neurite differentiation. Neuron type cells and lower percentage of oligodendrocyte populations. (cells strongly expressing BIII-tubulin) showed neurite for mation after differentiation, including an increase in the aver age number of neurites per neuron (from e.g. 1.38+0.1) The Example VI same pattern was observed in BIII-tubulin positive cells. Accordingly, the average neurite length (118.3-3.5 um) and Implication of BMP Signaling Pathway in the the number of branch points (3.28+0.3) per neuron also Reprogramming of HFFs increased. The differentiated neuron-like cells developed long neurites that were greater than three cell diameters in 0336. This study was designed to evaluate the role of Nog length with a growth cone at the end, expressed neuron gin in the process of de-differentiation of HFFs towards specific genes, and stopped proliferating after the induction of NSLCs. HFFs were cultured and treated with cytochalasin B differentiation. as described in Example III. After two days of treatment, cells 0333. Further differentiation was performed using an opti were transfected by Nucleofection as described in Example II mised medium that promoted the differentiation towards oli with the constructed vector Msi1/Ngn2. Briefly, after prepar godendrocyte lineage. NSLCs and hNPCs were cultured in ing the cells, they were mixed with 2 ug of total DNA (Msil/ NS-A differentiation medium as described previously Ngn2) and were co-transfect with MBD2 (24), by the supplemented with FGF-2 (10 ng/ml, Peprotech) and sonic Amaxa's NucleofectorTM according to the manufacturers hedgehog (SHH, 100 ng/ml, Peprotech) for 4 days. After 4 protocol. The samples were then transferred into a Laminin days medium was changed to NS-A differentiation medium (10 g/ml. Sigma) coated culture plate and cultured in the supplemented by T3 (60 ng/ml, Peprotech), IGF1 (10 ng/ml, presence of Neural Proliferation Medium (NeuroCultTM pro Peprotech), NT-3 (10 ng/ml, Peprotech), and PDGF (10 liferation Kit, StemCell Technologies) with recombinant ng/ml, Peprotech). Cells were cultured for 20 days at 37°C., hFGF (20 ng/ml, Peprotech), recombinant hEGF (20 ng/ml, 5% CO. Peprotech), and with or without the presence of Noggin (20 TABLE 1.8 The percentage of cells stained positive for neural stem cell and neuronal lineage markers in transfected and untransfected cells. NSLCs and hNPCs were cultured in differentiation medium supplemented with SHH (100 ng/ml, Peprotech), T3 (60 ng/ml, Peprotech), IGF1 (10 ng/ml, Peprotech), NT-3 (10 ng/ml, Peprotech), and PDGF (10 ng/ml, Peprotech) to induce different iation towards oligodendrocytes. The percentage of immunopositive cells was determined by Cellonics' and represented as meant SD (n = 5). % of positive cells Sox2 Nestin Musashi S100 O4 GABA hNPC 84.33.7 26.944 51.8 2.9 33.4 19 40.1 - 6.4 89.6 O.8 NSLC 693 - 4.4 24.3 2.5 45.1 111 51.6, 9.5 8.5 - O.6 76.91.4
0334 Quantification of the differentiation of hNPCs and ng/ml, Peprotech). Samples were collected at different time NSLCs revealed a population of cells that were positively points (1, 3, 4, 6, and 8 days) to analyze neuronal gene stained for O4. As shown in Table 18, the percentage of O4 expression by RT-PCR and protein expression levels by positive cells was more pronounced in differentiated hNPC immunohistochemistry. (40.1+6.4%) as compared to differentiated NSLCs (8.5+0. 0337 Fluorescent immunohistochemical staining was 6%) when using the above differentiation protocol. performed on samples after 4 days of transfection as previ 0335. This study showed that transfecting the cells with ously described in Example I. Transfected cells were stained one or two neurogenic transcription factors in the presence of and analyzed for expression of Sox2, the percentage of Sox2 a DNA demethylator or small molecules for epigenetic modi was 33.3+1.00% in the presence of Noggin compared to fication achieves stable reprogrammed cells (NSLCs). Like a 27.5+0.50% without the presence of nogginat day 4. RT-PCR DNA demethylator, epigenetic modification (inhibition of analysis of relative expression of neuronal precursor cell acetylation and methylation) are sometimes useful in boost markers such as nestin and Sox2 after transfection of HFFs ing the reprogramming process. These cells possess and with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5-MBD2 with retain neural stem cell properties as determined by: (1) the expression of neural stem cell genes and proteins, (2) the or without the presence of Noggin (20 ng/ml) was associated capacity to generate and grow as neurospheres starting from with an increase in nestin and Sox2 starting at day 3 and a single cell, and (3) to differentiate to neuronal lineages in maintained until day 8 (Table 19). No difference in the differentiation conditions. When differentiated to neurons, expression was noticed in the absence of Noggin. Inhibiting cells display one or more neural-specific morphological, the BMP signaling pathway by Noggin thus enhanced repro physiological and/or immunological features associated with gramming, but had no reprogramming effect on its own. US 2014/0038291 A1 Feb. 6, 2014 51
TABLE 19 RT-PCR analysis of relative expression of neuronal precursor cell markers such as nestin and Sox2 after transfection of HFF with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5 MBD2 with or without Noggin (20 ng/ml). Relative expression of Sox2, and nestin was increased after transfection with and without Noggin.
Rel. Std. Rel. Std. Rel. Std. Rel. Exp. Dev. Exp. Dew. Exp. Dew Exp. Dev 7.08 2.97 O.93 O.91 1.37 0.10
7.34 1.03 2.01 O.O8 1.28 O.18 O.6O O.10 O.98 O.OS
9.67 241 15.13 1.66 1.98 0.2O 6333.63 277.87 0.95 0.07
11.68 2.6S 194.07 25.22 4.19 O.S2 20231.33 1034.29 190 O.45
3.58 0.66 227.99 16.83 1.68 0.09 6298.51 289.84 O.96 O.17
10.89 O.S7 650.34 22.92 4.42 O.O3 18134.90 63.93 1.81 0.06
#7 Ctrl 1.01 (0.19 1.OO O.OS 1.OO O.O2 1.12 O.70 100 0.09 Ontransfected + Noggin Day1 #8 Msi1/Ngn2+ 2.79 0.83 1.62 O.19 O.99 O.08 1.28 O.25 O.75 O.O1 MBD2, -Noggin Day1 #9 Msi1/Ngn2+ 3.79 0.91 1.47 O.O8 1.23 O.08 1.36 O.O8 O.72 O.O7 MBD2, -Noggin Day2 #10 Msi1/Ngn2+ 6.18 O.S9 14.6O 1.85 2.62 O.30 10949.28 448.28 O.90 O.O1 MBD2, -Noggin Day3 #11 Msi1/Ngn2+ S.63 0.74 74.56 16.56 2.97 0.21 19623.99 3109.69 O.75 0.11 MBD2, -Noggin Day4 #12 Msi1/Ngn2+ 3.21 O.96 232.42 S.47 147 0.07 15311.64 1909.23 O.86 O.O3 MBD2, -Noggin Day6 #13 Msi1/Ngn2+ 3.82 O.S2 496.99 75.81 3.32 0.32 26892.31 1817. OS 2.0S 0.10 MBD2, -Noggin Day8 #14 Ctrl 1.08 O.S7 1.01 014 1.OO O.04 1.15 O.81 100 OOO Untransfected - Noggin Day1
Example VII dPBS/Ca"/Mg"/2 hours at 37° C.) coated tissue culture plates. When cells reached confluence (after 3 to 4 days), 0338 NSLCs Created from HFF Cells are not Skin-De proliferation media were replaced by differentiation medium rived Precursors (SKPs) consisting in DMEM/F12 (50:50), ITS (1:100), HEPES 0339. It’s known that cells termed skin-derived precursors (1:100), GlutaMAXTM (1:100), T3 (0.2 nM), Rosiglitasone (SKPs) may reside in adult human skin (Fernandes et al., 2004). These cells are capable of proliferating in response to (0.5 lug/ml), IBMX (100 LM) and Dexamethasone (1 uM). EGF and bFGF and express nestin, versican and fibronectin, Three days after, IBMX and dexamethasone were withdrawn and can differentiate into both neuronal and mesodermal from the differentiation medium. At day 10, cells were fixed progeny. In order to verify that NSLCs are distinct from with a 4% formaldehyde solution for 10 min and stained with SKPs, differentiation towards adipocyte cells was performed. Oil Red O (invitrogen) staining solution for 15 min. Staining Adipose derived stem cells (ADSC) were maintained in was removed and cells washed twice with PBS. Adipose cells StemProTM MSC serum free medium (Invitrogen) on flasks appeared red with lipid droplets specifically stained with Oil coated with CellStartTM (Invitrogen). CellStartTM was diluted Red O, however NSLCs were stained negative, with no pres 1:100 indPBS/Ca"/Mg" and the flaskincubated for 2 hours ence of lipid droplets in the cells, and the cells adopted neu at 37° C. Cells are passaged every 3 to 4 days using ronal cell morphology. AccutaseTM and medium was changed every 2 days. Three to 0340 Immunohistochemistry analysis confirmed that four days before initiating differentiation, ADSCs and NSLCs are distinct from SKPs: NSLCs stained positive for NSLCs were seeded in 6-wellplates in CellStartTM (1:100 in p75NTR and negative for fibronectin and versican, while US 2014/0038291 A1 Feb. 6, 2014 52
SKPs express fibronectin and versican and do not express TABLE 20-continued p75NTR (Fernandes et al., 2004). This study indicates that NSLCs represent a tripotent-like precursor cell and they are Quantification of BDNF release by Neural-Like Cells (NLCs) that had been differentiated for 55 days from Neural Stem-Like Cells (NSLCs) not a subpopulation of SKPs. that had been created from transfected HFFs. BDNF release from NLCs into the medium, at different time points, was measured by antigen Example VIII capture ELISA and compared to BDNF release of normal mature human neurons (Sciencell). (0341 BDNF Release from Neural-Like Cells (NLCs) (0342 Neural Stem-Like Cells (NSLCs) differentiated into Control medium Neurons NLC neuronal and glial cells were kept in culture for 55 days, and day 34 O.23 25.53 32.21 BDNF released in the conditioned medium was measured by day 41 0.27 19.02 22:43 antigen-capture ELISA at different time points and compared day 55 O.O2 20.73 30.01 to the release in mature neurons (ScienCell), undifferentiated Neural Human Normal Precursor cells (NHNP. Lonza) as 0344. In addition to adopting neuronal morphology crite well as to undifferentiated NSLCs and untransfected cells ria, the NLCs were functional and possessed the capacity to (HFF). Conditioned medium from each group was collected, release neurotrophic factor (BDNF). Generating repro centrifuged, and then stored at -80°C. until assaying. BDNF grammed neuronal-like cell lines that can locally deliver concentrations were measured by ELISA kits (BDNF E. these neurotrophic factors could be used as a method to treat Immunoassay System, Promega Corporation, USA), accord several neurological conditions and may offer crucial benefits ing to the manufacturers instructions. Briefly, 96-well ELISA in regeneration and functional recovery from brain and other immunoplates Were coated with Anti-BDNF injuries. (CatNbf{G700B) diluted 1/1000 in carbonate buffer (pH 9.7) and incubated at 4°C. overnight. The following day, all wells Example IX were washed with TBS-TweenTM 0.5% before incubation with Block/Sample buffer 1x at room temperature for one 0345 Reprogramming of Different Cell Types Towards hour without shaking. After blocking, standards and samples NSLCS: were added to the plates and incubated and shaken (450-100 0346. This study was performed to investigate the capacity rpm) for 2 hat room temperature. Subsequently, after wash of keratinocytes (Invitrogen), human Adipocytes Derived ing with TBS-TweenTM wash buffer, plates were incubated Stem Cells (ADSCs, Invitrogen) and human hematopoietic for 2 h with Anti-Human BDNF pab (1:500 dilution in Block stem cells (CD34", Invitrogen) cells into neural stem-like & Sample 1.x Buffer) at 4°C. After incubation, plates were cells. washed five times with TBS-TweenTM 0.5% wash buffer and (0347 Preparation of Human CD34" Cells, Human ADSC 100 ul of diluted Anti-IgYHRP Conjugate was added to each and Human Keratinocytes: well (1:200 dilution in Block & Sample 1.x Buffer) and incu (0348 Human mobilized peripheral blood CD34" cells bated for 1 hour at room temperature with shaking (450-100 were purchased from StemCell Technologies and expanded rpm). Then, plates were washed five times with TBS as a floating culture in Petri Dishes in complete StemProTM(R)- TweenTM 0.5% wash buffer and 100 ul of TMB One Solution 34 Serum-free Medium (Invitrogen) supplemented with Stem was added to each well. Following 10 minutes incubation at Cell Factor (SCF, 150 g/ml, Peprotech), Granulocyte Colony room temperature with shaking (450-100 rpm) for the BDNF Stimulating Factor (GM-CSF, 37.5 ng/ml, Peprotech) and plate, a blue color formed in the wells. After stopping the IL-3 (75 ng/ml, Peprotech). Medium supplemented with reaction by adding 100 ul of 1N hydrochloric acid, the absor cytokines was changed everyday 2-3 days after centrifugation bance was read at 450 nm on a microplate reader (Synergy of the cell suspension at 300xg for 10 min. Every other day 4TM) within 30 minutes of stopping the reactions. Concentra the cytokines were added directly to the culture without tion of released BDNF in the supernatants was determined changing the media. Cells were incubated at 37°C., 5% CO. according to the standard curves. For their passaging, cells were centrifugated, resuspended in 0343 ELISA results revealed that BDNF was released at the above medium plus cytokines and placed into the the same concentration from differentiated Neuron-Like adequate number of Petri dishes. Cells (NLCs differentiated from NSLCs) and normal Human (0349 Human Adipose-Derived Stem Cells (ADSC) were neuron cells starting at day 11 and remained until day 55 purchased from Invitrogen and expanded in complete Stem (Table 20), while no BDNF (except for tiny amounts in the ProTM MSC Serum-free medium (Invitrogen) on CellStartTM untransfected HFF group) was released in the other groups. (Invitrogen) coated flasks (diluted 1:100 in PBS containing Ca"/Mg") at a cell density of 1x10" cells/cm. Medium was TABLE 20 replaced every two days with fresh pre-warmed complete StemProTM MSC SFM. Cells were incubated at 37° C., 5% Quantification of BDNF release by Neural-Like Cells (NLCs) that had been differentiated for 55 days from Neural Stem-Like Cells (NSLCs) CO. Cells were sub-passaged when 80% confluent by incu that had been created from transfected HFFs. BDNF release from NLCs bation for 3-5 min in pre-warmed TrypLETMTM (Invitrogen) into the medium, at different time points, was measured by antigen and then collected in StemProTM MSC medium. After cen capture ELISA and compared to BDNF release of normal mature human trifugation at 1500 rpm for 5 min, cells were seeded on neurons (Sciencell). CellStartTM coated flasks as described above. Control medium Neurons NLC 0350 Primary human keratinocytes were purchased from Invitrogen and expanded in Defined Keratinocyte Serum-free day O day 11 1.55 30.25 22.99 medium on Coating matrix (Invitrogen) coated flasks (Invit day 18 O.33 29.49 25.15 rogen) at a cell density of 5x10 cells/cm. The cells were day 24 O.33 22O1 26.39 incubated at 37°C., 5% CO. Media was replaced with fresh, complete growth media every two to three days until subcul US 2014/0038291 A1 Feb. 6, 2014 53 ture. Once the cells had reached 70-80% confluency, media 0352 Further analysis and quantification of the repro was removed and the cells were incubated in VerseneTM (In grammed cells revealed a population of NSLCs engendered vitrogen) for 3-5 min at room temperature. VerseneTM was from keratinocyte and CD34" cells. RT-PCR Analysis removed, and pre-warmed 0.05% trypsin-EDTA (Invitrogen) revealed an increase of relative expression of neural stem cell markers such as Sox2, nestin, GFAP, and BIII-tubulin after was added to the flasks. After 5-10 min incubation, growth transfecting Keratinocyte and CD34" by Msil and Ngn2. medium containing Soybean Trypsin inhibitor (Invitrogen) Relative expression of nestin and GFAP was enhanced in was added to the flasks and the cells gently triturated. After NSLCs created from keratinocytes and CD34" cells as com centrifugation at 100xg for 10 min, cells were resuspended in pared to NSLCs from HFFs; however, the reverse was true for the desired Volume of pre-warmed, complete growth medium Sox2 and ACHE expression. BIII-tubulin (TUBB3) and on coated flasks as described above. Map2b expression was highest in NSLCs created from CD34" cells, followed by NSLCs created from HFF (Table 0351 Prior to transfection, cells were trypsinized and 21). This data shows that different types of NSLCs with transiently co-transfected with pCMV-Msil-Ngn2 and different gene expression profiles (and characteristics) can be pCMV6-XL5-MBD2 as previously described in Example IV created from different types of starting/source cells (and the using the Shuttle and plated into a culture plate coated with same has been observed for creating some other types of laminin (Sigma, 10 g/ml). Starting one day after transfec stem-like cells discussed in this application). The data is also tion, cells were treated with VPA (1 mM) for 4 days and the intriguing since it was not expected that keratinocytes (which medium was changed gradually to proliferation medium are derived from the ectodermjust as endogenous neural stem supplemented with FGF (20 ng/ml) and EGF (20 ng/ml) and cells) would have a lower expression than HFFs for all the were cultured for 18 days at 37° C., 5% CO, and 5% O. Cells genes analyzed except for Nestin (it was expected that kera were then analyzed for neural stem cell markers by RT-PCR tinocytes would be the easiest to reprogram into NSLCs since and Immunohistochemistry. they are derived from the ectoderm). TABLE 21
RT-PCR analysis was performed after one month of transfection of human fibroblasts (HFF), Keratinocytes, and CD34" cells with Msi1/Ngn2 (MSI1/NGN2), in the presence MBD2 with VPA treatment. Cells were cultured on coated culture plates in proliferation medium (StemCell Technologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml) for 18 days. Untransfected cells were considered as negative control.
NES TUBB3 ACHE GFAP SOX2
Rel. Rel. Std. Rel. Std. Std. Rel. Exp. Std. Dev. Exp. Dev. Exp. Dev. Rel. Exp Dew Exp. Std. Dev.
#1 Day12 1.OO O.O7 1.OO 1.OO O.O1 101 (0.15 1.OO O.O2 1.08 O.S9 Untransfected HFF #2 Day12 HFF 2.25 O.O3 2148 2.09 3.41 0.45 12.92 1.88 SS8.69 80.08 71513.12 14146.80 Msi1/Ngn2+ MBD2 #3 Day18 HFF 2.56 O.15 17.12 O.14 2.65 O.O2 4.13 0.64 75.96 8.82 84794.40 318.54 Msi1/Ngn2+ MBD2 i4 1.07 O.S4 1.OO 0.07 1.OO O.O2 101 (0.19 1.06 O.48 1.OO O.O1 Untransfected Keratinocytes #5 Day 12 11452.6S 1137.13 O.96 O.11 6.78 O.28 1.09 O.OS 5815.54 S10.91 975.81 7.47 Keratinocytes Msi1/Ngn2+ MBD2 #6 Day 18 12593.79 431.06 O.93 O.04 6.41 O.27 0.48 O.O3 1295.15 32.OS 1047.17 139.48 Keratinocytes Msi1/Ngn2+ MBD2 #7 1.OO O.04 1.01 O16 1.OO O.OO 100 OO1 1.10 O.66 1.01 O.21 Untransfected CD34+ #8 Day 18 839.57 13451 346.61 3 3.97 33.91 4.38 0.28 O.OO 2790.18 3O4.43 25080.35 35.93 CD34+ Msi1/Ngn2+ MBD2 hNPC 4.56 O.07 278.36 1 1...SO O.81 0.06 72.6S 1.83 1285.73 5.27 565552.30 41.717.72 US 2014/0038291 A1 Feb. 6, 2014 54
0353. Immunohistochemistry revealed positive staining 0354 NSLCs generated from keratinocytes and CD34" for GFAP, Sox2, and nestin. NSLCs developed from HFF cells were tested for tripotent capacity. Further differentiation yield a higher percentage of positive staining for Sox2 and studies were performed to induce differentiation of these GFAP (55.8+3.8 and 78.1+2.4) as compared to CD34" cells NSLCs towards neuronal lineage, using NeuroCultTM differ (42.8+2.7 and 24.2+4.4), and keratinocytes (47.1+2.1 and entiation medium (NeuroCult TM differentiation Kit, Stem 43.4+8.9). The percentage of nestin positive cells was high in Cell Technologies) supplemented with BDNF (20 ng/ml, Keratinocytes (77.8+10.7) and HFF (88.45+12.9) and lower Peprotech) and bFGF (40 ng/ml, Peprotech) as described in in CD34" cells (15.5+2.7) (Table 22). Sox2 and Nestin posi Example V. NSLCs generated from HFFs and hNPCs were tive staining was undetectable in ADSCs. used as controls, cultures were incubated at 37°C., 5% CO, 5% O for three weeks. Samples were collected or fixed at TABLE 22 Day 14 and 28 following differentiation for further analysis. The percentage of Sox2 and nestin positive cells for neural stem cell RT-PCR analysis revealed decrease ofundifferentiated genes markers after transfecting fibroblast, keratinocyte, and CD34" cells (Nestin and Sox2) and increased of differentiated genes with pCMV-Msil-Ngn2 in the presence of MBD2 and VPA. Cells were cultured on coated culture plates in proliferation medium (StemCell (Map2, BIII-tubulin, CNPase, and GFAP) as shown in Tables Technologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml) for 23A, 23B, 23C and 23D. 18 days. Untransfected cells were considered as negative control. The percentage of immunopositive cells was determined by Cellomics TM and represented as meant SD (n = 5). % positive Untransfected cells cells Fibroblasts Keratinocytes CD34 Sox2 1.5 - 1.7 55.83.8 47.1.2.1 42.8 2.7 GFAP 0.04+/- 0.2 78.12.4 43.48.9 24.2 + 4.4 Nestin 0.3 +/- 0.3 68.45 - 12.9 77.6 10.7 15.5 2.7
TABLE 23A RT-PCR analysis was performed on NSLCs generated from human fibroblasts (HFF), keratinocytes, and CD34" cells that were cultured on Poly-D-Lysin Laminin coated culture plates in differentiation medium for 28 days (StemCell Technologies) Supplemented with BDNF (20 ng/ml) and FGF (40 ng/ml). hNPCs (Lonza) were considered as a positive control. hNPCs had a much lower increase in ACHE, GFAP, and MAP2b (which actually decreased in hNPCs), but an increase in Nestin, compared to NSLCs under differentiation conditions.
NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.
hNPC Control OO O.O8 100 0.10 1.OO O.O8 1.01 (0.16 1.OO O.O9 1.01 O.16 100 0.12 OO O.09 Diff. h.NPC Day 3.86 0.2O O.6S O.OS 4.87 O.S7 O.74 O.S2 97.26 7.13 1.85 0.21 O.SO 0.04 143 O.OS 4 Diff. h.NPC Day 86 OO6 O.68 O.O2 3.67 O.13 1.33 O.09 102.74 1.89 1.29 O.O1 O.73 O.OS 37 O.O2 28 NSLC Control OO O.O4 100 OO4 1.OO O.04 1.OO O.O3 1.00 OO1 1.OO O.O1 100 OO2 OO O.OS Diff. NSLC Day 38 OO1 OO O.09 2.06 0.02 1.57 0.24 1.79 0.12 0.73 O.O1 O.S6 O.O1 31 O.OS 4 Diff. NSLC Day O62 O.O2 O.90 O.O8 S14 0.21 6.47 0.78 5.7O 0.15 1.30 O.O2 0.79 0.03 41 O.O1 28 HFF-NS OO O.OO OO O.OS OO O.O1 1.OO O.O7 1.00 OOO 1.OO O.O7 100 OO1 OO O.O2 Control Diff. HFF-NS 2.7O O.O8 3.08. O.12 324 O.14 59.93 5.85 478.97 O.27 2.90 O.32 0.81 O.O3 4.02 0.35 Day 14 Diff. HFF-NS 27 O.OS 48 0.11 S9 O.O3 24.62 100 Sf 6.80 20.98 1.52 O.OO O.86 0.08 2.74 0.23 Day 28 Kerat-NS OO OO6 OO O.O2 OO O.O3 1.OO 0.11 1.00 OO1 1.OO O.O7 100 OO2 1.OO O.O1 Control Diff. Kerat-NS 2.43 O.O6 3.48 O.O8 2.82 0.11 56.22 S.58 665.91 10.52 3.09 0.29 1.01 0.14 3.72 0.17 Day 14 Diff. Kerat-NS O.81 O.O3 72 O.OO 61 O.18 26.09 112 673.6S 11.34 1.29 O.O3 1.12 O.O3 2.02 O.OS Day 28 CD34+-NS 1.OO O.OS OO O.O7 OO O.04 1.OO O.08 1.00 OOO 1.OO O.O8 1.OO O.O2 1.OO O.O7 Control Diff. CD34+-NS 2.21 O.O4 3.47 0.07 2.75 O.O4 57.87 6.68 407.54 52.07 2.90 O.18 1.10 O.OS 3.54 0.02 Day 14 Diff. CD34+-NS O.79 0.04 148 OO1 83 0.37 26.92 3.73 485.51 10.66 1.02 0.04 120 0.09 2.34 O.OS Day 28 US 20 14/0038291 A1 Feb 6, 2014 55
TABLE 23B
RT-PCR analysis was performed on undifferentiated NSLCs generated from human fibroblasts (HFF), keratinocytes, and CD34" cells that were cultured on Laminin coated culture plates in Proliferation medium for 4 days (StemCell Technologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml). Relative expression calibrated to undifferentiated hNPCs.
NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP
iate 1.OO O.O8 100 0.10 1.OO O.O8 1.01 O.16 100 0.09 1.01 O.16 1.OO O.12 1.OO 0.09
iate 1.23 O.O5 O.12 O.OO 112 O.O4 O.09 OOO 2145 0.26 O65, O.O1 O.28 OO1 O.37 O.O2
iate O.94 O.OO O. 12 O.O1 O.92 O.O1 O.O3 O.OO O.38 O.OO O.37 O.O2 0.32 O.OO O.31 O.OO
iate 1.00 OO6 O.09 OOO O.97 O.O3 O.O3 O.OO O.23 O.OO O.38 O.O3 0.26 O.OO O.30 O.OO Kerat-NS
Ondifferen iate 1.10 O.O5 O.12 OO1 O.95 O.O4 O.04 OOO O.33 O.OO 0.44 O.04 0.26 O.OO O.3O O.O2 CD34+-NS Control Day 4
TABLE 23C RT-PCR analysis was performed on differentiated NSLCs generated from human fibroblasts (HFF), keratinocytes, and CD34" cells that were cultured on Poly-D-Lysin Laminin coated culture plates in differentiation medium for 14 days (StemCell Technologies) supplemented BDNF (20 ng/ml) and FGF (40 ng/ml). Relative expression calibrated to Day 14 differentiated hNPCs.
NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Diff. hNPC Day 1.OO O.OS 1.OO O.O7 1.OO O.12 1.15 O.80 1.OO O.O7 1.OO O.11 1.OO O.O8 1.OO O.O3 14 Diff. NSLC Day O44 OOO O.18 O.O2 O.47 O.OO O.22 O.O3 O.4O O.O3 0.26 O.OO 0.31 O.OO O.34 O.O1 14 Day 14 Diff. Kerat-NS O.63 O.O2 O.S1 O.O1 O.S6 O.O2 2.78 O.28 1.56 O.O2 0.64 OO6 O.S4 O.O8 O.79 0.04 Day 14 Day 14
TABLE 23D RT-PCR analysis was performed on differentiated NSLCs generated from human fibroblasts (HFF), keratinocytes, and CD34 cells that were cultured on Poly D-Lysin Laminin coated culture plates in differentiation medium for 28 days (StemCell Technologies) supplemented with BDNF (20 ng/ml) and FGF (40 ng/ml). Relative expression calibrated to Day 28 differentiated hNPCs.
NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Diff. hNPC Day 1.OO O.O3 1.00 OO2 1.OO O.O4 100 OO7 1.OO O.O2 100 OO1 100 0.07 1.OO O.O2 28 Diff. NSLC Day O41 OO1 O.15 O.O1 1.56 OO6 O.44 O.OS 1.19 O.O3 0.66 OO1 O.30 OO1 O-38 O.OO 28 US 2014/0038291 A1 Feb. 6, 2014 56
TABLE 23D-continued RT-PCR analysis was performed on differentiated NSLCs generated from human fibroblasts (HFF), keratinocytes, and CD34" cells that were cultured on Poly D-Lysin Laminin coated culture plates in differentiation medium for 28 days (StemCell Technologies) supplemented with BDNF (20 ng/ml) and FGF (40 ng/ml). Relative expression calibrated to Day 28 differentiated hNPCs.
NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP
Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Diff. HFF-NS O64 O.O3 0.26 O.O2 O.4O O.O1 O.59 O.O2 2.12 O.O8 O.43 O.OO 0.38 0.04 O.62 0.05 Day 28 Diff. Kerat-NS O44 O.O2 (0.24 OOO O42 (0.05 O.62 O.O3 15O O.O3 O-38 OO1 O.4O O.O1 O.44 0.01 Day 28 Diff. CD34+-NS O.47 O.O3 O.25 O.OO O.47 0.10 O.85 0.12 1.57 0.03 O.3S O.O1 O.43 O.O3 O.S2 O.O1 Day 28
0355 Fluorescent immunohistochemical staining was for HFF-NC, 59.55+9.12 for Keratinocyte NC, and 61.70+1. performed on samples after 14 days and 28 days of differen 48 for CD34"-NC). A high percentage for BIII-tubulin posi tiation. The expression of Sox2 and Nestin was decreased tive cells was generated from differentiated NSLCs generated time dependently in differentiated cells (HFF, keratinocyte, from HFF (57.83+4.49) as compared to BIII-tubulin positive and CD34). This decrease was associated with an increase of cells generated from Keratinocytes (23.27+2.91) and CD34' differentiated markers at day 28 such as GFAP (68.51+11.87 cells (39.15+7.99) (Table 24) TABLE 24
The percentage of cells stained positive for neural stem cell markers and neuronal lineage markers in hNPCs (Lonza) and transfected keratinocytes, HFF, and CD34' cells with plMsi1/Ngn2/MBD2. Transfected cells (NSLCs) were cultured in Proliferation medium or differentiation medium for 28 days at 37° C., 5% CO2, 5% O2. The percentage of immunopositive cells (SOX2, Nestin, GFAP, S100beta, and BIII-tubulin) was determined by Cellomics TM and represented as meant SD (n = 5).
Proliferation 14 days 28 days % positive cells conditions differentiation differentiation
hNPC Sox2 96.23 - 0.51 59.OS 3.01 41.43 6.05 Nestin 41.47 O.23 10.774.78 16.147.41 S100? 37.38, 7.85 49.51 2.39 n.d. BIII-tubulin 2.34 - 0.43 11.54 - 4.03 23.34 4.77 GFAP 1.16 O.14 23.42 2.51 48.04 8.30 Sox2 93.28 0.53 79.48 - 0.54 52.06 9.07 Nestin 29.294.72 1.15 O.46 218 1.96 S100? 13.51 - 0.28 80.7S 3.50 79.38 - 10.62 BIII-tubulin 3.91 O.33 42.16 15.O7 57.83 4.49 GFAP 8.41 - 0.73 59.66 - 11.48 68.51 - 11.87 Keratinocyte-NC Sox2 96.55 - 1.01 76.93 - S13 63.11 - 8.54 Nestin 40.10 - 8.41 2.67 + 1.61 3.57 0.48 S100? 13.584.97 76.69.72 74.75 - 11.21 BIII-tubulin 6.42 2.94 20.588.34 23.27 2.91 GFAP 9.36 O.34 43.43 - 244 59.SS 9.12 CD34-NC Sox2 95.492.6 81:18, 124 63465.14 Nestin 51.68 1427 12.64 - 1.27 846 - 4.6 S100? 30.1 - 1.03 72.40 4.5 79.57 8.52 BIII-tubulin 5.82 2.08 25.04 19.95 39.1S 7.99 GFAP 13.995.48 S1.79 - 13.68 61.7O 148
n.d. = not determined; + = standard deviation CD34'-NC: neuronal cells generated after differentiation of NSLCs generated from CD34 cells. Each data point represents the analysis of at least 1000 cells from at least 8 images. US 2014/0038291 A1 Feb. 6, 2014 57
0356. The % of Sox2 positive cells decreased faster, the% another 12 days at 37° C., 5% CO, and the medium was of Nestin positive cells generally decreased slower, and the '% changed every day. Samples were fixed to perform immuno of cells expressing one of the differentiation markers (S100B, histochemistry as described previously to detect Neuronal BIII-tubulin, GFAP) generally increased slower in hNPCs markers. The following antibodies were used: mouse anti than in the NSLCs during differentiation. Out of the three nestin 647 (1:100, BD) and anti-BIII-tubulin (1:200, Neuro types of created NSLC lines, the '% of cells expressing one of mics). No clear morphology change of the cells was observed the differentiation markers (S100B, BIII-tubulin, GFAP) gen within the CDM and the immunohistochemical analysis erally increased slowest in NSLCs created from keratinocytes failed to detect BIII-tubulin positive cells. Thus, inducing the and fastest in NSLCs created from HFFs. trans-differentiation of cells using only cytochalasin B and 0357 This study indicates that NSLCs can be created from chemically-defined neural medium was not sufficient to keratinocytes and CD34" blood cells, and these cells share reprogram the cells. morphology and markers similarly to NSLCs generated from 0360 Next, Day 6 CDM samples grown in LAS pre HFF. Similarly to hNPCs, NSLCs created from keratinocytes, coated plates at 37° C. and 5% CO, were exposed simulta CD34" cells, and HFFs had a tendency to differentiate more neously to cytocahlasin B (10 ug/ml) over 5 days, histone towards an astrocyte lineage than a neuronal lineage (except deacetylation inhibitor (VPA, 4 mM, Calbiochem) and NSLCs created from HFFs had an almost similar number of inhibitor of DNA methylation (5-AZacytidine, 5uM, Sigma). BIII-tubulin positive and GFAP positive cells) as shown by Four days later, the medium was changed to differentiation the high percentage of GFAP positive cells during differen medium consisting of a 3:1 ratio of CDM medium without the tiation, which was confirmed by S100beta staining. However, presence of EGFand NbActive medium (Brain BitsTM) the proportion of astrocyte and neuronal cells generated from supplemented with NT-3 (20 ng/ml, Peprotech) and BDNF hNPCs was lower in same culture conditions, indicating that (20 ng/ml, Peprotech). The ratio of the differentiation NSLCs generated from HFF, Keratinocytes, and CD34 cells medium was increased gradually day after day until reaching can give rise to a higher number of neuronal and astrocyte 100% of complete differentiation medium. After two weeks cells as compared to hNPCs. NSLCs, whether created from of treatment, cells were fixed for immunohistochemical HFFs, Keratinocytes or CD34" cells (or potentially even analysis to investigate the identity of the cells. Immun Some other cell), are tripotent cells and possess the capacity to ostained cells with fill-tubulin at day 7, indicating the de differentiate to neurons, astrocytes, and oligodendrocytes differentiation of fibroblast cells to neurons. However, one similarly to hNPCs. However, RT-PCR and immunohis week later, these trans-differentiated cells reverted back to tochemistry analysis of transfected ADSCs did not reveal any fibroblast cells and III-tubulin expression was lost. The loss significant expression of neural stem cell genes, indicating a of morphology and BIII-tubulin expression after withdrawal need to optimize conditions for turning ADSCs to NSLCs or of the priming agents indicate that complete conversion to to investigate the effect of others neurogenic factors that functional and stable reprogrammed cells did not occur. could turn these into NSLCs. 0361 Next CDM was treated with VPA (4 mM), 5-Aza (5 Example X uM) and cytochalasin B (10 g/ml) as above. After 2 days of chemical treatment, fibroblast cells within the CDM were transfected with DNA using Lipofectamine reagent (Invitro Fabrication 3D Extracellular Matrix (CDM) gen) as per the manufacturer's protocol. 15ug of the eukary 0358 Fibroblast cells were cultured in DMEM medium in otic DNA expression vectors pCMV6-XL5-Pax6, pCMV6 the presence of 10% FCS as described in Example I, followed XL5-Msil and pCMV6-XL4-Ngn2 (Origene) were used to by seeding onto 12-well plates pre-coated with laminin (10 transfect the cells. 24 hours later, the media was changed to ug/ml) at a concentration of 2x10 cells/ml in defined CDM Neural Progenitor Basal Medium (Lonza) supplemented with Medium consisting of a 3:1 ratio of Dulbecco's modified Noggin (50 ng/ml), EGF (20 ng/ml), and bFGF (20 ng/ml), Eagle medium (DMEM, high glucose (4.5 g/L) with and the cells were cultured at 37°C., 5% CO and 5% O2, and L-glutamine and sodium pyruvate) and Ham's F-12 medium the medium was changed every day. At day 6, differentiation supplemented with the following components: EGF (4.2x10 was initiated by adding gradually NBActive medium (Brain lo M), bFGF (2.8x10'M), ITS (8.6x10M), dexametha BitsTM) supplemented with NT-3 (20 ng/ml, Peprotech), all sone (1.0x107M), L-ascorbic acid phosphate magnesium trans-retinoic acid (ATRA, 5 uM, Spectrum), BDNF (20 salt n-hydrate (3.2x10"M), L-3.3',5-triiodothyronine (2.0x ng/ml, Peprotech), and bFGF (40 ng/ml, Peprotech). To char 10' M), ethanolamine (10M), GlutaMAXTM (4x10M), acterize the reprogrammed cells, immunohistochemical glutathione (3.3x10M), and 1% penicillin/streptomycin/ analysis and RT-PCR was performed at various time points amphotericin B. By culturing the fibroblast cells at hypercon according to the methods described in Example II using prim fluent density in this completely chemically defined medium ers for nestin, GFAP, MAP2b, and ACHE. In agreement with causes them to enter a high synthetic phase with a slow-down previous studies, un-transfected cells and cells transfected in proliferation, leading to the production of a living tissue with Pax6 did not expressed genes specific for neuronal lin equivalent (LTE) consisting of multiple layers of fibroblasts eages (Table 25). On the other hand, following transfection within de novo 3D extracellular matrix (CDM) that is com with Msil, levels of nestin and ACHE were increased to pletely synthesized by the fibroblasts themselves. 4-fold and 8-fold, respectively, and this expression was main Trans-Differentiation and Reprogramming of Cells within tained over the 12-day period. Also levels of GFAP mRNA CDM was enhanced time dependently by approximately 14 times. 0359 Day 14 CDM samples were treated with cytochalsin Likewise, the same pattern was observed in Ngn2 transfected B (100/ml, Caibiochem), with the concentration of cytoch cells. While expression of BIII-tubulin and MAP2b were alsin B reduced from 10 ug/ml to Oug/ml (none) over 5 days modestly increased following transfection with one neuro while at the same time switching the medium from CDM genic transcription factors the regulation of gene expression Medium to NbActive medium. Samples were cultured for after transfecting the cells with two neurogenic factors, Msil
US 2014/0038291 A1 Feb. 6, 2014 59
0362 Same pattern of gene expression was observed when tomized Neuronal Markers 2 TLDA. In order to identify the transfecting the cells with three transcription factors (Msi1. expression of genes related to neural stem cells, neuronal Ngn2, and Pax6), but the expression was less pronounced cells and glial cells, and growth factors expressed by the cells than in cells transfecting with just Msil and Ngn2. In terms of after transfection. The expression of oligodendrocyte genes, immunohistochemical analysis after the 12 days of the trans such as NKX2.2. olig2, and MAG was increased by Msil and fection, cells displayed neuronal markers after transfection Ngn2; however, the increased was more pronounced by Msil with Msil or Ngn2, as indicated by the expression of nestin as compared to Ngn2 (Table 26). Two markers for astrocytes and MAP2b. Cells transfected with pCMV-XL-PAX6 did not (GFAP and AQP4) were highly expressed after transfection Stain for Nestin and MAP2b. with Msil and Ngn2 in the presence of the DNA demethylator 0363 This study shows that transfecting cells within CDM with only one neurogenic factor (Msil or Ngn2) MBD2. Interestingly, several markers of early neuronal cells induces morphological changes and expression of one or were enhanced; 12 days after transfection, TDLA data more markers of neural stem cells and neuronal cells. Since revealed increases in specific markers for interneurons, such the reprogrammed cells expressed a key neurogenic factor, a as somatostatin and calbindin1. Doublecortin (DCX), which neuronal precursor marker, and a mature neuronal marker at is expressed by migrating immature cells during develop low percentage (10%), this suggests that cells within the ment, and acetylcholine (ACHE), an early marker of neuronal CDM were transformed to NSLCs and then started to differ cells, were highly expressed in reprogrammed cells (Table entiated through the various phases of the neuronal determi 26). Transfection with Msil or Ngn2 increased the expression nation and differentiation program induced in neural stem of dihydropyrimidinase-like 3 (DPYSL3), an early marker of cells. newborn neurons to five-fold with Msil and seven-fold with Ngn2. Expression of microtubule-associated protein 2 Example XI (MAP2), an essential marker for development and mainte 0364 Gene Expression Analysis of Reprogrammed Cells nance of early neuronal morphology, and neuronal cell adhe within COM sion molecule (NCAM) were highly expressed with Msil and 0365. This study was designed to test the effect of trans Ngn2. The expression of enolase-2, a marker of mature neu fecting cells with Msil and Ngn2 in the presence of MBD2 in rons, was 20-fold enhanced by Msil and Ngn2. Member of the reprogramming process. Cells were transfected after two the NeuroD family NeuroD1 was highly expressed after days of pre-treatment with cytocahlasin B with the DNA transfection with Mail to 84.22 fold and to 34.27 by Ngn2. expression vectors using Lipofectamine reagent as described Gene expression of growth factors such as IGF-1, IGF-2, in Example X. 15 lug of eukaryotic DNA expression vectors NPY and CSF-3 was enhanced following transfection with pCMV6-XL5-MusashiorpCMV6-XL4-Ngn2, and pCMV6 Msil or Ngn2. The expression of VEGF and GDNF genes XL5-MBD2 (Origene), were used to co-transfect cells. After were increased to almost five-fold and seven-fold by Msil 24 hours, the media was changed to CDM:Neural Progenitor and Ngn2, respectively. However in transfected cells, the Maintenance Medium (1:1) supplemented with Noggin (50 expression of BDNF, EGF, and bFGF were not activated and ng/ml), EGF (20 ng/ml), and bFGF (20 ng/ml). Medium was even down-regulated as compared to untransfected cells. The changed every day by increasing the percentage of NPBM expression of growth associated protein (GAP-43), a growth and decreasing CDM medium. Cells were cultured for 6 days and regeneration-associated marker of neurite extension, and at 37°C., 5% CO, and 5% O. After one week, differentiation expression ofnetrin, implicated in neuronal development and was initiated by gradually supplementing the NPBM Medium guidance, were highly expressed in transfected cells (Table with NT-3 (20 ng/ml, Peprotech), all-trans-retinoic acid 26). Expression of receptors for growth and neurotrophic (ATRA, 5uM, Spectrum), BDNF (20 ng/ml, Peprotech), and factors was increased, such as type III receptor tyrosine bFGF (40 ng/ml, Peprotech). Samples were collected at the kinase, Neurotrophic tyrosine kinase receptor, and neu end of the study (day 14) and data were analyzed by gene rotrophic tyrosine kinase. The fibroblast-specific markers array to identify genes that were reproducibly found to be vimentin and fibronectin were down-regulated in the repro specific for neuronal lineages, grammed cells. Gene Expression Analysis 0367 Transfection of HFF with only Mail and Ngn2in the 0366 Gene expression analysis on 8 samples was per presence of MBD2 increased the expression of glial cells and formed as previously described in Example I with the cus neuronal cells markers. TABLE 26 Gene array of CDM transfected with pMsi1 and pNgn2 following the pre-treatment with cytochalasin B (10 g/ml), VPA (4 mM) and 5-AZacytidine (5 M). Transfected cells were cultured in differentiation medium (Nb Active, BrainBits TM) supplemented by ATRA (5 M), bFGF (40 ng/ml) and BDNF (20 ng/ml). Gene array of CDM transfected with pMsi1 and pNgn2 following the pre-treatment with cytochalasin B (10 g/ml), VPA (4 nM) and 5-Azacytidine (SIM Relative Relative Expression Symbol Common name and description Company Gene ID Expression Msi 1 Ngn 2 Astrocytes and oligodendrocytes markers NKX2-2 Markers for oligodendrocyte progenitors NM OO2509.2 1.72 10.19 OLIG2 Oligodendrocyte lineage transcription factor 2 NM_0058.06.2 1.72 1.52 MBP Myelin-basic protein NM-OO1 O2SO90.1 1.72 1.52 GFAP Glial fibrillary acidic protein NM 002055.4 6.04 2.41 AQP4 Aquaporin 4 NM OO1650.4 1.72 1.52 US 2014/0038291 A1 Feb. 6, 2014 60
TABLE 26-continued Gene array of CDM transfected with plMsi1 and pNgn2 following the pre-treatment with cytochalasin B (10 g/ml), VPA (4 mM) and 5-AZacytidine (5 M). Transfected cells were cultured in differentiation medium (NbActive, BrainBitSTM) supplemented by ATRA (5 M), bFGF (40 ng/ml) and BDNF (20 ng/ml). Gene array of CDM transfected with plMsi1 and pNgn2 following the pre-treatment with cytochalasin B (10 g/ml), VPA (4 nM) and 5-Azacytidine (SIM Relative Relative Expression Symbol Common name and description Company Gene ID Expression Msi 1 Ngn 2 DIO2 Deiodinase iodothyronine NM O13989.3 8.29 10.61 NC markers SST Somatostatin, specific marker for NM OO1048.3 Very high Very high interneurons CALB1 Clabindin 1, interneuron marker NM 004929.2 1.72 1.52 Tubulin1A Are necessary for axonal growth NM OO6009.2 O.63 O.76 NES Precursor neurons (mestin) NM OO6617.1 2.42 2.86 DCX An early neuronal marker (Doublecortin) NM 178151.1 1.72 1.52 ACHE Acetylcholinesterase, marker of early NM O16831.2 10.68 20.37 neuronal development ENO2 A marker for neurons cells, enolase NM OO1975.2 0.55 O.S4 NEUROD1 Neural marker; expression gradually NMOO2500.2 1.72 1...SO increased from neural precursor to fully differentiated neuron DPYSL3 Dihydropyrimidinase-like 3, marker of NKM OO1387.2 O.62 O.71 immature neurons MAP2 Microtubule-associated protein 2, essential NM 002374.3 1.99 1.70 for development of early neuronal morphology and maintenance of adult neuronal morphology NCAM Neural cell adhesion molecule 1 NM 18135.3 3.11 5.72 CEND1 Cell cycle exit & neuronal differentiation, NM O16564.3 6.68 8.28 early marker of proliferating precursor cells that will differentiate into neurons Neuroregeneration and Survival genes FGF2 Fibroblast growth factor NMOO2006.4 1.19 1.26 EGF Epidermal growth factor HsOO153181 ml 28.37 52.13 IGF-1 Insulin growth factor-1 NM 000618.2 O.82 1.03 IGF-2 Insulin growth factor-2 NM 0000612.3 O.99 1.21 CSF3 Granulocyte colony stimulating factor NM 2219.1 Very high Very high BDNF Brain derived growth factor, neurogenesis NM-1992.31.1 8.54 7.84 GDNF Glial derived neurotrophic factor NM-OOO614.2 O.63 O.91 CNTF Ciliary neurotrophic factor NM 001025366.1 3.80 14.92 VEGF Vascular endothelial growth factor NM 130850.1 6.28 7.22 BMP-4 Bone morphogenetic protein 4 NMOO2253.1 1.17 1.34 KDR Type III receptor tryrosine kinase NM OO6180.3 113.85 43.87 NTRK2 Neurotrophic tyrosine kinase receptor NMOOO905.2 O.O2 O.O2 (TrkB) NPY Neuropeptide Y NMOO905.2 33.39 1.52 NTF-5 Neuortrophin 5 NM OO6179.3 4.43 5.93 PIK3CG Phosphinositide-3-kinase NM 002649.2 1.70 1...SO STAT3 Signal transduction transcription 3 NM 213662.1 3.15 2.24 Gap43 Growth associated protein 43 NM 002045.2 1.82 2.98 NTN1 Netrin1, implicated in neuronal NM 004.822.2 OSO O.29 development and guidance NTRK2 Neurotrophic tyrosine kinase, receptor, type 2 NM 006180.3 O.O2 O.O2 L1 CAM L cell adhesion molecule, associated with NM 024003.1 O.08 O.11 regenerating axons LIMK1 LIM domain kinase 1 NM 002314.2 2.88 2.96 Vimentin Radial glia and fibroblast marker NM-003380.2 O.21 O.2O Fibronectin Fibronectin is a marker for fibroblasts NM 212474.1 O.15 O.14 Transfected cells were cultured in differentiation medium (NbActive, BrainBits TM) supplemented by ATRA (5 uM), bFGF (40 ng/ml) and BDNF (20 ng ml),
Example XII MBD2 after 2 days of pre-treatment with or without cytocha 0368 Reprogramming of Cells within CDM by Lipo lasin B. In parallel, transfection was performed on fresh HFFs fectamine and Nucleofection after the 6 hours usingNucleofection as described in Example 0369. This study was designed to improve transfection of II, and transferred on top of the CDM when the lipofectamine CDM by combining lipofectamine and nucleofection and media was changed to fresh CDM medium. After 24 hours, using two vectors pCMV6-XL5-Msil and pCMV6-XL4 the medium was changed to Neural Progenitor Basal Medium Ngn2 individually or in combination together with pCMV (NPBM, Lonza) with the presence of Noggin (50 ng/ml, XL5-MBD2. Cells within Day 4 CDM were lipotransfected Peprotech), recombinant hFGF (20 ng/ml, Peprotech), and for 6 hours with Msi1/MBD2, Ngn2/MBD2 or Msi/Ngn2/ recombinant hEGF (20 ng/ml, Peprotech). Differentiation US 2014/0038291 A1 Feb. 6, 2014
was induced at day 7, by adding NSA-A differentiation transcription factor (Msil or Ngn2) express high levels of medium (StemCell Technologies) for 21 days. nestin and BIII-tubulin at day 8. The same pattern of expres sion was observed at day 15 and 21, while the expression was Gene Expression Analysis slightly decreased in the absence of cytochalasin B in cells 0370 Samples were collected at 8, 15, and 21 days to transfected with Ngn2. The expression of all genes, except the evaluate the nature of newly formed cells by analyzing the mature neuronal marker MAP2b, were remarkably increased expression of several neuronal marker genes using RT-PCR in cells transfected with both neurogenic transcription fac according to the methods previously described in Example I. tors. The upregulation of these genes was slightly reduced in As shown in Table 27, cells transfected with one neurogenic the absence of cytochalasin B, indicating its role in enhancing TABLE 27 RT-PCR analysis of relative expression of neuronal stem cell markers such as nestin, Sox2, and GFAP after transfection of fibroblast cells within the CDM with different combinations with or without the co-treatment with cytochalasin B. Relative expression of Sox2, nestin, and GFAP in NSLCs was increased after transfection with both transcription factors Ngn2 and Msi1 with MBD2 as the DNA demethylator.
MSI1 NGN2 TUBB3 GFAP NES MAP2
Rel. Std. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dew. Rel. Exp. Dew. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1 Day8 CDM - CytoB Control 1.11 O.21 1.33 O.2O 1O O.O2 O.91 O.O2 18 O.09 0.91 O.O2 #2 Day8 CDM - CytoB Control 1.11 0.17 O.65 O.O8 0.92 OO6 O.91 (0.11 O.82 OO1 O.91 O.11 #3 Day8 CDM - CytoB Control O.83 O.O1 O.71 O.86 0.99 0.04 121 O.OO O3 O.OO 1.21 O.OO #4 Day8 CDM + CytoB Control 7.42 0.35 1.52 O.S3 32 O.16 O.44 0.06 O4 O.O2 (0.44 0.06 #5 Day8 CDM + CytoB Control 7.01 (0.42 2.14 O.S8 23 O.O7 O.62 O.OS O2 OO6 0.62 0.05 #6 Day8 CDM + CytoB Control 9.15 0.48 O.76 O.O8 0.4O O.OS O.59 0.14 O.34. O.16 0.59 O.14 #7 Day 15 CDM - CytoB Control 1.45 0.07 1.53 O.33 32 O.O1 O.90 O.O7 31 O.O3 0.90 O.O7 #8 Day 15 CDM - CytoB Control O.79 0.02 2.01 149 0.91 O.O3 1.14 0.16 O.91 OO1 1.14 O.16 #9 Day 15 CDM - CytoB Control O.87 0.04 O.64 O.72 0.84 O.O8 O.98 0.15 O.84 O.O1 O.98 0.15 #10 Day 15 CDM + CytoB 127 0.14 O.99 O.66 .7O O.21 O.36 O.O2 O8 O.O8 0.36 0.02 Contro #11 Day 15 CDM + CytoB 1.39 O.04 0.97 O.65 2.65 0.38 O.44 0.06 97 (O.30 0.44 0.06 Contro #12 Day 15 CDM + CytoB 1.09 0.21 O49 O46 32 0.14 O.47 0.15 2.45 0.15 O.47 0.15 Contro #13 Day21 CDM - CytoB 1.21 O.OO 1.06 O.O6 1O O.O1 O.86 0.16 O7 O.O1 O.86 0.16 Contro #14 Day21 CDM - CytoB O.97 0.09 2.16 O.77 0.96 OO1 1.11 O.1O O.94 OO1 1.11 O.10 Contro #15 Day21 CDM - CytoB O.86 O.O2 1.01 127 0.94 O.OO 108 0.26 O.99 O.04 1.08 O.26 Contro #16 Day21 CDM + CytoB 141 0.21 1.29 1.64 2.46 O.O7 O.88 0.22 158 0.05 0.88 O.22 Contro #17 Day21 CDM + CytoB 2.24 O.OO O.35 O.O1 2.23 O.O3 O.S.S. O.16 1.57 O.O2 O.S.S. O.16 Contro #18 Day21 CDM + CytoB 2.18 0.14 O.77 O.06 2.29 O.12 O.54 0.04 147 O.04 0.54 O.04 Contro #19 Day8 CDM - CytoB 694.16 18.10 O.S1 O.OS 1.46 0.04 2.18 0.13 1.02 O.O3 2.18 O.13 Msi1 MBD2 #20 Day8 CDM - CytoB 2.38 0.29 4106.88 48.57 0.46 O.O2 1.88 O.14 O.99 O.O2 1.88 O.14 Ngn2/MBD2 #21 Day8 CDM - CytoB 365.04 6.71 27O2.81 SS.69 4.44 O.O2 2.95 0.38 S.11 O.OS 2.95 0.38
#22 Day8 CDM + CytoB 1262.OO 63.21 0.75 O.91 O-54 O.O3 248 0.11 116 O.OS 2.48 0.11
#23 Day8 CDM + CytoB 2.34 0.2O 10963.51 1989 O.S3 O.OO 2.27 0.26 100 OO6 2.27 0.26 Ngn2/MBD2 #24 Day8 CDM + CytoB 869.1S 65.33 64O1.28 87.1 2 458 0.01 3.65 0.13 3.15 O.OO 3.65 0.13 Msi1/Ngn2/MBD2 #25 Day 15 CDM - CytoB 41.07 1.74 2.58 O.36 1.43 O.OS O.S8 0.06 1.34 0.07 O.S8 O.06 Msi1 MBD2 #26 Day 15 CDM - CytoB O.73 0.02 2192.64 15.74 0.95 0.08 1.01 0.09 0.99 O.O3 1.01 0.09 Ngn2/MBD2 #27 Day 15 CDM - CytoB 45.59 2.33 33 18:42 S1.51 S.32 0.08 3.80 O.O1 432 O.O1 4.80 O.O1 Msi1/Ngn2/MBD2 #28 Day 15 CDM + CytoB 106.34 4.43 4.90 1.7O 147 O.O1 O.S7 0.10 1.19 O.O3 O.S7 O.10 Msi1 MBD2 #29 Day 15 CDM + CytoB 1.09 0.11 6715.95 SOS.86 1.30 O.OS O.7O 0.17 1.18 O.O7 O.7O 0.17 Ngn2/MBD2 #30 Day 15 CDM + CytoB 46.77 0.76 281 6.33 90.83 5.76 O.O2 4.52 0.09 3.6O O.O3 S.S2 0.09 Msi1/Ngn2/MBD2 #31 Day21 CDM - CytoB 22.94 109 10.09 2.72 1.08 O.O7 O.S8 O.O8 1.17 O.O2 O.S8 O.08 US 2014/0038291 A1 Feb. 6, 2014 62
TABLE 27-continued RT-PCR analysis of relative expression of neuronal stem cell markers such as nestin, Sox2, and GFAP after transfection of fibroblast cells within the CDM with different combinations with or without the co-treatment with cytochalasin B. Relative expression of Sox2, nestin, and GFAP in NSLCs was increased after transfection with both transcription factors Ngn2 and Msi1 with MBD2 as the DNA demethylator.
MSI1 NGN2 TUBB3 GFAP NES MAP2
Rel. Std. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Rel. Exp. Dew. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #32 Day21 CDM - CytoB O.78 O.O2 4450S6 255.75 1.OO O.O3 0.75 0.21 1.09 OO3 0.75 0.21 Ngn2/MBD2 #33 Day21 CDM - CytoB 24.02 O.86 2SO9.95 64.OO S.18 O.OS 4.74 0.16 4.37 0.06 3.74 0.16 Msi1/Ngn2/MBD2 #34 Day21 CDM + CytoB 54.17 141 8.31 3.32 1.42 O.OS O.7O O.22 1.71 O.O2 O.7O O.22 Msi1 MBD2 #35 Day21 CDM + CytoB 1.19 O.15 1180.19 27.29 1.21 O.06 1.03 0.34 131 O.O4 1.03 O.34 Ngn2/MBD2 #36 Day21 CDM + CytoB 81.66 1.34 7789.96 345.72 5.24 O.OS 5.84 O.10 4.37 O.OS 5.84 O.10
Immunohistochemical Analysis mixtures were incubated at 30°C. for 30 minutes for template synthesis, followed by a PCR procedure (95°C/15 min for 0371 Samples were collected at 4, 8, 14, and 21 days to initial denaturation, 94°C./30 sec, 59°C/30 sec, 72°C/1 min evaluate the nature of any reprogrammed cells by analyzing for 32 cycles) for amplification of the extended telomerase the expression of several neuronal markers using immunohis products. To detect telomerase activity, polyacrylamide gel tochemical analysis according to the methods previously electrophoresis (PAGE) was performed for the reaction prod described in Example I. The immunohistochemical analysis ucts on a 10% non-denaturing TBE gel. After electrophoresis, at various time points revealed that within the first 8 days the the gel was stained with SYBR(R) Green I Nucleic Acid Gel expression of nestin was induced in a large proportion of cells Stain for 30 minutes, followed by image capture using a and decreased time-dependently after inducing the differen Gel-Documentation System (Alpha Innotech). tiation. 0374. All 4 samples were telomerase positive (as indicated 0372. This study indicates that upon transfecting the cells by the TRAP product ladder). As expected, the Heat-treated with one or two neurogenic genes in the presence of cytocha control (AH) showed no Telomerase activity (Negative Con lasin B and MBD2, reprogrammed cells were stable in cul trol). A 36 bp internal control band (S-IC) is used to monitor ture, responded to environmental changes (proliferation VS PCR amplification (to distinguish false-negative results). differentiation), and expressed neuronal markers for at least This S-IC band was observed for all samples except for the 24 days in culture. test samples. This may have been due to the excessively high telomerase activity in the test samples; amplification of the Example XIII TRAP products and the S-IC control band are semi-competi tive. All controls gave expected results (No TRAP products Telomerase Activity of NSLCs for CHAPS ctrl, and TRAP ladder of products for the positive 0373 Telomerase is active in neural precursor cells and control cells and the TSR8 control). Suggest that its regulation is an important parameter for cel Example XIV lular proliferation to occur in the mammalian brain (Caporaso G. Let, 2003). This study was performed to evaluate telom Tumor Formation Assay erase activity in cell extracts of adherent NSLCs (NSLCs cultured on laminin-coated plates) as well as NSLCs in float 0375 Malignantly transformed cells show reduced ing neurospheres (NSLCs cultured in plates with a low-bind requirements for extracellular growth promoting factors, are surface) at early (P7) and late passage (P27). The telomerase not restricted by cell-cell contact, and are often immortal. activity of the 4 samples was measured by the PCR-based Anchorage-independent growth and proliferation is one of telomere repeat amplification protocol (TRAP) using the the hallmarks of malignant transformation, which is consid TRAPeze(R) Telomerase Detection Kit (Chemicon). Briefly, ered the most accurate and stringent in vitro assay for detect the cells were grown in 24-well plates, washed in PBS, and ing malignant transformation of cells. homogenized for 30 min on ice in buffer containing 10 mM 0376. Adherent and neurosphere NSLCs at early and late Tris-HCl, pH 7.5, 1 mM MgCl, 1 mM EGTA, 0.1 mM passage (P7 and P25), as well as normal human neuropro Benzamidine, 5 mM f-mercapthethanol, 0.5% CHAPS and genitor cells (hNPCs), were investigated for the anchorage 10% Glycerol (1x CHAPS Lysis Buffer, provided in kit) and independent growth. HFFs were used as a negative control RNase Inhibitor. The samples were spun down and the protein and cervical carcinoma HeLa cells were used as a positive concentration of the Supernatant was determined using the control. Cells were sedimented by centrifugation at 150xg for BCA Assay. 900 ng of protein from each cell extract was 3 min at room temperature (RT). The assay was performed added directly to the TRAP reaction mixture containing using the CytoSelectTM96-well cell transformation assay TRAP reaction buffer, dNTPs, template substrate (TS) (CellBiolabs). The base agar layer (1.2%) was dissolved in primer, TRAP primer mix and Tag polymerase. The reaction 2xDMEM/20% PBS solution and 50 ul of the agar solution US 2014/0038291 A1 Feb. 6, 2014
was added to the plate and incubated for 30 min at 4°C. to (Roche) at 42°C. overnight with a 1.2 kb Dig-labeled PCR solidify. Prior to adding the cell agar layer, the plate was probe amplified from the plasmid DNA using a set of primers. allowed to warm up for 15 minutes at 37°C. The cells were The membrane was washed twice at room temperature with resuspended at different density (20.000 and 5000 cells/well), 2xSSC, 0.1% SDS for 5 min per wash, twice with 0.5xSSC, except the hNPCs were resuspended only at 5000 cells/well 0.1% SDS at 65° C. for 15 min per wash. Hybridization due to a lack of enough cells. The cells were mixed with the signals of the membrane were detected using the CDP-StarTM 1.2% agar solution, 2xDMEM/20% PBS, and cell suspension substrate (Roche). The membrane was exposed to an X-ray (1:1:1), and 75 ul of the mixture was transferred to wells film for analysis. The signals were stripped from the mem already containing the Solidified base agar layer, and was then brane using stripping buffer (0.2 M NaOH, 0.1% SDS). The placed in 4°C. for 15 minutes to allow the cell agar layer to membrane was re-hybridized with a 0.9 kb Dig-labeled PCR solidify. 100 ul of proliferation medium (StemCell Technolo probe amplified from the plasmid DNA using a set of primers. gies) was added and the plate was incubated for 8 days at 37° (0380. The Southern blot analysis with the 1.2 kb. Dig C. and 5% CO, before being solubilized, lysed and detected labeled PCR probe revealed distinct signals in the positive by the CyOuantTMGR dye in a fluorescence plate reader. The control samples where the Msi1/Ngn2 plasmid DNA was fluorescence measurement was performed using the FlexSta spiked into HFF genomic DNA for the equivalence of 1, 10 or tionTM (Molecular Devices) with a 485/538 nm filter. 100 integrations per genome. There were a few weak and identical bands that appeared in the restriction enzyme TABLE 28 digested genomic DNA from HFF, NSLC samples #1 and #2, Fluorescence measurement (Relative Fluorescence Unit, RFU) indicate Suggesting that there is no plasmid DNA integration in the that under the same conditions only carcinoma. HeLa cells grow as an genomic DNA of NSLCs. These bands may represent the anchorage-independent colony, while both hNPCs and NSLCs (adherent endogenous Ngn2 gene since the 1.2 kb Dig-labeled PCR and floating neurospheres) were negative for tumor growth in the standard agar plate tumor formation assay (CytoSelectTM cell transformation probe contains a small part of the Ngn2gene. This data shows kit. Cell Biolabs Inc.). that no, or only a tiny number of NSLCs had plasmid inte gration into the host genome after transient transfection, and Cell density/Cell that the transfected genes are only present in the cell for a types Hela HFF NSLCS HNPCs short period of time (less than two weeks).
2O.OOO 6O.OS 8.7O 14.82 157 19:22, 1.85 1900 - 2.71 1O.OOO 39.03 - 3.97 13.73 - 1.05 14.99 - 1.12 21.61 - 9.95 Example XVII SOOO 24.70 - 3.89 11.65 0.57 12.29 O.79 12.45 0.73 (0381 Neuroprotective Effect of Transplanted hNSLCs in: 0377 As shown in Table 28, fluorescence measurement indicated that under the same conditions only carcinoma 1) Animal Model of Multiple Sclerosis. HeLa cells significantly grew and proliferated as anchorage 0382 Multiple Sclerosis (MS) is an incurable inflamma independent colonies, while both hNPCs and NSLCs (adher tory demyelinating disease of the central nervous system ent and floating neurospheres) were negative for tumor (CNS) (Frohman E Metal 2006). Therapies for MS rely on growth (same value as HFFs (negative control) for 5,000 and manipulation of the immune system, but with often modest 10,000 cells) in the standard agar plate tumor formation assay effectiveness on reducing clinical episodes or permanent neu by visual observation of cells by light microscopic observa rological disability, requiring frequent injections, and with tion using bright field at 10x confirm Fluorescence measure Sometimes-significant side effects (Langer-Gould A. et al ment. Thus the transient transfection method and genes used 2004). Experimental Allergic Encephalomyelitis (EAE) is an allows the reprogramming of cells without the neoplastic animal model of MS commonly used for studying disease transformation that generally occurs with stable transfection mechanisms and testing potential therapies. EAE can be or certain genes via a series of genetic and epigenetic alter induced in a variety of species and strains of animals mice, ations that yield a cell population that is capable of prolifer Rat, marmoset monkey, rhesus macaques using various CNS ating independently of both external and internal signals that antigens Myelin Oligodendrocyte Glycoprotein (MOG), normally restrain growth. proteolipid protein (PLP) and myelin basic protein (MBP). Example XVI 0383. After obtaining all appropriate animal approvals for the experiments, Female 7 to 8 weeks old C57BL/6 mice were 0378. No Genomic Integration of Plasmid DNA in NSLCs purchased from Charles Rivers, and housed at MISPRO ani from Transient Transfection mal facility for one week before experimentation for adaption 0379 The DNA plasmid Msi1/Ngn2 (designed and con to the new environment. C57BL/6 mice were injected s.c. structed in house) was used in transient transfection for gen with 100 ug MOG 35-55 in CFA (Sheldon Biotechnology, eration of NSLCs along with MBD2 (for sample 1), or 5-Aza McGill University) containing 5 mg/ml Mycobacterium and VPA (for sample 2). Two weeks after transfection, South tuberculosis H37Ra (Difco, inc), at 2 sites on the back. All ern blot was performed to test for possible genomic integra mice received 200 ng pertussis toxin (List Biological Labo tion of the plasmid DNA. 3 ug of genomic DNA extracted ratories, Inc) i.p. on day 0 and 2, while clinical scores were from the NSLC samples, as well as from HFF (a human calculated blindly daily during a 43 day period, according to fibroblast cell line) used as a negative control, was digested the 0-5 scale as follows: 1, limp tail or waddling gait with tail with several restriction enzymes including BgIII, PstI and tonicity: 2, waddling gait with limp tail (ataxia); 2.5, ataxia Stul, Subjected to electrophoresis on a 1% agarose gel and with partial limb paralysis; 3, full paralysis of 1 limb; 3.5, full transferred to a positively charged nylon membrane (Roche). paralysis of 1 limb with partial paralysis of second limb; 4. The membrane was hybridized in the DIGEasy HybTM buffer full paralysis of 2 limbs; 4.5, moribund; and 5, death. US 2014/0038291 A1 Feb. 6, 2014 64
Treatment of EAE Animal Model with and without the Cells: 0388 Beam Walking: 0384 hNSLC and hNPCs (1.5x10 cells in 200 ul PBS/ 0389 Beam walking measures hindlimb coordination by each mouse) were given by single injection i.v. via the tail means of distance traveled across 100 cm beam (2.3 cm in vein when the animals started to show symptoms of EAE (day diameter, 48 cm off the floor). Rats were systematically 13 i.v.). Both animals groups received cyclosporine (10 mg/kg/day) one day before the injection of cells and daily trained to walk along the elevated beam from start to finish from the day of transplantation to avoid any rejection of the with the aim of completing the task. A safe location, i.e., a flat human cells. Sham-treated age-, sex-, and strain-matched box, is placed at the end of the beam so that the rat is moti mice, injected i.p. with PBS alone, were used as controls. All vated to complete the task. groups of animals were observed for 43 days. Animals were sacrificed at 43 days p.t., brains and spiral cord were har vested in 30% sucrose in PBS. Statistical analysis of the clinical scores revealed that the clinical signs of EAE were Scale used for evaluation of beam-walking performance significantly attenuated in NSLC-injected animals as com pared to control and hNPCs-injected animals. Cumulative Scale Performance characteristic scores was significantly reduced in the NSLC transplanted 1 Animals fail to traverse the beam and do not place the animals and the treatment has no effect on body weight. hindlimb on the horizontal surface of the beam 2 Animals fail to traverse the beam, but place the hindlimb on the 2) Hemiplegic Animal Model (Unilateral Ablation of the Left horizontal Surface of the beam and maintain balance Sensorimotor Cortex in Adult Rats). 3 Animals traverse the beam while dragging the hindlimb Animals traverse the beam and place the hindlimb at least once 0385 After obtaining all appropriate animal approvals for during the traverse the experiments, 8 rats per group (Sprague-Dawley, 250-300 5 Animals traverse the beam using the hindlimb to aid less than 50% g, Charles River) were anaesthetized using ketamine of its steps on the beam (Bimeda-MTC)/xylazine (50/10 mg/kg, Novopharm) and 6 Animals traverse the beam using the left hindlimb to aid more placed onto a, Stereotaxic frame. A midline cranial incision than 50% of its steps on the beam was performed with a sterile Surgical scalpel blade, the cra Animals traverse the beam with no more than two foot slips nial vault exposed and the bregma identified. The skull above 8 Normal animals the sensorimotor cortex was opened and the sensorimotor cortex area 0.5-4.0 mm caudal to bregma and 1.8-3.8 mm lateral to the midline (Paxinos and Watson 1986) was care 0390 Before the surgery, all the animals fell at least once fully aspirated. After ablation, the treatments (AlginateTM, from the rotarod, not because they had a walking or coordi Alginate--hNPC, Alginate+NSLCs, RM+NSLCs, RM nation problem, but because the speed was high. After the Only, Fibrin Gel, or Saline) were applied directly on the brain Surgery (2 days), all the animals showed signs of significant after ablation. The opening in the skull was then filled with walking and coordination problems leading to an increase in Bone Wax. In case of a bleeding, small pieces of sterile the number of falls from the rotarod. Three weeks after the homeostatic tissue were inserted into the lesion in order to stop the bleeding. The sutures were performed using Ethi surgery, the number of falls was clearly reduced for the ani conTM monofilament suture /2 circle needle shape. Surgeries mals receiving NSLCs as treatment compared to controls. were performed in sterile clean rooms, and topical antibiotics 0391 Animals passed the beam-walking test before the (Cicatrin R, GlaxoSmithKline) were applied to the exposed surgery without any difficulty. The rats crossed the 100 cm skull and Scalp to limit local infection. Rats were immuno beam and got to the safe spot without failing off the beam. Suppressed by daily injection i.p. of cyclosporine A (10 Two days after Surgery, all groups completely failed to pass mg/kg/day) starting the day before the Surgery until the end of the test, and the animals were notable to stay in balance on the the study period. The purpose of the cyclosporine Ainjection beam. One week after the surgery, all the animals showed was to reduce the rat’s immune reaction to the treatment. The Some improvement in their walking capacity, but no signifi immune-suppression was Sustained until the end of the study cant difference was noticeable between the different treated to ensure that any potential failure of regeneration (if taken place) was not due to the immune reaction against the treat groups. From week 4 until week 26, the animals treated with ment. Functional scores were performed weekly, in all NSLCs as well as RM showed significant improvements in groups, sensorimotor impairment was evaluated based on the their walking capacity compared to the controls. behavioural tests as described below. 0386 Rotarod Test: Example XVIII 0387. The rotarod speed was manually calibrated for the 0392 Transfection of HFF by Various Combinations of 10 and 20 RPM speed before all procedures. Animals were Genes Using the Shuttle R Device and Treatment with Differ required to perch on the stationary rod for 30 sec to acclimate ent Small Molecules for Reprogramming to Mesendoderm themselves to the environment. During this time, if any ani Like Cells mal fell, it was placed back on the rod until it had achieved stationary capabilities for a period of 30 seconds. The animals 0393. HFF cells were cultured as described in CDM II were allowed 3 trials. The animals that were comfortable medium as described in Example I with only modifying EGF staying on the stationary rod for 30 sec were allowed to run (5 ng/ml) and FGF (10 ng/ml), and transfecting using the with a constant speed of 10 and 20 RPM for 60 sec, and the NucleofectorTM(R) 96-well Shuttle R Device (Lonza) follow number of falls were electronically recorded. ing the procedure described in Example IV. The cells were US 2014/0038291 A1 Feb. 6, 2014
transfected with various combinations of cDNA clones as 0394 Cells were collected on Day 4 by detaching with described in Table 29. After transfection, the cells were plated TrypLETM, followed by centrifugation at 80xg for 5 minutes. on 0.1% Gelatin-coated plates and incubated at 37° C., 5% Supernatant was aspirated and the cell pellet was frozen at CO, 5% O. Medium was changed every other day according -86° C. until ready for RNA Isolation. RNA isolation and to Table 30. Cells were analyzed at Day 4 by Quantitative quantification was performed as previously described in Real-time PCR. Example I. cDNA was prepared and quantitative real-time PCR was performed as previously described in Example II, TABLE 29 except the following TaqmanTM(R) Gene Expression Assays Various combinations of plasmids with potential to transfect the cells (Applied Biosystems) were used: towards mesendodern lineage. Da-2 to Day 0 Plasmids transfected at Day O'
Untreated Octa, FoxD3, MBD2 Gene Taqman TM(R) Assay ID Octa, T, MBD2 Octa, Mixl1, MBD2 Octa, Sox17, MBD2 GAPDH (housekeeper) S99999905 m FoxD3, T, MBD2 PPLA (housekeeper) S99999904 m. FoxD3, Mixl1, MBD2 FoxD3, Sox17, MBD2 FOXA2 S00232764 m T. Mix11, MBD2 T, Sox17 MBD2 SOX17 s00751752 s1 10 Mixl1, Sox17, MBD2 Endogenous T s00610.073 g1 13 Pre-treated with Octa, FoxD3 14 WPA & S-Aza FoxD3, T GSC s00418279 m 15 FoxD3, Mixl1 CXCR4 S00607978 S1 16 FoxD3, Sox17 17 Octa, FoxD3, T GATA4 s001714.03 m 18 Mixl1, Sox17, FoxA2 CER1 sOO193796 m 19 Octa, FoxD3, T 2O Mixl1, Sox17, FoxA2 CDH1 (E-cadherin) SO1023894 m p63 s00978340 m SOX2 SOX2 1078-ANY SOX17, FoxA2 = p(CMV6-XL5-FOXA2. All clones were purchased from Origene and prepared using the EndoFree Plasmid Maxi Kit (Qiagen).
TABLE 30
Medium composition from Day -2 to Day 10 Media Composition
Day 0 Day 1 Day 2 to Day 3 Day 4 to Day 7 Day 8 to Day 10
CDM II (3:1 of CDM II IMDMAF 12- MDMAF12 - IMDMAF12 DMEM: F12; (50%) + NEAA - ITS - NEAA - ITS - NEAA - ITS GltaMAXTM IMDMAF12 HSA- HSA+ bRGF + HSA+ bRGF + 100x, (50%) + bFGF- EGF - EGF- EGF - BMP4 Dexthamesone, NEAA - ITS - VPA- Activin A+ 19.7 g/ml, HSA- Activin A+ CHIR99021 Glutathione (500 g/ml, bFGF + EGF + CHIR99021 + BMP4 L-AScorbic WPA- BMP4 75 mg/ml, Activin A+ Selenious acid CHIR99021 2.5 g/ml, Insulin Solution 10 mg/ml, T3 675 ng/ml, ethanolamine 5OOX, bFGF 2.5ug/ml, and Egf (1.25 ugml) + Activin A+ HSA
*Supplements added to media at the following concentrations: Activin A (Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco, 1X), EGF (Peprotech, 5 mg/ml), bFGF(Peprotech, 10 ng/ml), CHIR99021 (Stemgent, 2 uM), VPA (Stemgent, 1 mM), 5-Aza (Sigma, 0.5uM), BMP4 (Peprotech, 10 mg/ml)
US 2014/0038291 A1 Feb. 6, 2014 67
TABLE 32-continued Expression of GATA4, CDH1 (E-cadherin), p.63, and SOX2 relative to untreated HFF control 4 days after transfecting HFF cells with various gene combinations with potential to reprogram cells into mesoendodern-like cells. CDH1 (E- GATA4 cadherin p63 SOX2
Std. Rel. Std. Rel. Std. Rel. Std. Rel. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Day4FoxD3/T/MBD2 121.36 11.68 26.85 0.02 2.22 0.04 16.99 4.74 Day4 13021 21.04 69.19 22.84 4.OS 3.56 1.52 O.O1 FoxD3, Mix1 MBD2 Day4 99.49 30.30 6.89 3.69 1.78 O.O1 15.19 9.08 FoxD3 Sox17 MBD2 Day4T/Mixl1/MBD2 110.30 3.SS 1.36 O.OO 1.36 O.OO 6.64 2.25 Day4T/Sox17/MBD2 53-19 402 2.69 186 18O1 O.54 14.21 S.21 Day4 16.53 16.50 2.91 2.13 13.44 6.68. 10.SS 3.27 Mix1 SOX17 MBD2 Day4 Octa/FoxD3 66.45 26.34 47.31 47.30 13.87 0.16. 23.87 14:31 Day4FoxD3/T 68.25 68.OO 39.08. 29.27 1945 1.51 19.4S 1.51 Day4FoxD3/Mixl1 78.18 78.OO 21.20 2.31 21.20 2.31 25.10 3.20 Day4FoxD3/Sox17 176.4S 93.54 15.64 O.60 15.64 O.6O 26.78 16.35 Day4 Octa/FoxD3/T 12.27 126 12.27 1.26 12.27 1.26 12.27 1.26 Day4 85.89 64.52 2006. 20.00 3.67 0.13 13.66 0.66 Mix1 SOX17 FoxA2 Day4 Octa/FoxD3/T 89.0S SOOO 10.40 8.14 1.52 O.O2 1.52 O.O2 Day4 6.16 6.1O 1.23 O.04 123 O.O4 1.23 O.04 Mix1 SOX17 FoxA2
0395. Identification of gene combinations that may induce 0396 Reprogramming HFFs into Pancreatic Progenitor the formation of Mesendoderm-like cells was investigated by Like Cells: transfection with combinations of Oct4, Sox17, FoxD3, T, MixI1, FoxA2, and MBD2. As shown in Table 25 and 26, the 0397 HFF cells were cultured as described in Example I, Relative Expression of CXCR4 and GATA4, both Mesendo and transfected using the NucleofectorTMR 96-well Shuttle(R) derm/Endoderm/Mesoderm markers, appear to be up-regu Device (Lonza) following the procedure described in lated in various combinations, most noticeably in FoxD3/ Example IV. The cells were transfected with various combi MixI1/MBD2 and FoxD3/Sox17/MBD2. Similarly, FOXA2, nations of cDNA clones as described in Table 27. After trans a marker for Endoderm and Mesoderm, was up-regulated fection, the cells were plated on Fibronectin-coated collagen FoxD3/Sox 17-transfected sample, although the expression is gels and incubated at 37°C., 5% CO., 5% O. Fibronectin still very low. Four days following transfection, SOX17 is still coated Collagen gel plates were prepared prior to transfec highly expressed in the SOX17-transfected samples (50,000 tion. Rat Tail Collagen I (Gibco) was diluted to 1.125 mg/ml to 400,000-fold as compared to the untreated HFF sample). using 10xPBS and distilled water, where 125ul was added to The SOX17 gene expression represents leftover plasmid each well of a 24-well plate and incubated in 37° C. for 40 DNA (exogenous SOX17) that still remains. 4 days post minutes. After rinsing with 1xPBS, Fibronectin (BD Bio transfection, and any endogenous SOX17 expression that Sciences) was added on top of the gel at a concentration of 1.9 may have been induced. Ectoderm markers CDH1, p63 and ug?well. Media was changed every other day according to Sox2 were also up-regulated in Some samples (e.g. Oct4/ Table 33. Cells were analyzed at Day 7 by Quantitative Real FoxD3/MBD2, Oct4/Sox17/MBD2). time PCR. TABLE 33 Plasmids and media composition from Day 0 to Day 14 Plasmids Media Composition transfected at Day O' Day 0 Day 1 to Day 3 Day 4 to Day 14 1 FoxD3, Sox17, Pdx1, CDMII- DMEMSF12- DMEMF12 + NEAA+ ITS + Activin A + NEA A + ITS + HSA - B27 - EGF 2 FoxD3, Sox17, Ngn3, HSA HSA - B27- bFGF + Retinoic Acid + EGF - bcR- FGF10 + Cyclopamine + 3 FoxD3, Mix11, Pdx1, Activin A+ Noggin CHIR99021 + Na 4 FoxD3, Mixl1, Ngn3, Butyrate 5 Sox17, Mix11, Pdx1, 6 Sox17, Mixl1, Ngn3, US 2014/0038291 A1 Feb. 6, 2014 68
TABLE 33-continued Plasmids and media composition from Day 0 to Day 14 Plasmids Media Composition transfected at Day O' Day 0 Day 1 to Day 3 Day 4 to Day 14 7 FoxD3, Sox17, Mix11 DMEMF 12 Px1 NEAA - ITS 8 FoxD3, Sox17, Mix11, HSA - B27 Ngn3 EGF - bcR - 9 FoxD3, Sox17, Pdx1, Activin A+ Ngn3 CHIR99021 + Na 10 FoxD3, Mix11, Pdx1, Butyrate + VPA + Ngn3 5-Aza 11 Sox17, Mix11, Pdx1, Ngn3 where FoxD3 = pCMV6-XL5-FoxD3, Sox17 = pCMV6-XL4-SOX17, Mixl1 = pCMV6-XL5-MLXL1, Pdx1 = pCMV6-XL5-Pdx1, and Ngn3 = pCMV6-XL5-Ngn3. All clones were purchased from Origene and prepared using the EndoFree Plasmid Maxi Kit (Qiagen). *Supplements added to media at the following concentrations: Activin A (Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco, 1X), B27 (Gibco, 1%), EGF (Peprotech, 5 ng/ml), bFGF (Peprotech, 10 ng/ml), CHIR99021 (Stemgent, 2 uM), Na Butyrate (Stemgent, 1 mM), VPA (Stemgent, 1 mM), 5-Aza (Sigma, 0.5 uM), Retinoic Acid (Sigma, 2 uM), FGF10 (Peprotech, 50 mg/ml), Cyclopamine (Stemgent, 2.5uM), Noggin (Peprotech, 50 ng ml)
0398 Cells were collected on Day 7 and RNA isolation Example XIX and quantification was performed as previously described in 0400 Reprogramming Human Adipocytes Derived Stem Example I. cDNA was prepared and quantitative real-time Cells (ADSC) to Pluripotent-Like Stem Cells (PLSC): PCR was performed as previously described in Example 11, 04.01 ADSCs (Invitrogen Corporation) were cultured in except the following TaqmanTM(R) Gene Expression Assays cell culture flasks with complete StemProTM-43 medium (In (Applied Biosystems) were used: vitrogen) at 37°C., 5% CO and the medium was changed 3 times per week. After 3 days in culture cells (passage 5) were trypsinized and counted to be transfected. Cells were tran siently transfected with one plasmid: pCMV6-Octá-2A Gene Taqman TM(R) Assay ID Klf4-2A-Nanog, pCMV-SalI4-2A-Octá-2A-Klf4-2A GAPDH (housekeeper) Hs99999905 m1 Nanog, pCMV-Dax 1-2A-Octá-2A-klf4 pCMV-FoxD3-2A PPLA (housekeeper) HS.99999904 m1 Octá-2A-klf4, pCMV-Octá-2A-Klf4-2A-SalA, pCMV FOXA2 HsOO232764 m1 MBD2-2A-Octá-2A-Klf4-2A, pCMV-AGR2-2A-Octá-2A SOX17 HsOO751752 s1 Klf4-2A, or Rex 1-EF-Octá-2A-Klf4 (2 ug); or by two GATA4 HsOO1714.03 m1 plasmids: pEF-Octánuc-IRES2-MBD2 with pCMV Endo PDX1 PDX1 1201. Sox2nuc-IREC-Lin28 orpCMV-Klf4nuc-IRES2-Tpt1nuc or SOX9 HsOO165814 m1 pEF-Stella-IRES2-NPM2, using NucleofectorTM as NGN3 HsO1875.204 S1 described in Example II. Following the transfection cells NKX2-2 HsOO159616 m1 were cultured in 6-well plates in suspension with 50:50 ratio PAX4 HsOO17301.4 m1 of adipocytes complete medium (StemProTM-43) and embry INS HsO2741908 m1 onic stem cells medium (mTeSR1). After two days in culture, CXCR4 HsOO607978 s1 cells were re-transfected with the same plasmids listed above and cells were plated in 96 well-plates coated with MatrigelTM (BD Biosciences) in the presence of mTesR complete 0399 Identification of gene combinations that may induce medium supplemented with thiazovivin (0.5uM), an ALK-5 the formation of Pancreatic Progenitor-like cells was investi inhibitor (SB 34.1542, Stemgent, 2 uM), and inhibitor of MEK (PD0325901, Stemgent, 0.5 uM). Medium was gated by transfection with combinations of FoxD3, Sox17, changed every day and cells were cultured for 22 days at 37° Pdx1 Ngn3, MixI1, and MBD2. FoxA2, a marker for Endo C., 5% CO, 5% O. Alkaline Phosphatase Detection Kit (AP, derm and Mesoderm, was slightly up-regulated for the Millipore) and immunohistochemistry were performed to FoxD3/Sox17/Ngn31 MBD2-transfected sample as com analyse the expression of pluripotency markers. ALP staining pared to the GFP mock-transfected control sample. Similarly, was performed using AP detection kit (Millipore) according CXCR4, also a marker for both endoderm and mesoderm, to manufacturers instructions. was slightly up-regulated (3-fold compared to GFP-ctrl) for 0402 Visual observation of reprogrammed cells was per the FoxD3/MixI1/Ngn3/MBD2-transfected sample. 7 days formed by CellomicsTM using a live staining for SSEA-4, following transfection, SOX17 can still be detected for the (BD Biosciences) and TRA-1-81ss (BD Biosciences) start samples transfected with SOX17 at varying levels (4 to 570 ing on Day 6 after transfection and every 5 days thereafter. fold up-regulation as compared to the GFP-ctrl). The highest Reprogrammed colonies of PLSCs, positively stained with SOX17 expression up-regulation is detected for the sample SSEA-4 and TRA1-81, was observed only with Plasmid transfected with Sox17/MixI1/Pdx1/Ngn3 (570-fold as com pCMV-SalI4-2A-Octá-2A-Klf4-2A-Nanog, pFF-Rex 1-EF pared to GFP-ctrl), which may suggest that this gene combi Octá-2A-Klf4-2A-RFP pEF-Octánuc-IRES1-MBD2 with nation may increase the amount of SOX17 RNA in cells. pCMV-Sox2nuc-IRES1-Lin28, and pEF-Octánuc-IRES1 US 2014/0038291 A1 Feb. 6, 2014 69
MBD2 with pCMV-Klf4nuc-IRES2-Tpt1nuc. These colo the change in pluripotent markers. 1-5% of total cells trans nies emerged around Day 6 and maintained in culture up to fected with Rex 1/Oct4 or Rex 1/Klf4 showed a SSEA4" and the end of the study period (Pay 22) with a stable morphology. TRA-1-81 phenotype, and this pattern was stable until the Among the plasmids cited above, pCMV-SalI4-2A-Octá-2A end of the study period (Day 22). The observation over time Klf4-Nanog and pPF-Rex 1-EF-Octá-2A-Klf4-2A-RFP gave showed that the phenotype of these colonies moved from an the highest number of colonies. Live staining showed that early SSEA-4 phenotype to a late Oct4"/Sox2/Nanog' phe these colonies express typical pluripotency markers, includ notype by Day 22, which is closer to the final reprogrammed ing SSEA-4 and TRA1-81, and further analysis of these colo state of a pluripotent-like stem cell. nies showed that the colonies also expressed other ESC mark 0404 Various genes were tested for their effect on repro ers such as alkaline phosphatase and Oct4. When the cultures gramming efficiency towards pluripotent-like cells. ADSC were treated with PD0325901 and SB431542 for up to 22 cells were cultured as described in Example IX with 2 days days, a 4-fold improvement in efficiency over the conven VPA and 5-AZA pre-treatment (1 mM and 0.5 LM respec tional method was obtained following the transfection of tively) in StemProTM MSC SFM medium. Cells were trans ADSCs with pCMV-SalI4-2A-Oct4-2A-Klf4-Nanog and fected using the NucleofectorTMR 96-well Shuttle R Device pEF-Rex1-EF-Octá-2A-Klf4-2A-RFP. (Lonza) following procedure described in Example IV and using the transfection program EW-104 with the DNA mixes 0403 Based on the previous study, the highest reprogram described in Table 34. Following transfection the cells were ming efficiency was observed using pEF-Rex1-EF-Octá-2A plated in StemProTM MSC SFM medium described in Klf4-2A-RFP and pCMV-SalI4-2A-Octá-2A-Klf4-2A example A on MatrigelTM (BD Biosciences) coated 24 well Nanog. Another study was designed to ascertain the effect of plates and incubated at 37° C., 5% CO, 5% O. On Day 1, pEF-Rex 1-EF-Octá-2A-Klf4-2A-RFP on the reprogram media was changed to a mix of 75% StemProTM MSC and ming efficiency and to investigate the effect of individual 25% hES cell medium; the percentage of StemProTM MSC pluripotent genes Rex 1, Oct4, and Klf4 in different combina was decreased every day over four days to have 100% hES tions. ADSCs were transfected as above with pPF-Rex 1-EF cell medium by Day 4. From then onwards the medium was Octá-2A-Klf4-2A-RFP pCMV6-XL5-Rex1, pCMV6-XL4 changed every two days. The hES cell medium consisted in Octá/pCMV6-XL5-Klf4, pCMV6-XL5-Rex.1/pCMV6 Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) XL4-Octa, or pCMV6-XL5-Rex 1/pCMV6-XL5-Klf4. After supplemented with 20%. Knockout TM Serum Replacement the second transfection, ADSC were cultured in 96-well (KSR, Invitrogen), 1 mM Gluth MAXTTM, 100 uM Non-es plates coated with MatrigelTM for 24 days in the presence of sential Amino acids, 100 uM 3-mercaptoethanol and 10 mTeSR1 medium supplemented with SB341542 and PD. ng/ml Fgf-2. Different inhibitors and growth factors were 0.325901 at 37° C., 5% CO 5%02. In order to characterize added through the course of the experiment; these are listed in Subpopulations of cells after transfection, live staining, Table 34. Cells were analysed at Day 7 and Day 14 by immu immunohistochemistry, and AP staining was used to follow nohistochemistry analysis and at Day 7 by RT-PCR. TABLE 34 Plasmids and media composition from Day 1 to Day 14.
From day 7 From day Plasmids to -2 transfected at From day 1 to From day 3 to day to day 0 day O day 3 day 7 14 1 WPA - S- CMV6-XL4- StemPro TM,ES StemPro TM,ES hES medium AZa pre- Octá + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + Sox2 + CHIR99021 (3 M) CHIR99021 (3 M) CMV6-XLS MBD2 2 VPA - S- CMV6-XL4- StemPro TM,ES StemPro TM,ES ES AZa pre- Octá + medium + medium + medium treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + FoxD3 -- CHIR99021 (3 M) CHIR99021 (3 M) CMV6-XLS MBD2 3 VPA - S- CMV6-XL4- StemPro TM,ES StemPro TM,ES hES medium AZa pre- Octá + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + UTF1 - CHIR99021 (3 M) CHIR99021 (3 M) CMV6-XLS MBD2 4 VPA - S- CMV6-XL4- StenPro TMES StemPro TM,ES hES medium AZa pre- Octá + medium + medium + treated pCMV6-XL4- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + DPPA4 - CHIR99021 (3 M) CHIR99021 (3 M) CMV6-XLS MBD2 S VPA - S- CMV6-XLS- StemPro TM,ES StemPro TM,ES hES medium AZa pre- Sox2 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + US 2014/0038291 A1 Feb. 6, 2014 70
TABLE 34-continued Plasmids and media composition from Day 1 to Day 14.
From day 7 From day Plasmids to -2 transfected at From day 1 to From day 3 to day to day 0 day O day 3 day 7 14 CHIR99021 (3 M) CHIR99021 (3 M)
WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) CHIR99021 (3 M)
WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) CHIR99021 (3 M)
WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) CHIR99021 (3 M)
WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) CHIR99021 (3 M)
10 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) CHIR99021 (3 M)
11 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) + CHIR99021 (3 M) WPA - S Aza 12 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) + CHIR99021 (3 M) WPA - S Aza 13 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) + CHIR99021 (3 M) WPA - S Aza 14 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) + CHIR99021 (3 M) WPA - S Aza 15 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) + CHIR99021 (3 M) WPA - S Aza 16 WPA - S StenPro TM,ES StenPro TM,ES hES medium AZapre medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) + CHIR99021 (3 M) WPA - S Aza US 2014/0038291 A1 Feb. 6, 2014 71
TABLE 34-continued Plasmids and media composition from Day 1 to Day 14.
From day 7 From day Plasmids to -2 transfected at From day 1 to From day 3 to day to day 0 day O day 3 day 7 14 17 WPA - S- CMV6-XLS- SteinPro TM,ES StenPro TM,ES hES medium AZa pre- Sox2 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + FoxD3 - CHIR99021 (3 M) + CHIR99021 (3 M) CMV6-XLS- VPA - S UTF1 Aza 18 VPA - S- CMV6-XLS- SteinPro TM,ES StenPro TM,ES hES medium AZa pre- Sox2 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + FoxD3 - CHIR99021 (3 M) + CHIR99021 (3 M) CMV6-XL4- VPA + S DPPA4 Aza 19 VPA - S- CMV6-XLS- SteinPro TM,ES StenPro TM,ES hES medium AZa pre- Sox2 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + UTF1 - CHIR99021 (3 M) + CHIR99021 (3 M) CMV6-XL4- VPA + S DPPA4 Aza 2O WPA - S- CMV6-XLS- StemPro TM,ES StenPro TM,ES hES medium AZa pre- FoxD3 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + UTF1 - CHIR99021 (3 M) + CHIR99021 (3 M) CMV6-XL4- VPA + S DPPA4 Aza 21 WPA - S- GFP StenPro TM,ES StenPro TM,ES hES medium AZa pre- medium + medium + treated ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 M) +for- CHIR99021 (3 M) WPA - S Aza
0405. In order to characterize subpopulations of cells after case of Oct4/FoxD3/MBD2 transfection. Colonies started to transfection, live staining, immunohistochemistry, and AP form on Day 7 and continued to form until Day 14 (the end of staining was performed to follow the change in pluripotent the study period). These colonies were positive for AP as well. markers. Cells transfected with either Octá/UTF1/MBD2, 0406. These results were confirmed by RT-PCR analysis Octá/Dppa4/MBD2, FoxD3/Dppa4/MBD2, showing up-regulation of Oct4 expression as shown in Table Octá1 FoxD31 Dppa4, or Sox2/FoxD3/UTF1 showed positive 35. Relative expression for SOX2 was also slightly up-regu colonies for TRA1-60, TRA1-81, and SSEA4. This observa lation in Day 7 after transfecting cells with Oct4/Foxd3/ tion indicated that MBD2 generally had no effect by itself on MBD2. There is also a trend of Sox2 up-regulation following reprogramming towards pluripotent-like cells, except in the transfection with Octá1SOX2/Foxd5 and Octa/Foxd5/Utf1. TABLE 35 Relative expression of Pluripotent genes after transfecting ADSCs with various combinations of vectors as described in Table 34. OCT4 Endogenous SOX2 Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. #1 Day 7, Octá/Sox2/MBD2 2S2O 1.89 3.89 2.06 #2 Day 7, Octá/Foxd3/MBD2 1.28 O.13 18.79 O.O3 #3 Day 7, Octá/Utf1/MBD2 2.01 O.20 2.93 1.73 #4 Day 7, Octá/Dppa4/MBD2 9.68 1.36 1.18 O.15 #5 Day 7, Sox2/Foxd5/MBD2 1.06 0.55 2.68 2.90 #6 Day 7, Sox2/Utf1/MBD2 O.66 O.10 3.36 O.68 #7 Day 7, Sox2/Dppa4/MBD2 O.74 O.OO S.O.3 4.73 #8 Day 7, Foxd5/Utf1/MBD2 4.6 O.61 4.15 2.92 #9 Day 7, Foxd5/Dppa4/MBD2 O.63 O.O2 3.90 2.17 #10 Day 7, Utf1/Dppa4/MBD2 O.96 O.04 4.97 1.92 #11 Day 7, Octa/Sox2/Foxd5 48.17 1.89 7.68 1.79 #12 Day 7, Octa/Sox2/Utf1 48.97 6.93 3.71 O.39 #13 Day 7, Octá/SOX2/Dppa4 32.40 2.74 4.61 2.37 #14 Day 7, Octa/Foxd5/Utfl 4.30 O.91 9.83 3.03 #15 Day 7, Octa/Foxd5/Dppa4 4.21 O.11 4.57 O.85