GENERATION OF INDUCED PLURIPOTENT STEM (iPS) CELLS

Abstract

The present invention relates to a method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox. Furthermore, the present invention relates to an induced pluripotent stem cell generated by the method of the invention and a method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell. Also, a method of generating a transgenic non-human animal and a composition comprising an iPS cell generated by the method of the present invention for gene therapy, regenerative medicine, cell therapy or drug screening are envisaged.

Description

[0001] The present invention relates to a method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stern cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox. Furthermore, the present invention relates to an induced pluripotent stem cell generated by the method of the invention and a method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell. Also, a method of generating a transgenic non-human animal and a composition comprising an iPS cell generated by the method of the present invention for gene therapy, regenerative medicine, cell therapy or drug screening are envisaged.

[0002] Several documents are cited throughout the text of this specification. The disclosure content of the documents cited herein (including manufacturer's specifications, instructions, etc.) is herewith incorporated by reference.

[0003] Pluripotent stem cells like embryonic stem (ES) cells are hallmarked by their ability to self-renew and differentiate into a wide variety of cell types. ES cells can be differentiated in vitro into specialized cell lineages of all three embryonic germ layers--ectodermal, mesodermal and endodermal--in the presence of physical inducing and biological inducing factors. So far, many promising studies have shown the therapeutic potential of differentiated derivatives of ESCs in ameliorating a range of disease in animal models. As a result, pluripotent stem cells have enormous potential for use in tissue engineering and transplantation therapy. If these cells can be induced to differentiate into a particular cell type, they may provide an almost unlimited source of cells for transplantation for the treatment of many devastating degenerative diseases such as diabetes, Parkinson's disease and Alzheimer's disease (Biswas et al., 2007; Kim et al., 2007; Zimmermann et al., 2007).

[0004] Only recently, it has been shown that somatic cells may be genetically modified to redifferentiate into a state that is in terms of pheno- and genotype as well as pluripotency similar to ES cells (Takahashi and Yamanaka, 2006: Okita et al., 2007; Wernig at al., 2007). The so-called "reprogramming" of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility to generate patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4.

[0005] Presently, although it is widely acknowledged that iPS cells have a great potential for medical applications such as, e.g., patient-specific regenerative cell therapy, the currently employed methods to generate iPS cells prevent their use in the medical field. Specifically, the retroviral vectors used to introduce and express the combination of several reprogramming factors randomly integrate into the genome in multiple copies, preferably into the vicinity or into active endogenous genes and hence may cause activating or inactivating mutations of cancer or tumor suppressor genes, respectively. Thus, the generation of iPS cells using a method that minimizes the degree of modification of the target cell's genome may boost the clinically safe application of this approach.

[0006] Accordingly, the present invention relates in a first embodiment to a method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox.

[0007] An "induced pluripotent stem (iPS) cell" is a cell that exhibits characteristics similar to embryonic stem cells (ESCs). Said characteristics include, for example, unlimited self renewal in vitro, a normal karyotype, a characteristic gene expression pattern including stem cell marker genes like Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) and stem cell-specific antigen 3 and 4 (SSEA3/4), and the capacity to differentiate into specialized cell types (Hanna, J., et al. (2007). Science 318(5858): 1920-3; Meissner, A., at al. (2007). Nat Biotechnol 25(10): 1177-81; Nakagawa, M., et al. (2007). Nat Biotechnol.; Okita, K., et al. (2007). Nature 448(7151): 313-7; Takahashi, K., et al. (2007Cell 131(5): 861-72; Wernig, M., et al. (2007). Nature 448(7151): 318-24; Yu, J., et al. (2007). Science 318(5858): 1917-20; Park, I. H., at al, (2008). Nature 451(7175): 141-6). The state of the art generation of iPS cells from fibroblast cultures has been described in Takahashi, Okita, Nakagawa, Yamanaka (2007) Nature Protocols 2(12). The pluripotency of murine iPS cells can tested, e.g., by in vitro differentiation into neural, glia and cardiac cells and the production of germline chimaeric mice through blastocyst injection. Human iPS cells lines can be analyzed through in vitro differentiation into neural, glia and cardiac cells and their in vivo differentiation capacity can be tested by injection into immunodeficient SCID mice and the characterisation of resulting tumors as teratomas.

[0008] iPS cells can generally be evaluated and classified according to the following cellular biological properties:

[0009] Morphology: iPS cells are morphologically similar to embryonic stem cells (ESCs). Each cell has a round shape, large nucleolus and scant cytoplasm. Colonies of iPS cells are also similar to that of ESCs. Human iPS cells form sharp-edged, flat, tightly-packed colonies similar to hESCs whereas mouse iPS cells form the colonies similar to mESCs, less flatter and more aggregated colonies than that of hESCs.

[0010] Growth properties: Doubling time and mitotic activity are cornerstones of ESCs, as stem cells must self-renew as part of their definition. iPS cells are mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs.

[0011] Stem cell markers: iPS cells express cell surface antigenic markers expressed on ESCs. Human iPSCs express the markers specific to hESC, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPS cells express SSEA-1 but not SSEA-3 nor SSEA-4, similarly to mESCs.

[0012] Stem Cell Genes: iPS cells express genes expressed in undifferentiated ESCs, including, e.g., Oct3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.

[0013] Telomerase activity: Telomerases are necessary to sustain cell division unrestricted by the Hayflick limit of ˜50 cell divisions. hESCs express high telomerase activity to sustain self-renewal and proliferation, and iPS cells also demonstrate high telomerase activity and express hTERT (human telomerase reverse transcriptase), a necessary component in the telomerase protein complex.

[0014] Pluripotency: lPS cells are capable of differentiation in a fashion similar to ESCs into fully differentiated tissues. For example, iPS cells injected into immunodeficient mice spontaneously form teratomas after nine weeks. Teratomas are tumors of multiple lineages containing tissue derived from the three germ layers endoderm, mesoderm and ectoderm; this is unlike other tumors, which typically are of only one cell type. Teratoma formation is a landmark test for pluripotency. Further, hESCs in culture spontaneously form ball-like embryo-like structures termed "embryoid bodies", which consist of a core of mitotically active and differentiating hESCs and a periphery of fully differentiated cells from all three germ layers. iPS cells also form embryoid bodies and have peripheral differentiated cells. Blastocyst Injection: hESCs naturally reside within the inner cell mass (embryoblast) of blastocysts, and in the embryoblast, differentiate into the embryo while the blastocyst's shell (trophoblast) differentiates into extraembryonic tissues. The hollow trophoblast is unable to form a living embryo, and thus it is necessary for the embryonic stem cells within the embryoblast to differentiate and form the embryo. iPS cells can be injected by micropipette into a trophoblast, and the blastocyst is transferred to recipient females. Chimeric living mouse pups can thus be created, i.e. mice with iPS cell derivatives incorporated all across their bodies with a varying degree of chimerism.

[0015] Promoter demethylation: Methylation is the transfer of a methyl group to a DNA base, typically the transfer of a methyl group to a cytosine molecule in a CpG site (adjacent cytosine/guanine sequence). Widespread methylation of a gene interferes with expression by preventing the activity of expression proteins or recruiting enzymes that interfere with expression. Thus, methylation of a gene effectively silences it by preventing transcription. Promoters of pluripotency-associated genes, including for example Oct3/4, Rex1, and Nanog, are demethylated in iPS cells, demonstrating their promoter activity and the active promotion and expression of pluripotency-associated genes in iPSCs.

[0016] Histone demethylation: Histones are compacting proteins that are structurally localized to DNA sequences that can effect their activity through various chromatin-related modifications. H3 histones associated with, e.g., Oct3/4, Sox2, and Nanog are demethylated, indicating the expression of Oct3/4, Sox2, and Nanog.

[0017] The term "introducing" as used in accordance with the present invention relates to the process of bringing the coding sequences into the target cell and subsequently incorporation of said coding sequences into the genomic DNA of the target cell. This process is generally known as stable transfection and methods for stable transfection are well-known to the person skilled in the art and described, e.g., in Bonetta, L., (2005), Nature Methods 2, 875-883. Due to the low rate of reprogramming events taking place in transfected cells it is advantageous to rely on an efficient stable transfection method. Hence, the coding sequences are preferably introduced into a target cell by a method achieving high transfection/infection efficiency. For example, transfection/infection efficiencies of at least 30%, at least 50%, or at least 80% are preferred. Suitable methods include, for example, lipofection, electroporation, nucleofection, magnetofection or viral vector infection. Preferably, retroviral vectors are used to achieve stable transfection of the target cells as said vectors not only mediate efficient entry of the coding sequences into the target cell but also their integration into the genomic DNA of the target cell. Retroviral vectors have shown to be able to transduce a wide range of cell types from different animal species, to integrate genetic material carried by the vector into target cells, to express the transduced coding sequences at high levels, and, advantageously, retroviral vectors do not spread or produce viral proteins after infection. Suitable retroviral vector systems are well-known to the person skilled in the art such as, e.g., retroviral vectors with the MoMuLV LTR, the MESV LTR, lentiviral vectors with various internal promoters like the CMV promoter, preferably with enhancer/promoter combinations that show silencing of transgene expression in embryonic/pluripotent cells. Episomal vector systems like adenovirus vectors, other non-integrating vectors, episomally replicating plasmids could also be used. Preferably, the retroviral MX vector system is used in the method of the invention (Kitamura et al., (2003), Exp Hematol., 31(11):1007-1014).

[0018] Target cells to be used in the method of the invention can be derived from existing cells lines or obtained by various methods including, for example, obtaining tissue samples in order to establish a primary cell line. Methods to obtain samples from various tissues and methods to establish primary cell lines are well-known in the art (see e.g. Jones and Wise, Methods Mol Biol. 1997). Suitable somatic cell lines may also be purchased from a number of suppliers such as, for example, the American tissue culture collection (ATCC), the German Collection of Microorganisms and Cell Cultures (DSMZ) or PromoCell GmbH, Sickingenstr. 63/65, D-69126 Heidelberg. In accordance with the method of the invention, a suitable target cell endogenously expresses factors selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, wherein said factors in combination with exogenously introduced factors selected from the complementary set of factors, i.e. Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, are capable to reprogram a non-pluripotent target cell into an iPS cell. The cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox. The person skilled in the art is well-aware of methods to determine whether at least two of the above-described factors are endogenously expressed in a target cell. Such methods include, e.g., western blotting, realtime-PCR or intercellular stainings. The skilled person is further capable to realize without further ado which exogenous factor(s) are needed to complement the set of endogenously expressed factors in order to generate a cell that expresses the combination of Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox to initiate reprogramming of the target cell into an iPS cell. The cell into which the coding sequence(s) in expressible form have been introduced thus expresses a set of factors consisting of Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox.

[0019] The invention also encompasses embodiments where a coding sequence is introduced that is already endogenously present in the target cell. This may be effected, e.g., in cases where the endogenous coding sequence is expressed only at a low level with the effect that the corresponding factor does not or not sufficiently contribute to the reprogramming of the target cell.

[0020] The term "coding sequence" relates to a nucleotide sequence that upon transcription gives rise to the encoded product. The transcription of the coding sequence in accordance with the present invention can readily be effected in connection with a suitable promoter. Preferably, the coding sequence corresponds to the cDNA sequence of a gene that gives rise upon transcription to a factor that contributes to the reprogramming of a target cell into an induced pluripotent stem cell, wherein the reprogramming factors in accordance with the method of the invention are selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors.

[0021] A "factor that contributes to the reprogramming of a target cell into an induced pluripotent stem cell" relates to a factor that is capable of contributing to the induction of the reprogramming of target cells into induced pluripotent stem cells, wherein the factor is selected from Oct3/4 and factors belonging to the Myc, Klf and Sox families of factors. Such reprogramming factors include, for example, Oct3/4, Sox2, Sox1, Sox3, c-Myc, n-Myc, l-Myc, Klf1, Klf2, Klf4, Klf 5, and the like, or mutants thereof with retained reprogramming capabilities. Said contribution to the reprogramming may be in the form of, for example, changing the methylation pattern of a cell to one similar to an embryonic stem cell, shifting the expression profile of a cell towards the expression profile of an embryonic stem cell or affecting conformation of the aggregated nuclear DNA by modulating the histone binding similar to that observed in an embryonic stem cell wherein each of said changes may be effected either alone or in combination by a suitable reprogramming factor. Apart from the above-recited factors, the skilled person is aware of methods to identify further suitable reprogramming factors such as, e.g., bisulphite genomic sequencing, RT-PCR, real-time FOR, microarray analysis, karyotype analysis, teratoma formation, alkaline phosphatase staining, all of which are well-known to the person skilled in the art and are, for example described in Okita, K., et al. (2007), Nature 448(7151): 313-7; Park, I. H., et al. (2008), Nature 451(7175): 141-6; Takahashi, K., et al. (2007), Cell 131(5): 861-72; Wernig, M., et al. (2007), Nature 448(7151): 318-24; Takahashi, K. et al. (2007), Nat Protoc 2(12): 3081-9; or Hogan, B., et al. (1994), "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbour Press.

[0022] Oct3/4 belongs to the family of octamer ("Oct") transcription factors, and plays a role in maintaining pluripotency. The absence of Oct3/4 in cells normally expressing Oct3/4, such as blastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation. Thus, the presence of Oct3/4 contributes to the pluripotency and differentiation potential of embryonic stem cells. Various other genes in the "Oct" family, including Oct1 and Oct6, fail to elicit induction, thus demonstrating the exclusiveness of Oct3/4 to the induction process. The term "Oct4" is used herein interchangeably with the term "Oct3/4".

[0023] The Sox family of genes is associated with maintaining pluripotency similar to Oct3/4, although it is associated with multipotent and unipotent stem cells in contrast to Oct3/4, which is exclusively expressed in pluripotent stem cells. Klf4 of the Klf family of genes was initially identified as a factor for the generation of mouse iPS cells and was demonstrated as a factor for generation of human iPS cells.

[0024] The genes belonging to the Myc family are proto-oncogenes implicated in cancer. It was demonstrated that c-Myc is a factor implicated in the generation of mouse iPS cells and that it was also a factor implicated in the generation of human iPS cells. Introduction of the "Myc" family of genes into target cells for the generation of iPS cells is troubling for the eventuality of iPS cells as clinical therapies, as 25% of mice transplanted with c-Myc-induced iPS cells developed lethal teratomas. N-Myc and l-Myc have been identified to replace c-myc with similar efficiency.

[0025] The term "reprogramming" as used in accordance with the present invention relates to the process of changing the geno- and phenotypical profile of a cell that results in a cell that is geno- and/or phenotypically similar to an embryonic stem cell. Said changes comprise, for example, changes in the methylation pattern, shifts in the expression profile or conformational changes of the aggregated nuclear DNA as described herein above.

[0026] The above applies mutatis mutandis to other embodiments described herein below.

[0027] The method of the invention is based upon the surprising finding that it is possible to obtain iPS cells by the introduction of only two reprogramming factors. Prior to this finding the dogma of the prior art was that viable iPS cells which are functional in in vivo experiments, i.e. capable of contributing to the three germlayers, could only successfully be generated by introducing at least three, but more effectively by introducing a combination of four reprogramming factors.

[0028] Exemplarily, it was demonstrated that murine neural stem cells (NSCs) could be reprogrammed by introducing a combination of four (4F), three (3F) and only two (2F) reprogramming factors as well as only one reprogramming factor using the retroviral MX vector system. The NSCs were established from adult OG2/Rosa26 heterozygous transgenic mice brain (Ryan, A. K. & Rosenfeld, M. G., Genes Dev 11, 1207-25 (1997); Do, J. T. & Scholer, H. R., Stem Cells 22, 941-9 (2004); Pollard, S. M., Conti, L., Sun, Y., Goffredo, D. & Smith, A., Cereb Cortex 16 Suppl 1, i112-20 (2006)), expressing GFP under the control of the Oct4 promoter (Oct4-GFP) and the lacZ transgene from the constitutive Rosa26 locus.

[0029] First observed were GFP+ colonies in NSC cultures infected with Oct4 and Klf4 (2F OK) and 1-2 weeks later in those infected with Oct4 and c-Myc (2F OM) (Table 1).

TABLE-US-00001 TABLE 1 Overview of the applied combinations of reprogramming factors, timing of GFP colony formation, and establishment of iPS cell lines Timing of GFP- Establishment of Transfected factors positive colonies iPS cell line OK 2-3 weeks + CM 3-4 weeks +

[0030] The 2F OM iPS cells were further analyzed and showed an ESC-like expression pattern as well as contributing to the three germ layers in teratomas.

[0031] 2F OK iPS cells were compared with 4F (generated using standard approach of introducing 4 reprogramming factors to generate iPS cells) iPS cells and ESCs. On day 14 post-infection, 5 GFP+ colonies were dissociated and propagated under ESC culture conditions (FIG. 1c, f), yielding 3 (i.e. 60%) 2F OK iPS cell clones (B-2, D-7 and F-4) that were morphologically indistinguishable from ESCs (FIG. 1d, g). No colonies had formed from NSCs infected with control virus (MX) (FIG. 1e, h). The reprogramming efficiencies were estimated from the number of Oct4-GFP+ colonies and transduction rates with MX-GFP control virus on NSCs for the 2F OK iPS and 4F iPS by time course (FIG. 1i, j). Thereby a reprogramming efficiency of 3.6% for 4F reprogramming of NSCs and 0.11% for the two factors approach was calculated, what is comparable to reprogramming of fibroblasts with selection (below 0.08%, Takahashi, K. & Yamanaka, S., Cell 126, 663-76 (2006); Okita, K., Ichisaka, T. & Yamanaka, S., Nature 448, 313-7 (2007); Wernig, M. et al., Nature 448, 318-24 (2007)) and without selection (0.5%; Meissner, A., Wernig, M. & Jaenisch, R., Nat Biotechnol 25, 1177-81 (2007)) (FIG. 1j). Transduction with all 4 factors had a positive impact on the timing and number of GFP+ colonies. Integration of the viral transgenes was confirmed by genotyping PCR. The viral transgenes of all 4 factors were detected in 4F iPS cells, while 2F OK iPS cells only contained the Oct4 and Klf4 transgenes.

[0032] 2F OK iPS cells stained positive for SSEA-1 and alkaline phosphatase, and exhibited ES cell marker genes expression patterns similar to 4F iPS cells and ESCs (FIG. 2a). qRT-PCR results demonstrated that expression of endogenous Oct4, Sox2, c-Myc, and Klf4 in 2F OK iPS cells was comparable to ESCs, and the silencing of the viral transcripts in 2F OK iPS cells with a 1000-fold reduction after 30 days. 2F iPS global gene expression also clusters close to ESCs and 4F iPS (FIG. 2b). Scatter plots of DNA microarray analyses demonstrated a higher similarity between 2F iPS cells and ESCs than between 2F iPS cells and NSCs (FIG. 2c, d). Thus, 2F iPS cells (clone F-4) seemed to be very similar to mouse ESCs at the global transcription level.

[0033] The differentiation ability of 2F OK iPS cells was confirmed by in vitro differentiation into embryoid bodies (EBs). These cells expressed the ectoderm (Tuj1), endoderm (α-fetoprotein), and mesoderm marker Flk1 (expressed by beating cells mimicking cardiomyocytes) (FIG. 3a). Teratomas contained derivatives of all three germ layers (FIG. 3b), and expressed markers of the three germ layer. No teratoma had formed from donor cells (NSCs). These data demonstrate that 2F OK iPS cells exhibit a pluripotent phenotype in in vitro and in vivo.

[0034] To investigate their developmental potential, 2F OK iPS cells were aggregated with 8-cell-stage embryos. iPS cells had contributed to the formation of the inner cell mass in developing blastocysts (FIG. 4a). After transferring aggregated blastocysts into pseudopregnant females, 16 live embryos were obtained on E13.5, of which 2 embryos showed germ cell contribution in the foetal gonads, judged from Oct4-GFP expression (FIG. 4b). X-gal staining (visualising the NSC donor cells that carry the Rosa β-geo26 (lacZ) transgene) of embryonic tissue from whole embryos revealed that in the resulting chimeras, 2F OK iPS cells contributed to the development of all three germ layers (FIG. 4c, e). The strictest test for developmental potency tetraploid (4N) embryo aggregation (n=122) resulted in 2 dead (arrested) embryos at E13.5 (FIG. 4d). This is within the normal rate for 4N embryo aggregation and was not related to deficient pluripotency of the introduced cells. These data demonstrate that iPS cells can give rise to all of the tissues of a late-stage embryo. In diploid (2N) aggregation, PCR genotyping showed that 2 out of 13 chimeras were positive for the Oct4-GFP allele of the donor cell (FIG. 4f and g (top panel)). To assess whether 2F OK iPS cells can contribute to the germline, chimeras were mated with CD-1 females. Two out of 12 pups had a Oct4-GFP allele and 1 out of 12 mice had a lacZ allele. Since the donor cells are derived from a heterogeneous mouse (Oct4+/-Rosa26+/-), they also have the Oct4 and Klf 4 transgenes (FIG. 4g (bottom panel)). No tumour formation was observed from adult chimeras and F1 mice by the age of 17 weeks and 3 weeks respectively. This finding indicates that 2F OK iPS cells can contribute the full term development of chimera, resulting in a next generation (F1) of viable pups and thus suggests that the iPS cells have a similar developmental property like ESCs.

[0035] As described in detail in Example 9 below, the inventors were able to also demonstrate conversion of human cells into pluripotent stem cells by the introduction of two or only one reprogramming factor. Said reprogramming factors were Oct4 or Oct4 and Klf4.

[0036] In conclusion, the above findings demonstrate the successful generation of iPS cells using two reprogramming factors or only one reprogramming factor. The advantage of the method of the invention lies in the use of only two or even only one retroviral vector for stable transfection of one or two reprogramming factors. The possibility of inducing iPS cells with a reduced number of retroviral vectors as compared to prior art approaches presents a major step towards the minimization of genetic modulation of the initial cell population to be reprogrammed. Accordingly, the risk of formation of aberrant and tumourigenic cells is significantly decreased, hence allowing the generation of iPS cells suitable for therapeutic purposes, inter alia.

[0037] In a preferred embodiment of the method of the invention, the factors belonging to the factor families of Myc, Klf and Sox and endogenously expressed by or encoded by the coding sequences to be introduced into the target cell are selected from the group consisting of l-Myc, n-Myc, c-Myc, Klf1, Klf 2, Klf4, Klf15, Sox1, Sox2, Sox3, Sox15 and Sox18.

[0038] The coding sequence of, for example, murine Oct3/4, Sox2, c-Myc, and Klf4 can be found in SEQ ID NOs: 1, 5, 9 and 13, respectively. The protein sequence of murine Oct3/4, Sox2, c-Myc and Klf4 can be found in SEQ ID NOs: 2, 6, 10 and 14, respectively. The coding sequence of human Oct3/4, Sox2, c-Myc and Klf4 can be found in SEQ ID NOs: 3, 7, 11 and 15, respectively. The protein sequence of human Oct314, Sox2, c-Myc and Klf4 can be found in SEQ ID NOs: 4, 8, 12 and 16, respectively. The skilled person is in the position to determine the coding sequences of reprogramming factors for any target species using methods well-known in the art. For example, he can retrieve data relating to sequence and function from databases such as, for example, the databases maintained by the National Center for Biotechnology Information (NCBI) and accessible via the World Wide Web under http://www.ncbi.nlm.nih.gov/. Further, databases for comparative genomics include without limitation, a database maintained also by the NCBI at http://www.dcode.org/, a database for protein annotations for all completely sequenced organisms accessible at http://supfam.org/SUPERFAMILY/, a database comprising genome information for various species accessible at http://www.cbs.dtu.dk/services/GenomeAtlas/, or a database comprising gene dusters accessible at http://phigs.jgi-psf.org/. Said databases allow the skilled person to identify coding sequences for reprogramming factors in other species starting from the sequences known for mice and humans by, for example, performing cross-species sequence alignments to identify homologous genes.

[0039] Several, only recently published scientific articles (Hanna, J., et al. (2007). Science 318(5858): 1920-3; Meissner, A., et al. (2007). Nat Biotechnol 25(10): 1177-81; Nakagawa, M., et al. (2007). Nat Biotechnol.; Akita, K, et al. (2007), Nature 448(7151): 313-7; Takahashi, K., et al. (2007), Cell 131(5): 861-72; Wernig, M., et al. (2007). Nature 448(7151): 318-24; Yu, J., et al. (2007). Science 318(5858): 1917-20; Park, I. H., et al. (2008). Nature 451(7175): 141-6) have shown that transcription factors belonging to the Oct, Sox, Klf and Myc families are capable of contributing to the induction of reprogramming in murine as well as human somatic cells.

[0040] In another preferred embodiment, the target cell does not endogenously express one of the factors encoded by the one or two coding sequences to be introduced into said target cell.

[0041] Methods of assessing endogenous expression of factors are well-known to the skilled person and described elsewhere in this specification. In order to generate iPS cells in accordance with the method of the invention the target cell may not endogenously express one of the factors encoded by the one or two coding sequences that are to be introduced into the target cell. For example, it could be demonstrated that Oct3/4 was not expressed in murine neural stem cells as target cells, whereas Sox2, Klf4 and c-Myc were endogenously expressed. Exogenous introduction of Oct3/4 and subsequent expression was sufficient to complement the quartet of reprogramming factors and induce generation of iPS cells.

[0042] In another preferred embodiment, the target cell is a rnultipotent stem cell. Multipotent stern cells can give rise to several other cell types, but those types are limited in number. This is in stark contrast to pluripotent stern cells being capable of differentiating into any cell type. An example of a multipotent stem cell is a hematopoietic cell, found e.g. in bone marrow, cord blood or circulation, that can develop into several types of blood cells, but cannot develop into other types of cells. Another example of multipotent cells are neural stem cells. Multipotent cells are particularly suitable as reprogramming target cells, since they already have reprogramming factors upregulated.

[0043] In more preferred embodiment, the multipotent stem cell is an ectodermal cell.

[0044] The ectoderm is the outermost of the three primary germ cell layers (the other two being the mesoderm and endoderm) that make up the very early embryo. It differentiates to give rise to many important tissues and structures including the outer layer of the skin and its appendages (the sweat glands, hair, and nails), the teeth, the lens of the eye, parts of the inner ear, neural tissue, brain, and spinal cord. Ectodermal cells as multipotent stem cells are particularly suitable as target cells, since ectodermal cells like neural stem cells already endogenously express reprogramming factors.

[0045] In another preferred embodiment, the target cell is a neural stem cell (NSC).

[0046] Neural stem cells exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including humans. Neural stem cells can also be derived from more primitive embryonic stem cells. The location of the adult stem cells and the brain regions to which their progeny migrate in order to differentiate remain unresolved, although the number of viable locations is limited in the adult (for a review see Gage, 2000). Neural stem cells are particularly suitable as target cells as they already endogenously express reprogramming factors.

[0047] In a more preferred embodiment, the coding sequence to be introduced encodes the factor Oct3/4.

[0048] As outlined herein above and demonstrated in the Examples below, the introduction of Oct3/4 alone into a neural stem cell was sufficient to generate iPS cells. As c-Myc increases tumourigenicity in chimera pups (Okita, K., Ichisaka, T. & Yamanaka, S., Nature 448, 313-7 (2007)), the recent studies demonstrating iPS cell generation without the c-Myc retroviral integration (Nakagawa, M. et al., Nat Biotechnol 26, 101-106 (2008); Wernig, M., Meissner, A., Cassady, J. P. & Jaenisch, R., Cell Stem Cells 2, 11-12 (2008)) present a significant improvement. However, the possibility of inducing iPS cells without c-Myc as presented in this embodiment in combination with the reduced number of retroviral vectors is a major step towards the minimization of genetic modulation of the initial cell population to be reprogrammed.

[0049] The same target cell could also be reprogrammed by the introduction of only two factors. Accordingly, in a different more preferred embodiment, the two coding sequences to be introduced encode factors Oct3/4 and c-Myc or Oct3/4 and Klf4.

[0050] In an even more preferred embodiment, the target cell endogenously expresses the factors c-Myc, Klf4 and Sox2.

[0051] It could be shown that the target cell when endogenously expressing the above combination of reprogramming factors was amenable to reprogramming upon introduction of one or two exogenous reprogramming factors, such as Oct3/4 alone or Oct3/4 and c-Myc or Oct3/4 and Klf4.

[0052] In an even more preferred embodiment, the target cell endogenously expresses the factors c-Myc, Klf4 and Sox2 at levels at least 10-fold lower or at most 10-fold higher as compared to the corresponding expression levels in embryonic stem cells of the same genus as the target cell.

[0053] It is advantageous in accordance with the method of the invention when the expression levels of the endogenous reprogramming factors are in a certain range as compared to the expression levels in ESCs of the same genus as the target cell. Preferably, the target cell endogenously expresses the reprogramming factors c-Myc, Klf4 and Sox2 at levels at least 10-fold lower or at most 10-fold higher as compared to the corresponding expression levels of said factors in ESCs. More preferred is the expression of Sox2 about two-fold higher, c-Myc about 10-fold higher and/or Klf4 about 8-fold lower than in ESCs belonging to the same genus as the target cells. The term "about" as used in the context of the present invention refers to an average deviation of maximum +/-20%, preferably +/-10%. Also envisaged is the expression at levels at least 8-, 6-, 5-, 4-, 3- or 2-fold lower or at most 8-, 6-, 5-, 4-, 3- or 2-fold higher or any arbitrary number in-between as compared to said ESCs.

[0054] In a more preferred embodiment, the target cell is a murine or a human neural stem cell.

[0055] Furthermore, the invention relates to an induced pluripotent stem cell generated by the method of the invention.

[0056] Pluripotent stem cells generated by the method of the invention may be useful in a variety of experimental as well as therapeutic settings. For example, the use of the iPS cells, of cells derived therefrom by differentiation or tissues generated from said iPS cells or cells derived therefrom as a therapeutic or diagnosticum, within gene or cell transplantation treatments, for the identification and validation of genomic targets as well as Drug screening approaches are envisaged.

[0057] The culture conditions for iPS cells are the same as established for embryonic stem cells of the corresponding species and are well-known to the person skilled in the art. Generally, cell culture methods, such as, for example, media constituents, marker choice and selection, cell quantification and isolation, are methods well-known in the art and described, for example, in "Practical Cell Culture Techniques", Boulton at Baker (ads), Humana Press (1992), ISBN 0896032140; "Human Cell Culture Protocols", Gareth E. Jones, Humana Press (1996), ISBN 089603335X and exemplarily in the example section. Methods for culturing and maintaining cells in culture are well-known in the art; growth media and other cell culture related material as well as instructions and methods for successful culturing of cells can, for example, be obtained at Sigma-Aldrich or Invitrogen.

[0058] Further, the invention relates to a method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell comprising the steps of: (a) reprogramming a target cell according to the method of the invention, wherein one coding sequence to be introduced is replaced by the compound to be tested; and (b) assessing whether iPS cells are formed in the presence and absence of the compound to be tested, wherein the formation of iPS cells from target cells in which the compound to be tested has been introduced is indicative of the compound contributing to the reprogramming of a target cell into an induced pluripotent stem cell.

[0059] In accordance with the invention the compound to be tested may be one or more nucleic acids, such as DNA, cDNA, RNA, dsRNA, siRNA, shRNA, miRNA, proteins, peptides, small molecules (organic or inorganic), chemicals or any combination thereof.

[0060] Reprogramming a target cell in accordance with the method of the invention has been described herein-above. Depending on the nature of the compound to be tested the method of the invention may need to be modified as regards the introduction step of the compound into the target cell. For example, if other transcription factors are to be evaluated the corresponding coding sequences may be introduced as described above without modification. In contrast, chemicals or small molecules may be introduced by exogenously adding the respective compound to the cell medium and taking advantage of passive or active cellular uptake mechanisms. The skilled person is well-aware of methods that allow the introduction of any compound to be tested into the cell, preferably into the nucleus, in order to test whether the compound can indeed substitute the factor it replaces and accordingly induce reprogramming of the target cell. Nucleic acids, such as DNA, cDNA, RNA, dsRNA, siRNA, shRNA, miRNA can be introduced by transfection or infection, small molecules (organic or inorganic), chemicals just be penetration throughout the membrane.

[0061] The skilled person is well aware of methods to assess whether iPS cells are formed in the presence and absence of the compound to be tested. Criteria for the classification of an iPS cell are known to the skilled person and have been described herein above. Depending on the criteria to be assessed the methods vary and may include, e.g., visual control by microscopy, expression analysis of markers, teratoma formation alone or in combination.

[0062] The finding of the invention that cells endogenously expressing a set of factors contributing to the reprogramming of said cell may be complemented by the exogenous addition of further factors resulting in a cell expressing a quartet of reprogramming factors, i.e. Oct3/4 and a factor of each family of factors Myc, Klf and Sox, leading to the induction of reprogramming of the target cell, significantly simplifies the identification of compounds that can replace a factor in the reprogramming process. As only one or two factors have to be introduced instead of three or the entire set of four factors known in the art to generate cells suitable for screening, a considerable reduction of time, costs and experimental difficulties is achieved. Also high throughput screening approaches for novel reprogramming factors will evidently be improved as regards time and efficiency with a reduced set of factors necessary to be introduced.

[0063] Also, the invention relates to a method of generating a transgenic non-human animal comprising the steps of: (a) introducing the induced pluripotent stem cell of the invention or generated by the method of the invention into a non-human preimplantation embryo; (b) transferring the embryo of step (a) into the uterus of a female non-human animal; and (c) allowing the embryo to develop and to be born.

[0064] The term "transgenic non-human animal" as used in accordance with the invention relates to an animal in which there has been effected a deliberate modification of its genome by methods described herein.

[0065] The method of the invention of generating a transgenic non-human animal is preferably carried out according to methods that have been established for generating transgenic non-human animals by the use of embryonic stem cells, however, replacing the embryonic stem cells with iPS cells of the invention. Said methods are well-known in the art (Hogan, B., R. Beddington, et al. (1994), "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbour Press; Hanna, J., et al. (2007), Science 318(5858): 1920-3; Meissner. A., et al. (2007), Nat Biotechnol 25(10): 1177-81; Nakagawa, M., et al. (2007), Nat Biotechnol.; Okita, K., et al. (2007), Nature 448(7151): 313-7; Takahashi, K., et al. (2007), Cell 131(5): 861-72; Wernig, M., et al. (2007), Nature 448(7151): 318-24; Yu, J., et al. (2007), Science 318(5858): 1917-20; Park, I. H., et al. (2008), Nature 451(7175): 141-6). In brief, introduction of the iPS cell into a non-human preimplantation embryo, like a morula or a blastocyst, is preferably effected by microinjection into a morula or blastocyst or by aggregation of iPS cells with 8-cell or morula embryos. Said chimaeric embryo is then transferred into the uterus of a pseudopregnant non-human female where it develops into an embryo that is finally born (cf. Example 8).

[0066] Generating a transgenic non-human animal line from iPS cells is based on the pluripotence of said iPS cells (i.e., their ability, once injected into a host developing embryo, such as a blastocyst or morula, to participate in embryogenesis and to contribute to the germ cells of the resulting animal). As outlined above, the blastocysts containing the injected iPS cells are allowed to develop in the uteri of pseudopregnant non-human females and are born as chimeras. The resultant transgenic non-human animals are chimeric for cells originating from iPS cells and are backcrossed to wildtype non-human animals and screened for animals carrying only the genetic content of an iPS cell so as to identify transgenic animals homozygous for the combination of DNA segments.

[0067] The transgenic non-human animals may, for example, be transgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, or cows. Preferably, said transgenic non-human animal is a mouse.

[0068] Accordingly, the invention also relates to a transgenic non-human animal generated by the method of the invention.

[0069] Finally, the invention relates to a composition comprising an iPS cell generated by the method of the invention for gene therapy, regenerative medicine, cell therapy or drug screening.

[0070] A composition as used herein relates to a composition that comprises iPS cells and preferably further constituents that maintain cell viability of said cell. Such constituents are well-known to the skilled person and comprise, for example, cell media constituents. Further, depending on the intended application the composition may comprise additional constituents, for example, constituents facilitating administration to a patient.

[0071] A composition comprising the iPS cells of the invention (as well as the iPS cells of the invention per se) can be used in a variety of experimental as well as therapeutic scenarios. The iPS cell of the invention having a comparatively low number of transgenic expression elements and an overall reduced risk of developing into cancerous cells are expected to be beneficial in gene therapy, regenerative medicine, cell therapy or drug screening.

[0072] Gene therapy, which is based on introducing therapeutic DNA constructs for correcting a genetic defect into germ line cells by ex vivo or in vivo techniques, is one of the most important applications of gene transfer. Suitable vectors and methods for in vitro or in vivo gene therapy are described in the literature and are known to the person skilled in the art (Davis PB, Cooper MJ., AAPS J. (2007), 19;9(1):E11-7; Li S, Ma Z., Curr Gene Ther. (2001),1(2):201-26). In accordance with the invention, cells obtained from a patient could, for example, be genetically corrected by methods known in the art and subsequently be reprogrammed into iPS cells having the pheno- and genotype of ES cells, by the method of the invention. This evidences the applicability of iPS cells in gene therapy and/or cell therapy. Regenerative medicine can be used to potentially cure any disease that results from malfunctioning, damaged or failing tissue by either regenerating the damaged tissues in vivo or by growing the tissues and organs in vitro and subsequently implanting them into the patient. The iPS cells of the invention being capable of differentiating into virtually any tissue (ectoderm, mesoderm, endoderm cells) can be used in any aspect of regenerative medicine and hence drastically reduce the need for ES cells.

[0073] The iPS cells of the invention can also be used to identify drug targets and test potential therapeutics hence reducing the need for ES cells and in vivo studies. Experimental setups and methods to identify and/or assess effects of a potential drug including, for example, target-site and -specificity, toxicity, bioavailability, are well-known to the person skilled in the art.

[0074] Further, the iPS cells may be used to study the prevention and treatment of birth defects or study cell differentiation.

[0075] Also, the iPS cells of the invention may be useful in an experimental setting--besides therapeutic applications--to study a variety of aspects related to dedifferentiation when inducing reprogramming of a target cell such as, e.g., spatiotemporal shifts in the expression pattern of genes or of methylation patterns, or the morphological changes leading to changes in aggregation behaviour. The iPS cells can further be subject to studies relating to, e.g., gene therapy, gene targeting, differentiation studies, tests for safety and efficacy of drugs, transplantation of autologous or allogeneic regenerated tissue, tissue repair (e.g., nervous system, heart muscle), diseases like, e.g., Parkinson's disease, heart attack, diabetes, cancer, leukemia or spinal cord injury, embryonal gene expression, genetic manipulation of embryonal genes, early embryology and fetal development, identification of embryonic cell markers, cell migration or apoptosis.

[0076] The figures show:

FIG. 1: Generation of 2F Oct4/Klf4 (OK) iPS Cells From Adult NSCs of OG2/Rosa26 Transgenic Mice.

[0077] a. RT-PCR and qRT-PCR analyses of Oct4, Nanog, Klf4, Sox2, and c-Myc in ESCs and NSCs. β-actin was used as loading control. b. Western blot analyses of the four factors in ESCs and NSCs. Anti-actin antibody was used as loading control. c. Morphology of 2F OK iPS cell colony on day 14 post-infection. An ESC-like colony expressing Oct4-GFP (f). d. Morphology of an established 2F OK iPS cells (clone F-4) on day 30 post-infection, grown on irradiated MEFs. Phase contrast and Oct4-GFP (g) are shown. e. Morphology of NSCs and mock infection on day 30 post-infection (h). i. Generation of GFP-positive colonies at day 7, 14, and 21 after 2F OK and 4F infection (n=3; error bars indicated s.d.). j. Reprogramming efficiency of generating 2F and 4F iPS cells (n=3). Indicated are the total numbers of GFP+ colonies per 50,000 plated NSCs at day 7, 14, and 21 after infection.

FIG. 2: Gene Expression Profile of iPS Cells.

[0078] a. RT-PCR analysis of ES cell marker gene expression in ESCs, 4F iPS cells (clone A-2c), 2F OK iPS cells (clones B-2, D-7 and F-4), and NSCs. Primers are specific for transcripts from the respective endogenous locus. β-actin was used as loading control. b. The heatmap of the different expressed genes among the NSC, 2F (OK) iPS, 4F iPS and ESC. The gene hierarchical cluster was performed with a cityblock distance and an average linkage. c. Global gene expression patterns were compared between 2F iPS cells (done F-4) and ESCs, and between 2F iPS cells (clone F-4) and NSCs with DNA microarrays. d. Black lines indicate two-fold changes in gene expression levels between the paired cell types. Genes overexpressed in 2F iPS cells (clone F-4) compared with NSCs or ESCs are shown in blue; those underexpressed are shown in red. Positions of pluripotency genes Oct4, Nanog, Sox2, c-Myc, Klf4 and Lin28 in scatter plots are indicated. The gene expression level is scaled in log2.

FIG. 3: 2F Oct4/Klf4 (OK) iPS Cells (Clone F-4) are Pluripotent and Differentiate In Vitro and In Vivo.

[0079] a. In vitro differentiation into all three germ layers. After embryoid body formation, aggregates were transferred onto gelatine-coated plates and allowed to differentiate for another 10 days. Cells were stained with anti-Tuj1, anti-α-fetoprotein (AFP), or anti-Flk1. Nuclei were stained with DAPI. b. Teratomas of F-4 iPS cells containing all three germ layers. F-4 iPS cells (1.5×10⁶ cells) were subcutaneously inoculated into nude mice. After 4 weeks, teratomas were stained with haematoxylin and eosin dyes. Shown is a teratoma containing a neural rosette (ectoderm), muscle (mesoderm), and columnar epithelium (endoderm).

FIG. 4: In Vivo Developmental Potential of 2F Oct4/Klf4 (OK) iPS Cells (Clone F-4).

[0080] a. The chimeric embryos of F-4 iPS cells developed to blastocysts after 24 hrs of aggregation. Fluorescence optics show Oct4-GFP cells located in the inner cell mass of blastocysts. b. Germline contribution of F-4 iPS cells to mouse embryonic development as shown by the expression of Oct4-GFP. Embryos were analyzed with a fluorescence microscope at E13.5. c, d. The 13.5 dpc chimeric embryos (control, 2N, and 4N) were stained with X-gal solution. e. Histological analysis of lacZ-stained 13.5 dpc chimeric embryo (2N). f. Chimeric mouse (8-week-old) generated by F-4 iPS cells. Agouti coat colour originated from F-4 iPS cells. g. PCR genotyping of chimeras derived from F-4 iPS cell. PCR analyses were performed for Oct4-GFP (top panel). Germline transmission of F-4 iPS cells. Genotyping of offspring from chimeric males mated with CD-1 females demonstrated the presence of Oct4-GFP and lacZ allele and Oct4 and Klf4 virus integrations (bottom panel). Abbreviation: Gastroint. tract.: gastrointestinal tract.

FIG. 5: One-Factor hNSC-Derived iPS (1F hNiPS) Cell Colony Formation and Cell Line Characterization.

[0081] (A) Morphology of hNSCs grown in NSC medium. (B) Colony formation of hOCT4-infected cells 10 weeks post-infection. (C) The colony grows hESC-like morphology but center of colony still remain unreprogrammed neural rosettes. (D) Typical hESC-like iPS colony growing on feeder after mechanical isolation at passage 1 (1F hNiPS clone C). (E) High magnification of iPS colony at passage 10. (F) 1F hNiPS colonies were stained for AP. Scale bars, 250 μm. (G) Immunocytochemical analysis of pluripotency markers (OCT4, SSEA4, TRA-1-60 and TRA-1-81) in 2F hNiPS (clone A) and 1F hNiPS (clone C) cells. Nuclei are stained with DAPI (blue). Scale bars, 250 μm.

FIG. 6: Expression Level of Pluripotent Markers and DNA Methylation Analysis in hNSC-Derived iPS (hNiPS) Cells.

[0082] (A) Quantitative PCR analysis for pluripotent markers in H1 hESCs, hNSCs, 2F hNiPS clones (A, B and C) and 1F hNiPS clones (A and C). Data are shown relative expression to H9 hESCs using primers specific for endogenous transcripts. RNA expression levels are shown on logarithmic scale. Transcripts levels were normalized to β-actin levels. Error bars indicate the s.d. from triplicates. (B) Bisulfite sequecing analysis of OCT4 and NANOG promoter regions in H9 hESCs, hNSCs, 2F hNiPS clones (A, B and C) and 1F hNiPS clones (A and C). Each row of circles for a given amplicon represents the methylation status of each CpG in one bacterial clone for that region. Open circles represent unmethylated CpGs, and closed circles represent methylated CpGs. Bottom numbers of each column indicate CpG dinucleotide locations, relative to the transcriptional start site (TSS; +1).

FIG. 7: In Vitro Differentiation of hNSC-Derived IPS (hNiPS) Cells Into All Three Germ Layers.

[0083] (A) Immunofluorescence analysis shows differentiation of 2F and 1F hNiPS cells into all three germ layers: endoderm (alpha-fetoprotein; AFP), mesoderm (alpha-smooth muscle actin; α-SMA) and ectoderm (β-tublin lllb; Tuj1). Nuclei are stained with DAPI (blue). Scale bars, 100 μm. (B) Quantitative PCR analyses of one-month embryoid bodies (EBs) differentiation derived from 2F hNiPS (clone A) and 1F hNiPS (clone C) cells. Endoderm (AFP, GATA6 and Sox17), mesoderm (FOXF1 and HAND1) and ectoderm (NCAM1, PAX6 and Sox1). Data are shown relative expression to each undifferentiated parental hNiPS cells. RNA expression levels are shown on logarithmic scale. Transcripts levels were normalized to β-actin levels.

FIG. 8: In Vivo Pluripotency and Global Gene Expression Profile of hNSC-Derived iPS (hNiPS) Cells.

[0084] (A) Teratoma formation after transplantation of 2F hNiPS (clone A) and 1F hNiPS (clone C) cells into SCID mice, and teratomas were sectioned and stained with hematoxylin and eosin at 6-8 weeks. Histological section of identified cells representing all three germ layers: endoderm (respiratory epithelium; r), mesoderm (skeletal muscle; m, cartilage; c) and ectoderm (neural epithelium; n). Enlargements of sections showing respiratory epithelium, muscle and neural epithelium indicated by arrows. Scale bars, 100 μm. (B) Heat map (left panel) and hierarchical cluster analysis (right panel) of global gene expression from hNSCs, 1F hNiPS (clone C), 2F hNiPS (clone A) H9 hESCs and H1 hESCs (left). (C) Scatter plots comparing global gene expression profiles between 1F hNiPS (clone C) and H9 hESCs (left panel), 2F hNiPS (clone A) and H9 hESCs (middle panel), and hNSCs and 1F hNiPS (clone C) (right panel). The black lines indicate twofold difference in gene expression levels between the paired cell populations. The transcript expression levels are on the log² scale.

The examples illustrate the invention:

Example 1: Generation of OG2 Mice

[0085] The OG2 strain was crossed with the ROSA26 transgenic strain (Do, J. T. & Scholer, H. R., Stem Cells 22, 941-9 (2004); Szabo, P. E., Hubner, K., Scholer, H. & Mann, J. R., Mach Dev 115, 157-60 (2002)) over several generations to produce compound homozygous mice for the neo/lacZ and Oct4-GFP transgenes. To derive NSCs, homozygous OG2×ROSA26 male mice were crossed with ICR females to produce heterozygous pups. Brain tissue was collected from 5-day-old OG2×ROSA26 heterozygous mice.

Example 2: Generation of Induced Pluripotent Stem Cells

[0086] iPS cells and ESCs were grown on irradiated MEFs and in ESC medium (DMEM supplemented with 15% FBS, nonessential amino acids, L-glutamine, penicillin/streptomycin, β-mercaptoethanol, and 1,000 U/ml leukemia inhibitory factor (LIF)). pMX-based retroviral vectors encoding the mouse cDNAs of Oct4, Sox2, Klf4, and c-Myc were separately cotransfected with packaging-defective helper plasmids into 293 cells using Fugene 6 transfection reagent (Roche). 48 hrs later, virus supernatants were collected as previously described (Zaehres, H. & Daley, G. Q., (2006), Methods Enzymol 420, 49-64). NSCs derived from OG2/Rosa26 transgenic mice were seeded at a density of 5×10⁴ cells per 6-well plate and incubated with virus-containing supernatants for the four factors (1:1:1:1) or for Oct4 and Klf4 (1:1) supplemented with 6 μg/ml protamine sulfate (Sigma) for 24 hrs. Transduction efficiencies were calculated with pMX-GFP control virus. Cells were replated in fresh neural expansion medium. Two days after infection, the cells were further subcultured on irradiated MEFs in ESC medium containing LIF without any further selection. Oct4-GFP--positive colonies were mechanically isolated, and individual cells were dissociated and subsequently replated onto MEFs. The colonies were selected for expansion.

Example 3: qRT-PCR analysis

[0087] Total RNA was extracted from cells using the MiniRNeasy Kit (Qiagen GmbH, Hilden, Germany; http://www.qiagen.com) according to the manufacturer's instructions. Complementary DNA synthesis was performed with the High Capacity cDNA Archive Kit (Applied Biosystems GmbH, Darmstadt, Germany; http://www.appliedbiosystems.com) following the manufacturer's instructions with a down-scaled reaction volume of 20 μl. Transcript levels were determined using the ABI PRISM Sequence Detection System 7900 (Applied BioSystems) and the ready-to-use 5'-nuclease Assays-on-Demand. For each real-time amplification, the template was equivalent to 5 ng of total RNA. Measurements were done in triplicate; a RT blank of each sample and a no-template blank served as negative controls. Amplification curves and gene expression were normalized to the housekeeping gene Hprt, used as internal standard.

[0088] Oligonucleotides were designed by the Taqman Assay-on-Demand for the detection of the following genes: Pou5f1 (Oct3/4) (Mm00658129_gH), Sox2(Mm00488369_s1), c-Myc (Mm00487803_m1), Klf4 (Mm00516104_m1) B-Act (Mm00607939_s1), and Hprt1 (Mm00446968_m1). Oligos for the detection of Nanog and the viral sequences were custom-designed. Quantification was normalized on the endogenous Hprt gene within the log-linear phase of the amplification curve obtained for each probe/primers set using the ΔΔCt method (ABI PRISM 7700 Sequence Detection System, user bulletin #2).

TABLE-US-00002 Primer sequences for viral-specific qRT-PCR (SEQ ID NO: 17) pMXs-Oct4 PF: 5'-TGGTACGGGAAATCACAAGTTTG, (SEQ ID NO: 18) PR: 5'-GTCATAGTTCCTGTTGGTGAAGTTCA, (SEQ ID NO: 19) Probe: 5'-6FAM-CTTCACCATGCCCCTCA-MGB (SEQ ID NO: 20) pMXs-Sox2 PF: 5'-GTGTGGTGGTACGGGAAATCAC, (SEQ ID NO: 21) PR: 5'-TTCAGCTCCGTCTCCATCATG, (SEQ ID NO: 22) Probe: 5'-6FAM-TGTACAAAAAAGCAGGCTTGT-MGB (SEQ ID NO: 23) pMXs-Klf4 PF: 5'-GTGTGGTGGTACGGGAAATCA, (SEQ ID NO: 24) PR: 5'-CGCGAACGTGGAGAAGGA, (SEQ ID NO: 25) Probe: 5'-6FAM-CTTCACCATGGCTGTCAG-MGB (SEQ ID NO: 26) pMXs-cMyc PF: 5'-TGGTACGGGAAATCACAAGTTTG, (SEQ ID NO: 27) PR: 5'-GTCATAGTTCCTGTTGGTGAAGTTCA, (SEQ ID NO: 28) Probe: 5'-6FAM-CTTCACCATGCCCCTCA-MGB (SEQ ID NO: 29) Nanog PF: 5'-AACCAGTGGTTGAATACTAGCAATG, (SEQ ID NO: 30) PR: 5'-CTGCAATGGAT GCTG GGATACT, (SEQ ID NO: 31) Probe: 5'-6FAM-TTCAGAAGGGCTCAGCAC-MGB

Example 4: Microarray Analysis

[0089] The microarray study was carried out using Affymetrix Mouse Genome 430 2.0 GeneChip arrays (Affymetrix, Santa Clara, Calif.) essentially as described before (Ruau, D. at al., (2008), Stem Cells). Briefly, total RNA was extracted from cells with RNAeasy kit including DNAse digestion (Qiagen, Hilden, Germany). Biotin-labelled cRNA was obtained from 3 μg of total RNA with the GeneChip One-Cycle labelling kit (Affymetrix). Fifteen micrograms of cRNA were fragmented and hybridized to Affymetrix 430 2.0 GeneChip arrays at 45° C. for 16 hrs. DNA chips were washed, stained and scanned using an Affymetrix Fluidics device and GCS3000 scanner, and the images obtained were analyzed using the GCOS software. The experiment was performed in triplicates for the ESCs and iPS cells and in duplicates for the NSCs. Normalization was calculated with RMA algorithm (Irizarry, R. A. et al., (2003), Nucleic Acids Res 31, e15) implemented in BioConductor.

Example 5: In Vitro Differentiation of iPS Cells

[0090] Oct4-GFP cells were harvested by FACS analysis and used for in-vitro differentiation in embryoid bodies (EBs), which was performed with hanging drop in ESC medium without LIF. After 3 days, EBs were plated onto gelatine-coated 4-well dishes for another 10 days. The cells were stained with anti-Tuj1 antibody (1:100; Chemicon), anti-α-fetoprotein (AFP) antibody (1:100; R&D Systems), or anti-Flk1 antibody (1:100; R&D Systems).

Example 6: Western Blot Analysis, SSEA-1 and AP Staining

[0091] Total cell lysates (2×10⁶) prepared from the ESC and NSC were subjected to western blot analysis for expression of Oct4 (Santa Cruz), Sox2 (Santa Cruz), Klf4 (Abcam), and c-Myc (Abcam). β-actin expression levels in all the samples were used as loading control (Abcam).

[0092] SSEA-1 and alkaline phosphatase (AP) staining was performed with the ES Cell Characterization Kit (Chemicon) according to the manufacturer's protocol.

Example 7: Teratoma Formation

[0093] iPS cells and NSCs cells (1.5×10⁶ cells/mice) were injected subcutaneously into the dorsal flank of nude mice. Four weeks after the injection, teratomas that had formed were fixed overnight in 4% PFA and embedded in paraffin. Sections were stained with haematoxylin and eosin dyes.

Example 8: Chimera Formation

[0094] iPS cells were aggregated and cultured with denuded post-compacted 8-cell-stage mouse embryos. Briefly, 2-cell-stage embryos were flushed from mice [(C57BL/6×C3H) F1 females×CD1 males] at 1.5 dpc and placed in M2 medium and cultured overnight in KSOM medium with 0.1% BSA overnight to 8-cell stage. Clumps of loosely connected iPS cells (10-20 cells) from short trypsin-treated day-2 cultures were selected and transferred into microdrops of KSOM medium with 10% FCS under mineral oil; each clump was placed in a depression in the microdrop. Meanwhile, batches of 30 to 40 embryos were briefly incubated with acidified Tyrode's solution until the zona pellucida had disintegrated. A single embryo was place onto the clump. All aggregates were assembled in this manner, and cultured at 37° C. in an atmosphere of 5% CO₂ in air. After 24 hours of culture, the majority of the aggregates had formed blastocysts. A total of 64 aggregated blastocysts (2.5 dpc) were transferred into the uterine horns of five pseudopregnant mice (CD-1 background).

Example 9: Reprogramming of Human Neural Stem Cells by Oct4

[0095] hNSCs that derived from human fetal brain tissue were expanded in serum-free NSC medium as described previously (cf. FIG. 5A) (Kim et al., Exp Neural 199, 222 (2006); Park et al., Nat Biotechnol 20, 1111 (2002)). hNSCs were first infected with pMXs encoding human OCT4 and KLF4 (2F) or OCT4 (1F). Then, infected hNSCs were maintained in NSC medium (Kim at al., Exp Neural 199, 222 (2006)) for up to 7 days. Day 8 post-infection, the cells were replated onto feeder cell layers in hESC medium containing 10 ng/ml bFGF and MEF-conditioned medium (CM) in a 1:1 ratio which culture continued to grow until the hESC-like colonies appeared. Within 10-11 weeks post-infection, the hESC-like IPS colonies were identified but the centre of the colonies still appears like a neural rosette (cf. FIG. 5B). The colony grew larger exhibiting typical hESC-like morphology within another 5-6 days but still the neural rosettes remain in the center of the colony (cf. FIG. 5C). The neural rosettes are removed from the colony. Then, a piece of the colony was transferred on a feeder cell layer by mechanical isolation (cf. FIG. 5D). We successfully established two clones out of three hESC-like colonies by picking from OCT4 infected hNSCs (1F hNiPS clone A and C, reprogramming efficiency 0,02%). Otherwise, we also established 3 clones out of five hESC-like colonies in 2F-infected hNSCs (2F hNiPS A, B and C, reprogramming efficiency, 0.15%) within 7-8 weeks post-infection. All of which could be expanded in hESC culture condition. The 1F hNiPS cells were morphologically similar to hESCs and stained positive for alkaline phosphatase (cf. FIG. 5E and F). Immunofluorescence staining confirmed that 2F and 1F hNiPS cells uniformly expressed the pluripotency markers, including OCT4, SSEA4, TRA-1-60 and TRA-1-81 (cf. FIG. 5G). These results demonstrate that human iPS cells can be generated from hNSCs by OCT4 and KLF4 as well as OCT4 alone.

[0096] Next, we tested mRNA expression levels of pluripotency marker genes in these iPS cells at molecular level by quantitative RT-PCR analysis. 2F and 1F hNiPS cells endogenously expressed the hESCs-specific markers, were similar to H9 and H1 hESCs and were markedly up-regulated compared with parental hNSCs (cf. FIG. 6A). Genotyping PCR showed 1F hNiPS clones have an OCT4 transgene only and 2F hNiPS clones have OCT4 and KLF4 transgenes in the genome. We also confirmed that the expression level of transgenic OCT4 or KLF4 was significantly silenced in 2F and 1F hNiPS clones, except the OCT4 expression from 2F hNiPS clone B. Southern blot analysis confirmed the integration of the OCT4 transgene in 2F and 1F hNiPS clones. To exclude the possibility that iPS clones arose through contamination from hESCs in the laboratory, DNA fingerprinting analysis was performed and confirmed that hNiPS cells precisely correlate to the donor hNSCs (cf. Table 2).

[0097] To confirm epigenetic remodelling of the OCT4 and NANOG promoters from reprogrammed cells, we performed bisulfite sequencing to determine the demethylation of both promoters. OCT4 and NANOG promoter regions were demethylatd in 2F and 1F hNiPS cells relative to the donor hNSCs and were similar to hESCs. Taken together, hNSCs can be reprogrammed into iPS cells that similar to hESCs at molecular level by transduction of OCT4 alone.

[0098] Next, we tested in vitro pluripotency of 2F and 1F hNiPS cells by embryoid body (EB) differentiation and direct differentiation. hNiPS cells readily differentiated into endoderm (AFP), medoderm (a-SMA) and ectoderm (Tuj1) by EB differentiation (cf. FIG. 5A) and we confirmed the expression of all three germ layer makers from direct differentiation by quantitative RT-PCR analysis (cf. FIG. 7B). To confirm in vivo pluripotency of these human iPS cells, the cells were subcutaneously transplanted into severe combined immunodeficient (SCID) mice. After 6-8 weeks injection, 2F and 1F hNiPS cells gave rise to teratomas containing all three germ layers, including respiratory tract, skeletal muscle, cartilage and neural epithelium (cf. FIG. 8A). These results indicate that 2F and 1F hNiPS cells have a pluripotency in vitro and in vivo alike hESCs.

[0099] Finally, we performed global gene expression analysis on hNSC, 2F and 1F hNiPS cells derived from hNSCs, H9 and H1 hESCs by cDNA microarrays. Heat map showed that 2F and 1F hNiPS cells similar to hESCs, otherwise parental hNSCs are isolated from pluripotent polulations (cf. FIG. 8B, left panel) and hierarchical clustering analysis showed that hNiPS cells clustered with hESCs and were distinct from parental hNSCs (cf. FIG. 8B, right panel). Scatter plots analysis showed that hNiPS cells are significantly more similar to hESCs as like between different hESCs than parental hNSCs (cf. FIG. 8C). 1F and 2F hNiPS cells also show similarity with H1 hESCs. These data indicate that hNiPS cells are similar to hESCs in global gene expression profiles. Our results demonstrated 1F and 2F hNiPS cells closely resemble hESCs in molecular level and pluripotency.

TABLE-US-00003 TABLE 2 STR analysis of hNSCs and hNiPS cells 2F 1F Genomic H9 NhiPS NhiPS loci hESCs hNSCs A B C A C Amelogenin X;X X;Y X;Y X;Y X;Y X;Y X;Y CSF1PO 11;11 11;13 11;13 11;13 11;13 11;13 11;13 D13S317 9;9 8;11 8;11 8;11 8;11 8;11 8;11 D16S539 12;13 9;9 9;9 9;9 9;9 9;9 9;9 D18S51 13;13 15;16 15;16 15;16 15;16 15;16 15:16 D21S11 30;30 31;32 31;32 31;32 31;32 31;32 31:32 D3S1358 13;16 16;16 16;16 16;16 16;16 16;16 16;16 D5S818 11;12 7;12 7;12 7;12 7;12 7;12 7;12 D7S820 9;11 11;11 11;11 11;11 11;11 11;11 11;11 D8S1179 8;14 12;14 12;14 12:14 12;14 12;14 12;14 FGA 26;28 23;24 23;24 23;24 23;24 23;24 23;24 Penta D 9;13 11;12 11;12 11;12 11;12 11;12 11;12 Penta E 11;14 11;18 11;18 11;18 11;18 11;18 11;18 TH01 9;9 7;7 7;7 7;7 7;7 7;7 7;7 TPOX 10;11 8;8 8;8 8;8 8;8 8;8 8;8 vWA 17;17 17;17 17;17 17;17 17;17 17;17 17:17

MATERIAL AND METHODS

Cell Culture

[0100] Human NSCs were derived from the telencephalon (HFT13), established as previously described (Kim et al., Exp Neural 199, 222 (2006)). Briefly, Telencephalon tissue was freshly dissected, dissociated in 0.1% trypsin for 30 min and seeded into 10 cm plates at a density of 200,000 cells/ml in NSC medium. These cells were cultured in NSC medium as previously described (Kim at al., Exp Neural 199, 222 (2006); Park at al., Nat Biotechnol 20, 1111 (2002)). Human ES and iPS cells were maintatined on mitomycin C-treated CF1 mouse feeder layers (Millipore) in human ESC medium, which contains knockout DMEM (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, penicillin/streptomycin and 10 ng/ml human basic fibroblast growth factor (bFGF) (Invitrogen) as previously described (Takahashi et al., Cell 131, 861 (2007)).

Induction of 1F hNiPS and 2F hNiPs Cells

[0101] The pMX-based retroviral vectors encoding the human cDNAs of OCT4 and KLF4 (Takahashi et Yamanaka, Cell 126, 663 (2006)) were cotransfected with packaging-defective helper plasmids into 293 cells using Fugene transfection reagent (Roche) to produce vesicular stomatitis virus (VSV) G protein pseudotyped virus as previously described (Zaehres et Daley, Methods Enzyrnol 420, 49 (2006)). Viral supernatants were collected and concentrated by ultracentrifugation 48 h post-transfection to infect human NSCs. For generation of iPS cells, human NSCs were seeded at a density of 5×10⁴ cells per 6-well plate and incubated with virus-containing supernatants for OCT4 or OCT4 and KLF4 supplemented with 6 μg/ml protamine sulfate (Sigma) for 24 h. On the next day, the medium was replaced with fresh NSC medium at 1 d post-infection and maintained up to 7 d post-infection. Cells were further cultured in human ESC medium from 8 d post-infection. The iPS colonies were mechanically isolated at 2 month or 2.5 month post-infection and were subsequently replated and maintained on CF1 mouse feeder layers (Millipore) in human ESC medium.

Quantitative RT-PCR

[0102] Total RNA was isolated from bulk cell culture samples or hand-picked undifferentiated colonies using RNeasy columns (Qiagen) with on-column DNA digestion. cDNA was produced using oligo-dT₁₅ priming and M-MLV reverse transcriptase (USB) according to the manufacturer's instructions at 42° C. for 1 h. About 50 ng of total RNA equivalent was typically used as template in 20 μl SYBR Green PCR reactions (40 cycles of 15'' 95° C./60'' 60° C. on Applied Biosystems 7300 instrumentation) that additionally contained 0.375 μM of each primer and 10 μl of SYBR Green PCR mix (ABI). All primers used were confirmed to amplify the predicted product at close-to-optimal efficiency without side products. Primer sequences are given in Table 3. Relative expression levels were calculated using the comparative Ct method, based on biological control samples and two housekeeping genes for normalization. Error bars reflect standard errors arising from biological replicates (marker gene expression data) or from using independent housekeeping genes for normalization (transgene silencing data).

Global Gene Expression Analysis

[0103] For transcriptional analysis, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit--Ambion) following the manufacturer's instructions (IVT: 10 h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto HumanRef-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials/instrumentation supplied/suggested by the manufacturer. The microarray data are available from the GEO (Gene Expression Omnibus) website under accession number GSE GSE15355.

Bisulfite Sequencing

[0104] Genomic DNA was isolated from bulk cell culture samples or hand-picked undifferentiated colonies using DNeasy columns (Qiagen). 300 ng was used as input for bisulfite conversion (EpiTect Bisulfite Kit--Qiagen). 50 ng of converted DNA was used as template for conventional nested PCRs amplifying 467 and 336 bp regions of the OCT4 and NANOG promoters, respectively. Primers were specific for conversion of the sense DNA strand and are given in Table 3. Purified PCRs were TA-cloned into pCR2.1-TOPO (Invitrogen). Insert sequences of randomly picked clones were analyzed using the BiQ Analyzer program, following its quality check-based suggestions to drop individual clones if appropriate. Data from one CpG site at position +20 relative to the OCT4 translation start codon is not shown as it was uninformative.

Short Tandem Repeat (STR) Analysis

[0105] Genomic DNA was isolated from cultured cell samples using DNeasy columns (Qiagen). This was used as template for STR analysis employing the PowerPlex 16 System (Promega) and ABI PRISM instrumentation, Numbers shown denote by lengths of the 15 autosomal fragments. The analysis was carried out at Eurofins Medigenomix, Martinsried, Germany.

Teratoma Formation

[0106] hNiPS cells and hNSCs (3-5×10⁶ cells/mice) were injected subcutaneously into the dorsal flank of SCID) mice. Teratomas were fixed in 4% PFA overnight and embedded in paraffin after 6-8 weeks injection. Sections were stained with haematoxylin and eosin dyes.

Alkaline Phosphatase (AP) and Immunofluorescence Staining

[0107] Alkaline phosphatase (AP) staining was performed with the ES Cell Characterization Kit (Chemicon) according to the manufacturer's protocol. Immunofluorescence staining was performed using the following primary antibodies; AFP (Sigma, 1:100), a-SMA (Sigma, 1:50), TuJ1 (Chemicon, 1:500), OCT4 (Santa Cruz, 1:200), SSEA4 (Chemicon, 1:200), TRA-1-60 (Chemicon, 1:200), TRA-1-81 (Chemicon, 1:200).

TABLE-US-00004 TABLE 3 Primers for Real-time PCR and Bisulfite sequencing. Gene Forward primer (5'-3') Reverse primer (5'-3') Real-time PCR Primers ACTB TCAAGATCATTGCTCCTCCTGAG ACATCTGCTGGAAGGTGGACA AFP AGCAGCTTGGTGGTGGATGA CCTGAGCTTGGCACAGATCCT CDH1 (E-CAD) TTGAGGCCAAGCAGCAGTACA ATCCAGCACATCCACGGTGA CDX2 TCACTACAGTCGCTACATCACCATC TTAACCTGCCTCTCAGAGAGCC DNMT3B GCTCACAGGGCCCGATACTT GCAGTCCTGCAGCTCGAGTTTA DPPA4 TGGTGTCAGGTGGTGTGTGG CCAGGCTTGACCAGCATGAA FGF2 GGCAAGATGCAGGAGAGAGGA GCCACGTGAGAGCAGAGCAT FOXF1 AAAGGAGCCACGAAGCAAGC AGGCTGAAGCGAAGGAAGAGG GAPDH CTGGTAAAGTGGATATTGTTGCCAT TGGAATCATATTGGAACATGTAAACC GATA6 TGTGCGTTCATGGAGAAGATCA TTTGATAAGAGACCTCATGAACCGACT GDF3 TTGGCACAAGTGGATCATTGC TTGGCACAAGTGGATCATTGC HAND1 TCCCTTTTCCGCTTGCTCTC CATCGCCTACCTGATGGACG KLF4 endo ACAGTCTGTTATGCACTGTGGTTTCA CATTTGTTCTGCTTAAGGCATACTTGG KLF4 viral GTCGGACCACCTCGCCTTAC TTTATCGTCGACCACTGTGCTG LIN28 GGAGGCCAAGAAAGGGAATATGA AACAATCTTGTGGCCACTTTGACA MYC CCAGCAGCGACTCTGAGGA GAGCCTGCCTCTTTTCCACAG NANOG CCTGTGATTTGTGGGCCTG GACAGTCTCCGTGTGAGGCAT NCAM1 TCATGTGCATTGCGGTCAAC ACGATGGGCTCCTTGGACTC OCT4 endo GGAAGGAATTGGGAACACAAAGG AACTTCACCTTCCCTCCAACCA OCT4 viral GGCTCTCCCATGCATTCAAAC TTTATCGTCGACCACTGTGCTG SOX17 TTCGTGTGCAAGCCTGAGATG GTCGGACACCACCGAGGAA SOX2 TGGCGAACCATCTCTGTGGT CCAACGGTGTCAACCTGCAT TDGF1 (Cripto) GGGATACAGCACAGTAAGGAGCTAA CACAAAAGGACCCCAGCATG ZNF206 TCACCATGGCCAGAGGAGAG GCAGGCCACGCCTTATTCTC ZNF589 TCGGGTGGCTAAATTACATCCAG CCCAAGGGAGTAAGGCAAACTG Primers for bisulfite sequencing OCT4 outer GAGGATAGGAATTTAAGATTAGTTTGGGTA AAATCCCCCACACCTCAAAACCTAACCCAA OCT4 inner GAGGTTGGAGTAGAAGGATTGTTTTGGTTT CCCCCCTAACCCATCACCTCCACCACCTAA OCT4 inner GAGGCTGGAGCAGAAGGATTGCTTTGGCCC CCCCCCTGGCCCATCACCTCCACCACCTGG unconverted NANOG outer TTAGTTTTTAGAGTAGTTGGGATTATAGA ATAATAACATAAAACAACCAACTCAATCCA NANOG inner TGGTTAGGTTGGTTTTAAATTTTTG AACCCACCCTTATAAATTCTCAATTA NANOG inner TGGCCAGGCTGGTTTCAAACTCCTG GACCCACCCTTGTGAATTCTCAGTTA unconverted

Southern Blot Analysis

[0108] BamHI digested genomic DNA from 1F hNiPS, hNSC and 2F hNiPS cells were separated on a 0.8% agarose gel and transferred to Biodyne B nylon membrane (PALL Life Sciences). DNA was hybridized with a 32P labeled fragment of OCT4 (Eco81I (Saul) human OCT4 cDNA fragment) using the DecaLabel® DNA Labeling Kit (Fermentas). Labeled Lambda HindIII digested DNA served as a marker.

In Vitro Differentiation of Human iPS Cells

[0109] For immunocytochemistry, embryoid bodies (EBs) were generated from iPS cells with the hanging drop method in MEF-conditioned medium. After 5 days, EBs were transferred to gelatin-coated plate and subsequent culturing for another 14 days in knockout DMEM (Invitrogen) supplemented with 20% FBS, 1mM L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, and penicillin/streptomycin. For qRT-PCR, iPS colonies were mechanically isolated and replated on Matrigel-coated plate in MEF-conditioned medium. After 2 d, medium replaced with each three germ layer differentiation medium. For endoderm differentiation, the cells maintained in RPMI1640 medium supplemented with 2% FBS, 100 ng/ml Activin A (R&D Systems). L-glutamine, and penicillin/streptomycin for 3 weeks (Huangfu et al., Nat Biotechnol 26, 1269 (2008)). For mesoderm differentiation, knockout DMEM supplemented with 100 uM ascorbic acid (Sigma), 20% FBS, 1mM L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, and penicillin/streptomycin for 3 weeks (Aasen et al., Nat Biotechnol 26, 1276 (2008)). For ectoderm differentiation, the cells maintained in N2B27 medium for 7 days and the medium replaced with N2 medium supplemented with 10 ng/ml bFGF2 (peprotech), 100 ng/ml Sonic Hedgehog (R&D Systems), 10 ng/ml PDFG (R&D Systems), L-glutamine, and penicillin/streptomycin for 2 weeks. The medium was changed every other day. Primer sequences are given in Table 3.

REFERENCES

[0110] Biswas, A. and Hutchins, R. (2007). Embryonic stem cells. Stem Cells Dev 16(2):213-22.

[0111] Davis, P. B., Cooper, M. J. (2007). Vectors for airway gene delivery. AAPS J 19;9(1):E11-7.

[0112] Do, J. T. & Scholer, H. R. (2004). Nuclei of embryonic stem cells reprogram somatic cells. Stem Cells 22:941-9.

[0113] Gage, F. H. (2000). Mammalian neural stem cells. Science 287(5457):1433-8.

[0114] Hanna, J., Wernig, M., Markoulaki, S., Sun, C. W., Meissner, A., Cassady, J. P., Beard, C., Brambrink, T., Wu, L. C., Townes, T. M. and Jaenisch, R. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318(5858):1920-3.

[0115] Hogan, B., R. Beddington, et al. (1994), "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbour Press.

[0116] Jones, G. E., Wise, C. J. (1997). Establishment, maintenance, and cloning of human dermal fibroblasts. Methods Mol Biol. 75:13-21.

[0117] Kim, D. S., Kim, J. Y., Kang, M., Cho, M. S., Kim, D. W. (2007). Derivation of functional dopamine neurons from embryonic stem cells. Cell Transplant 16(2):117-23.

[0118] Li, S. and Ma, Z. (2001). Nonviral gene therapy. Curr Gene Ther., 1(2):201-26.

[0119] Meissner, A., Wernig, M. and Jaenisch, R. (2007). Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 25(10): 1177-81.

[0120] Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N. and Yamanaka, S. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 26(1):101-6.

[0121] Okita, K., Ichisaka, T. and Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature 448(7151):313-7.

[0122] Park, I. H., Zhao, R., West, J. A., Yabuuchi, A., Huo, H., Ince, T. A., Lerou, P. H., Lensch, M. W., Daley, G. Q. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451(7175):141-6.

[0123] Pollard, S. M., Conti, L., Sun, Y., Goffredo, D. & Smith, A. (2006). Adherent neural stem (NS) cells from fetal and adult forebrain. Cereb Cortex 16 Suppl 1, i112-20.

[0124] Ryan, A. K. and Rosenfeld, M. G. (1997). POU domain family values: flexibility, partnerships, and developmental codes, Genes Dev 11:1207-25.

[0125] Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-76.

[0126] Takahashi, K., Okita, K., Nakagawa, M., and Yamanaka, S. (2007). Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2(12):3081-9.

[0127] Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861-72.

[0128] Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E. and Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318-24.

[0129] Wernig, M., Meissner, A., Cassady, J. P. and Jaenisch, R. (2008). c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cells 2:10-12.

[0130] Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Franc, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I. and Thomson, J. A. (2007). Induced pluripotent stern cell lines derived from human somatic cells. Science 318(5858):1917-20.

[0131] Zimmermann, W. H. and Eschenhagen, T. (2007). Embryonic stern cells for cardiac muscle engineering. Trends Cardiovasc Med 17(4):134-40.

[0132] H. T. Kim et al., Exp Neurol 199, 222 (May, 2006)

[0133] K. I. Park, Y. D. Teng, E. Y. Snyder, Nat Biotechnol 20, 1111 (Nov, 2002)

[0134] H. Zaehres, G. Q. Daley, Methods Enzymol 420, 49 (2006)

[0135] D. Huangfu et al., Nat Biotechnol 26, 1269 (Nov, 2008)

[0136] T. Aasen et al., Nat Biotechnol 26, 1276 (Nov, 2008)

Sequence CWU 1

9311059DNAmus musculus 1atggctggac acctggcttc agacttcgcc ttctcacccc caccaggtgg gggtgatggg 60tcagcagggc tggagccggg ctgggtggat cctcgaacct ggctaagctt ccaagggcct 120ccaggtgggc ctggaatcgg accaggctca gaggtattgg ggatctcccc atgtccgccc 180gcatacgagt tctgcggagg gatggcatac tgtggacctc aggttggact gggcctagtc 240ccccaagttg gcgtggagac tttgcagcct gagggccagg caggagcacg agtggaaagc 300aactcagagg gaacctcctc tgagccctgt gccgaccgcc ccaatgccgt gaagttggag 360aaggtggaac caactcccga ggagtcccag gacatgaaag ccctgcagaa ggagctagaa 420cagtttgcca agctgctgaa gcagaagagg atcaccttgg ggtacaccca ggccgacgtg 480gggctcaccc tgggcgttct ctttggaaag gtgttcagcc agaccaccat ctgtcgcttc 540gaggccttgc agctcagcct taagaacatg tgtaagctgc ggcccctgct ggagaagtgg 600gtggaggaag ccgacaacaa tgagaacctt caggagatat gcaaatcgga gaccctggtg 660caggcccgga agagaaagcg aactagcatt gagaaccgtg tgaggtggag tctggagacc 720atgtttctga agtgcccgaa gccctcccta cagcagatca ctcacatcgc caatcagctt 780gggctagaga aggatgtggt tcgagtatgg ttctgtaacc ggcgccagaa gggcaaaaga 840tcaagtattg agtattccca acgagaagag tatgaggcta cagggacacc tttcccaggg 900ggggctgtat cctttcctct gcccccaggt ccccactttg gcaccccagg ctatggaagc 960ccccacttca ccacactcta ctcagtccct tttcctgagg gcgaggcctt tccctctgtt 1020cccgtcactg ctctgggctc tcccatgcat tcaaactga 10592352PRTmus musculus 2Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly1 5 10 15Gly Gly Asp Gly Ser Ala Gly Leu Glu Pro Gly Trp Val Asp Pro Arg 20 25 30Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly Pro 35 40 45Gly Ser Glu Val Leu Gly Ile Ser Pro Cys Pro Pro Ala Tyr Glu Phe 50 55 60Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val Gly Leu Gly Leu Val65 70 75 80Pro Gln Val Gly Val Glu Thr Leu Gln Pro Glu Gly Gln Ala Gly Ala 85 90 95Arg Val Glu Ser Asn Ser Glu Gly Thr Ser Ser Glu Pro Cys Ala Asp 100 105 110Arg Pro Asn Ala Val Lys Leu Glu Lys Val Glu Pro Thr Pro Glu Glu 115 120 125Ser Gln Asp Met Lys Ala Leu Gln Lys Glu Leu Glu Gln Phe Ala Lys 130 135 140Leu Leu Lys Gln Lys Arg Ile Thr Leu Gly Tyr Thr Gln Ala Asp Val145 150 155 160Gly Leu Thr Leu Gly Val Leu Phe Gly Lys Val Phe Ser Gln Thr Thr 165 170 175Ile Cys Arg Phe Glu Ala Leu Gln Leu Ser Leu Lys Asn Met Cys Lys 180 185 190Leu Arg Pro Leu Leu Glu Lys Trp Val Glu Glu Ala Asp Asn Asn Glu 195 200 205Asn Leu Gln Glu Ile Cys Lys Ser Glu Thr Leu Val Gln Ala Arg Lys 210 215 220Arg Lys Arg Thr Ser Ile Glu Asn Arg Val Arg Trp Ser Leu Glu Thr225 230 235 240Met Phe Leu Lys Cys Pro Lys Pro Ser Leu Gln Gln Ile Thr His Ile 245 250 255Ala Asn Gln Leu Gly Leu Glu Lys Asp Val Val Arg Val Trp Phe Cys 260 265 270Asn Arg Arg Gln Lys Gly Lys Arg Ser Ser Ile Glu Tyr Ser Gln Arg 275 280 285Glu Glu Tyr Glu Ala Thr Gly Thr Pro Phe Pro Gly Gly Ala Val Ser 290 295 300Phe Pro Leu Pro Pro Gly Pro His Phe Gly Thr Pro Gly Tyr Gly Ser305 310 315 320Pro His Phe Thr Thr Leu Tyr Ser Val Pro Phe Pro Glu Gly Glu Ala 325 330 335Phe Pro Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser Asn 340 345 35031083DNAhomo sapiens 3atggcgggac acctggcttc ggatttcgcc ttctcgcccc ctccaggtgg tggaggtgat 60gggccagggg ggccggagcc gggctgggtt gatcctcgga cctggctaag cttccaaggc 120cctcctggag ggccaggaat cgggccgggg gttgggccag gctctgaggt gtgggggatt 180cccccatgcc ccccgccgta tgagttctgt ggggggatgg cgtactgtgg gccccaggtt 240ggagtggggc tagtgcccca aggcggcttg gagacctctc agcctgaggg cgaagcagga 300gtcggggtgg agagcaactc cgatggggcc tccccggagc cctgcaccgt cacccctggt 360gccgtgaagc tggagaagga gaagctggag caaaacccgg aggagtccca ggacatcaaa 420gctctgcaga aagaactcga gcaatttgcc aagctcctga agcagaagag gatcaccctg 480ggatatacac aggccgatgt ggggctcacc ctgggggttc tatttgggaa ggtattcagc 540caaacgacca tctgccgctt tgaggctctg cagcttagct tcaagaacat gtgtaagctg 600cggcccttgc tgcagaagtg ggtggaggaa gctgacaaca atgaaaatct tcaggagata 660tgcaaagcag aaaccctcgt gcaggcccga aagagaaagc gaaccagtat cgagaaccga 720gtgagaggca acctggagaa tttgttcctg cagtgcccga aacccacact gcagcagatc 780agccacatcg cccagcagct tgggctcgag aaggatgtgg tccgagtgtg gttctgtaac 840cggcgccaga agggcaagcg atcaagcagc gactatgcac aacgagagga ttttgaggct 900gctgggtctc ctttctcagg gggaccagtg tcctttcctc tggccccagg gccccatttt 960ggtaccccag gctatgggag ccctcacttc actgcactgt actcctcggt ccctttccct 1020gagggggaag cctttccccc tgtctccgtc accactctgg gctctcccat gcattcaaac 1080tga 10834360PRThomo sapiens 4Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly1 5 10 15Gly Gly Gly Asp Gly Pro Gly Gly Pro Glu Pro Gly Trp Val Asp Pro 20 25 30Arg Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly 35 40 45Pro Gly Val Gly Pro Gly Ser Glu Val Trp Gly Ile Pro Pro Cys Pro 50 55 60Pro Pro Tyr Glu Phe Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val65 70 75 80Gly Val Gly Leu Val Pro Gln Gly Gly Leu Glu Thr Ser Gln Pro Glu 85 90 95Gly Glu Ala Gly Val Gly Val Glu Ser Asn Ser Asp Gly Ala Ser Pro 100 105 110Glu Pro Cys Thr Val Thr Pro Gly Ala Val Lys Leu Glu Lys Glu Lys 115 120 125Leu Glu Gln Asn Pro Glu Glu Ser Gln Asp Ile Lys Ala Leu Gln Lys 130 135 140Glu Leu Glu Gln Phe Ala Lys Leu Leu Lys Gln Lys Arg Ile Thr Leu145 150 155 160Gly Tyr Thr Gln Ala Asp Val Gly Leu Thr Leu Gly Val Leu Phe Gly 165 170 175Lys Val Phe Ser Gln Thr Thr Ile Cys Arg Phe Glu Ala Leu Gln Leu 180 185 190Ser Phe Lys Asn Met Cys Lys Leu Arg Pro Leu Leu Gln Lys Trp Val 195 200 205Glu Glu Ala Asp Asn Asn Glu Asn Leu Gln Glu Ile Cys Lys Ala Glu 210 215 220Thr Leu Val Gln Ala Arg Lys Arg Lys Arg Thr Ser Ile Glu Asn Arg225 230 235 240Val Arg Gly Asn Leu Glu Asn Leu Phe Leu Gln Cys Pro Lys Pro Thr 245 250 255Leu Gln Gln Ile Ser His Ile Ala Gln Gln Leu Gly Leu Glu Lys Asp 260 265 270Val Val Arg Val Trp Phe Cys Asn Arg Arg Gln Lys Gly Lys Arg Ser 275 280 285Ser Ser Asp Tyr Ala Gln Arg Glu Asp Phe Glu Ala Ala Gly Ser Pro 290 295 300Phe Ser Gly Gly Pro Val Ser Phe Pro Leu Ala Pro Gly Pro His Phe305 310 315 320Gly Thr Pro Gly Tyr Gly Ser Pro His Phe Thr Ala Leu Tyr Ser Ser 325 330 335Val Pro Phe Pro Glu Gly Glu Ala Phe Pro Pro Val Ser Val Thr Thr 340 345 350Leu Gly Ser Pro Met His Ser Asn 355 3605960DNAmus musculus 5atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240tggaaacttt tgtccgagac cgagaagcgg ccgttcatcg acgaggccaa gcggctgcgc 300gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcggaa aaccaagacg 360ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420atggcgagcg gggttggggt gggcgccggc ctgggtgcgg gcgtgaacca gcgcatggac 480agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg 540ggctacccgc agcacccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac 600cgctacgacg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac 660ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac 900cagagcggcc cggtgcccgg cacggccatt aacggcacac tgcccctgtc gcacatgtga 9606319PRTmus musculus 6Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln1 5 10 15Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala Thr 20 25 30Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn 35 40 45Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu 50 55 60Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu65 70 75 80Trp Lys Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala 85 90 95Lys Arg Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr 100 105 110Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr 115 120 125Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly 130 135 140Val Gly Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp145 150 155 160Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met 165 170 175Gln Glu Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly 180 185 190Ala Ala Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln 195 200 205Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr 210 215 220Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly225 230 235 240Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val 245 250 255Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu 260 265 270Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro 275 280 285Ala Ala Pro Ser Arg Leu His Met Ala Gln His Tyr Gln Ser Gly Pro 290 295 300Val Pro Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met305 310 3157954DNAhomo sapiens 7atgtacaaca tgatggagac ggagctgaag ccgccgggcc cgcagcaaac ttcggggggc 60ggcggcggca actccaccgc ggcggcggcc ggcggcaacc agaaaaacag cccggaccgc 120gtcaagcggc ccatgaatgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc 180caggagaacc ccaagatgca caactcggag atcagcaagc gcctgggcgc cgagtggaaa 240cttttgtcgg agacggagaa gcggccgttc atcgacgagg ctaagcggct gcgagcgctg 300cacatgaagg agcacccgga ttataaatac cggccccggc ggaaaaccaa gacgctcatg 360aagaaggata agtacacgct gcccggcggg ctgctggccc ccggcggcaa tagcatggcg 420agcggggtcg gggtgggcgc cggcctgggc gcgggcgtga accagcgcat ggacagttac 480gcgcacatga acggctggag caacggcagc tacagcatga tgcaggacca gctgggctac 540ccgcagcacc cgggcctcaa tgcgcacggc gcagcgcaga tgcagcccat gcaccgctac 600gacgtgagcg ccctgcagta caactccatg accagctcgc agacctacat gaacggctcg 660cccacctaca gcatgtccta ctcgcagcag ggcacccctg gcatggctct tggctccatg 720ggttcggtgg tcaagtccga ggccagctcc agcccccctg tggttacctc ttcctcccac 780tccagggcgc cctgccaggc cggggacctc cgggacatga tcagcatgta tctccccggc 840gccgaggtgc cggaacccgc cgcccccagc agacttcaca tgtcccagca ctaccagagc 900ggcccggtgc ccggcacggc cattaacggc acactgcccc tctcacacat gtga 9548317PRThomo sapiens 8Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln1 5 10 15Thr Ser Gly Gly Gly Gly Gly Asn Ser Thr Ala Ala Ala Ala Gly Gly 20 25 30Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn Ala Phe 35 40 45Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu Asn Pro 50 55 60Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys65 70 75 80Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala Lys Arg 85 90 95Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro 100 105 110Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr Leu Pro 115 120 125Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly Val Gly 130 135 140Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp Ser Tyr145 150 155 160Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met Gln Asp 165 170 175Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly Ala Ala 180 185 190Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn 195 200 205Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser 210 215 220Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly Ser Met225 230 235 240Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val Val Thr 245 250 255Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu Arg Asp 260 265 270Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro Ala Ala 275 280 285Pro Ser Arg Leu His Met Ser Gln His Tyr Gln Ser Gly Pro Val Pro 290 295 300Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met305 310 31591320DNAmus musculus 9atgcccctca acgtgaactt caccaacagg aactatgacc tcgactacga ctccgtacag 60ccctatttca tctgcgacga ggaagagaat ttctatcacc agcaacagca gagcgagctg 120cagccgcccg cgcccagtga ggatatctgg aagaaattcg agctgcttcc caccccgccc 180ctgtccccga gccgccgctc cgggctctgc tctccatcct atgttgcggt cgctacgtcc 240ttctccccaa gggaagacga tgacggcggc ggtggcaact tctccaccgc cgatcagctg 300gagatgatga ccgagttact tggaggagac atggtgaacc agagcttcat ctgcgatcct 360gacgacgaga ccttcatcaa gaacatcatc atccaggact gtatgtggag cggtttctca 420gccgctgcca agctggtctc ggagaagctg gcctcctacc aggctgcgcg caaagacagc 480accagcctga gccccgcccg cgggcacagc gtctgctcca cctccagcct gtacctgcag 540gacctcaccg ccgccgcgtc cgagtgcatt gacccctcag tggtctttcc ctacccgctc 600aacgacagca gctcgcccaa atcctgtacc tcgtccgatt ccacggcctt ctctccttcc 660tcggactcgc tgctgtcctc cgagtcctcc ccacgggcca gccctgagcc cctagtgctg 720catgaggaga caccgcccac caccagcagc gactctgaag aagagcaaga agatgaggaa 780gaaattgatg tggtgtctgt ggagaagagg caaacccctg ccaagaggtc ggagtcgggc 840tcatctccat cccgaggcca cagcaaacct ccgcacagcc cactggtcct caagaggtgc 900cacgtctcca ctcaccagca caactacgcc gcacccccct ccacaaggaa ggactatcca 960gctgccaaga gggccaagtt ggacagtggc agggtcctga agcagatcag caacaaccgc 1020aagtgctcca gccccaggtc ctcagacacg gaggaaaacg acaagaggcg gacacacaac 1080gtcttggaac gtcagaggag gaacgagctg aagcgcagct tttttgccct gcgtgaccag 1140atccctgaat tggaaaacaa cgaaaaggcc cccaaggtag tgatcctcaa aaaagccacc 1200gcctacatcc tgtccattca agcagacgag cacaagctca cctctgaaaa ggacttattg 1260aggaaacgac gagaacagtt gaaacacaaa ctcgaacagc ttcgaaactc tggtgcataa 132010454PRTmus musculus 10Leu Asp Phe Leu Trp Ala Leu Glu Thr Pro Gln Thr Ala Thr Thr Met1 5 10 15Pro Leu Asn Val Asn Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp 20 25 30Ser Val Gln Pro Tyr Phe Ile Cys Asp Glu Glu Glu Asn Phe Tyr His 35 40 45Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp Ile 50 55 60Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser Arg65 70 75 80Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Ala Thr Ser Phe 85 90 95Ser Pro Arg Glu Asp Asp Asp Gly Gly Gly Gly Asn Phe Ser Thr Ala 100 105 110Asp Gln Leu Glu Met Met Thr Glu Leu Leu Gly Gly Asp Met Val Asn 115 120 125Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile 130 135 140Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu145 150 155 160Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Thr 165 170 175Ser Leu Ser Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu 180 185 190Tyr Leu Gln Asp Leu Thr Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser 195

200 205Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys 210 215 220Thr Ser Ser Asp Ser Thr Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu225 230 235 240Ser Ser Glu Ser Ser Pro Arg Ala Ser Pro Glu Pro Leu Val Leu His 245 250 255Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu 260 265 270Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Thr Pro 275 280 285Ala Lys Arg Ser Glu Ser Gly Ser Ser Pro Ser Arg Gly His Ser Lys 290 295 300Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His305 310 315 320Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala 325 330 335Ala Lys Arg Ala Lys Leu Asp Ser Gly Arg Val Leu Lys Gln Ile Ser 340 345 350Asn Asn Arg Lys Cys Ser Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn 355 360 365Asp Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu 370 375 380Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu385 390 395 400Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala 405 410 415Tyr Ile Leu Ser Ile Gln Ala Asp Glu His Lys Leu Thr Ser Glu Lys 420 425 430Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln 435 440 445Leu Arg Asn Ser Gly Ala 450111320DNAhomo sapiens 11atgcccctca acgttagctt caccaacagg aactatgacc tcgactacga ctcggtgcag 60ccgtatttct actgcgacga ggaggagaac ttctaccagc agcagcagca gagcgagctg 120cagcccccgg cgcccagcga ggatatctgg aagaaattcg agctgctgcc caccccgccc 180ctgtccccta gccgccgctc cgggctctgc tcgccctcct acgttgcggt cacacccttc 240tcccttcggg gagacaacga cggcggtggc gggagcttct ccacggccga ccagctggag 300atggtgaccg agctgctggg aggagacatg gtgaaccaga gtttcatctg cgacccggac 360gacgagacct tcatcaaaaa catcatcatc caggactgta tgtggagcgg cttctcggcc 420gccgccaagc tcgtctcaga gaagctggcc tcctaccagg ctgcgcgcaa agacagcggc 480agcccgaacc ccgcccgcgg ccacagcgtc tgctccacct ccagcttgta cctgcaggat 540ctgagcgccg ccgcctcaga gtgcatcgac ccctcggtgg tcttccccta ccctctcaac 600gacagcagct cgcccaagtc ctgcgcctcg caagactcca gcgccttctc tccgtcctcg 660gattctctgc tctcctcgac ggagtcctcc ccgcagggca gccccgagcc cctggtgctc 720catgaggaga caccgcccac caccagcagc gactctgagg aggaacaaga agatgaggaa 780gaaatcgatg ttgtttctgt ggaaaagagg caggctcctg gcaaaaggtc agagtctgga 840tcaccttctg ctggaggcca cagcaaacct cctcacagcc cactggtcct caagaggtgc 900cacgtctcca cacatcagca caactacgca gcgcctccct ccactcggaa ggactatcct 960gctgccaaga gggtcaagtt ggacagtgtc agagtcctga gacagatcag caacaaccga 1020aaatgcacca gccccaggtc ctcggacacc gaggagaatg tcaagaggcg aacacacaac 1080gtcttggagc gccagaggag gaacgagcta aaacggagct tttttgccct gcgtgaccag 1140atcccggagt tggaaaacaa tgaaaaggcc cccaaggtag ttatccttaa aaaagccaca 1200gcatacatcc tgtccgtcca agcagaggag caaaagctca tttctgaaga ggacttgttg 1260cggaaacgac gagaacagtt gaaacacaaa cttgaacagc tacggaactc ttgtgcgtaa 132012454PRThomo sapiens 12Leu Asp Phe Phe Arg Val Val Glu Asn Gln Gln Pro Pro Ala Thr Met1 5 10 15Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp 20 25 30Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr Gln 35 40 45Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp Ile 50 55 60Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser Arg65 70 75 80Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe Ser 85 90 95Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala Asp 100 105 110Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn Gln 115 120 125Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile 130 135 140Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val145 150 155 160Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser 165 170 175Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu Tyr 180 185 190Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser Val 195 200 205Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys Ala 210 215 220Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu Ser225 230 235 240Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His 245 250 255Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu 260 265 270Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro 275 280 285Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser Lys 290 295 300Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His305 310 315 320Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala 325 330 335Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile Ser 340 345 350Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn 355 360 365Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu 370 375 380Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu385 390 395 400Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala 405 410 415Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu 420 425 430Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln 435 440 445Leu Arg Asn Ser Cys Ala 450131425DNAmus musculus 13atggctgtca gcgacgctct gctcccgtcc ttctccacgt tcgcgtccgg cccggcggga 60agggagaaga cactgcgtcc agcaggtgcc ccgactaacc gttggcgtga ggaactctct 120cacatgaagc gacttccccc acttcccggc cgcccctacg acctggcggc gacggtggcc 180acagacctgg agagtggcgg agctggtgca gcttgcagca gtaacaaccc ggccctccta 240gcccggaggg agaccgagga gttcaacgac ctcctggacc tagactttat cctttccaac 300tcgctaaccc accaggaatc ggtggccgcc accgtgacca cctcggcgtc agcttcatcc 360tcgtcttccc cggcgagcag cggccctgcc agcgcgccct ccacctgcag cttcagctat 420ccgatccggg ccgggggtga cccgggcgtg gctgccagaa acacaggtgg agggctcctc 480tacagccgag aatctgcgcc acctcccacg gcccccttca acctggggga catcaatgac 540gtgagcccct cgggcggctt cgtggctgag ctcctgcggc cggagttgga cccagtatac 600attccgccac agcagcctca gccgccaggt ggggggctga tgggcaagtt tgtgctgaag 660gcgtctctga ccacccctgg cagcgagtac agcagccctt cggtcatcag tgttagcaaa 720ggaagcccag acggcagcca ccccgtggta gtggcgccct acagcggtgg cccgccgcgc 780atgtgcccca agattaagca agaggcggtc ccgtcctgca cggtcagccg gtccctagag 840gcccatttga gcgctggacc ccagctcagc aacggccacc ggcccaacac acacgacttc 900cccctggggc ggcagctccc caccaggact acccctacac tgagtcccga ggaactgctg 960aacagcaggg actgtcaccc tggcctgcct cttcccccag gattccatcc ccatccgggg 1020gccaactacc ctcctttcct gccagaccag atgcagtcac aagtcccctc tctccattat 1080caagagctca tgccaccggg ttcctgcctg ccagaggagc ccaagccaaa gaggggaaga 1140aggtcgtggc cccggaaaag aacagccacc cacacttgtg actatgcagg ctgtggcaaa 1200acctatacca agagttctca tctcaaggca cacctgcgaa ctcacacagg cgagaaacct 1260taccactgtg actgggacgg ctgtgggtgg aaattcgccc gctccgatga actgaccagg 1320cactaccgca aacacacagg gcaccggccc tttcagtgcc agaagtgtga cagggccttt 1380tccaggtcgg accaccttgc cttacacatg aagaggcact tttaa 142514474PRTmus musculus 14Met Ala Val Ser Asp Ala Leu Leu Pro Ser Phe Ser Thr Phe Ala Ser1 5 10 15Gly Pro Ala Gly Arg Glu Lys Thr Leu Arg Pro Ala Gly Ala Pro Thr 20 25 30Asn Arg Trp Arg Glu Glu Leu Ser His Met Lys Arg Leu Pro Pro Leu 35 40 45Pro Gly Arg Pro Tyr Asp Leu Ala Ala Thr Val Ala Thr Asp Leu Glu 50 55 60Ser Gly Gly Ala Gly Ala Ala Cys Ser Ser Asn Asn Pro Ala Leu Leu65 70 75 80Ala Arg Arg Glu Thr Glu Glu Phe Asn Asp Leu Leu Asp Leu Asp Phe 85 90 95Ile Leu Ser Asn Ser Leu Thr His Gln Glu Ser Val Ala Ala Thr Val 100 105 110Thr Thr Ser Ala Ser Ala Ser Ser Ser Ser Ser Pro Ala Ser Ser Gly 115 120 125Pro Ala Ser Ala Pro Ser Thr Cys Ser Phe Ser Tyr Pro Ile Arg Ala 130 135 140Gly Gly Asp Pro Gly Val Ala Ala Arg Asn Thr Gly Gly Gly Leu Leu145 150 155 160Tyr Ser Arg Glu Ser Ala Pro Pro Pro Thr Ala Pro Phe Asn Leu Gly 165 170 175Asp Ile Asn Asp Val Ser Pro Ser Gly Gly Phe Val Ala Glu Leu Leu 180 185 190Arg Pro Glu Leu Asp Pro Val Tyr Ile Pro Pro Gln Gln Pro Gln Pro 195 200 205Pro Gly Gly Gly Leu Met Gly Lys Phe Val Leu Lys Ala Ser Leu Thr 210 215 220Thr Pro Gly Ser Glu Tyr Ser Ser Pro Ser Val Ile Ser Val Ser Lys225 230 235 240Gly Ser Pro Asp Gly Ser His Pro Val Val Val Ala Pro Tyr Ser Gly 245 250 255Gly Pro Pro Arg Met Cys Pro Lys Ile Lys Gln Glu Ala Val Pro Ser 260 265 270Cys Thr Val Ser Arg Ser Leu Glu Ala His Leu Ser Ala Gly Pro Gln 275 280 285Leu Ser Asn Gly His Arg Pro Asn Thr His Asp Phe Pro Leu Gly Arg 290 295 300Gln Leu Pro Thr Arg Thr Thr Pro Thr Leu Ser Pro Glu Glu Leu Leu305 310 315 320Asn Ser Arg Asp Cys His Pro Gly Leu Pro Leu Pro Pro Gly Phe His 325 330 335Pro His Pro Gly Ala Asn Tyr Pro Pro Phe Leu Pro Asp Gln Met Gln 340 345 350Ser Gln Val Pro Ser Leu His Tyr Gln Glu Leu Met Pro Pro Gly Ser 355 360 365Cys Leu Pro Glu Glu Pro Lys Pro Lys Arg Gly Arg Arg Ser Trp Pro 370 375 380Arg Lys Arg Thr Ala Thr His Thr Cys Asp Tyr Ala Gly Cys Gly Lys385 390 395 400Thr Tyr Thr Lys Ser Ser His Leu Lys Ala His Leu Arg Thr His Thr 405 410 415Gly Glu Lys Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe 420 425 430Ala Arg Ser Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly His 435 440 445Arg Pro Phe Gln Cys Gln Lys Cys Asp Arg Ala Phe Ser Arg Ser Asp 450 455 460His Leu Ala Leu His Met Lys Arg His Phe465 470151413DNAhomo sapiens 15atggctgtca gcgacgcgct gctcccatct ttctccacgt tcgcgtctgg cccggcggga 60agggagaaga cactgcgtca agcaggtgcc ccgaataacc gctggcggga ggagctctcc 120cacatgaagc gacttccccc agtgcttccc ggccgcccct atgacctggc ggcggcgacc 180gtggccacag acctggagag cggcggagcc ggtgcggctt gcggcggtag caacctggcg 240cccctacctc ggagagagac cgaggagttc aacgatctcc tggacctgga ctttattctc 300tccaattcgc tgacccatcc tccggagtca gtggccgcca ccgtgtcctc gtcagcgtca 360gcctcctctt cgtcgtcgcc gtcgagcagc ggccctgcca gcgcgccctc cacctgcagc 420ttcacctatc cgatccgggc cgggaacgac ccgggcgtgg cgccgggcgg cacgggcgga 480ggcctcctct atggcaggga gtccgctccc cctccgacgg ctcccttcaa cctggcggac 540atcaacgacg tgagcccctc gggcggcttc gtggccgagc tcctgcggcc agaattggac 600ccggtgtaca ttccgccgca gcagccgcag ccgccaggtg gcgggctgat gggcaagttc 660gtgctgaagg cgtcgctgag cgcccctggc agcgagtacg gcagcccgtc ggtcatcagc 720gtcagcaaag gcagccctga cggcagccac ccggtggtgg tggcgcccta caacggcggg 780ccgccgcgca cgtgccccaa gatcaagcag gaggcggtct cttcgtgcac ccacttgggc 840gctggacccc ctctcagcaa tggccaccgg ccggctgcac acgacttccc cctggggcgg 900cagctcccca gcaggactac cccgaccctg ggtcttgagg aagtgctgag cagcagggac 960tgtcaccctg ccctgccgct tcctcccggc ttccatcccc acccggggcc caattaccca 1020tccttcctgc ccgatcagat gcagccgcaa gtcccgccgc tccattacca agagctcatg 1080ccacccggtt cctgcatgcc agaggagccc aagccaaaga ggggaagacg atcgtggccc 1140cggaaaagga ccgccaccca cacttgtgat tacgcgggct gcggcaaaac ctacacaaag 1200agttcccatc tcaaggcaca cctgcgaacc cacacaggtg agaaacctta ccactgtgac 1260tgggacggct gtggatggaa attcgcccgc tcagatgaac tgaccaggca ctaccgtaaa 1320cacacggggc accgcccgtt ccagtgccaa aaatgcgacc gagcattttc caggtcggac 1380cacctcgcct tacacatgaa gaggcatttt taa 141316469PRThomo sapiens 16Met Ala Val Ser Asp Ala Leu Leu Pro Ser Phe Ser Thr Phe Ala Ser1 5 10 15Gly Pro Ala Gly Arg Glu Lys Thr Leu Arg Gln Ala Gly Ala Pro Asn 20 25 30Asn Arg Trp Arg Glu Glu Leu Ser His Met Lys Arg Leu Pro Pro Val 35 40 45Leu Pro Gly Arg Pro Tyr Asp Leu Ala Ala Ala Thr Val Ala Thr Asp 50 55 60Leu Glu Ser Gly Gly Ala Gly Ala Ala Cys Gly Gly Ser Asn Leu Ala65 70 75 80Pro Leu Pro Arg Arg Glu Thr Glu Glu Phe Asn Asp Leu Leu Asp Leu 85 90 95Asp Phe Ile Leu Ser Asn Ser Leu Thr His Pro Pro Glu Ser Val Ala 100 105 110Ala Thr Val Ser Ser Ser Ala Ser Ala Ser Ser Ser Ser Ser Pro Ser 115 120 125Ser Ser Gly Pro Ala Ser Ala Pro Ser Thr Cys Ser Phe Thr Tyr Pro 130 135 140Ile Arg Ala Gly Asn Asp Pro Gly Val Ala Pro Gly Gly Thr Gly Gly145 150 155 160Gly Leu Leu Tyr Gly Arg Glu Ser Ala Pro Pro Pro Thr Ala Pro Phe 165 170 175Asn Leu Ala Asp Ile Asn Asp Val Ser Pro Ser Gly Gly Phe Val Ala 180 185 190Glu Leu Leu Arg Pro Glu Leu Asp Pro Val Tyr Ile Pro Pro Gln Gln 195 200 205Pro Gln Pro Pro Gly Gly Gly Leu Met Gly Lys Phe Val Leu Lys Ala 210 215 220Ser Leu Ser Ala Pro Gly Ser Glu Tyr Gly Ser Pro Ser Val Ile Ser225 230 235 240Val Ser Lys Gly Ser Pro Asp Gly Ser His Pro Val Val Val Ala Pro 245 250 255Tyr Asn Gly Gly Pro Pro Arg Thr Cys Pro Lys Ile Lys Gln Glu Ala 260 265 270Val Ser Ser Cys Thr His Leu Gly Ala Gly Pro Pro Leu Ser Asn Gly 275 280 285His Arg Pro Ala Ala His Asp Phe Pro Leu Gly Arg Gln Leu Pro Ser 290 295 300Arg Thr Thr Pro Thr Leu Gly Leu Glu Glu Val Leu Ser Ser Arg Asp305 310 315 320Cys His Pro Ala Leu Pro Leu Pro Pro Gly Phe His Pro His Pro Gly 325 330 335Pro Asn Tyr Pro Ser Phe Leu Pro Asp Gln Met Gln Pro Gln Val Pro 340 345 350Pro Leu His Tyr Gln Glu Leu Met Pro Pro Gly Ser Cys Met Pro Glu 355 360 365Glu Pro Lys Pro Lys Arg Gly Arg Arg Ser Trp Pro Arg Lys Arg Thr 370 375 380Ala Thr His Thr Cys Asp Tyr Ala Gly Cys Gly Lys Thr Tyr Thr Lys385 390 395 400Ser Ser His Leu Lys Ala His Leu Arg Thr His Thr Gly Glu Lys Pro 405 410 415Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp 420 425 430Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly His Arg Pro Phe Gln 435 440 445Cys Gln Lys Cys Asp Arg Ala Phe Ser Arg Ser Asp His Leu Ala Leu 450 455 460His Met Lys Arg His4651723DNAartificial sequence/note="Description of artificial sequence pMXs-Oct4 PF" 17tggtacggga aatcacaagt ttg 231826DNAartificial sequence/note="Description of artificial sequence pMXs-Oct4 PR" 18gtcatagttc ctgttggtga agttca 261917DNAartificial sequence/note="Description of artificial sequence pMXs-Oct4 Probe (6FAM attached 5', MGB attached 3')" 19cttcaccatg cccctca 172022DNAartificial sequence/note="Description of artificial sequence pMXs-Sox2 PF" 20gtgtggtggt acgggaaatc ac 222121DNAartificial sequence/note="Description of artificial sequence pMXs-Sox2 PR" 21ttcagctccg tctccatcat g 212221DNAartificial sequence/note="Description of artificial

sequence pMXs-Sox2 Probe (6FAM attached 5', MGB attached 3')" 22tgtacaaaaa agcaggcttg t 212321DNAartificial sequence/note="Description of artificial sequence pMXs-Klf4 PF" 23gtgtggtggt acgggaaatc a 212418DNAartificial sequence/note="Description of artificial sequence pMXs-Klf4 PR" 24cgcgaacgtg gagaagga 182518DNAartificial sequence/note="Description of artificial sequence pMXs-Klf4 Probe (6FAM attached 5', MGB attached 3')" 25cttcaccatg gctgtcag 182623DNAartificial sequence/note="Description of artificial sequence pMXs-cMyc PF" 26tggtacggga aatcacaagt ttg 232726DNAartificial sequence/note="Description of artificial sequence pMXs-cMyc PR" 27gtcatagttc ctgttggtga agttca 262817DNAartificial sequence/note="Description of artificial sequence pMXs-cMyc Probe (6FAM attached 5', MGB attached 3')" 28cttcaccatg cccctca 172925DNAartificial sequence/note="Description of artificial sequence Nanog PF" 29aaccagtggt tgaatactag caatg 253022DNAartificial sequence/note="Description of artificial sequence Nanog PR" 30ctgcaatgga tgctgggata ct 223117DNAartificial sequence/note="Description of artificial sequence Nanog Probe (6FAM attached 5', MGB attached 3')" 31tcagaagggc tcagcac 173223DNAartificial sequence/note="Description of artificial sequence ACTB PF" 32tcaagatcat tgctcctcct gag 233321DNAartificial sequence/note="Description of artificial sequence ACTB PR" 33acatctgctg gaaggtggac a 213420DNAartificial sequence/note="Description of artificial sequence AFP PF" 34agcagcttgg tggtggatga 203521DNAartificial sequence/note="Description of artificial sequence AFP PR" 35cctgagcttg gcacagatcc t 213621DNAartificial sequence/note="Description of artificial sequence CDH1 (E-CAD) PF" 36ttgaggccaa gcagcagtac a 213720DNAartificial sequence/note="Description of artificial sequence CDH1 (E-CAD) PR" 37atccagcaca tccacggtga 203825DNAartificial sequence/note="Description of artificial sequence CDX2 PF" 38tcactacagt cgctacatca ccatc 253922DNAartificial sequence/note="Description of artificial sequence CDX2 PR" 39ttaacctgcc tctcagagag cc 224020DNAartificial sequence/note="Description of artificial sequence DNMT3B PF" 40gctcacaggg cccgatactt 204122DNAartificial sequence/note="Description of artificial sequence DNMT3B PR" 41gcagtcctgc agctcgagtt ta 224220DNAartificial sequence/note="Description of artificial sequence DPPA4 PF" 42tggtgtcagg tggtgtgtgg 204320DNAartificial sequence/note="Description of artificial sequence DPPA4 PR" 43ccaggcttga ccagcatgaa 204421DNAartificial sequence/note="Description of artificial sequence FGF2 PF" 44ggcaagatgc aggagagagg a 214520DNAartificial sequence/note="Description of artificial sequence FGF2 PR" 45gccacgtgag agcagagcat 204620DNAartificial sequence/note="Description of artificial sequence FOXF1 PF" 46aaaggagcca cgaagcaagc 204721DNAartificial sequence/note="Description of artificial sequence FOXF1 PR" 47aggctgaagc gaaggaagag g 214825DNAartificial sequence/note="Description of artificial sequence GAPDH PF" 48ctggtaaagt ggatattgtt gccat 254926DNAartificial sequence/note="Description of artificial sequence GAPDH PR" 49tggaatcata ttggaacatg taaacc 265022DNAartificial sequence/note="Description of artificial sequence GATA6 PF" 50tgtgcgttca tggagaagat ca 225127DNAartificial sequence/note="Description of artificial sequence GATA6 PR" 51tttgataaga gacctcatga accgact 275221DNAartificial sequence/note="Description of artificial sequence GDF3 PF" 52ttggcacaag tggatcattg c 215321DNAartificial sequence/note="Description of artificial sequence GDF3 PR" 53ttggcacaag tggatcattg c 215420DNAartificial sequence/note="Description of artificial sequence HAND1 PF" 54tcccttttcc gcttgctctc 205520DNAartificial sequence/note="Description of artificial sequence HAND1 PR" 55catcgcctac ctgatggacg 205626DNAartificial sequence/note="Description of artificial sequence KLF4 endo PF" 56acagtctgtt atgcactgtg gtttca 265727DNAartificial sequence/note="Description of artificial sequence KLF4 endo PR" 57catttgttct gcttaaggca tacttgg 275820DNAartificial sequence/note="Description of artificial sequence KLF4 viral PF" 58gtcggaccac ctcgccttac 205922DNAartificial sequence/note="Description of artificial sequence KLF4 viral PR" 59tttatcgtcg accactgtgc tg 226023DNAartificial sequence/note="Description of artificial sequence LIN28 PF" 60ggaggccaag aaagggaata tga 236124DNAartificial sequence/note="Description of artificial sequence LIN28 PR" 61aacaatcttg tggccacttt gaca 246219DNAartificial sequence/note="Description of artificial sequence MYC PF" 62ccagcagcga ctctgagga 196321DNAartificial sequence/note="Description of artificial sequence MYC PR" 63gagcctgcct cttttccaca g 216419DNAartificial sequence/note="Description of artificial sequence NANOG PF" 64cctgtgattt gtgggcctg 196521DNAartificial sequence/note="Description of artificial sequence NANOG PR" 65gacagtctcc gtgtgaggca t 216620DNAartificial sequence/note="Description of artificial sequence NCAM1 PF" 66tcatgtgcat tgcggtcaac 206720DNAartificial sequence/note="Description of artificial sequence NCAM1 PR" 67acgatgggct ccttggactc 206822DNAartificial sequence/note="Description of artificial sequence OCT4 endo PF" 68ggaggaattg ggaacacaaa gg 226922DNAartificial sequence/note="Description of artificial sequence OCT4 endo PR" 69aacttcacct tccctccaac ca 227021DNAartificial sequence/note="Description of artificial sequence OCT4 viral PF" 70ggctctccca tgcattcaaa c 217122DNAartificial sequence/note="Description of artificial sequence OCT4 viral PR" 71tttatcgtcg accactgtgc tg 227221DNAartificial sequence/note="Description of artificial sequence SOX17 PF" 72ttcgtgtgca agcctgagat g 217319DNAartificial sequence/note="Description of artificial sequence SOX17 PR" 73gtcggacacc accgaggaa 197420DNAartificial sequence/note="Description of artificial sequence SOX2 PF" 74tggcgaacca tctctgtggt 207520DNAartificial sequence/note="Description of artificial sequence SOX2 PR" 75ccaacggtgt caacctgcat 207625DNAartificial sequence/note="Description of artificial sequence TDGF1 (Cripto) PF" 76gggatacagc acagtaagga gctaa 257720DNAartificial sequence/note="Description of artificial sequence TDGF1 (Cripto) PR" 77cacaaaagga ccccagcatg 207820DNAartificial sequence/note="Description of artificial sequence ZNF206 PF" 78tcaccatggc cagaggagag 207920DNAartificial sequence/note="Description of artificial sequence ZNF206 PR" 79gcaggccacg ccttattctc 208023DNAartificial sequence/note="Description of artificial sequence ZNF589 PF" 80tcgggtggct aaattacatc cag 238122DNAartificial sequence/note="Description of artificial sequence ZNF589 PR" 81cccaagggag taaggcaaac tg 228230DNAartificial sequence/note="Description of artificial sequence OCT4 outer PF" 82gaggatagga atttaagatt agtttgggta 308330DNAartificial sequence/note="Description of artificial sequence OCT4 outer PR" 83aaatccccca cacctcaaaa cctaacccaa 308431DNAartificial sequence/note="Description of artificial sequence OCT4 inner PF" 84gaggttggag taggaaggat tgttttggtt t 318530DNAartificial sequence/note="Description of artificial sequence OCT4 inner PR" 85cccccctaac ccatcacctc caccacctaa 308630DNAartificial sequence/note="Description of artificial sequence OCT4 inner unconverted PF" 86gaggctggag cagaaggatt gctttggccc 308730DNAartificial sequence/note="Description of artificial sequence OCT4 inner unconverted PR" 87cccccctggc ccatcacctc caccacctgg 308829DNAartificial sequence/note="Description of artificial sequence NANOG outer PF" 88ttagttttta gagtagttgg gattataga 298930DNAartificial sequence/note="Description of artificial sequence NANOG outer PR" 89ataataacat aaaacaacca actcaatcca 309025DNAartificial sequence/note="Description of artificial sequence NANOG inner PF" 90tggttaggtt ggttttaaat ttttg 259126DNAartificial sequence/note="Description of artificial sequence NANOG inner PR" 91aacccaccct tataaattct caatta 269225DNAartificial sequence/note="Description of artificial sequence NANOG inner unconverted PF" 92tggccaggct ggtttcaaac tcctg 259326DNAartificial sequence/note="Description of artificial sequence NANOG inner unconverted PR" 93gacccaccct tgtgaattct cagtta 26

Claims

1. A method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least one of the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox.

2. The method of claim 1, wherein the factors belonging to the factor families of Myc, Klf and Sox and endogenously expressed by or encoded by the coding sequences to be introduced into the target cell are selected from the group consisting of 1-Myc, n-Myc, c-Myc, Klf1, Klf2, Klf4, Klf15, Sox1, Sox2, Sox3, Sox15 and Sox18.

3. The method of claim 1, wherein the target cell does not endogenously express said at least one of the factors encoded by the one or two coding sequences to be introduced into said target cell.

4. The method of claim 1, wherein the target cell is a multipotent stem cell.

5. The method of claim 4, wherein the multipotent stem cell is an ectodermal cell.

6. The method of claim 1, wherein the target cell is a neural stem cell (NSC).

7. The method of claim 6, wherein the coding sequence to be introduced encodes the factor Oct3/4.

8. The method of claim 6, wherein the two coding sequences to be introduced encode factors Oct3/4 and c-Myc or Oct3/4 and Klf4.

9. The method of claim 7, wherein the target cell endogenously expresses the factors c-Myc, Klf4 and Sox2.

10. The method of claim 9, wherein the target cell endogenously expresses the factors c-Myc, Klf4 and Sox2 at levels at least 10-fold lower or at most 10-fold higher as compared to the corresponding expression levels in embryonic stem cells of the same genus as the target cell.

11. The method of claim 7, wherein the target cell is a murine neural stem cell.

12. An induced pluripotent stem cell generated by the method of claim 1.

13. A method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell comprising the steps of: (a) reprogramming a target cell according to the method of claim 1, wherein one coding sequence to be introduced is replaced by the compound to be tested; and (b) assessing whether iPS cells are formed in the presence and absence of the compound to be tested, wherein the formation of iPS cells from target cells in which the compound to be tested has been introduced is indicative of the compound contributing to the reprogramming of a target cell into an induced pluripotent stem cell.

14. A method of generating a transgenic non-human animal comprising the steps of: (a) introducing the induced pluripotent stem cell generated by the method of claim 1 into a non-human preimplantation embryo; (b) transferring the embryo of step (a) into the uterus of a female non-human animal; and (c) allowing the embryo to develop and to be born.

15. A transgenic non-human animal generated by the method of claim 14.

16. A composition comprising an iPS cell generated by the method of claim 1 for gene therapy, regenerative medicine, cell therapy or drug screening.

Images