WO2010105257A2

WO2010105257A2 - Generation of mouse induced pluripotent stem cells bytransient expression of a single non- viral polycistronic vector

Info

Publication number: WO2010105257A2
Application number: PCT/US2010/027316
Authority: WO
Inventors: Federico Gonzalez Grassi; Juan Carlos Izpisua Belmonte
Original assignee: The Salk Institute For Biological Studies
Priority date: 2009-03-13
Filing date: 2010-03-15
Publication date: 2010-09-16
Also published as: WO2010105257A3

Abstract

Provided herein are, inter alia, methods for the generation of iPS cell lines and compositions related to the same.

Description

GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS

BYTRANSIENT EXPRESSION OF A SINGLE NON- VIRAL

POLYCISTRONIC VECTOR

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/160,263, filed March 13, 2009, which is incorporated herein by reference in its entirety and for all purposes.

BACKGROUND OF THE INVENTION

[0002] Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types, in both mouse and human, by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28. Although different reprogramming protocols have been reported, the delivery of the original Oct4, Sox2, KIf 4, c-Myc (OSKM) transcription factor set remains the most commonly used method. Without wishing to be bound by theory, it has been believed that reprogramming requires the delivery of all the factors to the cell and adequate expression thereof for a period of time of approximately 8-12 days. The retroviral and (both constitutive and inducible) lentiviral vectors commonly used can meet these requirements, but permanent integration thereof into the genome limits the use of such vectors for eventual therapeutic applications due to the risk of both insertional mutagenesis and particularly the reactivation of the reprogramming factors leading to tumor formation. Presented herein are methods for solving these and other problems in the art.

BRIEF SUMMARY OF THE INVENTION [0003] There are provided herein methods and compositions for making and using an induced pluripotent stem (iPS) cell, including methods for generation of iPS cell lines with no evidence of integration of the reprogramming vector in the genome thereof.

[0004] In a first aspect, there is provided a method for preparing an induced pluripotent stem cell. The method includes transfecting a non-pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non- pluripotent cell is allowed to divide thereby forming an induced pluripotent stem cell. [0005] In another aspect, there is provided a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

[0006] In another aspect, there is provided a non-pluripotent cell which includes a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

[0007] In another aspect, there is provided an induced pluripotent stem cell prepared by transfecting a non-pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a S OX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide, thereby forming an induced pluripotent stem cell.

[0008] In another aspect, there is provided a method of treating a mammal in need of tissue repair. The method includes administering an induced pluripotent stem cell to the mammal. The induced pluripotent stem cell is allowed to divide and differentiate into somatic cells in the mammal, thereby providing tissue repair in the mammal. The induced pluripotent stem cell is prepared by transfecting a non-pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide thereby forming the induced pluripotent stem cell.

[0009] In another aspect, there is provided a method for producing a somatic cell, which method includes contacting an induced pluripotent stem cell with a cellular growth factor. The induced pluripotent stem cell is allowed to divide, thereby forming a somatic cell. The induced pluripotent stem cell is prepared by a process including transfecting a non- pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide thereby forming an induced pluripotent stem cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. IA depicts a CAG driven polycistronic plasmid expressing Oct4, Sox2, Klf4 and c-Myc (pCAG-OSKM). [0011] FIG. IB depicts a CAG driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc and GFP (pCAG-OSKMG). [0012] FIG. 1C depicts the results of a test of the functionality of pCAG-OSKM by transient transfection into mouse embryonic fibroblasts (MEFs) followed by real-time RT- PCR relative to GAPDH, as described herein and known in the art. T: transfected fibroblasts. NT: non-transfected fibroblasts. [0013] FIG. ID depicts the results of Western blot analysis to detect expression of the OCT4, SOX2 and KLF4 protein products, and additionally C-MYC and β-actin, after transient transfection of pCAG-OSKM into MEFs.

[0014] FIG. IE and FIG. IF depict timelines for nucleofection of MEFs by e.g. pCAG- OSKM, wherein MEFs were nucleofected once (FIG. IE) or twice (FIG. IF) following the indicated timelines and seeded onto irradiated MEFs (irMEFs).

[0015] FIG. 2 A depicts the linear representation of pCAG-OSKM showing the location of the probes used for Southern Blot (bars, A-D) and the approximate position and length of the amplicons generated by PCR (bars 1-22).

[0016] FIG. 2B depicts the results of Southern blot analysis of clones #6, #11, #16, #36, #38, #41, and mouse ES cells using probes against Oct4, Sox2, Klf4, and c-Myc. Black and grey arrowheads point respectively to specific and non-specific endogenous bands present in the genomic DNA of control ES cell. Extra bands are highlighted by an asterisk in clones #6, #16 and #36, indicating variable degrees of insertion of the transgene.

[0017] FIG. 2C depicts the results of PCR analysis of clones #6, #11, #16, #36, #38, #41. Consistent with Southern blot, clones #6 and #36 are positive for almost the full set of primer pairs, whereas clone # 16 is not positive for the backbone-specific primers 2 to 9. Clones #11 shows a weak signal for primer pairs 3, 9, 12 and 20, whereas clones #38 and #41 are negative for all primer pairs tested except faint bands for primer pairs 17 and 20. In another series of PCRs, MEFs were positive using these primer pairs 17 and 20 suggesting that non- specific amplification could be the reason of this signal (not shown).

[0018] FIG. 3 A depicts in the upper panel the results of RT-PCR of clones #6, #11 , #16, #36, #38 and #41 for Oct4, Sox2, Nanog, UTFl and ZFP42. FIG. 3 A depicts in the lower left panel the results of RT-PCR of clones #6, #11, #16, #36, #38 and #41 for Col6a2, Grem2 and Thyl. FIG. 3A depicts in the lower right panel the expression level of the transgene using a set of primers spanning the junction between the coding sequences of Sox2 and KIf 4. Legend (upper panel): clones and controls appear in the order #11, #16, #36, #38, #41, #6, Mefs, mSEc. Legend (lower panels): clones and controls appear in the order #6, #11, #16, #36, #38, #41, Mefs, mSEc.

[0019] FIG. 3B depicts the results of promoter methylation analysis by sodium bisulfite mutagenesis of the Oct4 promoter in iPS lines #1, #6, #38, #4, #11 and #41, and in MEF and ES cells.

[0020] FIG. 3C depicts the immunofluorescence for OCT4, SOX2, SSEAl and NANOG for clones #6, #11, #16, #36, #38 and #41.

[0021] FIG. 4A depicts the differentiation potential of iPS cell lines, depicting in vitro differentiation towards ectoderm (a,b,c) (TuJl -positive neuronal cells), mesoderm (d,e,f) (α- actinin-positive cardiac myocytes) and endoderm (g,h,i) (α -fetoprotein-positive; FOXA2- positive). Nuclear staining employed DAPI. Scale bars, 25 μm.

[0022] FIG. 4B depicts the differentiation potential of iPS cell lines and in vivo differentiation (teratomas) towards ectoderm (a,b,c) (TuJl -positive neuronal cells; glial fibrillary acidic protein (GFAP)-positive cells), mesoderm (d,e,f) (α - actinin-positive cardiac myocytes) and endoderm (g,h,i) (α -fetoprotein-positive; FOXA2 -positive). Scale bars, 50 μm; Upper and down left, 50 μm; Middle left, 25 μm.

[0023] FIG. 4C depicts a chimeric pup obtained by injection of clone #11 into a blastocyst. Legend: chimeric pup (arrow); control pup (no arrow).

[0024] FIG. 5 depicts the results of AP staining of iPS lines #11, #16, #36, #38 and #41. [0025] FIG. 6A depicts a real time RT-PCR results showing the level of silencing of the pCAG-OSKM in different iPS lines positive for integration by Southern Blot.

[0026] FIG. 6B depicts GFP expression analysis of MEFs nucleofected once with pCAG- OSKMG, plated on gelatin and analyzed using FACS at day 1, 3, 4, 7, 8 and 10 after transfection. Squares indicate the percentage of positive cells. [0027] FIG. 6C depicts fluorescent microscopy images of the putative iPS colonies appearing after a single nucleofection of pCAG-OSKMG in MEFs. All of them show detectable, though different expression levels of the reporter gene validating the integration- reporter system.

[0028] FIG. 7 depicts the CAG driven polycistronic plasmids pCAG-OSKM, pCAG- OSKMG and pCAG-OKTMG, as described herein. Symbols used herein for plasmid designation: O: Oct4; S: Sox2; K: KIf 4, M: c-Myc; T: nucleic acid encoding Large T antigen of the SV40 polyomavirus.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions [0029] The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0030] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.

[0031] The terms "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

[0032] The terms "identical" or percent "identity," in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as known in the art, or by manual alignment and visual inspection (see, e.g., the BLAST tools available at the National Center for Biotechnology Information (NCBI) web site or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As known in the art, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0033] The word "polynucleotide" refers to a linear sequence of nucleotides. The nucleotides can be ribonucleotides, deoxyribonucleotides, or a mixture of both. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including miRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. [0034] The words "protein", "peptide", and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.

[0035] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. The term "protein gene product" refers to a protein expressed from a particular gene. [0036] The word "expression" or "expressed" as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et ah, 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression" the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0037] The term "plasmid" refers to a nucleic acid molecule that encodes for genes and/or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0038] A "cell culture" is a population of cells residing outside of an organism. These cells are optionally primary cells isolated from a cell bank, animal, or blood bank, or secondary cells that are derived from one of these sources and have been immortalized for long-lived in vitro cultures.

[0039] A "stem cell" is a cell characterized by the ability of self-renewal through mitotic cell division and the potential to differentiate into a tissue or an organ. Among mammalian stem cells, embryonic and somatic stem cells can be distinguished. Embryonic stem cells reside in the blastocyst and give rise to embryonic tissues, whereas somatic stem cells reside in adult tissues for the purpose of tissue regeneration and repair .

[0040] The term "pluripotent" or "pluripotency" refers to cells with the ability to give rise to progeny that can undergo differentiation, under appropriate conditions, into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to tissues of a prenatal, postnatal or adult organism. A standard art-accepted test, such as the ability to form a teratoma in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population. However, identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells.

[0041] "Pluripotent stem cell characteristics" refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. Expression or non-expression of certain combinations of molecular markers are examples of characteristics of pluripotent stem cells. More specifically, human pluripotent stem cells may express at least some, and optionally all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-I -60, TRA-I -81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-I, Oct4, Lin28, Rexl, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics.

[0042] The term "induced pluripotent stem cell" refers to a pluripotent stem cell artificially derived from a non-pluripotent cell. A non-pluripotent cell can be a cell of lesser potency to self-renew and differentiate than a pluripotent stem cell. Cells of lesser potency can be, but are not limited to, somatic stem cells, tissue specific progenitor cells, primary or secondary cells. Without limitation, a somatic stem cell can be a hematopoietic stem cell, a mesenchymal stem cell, an epithelial stem cell, a skin stem cell or a neural stem cell. A tissue specific progenitor refers to a cell devoid of self-renewal potential that is committed to differentiate into a specific organ or tissue. A primary cell includes any cell of an adult or fetal organism apart from egg cells, sperm cells and stem cells. Examples of useful primary cells include, but are not limited to, skin cells, bone cells, blood cells, cells of internal organs and cells of connective tissue. A secondary cell is derived from a primary cell and has been immortalized for long-lived in vitro cell culture.

[0043] The term "reprogramming" refers to the process of dedifferentiating a non- pluripotent cell into a cell exhibiting pluripotent stem cell characteristics. [0044] The term "transfection" or "transfecting" refers to a process of introducing nucleic acid molecules into a cell. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Any appropriate transfection method is useful in the methods described herein. In particular, any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell is useful in the methods described herein. Exemplary transfection methods include without limitation calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art.

[0045] The term "treating" in the context of disease refers to ameliorating, suppressing, eradicating, and/or delaying the onset of the disease being treated.

II. Methods and Compositions

[0046] In one aspect, there is provided a method for preparing an induced pluripotent stem cell. The method includes transfecting a non-pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non- pluripotent cell is allowed to divide thereby forming an induced pluripotent stem cell.

[0047] The terms "Oct4," "OCT4," "Oct4 protein," "OCT4 protein" and the like refer to any of the naturally-occurring forms of the Octomer 4 transcription factor, or variants thereof that maintain Oct4 transcription factor activity (e.g. within at least 50%, 80%, 90% or 100% activity compared to Oct4 as measured by methods known in the art). In some embodiments, variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Oct4 polypeptide. In other embodiments, the Oct4 protein is the protein as identified by the NCBI reference gi:42560248 and girl 16235491 corresponding to isoform 1 and 2, respectively.

[0048] The terms "Sox2," "SOX2," "Sox2 protein," "SOX2 protein" and the like as referred to herein includes any of the naturally-occurring forms of the Sox2 transcription factor, or variants thereof that maintain Sox2 transcription factor activity (e.g. within at least 50%, 80%, 90% or 100% activity compared to Sox2 as measured by methods known in the art). In some embodiments, variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Sox2 polypeptide. In other embodiments, the Sox2 protein is the protein as identified by the NCBI reference gi:28195386.

[0049] The terms "KLF4," "KLF4 protein" and the like as referred to herein includes any of the naturally-occurring forms of the KLF4 transcription factor, or variants thereof that maintain KLF4 transcription factor activity (e.g. within at least 50%, 80%, 90% or 100% activity compared to KLF4 as measured by methods known in the art). In some embodiments, variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring KLF4 polypeptide. In other embodiments, the KLF4 protein is the protein as identified by the NCBI reference gi: 194248077. [0050] The terms "cMyc," C-MYC," "cMyc protein," "C-MYC protein" and the like as referred to herein includes any of the naturally-occurring forms of the cMyc transcription factor, or variants thereof that maintain cMyc transcription factor activity (e.g. within at least 50%, 80%, 90% or 100% activity compared to cMyc as measured by methods known in the art). In some embodiments, variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring cMyc polypeptide. In other embodiments, the cMyc protein is the protein as identified by the NCBI reference gi:71774083.

[0051] In one embodiment, there is provided a method for preparing an induced pluripotent stem cell, which method includes transfecting a non-pluripotent cell with a nucleic acid which consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide thereby forming an induced pluripotent stem cell.

[0052] In one embodiment, the induced pluripotent stem cell is a footprint-free pluripotent stem cell. The term "footprint-free induced pluripotent stem cell" refers to an induced pluripotent stem cell that is devoid of any detectable genomic integration event following transfection of the non-pluripotent cell. The genome of a footprint- free induced pluripotent stem cell does not contain any detectable parts of the nucleic acid molecules initially transfected into the non-pluripotent cell. In some embodiments, "detectable genomic integration event" refers to detectable integration of transfected nucleic acid molecules, or portions thereof, into the genome of an induced pluripotent stem cell. Any appropriate method of detecting integration may be employed, such as polymerase chain reaction and Southern Blot hybridization. In some embodiments, Southern Blot hybridization is used to detect integration. Where Southern Blot hybridization is used to detect integration, a "footprint-free induced pluripotent stem cell" refers to an induced pluripotent stem cell that is devoid of a genomic integration event as detected by Southern Blot hybridization. In some embodiments, a "footprint- free human induced pluripotent stem cell" refers to a human induced pluripotent stem cell devoid of any genomic integration.

[0053] In another embodiment, the transfecting is performed without the use of a viral transfection system, as described herein and as known in the art.

[0054] In another embodiment, the non-pluripotent cell is a mammalian cell. In another embodiment, the non-pluripotent cell is a human cell. In another embodiment, the non- pluripotent cell is a mouse cell.

[0055] In another embodiment, the nucleic acid consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

[0056] In another embodiment, the nucleic acid is a polycistronic nucleic acid.

[0057] In another embodiment, the nucleic acid encodes a ribosomal skipping sequence. [0058] In another embodiment, the ribosomal skipping sequence is a picornavirus ribosomal skipping sequence.

[0059] In another aspect, there is provided a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

[0060] In one embodiment, the nucleic acid consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein. Where a nucleic acid "consists essentially of or is "consisting essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, the nucleic acid does not encode other transcription factors known to be useful in iPS cell formation. In some embodiments, the nucleic acid does not encode other transcription factors. And in other embodiments, the nucleic acid does not encode other protein expressing genes.

[0061] In one embodiment, the nucleic acid consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein, C-MYC protein and the Large T antigen of SV40 polyomavirus. As known in the art, the large t antigen of SV40 (also commonly referred to as SV40 T antigen or Simian Vacuolating Virus 40, or Tag or "Large T") is a hexamer protein that is an oncogene derived from the polyomavirus S V40 which is capable of transforming a variety of cell types.

[0062] In another embodiment, the nucleic acid is a polycistronic nucleic acid. In another embodiment, the nucleic acid encodes a ribosomal skipping sequence. In another embodiment, the nucleic acid encodes a picornavirus ribosomal skipping sequence.

[0063] In another embodiment, the gene sequences encoding the OCT4 protein, the SOX2 protein and the KLF4 protein are not arranged such that the relative order of translation is OCT4 protein following by KLF4 protein followed by SOX2 protein.

[0064] In another aspect, there is provided a non-pluripotent cell which includes a nucleic acid including gene sequences encoding an OCT4 protein, a S OX2 protein, a KLF4 protein and a C-MYC protein. The non-pluripotent cell may be made by a method disclosed herein.

[0065] In one embodiment, the non-pluripotent cell is a mammalian cell. In another embodiment, the non-pluripotent cell is a human cell. In another embodiment, the non- pluripotent cell is a mouse cell. [0066] In another embodiment, the nucleic acid consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

[0067] In another embodiment, the nucleic acid is a polycistronic nucleic acid. In another embodiment, the nucleic acid encodes a ribosomal skipping sequence. In another embodiment, the ribosomal skipping sequence is a picornavirus ribosomal skipping sequence. [0068] In another aspect, there is provided an induced pluripotent stem cell prepared by transfecting a non-pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide, thereby forming an induced pluripotent stem cell. [0069] In one embodiment, there is provided an induced pluripotent stem cell prepared by transfecting a non-pluripotent cell with a nucleic acid which consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell, which transfected non-pluripotent cell is then allowed to divide, thereby forming an induced pluripotent stem cell. [0070] In another aspect, there is provided a method of treating a mammal in need of tissue repair. The method includes administering an induced pluripotent stem cell to the mammal. The induced pluripotent stem cell is allowed to divide and differentiate into somatic cells in the mammal, thereby providing tissue repair in the mammal. The induced pluripotent stem cell is prepared by transfecting a non-pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide thereby forming the induced pluripotent stem cell.

[0071] In one embodiment, there is provided a method of treating a mammal in need of tissue repair. The method includes administering an induced pluripotent stem cell to the mammal. The induced pluripotent stem cell is allowed to divide and differentiate into somatic cells in the mammal, thereby providing tissue repair in the mammal. The induced pluripotent stem cell is prepared by transfecting a non-pluripotent cell with a nucleic acid which consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell, which transfected non-pluripotent cell is allowed to divide thereby forming the induced pluripotent stem cell.

[0072] In another aspect, there is provided a method for producing a somatic cell, which method includes contacting an induced pluripotent stem cell with a cellular growth factor. The induced pluripotent stem cell is allowed to divide, thereby forming a somatic cell. The induced pluripotent stem cell is prepared by a process including transfecting a non- pluripotent cell with a nucleic acid which includes gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide thereby forming an induced pluripotent stem cell.

[0073] The term "cellular growth factor" refers to agents which cause cells to migrate, differentiate, transform or mature and divide. They are polypeptides which can usually be isolated from various normal and malignant mammalian cell types. Some growth factors can be produced by genetically engineered microorganisms, such as bacteria (e.g., E. colϊ) and yeasts. Cellular growth factors may be supplemented to the media and/or may be provided through co-culture with irradiated embryonic fibroblast that secrete such cellular growth factors. Examples of cellular growth factors include, but are not limited to, FGF, bFGF2, and EGF.

[0074] In one embodiment, the method includes contacting an induced pluripotent stem cell with a cellular growth factor. The induced pluripotent stem cell is allowed to divide, thereby forming a somatic cell. The induced pluripotent stem cell is prepared by a process including transfecting a non-pluripotent cell with a nucleic acid which consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein, thereby forming a transfected non-pluripotent cell. The transfected non-pluripotent cell is allowed to divide thereby forming an induced pluripotent stem cell.

EXAMPLES

[0075] Without wishing to be bound by theory, an initial requirement for direct reprogramming appears to be delivery of all four factors (Oct4, Sox2, KLF4 and c-Myc) to the same cell. In this context, advantage was taken of 2 A peptide sequences which are short sequences originally reported in picornaviruses (Ryan, M.D. and Drew, J., The EMBO journal, 13:928-933 (1994)) that, when cloned between open reading frames (ORFs), allow near stoichiometric expression of up to three proteins via a ribosomal skipping mechanism, which system was previously shown to work efficiently in embryonic stem cells (Hasegawa, K. et al., Stem cells (Dayton, Ohio), 25:1707-1712 (2007)). This strategy was employed in the cloning of a CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4 and c-Myc (pCAG-OSKM, FIG. IA). The functionality of the construct was confirmed by transient transfection into MEFs followed by real-time RT-PCR and western blot analysis. A primer pair designed to span the 2A peptide sequence junction between Sox2 and Klf4 was used for RT-PCR analysis, ensuring that the signal observed corresponded to the polycistronic transgene mRNA (FIG. IB). Western blot analysis detected expression of the Oct4, Sox2 and Klf4 protein products. Without wishing to be bound by theory, it is understood that over expression of c-Myc may not be detectable due to the high levels of this protein expressed endogenously in the fibroblast line transfected (FIG. 1C).

[0076] In order to determine whether an ORF cloned into the fourth position of the polycistron could be expressed, c-Myc was replaced with GFP in a test construct, and transfection of fibroblasts resulted in GFP expression (data not shown). Furthermore, a recent report describes the generation of mouse and human iPS cells by lenti viral delivery of a OSKM, 2A peptide sequence linked construct (Carey, B.W. et al., Proceedings of the National Academy of Sciences, 106:157-162 (2009)). Accordingly, it appears that the polycistronic construct efficiently expresses all four expected factors.

[0077] In order to reprogram MEFs by nucleofection of pCAG-OSKM, MEFs were nucleofected once or twice following the timelines depicted in FIG. IE and FIG. IF, respectively, and seeded onto irradiated MEFs (irMEFs). Approximately 12 days after seeding, colonies appeared in both treatments and were allowed to grow for 1 week, after which single clones were manually passaged onto fresh irMEFs. The colonies did not initially resemble mouse embryonic stem (mES) cells, but after two passages they had acquired typical mES cell morphology as known in the art. Accordingly, there were established 46 iPS cell lines from a total of 75 colonies picked.

[0078] All cell lines thus established were subjected to a Southern blot using a probe against Sox2 which resulted in the determination that 43 of them had integrated the transgene (data not shown). Probes A-D are provided in Table 2 herein. The three remaining lines (#11, #38 and #41) were further analyzed together with three integrated lines (#6, #16 and #36) using probes against Oct4, Klf4 and c-Myc (probes A, B, C, and D). Clones #11, #38 and #41 were undistinguishable from wild type control ES analyzed with the same probes (FIG. 2B). As expected, clones #6, #16 and #36 showed additional bands also for this other set of probes. In order to exclude integrations of small portions of pCAG-OSKM not revealed by Southern blot, we analyzed those lines by PCR using a set of 22 primer pairs spanning the entire vector (primer pairs 1-22). Consistently, clones #6 and #36, positive for probes A, B, C and D by Southern blot were positive for most PCR reactions, whereas line #16 (only positive for probes B and D) was negative for a subset of backbone-specific primers. Clones #11, #38 and #41 resembled the pattern of control wt MEFs with bands appearing only for PCR conditions allowing the amplification of endogenous loci (PCR 15, 18, 19). However, a careful examination of the gel revealed that PCR 3, 9, 12 and 20 were positives in clone # 11 (FIG. 2C). Although we cannot strictly rule out the integration of small segments of the plasmid into the genome, these data strongly suggest that these three iPS clones do not contain the original transgene used to reprogram them.

[0079] Real-time RT-PCR on the iPS clones #6, #11, #16, #36, #38 and #41 revealed that they all had reactivated a set of endogenous pluripotent-specific genes (Oct4, Sox2, Nanog, UTFl and ZFP42) to levels similar to mES cells, as known in the art. Probes useful for the methods described herein, including quantitative RT-PCR, are provided in Table 4. Furthermore and consistently, the fibroblast specific markers Thyl and Col6a2 were down- regulated (FIG. 3A, upper and middle panels). The term "Nanog protein" as referred to herein includes any of the naturally-occurring forms of the Nanog transcription factor, or variants thereof that maintain Nanog transcription factor activity (e.g. within at least 50%, 80%, 90% or 100% activity compared to Nanog). In some embodiments, variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Nanog polypeptide. In other embodiments, the Nanog protein is the protein as identified by the NCBI reference gi:153945816. The term "UTFl" and like terms refer to undifferentiated embryonic cell transcription factor 1 , as known in the art. The term "ZFP42" and like terms refer to zinc fmger protein 42, as known in the art.

[0080] The expression level of the transgene was tested using a set of primers spanning the junction between the coding sequences of Sox2 and Klf4 of pCAG-OSKM (FIG. 3 A, lower panel). As expected, clones #6 and #36 (positive by Southern blot analysis), showed significant expression of the polycistronic transcript, whereas the expression could not be detected for clones #11, #38 and #41 (all negative by Southern blot analysis).

[0081] Promoter methylation analysis by sodium bisulfite mutagenesis revealed demethylation of the Oct4 promoter in the iPS lines #38 and #41 , consistent with the endogenous expression of Oct4 observed in these clones (FIG. 3B). Regardless of their integration status, the iPS clones analysed were differentially demethylated, clustering either as ES-like (#1, #6 and #11), intermediate (#41) or MEF-like (#4, #38). However, in all cases Oct4 promoter methylation was lower than in control MEFs consistent with the endogenous reactivation of Oct4 observed in those clones by Real time RT-PCR (Figure 3B).

Furthermore, the clones were positive by immunofluorescence for OCT4, SOX2, SSEAl, and NANOG (Fig. 3C) and stained positive for Alkaline Phosphatase (FIG. 5). Taken together, these data show that, regardless of integration status, the iPS clones resulting from the methods provided herein display the molecular hallmarks of mES cells. [0082] The differentiation potential of clones #4 (positive by Southern blot), #38, and #41 was then tested by generating embryoid bodies (EBs) and differentiating them to cell derivates representative of the three germ layers. All iPS clones tested showed positive immunofluorescence staining for markers of ectoderm (TUJl), mesoderm (α-S ARCOMERIC-ACTIN) and endoderm (α-1 -FETOPROTEIN and FOXA2) (FIG. 4A). Furthermore, clones #36, #38, and #41 were injected into SCID mice, resulting in the formation of teratomas containing tissues belonging to the three germ layers (FIG. 4B). Finally, clones #11, #38 and #41 were injected into blastocysts, and there obtained chimeric pups (e.g., Figure 4C, clone #11) with all tested clones, confirming the pluripotency of the iPS cell lines generated with our non-integrative approach. [0083] A construct expressing Oct4, Sox2, KIf 4 and c-Myc from a single transcription unit has been designed and tested. This design allows for the expression of four factors required for reprogramming the transfected cell. The construct was delivered to MEFs by one or two nucleofections and succeeded in generating iPS lines. The iPS clones were morphologically similar to niES cells, expressed endogenous markers of pluripotency and were capable of differentiation into the three germ layers both in vitro and in vivo. In the Southern blot analysis the probes designed against the four factors were always able to recognize their endogenous targets, present at 2 copies per genome. If an iPS line had integrated the transgene, it would have at least 1 copy per genome, resulting in half the intensity of the endogenous signal. Among the clones that had integrated the transgene, band numbers and signal intensities consistent with 1 to 3 copies were observed. Interestingly, 3 of the 4 clones that were negative by Southern blot had indeed integrated some transgene elements when genotyped by PCR. Without wishing to be bound by theory, it is possible that these lines, despite being picked from single and isolated outgrowths, are chimeric, i.e., containing a small proportion of cells that have incorporated elements of the transgene. However, iPS clone # 41 showed no evidence of integration of the transgene into the genome by either Southern blot or PCR analysis. Accordingly, these results suggest that reprogramming can be achieved by transient expression of the OSKM factor set. [0084] Additional constructs are provided herein. For example, as described herein there has been designed and tested a construct expressing Oct4, Sox2, Klf4 and c-Myc from a single transcription unit (pCAG-OSKM). Additionally, a construct expressing Oct4, Sox2, KIf 4, c- Myc and eGFP from a single transcription unit (pCAG-OSKMG), and a construct expressing Oct4, Sox2, SV40 Large-T, KIf 4, c-Myc and eGFP from a single transcription unit (pCAG- OSTKMG) have been provided. See FIG. 7. In these constructs, advantage was taken of the 2A peptide sequence originally reported in picornaviruses. This peptide allows efficient, near stoichiometric production of up to three discrete protein products, when cloned between open reading frames (ORFs), via a ribosomal skipping mechanism. As described herein, pCAG- OSKM allows expressing the four factors required for reprogramming in each transfected cell.

[0085] pCAG-OSKMG allows expressing the four factors required for reprogramming and an eGFP reporter gene in each transfected cell. It was determined if pCAG-OSKMG was able to express GFP by nucleofecting this construct once in MEFs and monitoring GFP expression by FACS over the time period required for reprogramming. At day 1 after nucleofection, 37 % of the cells were GFP positives and 6 % were still labeled at 10 days (FIG. 6B). MEFs were again nucleofected with pCAG-OSKMG and plated on feeder cells. This pilot experiment provided 12 iPS-like colonies, all of them strongly expressing GFP. See Figure 6C. [0086] Without wishing to be bound by theory, it appears that pCAG-OSTKMG allows the expression of four factors required for reprogramming, the Large T antigen of the SV40 polyomavirus and an eGFP reporter gene in each transfected cell. The constructs provided herein (pCAG-OSKM, pCAG-OSKMG and pCAG-OSTKMG) were delivered by nucleofection which succeeded in generating integration-free iPS lines from mouse and human fibroblasts following the strategy depicted in FIG. IF. These non-integrative iPS (NiPS) are morphologically similar to ES cells, express endogenous markers of pluripotency and are capable of differentiation into the three germ layers both in vitro and in vivo. Moreover, when large T is included in the reprogramming construct, the efficiency of reprogramming is increased 100 times, facilitating the isolation of non-integrative colonies.

Materials and Methods pCAG-OSKM plasmid construction and delivery

[0087] The Oct4 cDNA was amplified using a reverse primer eliminating the Oct4 stop codon and adding a BspEI site and cloned into pcRII (Invitrogen) to give pcRII-Oct4. Primers useful for the generation of constructs by the methods described herein are provided in Table 5. The Sox2 cDNA was amplified using a forward primer containing an Agel site followed by P2A peptide sequence and a reverse primer eliminating the Sox2 stop codon and containing a BspEI site; this fragment was cloned in pCRII to give pCRII-Age-Sox2-Bsp (oriented NotI-5'cDNA3'-Acc65I). pCRII-Age-Sox2-Bsp was cut Agel and Acc65I and cloned into pCRII-Oct4-Bsp cut BspEI- Acc65I producing pCRII-Oct4-P2A-Sox2-BspEI.

The strategy of cloning was repeated twice, to incorporate Klf4 and c-Myc (producing pCRII- OSKM) or three times, to incorporate Klf4, c-Myc and GFP (producing pCRII-OSKMG). The last ORF was amplified in order to preserve the Stop codon. These constructs were cut with EcoRI and cloned into an EcoRI site immediately downstream of the CAG promoter and upstream of the Bgh pA previously cloned into pCRII or pBSKII vectors respectively giving rise to pCAGOSKM and pCAG-OSKMG. The plasmids were purified under endotoxin free conditions (Quiagen) for subsequent nucleofection.

Reprogramming protocol

[0088] P2 MEFs from E13.5 C57BL/6 embryos were nucleofected using the MEF2 Nucleofector Kit (Amax) following the manufacturer's instructions with detailed reprogramming protocols (as described for FIG. IE and FIG. IF) provided in Table 1 herein. The term "ES media" refers to the following composition: DMEM high glucose supplemented with 15% fetal bovine serum (FBS, Hyclone, Cultek), 50 U/ml Penicillin : 50 μg/ml Streptomycin, MEM NEAA (Cambrex, Lonza), 2 rnM GlutaMAX, 1 mM Sodium pyruvate (GIBCO, Invitrogen), lOOμM 2-Mercaptoethanol (GIBCO, Invitrogen), and 100 U

ESGRO (Chemicon, Millipore). The term "ES conditioned media" refers to the composition made by the following procedure: 10 cm2 gelatinized dishes were seeded with 4 million iMEFs; the day after, G4 media was added to the dishes, and culture media was collected 24h later, filtered and stored at 4 ⁰C. The iMEFs are used for 7 days.

Table 1. Detailed reprogramming protocol employing one or two nucleofections.

Western blot [0089] Cells were resuspended in RIPA buffer containing 1 x protein inhibitor mixture (Roche), incubated on ice for 20 minutes and cleared by centrifugation. Protein concentration was determined by Bradford assay (Pierce). Twenty-five micrograms of total protein were subjected to electrophoresis on 4-12% BIS-TRIS denaturing gels (Invitrogen). Proteins were transferred to Immobilon-P membranes (Millipore). The membranes were blocked in PBS containing 10% non fat powdered milk and 0,02% Tween 20. Primary antibodies were against OCT4 (Santa Cruz), SOX2 (Chemicon), C-MYC (Sigma), KLF4 (Santa Cruz) and ACTIN (Abeam). Integration analysis

[0090] For Southern blot analysis, 5 micrograms of genomic DNA were cut with Xbal (for blots probed for Oct4, Klf4, and c-Myc) or HindIII (for blot probed with Sox2), separated on a 1% agarose gel and transferred to a nylon membrane (Amersham). Dioxigenin-dUTP labeled probes were synthesized using a PCR DIG Probe Synthesis Kit (Roche) according to manufacturer's instructions. For primers see Table 2. Blots were blocked, hybridized, washed and then developed using Anti-Digoxigenin-AP Fab fragments (Roche). For PCR, 50 ng of genomic DNA were amplified with Taq polymerase (Roche) using a set of 22 primer pairs spanning the pCAG-OSKM sequence. See Table 3 for primer sequences.

RT-PCR analysis [0091] Total mRNA was isolated using TRIZOL (Invitrogen), treated with DNAseI to remove contaminating genomic DNA, and 1 microgram was used to synthesize cDNA using the Cloned AMV First-Strand Synthesis Kit (Invitrogen). Quantitative PCR analysis was done in triplicate on 500 ng using Platinum Syber Green qPCR Super Mix (Invitrogen) in an ABI Prism 7000 thermocycler (Applied Biosystems), and values were normalized to Gapdh. For primers see Table 4.

AP-Staining

[0092] iPS lines were seeded at low density on irMefs and allowed to grow for three days in ES cells medium. Cells were washed with PBS, fixed 1 minute with cold 4% PFA and washed again with PBS. They were stained with Alkaline Phosphatase Blue Membrane Substrate Solution (Sigma) according to manufacturers guidelines.

In vitro differentiation

[0093] iPS cells were cultured on irMEFs in Knock-Out DMEM (Gibco) supplemented with 15% FBS, 1000 U/ml leukaemia inhibitory factor (LIF), 2 mM L-glutamine, 0,1 niM non-essential amino acids (NEAA), 1000 U/ml penicillin, streptomycin, and 100 mM β-mercaptoethanol. For the generation of EBs, the hanging droplet culture method was used. Briefly, single cells were harvested by trypsinization and diluted to 40 cells per μl of ES cell media without LIF. Hanging droplets of 20 μl (800 cells per droplet) were suspended on the underside of 100 mm Petri-dish lids and maintained at 37°C, 5% CO₂. To induce differentiation, EBs were collected after 3 days in hanging droplet culture and transferred to differentiation medium. For neural differentiation: DMEM/F12 (Invitrogen), N2 and B27 supplements (Invitrogen), 1 mM 1-glutamine, 1% non-essential amino acids, 0.1 mM beta- mercaptoethanol for 3 days, after which they were seeded onto matrigel chamber-slides with the same media plus 10^"6 M retinoic acid. Media was changed daily. For cardiomyocyte differentiation, EBs were replated onto gelatin-coated chambers-slide. Cultures were maintained in ES cell media without LIF supplemented with 100 μM ascorbic acid. Media was changed every two days. For endoderm differentiation, EBs were replated onto gelatin- coated chambers-slide in ES cell media without LIF. Media changes were performed every two days.

Teratoma formation

[0094] iPS cells (1 x 10⁵ cells) were resuspended in 20-40 μl of mES medium were injected into the testis of severe combined immunodeficient (SCID) Biedge mice (Charles River Laboratories). Mice were euthanized 5 weeks after cell injection and tumors were processed and analyzed by immunofluorescence.

Immunofluorescent Staining

[0095] For immunostainings, samples were fixed with 4% paraformaldehyde (PFA) for 15 min, washed with PBS and blocked. Teratomas were fixed with 4% PFA, washed, dehydrated, included in parafin, sectioned and blocked. Primary antibodies used were: anti Oct3-4 (Santa Cruz SC-5279), anti-Nanog (Abeam ab21603) , anti-Sox2 (M&D MAB 2018), anti-SSEAl (DSHB, MC-480), anti-B-tubulin III (Tujl) (Covance, MMS- 435P-O), anti- GFAP (Advanced ICI, 31223), anti-sarcomeric-actin (Sigma, 2172), antialpha-1 -fetoprotein (Dako, A0008), FOXA2 (HNF3-B, RD Systems, AF2400). After over night primary antibody incubation, samples were washed with PBS and incubated with secondary antibodies (Cyanine Series, all from Jackson Immunoresearch). Samples were also counterstained with DAPI (Invitrogen, 21490). Images were taken using Leica AOBS confocal microscope.

Oct4 promoter methylation analysis

[0096] Genomic DNA was extracted from 0.5 x 10⁶ cells, and DNA mutagenesis was performed with EpiTect DNA mutagenesis kit (Quiagen) according to manufacturer's specifications. The Oct4 promoter was amplified by two subsequent PCRs using primers previously described (Blelloch, R. et al, Stem cells (Dayton, Ohio), 24:2007-2013 (2006)). The resulting amplified products were cloned into PCR 2.1 or pGEM Easy plasmids, amplified in DH5α cells, purified and sequenced. Flow cytometry analysis

[0097] All analyses were performed on a MoFIo cell sorter (DakoCytomation) running Summit software. To measure the percentage of GFP positive cells, cells were plated on gelatin and trypsinized at different time points post-nucleofection, washed with PBS and resuspended in PBA (phosphate-buffered salt solution with 0.1 % BSA and 0.01 % sodium azide) + 2 μg/mL propidium iodide. Flow cytometric analysis was done with an EPICS ELITE-ESP cytometer (Coulter, Miami, FL).

Nucleic acid primers

[0098] Primers useful in the methods provided herein include the following: Table 2. Primers for probe generation.

Table 3. PCR Primers to detect integration of the transgene.

Table 4. Primers for quantitative RT-PCR.

Table 5. Primers for construct generation.

Claims

WHAT IS CLAIMED IS:

L A method for preparing an induced pluripotent stem cell comprising: (i) transfecting a non-pluripotent cell with a nucleic acid comprising gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein thereby forming a transfected non-pluripotent cell; and (ii) allowing said transfected non-pluripotent cell to divide thereby forming said induced pluripotent stem cell.

2. The method of claim 1, wherein said induced pluripotent stem cell is a footprint-free pluripotent stem cell.

3. The method of claim 1, wherein said transfecting is performed without the use of a viral transfection system.

4. The method of claim 1, wherein said non-pluripotent cell is a mammalian cell.

5. The method of claim 1 , wherein said non-pluripotent cell is a human cell.

6. The method of claim 1 , wherein said non-pluripotent cell is a mouse cell.

7. The method of claim 1, wherein said nucleic acid consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

8. The method of claim 1 , wherein said nucleic acid is a polycistronic nucleic acid.

9. The method of claim 1, wherein said nucleic acid encodes a ribosomal skipping sequence.

10. The method of claim 9, wherein said ribosomal skipping sequence is a picornavirus ribosomal skipping sequence.

11. The method of claim 1 , wherein said nucleic acid further comprises a gene sequence encoding an EGFP protein.

12. The method of claim 1 , wherein said nucleic acid further comprises a gene sequence encoding the Large T antigen of the SV40 polyomavirus.

13. A nucleic acid comprising gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

14. The nucleic acid of claim 13, consisting essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

15. The nucleic acid of claim 13, consisting essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein, C-MYC protein and the Large T antigen of the SV40 polyomavirus.

16. The nucleic acid of claim 13, wherein said nucleic acid is a polycistronic nucleic acid.

17. The nucleic acid of claim 13, wherein said nucleic acid encodes a ribosomal skipping sequence.

18. The nucleic acid claim 17, wherein said ribosomal skipping sequence is a picornavirus ribosomal skipping sequence.

19. The nucleic acid of claim 13, wherein said gene sequences encoding said OCT4 protein, said SOX2 protein and said KLF4 protein are not arranged such that the relative order of translation is OCT4 protein followed by KLF4 protein followed by SOX2 protein.

20. The nucleic acid of claim 13, wherein said nucleic acid further comprises a gene sequence encoding an EGFP protein.

21. The nucleic acid of claim 13, wherein said nucleic acid further comprises a gene sequence encoding the Large T antigen of the SV40 polyomavirus.

22. A non-pluripotent cell comprising a nucleic acid comprising gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

23. The non-pluripotent cell of claim 22, wherein said non-pluripotent cell is a mammalian cell.

24. The non-pluripotent cell of claim 22, wherein said non-pluripotent cell is a human cell.

25. The non-pluripotent cell of claim 22, wherein said non-pluripotent cell is a mouse cell.

26. The non-pluripotent cell of claim 22, wherein said nucleic acid consists essentially of gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein.

27. The non-pluripotent cell of claim 22, wherein said nucleic acid is a polycistronic nucleic acid.

28. The non-pluripotent cell of claim 22, wherein said nucleic acid encodes a ribosomal skipping sequence.

29. The non-pluripotent cell of claim 28, wherein said ribosomal skipping sequence is a picornavirus ribosomal skipping sequence.

30. The non-pluripotent cell of claim 22,wherein said nucleic further comprises a gene sequence encoding an EGFP protein.

31. The non-pluripotent cell of claim 22,wherein said nucleic further comprises a gene sequence encoding the Large T antigen of the SV40 polyomavirus.

32. An induced pluripotent stem cell prepared by a process comprising the steps of: (i) transfecting a non-pluripotent cell with a nucleic acid comprising gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein thereby forming a transfected non-pluripotent cell; and (ii) allowing said transfected non-pluripotent cell to divide thereby forming said induced pluripotent stem cell.

33. The induced pluripotent stem cell of claim 32, wherein said nucleic acid further comprises a gene sequence encoding an EGFP protein.

34. The induced pluripotent stem cell of claim 32, wherein said nucleic acid further comprises a gene sequence encoding the Large T antigen of the SV40 polyomavirus.

35. A method of treating a mammal in need of tissue repair comprising: (i) administering an induced pluripotent stem to said mammal;

(ii) allowing said induced pluripotent stem cell to divide and differentiate into somatic cells in said mammal, thereby providing tissue repair in said mammal; wherein said induced pluripotent stem cell is prepared by a process comprising the steps of: (a) transfecting a non-pluripotent cell with a nucleic acid comprising gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein thereby forming a transfected non-pluripotent cell; and (b) allowing said transfected non-pluripotent cell to divide thereby forming said induced pluripotent stem cell.

36. The method of claim 35, wherein said nucleic acid further comprises a gene sequence encoding an EGFP protein.

37. The method of claim 35, wherein said nucleic acid further comprises a gene sequence encoding the Large T antigen of the SV40 polyomavirus.

38. A method for producing a somatic cell comprising: (i) contacting an induced pluripotent stem cell with a cellular growth factor; and (ii) allowing said induced pluripotent stem cell to divide, thereby forming said somatic cell; wherein said induced pluripotent stem cell is prepared by a process comprising the steps of: (a) transfecting a non-pluripotent cell with a nucleic acid comprising gene sequences encoding an OCT4 protein, a SOX2 protein, a KLF4 protein and a C-MYC protein thereby forming a transfected non-pluripotent cell; and (b) allowing said transfected non-pluripotent cell to divide thereby forming said induced pluripotent stem cell.

39. The method of claim 38, wherein said nucleic acid further comprises a gene sequence encoding an EGFP protein.

40. The method of claim 38, wherein said nucleic acid further comprises a gene sequence encoding the Large T antigen of the SV40 polyomavirus.