WO2013181641A1 - Totipotent stem cells - Google Patents

Abstract

Provided herein are, inter alia, methods of forming, identifying and isolating totipotent stem cells. The methods and compositions provided herein are particularly useful for reprogramming somatic cells and tissue regeneration. Further provided are totipotent stem cells formed using the methods provided herein as well as non-totipotent cells capable of forming totipotent stem cells.

Description

TOTIPOTENT STEM CELLS CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 61/654,776 filed June 1, 2012, which is hereby incorporated in its entirety and for all purposes. BACKGROUND OF THE INVENTION

[0002] The zygote and its daughter cells are totipotent because they are able to develop into all embryonic and extraembryonic cell types^1,2. The progeny of these first two daughter cells become progressively more fate restricted as they activate distinct patterns of gene expression that first direct them towards one of three broad lineages: Oct4⁺ Sox2⁺Nanog⁺ epiblast cells that give rise to the embryo, Gata4⁺/6⁺ primitive endoderm cells that contribute to extraembryonic membranes that encase the embryo, and Cdx2⁺ trophectoderm cells that form a large part of the placenta³. These early cell-fate decisions represent a major and relatively recent advance in mammalian evolution in which the placenta and extraembryonic tissues that support the intrauterine nourishment of the fetus allow development to progress further before birth. The epigenetic landscape of the zygote changes markedly during the first cell divisions. Shortly after fertilization, the oocyte maternal transcripts are replaced with newly synthesized RNAs generated by activating transcription of the zygotic genome^4–6. The unique transcriptional profile of the zygote and its daughter cells defines a brief period when the cells are totipotent. [0003] To date totipotent stem cells have not been availble in sufficient quantities to be used for nuclear reprogramming and in vitro tissue regeneration. Therefore there is a need in the art for methods and composition that provide for sufficeint quantities of totipotent stem cells to be used for tissue regeneration and nuclear reprogramming. The present invention solves these and other problems in the art. BRIEF SUMMARY OF THE INVENTION

[0004] In one aspect, a method of forming a totipotent stem cell is provided. The method includes transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell and allowing the transfected non-totipotent cell to form a totipotent stem cell. [0005] In another aspect, a method of forming a totipotent stem cell is provided. The method includes contacting a non-totipotent cell with a zygote-specific gene repressor inhibitor, thereby forming an inhibited non-totipotent cell and allowing the inhibited non-totipotent cell to form a totipotent stem cell. [0006] In another aspect, a totipotent stem cell prepared according to the methods provided herein including embodiments thereof is provided. [0007] In one aspect, a non-totipotent cell including an exogenous nucleic acid encoding a zygote-specific protein is provided. [0008] In another aspect, a zygote-specific reporter construct including a MuERV-L promoter sequence, a MuERV-L primer-binding sequence and a MuERV-L Gag protein coding sequence operably linked to a reporter sequence is provided. [0009] In one aspect, an isolated totipotent stem cell including a zygote-specific reporter construct according to the embodiments provided herein is provided. [0010] In another aspect, a method of identifying a totipotent stem cell is provided. The method includes transfecting a plurality of cells with the zygote-specific reporter construct provided herein including embodiments thereof. The plurality of cells includes totipotent stem cells and non-totipotent cells and the plurality of cells is allowed to divide, thereby forming a cell expressing a zygote-specific reporter phenotype. The cell expressing the zygote-specific reporter phenotype is detected and thereby the totipotent stem cell is identified. [0011] In one aspect, a method of isolating a totipotent stem cell is provided. The method includes transfecting a plurality of cells with the zygote-specific reporter construct provided herein including embodiments thereof. The plurality of cells includes totipotent stem cells and non-totipotent cells and the plurality of cells is allowed to divide thereby forming a cell expressing a zygote-specific reporter phenotype. The cell expressing the zygote-specific reporter phenotype is detected and separated from cells not expressing the zygote-specific reporter phenotype, thereby isolating the totipotent stem cell. [0012] In another aspect, a method of producing a somatic cell is provided. The method includes contacting a totipotent stem cell with a cellular growth factor and allowing the totipotent stem cell to divide, thereby forming the somatic cell. For the method provided the totipotent stem cell is prepared by a process including the steps of transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell, and allowing the transfected non-totipotent cell to form a totipotent stem cell. [0013] In another aspect, a method of treating a mammal in need of tissue repair is provided. The method includes administering a totipotent stem cell to a mammal and allowing the totipotent stem cell to divide and differentiate into somatic cells in the mammal, thereby providing tissue repair in the mammal. For the method provided the totipotent stem cell is prepared by a process including the steps of transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell and allowing the transfected non-totipotent cell to form a totipotent stem cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The MuERV-L retrovirus and a reporter driven by its LTR marks the 2C state. Figure 1a, Comparison of gene expression between oocytes and 2C embryos. Genes generating junctions to MuERV-L are shown, with those in dark gray denoting significant change in expression. Figure 1b, ORF status of predicted MuERV-L-linked chimaeric transcripts. Figure 1c, Gene Ontology (GO) analysis of MuERV-L-linked protein-coding transcripts. The number of genes from the ten most enriched GO categories are shown. Figure 1d, Figure 1e, 2C Figure 1 (d) and blastocyst Figure 1 (e) embryos were mixed and immunostained with MuERV-L-Gag and Oct4 antibodies. Scale bars, 20 μm. Figure 1f, Zygotes were injected with the 2C::tdTomato transgene, and allowed to develop in vitro for 48 h before imaging. DIC, differential interference contrast. Scale bar, 50 μm. Figure 1g,

2C::tdTomato⁺ ES cells express MuERV-L-Gag protein, as detected by immunofluorescence. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 50 μm. Figure 1h, Microarray analysis of 2C::tdTomato⁺ and 2C::tdTomato^– cells. Dark gray indicates genes with a greater than fourfold change in expression. Figure 1i, 2C::tdTomato⁺ MuERV-L-Gag⁺ ES and iPS cells lack Oct4 protein, as determined by immunofluorescence. Scale bars, 20 μm. [0015] Figure 2 ES cells enter the 2C state regularly, but remain in the state transiently owing to cell intrinsic and extrinsic factors. Figure 2a, FACS analysis of 2C::ERT2-Cre- ERT2, ROSA::LSL-tdTomato ES cells at increasing passage (P) in the presence of 4HT. The percentage of tdTomato⁺ cells is indicated. Figure 2b, 2C::ERT2-Cre-ERT2, ROSA::LSL- LacZ ES cells were cultured in the presence of 4HT, and at increasing passage, cells were fixed and immunostained with anti-ȕ-galactosidase antibodies and counterstained with DAPI. Scale bars, 50 μm. Figure 2c, 2C::tdTomato⁺ and 2C::tdTomato^– cells were collected by FACS and plated before imaging 48 h later. Scale bars, 50 μm. Figure 2d, 2C::tdTomato ES cells were cultured in 20% O₂ (normoxia) or 5% O₂ (hypoxia) for 48 h, and the percentage of tdTomato⁺ cells was determined by FACS. Error bars represent s.d., n = 3 Figure 2e, 2C::tdTomato ES cells at the indicated passage were cultured in media containing 15% fetal calf serum (FCS), 20% knockout serum replacement (KOSR) or N2B27 media containing 3 mM glycogen synthase kinase 3ȕ (GSK3ȕ) and mitogen-activated protein-kinase kinase (MEK) inhibitors (2i) for 48 h before counting the percentage of 2C::tdTomato⁺ cells by FACS. Error bars represent s.d., n = 3. [0016] Figure 3 The 2C state is associated with an active epigenetic signature and is antagonized by repressive chromatin-modifying enzymes. Figure 3a, 2C::tdTomato⁺ (+) and 2C::tdTomato^– (–) cells were collected by FACS and subjected to immunoblot analysis with indicated antibodies. H3K4me2, histone H3 dimethyl Lys 4; AcH3, acetylated histone H3. Figure 3b, Pairwise comparisons of the number of genes activated in Kap1, G9a and Kdm1a mutant ES cells compared with genes activated in 2C embryos. c, ES cell lines homozygous for mutant alleles of Kdm1a, Kap1 and G9a, and corresponding wild-type (WT) ES lines were immunostained with MuERV-L-Gag antibodies and counterstained with DAPI. GT, gene trap; KO, knockout. Scale bars, 50 μm. Figure 3d, 2C::tdTomato ES cells were treated with 40 nM trichostatin A (TSA) for 24 h before imaging. Scale bars, 50 μm. Figure 3e, Kdm1a^fl/fl; Cre- ERT ES cells containing a stably integrated 2C::tdTomato transgene were treated with vehicle or 4HT and subject to FACS analysis to determine the percentage of tdTomato⁺ cells. Figure 3f, 2C::tdTomato; Kdm1a^fl/fl; Cre-ERT ES cells were treated with 4HT or vehicle for 24 h, then passaged for 72 h before collecting tdTomato^– cells by FACS. The percentage of tdTomato⁺ cells was plotted after increasing hours in culture. Error bars represent s.d., n = 3. [0017] Figure 4 Activation of the 2C state is associated with expanded potency in chimaeric embryos towards extraembryonic lineages. Figure 4a, 2C::tdTomato⁺ or 2C::tdTomato^– cytomegalovirus (CMV)–GFP ES cells were injected into morula-stage embryos, which were then grown in vitro. The resulting blastocysts were imaged to visualize the position of injected cells in either the trophectoderm (TE) or ICM. Scale bars, 20 μm. Figure 4b, 2C::tdTomato⁺ or 2C::tdTomato^–, Ef1a::GFP⁺ cells were injected into blastocysts that were then implanted into pseudopregnant females to generate chimaeric embryos. Arrows indicate 2C::tdTomato⁺, GFP⁺ cells contributing to the yolk sac and placenta. Bright denotes bright-field microscopy. Figure 4c, 2C::tdTomato⁺, Ef1a::GFP cells contribute to embryonic endoderm, mesoderm, ectoderm, yolk sac, placental tissues (including giant trophoblast cells, white arrows) and primordial germ cells (PGCs, colabelled with anti-Ddx4 antibody in red, blue arrows). Scale bars, 500 μM (endoderm, mesoderm, ectoderm and yolk sac) and 50 μm (placenta and PGCs). Figure 4d, Heterozygous (+/GT) or homozygous (GT/ GT) Kdm1a-ȕ-geo gene-trap ES cells were injected into wild-type blastocysts that were implanted into pseudopregnant females. (ȕ- geo is a fusion of b-galactosidase and neomycin-resistance genes.) Embryonic (E) and extraembryonic (X) tissues were separated from chimaeric embryos, and subject to

semiquantitative PCR with ȕ-geo primers to determine the relative contribution of the injected cells to these lineages. Error bars represent s.e.m. Figure 4e, A 1:1 mixture of Kdm1a^fl/fl control and Kdm1a knockout (KO) ES cells were co-injected into wild-type blastocysts. At embryonic day 12.5, chimaeric embryonic (E) tissue was separated from placenta (P), yolk sac (Y) and amnion (A) and subject to PCR to detect the floxed (fl) and knockout alleles of the injected cells relative to wild-type alleles of the resident injected embryo. M denotes PCR marker. Figure 4f, Kdm1a^GT/GT, Ef1a::GFP⁺ cells contribute to embryonic endoderm, mesoderm, ectoderm, yolk sac, placental tissues (including giant trophoblast cells, white arrow) and primordial germ cells (PGCs, colabelled with anti-Ddx4 antibody, see arrow). Scale bars, 500 μM (endoderm, mesoderm, ectoderm and yolk sac) and 50 μm (placenta and PGCs). [0018] Figure 5 Model of the role of the MuERV-L-LTR-linked 2C gene network in regulating embryonic potency. Figure 5a, During zygote genome activation, a network of genes that use MuERV-L-LTRs as promoters is activated. This stage correlates with a period in which blastomeres are totipotent. As development progresses, the MuERV-L-LTR-linked 2C gene network is silenced by chromatin repressors, as the ICM segregates from the trophectoderm and primitive endoderm (PrE). HDACs, histone deacetylases. Figure 5b, During the derivation of ES cells from blastocysts, a rare transient population of cells marked by the 2C::tdTomato reporter expresses high levels of 2C genes and low levels of pluripotency markers. In mouse chimaera assays, these cells contribute to embryonic and extraembryonic tissues (shown as dotted gray areas). Increasing the oxidative tension of ES cell cultures or deletion/inhibition of repressive histone-modifying enzymes alters the equilibrium between the 2C and ES states. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0019] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. 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. [0020] As used herein, the singular form“a”,“an”, and“the” includes plural references unless otherwise indicated or clear from context. For example, as will be apparent from context,“a” analog can include one or more analogs. The term“about” in the context of a numeric value refers to +/- 10% of the numeric value. [0021] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl

ribonucleotides, peptide-nucleic acids (PNAs). [0022] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. [0023] A particular nucleic acid sequence also implicitly encompasses“splice variants.” Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. An example of potassium channel splice variants is discussed in Leicher, et al., J. Biol. Chem.

273(52):35095-35101 (1998). [0024] Construction of suitable vectors containing the desired therapeutic gene coding and control sequences may employ standard ligation and restriction techniques, which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthesized oligonucleotides may be cleaved, tailored, and re-ligated in the form desired. [0025] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. [0026] The terms“identical” or percent“identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or 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 algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site

http://www.ncbi.nlm.nih.gov/BLAST/ 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 described below, 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. [0027] The term“recombinant” when used with reference, e.g., to a cell, virus, nucleic acid, protein, or vector, indicates that the cell, virus, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. [0028] 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. Further, a "protein gene product" is a protein expressed from a particular gene. [0029] The phrase“stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in

Biochemistry and Molecular Biology--Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10^oC lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42^oC, or, 5x SSC, 1% SDS, incubating at 65^oC, with wash in 0.2x SSC, and 0.1% SDS at 65^oC. [0030] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary“moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37^oC, and a wash in 1X SSC at 45^oC. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley & Sons. [0031] For PCR, a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). [0032] A "short hairpin RNA" or "small hairpin RNA" is a ribonucleotide sequence forming a hairpin turn which can be used to silence gene expression. After processing by cellular factors the short hairpin RNA interacts with a complementary RNA thereby interfering with the expression of the complementary RNA. [0033] The term“amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, Ȗ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an Į carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0034] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. [0035] The terms“polypeptide,”“peptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. [0036] A "dominant negative protein" is a modified form of a wild-type protein that adversely affects the function of the wild-type protein within the same cell. As a modified version of a wild-type protein the dominant negative protein may carry a mutation, a deletion, an insertion, a post-translational modification or combinations thereof. Any additional modifications of a nucleotide or polypeptide sequence known in the art are included. The dominant-negative protein may interact with the same cellular elements as the wild-type protein thereby blocking some or all aspects of its function. [0037] The term "isolated," when applied to a protein, denotes that the protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as

polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term "purified" denotes that a protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. [0038] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral methods of transfection include 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. Exemplary non-viral transfection methods include 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. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms ƎtransfectionƎ or ƎtransductionƎ also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20. [0039] 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 al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). [0040] 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. Expression of a transfected gene can further be accomplished by transposon-mediated insertion into to the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow insertion into the host genome as well as subsequent excision. [0041] 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. [0042] The term "episomal" refers to the extra-chromosomal state of a plasmid in a cell.

Episomal plasmids are nucleic acid molecules that are not part of the chromosomal DNA and replicate independently thereof. [0043] The term“exogenous” refers to a molecule or substance (e.g., nucleic acid or protein) that originates from outside a given cell or organism. Conversely, the term“endogenous” refers to a molecule or substance that is native to, or originates within, a given cell or organism. [0044] A "vector" is a nucleic acid that is capable of transporting another nucleic acid into a cell. A vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. [0045] A "viral vector" is a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. [0046] 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. [0047] The terms“culture,”“culturing,”“grow,”“growing,”“maintain,”“maintaining,” “expand,”“expanding,” etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. The term does not imply that all cells in the culture survive or grow or divide, as some may naturally senesce, etc. Cells are typically cultured in media, which can be changed during the course of the culture. [0048] The terms“media” and“culture solution” refer to the cell culture milieu. Media is typically an isotonic solution, and can be liquid, gelatinous, or semi-solid, e.g., to provide a matrix for cell adhesion or support. Media, as used herein, can include the components for nutritional, chemical, and structural support necessary for culturing a cell. [0049] The term“derived from,” when referring to cells or a biological sample, indicates that the cell or sample was obtained from the stated source at some point in time. For example, a cell derived from an individual can represent a primary cell obtained directly from the individual (i.e., unmodified), or can be modified, e.g., by introduction of a recombinant vector, by culturing under particular conditions, or immortalization. In some cases, a cell derived from a given source will undergo cell division and/ or differentiation such that the original cell is no longer exists, but the continuing cells will be understood to derive from the same source. [0050] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including

biomolecules, or cells) to become sufficiently proximal to react, interact or physically touch. The two species may be a cell (e.g., a transfected non-totipotent cell, a non-totipotent cell, a totipotent cell) as described herein and an inhibitor (e.g., zygote-specific gene repressor inhibitor, growth factors) as described herein. In some embodiments, contacting may involve a transfected non-totipotent cell as described herein and a zygote-specific gene repressor inhibitor. In other embodiments, contacting may involve a non-totipotent cell as described herein and a zygote- specific gene repressor inhibitor. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. [0051] A "somatic cell" is a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germline cells. [0052] A“primary cell” is a cell taken directly from living tissue (e.g., via biopsy) and is established for growth in vitro. Such cells may be representative of the main function component of the tissue from which they are derived. Primary cell types include but are not limited to fibroblasts, including mouse embryonic fibroblasts (MEF), keratinocytes, melanocytes, myoblasts, mesenchymal cells endothelial cells, epithelial cells, fat cells and stromal cells. [0053] 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. [0054] "Self-renewal" refers to the ability of a cell to divide and generate at least one daughter cell with the self-renewing characteristics of the parent cell. The second daughter cell may commit to a particular differentiation pathway. For example, a self-renewing hematopoietic stem cell can divide and form one daughter stem cell and another daughter cell committed to differentiation in the myeloid or lymphoid pathway. A committed progenitor cell has typically lost the self-renewal capacity, and upon cell division produces two daughter cells that display a more differentiated (i.e., restricted) phenotype. Non-self-renewing cells refer to cells that undergo cell division to produce daughter cells, neither of which have the differentiation potential of the parent cell type, but instead generate differentiated daughter cells. [0055] 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. [0056] "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-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rex1, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics. [0057] A "totipotent stem cell" as provided herein is a cell characterized by the ability to differentiate into embryonic and extraembryonic cells. A totipotent cell includes a zygote and its daughter cells as well as cells expressing totipotent cell characteristics. A totipotent stem cell is a cell that develops and differentiates into cell types that exhibit characteristics associated with embryonic cell types (endoderm, mesoderm and ectoderm) and extraembryonic cell types.

Extraembryonic cell types as provided herein include primitive endoderm cells encasing the embryo (e.g., cells of the amnion the yolk sac, the chorine) and trophectoderm cells that form part of the placenta. Non-limiting examples of extraembryonic cell surface markers are Gata4⁺, Gata6⁺, and Cdx2⁺. Embryonic cells include endodermal cells, mesodermal cells and ectodermal cells. Thus, totipotent stem cells can differentiate into endodermal cells, mesodermal cells and ectodermal cells as well as into primitive endodermal cells and trophectodermal cells. [0058] "Totipotent stem cell characteristics" refer to characteristics of a cell that distinguish totipotent 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, totipotent stem cells may express at least some, and optionally all, of the markers from the following non-limiting list: ZSCAN4, EIF1A, THOC4, TDPOZ1, TDPOZ4 and ZFP 352. In some embodiments, the totipotent stem cell characteristics include lack of detectable expression of Oct4, Nanog, and Sox2. In some embodiments, the totipotent stem cell characteristics include expression of the cell surface markers are Gata4⁺, Gata6⁺, and Cdx2⁺. Totipotent stem cell characteristics include the ability (potency) of a cell to form cells of extraembryonic tissue and embryonic tissue. Therefore, a cell has totipotent stem cell characteristics when it is able to differentiate into embryonic ectoderm, embryonic mesoderm and embryonic endoderm as well as into primitive endoderm and extraembryonic trophectoderm. [0059] Identification of the induced totipotent stem cell may include, but is not limited to the evaluation of the afore mentioned totipotent stem cell characteristics. Such totipotent stem cell characteristics include without further limitation, the expression or non-expression of certain combinations of molecular markers. Further, cell morphologies associated with totipotent stem cells are also totipotent stem cell characteristics. [0060] The term "zygote" as provided herein refers to the diploid cell formed when two haploid gamete cells, a sperm cell for the male gamete and an oocyte for the female gamete, respectively, are joined by means of sexual reproduction. In multicellular organisms, the zygote is the earliest developmental stage of the embryo. The zygote and its daughter cells are capable of differentiating and developing into all cells of an embryo as well as the extraembryonic cell types. [0061] A "non-totipotent cell" refers to a cell that lacks the ability to produce extraembryonic cell types and embryonic cell types. A non-totipotent cell therefore is of lesser potency to self- renew and differentiate than a totipotent stem cell. Cells of lesser potency can be, but are not limited to pluripotent stem cells, 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, fat 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. [0062] An "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. [0063] The term "reprogramming" refers to the process of dedifferentiating a non-pluripotent or a non-totipotent cell into a cell exhibiting pluripotent or totipotent stem cell characteristics. [0064] A "zygote-specific gene" as provided herein refers to a nucleic acid sequence encoding a zygote-specific protein. Non-limiting examples of zygote specific genes are genes encoding for ZSCAN4, EIF1A, THOC4, TDPOZ1, TDPOZ4, and ZFP 352. Zygote-specific genes are characterized by their differential expression in totipotent stem cells relative to non-totipotent cells. In non-totipotent cells zygote-specific genes may be repressed (i.e. transcriptionally inactive) or expressed at lower levels relative to totipotent stem cells. Non-limiting examples of zygote-specific genes are listed in Table 3, 4, and 5. In some embodiments a zygote-specific gene is controlled by a MuERVL promoter sequence. [0065] A "zygote-specific protein" as provided herein refers to a protein which is expressed by a totipotent stem cell. A zygote-specific protein when expressed in a non-totipotent cell conveys totipotent stem cell characteristics to said non-totipotent cell. Upon expression of one or more zygote-specific proteins a non-totipotent acquires totipotent stem cell characteristics and is thereby reprogrammed into a totipotent stem cell. In some embodiments, the zygote specific protein is not expressed by a non-totipotent cell. In some embodiments, the zygote specific protein expression is repressed in a non-totipotent cell relative to a totipotent stem cell. Where the zygote specific protein expression is repressed in a non-totipotent cell the expression levels of the zygote specific protein are decreased relative to a totipotent stem cell. The decrease of expression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less than the expression in a totipotent stem cell. In certain instances, the decrease is 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, or more in comparison to a totipotent stem cell. In other embodiments, the zygote specific protein is expressed in a totipotent cell at an increased level relative to a non-totipotent cell. The increase of expression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than the expression in a non-totipotent cell. In certain instances, the increase is 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a non-totipotent cell. [0066] For specific proteins described herein (e.g., ZSCAN4, THOC4, EIF1A), the named protein includes any of the protein’s naturally occurring forms, or variants that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference or functional fragment thereof. [0067] A "ZSCAN4 protein" as referred to herein includes any of the recombinant or naturally-occurring forms of the zinc finger and SCAN domain containing 4 protein (ZSCAN4), or variants thereof that maintain ZSCAN4 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ZSCAN4). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ZSCAN4 polypeptide. In some aspects, the ZSCAN4 protein is the protein as identified by the NCBI reference gi:119592929 or a variant having substantial identity thereto. [0068] A "EIF1A protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the eukaryotic translation initiation factor 1A protein (EIF1A), or variants thereof that maintain EIF1A protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to EIF1A). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring EIF1A polypeptide. In some aspects, the EIF1A protein is the protein as identified by the NCBI reference gi:4503499 or a variant having substantial identity thereto. [0069] A "THOC4 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the THO complex subunit protein 4 (THOC4), or variants thereof that maintain THOC4 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to THOC4). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring THOC4 polypeptide. In some aspects, the THOC4 protein is the protein as identified by the NCBI reference gi:48429165 or a variant having substantial identity thereto. [0070] A "TDPOZ1 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the TD and POZ domain containing protein 1 (TDPOZ1), or variants thereof that maintain TDPOZ1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to TDPOZ1). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TDPOZ1 polypeptide. In some aspects, the TDPOZ1 protein is the protein as identified by the NCBI reference gi:33333718 or a variant having substantial identity thereto. [0071] A "TDPOZ4 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the TD and POZ domain containing protein 4 (TDPOZ4), or variants thereof that maintain TDPOZ4 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to TDPOZ4). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TDPOZ4 polypeptide. In some aspects, the TDPOZ4 protein is the protein as identified by the NCBI reference gi: 34766460 or a variant having substantial identity thereto. [0072] A "ZFP 352 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the zinc finger protein 352 (ZFP 352), or variants thereof that maintain ZFP 352 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ZFP 352). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ZFP 352 polypeptide. In some aspects, the ZFP 352 protein is the protein as identified by the NCBI reference gi:33333718 or a variant having substantial identity thereto. [0073] A "KDM1A protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the lysine-specific histone demethylase (KDM1A), or variants thereof that maintain KDM1A protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to KDM1A). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring KDM1A polypeptide (e.g. NCBI reference gi:58761544 corresponding to KDM1A isoform a and NCBI reference gi:58761546 corresponding to KDM1A isoform b). [0074] A "G9A protein " as referred to herein includes any recombinant or naturally-occurring forms of the histone-lysine N-methyltransferase G9A (G9A), or variants thereof that maintain G9A protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to G9A). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring G9A polypeptide. (e.g. NCBI reference gi:156142197 corresponding to G9A isoform a and NCBI reference gi:156142199 corresponding to G9A isoform b). [0075] A "KAP1 protein" as referred to herein includes any recombinant or naturally- occurring forms of the KRAB (Krueppel-associated-box) associated protein 1 (KAP1), or variants thereof that maintain KAP1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to KAP1). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring KAP1 polypeptide. (e.g. NCBI reference gi:3183179) [0076] An "OCT4 protein" as referred to herein includes any of the recombinant or 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%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Oct4). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Oct4 polypeptide. In other aspects, the Oct4 protein is the protein as identified by the NCBI reference gi:42560248 corresponding to isoform 1, gi:116235491 and gi:291167755 corresponding to isoform 2, or a variant having substantial identity thereto. [0077] A "SOX2 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the SOX2 transcription factor, or variants thereof that maintain SOX2 transcription factor activity (e.g. at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Sox2). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion, e.g., the DNA-binding region) compared to a naturally occurring Sox2 polypeptide. In some aspects, the SOX2 protein is the protein as identified by the NCBI reference gi:28195386 or a variant having substantial identity thereto. [0078] A "Nanog protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the Nanog transcription factor, or variants thereof that maintain Nanog transcription factor activity (e.g. at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Nanog). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion, e.g., the DNA-binding region) compared to a naturally occurring Nanog polypeptide. In some aspects, the Nanog protein is the protein as identified by the NCBI reference gi:47716683 or a variant having substantial identity thereto. [0079] The terms "murine endogenous retrovirus-like sequence", "MERVL sequence", or "Mu-ERV-L sequence" are used herein interchangeably and refer to any recombinant or naturally-occurring form of the murine endogenous retrovirus-like nucleic acid sequence, or variants, alleles, mutants, and interspecies homologs. Said variants may specifically hybridize under stringent hybridization conditions to a nucleic acid encoding the murine endogenous retrovirus-like sequence and/or have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a reference nucleic acid sequence, including a reference nucleic acid encoding the murine endogenous retrovirus-like sequence. A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate (e.g., human), rodent (e.g., rat, mouse, hamster), cow, pig, horse, sheep, or any mammal. In some embodiments, the variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the long terminal repeat (LTR) of the murine endogenous retrovirus-like sequence. In other

embodiments, the variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the primer-binding sequence of the murine endogenous retrovirus-like sequence. In other embodiments, the variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the gag gene sequence of the murine endogenous retrovirus-like sequence. In other embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the long terminal repeat (LTR), the primer-binding sequence and the gag gene of the Mu-ERV-L sequence. In some aspects, the murine endogenous retrovirus-like sequence is the nucleic acid sequence as identified by the NCBI reference gi: 2065208 or a variant having substantial identity thereto. [0080] The terms "inhibitor," "repressor" or "antagonist" or "downregulator" interchangeably refer to a substance that results in a detectably lower expression or activity level as compared to a control. The inhibited expression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the inhibition is 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control. As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein (e.g. a demethylase, a methyltransferase, a transcriptional repressor) relative to the activity or function of the protein in the absence of the inhibitor. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity. Similarly an "inhibitor" is a compound or small molecule that inhibits protein activity (e.g., demethylation, deacetylation, transcriptional repression) e.g., by binding, partially or totally blocking stimulation, decrease, prevent, or delay activation, or inactivate, desensitize, or down-regulate signal transduction or enzymatic activity necessary for protein activity. Inhibition as provided herein may also include decreasing or blocking a protein activity (e.g., demethylation, deacetylation, transcriptional repression) by expressing a mutant form of said protein thereby decreasing or blocking its activity. [0081] A "transcriptional repressor" as used herein refers to any protein capable of interfering with the transcription of a gene. For example, the transcriptional repressor may bind to specific regulatory sites of a gene sequence (e.g., promoter) to prevent expression of said gene. In some embodiments, the transcriptional repressor may bind to one or more transcriptional activators of a gene thereby preventing interaction of the one or more activators with the promoter of said gene and subsequent transcription of said gene. [0082] A“small molecule inhibitor” as used herein refers to any organic, bioorganic, or inorganic compound that alters the activity or function of a protein, nucleic acid, or

polysaccharide. [0083] A“control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample or condition. For example, a test sample can include cells exposed to a test condition or a test agent, while the control is not exposed to the test condition or agent (e.g., negative control). The control can also be a positive control, e.g., a known primary cell or a cell exposed to known conditions or agents, for the sake of comparison to the test condition. A control can also represent an average value gathered from a plurality of samples, e.g., to obtain an average value. For therapeutic applications, a sample obtained from a patient suspected of having a given disorder or deficiency can be compared to samples from a known normal (non-deficient) individual. A control can also represent an average value gathered from a population of similar individuals, e.g., patient having a given deficiency or healthy individuals with a similar medical background, same age, weight, etc. A control value can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to the disorder or deficiency, or prior to treatment. One of skill will recognize that controls can be designed for assessment of any number of parameters. [0084] One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. [0085] The terms“therapy,”“treatment,” and“amelioration” refer to any reduction in the severity of symptoms, e.g., of a neurodegenerative disorder or neuronal injury. As used herein, the terms“treat” and“prevent” are not intended to be absolute terms. Treatment can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, improved cognitive function or coordination, increase in survival time or rate, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques. [0086] The term“therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate a given disorder or symptoms. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as“-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. [0087] “Subject,”“patient,”“individual in need of treatment” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. [0088] In the context of the present invention, i.e., methods for generating totipotent stem cells or somatic cells derived from totipotent stem cells, a subject in need of treatment can refer to an individual that is deficient of one or more somatic cell populations. The deficiency can be due to a genetic defect, injury, or pathogenic infection. II. Methods

[0089] Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Applicants have identified a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. Applicants genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. Applicants show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals. [0090] Provided herein are, inter alia, methods of forming, identifying and isolating totipotent stem cells. The methods and compositions provided herein are particularly useful for reprogramming somatic cells and tissue regeneration. Further provided are totipotent stem cells formed using the methods provided herein as well as non-totipotent cells capable of forming totipotent stem cells. [0091] In one aspect, a method of forming a totipotent stem cell is provided. The method includes transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell and allowing the transfected non-totipotent cell to form a totipotent stem cell. In some embodiments, the allowing includes culturing the transfected non-totipotent cell. The allowing may include culturing the transfected non- totipotent cell to undergo cell division. The allowing may further include culturing the transfected non-totipotent cell under conditions suitable for cell reprogramming, thereby preparing a totipotent stem cell. Suitable culture conditions are described herein, and can include standard tissue culture conditions. For example, the transfected non-totipotent cell can be cultured in a buffered media that includes amino acids, nutrients, growth factors, etc., as will be understood in the art. In some aspects, the culture includes feeder cells (e.g., fibroblasts), while in others, the culture is devoid of feeder cells. Cell culture conditions are described in more detail, e.g., in Picot, Human Cell Culture Protocols (Methods in Molecular Medicine) 2010 ed. and Davis, Basic Cell Culture 2002 ed. Culturing the transfected non-totipotent cell may occur in the presence of suitable media and cellular growth factors. Cellular growth factors are agents which cause cells to migrate, differentiate, transform or mature and divide. Cellular growth factors are polypeptides which can usually be isolated from various normal and malignant mammalian cell types. Some growth factors can also be produced by genetically engineered microorganisms, such as bacteria (E.coli) 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. [0092] Where appropriate the transfected non-totipotent cell may be subjected to a process of selection. A process of selection may include a selection marker introduced into a non-totipotent cell upon transfection. A selection marker may be a gene encoding for a polypeptide with enzymatic activity. The enzymatic activity includes, but is not limited to, the activity of an acetyltransferase or a phosphotransferase. In some embodiments, the enzymatic activity of the selection marker is the activity of a phosphotransferase. The enzymatic activity of a selection marker may confer to a transfected neural stem cell the ability to expand in the presence of a toxin. Such a toxin typically inhibits cell expansion and/or causes cell death. Examples of such toxins include, but are not limited to, hygromycin, neomycin, puromycin and gentamycin. In some embodiments, the toxin is hygromycin. Through the enzymatic activity of a selection maker a toxin may be converted to a non-toxin which no longer inhibits expansion and causes cell death of a transfected neural stem cell. Upon exposure to a toxin a cell lacking a selection marker may be eliminated and thereby precluded from expansion. [0093] In other embodiments, the allowing further includes expressing the zygote-specific protein in the transfected non-totipotent cell. Expression of the zygote-specific protein may be controlled by exogenous or endogenous regulatory sequences (e.g. promoters) or exogenous or endogenous factors (e.g. transcription factors, translation factors). In some embodiments, the zygote-specific protein expression is controlled by an endogenous retroviral sequence. In some embodiments, the endogenous retroviral sequence is a murine endogenous retrovirus-like (MuERV-L) sequence. In other embodiments, the endogenous retroviral sequence is a human endogenous retrovirus-like (hERV-L) sequence. In some embodiments, the nucleic acid encoding the zygote-specific protein is operably linked to a murine endogenous retrovirus-like (MuERV-L) sequence. In other embodiments, the MuERV-L sequence includes a MuERV-L promoter sequence and a MuERV-L gag protein encoding sequence. In some embodiments, the nucleic acid encoding the zygote-specific protein is operably linked to a human endogenous retrovirus-like (hERV-L) sequence. In other embodiments, the hERV-L sequence includes a hERV-L promoter sequence and a hERV-L gag protein encoding sequence. [0094] The zygote-specific protein provided herein is a protein expressed by a zygote and upon expression in a non-totipotent cell conveys totipotent stem cell characteristics to said non- totipotent cell, thereby reprogramming said non-totipotent cell into a totipotent stem cell. Upon expression of one or more zygote-specific proteins a non-totipotent acquires totipotent stem cell characteristics and is thereby reprogrammed into a totipotent stem cell. In some embodiments, the zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein or a Zinc finger protein (ZFP) 352 protein. In other embodiments, the zygote-specific protein is encoded by a gene as set forth by Tables 4 or 5. [0095] In some embodiments, the method further includes contacting the non-totipotent cell or the transfected non-totipotent cell with a zygote-specific gene repressor inhibitor. A "zygote- specific gene repressor inhibitor" as provided herein refers to an agent capable of inhibiting zygote-specific gene repression. A zygote-specific gene repressor inhibitor may activate zygote- specific gene expression by directly or indirectly interacting with a repressor of zygote-specific gene expression. Non-limiting examples of zygote-specific gene repressors are histone modifying enzymes such as methyltransferases (e.g., G9a), demethylases (e.g., Kdm1a), deacetylases, transcriptional repressors (e.g., Kap1). The zygote-specific gene repressor inhibitor may be a molecule that reduces zygote-specific gene repressor activity and expression. In some embodiments, the zygote-specific gene repressor inhibitor reduces the activity of a zygote-specific gene repressor. In other embodiments, the zygote-specific gene repressor inhibitor reduces the expression of a zygote-specific gene repressor gene. In some embodiments, the zygote-specific gene repressor inhibitor reduces the activity of a zygote-specific gene repressor protein and the expression of a zygote-specific gene repressor gene. Examples of a zygote-specific gene repressor inhibitor include, but are not limited to nucleic acids, proteins, dominant negative proteins, peptides, oligosaccharides, polysaccharides, lipids, phospholipids, glycolipids, monomers, polymers, small molecules and organic compounds. The zygote-specific gene repressor inhibitor may be a polynucleotide. In some embodiments, the zygote-specific gene repressor inhibitor is a short hairpin RNA. In other embodiments, the zygote-specific gene repressor inhibitor is a small interfering RNA. The zygote-specific gene repressor inhibitor may be a protein. In some embodiments, the zygote-specific gene repressor inhibitor is a dominant negative protein. In some embodiments, the zygote-specific gene repressor inhibitor is a zygote- specific gene repressor inhibitor gene. In some further embodiments, the zygote-specific gene repressor inhibitor gene is transfected into a non-totipotent cell. In some other further embodiments, the zygote-specific gene repressor inhibitor gene is transfected into a transfected non-totipotent cell. [0096] In some embodiments, the zygote-specific gene repressor inhibitor is a histone modification inhibitor. In other embodiments, the histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor. In some embodiments, the histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor. In some embodiments, the methyltransferase inhibitor is a G9a inhibitor. In other embodiments, the histone demethylase inhibitor is a Kdm1a inhibitor. [0097] In some embodiments, the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. In some embodiments, the transcriptional repressor inhibitor is a Krueppel- associated protein (Kap) 1 inhibitor. In some embodiments, the zygote-specific gene repressor inhibitor is a small molecule. In some embodiments, the zygote-specific gene repressor inhibitor is a deacetylase inhibitor. In some embodiments, the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. In other embodiments, the deacetylase inhibitor is trichostatin A. Any histone modification inhibitor known in the art may be applicable to the invention provided herein including embodiments thereof. [0098] Non-totipotent cells useful for the methods provided herein including embodiments thereof are cells that are of lesser potency than totipotent cells. A cell of lesser potency than a totipotent cell, is a cell that does not have the ability to form extraembryonic and embryonic cells. In some embodiments, the non-totipotent cell is a primary cell. In other embodiments, the primary cell is a fibroblast. In some embodiments, the primary cell is a fat cell. In other embodiments, the non-totipotent cell is a pluripotent cell. In some embodiments, the pluripotent cell is an induced pluripotent stem cell or an embryonic stem cell. In some embodiments, the pluripotent cell is an induced pluripotent stem cell. In other embodiments, the pluripotent cell is an embryonic stem cell. [0099] In another aspect, a method of forming a totipotent stem cell is provided. The method includes contacting a non-totipotent cell with a zygote-specific gene repressor inhibitor, thereby forming an inhibited non-totipotent cell and allowing the inhibited non-totipotent cell to form a totipotent stem cell. For the method provided any zygote-specific gene repressor inhibitor described herein may be used (see above). Thus, in some embodiments, the zygote-specific gene repressor inhibitor is a histone modification inhibitor. In some embodiments, the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. In some embodiments, the zygote-specific gene repressor inhibitor is a small molecule. In some embodiments, the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. In other embodiments, the deacetylase inhibitor is trichostatin A. [0100] In some embodiments, the method further includes transfecting the non-totipotent cell or the inhibited non-totipotent cell with a nucleic acid encoding a zygote-specific protein. The zygote-specific protein may be any zygote-specific protein provided herein (see above). III. Compositions

[0101] The compositions provided herein are useful for reprogramming somatic nuclei and tissue regeneration. In one aspect, a totipotent stem cell prepared according to the methods provided herein including embodiments thereof is provided. According to the methods described above the totipotent stem cell may be prepared by transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell and allowing the transfected non-totipotent cell to form a totipotent stem cell. The totipotent stem cell may further be prepared by contacting a non-totipotent cell with a zygote-specific gene repressor inhibitor, thereby forming an inhibited non-totipotent cell and allowing the inhibited non-totipotent cell to form a totipotent stem cell. [0102] In some embodiments, the totipotent stem cell does not include detectable amounts of an Oct-4 polypeptide, a Sox-2 polypeptide or a Nanog polypeptide. Detectable amounts are protein amounts that can be detected by standard protein detection methods well known in the art. In other embodiments, the totipotent stem cell includes a Gag polypeptide. In some embodiments the totipotent stem cell includes a functional fragment of a Gag polypeptide. In some embodiments, the totipotent stem cell forms extraembryonic tissue or embryonic tissue. In other embodiments, the totipotent stem cell forms extraembryonic tissue and embryonic tissue. [0103] In one aspect, a non-totipotent cell including an exogenous nucleic acid encoding a zygote-specific protein is provided. In some embodiments, the zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein or a Zinc finger protein (ZFP) 352 protein. In some

embodiments, the zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein and a Zinc finger protein (ZFP) 352 protein. In some embodiments, the zygote-specific protein is a protein encoded by a gene set forth by Tables 4 or 5. In some embodiments, the level of expression of the zygote-specific protein in the non-totipotent cell is increased compared to a control level. A control level as provided herein may be the level of expression of the zygote-specific protein in the absence of the exogenous nucleic acid. [0104] In some embodiments, the non-totipotent cell further includes a zygote-specific gene repressor inhibitor. In other embodiments, the zygote-specific gene repressor inhibitor is a histone modification inhibitor. In some embodiments, the histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor. In some embodiments, the histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor. In some embodiments, the methyltransferase inhibitor is a G9a inhibitor. In some embodiments, the histone demethylase inhibitor is a Kdm1a inhibitor. [0105] In some embodiments, the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. In other embodiments, the transcriptional repressor inhibitor is a Krüppel- associated protein (Kap) 1 inhibitor. In some embodiments, the zygote-specific gene repressor inhibitor is a small molecule. In some embodiments, the histone modification inhibitor is a deacetylase inhibitor. In some embodiments, the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. [0106] In another aspect, a non-totipotent cell including a zygote-specific gene repressor inhibitor is provided. The zygote-specific gene repressor inhibitor may be any inhibitor as described herein. Thus, in some embodiments, the zygote-specific gene repressor inhibitor is a histone modification inhibitor. In some embodiments, the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. In some embodiments, the zygote-specific gene repressor inhibitor is a small molecule. In some embodiments, the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. In other embodiments, the deacetylase inhibitor is trichostatin A. In some embodiments, the non-totipotent cell further includes a nucleic acid encoding a zygote-specific protein. The zygote-specific protein may be any zygote-specific protein provided herein (see above). [0107] In another aspect, a zygote-specific reporter construct including a MuERV-L promoter sequence, a MuERV-L primer-binding sequence and a MuERV-L Gag protein coding sequence operably linked to a reporter sequence is provided. In some embodiments, the reporter sequence encodes a fluorescent protein. In some embodiments, the MuERV-L promoter sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the long terminal repeat (LTR) of the murine endogenous retrovirus-like sequence. In other

embodiments, the MuERV-L primer-binding sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the primer-binding sequence of the murine endogenous retrovirus-like sequence. In other embodiments, the MuERV-L Gag protein coding sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the gag gene sequence of the murine endogenous retrovirus-like sequence. In some embodiments, the Mu-ERV-L promoter sequence includes nucleotide 1-495 of the nucleic acid sequence as identified by the NCBI reference gi: 2065208 or a functional fragment thereof. In some embodiments, the Mu-ERV-L promoter sequence has the sequence of nucleotide 1-495 of the nucleic acid sequence as identified by the NCBI reference gi: 2065208. In other

embodiments, the Mu-ERV-L primer-binding site includes nucleotide 499-513 of the nucleic acid sequence as identified by the NCBI reference gi: 2065208 or a functional fragment thereof. In other embodiments, the Mu-ERV-L primer-binding site has the sequence of nucleotide 499- 513 of the nucleic acid sequence as identified by the NCBI reference gi: 2065208. In some embodiments, the Mu-ERV-L Gag protein coding sequence includes nucleotide 538-2283 of the nucleic acid sequence as identified by the NCBI reference gi: 2065208 or a functional fragment thereof. In other embodiments, the Mu-ERV-L Gag protein coding sequence has the sequence of nucleotide 538-2283 of the nucleic acid sequence as identified by the NCBI reference gi:

2065208. [0108] In some embodiments, the fluorescent protein is a red fluorescent protein. In other embodiments, the fluorescent protein is a green fluorescent protein. In some embodiments, the MuERV-L promoter sequence, the MuERV-L primer-binding sequence, the MuERV-L Gag protein coding sequence and the reporter sequence form part of the same nucleic acid. In some embodiments, the MuERV-L promoter sequence is operable linked to a MuERV-L primer- binding sequence, thereby forming a MuERV-L regulator sequence. In some further embodiment, the MuERV-L regulator sequence is operably linked to a MuERV-L Gag protein coding sequence or a functional fragment thereof, thereby forming a MuERV-L sequence. In some further embodiments, the MuERV-L sequence is operably linked to a reporter sequence. [0109] In some aspects, the invention involves recombinant methods, e.g., for construction of vectors encoding a MuERV-L sequence as described herein. Standard recombinant methods are used for cloning, DNA and RNA isolation, amplification and purification. Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007 with updated through 2010) Current Protocols in Molecular Biology, among others known in the art. [0110] In some aspects, amplification of known sequences may be desirable, e.g., for cloning into appropriate expression vectors. Such methods of amplification are well known to those of skill in the art. Detailed protocols for PCR are provided, e.g., in Innis et al. (1990) PCR

Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). The known nucleic acid sequences for the genes listed herein is sufficient to enable one of skill to routinely select primers to amplify any portion of the gene. [0111] In one aspect, an isolated totipotent stem cell including a zygote-specific reporter construct according to the embodiments provided herein is provided. In some embodiments, the totipotent stem cell is derived from an induced-pluripotent stem cell. In other embodiments, the totipotent stem cell is derived from an embryonic stem cell. In other embodiments, the totipotent stem cell is derived from a primary cell. In some embodiments, the totipotent stem cell forms extraembryonic tissue and embryonic tissue. [0112] In another aspect, a method of identifying a totipotent stem cell is provided. The method includes transfecting a plurality of cells with the zygote-specific reporter construct provided herein including embodiments thereof. The plurality of cells includes totipotent stem cells and non-totipotent cells and the plurality of cells is allowed to divide, thereby forming a cell expressing a zygote-specific reporter phenotype. The cell expressing the zygote-specific reporter phenotype is detected and thereby the totipotent stem cell is identified. In some embodiments, the zygote-specific reporter phenotype is a decreased expression of an Oct-4 polypeptide, a Sox- 2 polypeptide or a Nanog polypeptide relative to a standard control. A standard control as provided herein is the expression of an Oct-4 polypeptide, a Sox-2 polypeptide or a Nanog polypeptide in the absence of the zygote-specific reporter construct. In other embodiments, the zygote-specific reporter phenotype is expression of a Gag polypeptide. [0113] In one aspect, a method of isolating a totipotent stem cell is provided. The method includes transfecting a plurality of cells with the zygote-specific reporter construct provided herein including embodiments thereof. The plurality of cells includes totipotent stem cells and non-totipotent cells and the plurality of cells is allowed to divide thereby forming a cell expressing a zygote-specific reporter phenotype. The cell expressing the zygote-specific reporter phenotype is detected and separated from cells not expressing the zygote-specific reporter phenotype, thereby isolating the totipotent stem cell. The separation of the cell expressing the zygote-specific reporter phenotype from the remainder of the cell population can be performed using cell separation techniques known in the art (differential size fractionation, FACS-based cell sorting, or affinity based methods such as magnetic or chromatographic separation). In some embodiments, the separating is carried out 4 or more days after said contacting. In some embodiments, the separating is carried out 7 days after said contacting. In other embodiments, the separating is carried out 8 or more days after the contacting. [0114] In another aspect, a method of producing a somatic cell is provided. The method includes contacting a totipotent stem cell with a cellular growth factor and allowing the totipotent stem cell to divide, thereby forming the somatic cell. For the method provided the totipotent stem cell is prepared by a process including the steps of transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell, and allowing the transfected non-totipotent cell to form a totipotent stem cell. [0115] In another aspect, a method of treating a mammal in need of tissue repair is provided. The method includes administering a totipotent stem cell to a mammal and allowing the totipotent stem cell to divide and differentiate into somatic cells in the mammal, thereby providing tissue repair in the mammal. For the method provided the totipotent stem cell is prepared by a process including the steps of transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell and allowing the transfected non-totipotent cell to form a totipotent stem cell. IV. Examples

[0116] Mouse ES cells are isolated from the inner cell mass (ICM) of blastocysts that have already become a separate lineage from the trophectoderm^7,8. ICM-derived ES cells are regarded as pluripotent because they have the capacity to generate tissues of the fetus, but they are extremely inefficient at colonizing the extraembryonic tissues⁹. The rare contribution of ES cells to extraembryonic tissues could be explained by contamination of ES cultures with

trophectoderm or primitive endoderm-committed cells, or may occur because rare ES cells have acquired the ability to produce extraembryonic tissues in addition to embryonic tissues. This latter possibility is intriguing, because recent evidence shows that ES cell cultures are a heterogeneous mixture of metastable cells with fluctuating expression of genes such as Zscan4, stella (also known as Dppa3), Nanog, Sox17 and Gata6, which could account for special attributes of individual cells^10–14. [0117] A large number of retrotransposons are expressed when the zygotic genome is first transcribed, including the endogenous retroviruses (ERVs), long interspersed nuclear element-1 (LINE-1), and the non-autonomous short interspersed elements (SINEs)¹⁵. At the 2C stage, murine endogenous retrovirus-like (MuERV-L, also known as MERVL and Erv4) elements are transiently derepressed and produce 3% of the transcribed messenger RNAs^15–17. After the 2C stage, MuERV-L retroelement expression is silenced^18,19. Applicants discovered that this regulated pattern of MuERV-L expression overlapped with more than 1002C-specific genes that have co-opted regulatory elements from these foreign retroviruses to initiate their transcription. Applicants exploited the regulated activity of these 2C virus-derived promoters to label cells, and found that both ES and iPS cell cultures contain a small but relatively constant fraction of cells that has entered into the 2C-transcriptional state. Purification of these 2C-like cells shows that they have unique developmental characteristics and efficiently produce progeny for

extraembryonic and embryonic lineages. [0118] Identification of a 2C-like state within ES cultures [0119] To identify zygotically activated genes Applicants performed deep RNA sequencing (RNA-seq) on mouse oocytes and 2C-stage embryos. A comparison of the transcripts in these cells identified a large number of genes and retrotransposons that became expressed in the 2C embryo, as well as numerous transcripts that were downregulated (Fig. 1a and Table 1). The most highly activated repeat was the MuERV-L family of retroviruses and their corresponding long terminal repeat (LTR) promoters (Mt2_mm), which were activated more than 300-fold (Table 1). Sequence alignments showed that more than 25% of the nearly 700 copies of MuERV-L elements were activated, and that 307 genes generated chimaeric transcripts with junctions to MuERV-L elements (Fig. 1a), including 10 that were previously described¹⁵. Of the 626 chimaeric transcripts generated, >90% were 5′ LTR–exon fusions that generated open reading frames (ORFs), suggesting that these LTRs had become functional promoters for protein-coding genes (Fig. 1b). The most significantly enriched Gene Ontology categories representing these chimaeric proteins were regulation of transcription, ion binding, translation, nucleotide binding and mRNA transport (Fig. 1c). Two notable transcription factors that used alternate MuERV-L-LTR promoters were Gata4 and Tead4, which are important for the specification of primitive endoderm and trophectoderm, respectively^20–22. [0120] Because more than 300 of the nearly 700 copies of the MuERV-L endogenous retroviruses still encode Gag viral protein, Applicants stained 2C and early blastocyst embryos to confirm that viral Gag was expressed and developmentally regulated. Applicants found that 2C embryos express Gag but lack the pluripotency marker Oct4, whereas blastula cells lack Gag but express Oct4 (Fig. 1d, e). Thus, MuERV-L activity is developmentally regulated and these retroviral promoters have been co-opted by many cellular genes to impose tight control over their expression. [0121] Next Applicants asked whether it was possible to use the regulatory sequences from MuERV-L elements to label 2C cells. Applicants cloned the MuERV-L 5′ LTR, the primer- binding site, and a portion of the gag gene upstream of the red fluorescent protein tandem dimeric Tomato (tdTomato). Applicants injected fertilized eggs with the 2C::tdTomato construct and monitored the expression of tdTomato during culture in vitro. tdTomato expression was highest in arrested zygotes and 2C embryos and became downregulated at the morula stage (Fig. 1f). Notably, when Applicants introduced the 2C::tdTomato construct into ES cells and selected for clonal stable integrants, Applicants found several colonies that contained 1–5 cells that were strongly labelled with tdTomato among cells lacking expression of the reporter (Fig. 1g).

Applicants also found that rare ES cells expressed MuERV-L mRNA and Gag protein, and that these overlapped with 2C::tdTomato⁺ cells (Fig. 1g). The correspondence between the

2C::tdTomato reporter and MuERV-L expression was further confirmed by immunoblotting, and electron microscopy imaging of viral epsilon particles encoded by MuERV-L within the endoplasmic reticulum of tdTomato⁺ cells but not tdTomato^– cells. Thus, MuERV-L expression is restricted in vivo to 1–4-cell-stage embryos and is reactivated within a small subpopulation of ES cells derived from blastocysts. [0122] To characterize the unexpected 2C::tdTomato-labelled cells within ES cultures, Applicants sorted tdTomato⁺ and tdTomato^– cells and performed microarray and mRNA sequencing analyses (Fig. 1h and Tables 2 and 3). tdTomato⁺ cells expressed 55-fold higher levels of MuERV-L transcripts than tdTomato^– cells, but the vast majority of other

retrotransposons were unaffected (Table 2). Notably, tdTomato⁺ cells had 165 transcripts activated more than fourfold, and no genes repressed more than fourfold compared with tdTomato^– cells (Fig. 1h). Among the genes that were highly enriched in tdTomato⁺ cells, several were previously shownto be restricted to the 2–4-cell stage of development, including Zscan4, Tcstv1/3, Eif1a, Gm4340 (also known as Thoc4), Tdpoz1–5 and Zfp352 (refs 23–25). In total, 525 genes that were enriched in 2C::tdTomato⁺ cells were also activated at the 2Cstage, including 52 genes that generated chimaeric transcripts linked to MuERV-L elements (Tables 4 and 5). [0123] A hallmark of the ICM and ES cells is their expression of Oct4, Sox2 and Nanog, whereas totipotent 2C embryos do not express Oct4 (Fig. 1d, e). Applicants found that

2C::tdTomato⁺ cells within ES cultures also lacked Oct4, Sox2 and Nanog (Fig. 1i). The reduction in Oct4, Sox2 and Nanog protein labelling occurred despite changes in

theirmRNAlevels, suggesting that the regulation is occurring post-transcriptionally (Fig. 1h). In summary, 2C::tdTomato labels a subset of ES cells that share transcriptional and proteomic features of 2C embryos and display markedly different patterns of pluripotency markers from most ES cells in culture. [0124] ES cells cycle in and out of the 2C state [0125] Applicants considered the possibility that the expression of the 2C::tdTomato reporter and MuERV-L-Gag protein in sporadic cells within ES cultures might arise from contamination with trophectoderm or primitive endoderm. To exclude this possibility, Applicants examined iPS cells derived from mouse fibroblasts because they should not be contaminated with cells from blastocyst embryos. Similar to ES cells, Applicants found that sporadic iPS cells express the MuERV-L-Gag protein and lack Oct4 (Fig. 1i). Thus, the heterogeneity within ES cultures is a property that is shared with iPS cell cultures and is unlikely to arise from a cell contaminant. [0126] Next Applicants examined whether the 2C::tdTomato⁺ cells represent a stable cell population or whether ES cells transition in and out of this 2C-like state. Applicants used a Cre/loxP fate-mapping strategy to mark cells indelibly that had expressed 2C genes. Applicants generated a transgenic mouse line using the MuERV-L regulatory elements driving expression of a tamoxifen-inducible Cre recombinase (2C::ERT2-Cre-ERT2). These mice were then mated with Cre-responsive reporter lines (ROSA::LSL-tdTomato and ROSA::LSL-LacZ). ES cell lines were derived from double-positive transgenic blastocysts. After addition of 4-hydroxytamoxifen (4HT) to the ES cultures Applicants detected nuclear Cre expression inMuERV-L-Gag⁺ cells. When ES cultures were grown for 2–6 days with 4HT Applicants found a steady increase in the percentage of reporter-positive cells (Fig. 2a). Remarkably, over extended passages nearly every ES cell activated the reporter (Fig. 2b), demonstrating this transient state is regularly entered by ES cells. [0127] To monitor the kinetics of the interconversion between 2C::tdTomato⁺ and

2C::tdTomato^– cells Applicants performed flow cytometry to collect tdTomato⁺ and tdTomato^– cells. When these purified subpopulations were cultured Applicants found that tdTomato⁺ cells produced tdTomato^– cells and vice versa (Fig. 2c). Within 24 h nearly 50%of the tdTomato⁺ cells convert to tdTomato^–, independently of the starting percentages of the two different cell populations (Fig. 2c and data not shown). Under hypoxic conditions (5% O₂), the percentage of cells expressing the 2C::tdTomato reporter was decreased, which could be reversed by shifting the cultures back to 20%O₂ (Fig. 2d). Applicants also found that growing cells for 48 h in ‘ground-state’ media conditions (2i media²⁶) reduced but did not eliminate the presence of tdTomato⁺ cells relative to media containing knockout serum replacement, suggesting extrinsic and intrinsicmechanisms regulate theMuERV-L and 2C gene network (Fig. 2e). [0128] The 2C-ES switch is regulated by histone modification [0129] After activation of the zygotic genome in mouse development, histone deacetylation and histone H1 synthesis lead to the formation of repressive chromatin that is thought to limit the broad pattern of transcription present in 2C embryos^27,28. Using indirect immunofluorescence, Applicants found that tdTomato⁺ cells had markedly higher levels of active histone marks, including methylation of histone 3 lysine 4 (H3K4) and acetylation of H3 and H4, a finding confirmed using immunoblot analysis of sorted cell populations (Fig. 3a). This type of chromatin mirrors that found in 2C embryos²⁸. Next Applicants tested whether tdTomato⁺ cells had different levels of DNA methylation compared with non-labelled ES cells. Applicants found that the MuERV-L sequences were hypomethylated in tdTomato⁺ cells compared with tdTomato^– cells. In contrast to the MuERV-L sequences, intracisternal A-type particle retroviruses were highly methylated in both tdTomato⁺ and tdTomato^– cells, suggesting the altered pattern of methylation was not uniform across the genome. In summary, these data suggest that as ES cells (re)enter into the 2C state, their chromatin and DNA is altered to favour transcription in a way that mirrors the 2C embryo. [0130] Applicants previously demonstrated that MuERV-L and 2C-specific genes were increased in mutant ES cells lacking the histone lysine-specific demethylase gene Kdm1a (also known as LSD1)29. To test whether other proviral co-repressors and histone-modifying enzymes also influence 2C-specific gene expression Applicants profiled the transcriptome of ES cells with homozygous mutations in the KRAB (Kruppelassociated box)-associated

transcriptional repressor Kap1 and the H3K9 histone methyltranferase G9a^29–31. Applicants found that MuERV-L and several 2C genes were significantly upregulated in Kdm1a, Kap1 and G9a mutant ES cells (Table 5). These findings were confirmed using in situ hybridization and immunofluorescence microscopy. Treatment of 2C::tdTomato ES lines with the histone deacetylase inhibitor trichostatin A also increased the number of tdTomato⁺ cells fourfold (Fig. 3d). To understand better how 2C gene regulation is controlled when chromatin repressors are acutely eliminated Applicants used a stably integrated 2C::tdTomato ES line that is homozygous for a floxed allele of Kdm1a and contains a Cre-ERT transgene that can be activated with 4HT. Within 24 h of deleting Kdm1a Applicants found a tenfold increase in tdTomato⁺ cells thatwas steadily maintained (Fig. 3e). In addition, fluorescence-activated cell sorting (FACS)-purified tdTomato^– cells more rapidly became tdTomato⁺ in the absence of Kdm1a (Fig. 3f), and stayed in this state longer. These findings suggest that Kdm1a, Kap1, G9a and histone deacetylases all contribute to the repression of 2C genes in ES cells, and that they function by altering the equilibrium between the 2C::tdTomato⁺ and 2C::tdTomato^– states. [0131] 2C-like ES cells have expanded fate potential [0132] Because 2C-like cells within ES cultures express high levels of 2C- restricted genes found in totipotent blastomeres and reduced levels of pluripotency-associated proteins,

Applicants reasoned that this subpopulation of ES cells might have distinct functional characteristics. Applicants tested whether 2C::tdTomato⁺ cells have acquired the ability to produce extraembryonic tissues, a characteristic that ES cells lack. Applicants used FACS to collect tdTomato⁺ and tdTomato- cells from a 2C::tdTomato ES line, and injected four cells into morula-stage embryos. The tdTomato- cells contributed exclusively to the ICM of all five chimaeric blastocysts analysed (Fig. 4a). By contrast, the tdTomato⁺ cells contributed to the trophectoderm (in four out of five chimaeric embryos) in addition to the ICM (in three out of five chimaeric embryos) (Fig. 4a). To track the fate of the 2C::tdTomato ES cells later in development, Applicants injected blastocysts with tdTomato⁺ or tdTomato- cells that were pre- infected with a lentivirus encoding green fluorescent protein (GFP) from a constitutively active Ef1a promoter (Ef1a::GFP). tdTomato- GFP⁺ cells contributed exclusively to embryonic tissues, whereas tdTomato⁺GFP⁺ cells contributed to embryonic endoderm, ectoderm, mesoderm, the germ lineage as well as the yolk sac and placenta (Fig. 4b, c). The extraembryonic contribution of the tdTomato⁺GFP⁺ cells included giant trophoblast cells of the placenta (Fig. 4c). Thus, the developmental potential of 2C::tdTomato⁺ cells includes embryonic plus extraembryonic tissues in contrast to most ES cells in culture, which are restricted to generating only embryonic cell types. [0133] Applicants next examined whether Kdm1a mutant ES lines, which contain higher frequencies of 2C::tdTomato⁺ cells, also had increased potency in mouse chimaera assays. As expected, Kdm1a heterozygous ES cells contributed exclusively to embryonic tissues (in five out of five chimaeric embryos) but never to extraembryonic tissues (Fig. 4d). By contrast, Kdm1a homozygous mutant ES cells generated both embryonic (in four out of six chimaeric embryos) and extraembryonic (in five out of six chimaeric embryos) tissues (Fig. 4d). To confirm the increased potential of Kdm1a mutant ES cells, Applicants used a competition chimaera assay. Applicants co-injected a 1:1 mixture of control loxP-flanked (floxed) Kdm1a^fl/fl and homozygous Kdm1a knockout ES cells into five wild-type blastocysts. PCR was then used to detect the appearance of Kdm1a^fl/fl or knockout cells in dissected tissues. Applicants detected Kdm1a^fl/fl ES cells in the embryonic tissues and amnion, but not the yolk sac or placenta (Fig. 4e). By contrast, Kdm1a mutant ES cells contributed to embryonic tissues, the amnion, yolk sac and placental tissues, including giant trophoblast cells and primordial germ cells (Fig. 4e, f). Thus, the artificial activation of 2C genes achieved by removing Kdm1a is associated with expanded fate potential. [0134] Applicants have shown that 2C::tdTomato⁺ cells within ES cultures have increased potency, but it is unclear whether entrance into this state is essential for their long-term pluripotency. To test this possibility, Applicants performed serial depletion of 2C-like ES cells by genetic ablation with diptheria toxin (DTA). Applicants generated ES lines by crossing 2C::ERT2-Cre-ERT2 mice with a Cre-responsive DTA allele (ROSA::LSL-DTA) and treated the ES line with 4HT for 20 passages. Applicants found that these 2C-depleted ES cultures were still capable of generating high contribution chimaeras, although their differentiation was biased towards mesoderm and ectoderm lineages in vitro. These data suggest that occasional entry into the 2C-like state might help to preserve the broad embryonic fate potential of ES cells. [0135] In mammalian development, the zygote and its daughter cells progress from totipotent cells capable of generating an entire mouse to more lineage-restricted inner and outer cells of the morula capable of generating embryonic or extraembryonic lineages, respectively. A key transcriptional feature of the totipotent cells is the onset of zygote genome activation in which the embryo switches from a maternal to a zygotic transcriptome. To mark cells at this early stage of embryonic development, Applicants generated a reporter with the regulatory elements from the endogenous retrovirus MuERV-L, which is highly restricted to the zygote/2C stage.

Surprisingly, Applicants found that rare ES and iPS cells expressed the reporter. When

Applicants characterized these cells, Applicants found that they lacked expression of the pluripotency proteins Oct4, Sox2 and Nanog. Instead, these rare cells expressed a large number of genes restricted to the 2C stage, and most importantly, were capable of forming both embryonic and extraembryonic lineages (Fig. 5a, b). Applicants’ studies identify a rare 2C-like cell in ES cultures that has expanded fate potential. [0136] Although it is unclear how MuERV-L and other 2C genes regulate potency, several lines of evidence indicate that 2C-like cells are required for the health and maintenance of ES cultures. First, Applicants found that nearly all cells enter into the 2C-like state over increasing passage. Second, when Applicants depleted 2C-like ES cells from cell cultures Applicants found that their differentiation characteristics were altered to generate more ectoderm and mesoderm derivatives. Third, functional studies of the Zscan4 gene, found adjacent to a full-length MuERV-L element and highly enriched within 2C::tdTomato⁺ cells, have shown that it is required for the maintenance of telomeres within ES cultures¹⁴. Another important question that remains is whether the selection of these special ES cells can be used for practical purposes, such as reprogramming somatic nuclei. This idea is supported by the finding that 2C genes are not properly activated in cloned embryos, and that reprogramming efficiency is enhanced by inhibition of histone deacetylases and Kdm1a, which repress the 2C state^32-34. Thus, overexpression of one or several MuERV-L-linked 2C genes or inhibition of other 2C gene repressors may be useful strategies to facilitate reprogramming. This possibility is supported by the recent finding that forced Zscan4 expression in fibroblasts enhances their iPS cell reprogramming efficiency³⁵. [0137] Transposable elements are a major driving force of evolution. Applicants’ findings support the notion that the co-option of retrotransposable elements by cellular genes can act as an evolutionary mechanism for coordinately linking the expression of many genes^15,29,36.

Transposon sequences have recently been shown to have a crucial role in rewiring gene regulatory networks in ES cells and in the endometrium that contributed to the evolution of pregnancy in mammals^37,38. It has also been speculated that ERVs were involved in the evolution of the placenta by providing fusogenic envelope genes adapted for formation of the syncytiotrophoblasts³⁹. Applicants suggest that endogenous retroviruses, which are found in all placental mammals⁴⁰, may have had an equally important gene regulatory role in early mammalian development, by contributing to the specification of cell types and leading to the formation of placental tissues. [0138] Material and Methods [0139] 2C::tdTomato was created by digesting the MuERV-L-LTR-Gag clone 9 (ref. 29) with MluI and HindIII, resulting in MuERV-L-LTR 1-730, and was ligated into pcDNA3 hygro tdTomato with the cytomegalovirus (CMV) promoter removed. To generate 2C::tdTomato ES cells, Kdm1a^fl/fl; Cre-ERT ES cells were transfected with 2C::tdTomato using Lipofectamine 2000 (Invitrogen) and selected with 150 μgml^–1 hygromycin for 7 days. Colonies containing tdTomato⁺ cells were then picked and expanded. 2C::ERT2-Cre-ERT2 was generated by replacing tdTomato with an ERT2-Cre-ERT2 insert using EcoR1 and Not1 sites. DNA was linearized with Mlu1 and AvrII sites before injection into embryos to generate transgenic mice. The resulting mice were mated with ROSA::LSL-tdTomato mice (JAX 007905), ROSA::LSL- DTA mice (JAX 010527) or ROSA::LSL-LacZ mice (gift from D. Anderson laboratory), and ES lines were derived using standard procedures. Kdm1a^GT/GT, Kdm1a^fl/fl, Kap1 and G9a mutant ES cells were described previously^29–31. RNA-seq from oocytes and 2C embryos was performed by lysing litters of embryos (5–10 embryos) in prelude direct lysis buffer (Nugen), and amplifying RNA using the ovation RNA-seq system (Nugen) before library construction using the Tru-seq RNA sample prep kit (Illumina). Microarray, quantitative PCR with reverse transcription (qRT– PCR), immunostaining and chimaeric mouse injections were performed as described²⁹. [0140] RNA-Seq [0141] For RNA-Seq analysis of early stage embryos, three independent litters of

superovulated oocytes or naturally fertilized superovulated oocytes were collected and lysed directly in 2 ul of Prelude Direct Lysis buffer (Nugen). RNA was then subject to amplification using the Ovation RNA-Seq System (Nugen). Amplified cDNA was fragmented using Covaris, and single end (oocytes) or paired end (2C embryos) libraries were then constructed using the mRNA-Seq sample prep kit (Illumina) or Tru-Seq RNA library construction kit (Illumina) starting with end repair. Sequencing was performed on either Illumina Genome Analyzer (oocytes) or Hi-Seq (2C embryos). 72 bp single-end reads (oocytes) or 100 bp paired-end reads (2C embryos) were aligned to the mouse genome using Bowtie, allowing up to three mismatches per alignment and up to 20 alignments per read, filtering out any read aligning in more than 20 locations. To compare the Oocyte data to the 2C data, read lengths were cut down to 72 bp (from the 3’end). The Oocyte data had an average of 33 million alignments per sample while the 2C data had an average of 49 million alignments per sample. Read counts were quantified using a custom gene reference based on UCSC's Knowngene reference. At each gene locus, all isoforms belonging to a single gene were fused into one transcript containing all exons from each isoform. Counts aligning in multiple locations were counted as a fraction of their total number of alignments at each location. Differential expression testing was performed with DESeq ⁴¹. Genes with adjusted p-values less than 0.05 were marked as significant. Chimeric transcripts were identified utilizing the spliced alignment data produced by Tophat. Tophat identifies exons based on alignment pileup and it follows by aligning previously unaligned reads across potential splice junctions. Applicants split the junction information into two lists, left and right side of each junction, and compared to both the UCSC known gene database for mm9 and to the RepeatMasker database, also from UCSC's database. Only junctions that hit an exon of a known model on one end and a repeat element on the other were retained. GO analysis was performed using the David Bioinformatic Resource (on the Worl Wide Web at:

www.http://david.abcc.ncifcrf.gov/) ⁴². [0142] For RNA-Seq of 2C::tomato⁺ and^– cells, Kdm1a KO ES cells, Kap1 KO ES cells, and G9a KO ES cells, sample libraries were prepared from 500-5ug of total RNA using the mRNA- Seq sample prep kit (Illumina) or Tru-Seq RNA library construction kit. Library samples were amplified on flow cells using cluster generation kit (Illumina) and then sequenced using consecutive 36 cycle sequencing kit on the Genome Analyzer (Illumina) or 100bp paired end reads on the Hi-Seq (Illumina). Raw sequence data was then aligned to the mouse genome using the short read aligner Bowtie and the default setting (2 mismatches per 25 bp and up to 40 genomic alignments) (on the Worl Wide Web at: www.http://bowtie- bio.sourceforge.net/index.shtml). RPKM values were also determined by Bowtie. For repetitive sequences, Applicants aligned sequencing reads to the Repbase database using Bowtie (on the Worl Wide Web at: www.http://www.girinst.org/repbase/index.html). [0143] ES culture and generation of 2C::tomato and 2C::ERT2-Cre-ERT2 ES lines The derivation and culture of Kdm1a GT/GT, Fl/Fl, and Fl/Fl, Cre-ERT ES cells were described previously ²⁹. The 2C::tomato construct was created by digesting the MERVL LTR-Gag clone #9 in pGL3 basic with MluI and HindIII , resulting in MERVL LTR 1-730, and was ligated into pcDNA3 hygro tdtomato digested with MluI and HindIII (to remove CMV promoter). To generate 2C::tomato ES cells, Kdm1a Fl/Fl; Cre:ERT2 ES cells were transfected with

2C::tomato using Lipofectamine 2000 (Invitrogen) and selected with 150ug/ml hygromycin for 7 days. Colonies containing tomato positive cells were then picked and expanded. 2C::tomato ES cells were also derived from a transgenic mouse generated by pronuclear injection of the 2C::tomato ES line. 2C::ERT2-Cre-ERT2 was generated by replacing tdtomato with an ERT2- Cre-ERT2 insert using EcoR1 and Not1 sites. DNA was linearized with Mlu1 and AvrII sites before injection into embryos to generate transgenic mice. The resulting mice were mated with ROSA::LSL-tomato mice (JAX 007905), ROSA::LSL-DTA mice (JAX 010527), or

ROSA::LSL-LacZ mice (Gift of Anderson lab) and ES lines were derived using standard procedures. [0144] KAP1 ES3Cre and G9A TT2 ES cells were described previously ^30,31. To recombine and delete the Kdm1a, KAP1, and G9A floxed alleles, cells were treated with 1uM 4OHT for 24 hours. Cells were then harvested at a minimum of 48 hours later to allow for loss of residual protein. To activate the 2C::ERT2-Cre-ERT2 transgene, cells were maintained in 1uM 4OHT and fed daily. 2C::tomato positive and negative cells were counted using a FACScan and sorted using a FACSDiVA. For differentiation assays, ES cells were grown in suspension in the absence of Lif as described29. [0145] Immunofluorescence Staining and Microscopy [0146] ES cells and iPS cells were plated on gelatinized glass coverslips on PMEFs. Cells were fixed with 4% PFA for 10 minutes, followed by washing with PBS-T (0.05% tween). Cells were then blocked in PBS-T containing 3% BSA for ten minutes and stained with primary antibody for 1 hour at room temperature. Antibodies used: mouse anti KAP1 (Abcam), 1:1000; mouse anti OCT3/4, Santa Cruz sc-5279, rabbit anti MERVL-GAG, gift of Heidmann lab, 1:2000; rat anti E-Cadherin, Abcam ab11512, 1:500, rabbit anti Pan Acetylated histone H3, Upstate #06-599, 1:1000; rabbit anti Pan acetyl H4, Upstate #06-598, 1:1000; and rabbit anti H3 DiMeK4, clone AW30, Abcam, 1:1000. After washing 3 times for 10 minutes with PBS-T, cells were stained with secondary antibody (1:1000 anti mouse, rat, or rabbit IgG Alexa fluor 488, 555, or 647) for one hour at room temperature and washed again 3 times with PBS-T. Coverslips were stained with DAPI in PBS for 5 minutes before inverting onto slides in mounting medium. Cells were then imaged using either an Olympus FV1000 confocal microscope and 60X oil objective, or a Zeiss Axioskop 2 epifluorescence microscope and 40X objective. Quantification of histone stains were performed with Fluoview. Preimplantation embryos were stained as described with minor modifications. Embryos were fixed in 4% PFA for 30 minutes and permeabilized in 0.25% Triton for 20 minutes, prior to blocking in 10% FBS for 1 hour in 0.1% Triton-PBS. Primary antibodies were incubated overnight at 4 degrees C in blocking buffer. Subsequent washes and secondary antibody incubations were at room temperature in 0.1% Triton-PBS. [0147] In situ hybridization [0148] A MERVL probe was generated by PCR from mouse ES cDNA using the forward primer 5’ ccatccctgtcattgctca 3’ and reverse primer 5’ ccttttccaccccttgatt 3’ and cloned into the PCR2.1 TOPO vector. A DIG labeled probe was prepared using in vitro transcription with the T7 polymerase. ES samples were fixed in 4% PFA, digested for two minutes with proteinase K, washed with PBS, acetylated, and hybridized with denatured probe overnight at 68 degrees. After washing with 5X SSC and 0.2 X SSC, DIG labeled probe was visualized using an anti DIG antibody coupled to alkaline phosphatase. [0149] Immunoblotting [0150] Whole cell extracts were prepared by pelleting ES cells at 200 X g and resuspending in 1:5 volume of 1% NP40 lysis buffer containing 10mM Tris, 150mM NaCl, and 1X protease inhibitors. To solubilize histones, extracts were also sonicated using a bioruptor on the high setting for 10 minutes. 10-50ug of total protein in LDS sample buffer (Invitrogen) was then loaded onto a 4-12% NuPage gel (Invitrogen), electrophoresed at 200V for 60 minutes, and transferred to nitrocellulose membranes at 30V for 90 minutes. Membranes were blocked in PBS-T containing 5% nonfat dry milk. Primary antibodies were incubated overnight at 4 degrees. Antibodies utilized: rabbit anti GAPDH, Santa Cruz sc25-778, 1:1000, rabbit and MERVL-GAG, gift of Heidmann lab, 1:1000, anti Pan AcH3, Upstate #06-599, 1:1000; anti Pan AcH4, Upstate #06-598, 1:1000; anti H3 DiMeK4 clone AW30, Abcam, 1:500; anti H4, Novus ab10158, 1:1000; anti H3, Novus, NB 500-171, 1:500. After washing extensively with PBS-T, secondary antibodies (anti rabbit or mouse HRP conjugate, 1:10,000 dilution) were incubated for one hour at room temperature. After washing extensively with PBS-T and water, blots were developed using ECL plus detection system (Amersham). [0151] Bisulfite Sequencing [0152] ES cells were lysed in tail lysis buffer (0.1M Tris pH8.5, 5mM EDTA, 0.2% SDS, 0.2M NaCl,) containing proteinase K (Roche) for 1 hour at 55 degrees C, followed by treatment with DNase free RNase for 30 minutes at 37 degrees C. DNA was then sonicated briefly and purified using Qiagen PCR purification columns. Bisulfite conversion of genomic DNA was carried out using the Epitect Bisulfite Kit (Qiagen). Bisulfite converted DNA was then PCR amplified using Accuprime Taq polymerase (Invitrogen) followed by TOPO TA cloning (Invitrogen). At least 10 individual clones per primer pair were sequenced (Eton Bio). Primer sequences were described previously ²⁹. [0153] QRT-PCR [0154] For QRT-PCR Analysis, first strand cDNA was generated from up to 5ug total RNA using Superscript III (Invitrogen) and polydT or random hexamer priming. QPCR was performed using SYBR green master mix (Applied Biosystems) in 96 well dishes in triplicate and repeated at least two times. Standard curves were generated for each primer pair (described previously ²⁹) and expression levels were plotted relative to Gapdh (in arbitrary units). [0155] Microarray [0156] Total RNA was prepared from 2C::tomato positive and negative cells using RNEasy kits (Qiagen). Labeling of 100ng of total RNA was performed using the Whole Transcript (WT) Sense Target Labeling Assay kit (Affymetrix) before hybridization to Genechip Mouse Gene 1.0 ST Arrays. Probeset normalization and summarization were prepared using Robust Multichip Analysis (RMA) in Expression Console (Affymetrix). [0157] Mouse Chimera Assay [0158] ES cells were injected into either E2.5 or E3.5 C57Bl/6J embryos and cultured in vitro or implanted into pseudopregnant females. For PCR assays dissected tissues were placed in lysis buffer (1% SDS, 150mM NaCl, 10mM TriS pH8.0, 1mM EDTA pH 8.0) containing proteinase K overnight at 55 degrees C. DNA was then isolated by phenol chloroform extraction and ethanol precipitation, followed by PCR analysis with primers designed to amplify the Betageo cassette or the wild type Kdm1a foxed allele. For embryo imaging, chimeric mice were harvested between E9.5 and E12.5 and fixed with 4% PFA for two hours, washed extensively in PBS overnight, incubated in 30% sucrose for 4 hours, and frozen on dry ice in OCT. Cryosections were then taken and stained with DAPI before imaging. V. References

[0159] 1. Tarkowski, A. K. Experiments on the development of isolated blastomers of mouse eggs. Nature 184, 1286–1287 (1959).

[0160] 2. Papaioannou, V. E., Mkandawire, J. & Biggers, J. D. Development and phenotypic variability of genetically identical half mouse embryos. Development 106, 817–827 (1989).

[0161] 3. Cockburn, K. & Rossant, J. Making the blastocyst: lessons from the mouse. J. Clin. Invest. 120, 995–1003 (2010).

[0162] 4. Latham, K. E. & Schultz, R. M. Embryonic genome activation. Front. Biosci. 6, d748–d759 (2001).

[0163] 5. Schultz, R. M. The molecular foundations of the maternal to zygotic transition in the preimplantation embryo. Hum. Reprod. Update 8, 323–331 (2002).

[0164] 6. Kan?ka, J. Gene expression and chromatin structure in the pre-implantation embryo. Theriogenology 59, 3–19 (2003).

[0165] 7. Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

[0166] 8. Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981).

[0167] 9. Beddington, R. S. & Robertson, E. J. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733–737 (1989).

[0168] 10. Niakan, K. K. et al. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev. 24, 312–326 (2010).

[0169] 11. Hayashi, K., Lopes, S. M., Tang, F. & Surani, M. A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3, 391–401 (2008).

[0170] 12. Singh, A. M., Hamazaki, T., Hankowski, K. E. & Terada, N. A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25, 2534–2542 (2007).

[0171] 13. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007).

[0172] 14. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

[0173] 15. Peaston, A. E. et al. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev. Cell 7, 597–606 (2004).

[0174] 16. Evsikov, A. V. et al. Systems biology of the 2-cell mouse embryo. Cytogenet. Genome Res. 105, 240–250 (2004).

[0175] 17. Kigami, D., Minami, N., Takayama, H. & Imai, H. MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos. Biol. Reprod. 68, 651–654 (2003).

[0176] 18. Svoboda, P. et al. RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev. Biol. 269, 276–285 (2004). [0177] 19. Ribet, D. et al. Murine endogenous retrovirus MuERV-L is the progenitor of the “orphan” epsilon viruslike particles of the early mouse embryo. J. Virol. 82, 1622–1625 (2008).

[0178] 20. Soudais, C. et al. Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro. Development 121, 3877–3888 (1995).

[0179] 21. Yagi, R. et al. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 134, 3827–3836 (2007).

[0180] 22. Nishioka, N. et al. Tead4 is required for specification of trophectoderm in pre- implantation mouse embryos. Mech. Dev. 125, 270–283 (2008).

[0181] 23. Choo, K. B., Chen, H. H., Cheng, W. T., Chang, H. S. & Wang, M. In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes. Mol. Reprod. Dev. 59, 249–255 (2001).

[0182] 24. Huang, C. J., Chen, C. Y., Chen, H. H., Tsai, S. F. & Choo, K. B. TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains. Gene 324, 117–127 (2004).

[0183] 25. Zhang, W. et al. Zfp206 regulates ES cell gene expression and differentiation. Nucleic Acids Res. 34, 4780–4790 (2006).

[0184] 26. Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

[0185] 27. Ma, J., Svoboda, P., Schultz, R. M. & Stein, P. Regulation of zygotic gene activation in the preimplantation mouse embryo: global activation and repression of gene expression. Biol. Reprod. 64, 1713–1721 (2001).

[0186] 28. Wiekowski, M., Miranda, M., Nothias, J. Y. & DePamphilis, M. L. Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription. J. Cell Sci. 110, 1147–1158 (1997).

[0187] 29. Macfarlan, T. S. et al. Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A. Genes Dev. 25, 594–607 (2011).

[0188] 30. Rowe, H. M. et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, 237–240 (2010).

[0189] 31. Yokochi, T. et al. G9a selectively represses a class of late-replicating genes at the nuclear periphery. Proc. Natl Acad. Sci. USA 106, 19363–19368 (2009).

[0190] 32. Suzuki, T., Minami, N., Kono, T. & Imai, H. Zygotically activated genes are suppressed in mouse nuclear transferred embryos. Cloning Stem Cells 8, 295–304 (2006).

[0191] 33. Shao, G. B. et al. Effect of trychostatin A treatment on gene expression in cloned mouse embryos. Theriogenology 71, 1245–1252 (2009).

[0192] 34. Li, W. et al. Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2. Stem Cells 27, 2992–3000 (2009).

[0193] 35. Hirata, T. et al. Zscan4 transiently reactivates early embryonic genes during the generation of induced pluripotent stem cells. Sci. Rep. 2, 208 (2012). [0194] 36. Feschotte, C. Transposable elements and the evolution of regulatory networks. Nature Rev. Genet. 9, 397–405 (2008).

[0195] 37. Kunarso, G. et al. Transposable elements have rewired the core regulatory network of human embryonic stem cells. Nature Genet. 42, 631–634 (2010).

[0196] 38. Lynch, V. J., Leclerc, R. D., May, G. & Wagner, G. P. Transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals. Nature Genet. 43, 1154–1159 (2011).

[0197] 39. Dupressoir, A. et al. Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene. Proc. Natl Acad. Sci. USA 106, 12127–12132 (2009).

[0198] 40. Bénit, L., Lallemand, J. B., Casella, J. F., Philippe, H. & Heidmann, T. ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals. J. Virol. 73, 3301–3308 (1999).

VI. Tables

[0199] Table 1. List of genes/repeat elements differentially expressed between oocytes and two-cell stage embryos.

[0200] Table 2. Retrotransposable elment Expression 2C::tomato+/- cells.

[0201] Table 3. Genes significantly mis-expressed in MERVL::tomato + vs. - cells (RNA- Seq).

[0202] Table 4. Genes activated during ZGA (the 2C stage) that are enriched in 2C::tomato+ cells. Genes containing MERVL chimeric transcripts are indicated.

[0203] Table 5. Genes mis-expressed in overlapping datasets.

VII. Embodiments

[0204] Embodiment 1. A method of forming a totipotent stem cell, said method comprising: (i) transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell; and (ii) allowing said transfected non-totipotent cell to form a totipotent stem cell. [0205] Embodiment 2. The method of claim 1, wherein said allowing comprises culturing said transfected non-totipotent cell. [0206] Embodiment 3. The method of claim 1 or 2, wherein said allowing further comprises expressing said zygote-specific protein in said transfected non-totipotent cell. [0207] Embodiment 4. The method of any one of claims 1-3, wherein said zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein or a Zinc finger protein (ZFP) 352 protein. [0208] Embodiment 5. The method of any one of claims 1-4, wherein said nucleic acid encoding said zygote-specific protein is operably linked to a murine endogenous retrovirus-like (MuERV-L) sequence. [0209] Embodiment 6. The method of claim 5, wherein said MuERV-L sequence comprises a MuERV-L promoter sequence and a MuERV-L gag protein encoding sequence. [0210] Embodiment 7. The method of any one of claims 1-6, further comprising contacting said non-totipotent cell or said transfected non-totipotent cell with a zygote-specific gene repressor inhibitor. [0211] Embodiment 8. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a histone modification inhibitor. [0212] Embodiment 9. The method of claim 8, wherein said histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor. [0213] Embodiment 10. The method of claim 9, wherein said histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor [0214] Embodiment 11. The method of claim 10, wherein said methyltransferase inhibitor is a G9a inhibitor. [0215] Embodiment 12. The method of claim 9, wherein said histone demethylase inhibitor is a Kdm1a inhibitor. [0216] Embodiment 13. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. [0217] Embodiment 14. The method of claim 13, wherein said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor. [0218] Embodiment 15. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a small molecule. [0219] Embodiment 7. The method of claim 9, wherein said zygote-specific gene repressor inhibitor is a deacetylase inhibitor. [0220] Embodiment 17. The method of claim 16, wherein said deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. [0221] Embodiment 18. The method of claim 17, wherein said deacetylase inhibitor is trichostatin A. [0222] Embodiment 19. The method of any one of claims 1-18, wherein said non-totipotent cell is a primary cell. [0223] Embodiment 20. The method of claim 19, wherein said primary cell is a fibroblast. [0224] Embodiment 21. The method of claim 19, wherein said primary cell is a fat cell. [0225] Embodiment 22. The method of any one of claims 1-18, wherein said non-totipotent cell is a pluripotent cell. [0226] Embodiment 23. The method of claim 22, wherein said pluripotent cell is an induced pluripotent stem cell or an embryonic stem cell. [0227] Embodiment 24. A totipotent stem cell prepared according to the method of any one of claims 1-23. [0228] Embodiment 25. The totipotent stem cell of claim 24, wherein said totipotent stem cell does not comprise detectable amounts of an Oct-4 polypeptide, a Sox-2 polypeptide or a Nanog polypeptide. [0229] Embodiment 26. The totipotent stem cell of claim 24 or 25, wherein said totipotent stem cell comprises a Gag polypeptide. [0230] Embodiment 27. The totipotent stem cell of any one of claims 24-26, wherein said totipotent stem cell forms extraembryonic tissue or embryonic tissue. [0231] Embodiment 28. The totipotent stem cell of any one of claims 24-26, wherein said totipotent stem cell forms extraembryonic tissue and embryonic tissue. [0232] Embodiment 29. A non-totipotent cell comprising an exogenous nucleic acid encoding a zygote-specific protein. [0233] Embodiment 30. The non-totipotent cell of claim 29, wherein said zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein or a Zinc finger protein (ZFP) 352 protein. [0234] Embodiment 31. The non-totipotent cell of claim 29 or 30, wherein the level of expression of said zygote-specific protein in said non-totipotent cell is increased compared to a control level. [0235] Embodiment 32. The non-totipotent cell of any one of claims 29-31, further comprising a zygote-specific gene repressor inhibitor. [0236] Embodiment 33. The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a histone modification inhibitor. [0237] Embodiment 34. The non-totipotent cell of claim 33, wherein said histone

modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor. [0238] Embodiment 35. The non-totipotent cell of claim 34, wherein said histone

methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor. [0239] Embodiment 36. The non-totipotent cell of claim 35, wherein said methyltransferase inhibitor is a G9a inhibitor. [0240] Embodiment 37. The non-totipotent cell of claim 34, wherein said histone demethylase inhibitor is a Kdm1a inhibitor. [0241] Embodiment 38. The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. [0242] Embodiment 39. The non-totipotent cell of claim 38, wherein said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor. [0243] Embodiment 40. The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a small molecule. [0244] Embodiment 41. The non-totipotent cell of claim 34, wherein said histone

modification inhibitor is a deacetylase inhibitor. [0245] Embodiment 42. The non-totipotent cell of claim 41, wherein said deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. [0246] Embodiment 43. A zygote-specific reporter construct comprising a MuERV-L promoter sequence, a MuERV-L primer-binding sequence and a MuERV-L Gag protein coding sequence operably linked to a reporter sequence. [0247] Embodiment 44. The zygote-specific reporter construct of claim 43, wherein said reporter sequence encodes a fluorescent protein. [0248] Embodiment 45. An isolated totipotent stem cell, comprising a zygote-specific reporter construct of one of claims 43 or 44. [0249] Embodiment 46. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from an induced-pluripotent stem cell. [0250] Embodiment 47. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from an embryonic stem cell. [0251] Embodiment 48. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from a primary cell. [0252] Embodiment 49. The totipotent stem cell of claim 45, wherein said totipotent stem cell forms extraembryonic tissue and embryonic tissue. [0253] Embodiment 50. A method of identifying a totipotent stem cell, said method comprising: (i) transfecting a plurality of cells with the zygote-specific reporter construct of claim 43, wherein said plurality of cells comprises totipotent stem cells and non-totipotent cells; (ii) allowing said plurality of cells to divide, thereby forming a cell expressing a zygote- specific reporter phenotype; (iii) detecting said cell expressing said zygote-specific reporter phenotype, thereby identifying said totipotent stem cell. [0254] Embodiment 51. The method of claim 50, wherein said zygote-specific reporter phenotype is a decreased expression of an Oct-4 polypeptide, a Sox-2 polypeptide or a Nanog polypeptide relative to a standard control. [0255] Embodiment 52. The method of claim 50 or 51, wherein said zygote-specific reporter phenotype is expression of a Gag polypeptide. [0256] Embodiment 53. A method of isolating a totipotent stem cell, said method comprising: (i) transfecting a plurality of cells with the zygote-specific reporter construct of claim 43, wherein said plurality of cells comprises totipotent stem cells and non-totipotent cells; (ii) allowing said plurality of cells to divide thereby forming a cell expressing a zygote- specific reporter phenotype; (iii) detecting said cell expressing said zygote-specific reporter phenotype; and (iv) separating said cell expressing said zygote-specific reporter phenotype from cells not expressing said zygote-specific reporter phenotype, thereby isolating said totipotent stem cell. [0257] Embodiment 54. A method of producing a somatic cell comprising: (a) contacting a totipotent stem cell with a cellular growth factor; and (b) allowing said totipotent stem cell to divide, thereby forming said somatic cell; wherein said totipotent stem cell is prepared by a process comprising the steps of: (i) transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell; and (ii) allowing said transfected non-totipotent cell to form a totipotent stem cell. [0258] Embodiment 55. A method of treating a mammal in need of tissue repair comprising: (a) administering a totipotent stem cell to a mammal; (b) allowing said totipotent stem cell to divide and differentiate into somatic cells in said mammal, thereby providing tissue repair in said mammal; wherein said totipotent stem cell is prepared by a process comprising the steps of: (i) transfecting a non-totipotent cell with a nucleic acid encoding a zygote-specific protein, thereby forming a transfected non-totipotent cell; and (ii) allowing said transfected non-totipotent cell to form a totipotent stem cell.

Claims

WHAT IS CLAIMED IS: 1. A method of forming a totipotent stem cell, said method comprising: (i) transfecting a non-totipotent cell with a nucleic acid encoding a zygote- specific protein, thereby forming a transfected non-totipotent cell; and

(ii) allowing said transfected non-totipotent cell to form a totipotent stem cell. 2. The method of claim 1, wherein said allowing comprises culturing said transfected non-totipotent cell. 3. The method of claim 2, wherein said allowing further comprises expressing said zygote-specific protein in said transfected non-totipotent cell. 4. The method of claim 1, wherein said zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein or a Zinc finger protein (ZFP) 352 protein. 5. The method of claim 1, wherein said nucleic acid encoding said zygote- specific protein is operably linked to a murine endogenous retrovirus-like (MuERV-L) sequence. 6. The method of claim 5, wherein said MuERV-L sequence comprises a MuERV-L promoter sequence and a MuERV-L gag protein encoding sequence. 7. The method of claim 1, further comprising contacting said non-totipotent cell or said transfected non-totipotent cell with a zygote-specific gene repressor inhibitor. 8. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a histone modification inhibitor. 9. The method of claim 8, wherein said histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor. 10. The method of claim 9, wherein said histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor.

1 11. The method of claim 10, wherein said methyltransferase inhibitor is a G9a inhibitor. 12. The method of claim 9, wherein said histone demethylase inhibitor is a Kdm1a inhibitor. 13. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. 14. The method of claim 13, wherein said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor. 15. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a small molecule. 16. The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a deacetylase inhibitor. 17. The method of claim 16, wherein said deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. 18. The method of claim 17, wherein said deacetylase inhibitor is trichostatin A. 19. The method of claim 1, wherein said non-totipotent cell is a primary cell. 20. The method of claim 19, wherein said primary cell is a fibroblast. 21. The method of claim 19, wherein said primary cell is a fat cell. 22. The method of claim 1, wherein said non-totipotent cell is a pluripotent cell. 23. The method of claim 22, wherein said pluripotent cell is an induced pluripotent stem cell or an embryonic stem cell. 24. A totipotent stem cell prepared according to the method of any one of claims 1-23.

1 25. The totipotent stem cell of claim 24, wherein said totipotent stem cell does not comprise detectable amounts of an Oct-4 polypeptide, a Sox-2 polypeptide or a Nanog polypeptide. 26. The totipotent stem cell of claim 24, wherein said totipotent stem cell comprises a Gag polypeptide. 27. The totipotent stem cell of claim 24, wherein said totipotent stem cell forms extraembryonic tissue or embryonic tissue. 28. The totipotent stem cell of claim 24, wherein said totipotent stem cell forms extraembryonic tissue and embryonic tissue. 29. A non-totipotent cell comprising an exogenous nucleic acid encoding a zygote-specific protein. 30. The non-totipotent cell of claim 29, wherein said zygote-specific protein is a zinc finger and SCAN domain containing (ZSCAN) 4 protein, a eukaryotic translation initiation factor (EIF) 1A protein, a THO complex subunit (THOC) 4 protein, a TD and POZ domain containing (TDPOZ) 1 protein or a Zinc finger protein (ZFP) 352 protein. 31. The non-totipotent cell of claim 30, wherein the level of expression of said zygote-specific protein in said non-totipotent cell is increased compared to a control level. 32. The non-totipotent cell of claim 29, further comprising a zygote-specific gene repressor inhibitor. 33. The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a histone modification inhibitor. 34. The non-totipotent cell of claim 33, wherein said histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor. 35. The non-totipotent cell of claim 34, wherein said histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor.

1 36. The non-totipotent cell of claim 35, wherein said methyltransferase inhibitor is a G9a inhibitor. 37. The non-totipotent cell of claim 34, wherein said histone demethylase inhibitor is a Kdm1a inhibitor. 38. The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor. 39. The non-totipotent cell of claim 38, wherein said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor. 40. The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a small molecule. 41. The non-totipotent cell of claim 34, wherein said histone modification inhibitor is a deacetylase inhibitor. 42. The non-totipotent cell of claim 41, wherein said deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid. 43. A zygote-specific reporter construct comprising a MuERV-L promoter sequence, a MuERV-L primer-binding sequence and a MuERV-L Gag protein coding sequence operably linked to a reporter sequence. 44. The zygote-specific reporter construct of claim 43, wherein said reporter sequence encodes a fluorescent protein. 45. An isolated totipotent stem cell, comprising a zygote-specific reporter construct of one of claims 43 or 44. 46. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from an induced-pluripotent stem cell. 47. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from an embryonic stem cell.

1 48. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from a primary cell. 49. The totipotent stem cell of claim 45, wherein said totipotent stem cell forms extraembryonic tissue and embryonic tissue. 50. A method of identifying a totipotent stem cell, said method comprising: (i) transfecting a plurality of cells with the zygote-specific reporter construct of claim 43, wherein said plurality of cells comprises totipotent stem cells and non-totipotent cells;

(ii) allowing said plurality of cells to divide, thereby forming a cell expressing a zygote-specific reporter phenotype;

(iii) detecting said cell expressing said zygote-specific reporter phenotype, thereby identifying said totipotent stem cell. 51. The method of claim 50, wherein said zygote-specific reporter phenotype is a decreased expression of an Oct-4 polypeptide, a Sox-2 polypeptide or a Nanog polypeptide relative to a standard control. 52. The method of claim 50, wherein said zygote-specific reporter phenotype is expression of a Gag polypeptide. 53. A method of isolating a totipotent stem cell, said method comprising: (i) transfecting a plurality of cells with the zygote-specific reporter construct of claim 43, wherein said plurality of cells comprises totipotent stem cells and non-totipotent cells;

(ii) allowing said plurality of cells to divide thereby forming a cell expressing a zygote-specific reporter phenotype;

(iii) detecting said cell expressing said zygote-specific reporter phenotype; and (iv) separating said cell expressing said zygote-specific reporter phenotype from cells not expressing said zygote-specific reporter phenotype, thereby isolating said totipotent stem cell. 54. A method of producing a somatic cell comprising:

(a) contacting a totipotent stem cell with a cellular growth factor; and 3 (b) allowing said totipotent stem cell to divide, thereby forming said somatic 4 cell;

wherein said totipotent stem cell is prepared by a process comprising the steps of: (i) transfecting a non-totipotent cell with a nucleic acid encoding a zygote- specific protein, thereby forming a transfected non-totipotent cell; and

(ii) allowing said transfected non-totipotent cell to form a totipotent stem cell. 55. A method of treating a mammal in need of tissue repair comprising: (a) administering a totipotent stem cell to a mammal;

(b) allowing said totipotent stem cell to divide and differentiate into somatic cells in said mammal, thereby providing tissue repair in said mammal;

wherein said totipotent stem cell is prepared by a process comprising the steps of: (i) transfecting a non-totipotent cell with a nucleic acid encoding a zygote- specific protein, thereby forming a transfected non-totipotent cell; and

(ii) allowing said transfected non-totipotent cell to form a totipotent stem cell.