WO2013181641A1 - Cellules souches totipotentes - Google Patents

Cellules souches totipotentes Download PDF

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WO2013181641A1
WO2013181641A1 PCT/US2013/043793 US2013043793W WO2013181641A1 WO 2013181641 A1 WO2013181641 A1 WO 2013181641A1 US 2013043793 W US2013043793 W US 2013043793W WO 2013181641 A1 WO2013181641 A1 WO 2013181641A1
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cell
totipotent
cells
inhibitor
zygote
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PCT/US2013/043793
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Samuel L. PFAFF
Todd S. MACFARLAN
Wesley D. GIFFORD
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Salk Institute For Biological Studies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • 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 placenta 3 .
  • a method of forming a totipotent stem cell 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.
  • a method of forming a totipotent stem cell 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.
  • a totipotent stem cell prepared according to the methods provided herein including embodiments thereof is provided.
  • a non-totipotent cell including an exogenous nucleic acid encoding a zygote-specific protein is provided.
  • 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.
  • an isolated totipotent stem cell including a zygote-specific reporter construct according to the embodiments provided herein is provided.
  • a method of identifying a totipotent stem cell 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m.
  • 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 ⁇ m.
  • Figure 1g Figure 1g,
  • 2C::tdTomato + ES cells express MuERV-L-Gag protein, as detected by immunofluorescence.
  • DAPI 4′,6-diamidino-2-phenylindole. Scale bars, 50 ⁇ m.
  • 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 ⁇ m.
  • 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- ⁇ -galactosidase antibodies and counterstained with DAPI. Scale bars, 50 ⁇ m.
  • Figure 2c, 2C::tdTomato + and 2C::tdTomato – cells were collected by FACS and plated before imaging 48 h later. Scale bars, 50 ⁇ m.
  • Figure 2d, 2C::tdTomato ES cells were cultured in 20% O 2 (normoxia) or 5% O 2 (hypoxia) for 48 h, and the percentage of tdTomato + cells was determined by FACS.
  • 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.
  • FIG. 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 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 ⁇ m.
  • FIG. 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.
  • FIG. 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 ⁇ M (endoderm, mesoderm, ectoderm and yolk sac) and 50 ⁇ m (placenta and PGCs).
  • 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 ⁇ M (endoderm, mesoderm, ectoderm and yolk sac) and 50 ⁇ m (placenta and PGCs).
  • Figure 5 Model of the role of the MuERV-L-LTR-linked 2C gene network in regulating embryonic potency.
  • FIG. 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.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • 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.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl
  • ribonucleotides peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • 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.
  • 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.
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • a particular nucleic acid sequence also implicitly encompasses“splice variants.”
  • 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.
  • 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.
  • nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • 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.
  • "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.
  • 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.
  • the preferred algorithms can account for gaps and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • stringent conditions are selected to be about 5-10 o C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • 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.
  • 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 o C, or, 5x SSC, 1% SDS, incubating at 65 o C, with wash in 0.2x SSC, and 0.1% SDS at 65 o C.
  • 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.
  • 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 o C, and a wash in 1X SSC at 45 o C.
  • a positive hybridization is at least twice background.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ -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 ⁇ 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art.
  • any useful viral vector may be used in the methods described herein.
  • viral vectors examples include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors.
  • the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art.
  • the terms ⁇ transfection ⁇ or ⁇ transduction ⁇ 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.
  • 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).
  • 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.
  • transfected gene 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.
  • 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.
  • 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.
  • the term“exogenous” refers to a molecule or substance (e.g., nucleic acid or protein) that originates from outside a given cell or organism.
  • the term“endogenous” refers to a molecule or substance that is native to, or originates within, a given cell or organism.
  • 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.
  • 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.
  • 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.
  • 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 can include the components for nutritional, chemical, and structural support necessary for culturing a cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • contacting may involve a transfected non-totipotent cell as described herein and a zygote-specific gene repressor inhibitor.
  • contacting may involve a non-totipotent cell as described herein and a zygote- specific gene repressor inhibitor.
  • 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.
  • 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.
  • fibroblasts including mouse embryonic fibroblasts (MEF), keratinocytes, melanocytes, myoblasts, mesenchymal cells endothelial cells, epithelial cells, fat cells and stromal cells.
  • MEF mouse embryonic fibroblasts
  • keratinocytes melanocytes
  • myoblasts mesenchymal cells endothelial cells
  • epithelial cells fat cells and stromal cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • pluripotent 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.
  • 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.
  • 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.
  • totipotent stem cells can differentiate into endodermal cells, mesodermal cells and ectodermal cells as well as into primitive endodermal cells and trophectodermal cells.
  • 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.
  • 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.
  • zygote 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.
  • 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.
  • 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.
  • a secondary cell is derived from a primary cell and has been immortalized for long-lived in vitro cell culture.
  • 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.
  • telomere-specific gene 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.
  • 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.
  • a zygote-specific gene is controlled by a MuERVL promoter sequence.
  • 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.
  • a non-totipotent 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.
  • the zygote specific protein is not expressed by a non-totipotent cell.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • the ZSCAN4 protein is the protein as identified by the NCBI reference gi:119592929 or a variant having substantial identity thereto.
  • 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.
  • 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.
  • the EIF1A protein is the protein as identified by the NCBI reference gi:4503499 or a variant having substantial identity thereto.
  • 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.
  • THOC4 protein 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 THOC
  • the THOC4 protein is the protein as identified by the NCBI reference gi:48429165 or a variant having substantial identity thereto.
  • 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).
  • 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.
  • TDPOZ1 protein is the protein as identified by the NCBI reference gi:33333718 or a variant having substantial identity thereto.
  • 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).
  • 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.
  • the TDPOZ4 protein is the protein as identified by the NCBI reference gi: 34766460 or a variant having substantial identity thereto.
  • 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.
  • 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.
  • the ZFP 352 protein is the protein as identified by the NCBI reference gi:33333718 or a variant having substantial identity thereto.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • OCT4 protein 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • the SOX2 protein is the protein as identified by the NCBI reference gi:28195386 or a variant having substantial identity thereto.
  • 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.
  • Nanog protein is the protein as identified by the NCBI reference gi:47716683 or a variant having substantial identity thereto.
  • murine endogenous retrovirus-like sequence refers 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.
  • 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.
  • LTR long terminal repeat
  • 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.
  • LTR long terminal repeat
  • 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.
  • 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.
  • inhibition 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.
  • 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.
  • 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.
  • a "transcriptional repressor” as used herein refers to any protein capable of interfering with the transcription of a gene.
  • the transcriptional repressor may bind to specific regulatory sites of a gene sequence (e.g., promoter) to prevent expression of said gene.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the terms“therapy,”“treatment,” and“amelioration” refer to any reduction in the severity of symptoms, e.g., of a neurodegenerative disorder or neuronal injury.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • iPS induced pluripotent stem
  • 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.
  • 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.
  • totipotent stem cells formed using the methods provided herein as well as non-totipotent cells capable of forming totipotent stem cells.
  • a method of forming a totipotent stem cell 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.
  • 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.
  • 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.
  • the culture includes feeder cells (e.g., fibroblasts), while in others, the culture is devoid of feeder cells.
  • feeder cells e.g., fibroblasts
  • 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.
  • E.coli bacteria
  • 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.
  • 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.
  • toxins include, but are not limited to, hygromycin, neomycin, puromycin and gentamycin.
  • the toxin is hygromycin.
  • a toxin may be converted to a non-toxin which no longer inhibits expansion and causes cell death of a transfected neural stem cell.
  • a cell lacking a selection marker may be eliminated and thereby precluded from expansion.
  • 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.
  • the zygote-specific protein expression is controlled by an endogenous retroviral sequence.
  • the endogenous retroviral sequence is a murine endogenous retrovirus-like (MuERV-L) sequence.
  • the endogenous retroviral sequence is a human endogenous retrovirus-like (hERV-L) sequence.
  • the nucleic acid encoding the zygote-specific protein is operably linked to a murine endogenous retrovirus-like (MuERV-L) sequence.
  • the MuERV-L sequence includes a MuERV-L promoter sequence and a MuERV-L gag protein encoding sequence.
  • the nucleic acid encoding the zygote-specific protein is operably linked to a human endogenous retrovirus-like (hERV-L) sequence.
  • the hERV-L sequence includes a hERV-L promoter sequence and a hERV-L gag protein encoding sequence.
  • 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.
  • a non-totipotent 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.
  • 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.
  • the zygote-specific protein is encoded by a gene as set forth by Tables 4 or 5.
  • 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.
  • the zygote-specific gene repressor inhibitor is a short hairpin RNA.
  • the zygote-specific gene repressor inhibitor is a small interfering RNA.
  • the zygote-specific gene repressor inhibitor may be a protein.
  • 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.
  • the histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor.
  • the histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor.
  • the methyltransferase inhibitor is a G9a inhibitor.
  • the histone demethylase inhibitor is a Kdm1a inhibitor.
  • the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor.
  • the transcriptional repressor inhibitor is a Krueppel- associated protein (Kap) 1 inhibitor.
  • the zygote-specific gene repressor inhibitor is a small molecule.
  • the zygote-specific gene repressor inhibitor is a deacetylase inhibitor.
  • the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid.
  • 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.
  • 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.
  • the non-totipotent cell is a primary cell.
  • the primary cell is a fibroblast.
  • the primary cell is a fat cell.
  • the non-totipotent cell is a pluripotent cell.
  • the pluripotent cell is an induced pluripotent stem cell or an embryonic stem cell.
  • the pluripotent cell is an induced pluripotent stem cell.
  • the pluripotent cell is an embryonic stem cell.
  • a method of forming a totipotent stem cell 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.
  • any zygote-specific gene repressor inhibitor described herein may be used (see above).
  • the zygote-specific gene repressor inhibitor is a histone modification inhibitor.
  • 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
  • compositions provided herein are useful for reprogramming somatic nuclei and tissue regeneration.
  • a totipotent stem cell prepared according to the methods provided herein including embodiments thereof is provided.
  • 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.
  • 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.
  • the totipotent stem cell includes a Gag polypeptide.
  • the totipotent stem cell includes a functional fragment of a Gag polypeptide.
  • the totipotent stem cell forms extraembryonic tissue or embryonic tissue. In other embodiments, the totipotent stem cell forms extraembryonic tissue and embryonic tissue.
  • a non-totipotent cell including an exogenous nucleic acid encoding a zygote-specific protein is provided.
  • 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.
  • ZSCAN zinc finger and SCAN domain containing
  • EIF eukaryotic translation initiation factor
  • THOC THO complex subunit
  • TDPOZ Zinc finger protein
  • 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.
  • the zygote-specific protein is a protein encoded by a gene set forth by Tables 4 or 5.
  • 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.
  • the non-totipotent cell further includes a zygote-specific gene repressor inhibitor.
  • the zygote-specific gene repressor inhibitor is a histone modification inhibitor.
  • the histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor.
  • the histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor.
  • the methyltransferase inhibitor is a G9a inhibitor.
  • the histone demethylase inhibitor is a Kdm1a inhibitor.
  • the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor.
  • the transcriptional repressor inhibitor is a Krüppel- associated protein (Kap) 1 inhibitor.
  • the zygote-specific gene repressor inhibitor is a small molecule.
  • the histone modification inhibitor is a deacetylase inhibitor.
  • the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid.
  • 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.
  • the zygote-specific gene repressor inhibitor is a histone modification inhibitor.
  • the zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor.
  • the zygote-specific gene repressor inhibitor is a small molecule.
  • the deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid.
  • the deacetylase inhibitor is trichostatin A.
  • 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).
  • 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.
  • the reporter sequence encodes a fluorescent protein.
  • 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.
  • LTR long terminal repeat
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • the fluorescent protein is a red fluorescent protein. In other embodiments, the fluorescent protein is a green fluorescent protein.
  • 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.
  • the MuERV-L promoter sequence is operable linked to a MuERV-L primer- binding sequence, thereby forming a MuERV-L regulator sequence.
  • 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.
  • the MuERV-L sequence is operably linked to a reporter sequence.
  • 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.
  • 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
  • an isolated totipotent stem cell including a zygote-specific reporter construct according to the embodiments provided herein is provided.
  • the totipotent stem cell is derived from an induced-pluripotent stem cell.
  • the totipotent stem cell is derived from an embryonic stem cell.
  • the totipotent stem cell is derived from a primary cell.
  • the totipotent stem cell forms extraembryonic tissue and embryonic tissue.
  • a method of identifying a totipotent stem cell 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.
  • 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.
  • the zygote-specific reporter phenotype is expression of a Gag polypeptide.
  • 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).
  • 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.
  • a method of producing a somatic cell includes contacting a totipotent stem cell with a cellular growth factor and allowing the totipotent stem cell to divide, thereby forming the somatic cell.
  • 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.
  • 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.
  • 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
  • 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 tissues 9 .
  • the rare contribution of ES cells to extraembryonic tissues could be explained by contamination of ES cultures with
  • 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 .
  • Zscan4, stella also known as Dppa3
  • Nanog Nanog
  • Sox17 Gata6
  • 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 .
  • ERPs endogenous retroviruses
  • LINE-1 long interspersed nuclear element-1
  • SINEs non-autonomous short interspersed elements
  • MuERV-L murine endogenous retrovirus-like elements
  • MERVL and Erv4 murine endogenous retrovirus-like elements
  • 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
  • RNA-seq deep RNA sequencing
  • MuERV-L-LTR promoters 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 .
  • Gata4 and Tead4 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 .
  • Gag4 and Tead4 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 .
  • 2C embryos express Gag but lack the pluripotency marker Oct4
  • blastula cells lack Gag but express Oct4 (Fig. 1d, e).
  • 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.
  • 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).
  • tdTomato red fluorescent protein tandem dimeric Tomato
  • tdTomato expression was highest in arrested zygotes and 2C embryos and became downregulated at the morula stage (Fig. 1f).
  • 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.
  • 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.
  • 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
  • tdTomato + cells had 165 transcripts activated more than fourfold, and no genes repressed more than fourfold compared with tdTomato – cells (Fig. 1h).
  • 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).
  • 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.
  • ES cells cycle in and out of the 2C state
  • ES cell lines were derived from double-positive transgenic blastocysts.
  • 4-hydroxytamoxifen (4HT) to the ES cultures Applicants detected nuclear Cre expression inMuERV-L-Gag + cells.
  • 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% O 2 ), the percentage of cells expressing the 2C::tdTomato reporter was decreased, which could be reversed by shifting the cultures back to 20%O 2 (Fig. 2d).
  • 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 embryos 28 .
  • Fig. 3a This type of chromatin mirrors that found in 2C embryos 28 .
  • the MuERV-L sequences were hypomethylated in tdTomato + cells compared with tdTomato – cells.
  • 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).
  • 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).
  • GFP green fluorescent protein
  • 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).
  • 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.
  • Kdm1a mutant ES lines which contain higher frequencies of 2C::tdTomato + cells, also had increased potency in mouse chimaera assays.
  • Kdm1a heterozygous ES cells contributed exclusively to embryonic tissues (in five out of five chimaeric embryos) but never to extraembryonic tissues (Fig. 4d).
  • 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).
  • Kdm1a fl/fl 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).
  • 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.
  • 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 syncytiotrophoblasts 39 . Applicants suggest that endogenous retroviruses, which are found in all placental mammals 40 , 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.
  • 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.
  • CMV cytomegalovirus
  • Kdm1a fl/fl ; Cre-ERT ES cells were transfected with 2C::tdTomato using Lipofectamine 2000 (Invitrogen) and selected with 150 ⁇ gml –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.
  • 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 described 29 . [0140] RNA-Seq [0141] For RNA-Seq analysis of early stage embryos, three independent litters of
  • RNA-Seq sample prep kit Illumina
  • Tru-Seq RNA library construction kit Illumina
  • 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:
  • 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.
  • Applicants aligned sequencing reads to the Repbase database using Bowtie (on the Worl Wide Web at: www.http://www.girinst.org/repbase/index.html).
  • 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).
  • 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
  • KAP1 ES3Cre and G9A TT2 ES cells were described previously 30,31 .
  • ES cells were grown in suspension in the absence of Lif as described29.
  • 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.
  • 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.
  • In situ hybridization 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • RMA Robust Multichip Analysis
  • ES cells were injected into either E2.5 or E3.5 C57Bl/6J embryos and cultured in vitro or implanted into pseudopregnant females.
  • 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.
  • 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
  • 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).
  • 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).
  • Table 1 List of genes/repeat elements differentially expressed between oocytes and two-cell stage embryos.
  • 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.
  • Embodiment 2. The method of claim 1, wherein said allowing comprises culturing said transfected non-totipotent cell.
  • 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.
  • 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.
  • 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.
  • said histone modification inhibitor is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase inhibitor.
  • said histone methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor
  • Embodiment 11 The method of claim 10, wherein said methyltransferase inhibitor is a G9a inhibitor.
  • Embodiment 12 The method of claim 9, wherein said histone demethylase inhibitor is a Kdm1a inhibitor.
  • Embodiment 13 Embodiment 10.
  • said zygote-specific gene repressor inhibitor is a transcriptional repressor inhibitor.
  • said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor.
  • Embodiment 15 The method of claim 7, wherein said zygote-specific gene repressor inhibitor is a small molecule.
  • Embodiment 7. The method of claim 9, wherein said zygote-specific gene repressor inhibitor is a deacetylase inhibitor.
  • said deacetylase inhibitor is a hydroxymate, a depsipeptide, a benzamide, a phenylbutyrate, trichostatin A or a valproic acid.
  • Embodiment 18 The method of claim 17, wherein said deacetylase inhibitor is trichostatin A.
  • Embodiment 19 The method of any one of claims 1-18, wherein said non-totipotent cell is a primary cell.
  • Embodiment 20 The method of claim 19, wherein said primary cell is a fibroblast.
  • Embodiment 21 The method of claim 19, wherein said primary cell is a fat cell.
  • Embodiment 22 Embodiment 22.
  • Embodiment 23 The method of claim 22, wherein said pluripotent cell is an induced pluripotent stem cell or an embryonic stem cell.
  • Embodiment 24 A totipotent stem cell prepared according to the method of any one of claims 1-23.
  • 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.
  • Embodiment 26 Embodiment 26.
  • Embodiment 27 The totipotent stem cell of any one of claims 24-26, wherein said totipotent stem cell forms extraembryonic tissue or embryonic tissue.
  • Embodiment 28 The totipotent stem cell of any one of claims 24-26, wherein said totipotent stem cell forms extraembryonic tissue and embryonic tissue.
  • Embodiment 29 A non-totipotent cell comprising an exogenous nucleic acid encoding a zygote-specific protein.
  • Embodiment 30 A non-totipotent cell comprising an exogenous nucleic acid encoding a zygote-specific protein.
  • ZSCAN zinc finger and SCAN domain containing
  • EIF eukaryotic translation initiation factor
  • THOC THO complex subunit
  • TDPOZ Zinc finger protein
  • Embodiment 33 The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a histone modification inhibitor.
  • Embodiment 34 The non-totipotent cell of claim 33, wherein said histone
  • Embodiment 35 The non-totipotent cell of claim 34, wherein said histone
  • methyltransferase inhibitor is a histone 3 lysine 9 (H3K9) methyltransferase inhibitor.
  • Embodiment 36 The non-totipotent cell of claim 35, wherein said methyltransferase inhibitor is a G9a inhibitor.
  • Embodiment 37 The non-totipotent cell of claim 34, wherein said histone demethylase inhibitor is a Kdm1a inhibitor.
  • Embodiment 39 Embodiment 39.
  • the non-totipotent cell of claim 38 wherein said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor.
  • said transcriptional repressor inhibitor is a Krüppel-associated protein (Kap) 1 inhibitor.
  • Embodiment 40 The non-totipotent cell of claim 32, wherein said zygote-specific gene repressor inhibitor is a small molecule.
  • Embodiment 41 The non-totipotent cell of claim 34, wherein said histone
  • 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.
  • 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.
  • Embodiment 44 The zygote-specific reporter construct of claim 43, wherein said reporter sequence encodes a fluorescent protein.
  • Embodiment 45 An isolated totipotent stem cell, comprising a zygote-specific reporter construct of one of claims 43 or 44.
  • Embodiment 46 The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from an induced-pluripotent stem cell.
  • Embodiment 47 The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from an embryonic stem cell.
  • Embodiment 48. The totipotent stem cell of claim 45, wherein said totipotent stem cell is derived from a primary cell.
  • Embodiment 49 Embodiment 49.
  • 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.
  • Embodiment 51 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; (
  • a method of isolating a totipotent stem cell 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.
  • Embodiment 54 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.
  • 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.

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Abstract

L'invention concerne, notamment, des procédés de réalisation, d'identification, et d'isolement de cellules souches totipotentes. Les procédés et compositions de l'invention conviennent particulièrement à la reprogrammation des cellules somatiques et à la régénération de tissus. L'invention concerne également, non seulement des cellules souches totipotentes réalisées selon les procédés de l'invention, mais aussi des cellules non totipotentes capables de former des cellules souches totipotentes.
PCT/US2013/043793 2012-06-01 2013-05-31 Cellules souches totipotentes WO2013181641A1 (fr)

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WO2018172335A1 (fr) * 2017-03-20 2018-09-27 Ifom Fondazione Istituto Firc Di Oncologia Molecolare Procédé de génération de cellules souches de type 2c
EP3630952A4 (fr) * 2017-05-29 2021-04-07 Agency for Science, Technology and Research Inhibiteurs de totipotence et méthodes d'utilisation

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WO2018172335A1 (fr) * 2017-03-20 2018-09-27 Ifom Fondazione Istituto Firc Di Oncologia Molecolare Procédé de génération de cellules souches de type 2c
EP3630952A4 (fr) * 2017-05-29 2021-04-07 Agency for Science, Technology and Research Inhibiteurs de totipotence et méthodes d'utilisation

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