WO2009114949A1 - Methods for deprogramming somatic cells and uses thereof - Google Patents

Abstract

The invention is concerned with methods for remodeling cell chromatin and with methods of obtaining pluripotent, multipotent and/or unipotent cells. The methods comprise contacting somatic cells with at least one polynucleotide encoding an oocyte-deprogramming polypeptide. Expression of an oocyte-deprogramming polypeptide in the somatic cell modifyies the DNA of the somatic cell, modifies the chromatin of the somatic cell and/or remodels its chromatin. Also described are methods for deprogramming a somatic cell, and methods for conditioning somatic cells to differentiation into a pluripotent, multipotent or unipotent cell. In addition, the invention encompasses cells and cell lines obtained according to these different methods.

Description

METHODS FOR DEPROGRAMMING SOMATIC CELLS AND USES THEREOF

Related application

[0001] The present application claims priority of US application serial No. 61/038,107 filed on March 20, 2008, the content of which is incorporated herein by reference in its entirety.

Field of the Invention

[0002] The present invention relates to the field of eukaryotic cell reprogramming, and particularly to cell dedifferentiation. The invention is also concerned with methods for remodeling cell chromatin and with methods of obtaining pluripotent, multipotent and/or unipotent cells.

Background of the Invention

[0003] The nature and function of any given cell depends on the particular set of genes being expressed (e.g., transcribed and translated). Gene expression is directly mediated by sequence-specific binding of gene regulatory proteins that can effect either positive or negative regulation. However, ability of any of these regulatory proteins to directly mediate gene expression depends on the accessibility of their binding site within the cellular DNA. Since the cellular DNA generally exists in the form of chromatin, a complex comprising nucleic acid and protein, gene expression often depends on the structure of cellular chromatin within which cellular DNA is packaged. It has been shown that the structure of chromatin can be altered through the activity of macromolecular assemblies known as chromatin remodeling complexes, and it has been suggested in US patent 7,053,264 that chromatin remodeling ATPases may be used for reprogramming nuclei and facilitating dedifferentiation of cells.

[0004] Because they retain the ability to renew themselves and can differentiate into a diverse range of specialized cell types, pluripotent stem cells such as embryonic stem cells have the potential to change radically the treatment of human disease. However, there exists a widespread controversy over their use, especially human embryonic stem (ES) cells derived from cloned-embryo (i. e. therapeutic cloning) such that some scientists are now focusing their efforts in creating induced-pluripotent stem cells (iPS) which are derived from an adult somatic cell. Strong advancements have been made in recent years in efforts to control genome reprogramming and in creating induced- pluripotent stem cells. Two groups have successfully deprogrammed somatic cells into induced-pluripotent stem cells by retroviral transfection of a combination of only four factors OCT4, SOX2, KLF4, cMYC/LIN28 (Takahashi et al., 2007; Yu et al., 2007). Also, US patent publication 2009/0047263 (Yanamaka et al.) teaches nuclear reprogramming methods to derive induced pluripotent stem cells.

[0005] The oocyte is the only cell which possesses the impressive capacity to reprogram an entire genome. This laborious task is achieved through at least three key mechanisms: global transcriptional silencing, major chromatin remodeling (including centromeric silencing), and rapid cell cycles (Schier, 2007). The unique capacity of an oocyte to deprogram a genome of a differentiated cell was demonstrated by cloning, i. e. somatic cell nuclear transfert into an enuclated oocyte. Although many different genes are known to be conserved and preferentially expressed in the oocyte (Vallee et al., 2006), no one have yet proven the possibility of using mammalian oocyte genes for controlling genome deprogrammation and for creating induced-pluripotent stem cells.

[0006] In view of the above, there is thus a need for methods for facilitating reprogrammation of somatic cells. There is also a need for methods allowing dedifferentiation of somatic cells and for methods of obtaining pluripotent cells, such as induce-pluripotent stem cells. There is also a need for remodeling chromatin of somatic cells.

Summary of the Invention

[0007] The present invention relates a method for deprogramming a somatic cell, the method comprising contacting the somatic cell with a polynucleotide encoding an oocyte- deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide is expressed in the somatic cell and causes modifications to the somatic cell chromatin and/or to the somatic cell DNA. In a related aspect, the invention relates to a method for deprogramming a somatic cell, the method comprising contacting chromatin and/or DNA of the somatic cell with an oocyte-deprogramming polypeptide, wherein the oocyte- deprogramming polypeptide causes modifications to the somatic cell chromatin and/or to the somatic cell DNA.

[0008] The present invention also relates to a method for remodeling chromatin of a somatic cell, the method comprising contacting the somatic cell with a polynucleotide encoding an oocyte-deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide is expressed in the somatic cell and remodels the cell chromatin. In a related aspect, the invention relates to a method for remodeling chromatin of a somatic cell, the method comprising contacting chromatin of the somatic cell with an oocyte- deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide remodels the cell chromatin. [0009] In preferred embodiments the polynucleotide encoding an oocyte- deprogramming polypeptide is selected among AICDA₁ STELLA, PAIP1 , PTTG1 , GMNN₁ NPM2 and combinations thereof. Preferred combinations include, but are not limited to: i) NPM2 and AICDA; ii) NPM2 and STELLA; iii) NPM2 and GMNN; iv) NPM2, AICDA and STELLA; v) NPM2, GMNN and STELLA; vi) NPM2, AICDA and GMNN; and vii) NPM2, AICDA, GMNN and STELLA.

[0010] In preferred embodiments the contacting step is carried out by introducing (e.g. by transfection) an expression vector into the somatic cell, the vector comprising a polynucleotide encoding the oocyte-deprogramming polypeptide.

[0011] According to a further aspect, the invention relates a cell positive for expression of one or more pluripotent gene markers (e.g. NANOG, OCT4, SOX2, etc.), that cell comprising an exogenous polynucleotide encoding an oocyte-deprogramming polypeptide. In some embodiments, the pluripotent gene marker is only expressed transiently while in other embodiment it is expressed permanently.

[0012] Yet, another aspect of the invention relates to a method for conditioning a somatic cell to dedifferentiation into a pluripotent, multipotent or unipotent cell. The method comprising contacting a somatic cell with a polynucleotide encoding an oocyte- deprogramming polypeptide. Expression of the oocyte-deprogramming polypeptide in the somatic cell deprograms and conditions the somatic cell to dedifferentiation into a pluripotent, multipotent or unipotent cell.

[0013] A related aspect concerns a method for obtaining a pluripotent cell having desirable morphological and functional characteristics, comprising: deprogramming a somatic cell as defined hereinbefore; and exposing the somatic cell to at least one pluripotent factor. The pluripotent factor(s) is(are) selected for inducing development of the desirable morphological and functional characteristics, and the somatic cell is exposed to this(these) factor(s) for a sufficient period of time and under suitable conditions to obtain the morphological and functional characteristics so desired.

[0014] A further aspect relates to a method for transforming a somatic cell from a first cell type to a second cell type showing morphological and functional characteristics of a different specific cell lineage. In one embodiment the method comprises deprogramming the somatic cell from a first cell type according any of the method of the invention described herein, and exposing the deprogrammed cell to one or more differentiation factor(s) and/or one or more transcription factor(s). The differentiation factor(s) and transcription factor(s) is(are) selected for inducing development of morphological and functional characteristics specific of the second cell type. The cell is exposed to the factor(s) and/or transcription factor(s) for a sufficient period of time and under suitable conditions to obtain the morphological and functional characteristics desired in the second cell type.

[0015] An advantage of the present invention is that it provides effective means for reprogramming differentiated cells in view of generating personalized pluripotent, multipotent and/or unipotent cells for tissues replacement and repair. The invention also provides effective means for facilitating the generation of autologous pluripotent stem cells, without requiring the production of an embryo.

[0016] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.

Brief Description of the Drawings

[0017] FIGURE 1 shows pictures of immunoblots confirming expression of selected proteins in cells transfected with an empty vector (CTRL, left) and in cells transfected with genes coding for selected proteins (TRF, right). The genes which were transfected are identified to the left of each blot.

[0018] FIGURES 2A and 2B are plot graphs showing RNA neosynthesis in HEK293 cells transfected with the following genes: AICDA, STELLA, NPM2, GMNN, PAIP1 , PTTG1 , H1 FOO. For each gene selected, five (5) transfection replicates were analyzed. ³H-uridine measurements were normalized to total RNA (Figure 2A) and DNA (Figure 2B) and treated as fold increase/decrease relative to CTRL=L Data were log transformed and analyzed using one-way ANOVA with Dunnett post-hoc tests, P < 0.05*, N=5.

[0019] FIGURES 3A, 3B 3C and 3D are bar graphs and pictures of immunoblots showing global patterns of modified histones in transfected cells. Fluctuations in post- translational histone modifications associated to euchromatin: (H3K4Me3 (Fig. 3A); H3K9Ac (Fig. 3B) and heterochromatin (H3K9Me3 (Fig. 3C); H4K20Me3 (Fig. 3D)) in HEK293 cells transfected with one of the selected genes (day 3). Relative band intensity bar graphs (top) and immunoblots (bottom) are shown. Relative band intensity was evaluated as the histone band intensity normalized to β-actin. Intensity ratios were treated as fold increase/decrease relative to CTRL=L Data were log transformed and analyzed using one-way ANOVA with Dunnett post-hoc tests, P < 0.05^*, N=5.

[0020] FIGURE 4 are colour pictures showing immunofluorescence for DNA methylation of selected genes in HEK293 transfected cells (Day 4). Immunofluorescence appears in red for methylated DNA (5-MeC) and in blue for DAPI. N=3.

[0021] FIGURE 5 is a bar graph and gel bands picture showing DNA methylation intensity based on the intensity observed following southwestern analysis (3 replicates) for methylated DNA in HEK293 transfected cells. Each stained 5MeC DNA smear corresponds to an intensity bar in the lower graph. Intensity was measured with Adobe Photoshop™ CS3. Data were analyzed using one-way ANOVA with Dunnett post-hoc tests, considering ratio as log values, P < 0.05^*, N=3.

[0022] FIGURES 6A, 6B and 6C are bar graphs showing pluripotency-associated transcription factor expression in HEK293 transfected cells. NANOG (Fig. 6A), OCT4 (Fig. 6B), and SOX2 (Fig. 6C) expression was assessed in HEK293 cells transfected with one of the selected genes (day 7). For each gene selected, three GFP-sorted transfection replicates were analyzed. Intensities represented in the bar graphs were obtained by quantitative PCR and they were normalized on a correction factor (geNorm™ and three housekeeping genes (CYCLO, GAPDH and β-ACTIN)) for GAPDH and β-actin intensities. Data were log transformed and analyzed using one-way ANOVA with Dunnett tests, P < 0.05^*, N=3.

Detailed Description of the Invention

[0023] As used herein and in the appended claims, the singular forms "a," "an", and "the", include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a cell" includes one or more of such cells or a cell line derived from such a cell, reference to "an oocyte-deprogramming polypeptide" includes one or more of such different polypeptides, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

Oocyte-deprogramming nucleotide sequences (e.g. genes) and polypeptides [0024] The practice of the invention employs oocyte-deprogramming nucleotide sequences (e.g. genes) and polypeptides encoded by such nucleotide sequences. As used herein, the term "oocyte-deprogramming polypeptide" refers to a polypeptide which is coded by a mammalian gene: (i) preferentially expressed (e.g. transcribed or translated) in mammalian oocytes and (ii) highly conserved in vertebrates. "Vertebrate" include, but is not limited to, mammals (e.g. bovine, human, mouse), and amphibians. As used herein, in some embodiments the term "preferentially expressed in mammalian oocytes" refers to cellular RNA levels at least 5x, 1Ox, 5Ox, 100x, 25Ox, 50Ox or higher in mammalian oocytes, when compared to the RNA levels of a somatic cell or tissue.

[0025] Methods for identifying genes highly conserved in vertebrates and preferentially or specifically transcribed or translated in mammalian oocytes are described in Vallee et al. (Reproduction, May 2008, in press). The oocyte- deprogramming nucleotide sequences and polypeptides of the invention may be selected from the oocyte-preferentially expressed genes (or so-called oocyte-specific genes) conserved through evolution which are listed herein in Table 1-A. In some embodiments, the oocyte-deprogramming nucleotide sequences and polypeptides of the invention are selected from potential factors playing a role in chromatin remodeling as described in the art listed herein in Table 1-B. In other embodiments the invention comprises the use of one or more of the following nucleotide sequences and polypeptides: AICDA, STELLA, PAIP1 , PTTG1 , GMNN, H1 FOO, and NPM2 (see Table 2). In other embodiments, the invention comprises the use of a combination of oocyte- deprogramming nucleotide and polypeptides sequences including but not limited to: i) NPM2 and AICDA; ii) NPM2 and STELLA; iii) NPM2 and GMNN; iv) NPM2, AICDA and STELLA; v) NPM2, GMNN and STELLA; vi) NPM2, AICDA and GMNN; and vii) NPM2, AICDA, GMNN and STELLA.

[0026] TABLE 1 : List of genes preferentially expressed in oocyte and conserved through evolution, and of potential factors playing a role in chromatin remodelling.

[0027] The invention encompass all of the possible combinations of 2, 3, 4, 5, 6 or more genes or oocyte-deprogramming nucleotide sequences, polypeptides, and functional fragments thereof listed in Table 1 hereinbefore or Table 2 hereinafter. [0028] In one embodiment, the combination consists of the gene NPM2 and the gene AICDA. In another embodiment, the combination consists of the gene NPM2 and the gene STELLA. In another embodiment, the combination consists of the gene NPM2 and the gene GMNN. In another embodiment, the combination consists of the genes NPM2, AICDA and the gene STELLA. In another embodiment, the combination consists of the genes NPM2, GMNN and the gene STELLA. In another embodiment, the combination consists of the genes NPM2, AICDA and the gene GMNN. In another embodiment, the combination consists of the genes NPM2, AICDA, GMNN and the gene STELLA. The Unigene database accession number of those genes is provided in Table 2 hereinafter. In some embodiments the invention comprises the use of a cDNA sequence corresponding to any one of the nucleotide sequences of SEQ ID NO: 1-10 (see Table 2) and combinations thereof. Those skilled in the art will appreciate that the invention encommasses these nucleotide sequences and any additional sequences (e.g. sequences modified by mutation, deletion, substitution, etc.) that will give rise to a functional polypeptide having an oocyte-deprogramming activity. Those skilled in the art will also appreciate that the present invention is not limited to humans and that some embodiments encompasses corresponding sequences in addtional mammalian species (e.g. bovine, mouse).

[0029] Table 2: Selected oocyte-deprogramming factors of the invention

[0030] A "gene," for the purposes of the present disclosure, includes a DNA region encoding a gene product (see hereinafter), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. [0031] "Gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. As used herein, "an oocyte-deprogramming polypeptide" is considered as the gene product of an oocyte-deprogramming polynucleotide. In preferred embodiments the oocyte- deprogramming polypeptide is only expressed transiently, but in some embodiments it is expressed permanently.

[0032] Therefore, a related aspect of the present invention concerns isolated oocyte- specific polypeptides and polynucleotides encoding such polypeptides, compositions comprising the same and uses thereof, particularly for use according to the methods of the invention. As used herein, the term "polynucleotide" refers to any DNA, RNA sequence or molecule, including nucleotide sequences encoding a complete gene. The term is intended to encompass all polynucleotides whether occurring naturally or non- naturally in a particular cell, tissue or organism. This includes DNA and fragments thereof, RNA and fragments thereof, cDNAs and fragments thereof, expressed sequence tags, artificial sequences including randomized artificial sequences.

Delivery of Polynucleotides

[0033] In certain embodiments, the invention concerns the use of polynucleotides, e.g. a polynucleotide encoding an oocyte-deprogramming polypeptide. Means for introducing polynucleotides into a cell are well known in the art. For instance a polynucleotide encoding an oocyte-deprogramming polypeptide can be cloned into intermediate vectors for transfection in eukaryotic cells for replication and/or expression. Intermediate vectors for storage or manipulation of the nucleic acid or production of protein can be prokaryotic vectors, (e.g., plasmids), shuttle vectors, insect vectors, or viral vectors for example. A polynucleotide encoding an oocyte-deprogramming polypeptide can also cloned into an expression vector, for administration to a bacterial cell, fungal cell, protozoal cell, plant cell, or animal cell, preferably a mammalian cell, more preferably a human cell. An oocyte-deprogramming polypeptide can also be encoded by a fusion nucleic acid.

[0034] To obtain expression of a cloned nucleic acid, it is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook and Russell (Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press). Bacterial expression systems are available in E. coli, Bacillus sp., and Salmonella. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available, for example, from Invitrogen (Carlsbad, Calif.) and Clontech (Palo Alto, Calif.).

[0035] The promoter used to direct expression of the nucleic acid of choice depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification. In contrast, when a dedifferentiation protein is to be used in vivo, either a constitutive or an inducible promoter is used, depending on the particular use of the protein. In addition, a weak promoter can be used, such as HSV TK or a promoter having similar activity. The promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Ga14 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system.

[0036] In addition to a promoter, an expression vector typically contains a transcription unit or expression cassette that contains additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence, and signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding, and/or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

[0037] The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the resulting oocyte- deprogramming polypeptide. Standard bacterial expression vectors include plasmids such as pBR322, pBR322-based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.

[0038] Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0039] Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High-yield expression systems are also suitable, such as baculovirus vectors in insect cells, with a dedifferentiation nucleic acid sequence under the transcriptional control of the polyhedrin promoter or any other strong baculovirus promoter.

[0040] Elements that are typically included in expression vectors also include a replicon that functions in E. coli (or in the prokaryotic host, if other than E. coli), a selective marker, e.g., a gene encoding antibiotic resistance, to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the vector to allow insertion of recombinant sequences.

[0041] Standard transfection methods can be used to produce bacterial, mammalian, yeast, insect, or other cell lines that express large quantities of dedifferentiation proteins, which can be purified, if desired, using standard techniques. Transformation of eukaryotic and prokaryotic cells is performed according to standard techniques.

[0042] Any procedure for introducing foreign nucleotide sequences into host cells can be used. These include, but are not limited to, the use of calcium phosphate transfection, DEAE-dextran-mediated transfection, polybrene, protoplast fusion, electroporation, lipid-mediated delivery (e.g., liposomes), microinjection, particle bombardment, introduction of naked DNA, plasmid vectors, viral vectors (both episomal and integrative) and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.

[0043] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding reprogramming polypeptides to cells in vitro. Preferably, nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

[0044] Methods of non-viral delivery of nucleic acids include lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are known. Nucleic acid can be delivered to cells (ex vivo administration) or to target tissues (in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art.

[0045] The use of RNA or DNA virus-based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, wherein the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery include retroviral, lentiviral, poxviral, adenoviral, adeno-associated viral, vesicular stomatitis viral and herpesviral vectors. Integration in the host genome is possible with certain viral vectors, including the retrovirus, lentivirus, and adeno- associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0046] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, allowing alteration and/or expansion of the potential target cell population. Lentiviral vectors are retroviral vector that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors have a packaging capacity of up to 6 10 kb of foreign sequence and are comprised of cis-acting long terminal repeats (LTRs). The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV)₁ simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0047] Adeno-associated virus (AAV) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures. Recombinant adeno-associated virus vectors based on the defective and nonpathogenic parvovirus adeno-associated virus type 2 (AAV-2) are a promising gene delivery system. Exemplary AAV vectors are derived from a plasmid containing the AAV 145 bp inverted terminal repeats flanking a transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system.

[0048] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials. In applications for which transient expression is preferred, adenoviral- based systems are useful. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and are capable of infecting, and hence delivering nucleic acid to, both dividing and non-dividing cells. With such vectors, high titers and levels of expression have been obtained. Adenovirus vectors can be produced in large quantities in a relatively simple system.

[0049] In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. A viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The same principles can be applied to non-viral vectors.

[0050] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

[0051] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art.

[0052] In one embodiment, hematopoietic stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ stem cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFNK and TNFσ are known (Inaba et al. (1992) J. Exp. Med. 176:1693 1702). Stem cells are isolated for transfection and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T-cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells). See Inaba et al., supra.

[0053] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids can be also administered directly to the organism for transfection of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0054] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

Delivery of Polypeptides

[0055] In most, if not all the methods described herein, an alternative possibility consists of bypassing the use of a polynucleotide and contacting the somatic cell directly with an oocyte-deprogramming polypeptide. In other embodiments, for example in certain in vitro situations, the target cells are cultured in a medium containing one or more functional oocyte-deprogramming polypeptides.

[0056] Therefore, an additional aspect of the invention concerns a method for deprogramming a somatic cell, the method comprising contacting chromatin and/or DNA of said somatic cell with an oocyte-deprogramming polypeptide, wherein the oocyte- deprogramming polypeptide causes modifications to the somatic cell chromatin and/or DNA.

[0057] Another aspect concerns a method for remodeling chromatin of a somatic cell, the method comprising contacting chromatin of said somatic cell with an oocyte- deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide remodels the cell chromatin.

[0058] An important factor in the administration of polypeptide compounds is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins, lipids and other compounds, which have the ability to translocate polypeptides across a cell membrane, have been described. For example, "membrane translocation polypeptides" have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. An oocyte-deprogramming polypeptide according to the invention can be linked to suitable peptide sequences for facilitating its uptake into cells. Other suitable chemical moieties that provide enhanced cellular uptake can also be linked, either covalently or non-covalently, the oocyte-deprogramming polypeptides of the invention. Other suitable having the ability to transport polypeptides across cell membranes may also be used.

[0059] A suitable polypeptide can also be introduced into an animal cell, preferably a mammalian cell, via liposomes and liposome derivatives such as immunoliposomes. The term "liposome" refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase. The aqueous phase typically contains the compound to be delivered to the cell. In certain embodiments, it may be desirable to target a liposome using targeting moieties that are specific to a particular cell type, tissue, and the like. Targeting of liposomes using a variety of targeting moieties (e.g., ligands, receptors, and monoclonal antibodies) has been previously described.

Methods and applications

[0060] As described in the exemplification section, the inventors have successfully induced the expression of selected genes highly conserved in vertebrates and specifically transcribed in mammalian oocytes. Expression of those genes in somatic cells resulted in a reduction of RNA neosynthesis and/or epigenetic modifications and/or expression of sternness genes and/or DNA demethylation, those four parameters being recognized indicators of chromatin remodeling and cell deprogrammation.

[0061] Accordingly, disclosed herein are methods for (1) remodeling chromatin of a somatic cell; (2) deprogramming, or at least facilitating deprogrammation, of a somatic cell; (3) conditioning a somatic cell to dedifferentiation into a pluripotent, multipotent and/or unipotent cells; (4) transforming a somatic cell from a first cell type to a second cell type showing morphological and functional characteristics of a specific cell lineage different than the first cell type; (5) obtaining pluripotent cells, (6) reprogramming a somatic cell; (7) facilitating nuclear transfer.

A) Deprogramming somatic cells and remodeling chromatin,

[0062] In some aspects, the invention relates to methods for remodeling chromatin of a somatic cell and to methods for deprogramming, or at least facilitating deprogrammation, of a somatic cell.

[0063] "Chromatin" or "cellular chromatin" refers to the nucleoprotein structure comprising nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins. The majority of eukaryotic chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of

DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and a segment of linker DNA (of variable length depending on the organism) extends between nucleosome cores. A molecule of histone H1 is generally associated with the linker DNA. For the purposes of the present disclosure, the term "chromatin" is meant to encompass all types of eukaryotic cellular nucleoproteins. [0064] As used herein, "remodeling chromatin" or "remodeling cellular chromatin" refers to dynamic structural changes to the chromatin. These changes can range from local changes necessary for transcriptional regulation to global changes necessary for chromosome segregation. In some embodiments, it refers more specifically to epigenic modifications such as acetylation, methylation and/or demethylation of histones and DNA methylation.

[0065] As used herein, the term "somatic cell" refers to any cell forming the body of an organism, apart from germline cells (i.e. ovogonies and spermatogonies) and the cells derived therefrom (e.g. oocyte, spermatozoa). For instance, internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. Somatic cells according to the invention can be differentiated cells isolated from adult or can be foetal somatic cells. Somatic cells are obtained from animals, preferably human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art.

[0066] "Deprogramming a cell": refers to changes in the expression of the genome of cell that occurs without directly affecting the sequence of the DNA. Cell deprogrammation can occur because of epigenetic modifications to the cell chromatin, or DNA demethylation. According to preferred embodiments of the invention, deprogrammation of the cell causes a dedifferentiation of the differentiated cell into an undifferentiated cell or pluripotent multipotent or unipotent cell.

[0067] In one aspect of the invention is directed to a method of deprogramming adult somatic cells, wherein the method comprises one or more of the steps of acetylation, methylation, and/or demethylation of histones. In particular embodiment, those epigenetic modifications occurs on the N-terminal tail of histones H3 and H4.

[0068] In some embodiments, adult somatic cells may be treated to remove or reverse the affects of tissue specific epigenetic changes in chromosome architecture and patterns of gene expression. Examples of epigenetic changes include DNA methylation,

DNA demethylation, bound transcription activators or repressors, deacetylated histones or demethylated histones. Therefore, the present invention is also directed to a method of deprogramming adult somatic cells, wherein the method comprises one or more of the steps of DNA methylation, DNA demethylation, wherein methyl group(s) are removed or added on the CpG dinucleotides comprising the cell DNA.

[0069] The resulting reprogrammed cells may resemble embryonic stem cells in phenotype and/or patterns of gene expression and/or defining properties (e. g. .

pluripotency, self-renewal). These cells can be continuously passaged and survive cryopreservation.

[0070] The invention also envision the use of reagents that may be used in any of the methylation, demethylation and/or acetylation step, including, for example, methylase specific antibodies or other inhibitors of methylases, inhibition of or reversal of histone deacetylation using compounds such as trichostatin A, sodium butyrate, and other compounds known in the art. The cells of the invention may also be treated with a chromatin remodeling protein such as ISWI (see US 7,053,264), related chromatin remodeling ATPases, histone modifiers (e. g. AOF2, Mellor, J. 2006. Ce// 126(1 ):22-4). It is envisioned that cells may be treated with any chromatin remodeling enzyme, reagent, intercalating agent, or combination thereof, which are known in the art, which facilitates the removal of transcription repressors and nuclear remodeling.

[0071] The methods of the invention may also comprises the use of compounds facilitating dedifferentiation of somatic nuclei, for example, to render chromosomal sequences more accessible to regulatory factors (i.e., formation of "open" chromatin) or to make chromosomal sequences less accessible (i.e., formation of "closed" chromatin). Such modifications can include, for example, removal of nucleosomes from DNA, deposition of new nucleosomes onto DNA, repositioning of nucleosomes, changes in nucleosome spacing, changes in nucleosome density, changes in the degree and/or nature of the interaction between DNA and histones in the nucleosome, changes in the path of DNA along the surface of the nucleosome, and/or changes in higher-order chromatin structure such as, for example, unwinding of the chromatin solenoid.

[0072] The cells and methods disclosed herein can be used to facilitate a number of processes involving cellular chromatin. These processes include, but are not limited to, dedifferentiation or differentiation of a target cell, cloning, creation of cell lineages, immortalization of cell, transcription, replication, recombination, repair, integration, maintenance of telomeres, and processes involved in chromosome stability and disjunction. Accordingly, the methods and cells disclosed herein can be used to affect any of these processes, as well as any other process which can be influenced by the effect of the presence in somatic cells of one or more active oocyte-deprogramming polypeptide. B) Generation of a pluripotent, multipotent and/or unipotent cells, and differentiated cells

[0073] The invention ability to remodel chromatin and modulate gene expression (e.g. RNA neosynthesis, reactivation of pluripotent marker genes, alteration of histone post-translational modifications and DNA methylation) has profound implications for development of in vitro and in vivo systems of dedifferentiation. Thus, in certain embodiments, the invention concerns methods and cells to transform or deprogram a given somatic cell into pluripotent, multipotent and/or unipotent cells.

[0074] Therefore, some aspects of the invention relates to methods for conditioning a somatic cell to dedifferentiation into a pluripotent, multipotent or unipotent cell. Yet, a further related aspect of the invention concerns a method for transforming a somatic cell from a first cell type to a second cell type, or at least facilitating such transformation reprogrammation. The invention encompasses pluripotent, multipotent and unipotent cells, cells lines, and purified cells preparations derived from any of these methods.

[0075] According to a particular aspect, the method comprises contacting the somatic cell with a polynucleotide encoding an oocyte-deprogramming polypeptide and expressing the oocyte-deprogramming polypeptide within the somatic cell. Expression of the oocyte-deprogramming polypeptide deprograms, or at least participates in deprogramming, the cell. The cell deprogrammation can occur rapidly after some hours of treatment with the oocyte specific polypeptides or corresponding RNA or DNA, or may take some weeks depending on the technique applied and oocyte deprogramming polypeptide. Preferably, cell deprogrammation causes the dedifferentiation of the cells, therefore becoming phenotypically, immunologically, morphologically, functionally and/or genetically pluripotent, multipotent, or unipotent. That cell is then cultured using well known techniques to maintain the cell in a pluripotent, multipotent or unipotent state. Pluripotent cells can be cultured following standard culture protocols for mouse, human or others ES cells available in the art. For example, mouse derived-pluripotent cells of the invention can be maintained in an undifferentiated state (i. e. inhibition of differentiation) by culturing with mitotically inactivated feeder cells (e. g. STO mouse fibroblast cell line or primary embryo fibroblast) or in the presence of leukemia inhibitory factor (LIF). Cells can be cultured in a medium consisting in DMEM, glutamine, β- mercaptoethanol, 10% calf serum and 10% foetal calf serum, non-essential amino acids and antibiotics if desired. Human derived-pluripotent cells of the invention can be maintained in an undifferentiated state by culturing on all feeder layers suitable for hES cells as Matrigel™ matrix (i.e. commercially available basement membrane extract), human embryonic fibroblasts, adult Fallopian tube epithelium, foreskin fibroblasts. Feeder layer-free culture system can also be used with fibronectin-covered plates. Human derived-pluripotent cells can be cultured in the presence of 100% mouse embryonic fibroblast (MEF)-conditioned medium. Cells can also be cultured in hES serum-free medium consisting in knockout DMEM, serum replacement (SR), nonessential amino acids, glutamine, /?-mercaptoethanol, TGF/?1 , LIF, basic fibroblast growth factor (bFGF) or other suitable well-defined hES cell growth media.

[0076] Pluripotent cells of the invention can be cultured following standard protocols known in the art to differentiate ES cells into a variety of terminally differentiated somatic cells via precursor cells. The most common method is to induce the formation of 3D cells aggregates termed embryoid bodies (EBs) by culturing in suspension in the absence of LIF, bFGF or other factors which inhibits the differentiation. EBs are then collected, further cultivated in suspension and plated onto coated-dishes to further differentiation. Spontaneous or directed differentiation can occurr. According to the cell lineage desired, different approaches can be followed to induce differentiation: gene targeting (knockin and knockout specific relevant receptors or transcription factors), exogenic factors (administrating the cell culture with soluble growth and differentiation factors), matrix- based cultures or co-culture with a specific cell line. For example, pluripotent cells can differentiate in cardiac tissue using the spontaneous cardiomyocyte-differentiating system reported by Kehat and colleagues (Kehat, Kenyagin-Karsenti et al. 2001). Pluripotent cells can differentiate into neuronal precurseurs by cultivating in a standard differentiation medium in the presence of rh-FGF-2 and noggin (Itsykson, llouz et al. 2005). The following multistep protocol induce differentiation into pancreatic /?-cell : culture in the presence of Activin A and Bmp4, next hepatocyte growth factor (HGF), Exendin-4 and /?-cellulin, next FGF18, epidermal growth factor (EGF), transforming growth factor β (TGF- β), insulin growth factor-1 (IGF-1 ), IGF-2 and vascular EGF (Phillips, Hentze et al. 2007).

[0077] The cell reprogrammation may further be induced or stimulated with the use of one or more nuclear reprogramming factors such as those described in US patent publication 2009/0047263 (Yanamaka et al.); Takahashi and Yamanaka, (Cell (2006), 126:663-676); Okita et al. (Nature (2007), 448:313-317); Wernig et al. (Nature (2007), 448:318-324), and Takahashi er a/. (Cell (2007), 131 :861-872).

[0078] The ability of the pluripotent cells of the invention to differentiate into the three embryonic germ layers can be tested using the three traditionnal approaches available in the art. Pluripotency can be demonstrated in vitro by the formation of embryoid bodies when cells are cultured in suspension without differentiation inhibiting factors. Pluripotency can be demonstrated in vivo by the formation of teratomas (i. e. benign tumors consisting of cell types representing all three germ layers) when cells are injected into immunodeficiency mice. In the case of mouse derived-pluripotent cells, cells can be transferred into the inner cell mass (ICM) of an early mouse embryos where they contribute to all somatic lineages and produce germ line chimerism.

[0079] As used herein, "Stem cell" refers to those cells which retain the ability to renew themselves through mitotic cell division and which can differentiate into a diverse range of specialized cell types. It includes both embryonic stem cells that are found in blastocysts, and adult stem cells that are found in adult tissues. "Totipotent cells" refers to cells that have the ability to develop into cells derived from all three embryonic germ layers (mesoderm, endoderm and ectoderm) and an entire organism (e.g., human being if placed in a woman's uterus in the case of humans). Totipotent cells may give rise to an embryo, the extra embryonic membranes and all post-embryonic tissues and organs. The term "pluripotent" as used herein is intended to mean the ability of a cell to give rise to differentiated cells of all three embryonic germ layers. "Induced-pluripotent stem cells", commonly abbreviated as iPS cells or iPSCs, refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inserting certain genes. "Multipotent cells" refers to cells that can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). "Unipotent cells" refers to cells that have the capacity to develop/differentiate into only one type of tissue/cell type (e.g. skin cells). The term "differentiated cell" refers to a cell that has developed from a relatively unspecialized phenotype to a more specialized phenotype. For example, a progenitor cell type such as a hematopoietic stem cell can give rise to a more differentiated cell such as a monocyte or an erythrocyte. Differentiated cells can be isolated from adult or foetal somatic cells using techniques known in the art. The terms "dedifferentiated cell", or "deprogrammed cell" are used interchangeably to refer to a cell that had formerly attained a particular degree of differentiation, but has subsequently been immortalized or has regained the ability to differentiate into one or more specialized cells (e.g., has become pluripotent, multipotent or totipotent). Using the method and compositions described herein, differentiated cells can be deprogrammed into pluripotent, multipotent and unipotent cells.

[0080] The terms "transform", or "reprogram" are used interchangeably to refer to the phenomenon in which a differentiated cell is dedifferentiated to become pluripotent, multipotent and/or unipotent, the dedifferentiated cell being subsequently redifferentiated into a different type of cell. Cells can be reprogrammed or converted to varying degrees. For example, it is possible that only a small portion of cells are converted or that an individual cell is reprogrammed to be multipotent but not necessarily pluripotent. Thus, the terms "transforming" or "reprogramming " methods refer to methods wherein it is possible to reprogram a somatic cell such that the "new" cell shows morphological and functional characteristics of a different specific cell lineage (e.g. the transformation of fibroblasts cells into neuronal cells).

[0081] Accordingly, a related aspect of the invention concerns a method for transforming a somatic cell from a first cell type to a second cell type. Examples of adult somatic cells which may be used as the starting material (i.e. cells from the first type) in the methods of the invention include, but are not limited to, dermal fibroblasts, epidermal cells, keratinocytes, hair outer root sheath cells, and peripheral blood monocytes. Examples of cells that can be produced according to the methods of the invention include, but are not limited to, Sertoli cells, endothelial cells, granulosa epithelial, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, and other muscle cells, etc.

[0082] A related method concerns obtaining pluripotent, multipotent and/or unipotent cells. Those skilled in the art know to evaluate whether a cell is pluripotent, multipotent and/or unipotent. Evaluation criteria include morphology, proliferation, markers (e.g. surface antigens), gene expression, epigenic status and telomerase activity (see Takahashi et al, supra). In one embodiment the cells of the invention are positive in the expression of one or more pluripotent gene markers. "Pluripotent gene markers" refers to factors associated with pluripotency (e.g. pluripotent genes, pluripotent surface markers, transcription factors, etc.), including those expressed consistently and enriched in ES cells. The transcription factors Oct 3/4, Sox 2 and Nanog are usually used in the art as pluripotent gene markers to characterize pluripotent cells. Others pluripotent genes and markers are Tert, UtPI , Rex1 , Fgf4, UtM , Cx45, Cx43, Bcrp-1 , gp130, Stat3, etc. The triad Oct 3/4, Sox 2 and Nanog regulates the expression of over 1600 genes affecting cell cycle, chromatin remodeling, apoptosis, miRNAs and much more. (Campbell et al, 2007 (PLoS ONE), Boyer et al, 2005 (Cell)). "Pluripotent surface markers" refers more specifically to pluripotent cell surface antigens, preferably those that can be detected by antibodies in ES cells. SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 , TRA-2-54 and GCTM-2 are examples of mouse and/or human ES cell surface markers.

[0083] In one embodiment, adult somatic cells are treated with one or more oocyte- deprogramming polypeptides to reverse the epigenetic changes that occur during differentiation, resulting in cells that are pluripotent, multipotent or unipotent. Those cells may be cultured many times while maintaining an undifferentiated state and while retaining the capacity to differentiate into a variety of cell and tissue types.

[0084] In another embodiment, the method comprises reprogramming a somatic cell, or at least facilitating its deprogrammation, by causing modifications to the somatic cell chromatin and/or to the somatic cell DNA using an oocyte-deprogramming polypeptide, polynucleotide or gene as described herein, and exposing the deprogrammed somatic cell to at least one differentiation factor for a sufficient period of time and under suitable conditions such that desirable morphological and functional characteristics of a target reprogrammed cell are reached. The differentiation factor is selected according to the morphological and functional characteristics desired. For instance, to induce differentiation of pluripotent cells into neural cells via embyoid bodies, one can use well- established protocols and the following differentiation factors: bFGF and/or EGF then FGF8, sonic hedgehog (SHH), and ascorbic acid. On the other hand, sequential addition of dexamethasone, oncostatin M, acidic FGF and hepatocyte growth factor (HGF) can be used to induce differentiation into hepatocytes. In preferred embodiments, the deprogrammation or chromatin remodeling precedes the differentiation step but it is conceivable that those steps can be carried out simultaneously (e.g. in the same experimental procedure).

C) Nuclear transplantation and cloning

[0085] The methods described herein also allow for increased ease and efficiency in cloning and transfection. For example, cloning of domestic animals has typically been attempted by transplanting a somatic nucleus into an enucleated oocyte. Such nuclear transplant techniques have a low success rate, presumably because the somatic chromatin is not always properly remodelled and because the amount and duration of the exposure to oocyte factors in an enucleated oocyte might be limited. Use of the polynucleotides, polypeptides, cells and methods described herein allows for increased efficiency of nuclear transfer, particularly for somatic cell nuclei. The methods of the invention allows reprogrammation and/or dedifferentiation of cells to varying degrees prior to, or coincident with, their transplantation for cloning purposes, thereby increasing the likelihood of a live birth. An additional aspect of the present invention relates to methods for producing mammalian, preferably human, pluripotent, multipotent or unipotent cell lines from any adult somatic cell without requiring therapeutic cloning (i.e. no use of foetal or embryonic tissues).

D) Cells and Cell Lines

[0086] The invention encompasses the cells, cells lines, stem cells and purified cells preparations derived from any of the methods described herein. In some embodiments, the cells, cells lines, stem cells and purified cells preparations of the invention are of mammalian origins (e.g. bovine, human, mouse). In preferred embodiments, they originate from a human.

[0087] Accordingly, another aspect of the invention relates to modified cells, cells lines, pluripotent, multipotent or unipotent cells and purified cells preparations, the cells of the invention comprising an exogenous polynucleotide encoding an oocyte- deprogramming polypeptide. In one embodiment the cell is positive for expression of a pluripotent gene marker (e.g. NANOG, OCT4, SOX2, etc.). In one embodiment the cell transiently expresses the polynucleotide encoding the oocyte-deprogramming polypeptide.

[0088] As used herein, the term "Exogenous polynucleotide" refers to a nucleic acid molecule (such as cDNA, cDNA fragments, genomic DNA fragments, antisense RNA, oligonucleotide, etc.) which is not naturally expressed in a given cell. The "exogenous polynucleotide" may be from a different cell type, a different organism or purely synthetic. For instance, a polynucleotide encoding an oocyte-deprogramming polypeptide according to the invention which has been transfected into a fibroblast is a polynucleotide exogenous to that cell because fibroblasts do not normally express, or merely expresses at a low level, oocyte-specific/preferential genes at that stage of their differentiation.

[0089] The invention further encompasses cells derived from the transdifferentiation of a somatic cell to a "different" cell type or lineage expressing one or more morphological, physiological and/or immunological features specific of a differentiated cell found in animals and human.

[0090] As described herein some of the methods of the invention concerns generating, or at least facilitating generation of, somatic pluripotent, multipotent or unipotent cells by deprogramming and conditioning somatic cells to dedifferentiation into a pluripotent, multipotent or unipotent cell. In some embodiments, the cells of the invention are somatic cells positive for expression of pluripotent gene markers. Preferably, the methods of the invention concerns the generation of somatic pluripotent cells. The expression "somatic pluripotent cells" as used herein is intended to mean non-embryonic stem cells that are not derived from gametes (e.g. oocyte or spermatogonia cells). The cells obtained with the methods of the invention may resemble embryonic stem cells in morphology and in biochemical histotype. These cells may be passaged several times in culture, maintained for several months in culture and/or survive cryopreservation.

[0091] The phenotype of the cells of the invention may be determined as commonly described in the art. It is envisioned that the cells of the invention may express several molecular markers that are also expressed by embryonic stem (ES) cells. In particular embodiments, the cell of the invention expresses one or more pluripotent gene markers including but not limited to NANOG, OCT4, and SOX2. Gene expression may be determined by one of many art recognized methods, such as reverse transcription- polymerase chain reaction (RT-PCR). There are also various cell surface markers that may be employed under the current invention to isolate, identify and define the characteristics of the cells created under the current invention including, but not limited to, those described on Table 3 of patent application US 2003/0113910 which is incorporated herein by reference.

[0092] In some embodiments, the cells of the invention may have one or more of the characteristics and properties: self-renewal, multilineage differentiation in vitro and in vivo, clonogenicity, a normal karyotype, extensive proliferation in vitro under well defined culture conditions, and the ability to be frozen and thawed, as well as any of the commonly known and/or desired properties or characteristics typical of ES cells. The cells of the invention may further express molecular markers of pluripotent cells (i.e. gene and surface markers as defined previously).

[0093] Another aspect of the invention relates to the production of tissue specific autologous (self) progenitor cells derived from in vitro derived adult pluripotent stem cells. These progenitor cells may be used in cell therapy applications to treat diseases of cellular degeneration. Diseases of cellular degeneration include for example neurodegenerative diseases such as stroke, Alzheimer's disease Parkinson's disease, multiple sclerosis, Amyotrophic lateral sclerosis, macular degeneration, osteolytic diseases such as osteoporosis, osteoarthritis, bone fractures, bone breaks, diabetes and liver injury and degenerative diseases, myocardial infarct, burns and cancer. It is envisioned that in vitro derived adult pluripotent stem cells or fully differentiated cells derived from these cells may be implanted or transplanted into a host. An advantage of the invention is that large numbers of autologous stem cells can be produced for implantation without the risk of immune system mediated rejection. Those cells can lead to production of tissue suitable for transplant into the individual. Since the tissue is derived from the transplant recipient, it will not stimulate an immune response, as would tissue from an unrelated donor. Such transplants can constitute tissues (e.g. vein, artery, skin, muscle), solid organ transplants (e.g., heart, liver, kidney) or bone marrow, neuronal cell transplants such as are used in the treatment of various malignancies such as, for example, leukemias and lymphomas. Such transplants can also be used in the treatment of, for example, neurological disorders, diabetes and the like.

[0094] Another aspect of the invention relates to a method to produce ex vivo engineered tissues for subsequent implantation or transplantation into a host, wherein the cellular components of those engineered tissues are the in vitro derived adult pluripotent stem cells or cells derived therefrom.

[0095] For example, expanded cultures of the cells of the invention may be differentiated by in vitro treatment with growth factors and/or morphogens. Populations of differentiated cells are then implanted into the recipient host near the site of injury or damage, or cultured in vitro to generate engineered tissues, as described.

[0096] In another embodiment, the cells of the present invention are genetically modified to express one or more specific genes-of-interest or to disrupt the expression of specific genes. As used herein, the phase "genetically modified" means any modification or alteration in the sequence of any portion of the entire genomic sequence of a cell, including the mitochondrial as well as nuclear genome, and further including the addition of ectopic nucleic acids to the cell as in a plasmid or artificial chromosome or portion thereof. Exogenous DNA may be transferred to the cells by electroporation, calcium phosphate, microinjection, lipofection, retroviral or other viral or microbial vectors or other means commonly known in the art. The genetically modified cells could be used in bioreactors to produce pharmaceutical products, or in cell therapy treatments for genetic diseases such as cancer, Cystic Fibrosis, adenosine deaminase deficiency ("ADA"), Osteogenesis imperfecta, Hemophilia, or Tay-Sachs disease, for example. In the treatment of a genetic disease, genetically modified cells of the present invention may be administered to the patient near the site of the defect. Accordingly, another aspect of the invention relates to a method of producing a useful pharmaceutical product, wherein in vitro derived adult pluripotent stem cells or cells derived therefrom are transformed with a gene of interest which encodes a useful gene product. It is envisioned that such transformed cells may be grown in vitro in a bioreactor to produce the useful gene product. Alternatively, the transformed cells may be implanted into a host, preferably a human suffering from a disease of genetic deficiency.

[0097] The methods and cells of the invention described herein can be used to immortalize cells, for example to generate a cell line. Using the methods disclosed herein, a somatic cell can be transformed into one possessing a dedifferentiated phenotype, thereby facilitating the generation of cell lines from a variety of tissues. Therefore, the invention encompasses such immortalized cells. Certain cells, for example neoplastic cancer cells, are believed to be inappropriately dedifferentiated or immortalized in vivo. Immortalization of these cells allows them to divide indefinitely. Thus, the methods and cells described herein also find use in facilitating differentiation of a target cell, for example by introducing into the cell a polynucleotide encoding an oocyte-deprogramming polypeptide. Further, it is conceivable to use the oocyte- deprogramming polypeptides and polynucleotides of the invention to create fusion polynucleotides and fusion polypeptides, the latter having desirable properties such as having a higher affinity to bind DNA, having an improved nuclear localization, and/or having greater measurable effects on dedifferentiation and/or differentiation.

[0098] The methods and the cells according to the invention have advantages relative to other pluripotent cell lines. The cells of the invention also have characteristics and properties that make them more attractive alternative when compared with embryonic stem cells created under current technologies. An important advantage of this invention is that the cellular reprogramming procedure does not involve the use of human embryonic stem cells, human embryonic carcinoma cells, or human germ cells. Another advantage is that the reprogramming procedure does not involve the creation of human embryos or human/animal chimeras through nuclear transfer, or fusion of somatic cells with oocytes. The cells of the invention may also be considered as a more attractive alternative when compared with mutlipotent/adult stem cells produced or secured from other sources such as in vivo, umbilical cords and other limited sources.

[0099] In addition, the methods the cells according to the invention, may be helpful in scientific and therapeutic applications including, but not limited to, (a) scientific discovery and research involving cellular development and genetic research (e.g. uses in lieu of human embryonic stem cells as a model cell line to study the differentiation of human cells), (b) drug development and discovery (e.g., screening for efficacy and toxicity of certain drug candidates and chemicals, screening for prospective drugs or agents which mediate the differentiation of cells), (c) gene therapy (e.g., as a delivery device for gene therapy), and (d) treatment of diseases and disorders including, but not limited to, Parkinson's, Alzehimer's, Huntington's, Tay-Sachs, Gauchers, spinal cord injury, stoke, burns and other skin damage, heart disease, diabetes, Lupus, osteoarthritis, liver diseases, hormone disorders, kidney disease, leukemia, lymphoma, multiple sclerosis, rheumatoid arthritis, Duchenne's Musclar Dystrophy, Ontogenesis Imperfecta, birth defects, infertility, pregnancy loss, and other cancers, degenerative and other diseases and disorders.

[00100] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting.

Examples

[00101] The examples set forth herein below provide exemplary methods for remodeling chromatin and deprogramming somatic cells. Also provided are exemplary protocols, molecular tools, probes, primers and techniques.

EXAMPLE 1 : Deprogramming somatic cells by transient transfection of genes preferentially expressed in oocyte and conserved in vertebrates.

[00102] As the oocyte contains all the resources needed to sustain embryo development until embryonic genome activation, it was hypothesized that the recipe to deprogrammation resides in oocyte genes. As shown hereinafter, some reprogramming mechanisms were reproduced in somatic cells by transient transfection of selected oocyte-deprogramming factors of the present invention .

Materials and Methods

Cell Culture and Transfection

[00103] HEK293 (provided by ATCC) were grown without agitation in MEM++ medium supplemented with 10% FCS and 0.5% penicillin/streptomycin (Gibco) in a humidified incubator with 5% CO₂ at 37°C. Cells were washed with phosphate-buffer saline (PBS), trypsinized and transferred to a 6-well plate (Corning) 24h prior to transient transfection. [00104] HEK293 cells were transfected with Lipofectamine LTX™ (Invitrogen). 1.5μg of plasmid DNA was first incubated 5 min at room temperature in 500μl OPTIMEM™ supplemented with 2μl PLUS™ Reagent. Then 6μl Lipofectamine LTX was added to the preparation and incubated for 30 min at room temperature. HEK293 medium (MEM++) was substituted with MEM 10% FCS without antibiotics prior to the addition of transfection mixture. Five (5) transfection replicates were made for each gene, at different times.

Plasmids

[00105] PCMS-eGFP™ (Clontech) was used to clone the complete coding sequences of selected oocyte-deprogramming factors. This vector is composed of two promoters, CMV, which confers high expression of the target gene in mammalian cells, and SV40, which drives the GFP expression. Full-length sequence inserts were amplified or digested from one of the following templates: MGC clone (Open biosystems: H1 FOO, AICDA); human oocyte cDNA library (Sylvestre et al., in progress: NPM2, STELLA, GMNN, PTTG1 ; or bovine oocyte cDNA library, (PAIP1 (98% homology with human protein)). In the case of H1 FOO and AICDA, MGC clone was purified (Miniprep™ kit, Sigma) and digested with EcoRI-Xbal. For all other candidates, primers with restriction sites (bold) are described in the table below. Full-length sequences were amplified in 50μl reaction with 45μl Platinum supermix™ (Invitrogen), 2μl DMSO, 1 μl of each primer (10μM) and 1 μl of template. The PCR parameters were determined as follows: denaturation at 95⁰C for 2 min, then 35 cycles of 45 s denaturation at 95°C, 30 s of annealing depending of the primers Tm, extension at 68°C for up to 3 min, and a final extension at 68°C of 10 min.

[00106] TABLE 3: Directional cloning of gene highly expressed in oocytes into pCMS-eGFP vector

Gene Unigene 5' Primer Tm (⁰C) 3' Primer or MGC clone number Amplicon

(bp)

AICDA Hs.149342 4054915 GMNN Hs.234896 TCGTCTCGAGATG AAG CAG AAA CAA GAA G 53

(SEQ ID NO: 10) 669

7GΛ7TCTAGAGTG GAG GTA AAC TTC GGC AGT A

(SEQ ID NO: 11 )

H1FOO Hs.97358 5742122 NPM2 Hs.131055 TCG TCTCGAG ATG AAT CTC AGT AGC GCC AGT A 55

(SEQ ID NO: 12) 645

TGA 7TCTAGATCA TTT CTT GAA TCC TGG CT (SEQ ID NO: 13)

PAIP1 Bt.17115 TCGTCTCGAGCAT GTC GGA CGG TTT CGA CC 58

(SEQ ID NO: 14) 1203

TGA TTCTAG ATTA CTG TTT TCG CTT ACG CTC TGA (SEQ ID NO: 15)

PTTG1 Hs.350966 TCG 7^"CTCGAGATC CAG AAT GGC TAC TCT GAT C 55

(SEQ ID NO: 16) 615

7G/17ACGCGTTTA MT ATC TAT GTC ACA GCA AAC AGG (SEQ ID NO: 17)

STELLA Hs.131358 7^"CG TCTCG AGTTG TGT CAA GAC GCC GAT GGA 58

C (SEQ ID NO: 18) 595

TGA 7TCTAGATTA TGG CTG AAG TGG CTT GGT G (SEQ ID NO: 19)

Letters in bold correspond to the added restriction site Letters in italic correspond to additional bases to promote digestion at the restriction site

[00107] Fragment and vector ligation was performed using T4 DNA ligase (NEB), and incubated overnight at 16°C. The ligation product was then deposited on nitrocellulose membrane for 20 min to remove salts. 35μl of electrocompetent DH5-α were electroporated with 3.5μl of ligation product, and incubated 1 hour at 37°C prior to incubation on LB-Agar plates (Ampicilline 100μg/ml) overnight. The following vector primers were used to verify the presence of the insert (5': GTG TCC ACT CCC AGT TCA, 3': CCC CTG AAC CTG AAA CAT) by PCR with AmpliTaq Gold™ polymerase (Applied Biosystems). All clone inserts were sequenced using an automated sequencer and checked for their accurate coding sequence. Plasmid DNA of the selected recombinant clones were purified with Plasmid Maxi Kit™ (Qiagen).

3H-uridine incorporation

[00108] 72h after HEK293 transfection, cells were starved for 16h to synchronize cell cycle between treatments (using serum-free culture medium). The same treatment was applied for immunoblots analysis. 3H-uridine (1 μCi/ml, GE Healthcare) was added to fresh MEM++, and cells cultured in 6-well plates were incubated at 37⁰C for 3h. Cells were then washed with PBS and trypsinized. RNA and DNA were simultaneously extracted and purified with Allprep™ DNA/RNA Mini Kit (Qiagen). RNA neosynthesis was calculated as ratio scintillation counts/total RNA. A total of 5 replicates were analyzed.

Western Blots

[00109] 20 000 HEK293 cells were resuspended in protein loading buffer (2% SDS- 20% glycerol, 2mM Tris-HCI pH:8.0, 2μM EDTA₁ 15 mg/ml DTT) and heated at 95°C prior to each immunoblot. Samples were subjected to SDS-PAGE, using 15% resolving gels and 5 % stacking gels. Proteins were transferred to nitrocellulose membrane. Membranes were blocked with 4% ECL Advance Blocking Agent™ (GE Healthcare) in TBST (Tris buffer saline + Tween 0.01 %). Rabbit polyclonal primary antibodies were diluted in 4% ECL, as follows: H3K9Me3 (1/10 000, Abeam), H3K4Me3 (1/40 000, Abeam), H3K9Ac (1/2500, Abeam), H4K20Me3 (1/10 000, Abeam). Each membrane was also incubated with rabbit polyclonal to β-actin (1/10 000, Cell signaling technology). Goat anti-rabbit IgG Horseradish peroxidase was diluted 1/300 000 in non-fat milk/TBST, and incubated with membrane 1 h at room temperature. The signals were detected using ECL Advance System™ (GE Healthcare). Average band intensity was measured with Adobe Photoshop CS3™. The band intensity values for modified core histone were normalized on β-Actin intensity for further analysis.

Reverse transcriptase and Real-time PCR

[00110] 100 000 HEK293 cells were washed and pelleted at day 3 and 7 of transfection, and frozen at -80⁰C. RNA was extracted and purified with Picopure™ extraction kit (Arcturus). Reverse transcription was performed at 55 and 50⁰C for 30 min each, in 20μl with 4μl of 50μM oligo dTs (18), 10U of Transcriptor™ Reverse Transcriptase (Roche Diagnostics), RT buffer 5X, 2OU of RNase inhibitor and 1 mM each dNTP. For each gene amplified, a standard curve consisting of purified PCR products (PCR gel extraction, Qiagen) was established. The reaction was performed in capillaries and in a volume of 20 μl (Roche). Each capillary contained the cDNA of 7,500 HEK293 cells as startup material for amplification. We added 0.5 μl of 10 μM of each primer, 1.6 μl of 25 mM MgCI₂ (final concentration of 3 mM), 2 μl of the SYBR™ green mix containing dNTPs, FastStart™ DNA polymerase enzyme, and buffer (Roche). PCR conditions consisted of 10 min denaturing cycle at 95°C, 40-50 PCR cycles (denaturing: 95°C for 1 sec; annealing temperature (Table 3) for 5 sec; elongation: 72°C for 20 sec), a melting cycle consisting of 95°C for 1 sec, 72⁰C for 30 sec, and a step cycle starting at 72°C up to 95°C. To quantify SYBR™ green in amplification with the standard curve, we used Lightcycler™ Software Version 3.5 (Roche). We analyzed three pluripotency- associated transcription factors (NANOG, 0CT4, SOX2), and three housekeeping genes (β-actin, cyclophilin-B, GAPDH).

TABLE 4: Q-PCR primer sets used to amplify pi uri potency-associated transcription factors (NANOG, OCT4, SOX2) and housekeeping genes (β-ACTIN, CYCLO, GAPDH) in transfected HEK293 cells.

Gene Unigene Primer 5' Tm TAq Amp Primer 3' (⁰C) (⁰C) Size (bp)

P-ACTIN Hs.520640 CGT GAC ATT AAG GAG AAG CTG 57 89 374

TGC (SEQ ID NO : 20)

CTC AGG AGG AGC AAT GAT CTT GAT 59 84 166

(SEQ ID NO : 21) CYCLO Hs.434937 TGC TGG ACC CAA CAC AAA TGG TTC

(SEQ ID NO : 22)

TGG TGA TCT TCT TGC TGG TCT TGC

(SEQ ID NO : 23) GAPDH Hs.544577 AAC ACA GTC CAT GCC ATC AC (SEQ 57 89 452

ID NO : 24)

TCC ACC ACC CTG TTG CTG TA (SEQ

ID NO : 25) NANOG Hs.661360 TCC CAAAGG CAAACAACC CAC T 60 82 290

(SEQ ID NO : 26)

TTC TGC GTC ACA CCA TTG CT (SEQ

ID NO : 27) OCT4 Hs.249184 GAC AGG GGG AGG GGA GGA GCT 63 86 143

AGG (SEQ ID NO : 28)

CTT CCC TCC AAC CAG TTG CCC CAA

AC (SEQ ID NO : 29) SOX2 Hs.518438 TGA ATG CCT TCA TGG TGT GGT C 58 77 345

(SEQ ID NO : 30)

TAA CTG TCC ATG CGC TGG TT (SEQ

ID NO : 31 )

Tm = optimal melting temperature specific to the primer pair TAq= temperature of fluorescence acquisition Amp Size= amplicon size Immunofluorescence

[00111] To evaluate the level of DNA methylation in transfected cells, we transferred transfected HEK293 in 8-well Permanox™ slides (Nunc), using 35,000 GFP-sorted HEK293 cells per well (day 3), in which the cells were let grown for 24 hours. Cells were then washed with PBS and fixed with methanol-acetone 3:1 (-2O⁰C) for 20 min. Cells were permeabilized in TBS 0.5% Triton X-100™ for 1 hour at room temperature. As 5- MeC reacted with single-stranded DNA, cells were exposed to UV light in PBS for 14-16 hours under a germicidal lamp according to the procedure of Miller et al. Cells were then washed repeatedly in PBS, and TBST 0.1% with 5% goat serum and 1% BSA was used as blocking solution for 1 hour at room temperature. Primary antibody mouse monoclonal anti-5Me-C (Calbiochem; 1/2500) was diluted in fresh blocking solution, and slides were incubated overnight at 4⁰C. The next day, slides were washed with TBST 1% serum, prior to 1-hour incubation at room temperature with the secondary antibody, Alexa 555™ conjugated goat anti-mouse IgG (H+L) (Molecular Probes) diluted 1/200 in blocking solution. Cells were then washed twice in TBST and stained with DAPI (2 μg/ml) in TBS for 1 min at room temperature. Prolong™ (Invitrogen) was used to fix coverslips on slides, lmmunostaining was observed by epifluorescence with a Nikon™ fluorescence microscope, pictures were taken with 100x objective.

Southwestern analysis

[00112] Cells were sorted at day 3 and allowed to grow one more day to repeat the immunofluorescence conditions. Cells were then washed with PBS and pelleted. DNA was purified with DNeasy™ kit (Qiagen), and 2μg of DNA for each treatment was digested with restriction enzymes EcoRI-Xhol (3h at 37°C) before being loaded onto a 0.7% TAE gel with EtBr. DNA was then migrated at 75v for 3h. The gel was then placed under UV light to see that bands of digested DNA were equally intense. The gel was then exposed to 0.25M HCL for 7 min, 1.5M NaCI; 0.5M NaOH twice for 20 min and 1.5M NaCI; 1 M Tris pH 7.5 for an additional 20 min, to depurinate, denaturate DNA and neutralize the gel. DNA was then transferred overnight by capillarization of 10X SSC on a positively charged nylon membrane. The membrane was then cross-linked under UV light for 3 min, and stained with methylene blue 0.04% to verify that DNA had transferred. The membrane was then treated as described in the western protocol, we used primary antibody mouse monoclonal anti-5Me-C (Calbiochem; 1/5000) with goat anti-mouse IgG horseperoxidase (1/200 000). Relative band intensity was quantified with Adobe Photoshop™ CS3. Statistics

[00113] For each replicate, treated cells were compared to control (pCMS) as log (TRT/ pCMS) vs log(1 ). We used unilateral ratio T-test in Graphpad Prism 5.0™, as data and control were paired by a common denominator. Then the hypothesis to test was: no change is zero (the logarithm of 1.0), increases are positive and decreases are negative (Graphpad Prism 5.0™ Key concepts). A comparison resulting in a P value inferior to 0.05 was considered significantly different from control. In DNA methylation analysis, the intensity of 30 to 45 cell was measured, and analyzed with ANOVA (with post hoc Dunnett test).

Results

[00114] Expression of the transgene. As shown in Figure 1 , all genes transfected are expressed at the protein level

RNA Neosynthesis

[00115] Measuring RNA neosynthesis, by incorporating 3H-uridine into newly synthesized RNA, allows determining if transcription rates vary between cells transfected with the gene of interest and cells transfected with an empty vector used as negative control.

[00116] As shown in Figure 2, NPM2 and AICDA significantly reduced neosynthesis in HEK293 cells when calculated both on total RNA and DNA, 3 days following transfection (P=0.0172).

Western blots

[00117] Off the four used antibodies, two are associated to active euchromatin (H3K4Me3, H3K9Ac), two to heterochromatin (H3K9Me3, H4K20Me3. The results are shown in Figure 3.

[00118] Three candidates significantly affected histone core modifications (AICDA, GMNN, NPM2). In our study, NPM2 had a significant impact on euchromatin-related modified histones, inducing an approximately 50% reduction in H3K9Ac (P=O.005) and a statistically significant reduction of H4K20Me3 levels (P=O.048). GMNN also affected chromatin configuration. GMNN and AICDA both showed significant variations for heterochromatin-related modifications. Ovexpression of each candidate induced a significant reduction in H3K9Me3 levels (GMNN, P= 0.0322; AICDA, P=0.015) while GMNN-transfected cells also showed a significant increase in H4K20Me3 levels, which varied from 1.5- to 4-fold increase (P=O.005). The reduction in trimethylated H3K9 following AICDA induction is consistent with the observed DNA demethylation following AICDA overexpression in immunofluorescence analysis. Although overexpression of the other candidates yielded some interesting epigenetic changes, such as an increase in trimethylated H3K4 following PAIP1 expression cells and an increase in acetylated H3K9 for cells overexpressing PTTG1 , those changes only occurred in 3 out of the 5 replicates. Since we considered all replicates as we were looking for repeatable changes in transfected HEK293 cells, some treatments did not reach the 0.05 level.

DNA methylation

[00119] Immunofluorescence for DNA methylation showed that most cells overexpressing NPM2 and AICDA were only weakly stained compared to control cells, which showed intense and relatively uniform staining as shown in Figure 4. As the difference in staining was drastic and the DNA seemed to be mostly demethylated in AICDA and NPM2 transfected cells, we also performed south-western blots with control cells and the two candidates to confirm what we observed in immunofluorescence analysis, which also yielded significant difference with the control cells. As shown in Figure 5, CTRL 5Me-C staining on the DNA smear is much stronger in control cells than following AICDA and NPM2 overexpression. NPM2 and AICDA not only have an impact on histone core modifications, but also seems to alter DNA methylation in somatic cells.

Real-Time PCR

[00120] Reactivation of pluripotent marker genes is a concrete and major step in deprogramming somatic cells. In the mammalian early embryo, pluripotency-associated factors are massively activated following deprogrammation events, so we assumed that if there was a change in the activity of those factors, it would be detectable at least at day 7.

[00121] As shown on Figure 6, for each of the pluripotency-associated transcription factor, we found significantly increased expression following transfection of at least one of our candidates. OCT4 was the least stable of the genes, and was significantly overexpressed in NPM2-, PTTG 1-, and STELLA-transfected cells (P=0.029, 0.043, and 0.027 respectively). For PTTG1 , the fold increase ranged from 3 to 10 compared to control values. The other pluripotency-associated transcription factors also showed increased activity. We also found an approximately 3-fold SOX2 overexpression in GMNN-transfected cells (P=O.015), while STELLA-transfected cells showed a 3-fold increase in NANOG expression (P=0.012).

Conclusion

[00122] As demonstrated herein, factors preferentially expressed in oocyte are capable of inducing most of the major changes observed during embryonic deprogrammation to a certain extent. The oocyte factors studied herein induced transcription reduction, chromatin remodeling, expression of pluripotent gene markers and/or DNA demethylation. Results are recapitulated in Table 6. Those results confirm that it is possible to induce cell deprogramming events with only a few genes preferentially expressed in oocyte. Only a few DNA demethylase present in oocyte have been identified and studied, and this is the first time that overexpression of NPM2 is associated with DNA methylation loss. The present results support the use of oocyte deprogramming factors to increase the cell receptivity toward pluripotency induction to promote reset of somatic cells

[00123] Table 6. Recapitulative table of deprogramming impacts observed in HEK293 cells overexpressing one of the selected genes

Day AICDA GMNN H1FOO NPM2 PAIP1 PTTG1 STELLA

Euchromatin 3

Hetero- 3 +/- chromatin

DNA 4 methylation

RNA 3 neosynthesis

Doubling 3-

Time

OCT4 7 +

NANOG 7 +

SOX2 7

+ = increase; - = decrease [00124] The oocyte-deprogramming polypeptides and polynucleotides of the invention could thus strongly improve creation of pluripotent, multipotent or unipotent cells and, moreover, increase yield in somatic nuclear cloning.

EXAMPLE 2: Generation of pluripotent cells from HEK293 cells [00125] It is envisioned that deprogrammed HEK293 cells present characteristics and functional properties of pluripotent cells and can be maintained in culture in an undifferentiated state as follows.

HEK293 transient transfection with oocyte-deprogramming genes and derivation of ES cell-like cells

[00126] HEK293 are cultured and transiently transfected with a combination of oocyte-deprogramming genes, NPM2, STELLA and GMNN for example, cloned into pCMS-eGFP™ as described in Example 1. Following deprogramming step, cells exhibiting ES cell-like morphology (i. e. small and round shape, high nucleus-to- cytoplasm ratio, one or more large nuclei) and/or expressing Oct4, Nanog and Sox2 genes are selected to continue cultivating. To derive single-cell clones, each ES cell-like selected cell is trypsinized, plated in a well of 96-well plates and cultured on primary mouse embryo fibroblast (PMEF) monolayers in standard hES cell derivation then growth medium containing knockout-DMEM, serum replacement (SR), plasmanate, foetal bovine serum (FBS), human LIF and bFGF.

Characterization and demonstration of pluripotency of deprogrammed HEK293

Expression of pluripotent gene and surface markers expression

[00127] Expression of pluripotent gene markers is further assessed by RT-PCR amplification on Tert1 , Utf1 , Rex1 , Fgf4 genes, lmmunocytochemistry experiments are performed to detect surface markers. Briefly, ES cell-like cells are passage into Laminin- coated chamber slides and cultured for 7 days. Then cells are incubated with anti- SSEA-1 , SSEA-4, TRA-1-60 and TRA-1-81 primary antibody at 37°C for 30 min. After washes in DMEM, cells are fixed in 2% paraformaldehyde for 15 min, then washed in PBS. 5% goat serum in PBS is used as blocking solution and cells are incubated with Alexa Fluor™ 555-conjugated goat anti-mouse IgG 30 min at room temperature. Slides are mounted and clone staining is observed. Detection of alkaline phosphatase

[00128] To further characterize pluripotency markers in selected cells, detection of alkaline hosphatase is performed. Briefly, ES cell-like cells is passaged in Laminin- coated chamber slides and cultured for 7 days. Following fixation in 4% paraformaldehyde for 15 min, cells are washed in PBS and incubated with an alkaline phosphatase substrate at room temperature in the dark for one hour. After gently rinsing in 100% ethanol, slides are mounted and positive cells are selected as ES-like cells.

Analysis of deprogramming cell transcriptome

[00129] Global gene expression profiles of ES cell-like selected clones, HEK293 and hES cells are compared using Affymetrix™ technology. Microarray experiments and analysis are performed in a GeneChips™ Platform. The samples are processed following the Small Sample Labelling Protocol™ version Il from Affymetrix. Briefly, a first cycle of in vitro transcription (IVT) amplification is performed using the MEGAscript™ T7 Kit (Ambion, Austin, TX, USA), followed by a second cycle using the T7 BioArray High Yield RNA Transcript Labelling Kit™ (Enzo Diagnostics, Farmingdale USA) to produce biotinylated cRNA. Labelled cRNA are then purified, fragmented and hybridized in triplicate to the Human Genome U 133 Plus 2.0™ Array (Genechip, Affymetrix, Santa Clara, CA) for 16 h at 45°C with constant rotation. The arrays are then processed using the Affymetrix GeneChip Fluidic Station 450™ according to the manufacturer's standard protocols, and stained with streptavidin-conjugated phycoerythrin (SAPE) (Invitrogen), followed by an amplification with a biotinylated anti-streptavidin antibody (Vector Laboratories, Burlington Canada), and by a second round of SAPE. The arrays are scanned using a GeneChip Scanner 3000 G7™ (Affymetrix) and images are extracted with the GeneChip™ Operating Software (Affymetrix GCOS™ v1.2). Data are analyzed with Microarray Suite™ version 5.0 software (Affymetrix). It is expected that transcriptome of deprogrammed HEK293 cells is more similar to global gene expression in hES than in HEK293.

Embrvoid Body-Mediated Differentiation of deprogrammed cells

[00130] To test in vitro the capacity of ES cell-like selected clones to differentiate into the three embryonic germ layers, embryoid-bodies (EBs) are generated as follows: cells are incubated at 37 ⁰C for 5 min with collagenase IV (200 units/ml) into six-well plates. After washing, 2 ml of differentiation medium consisting in 20%c FBS, SR, non-essential amino acids, glutamin and /?-meraptoethanol is added. Then cells are scraped, transferred in a well of a low-attachment plate and further 2 ml of differentiation medium is added. After overnight culture in suspension, floating aggregates called EBs-like structures are expected to form. These EBs-like structures are transferred onto gelatin- coated plates and cultivated for 8 days. A variety of spontaneous differentiated somatic cell types may be yielded which exhibit morphologies resembling to neuronal cells and epithelial cells. Expression of lineage-specific markers representative of the three embryonal germ layers is assessed by RT-PCR and immunocytochemistry. /?lll-tubulin, glial fibrillary acidic protein (GFAP) (by immunocytochimie) and microtubule-associated protein 2 (MAP2), paired box 6 (PAX6) (by RT-PCR) are used as ectoderm markers; σ-smooth muscle actin (σ-SMA), desmin (by immunocytochimie) and BRACHYURY, Msh homeobox 1 (MSX1) (by RT-PCR) are used as mesoderm markers ; σ-fetoprotein (AFP) (by immunocytochimie) and forkhead box A2 (FOXA2), AFP, cytokeratin 8 and 18 (by RT-PCR) are used as endoderm markers.

Formation of teratomas

[00131] To test in vivo the pluripotency properties of ES cell-like selected clones, formation of teratomas is examined. Confluent cells are harvested by incubation in 200 units/ml of collagenase IV at 37°C for 10 min, then washed in PBS and resuspended at 1 x 1O⁸AnI in PBS. Cells are injected intramuscularly into severe-combined immunodeficiency mice (-5x10⁶ cells in 50 μl per site). It is expected to observe teratoma formation. When teratomas become visible (~ 60 to 90 days), they are excised and histological analysis are performed. The tumor is expected to contain various tissues representatives of ectoderm (nervous tissue and skin), mesoderm (muscle, bone) and endoderm (liver, intestine) origins.

[00132] Deprogrammed HEK293 cell exhibiting ES cell-like phenotype and molecular markers and able to differentiate into representative tissues of the three embryonic germ layers in vitro and in vivo are selected to continue cultivating. Such cells are defined as pluripotent cells.

Maintenance of pluripotent cell lineage generated from HEK293

[00133] In the perspectives of therapeutic applications, pluripotent cells are cultured in feeder-free and serum-free (i.e. animal-free) conditions. Such culture system has recently been reported by Amit et al. (Amit, Shariki et al. 2004). Briefly, pluripotent cells are cultured with mouse embryonic fibroblast (MEF) for several passages, and are then transferred to 50 μg per 10 cm² human cellular fibronectin-covered plates. The cells are grown on MEFs in a culture medium consisting of 85% Knockout -DMEM supplemented with 15% SR, glutamine, /?-mercaptoethanol, non-essential amino acids and bFGF. Cells are then transferred in a feeder layer-free culture system with transforming growth factor β1 (TGFβi ), LIF and bFGF. Pluripotent cells are expected to grow in these conditions for a prolonged culture and maintain their characteristics. To verify that spontaneous differentiation does not occur, periodically assessment of the cell population is performed over time. Pluripotent gene and surface marker expression and pluripotency are tested as described above.

EXAMPLE 3 : Transformation of adult fibroblast cells into dopamine neurons

[00134] It is envisioned that the present invention may be used for lineage-directed differentiation. As example, the following protocol based on Cho and colleagues's procedure may be suitable to transform fibroblast cells to dopamine neurons (Kim, Kim et al. 2007). Briefly, adult fibroblast cells are deprogrammed into pluripotent cells as described in Example 2 with modifications relatives to the different cell type used. Embryoid bodies are transferred onto Matrigel-coated plates and cultured in the presence of 0.5% N₂ supplement for the selection of neural precurseurs (NPs). Nestin (i.e. NPs marker)-positive cells are expanded with bFGF and N_2. Neural aggregates are expected to form and then they are cultured in suspension with bFGF and N₂ to form spherical neural masses (SNMs). To induce differentiation into neurons, SNMs are transferred onto Matrigel-coated plates and cultured in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Cells are then treated with ascorbic acid (AA) to induce dopamine neuron maturation. Characterization and functional analysis of the derived neurons can be performed as described in Cho et al.

EXAMPLE 4 : Use of pluripotent cell-derived cardiomvocvtes to treat heart disease [00135] It is envisioned that the deprogramming method of the present invention, as described in Example 1 , can be applied to a patient to transform patient's epidermal cells to cardiomyocytes. Such patient derived-cardiomyocytes can be transplanted into the said patient to treat his heart disease. The following protocol based on the art can be used. Firstly, a primary culture of epidermal cells can be established from the patient following the protocol described in Chai et al. (Chai, Sheng et al. 2007). A biopsy of skin is removed, washed thoroughtly and digested with 0.25 % dispase Il at 4⁰C for 10 to 16 hours. The epidermises are isolated and digested with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid for 10 min. After filtration, a cell suspension is prepared by adding serum-free DMEM and inoculated in plates. It is envisioned that protocol reported by Kehat and colleagues to differentiate hESC into cardiomyocytes can be used (Kehat, Kenyagin-Karsenti et al. 2001 ). During culture in suspension, embryoid bodiesare produced and then plated on gelatin-coated plates. Spontaneous differentiation is observed microscopically with the appearance of a contracting area in embryoid bodies 4 to 22 days after plating. To characterize cardiomyocyte-derived pluripotent cells, structural analysis by electron microscopy can be performed, expression of several cardiomyocytes specific markers can be studied (e. g. cardiac myosin heavy chain and α-actinin by immunohistochemistry, cardiac troponin T and atrial myosin light chain by RT-PCR). Functional properties can be assessed by intracellular Ca²⁺ measurements and extracellular electrophysiological recording as described in Kehat et al. An enriched population of cardiomyocytes can be obtained following strategies based on gene transfer or microenvironnement modifications.

[00136] During the last 20 years, extensive researches were accomplished on mouse ESC-derived cardiomyocytes and it was demonstrated that ESC-derived cardiomyocytes formed stable intracardiac grafts when transplanted into adult dystrophic mice hearts. In addition, mouse fetal cardiomyocytes formed a functional syncycium with the host cardiomyocytes. In human, Dai and colleagues have recently reported that human ESC- derived cardiomyocytes can survive and mature in rat heart up to 4 weeks after transplantation (Dai, Field et al. 2007). Therefore, according to the art, it is now reasonable to envision that human pluripotent cells-derived cardiomyocytes generated using the present invention could be used to cell-based therapy and transplantation. An important point is that cell graft is derived from the patient and would prevent the immunological rejection of the transplanted tissue.

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[00162] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims

CLAIMS:

1. A method for deprogramming a somatic cell, the method comprising contacting said somatic cell with a polynucleotide encoding an oocyte-deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide is expressed in the somatic cell and causes modifications to the somatic cell chromatin and/or to the somatic cell DNA.

2. A method for remodeling chromatin of a somatic cell, the method comprising contacting said somatic cell with a polynucleotide encoding an oocyte-deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide is expressed in the somatic cell and remodels the cell chromatin.

3. The method of claim 1 or 2, wherein said polynucleotide encoding an oocyte- deprogramming polypeptide is selected from the group consisting of STELLA, PAIP1 , PTTG1 , GMNN, H1 FOO, NPM2 and combinations thereof.

4. The method of claim 3, wherein said combination is is selected from the group consisting of: i) NPM2 and AICDA; ii) NPM2 and STELLA; iii) NPM2 and GMNN; iv) NPM2, AICDA and STELLA; v) NPM2, GMNN and STELLA; vi) NPM2, AICDA and GMNN; and vii) NPM2, AICDA, GMNN and STELLA.

5. The method of any one of claims 1 to 4, wherein said contacting comprises introducing into said somatic cell an expression vector comprising a polynucleotide encoding said oocyte-deprogramming polypeptide.

6. The method of any one of claims 1 to 5, wherein expression of the oocyte- deprogramming polypeptide in the somatic cell causes one or more of the following:

- reduction of RNA neosynthesis when compared to a somatic cell in which the oocyte-deprogramming polypeptide is not expressed; - modification(s) in acetylation, methylation and/or demethylation of histones; - modification(s) in methylation patterns of the somatic cell chromatin and/or somatic cell DNA;

- reactivation of expression of one or more pluripotent gene markers.

7. The method of any one of claim 6, wherein the pluripotent gene marker is selected from the group consisting of NANOG, 0CT4, and SOX2.

8. A cell line obtained using any one of the methods of claims 1 to 7.

9. A cell positive for expression of a pluripotent gene marker, wherein said cell comprises an exogenous polynucleotide encoding an oocyte-deprogramming polypeptide.

10. The cell of claim 9, wherein said cell transiently expresses said oocyte- deprogramming polypeptide.

11. The cell of claim 9 or 10, wherein said pluripotent gene marker is selected from the group consisting of NANOG, OCT4, and SOX2.

12. A method for conditioning a somatic cell to differentiation into a pluripotent, multipotent or unipotent cell, the method comprising contacting a somatic cell with a polynucleotide encoding an oocyte-deprogramming polypeptide, wherein the oocyte- deprogramming polypeptide is expressed into the somatic cell, thereby deprogramming and conditioning the somatic cell to differentiation into a pluripotent, multipotent or unipotent cell.

13. A method for transforming a somatic cell from a first cell type to a second cell type showing morphological and functional characteristics of a different specific cell lineage, comprising: - deprogramming a somatic cell according to the method of any one of claims 1 ,

3, 4, 5 and 6, wherein said somatic cell is from a first cell type; and

- exposing the somatic cell from the first cell type to at least one differentiation factor and/or to at least one transcription factor; wherein said at least one factor is selected for inducing development of morphological and functional characteristics specific of the second cell type, and wherein said somatic cell is exposed to said at least one factor for a sufficient period of time and under suitable conditions to obtain said morphological and functional characteristics.

14. The method of claim 13, wherein the somatic cell from a first cell type is selected from the group consisting of dermal fibroblasts, epidermal cells, keratinocytes, hair outer root sheath cells, and peripheral blood monocytes.

15. The method of claim 13 or 14, wherein the second cell type is selected from the group consisting of Sertoli cells, endothelial cells, granulosa epithelial, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B lymphocytes T lymphocytes, erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, muscle cells and cardiac cells.

16. A method for obtaining a pluripotent cell having desirable morphological and functional characteristics, comprising: - deprogramming a somatic cell according to the method of any one of claims 1 ,

3, 4, 5 and 6; and

- exposing the somatic cell to at least one pluripotent factor; wherein said at least one pluripotent factor is selected for inducing development of said desirable morphological and functional characteristics, and wherein said somatic cell is exposed to said at least one pluripotent factor for a sufficient period of time and under suitable conditions to obtain said morphological and functional characteristics.

17. A method for deprogramming a somatic cell, the method comprising contacting chromatin and/or DNA of said somatic cell with an oocyte-deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide causes modifications to the somatic cell chromatin and/or to the somatic cell DNA.

18. A method for remodeling chromatin of a somatic cell, the method comprising contacting chromatin of said somatic cell with an oocyte-deprogramming polypeptide, wherein the oocyte-deprogramming polypeptide remodels the cell chromatin.