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WO2009018832A1 - Method for increasing the plasticity level of a cell - Google Patents

Method for increasing the plasticity level of a cell Download PDF

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Publication number
WO2009018832A1
WO2009018832A1 PCT/DK2008/050197 DK2008050197W WO2009018832A1 WO 2009018832 A1 WO2009018832 A1 WO 2009018832A1 DK 2008050197 W DK2008050197 W DK 2008050197W WO 2009018832 A1 WO2009018832 A1 WO 2009018832A1
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cell
factor
cells
gene
plasticity
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PCT/DK2008/050197
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French (fr)
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Roberto Oliveri
Claus Yding Andersen
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Rigshospitalet
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Publication of WO2009018832A1 publication Critical patent/WO2009018832A1/en

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the reprogramming of partly or fully differentiated mammalian cells, so that they can act like stem cells.
  • the present invention provides a method for such reprogramming by increasing the plasticity level of a cell through changes in the pattern of gene expression in the partly or fully differentiated cell.
  • the present invention relates to plasticity modifying factor(s), said plasticity modifying factor(s) being selected from the group consisting of plasticity modifying factors increasing DNA demethylation, plasticity modifying factors inhibiting maintenance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones.
  • any type of cell depends on the specific set of genes expressed (e.g. transcribed and translated). Accordingly, development of cells exhibiting a high plasticity level (i.e. pluripotent or totipotent cells) into more specialized phenotypes exhibiting a decreased plasticity level (for example end- stage-committed cells) is determined by the type of genes expressed during development. Gene expression is directly mediated by sequence specific binding of gene regulatory proteins that can effect positive or repressive regulation. The ability of any of these regulatory proteins to directly mediate gene expression however, appear to depend on the accessibility of their binding site within the cellular DNA.
  • oogenesis In mammals, the culmination of oogenesis is the creation of a mature metaphase II (Mil) oocyte capable of sustaining fertilization, reprogramming of the parental genomes, and supporting early embryonic development.
  • the oocyte cytoplasm rapidly starts to modify the sperm genome upon fertilization resulting in decondensation of paternal chromatin, substitution of protamines with highly acetylated histones, and active demethylation of DNA (Santos et al. 2002; Spinaci et al. 2004).
  • the oocyte-derived maternal chromatin seems more protected from this extensive reprogramming activity undergoing DNA demethylation at a more passive level during subsequent cleavages.
  • the observed asymmetry in parental genome reprogramming is believed to result from marked differences in the original nucleosomal structure of the maternal and paternal chromatin, respectively.
  • a condensed chromatin structure is associated with an inaccessible and gene repressive state whereas more decondensed and open chromatin is more amenable to gene activation (Turner 2001).
  • transcription levels may be affected by covalent modifications of the DNA helix and core histones comprising the nucleosome, the core unit of chromatin.
  • DNMTs DNA methyltransferases
  • MeCPs methyl-CpG-binding proteins
  • HATs histone acetyltransferases
  • HDACs histone deacetylases
  • HDMs histone demethylases/deiminases
  • Covalent DNA methylation which is the most abundant form of epigenetic modification, occurs at cytosines in the pyrimidine ring of CpG dinucleotides dispersed throughout the genome and is generally associated with gene silencing.
  • the repressive effect of DNA methylation is mediated by the MeCPs, which again recruit a wide range of co-repressors, including HDACs.
  • Nucleosome core histones are covalently modified at their N-terminal tails, which are highly charged and tightly associated with the DNA helix.
  • acetylation and methylation have been associated with regulation of gene expression and it has been suggested that the vast number of combinations may actually form a specific histone code, which could determine the binding patterns of regulatory proteins.
  • ChRs do not interact covalently with DNA in a sequence specific manner but are rather recruited to promoters of target genes by sequence specific transcription factors.
  • the catalytic sub-units of the ChRs (ChR- ATPases) cause an altered conformation and position of nucleosomes by ATP- hydrolysis. This leads to weakened DNA-histone contacts resulting in histone octamers changing positions along the DNA helix, thus either increasing or reducing the binding of transcription factors to promoter regions.
  • the groups of proteins important to epigenetic modifications and chromatin remodeling do not act independently, but are closely coordinated, often by forming large multiprotein complexes.
  • the highly specialized ability of the Mil oocyte cytoplasm to reprogram a sperm genome extends to a comparable level of reprogramming of a diploid somatic nucleus from adult tissue, i.e. somatic cell nuclear transfer (SCNT) or cloning.
  • SCNT somatic cell nuclear transfer
  • GV germinal vesicle
  • Takahashi et al. 2006 demonstrated induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four transcription factors Oct3/4, Sox2, c-Myc and Klf4 under ES cell culture conditions.
  • the use of retroviral expression systems resulted in only a small fraction of cells expressing the four transcription factors to become induced pluripotent stem cells (iPS).
  • iPS induced pluripotent stem cells
  • Wernig et al. 2007 reported that reprogramming of fibroblasts to a pluripotent state may be induced in vitro through ectopic expression of the four previously mentioned transcription factors (Oct4 (Oct3/4 or Pou5fl), Sox2, c-Myc and Klf4).
  • the authors showed that the DNA methylation, gene expression and chromatin state in induced reprogrammed cells were similar to those of ES-cells.
  • the inventors have investigated further factors involved in early embryonic reprogramming. They discovered and identified a number of genes involved in epigenetic reprogramming.
  • the present invention relates to the construction of a cell having an increased level of plasticity.
  • a cell is termed a "supraplastic cell" and is capable of differentiating into a variety of end-stage-commited cell types, including the cell types similar to the cell type of the donor cell from which the supraplastic cell was derived and cell types different from the cell type of the donor cell from which the supraplastic cell was derived.
  • the present invention describes a method for increasing the plasticity level of a donor cell, said method comprising providing a mammalian, normoploid somatic cell as the donor cell and increasing the plasticity level in said cell by contacting said donor cell with, or introducing into said donor cell, one or more plasticity modifying factor(s), said plasticity modifying factor(s) being selected from the group consisting of plasticity modifying factors increasing DNA demethylation, plasticity modifying factors inhibiting maintenance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones, thereby obtaining a supraplastic cell with the ability to develop into at least two different end-stage-commited cell types.
  • the invention relates to a supraplastic cell obtained by any of the methods described herein.
  • Such supraplastic cell may be used in several different aspects.
  • the supraplastic cell may be used for various kinds of cell therapy.
  • One type of cell therapy comprises using the supraplastic cell or cells derived from a supraplastic cell for cell replacement by transplantation, for example replacement of bone marrow in patients suffering from diseases and conditions affecting hematopoietic and mesenchymal stem cells in disorders such as anemia, hypovolemic blood loss, hemoglobinopathies, and platelet disorders.
  • Cell therapy may as described in the present invention also comprise delivery of natively and non-natively produced compounds in situ in patients, said compounds being produced by the transplanted supraplastic cell or cells derived therefrom.
  • the present invention discloses a method for increasing the plasticity level in a donor cell by introducing one or more plasticity modifying factor(s), it is an aspect of the present invention to provide a composition for mediating an increase in the plasticity level of a donor cell, said composition comprising one or more plasticity modifying factors.
  • the supraplastic cell is used for the production of target cells exhibiting the phenotype of end-stage-committed cell types including unipotent pre-cursor cells.
  • Stem cells are the master cells that replenish all the body's tissues, from skin and blood to the brain and heart. In children and adults, particular types of stem cells are distributed throughout the body, and are specialized to create particular types of tissues. However, in the early embryo, inner cell mass cells can make derivatives of all three primary germ layers including germ cells, a quality known as pluripotency.
  • the "gold standard" test for pluripotency is the ability of a cell to contribute extensively to all adult cell types, including the germ line.
  • the goal for epigenetic reprogramming or production of stem cells or stem-like cells is mainly for regenerative medicine purposes and for understanding the molecular mechanisms behind cellular differentiation and de-differentiation. It is of course preferred to do this in e.g. murine cells for ethical reasons.
  • the method to assess pluripotency includes as explained injecting these cells into early mouse embryos and observe that these cells differentiate into all normal adult cell types.
  • formation of teratomas in severe combined immunodeficient (SCID) mice and/or embryoid body formation in vitro is considered proof of pluripotency (Thomson et al. 1998).
  • SCID severe combined immunodeficient
  • the present inventors describe various plasticity modifying factors that enable reprogramming of partly or fully differentiated cells so they can act like stem cells or stem-like cells.
  • pluripotency can be induced in mouse embryonic or adult fibroblasts by introducing four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4, under embryonic stem (ES) cell culture conditions (Takahashi et al. 2006).
  • the present invention provides an alternative method for such reprogramming by increasing the plasticity level of a cell through changes in the pattern of gene expression by use of new plasticity modifying factors.
  • the present application describes methods for increasing the plasticity level of a donor cell, said method comprising, providing a donor cell and increasing the plasticity level in said cell by contacting said cell with, or introducing into said cell, one or more plasticity modifying factor(s), said plasticity modifying factor(s) being selected from the group consisting of plasticity modifying factors increasing DNA demethylation, plasticity modifying factors inhibiting maintenance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones, thereby obtaining a supraplastic cell.
  • the one or more plasticity modifying factor(s) is selected from the group consisting of plasticity modifying factors increasing DNA demethylation and plasticity modifying factors inhibiting maintainance DNA methylation. In another embodiment, the one or more plasticity modifying factor(s) is selected from the group consisting of plasticity modifying factors increasing DNA demethylation and plasticity modifying factors mediating hyperacetylation of histones.
  • the one or more plasticity modifying factor(s) is selected from the group consisting of plasticity modifying factors inhibiting maintainance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones.
  • the plasticity modifying factor is a factor increasing DNA demethylation.
  • the plasticity modifying factor is a factor i inhibiting maintainance DNA methylation.
  • the plasticity modifying factor is a factor foring hyperacetylation of histones.
  • the present invention relates to a method for creating a stem cell comprising, obtaining a partly or fully differentiated donor cell from an individual, inducing in said cell epigenetic reprogramming by altering the level of expression of mediators of covalent modifications of DNA and/or histones and/or mediators of chromatin remodelling, and introducing the resulting stem cell into an individual by transplantation.
  • the invention thus also relates to use of a transplanted supraplastic cell or a cell derived herefrom for in vivo delivery of a therapeutic compound, said cell either natively producing the compound or being engineered by gene technology to produce the compound.
  • Plasticity a transplanted supraplastic cell or a cell derived herefrom for in vivo delivery of a therapeutic compound, said cell either natively producing the compound or being engineered by gene technology to produce the compound.
  • plasticity refers to the ability of a cell to develop into at least two end- stage-committed cell types. It is the object of the present invention to provide a donor cell and increasing in said donor cell the level of plasticity to obtain a supraplastic cell. Depending on the increase in the level of plasticity, the supraplastic cell may develop into a variety of target cell types.
  • the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives from one of the three germ layers including germ cells.
  • the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives from one of the three germ layers.
  • the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives from one of the representing two germ layers.
  • the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives of the three or two germs layers.
  • one of said germ layers may be of the same developmental origin as the donor cell from which the supraplastic cell was obtained.
  • the supraplastic cell has the ability of differentiating into target cells derived from one germ layer, said germ layer may be of the same developmental origin as the donor cell from which the supraplastic cell was obtained.
  • plasticity refers to the ability of a cell to differentiate into at least one end-stage-committed cell type different from the cell type from which said cell originated.
  • plasticity level refers to the number of end-stage-commited cell types into which said cell is capable of differentiating.
  • a totipotent stem cell comprises a higher plasticity level than a pluripotent stem cell
  • a totipotent stem cell in the present context refers to a cell capable of dividing and producing all types of differentiated cells in an organism, but also the strict extra-embryonic tissue (i.e. outer chorion)
  • a pluripotent stem cell in the present context refers to a cell capable of differentiating into all cell types comprising the adult human body including some tissues of the placenta (inner chorion, yolk sac, amnion, allantois), but not the strict extra-embryonic tissues derived from trophoblast cells (i.e. outer chorion).
  • the pluripotent stem cell thus comprises a higher plasticity level than a multipotent stem cell, which in the present context refers to a cell capable of dividing and producing only cell types representing the same germ layer, e.g. the hematopoietic stem cell within the mesoderm.
  • a method for increasing the plasticity level of a donor cell provides the potential of epigenetic reprogramming.
  • epigenetic reprogramming refers to the erasure and remodeling of epigenetic marks, such as, but not limited to, covalent DNA methylation, covalent histone modifications and non-covalent chromatin remodeling.
  • reprogram refers to the phenomenon, in which a partly or fully differentiated donor cell acquires an increase in the level of plasticity, hence becomes multipotent, pluripotent or totipotent.
  • Donor cells may be reprogrammed to varying degrees as it is possible that an individual donor cell is reprogrammed to be multipotent or pluripotent or even totipotent.
  • the increased level of plasticity obtained by the method disclosed in the present invention may vary and may thus be increased into different levels.
  • reprogrammed cell refers to a cell that had formerly attained a particular degree of differentiation i.e. a particular level of plasticity, but has subsequently regained the ability to differentiate into at least two types of specialized cells (e.g., has become multipotent, pluripotent or totipotent), it has been de-differentiated. It is in general highly unlikely that differentiated cells (i.e cells comprising a decreased level of plasticity) will revert into their precursor cells (i.e., reprogram) in vivo. However, using the method of the present invention, differentiated cells may be reprogrammed or de-differentiated into multipotent, pluripotent or totipotent cells both in vitro and in vivo.
  • differentiated cell and “cell comprising a decreased level of plasticity” are used interchangeably to refer to a cell comprising a poorer ability of differentiating into different specialized cell types when compared to its counterparts termed "undifferentiated cell”, “supraplastic cell” and “cell exhibiting an increased level of plasticity” (the latter terms also being used herein interchangeably).
  • the plasticity level of said cell is compared to the plasticity level of the donor cell by subjecting said supraplastic and donor cell to one or more differentiation protocols and determining the number and/or type of target cells into which the supraplastic cell and donor cell as well as cells derived herefrom are able to differentiate.
  • a cell is determined as having an increased level of plasticity (i.e. to be a supraplastic cell), if the number of target cell types into which said cell may develop is larger than the number of target cell types into which the donor cell may develop; or if a supraplastic cell derived from a donor cell is capable of developing into at least one type of target cell into which the donor cell cannot develop.
  • differentiated cell refers to a cell that has developed from a relatively unspecialized phenotype (e.g. multipotent stem cell) to a more specialized phenotype (e.g. nerve cell, pancreatic beta-cell, cardimyocyte).
  • a relatively unspecialized phenotype e.g. multipotent stem cell
  • a more specialized phenotype e.g. nerve cell, pancreatic beta-cell, cardimyocyte
  • an increase in the plasticity level is desirable, as a cell exhibiting a high level of plasticity has the potential to develop into a number of different cell types, i.e. shows an increased differentiation potential or developmental potential.
  • the present inventors have investigated several factors involved in early embryonic reprogramming, and thus in one embodiment relates to one or more plasticity modifying factor(s) increasing DNA demethylation.
  • plasticity modifying factor(s) increasing DNA demethylation.
  • examples given herein comprises MBD2 factor(s) and MBD4 factor(s)
  • the MBD2 factor is selected from the group consisting of DNA sequences encoded by the MBD2 gene, proteins encoded by the MBD2 DNA sequence, compounds increasing the transcription of the native MBD2 gene, compounds increasing the translation of MBD2 mRNA species and compounds increasing the activity of the proteins encoded by the MBD2 gene.
  • MBD2 also covers any well known variants (e.g. MBD2a, MBD2b and MBD2t) as well as any other putative splice variant isoforms hereof.
  • the MBD4 factor is selected from the group consisting of DNA sequences encoded by the MBD4 gene, proteins encoded by said DNA sequence, compounds increasing the transcription of the native MBD4 gene, compounds increasing the translation of MB42 mRNA species and compounds increasing the activity of the proteins encoded by the MBD4 gene.
  • MBD4 covers any well known variants as well as any other putative splice variant isoforms hereof.
  • the one or more plasticity modifying factor(s) inhibiting maintenance DNA methylation comprise DNMTl factor(s).
  • the DNMTl factor is selected from the group consisting of compounds decreasing the transcription of the native DNMTl gene, compounds decreasing the translation degradation of DNMTl mRNA species, compounds increasing the translation degradation of DNMTl mRNA species and compounds inhibiting the DNA methyltransferase activity of the proteins encoded by DNMTl .
  • the term "DNMTl” also covers any well known variants (e.g. DMNTIs and DMNTIo) as well as any other putative splice variant isoforms hereof.
  • the compounds decreasing the translational degradation of DNMTl mRNA species does according to the present invention also include siRNA and microRNA.
  • DNMTl, DNMT3a, DNMT3b (DNA methyltransferase 1, 3a, and 3b) carry out the process of DNA methylation wherein DNMTl is the proposed maintenance DNA methyltransferase that is responsible for replicating DNA methylation patterns to the daughter strands during DNA replication.
  • the present invention relates to epigenetic reprogramming or de-differentiation by increasing the plasticity level of a donor cell, such as inducing pluripotency through plasticity modifying factors.
  • plasticity modifying factors of the present invention such as but not limited to MBD2 factor(s), MBD4 factor(s) and DNMTl factor(s) is used in combination with any of the factors Oct4, Sox2, c-Myc, and Klf4 for the purpose of de-differenctiation.
  • the present invention relates to epigenetic reprogramming by increasing the plasticity level of a donor cell contacting said donor cell with, or introducing into said donor cell, one or more DNMTl factor(s) and/ or MBD2 /actor(s) and/or MBD4 factor(s) combined with the induction of expression of any of the transcription factors selected from the group consisting of OCT4, SOX2, C- MYC and KLF4.
  • the one or more plasticity factors mediating hyperacetylation of histones is encoded by one or more of the genes selected from the group of HATs consisting of but not limited to ATF2, CDYL,
  • the one or more plasticity modifying factor(s) disclosed in the present invention may be selected from the group consisting of small chemical entities, peptides, proteins, RNA species including but not limited to siRNA, microRNA, shRNA, peptide aptamers, modified nucleic acids and DNA species.
  • RNA species including but not limited to siRNA, microRNA, shRNA, peptide aptamers, modified nucleic acids and DNA species.
  • an inhibition of maintenance DNA methylation or mediate hyperacetylation of histones the level of gene expression of the genes encoding DNA demethylation, DNA methylation, or hyperacetylation of histones may be altered at the DNA or mRNA level.
  • the plasticity modifying factor is directed towards the mRNA of the mediators of covalent modifications of DNA and histones and mediators of chromatin remodeling, the plasticity modifying factor being selected from the group consisting of an antisense oligonucleotide, a siRNA, shRNA and microRNA.
  • the production of the mediators of covalent modifications of DNA and histones and mediators of chromatin remodeling is targeted.
  • said plasticity modifying factor may either increase or decrease the expression of the genes encoding the mediators of covalent modifications of DNA and histones and mediators of chromatin remodelling. It is an object of the present invention to increase the level of gene expression of MBD2 and/or MBD4 whereas it is an object to decrease the level of gene expression of DNMTl .
  • a preferred plasticity modifying factor is an antisense oligonucleotide that may be specifically directed to the mRNA encoded by the gene of interest by way of complementarity.
  • the antisense oligonucleotide is capable of mediating RNase H cleavage of the target mRNA.
  • the antisense oligonucleotide comprises a stretch of DNA residue and/or DNA analogue residues to make the mRNA/DNA hybrid substrate for RNase H.
  • the antisense oligonucleotide is of the so-called gapmer structure, which is well known to a person skilled in the art in the field of antisense oligonucleotides.
  • siRNAs are short double stranded RNA complexes of typically 20-22 nucleotides with a overhang of 1-2 nucleotides at the 3'end. siRNAs are capable of activating a sophisticated cellular machinery ultimately leading to degradation or translational inhibition of mRNAs that are complementary to the guide strand of the siRNA complex. siRNAs are typically not produced in the cell, but are introduced into the cell to mediate RNAi (RNA interference).
  • RNA interference RNA interference
  • shRNAs short hairpin RNAs
  • shRNA may be produced from a gene introduced into a cell and may thus be viewed as vector mediated RNAi.
  • MicroRNAs are small (typically 21-23 nucleotides in length) and noncoding RNAs which regulates gene expression by mediating sequence-specific repression of mRNA translation. miRNA may be overexpressed in a cell using transfection of synthetic miRNAs or miRNA-expressing plasmids or by down- regulation using transfection of miRNA inhibitors.
  • the plasticity modifying factor interferes with the mediators of covalent modifications of DNA and histones and mediators of chromatin remodelling production at the protein level.
  • said plasticity modifying factor is selected from the group consisting of a protein such as but not limited to a monoclonal antibody and a polyclonal antibody, an aptamer, a peptide, and a small chemical entity acting as inhibitor or activator of protein function. It is an object of the present invention to increase the level of activity of products encoded by MBD2 and/or MBD4 whereas it is an object to decrease the level of activity of products encoded of DNMTl .
  • the mediator of covalent modification of DNA, covalent modification of histones and/or modification of chromatin remodelling may be encoded by the genes selected from the group consisting of DNMTl, DNMT3A, DNMT3B, MBDl, MBD2, MBD4, MECP2, ZBTB33, ZBTB38, ZBTB4, ATF2, CDYL, CREBBP, ELP3, EP300, GCN5L2, GTF3C1, HATl, HTATIP, MYSTl, MYST2, MYST3, MYST4, NCOAl, NCOA2, NCOA3, OGT, PCAF, TAFl, HDACl, HDAC2,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7A, HDAC8, HDAC9, HDAClO, HDACIl, SIRTl, SIRT2, SIRT3,
  • an embodiment of the present invention discloses that the level of expression of at least one of the genes MBD2 and/or MBD4 or any well known variants ⁇ MBD2a, MBD2b, MBD2t), as well as putative splice variant isoforms hereof is increased.
  • the level of expression of the genes selected from the group consisting of DN MT3 A, DNMT3B, MBD2, MBD4, ZBTB33, ATF2, CDYL, EP300, NCOAl, NCOA2, OGT, TAFl, HDAC2, HDAC3, HDAC9, SIRTl, SIRT4, DOTlL, EHMTl, PRMTl, PRMT3, SETD2, SETD8 and SUV39H2 are increased.
  • the level of expression of the genes selected from the group consisting of MBD2, MBD4, ZBTB33 and SIRT4 are increased.
  • the level of expression of the gene DNMTl or any well known variants (DNMTIs and DMNTIo), as well as putative splice variant isoforms hereof, is repressed.
  • the level of expression of the genes selected from the group consisting DMNTl, MBDl, ELP3,GCN5L2, HTATIP, MYSTl, HDAC6, SIRT5, SIRT7, PRMT7, SMYD3, SUV39H1, SMARCA2, SMARCA4 are repressed.
  • donor cell refers to a normoploid somatic cell from the group consisting of end-stage-committed cell types including unipotent pre-cursor cells, and multipotent stem cells, e.g. isolated from adults, newly born and foetuses.
  • an end-stage-committed somatic cell belonging to any of the three germ layers may be isolated using techniques known in the art, and is selected from the group comprising of keratinizing epithelial cells, wet stratified barrier epithelial cells, exocrine secretory epithelial cells, hormone secreting cells, gut-, exocrine-, glands- and urogenital tract cells, metabolism and storage cells, barrier function cells (lung, gut, exocrine glands and urogenital tract), epithelial cells lining closed internal body cavities, ciliated cells with propulsive function, extracellular matrix secretion cells, contractile cells, blood and immune system cells, sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells and nurse cells.
  • Presently preferred cells are cells readily obtainable i.e. cell which may be obtained without penetration of the body. Such cell may be but is not limited to epithelial cells,
  • an adult stem cell refers to a partly differentiated cell characterized by its ability to divide or self-renew indefinitely and generate a number of cell types of the same germ layer from which it originates.
  • Adult stem cells may be found throughout the foetal or adult body and may thus be obtained from already developed tissue using techniques known in the art, said tissues being selected from the group consisting of, but not limited to, brain tissue, heart tissue, adipose tissue, skin, liver and bone marrow tissue.
  • the adult stem cells of the present invention are obtained from adipose tissue, skin and bone marrow tissue.
  • the source of the donor cell of the present invention comprises all mammalian species. In one embodiment, the source of the donor cell is human.
  • the isolated donor cell itself may exhibit a range of plasticity levels. Accordingly, the donor cell of the present invention may be selected from the group consisting of end-stage-committed cell types including unipotent pre-cursor cells, and multipotent stem cells, e.g. isolated from adults, newly born and foetuses.
  • the donor cell of the present invention is a cell, in which the plasticity level is to be increased.
  • supraplastic cell which refers to a donor cell in which the plasticity level has been increased.
  • target cell relates in the present context to a cell developed by differentiation from a supraplastic cell.
  • Target cells may for instance exhibit a phenotype different from the phenotype exhibited by the donor cell from which the supraplastic cell was derived.
  • the target cell may be selected from the group consisting of end-stage-committed cells including unipotent pre-cursor cells, oligopotent cells, multipotent cells, and pluripotent cells.
  • End-stage-committed cell refers to all differentiated cell types including tissue-specific unipotent pre-cursor cells with the ability to generate by proliferation and differentiation the specific type of tissue.
  • plasticity modifying factor(s) into a cell
  • introduction of one or more plasticity mediating factor(s) into a cell and the induction of epigenetic reprogramming may be performed employing various methods. Accordingly, the terms introduced herein will be used interchangeably.
  • plasticity modifying factors presented in the present application may be mediated through any means for regulating gene expression, such as, but not limited to, transfection of naked DNA, viral vectors (both episomal and integrative), antisense oligonucleotides, siRNA, shRNA, microRNA, antibodies and aptamers into the cell. Said transfection may either increase or repress the level of selected factors.
  • the viral vector and/or plasmid vector are employed to deliver gene constructs such as a promoter/enhancer, a reporter gene and/or selection marker into the cell.
  • Transfection may also be performed by various methods all well known to the person skilled in the art. Said methods 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 for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into the cell, which is to be reprogrammed.
  • the transfection may be either transient or stable.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vesicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery into the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, nakes DNA, artificial virolons and agent enhanced uptake of DNA.
  • the level of reprogramming or increase in plasticity may be assessed by various methods all of which are known to the skilled person.
  • the methods includes but are not limited to the measurement of global DNA methylation patterns (e.g. using methylation chips), down regulation of cell type-specific markers and up- regulation of pluripotency markers.
  • the supraplastic cells of the present invention may e.g. be characterised by biological markers for cellular multipotency, pluripotency or totipotency, e.g any of these pluripotency markers such as, but not limited to, NANOG, Oct-4 (POU5F1), S0X2, or REXl.
  • Example 17 demonstrates that altering the expression profiles of one or more of the genes (MBD2, MBD4 and DNMTl) may be a valid method of increasing the plasticity of cells through an upragulation of Oct-4 (POU5F1),
  • the cells disclosed in the present invention are mammalian cells. However, in a presently preferred embodiment the cells are human cells.
  • the present invention describes a method for cell therapy based upon transplantation of supraplastic cells or derivatives of the supraplastic cells for cell or tissue replacement or for delivery of compounds produced by the transplanted cells following transplantation into an individual.
  • differentiated derivatives of human embryonic stem cell lines for cell-based therapy involving transplantation of the cells to an individual with the aim of either replacing lost cells or using the transplanted cells to deliver biologically active compounds to an individual.
  • the pluripotency of embryonic stem cell represents a risk of the recipient developing tumours originating from the embryonic stem cells, socalled teratomas. It is therefore desireable that the cells to be used in such transplation- based treatment have a desired differentiation potential, which precludes the risk of teratoma formation.
  • the present invention allows for therapeutic use of supraplastic cells or derivatives thereof with various degrees of differentiation potential compared to embryonic cells.
  • the supraplastic cells of the present invention may be used for the production of derivatives of the supraplastic cell, such derivatives comprising a target cell with a desired phenotype as well as a partly differentiated supraplastic cell.
  • derivatives of supraplastic cells may be used as described above in transplantation therapy for cell replacement and/or tissue regeneration or for delivery of compounds into an individual of compounds produced by the transplanted cells.
  • An additional problem in cell-based therapy relates to the need for histocompatibility between the individual receiving a transplantation and the cells being transplanted to the individual.
  • the proposed therapeutic uses of non-histocompatible fetal stem cells in cell replacement treatment of Parkinson's disease or in cell delivery of brain cell growth factors to cure amyotrophic lateral sclerosis is only feasible due to the fact that the target tissue (brain) is immunologically sequestered.
  • the invention allows for therapeutic use of fully histocompatible, i.e. autologous, supraplastic cells and cells derived from the supraplastic cells exhibiting various degrees of differentiation potential compared to embryonic cells thus broadening the range of diseases and conditions amenable to cell-based therapy.
  • autologous stem cells may be isolated from tissues such as bone marrow from an adult individual, the number of such cells is low, and the adult stem cells are difficult to isolate for expansion. Importantly, as described in this application, this quantitative hurdle may be overcome by employing the strategy of epigenetic reprogramming of partly or fully differentiated cells from the individual to be treated.
  • Another source of autologous stem cells is umbilical cord blood, umbilical cord tissues as well as amniotic fluid, amnion epithelium, and placenta sampled at the time of birth or by means of ante-natal diagnostic invasive procedures and kept in storage under suitable conditions until the individual in question requires cell- based therapy.
  • the use of such cells depends upon their suitability for therapy after prolonged storage, and the use is limited to individuals who have had samples of umbilical blood, umbilical cord tissues or amniotic fluid taken at the time of birth or ante-natal diagnostic procedures.
  • the present invention relates to a method for cell therapy in which the transplantation of supraplastic cells or derivatives thereof is done to restore a tissue such as, but not limited to, bone marrow, liver, skin or cartilage.
  • the cells for replacement therapy may be transplanted as such by intravenous injection or by depositing the cells into a specific tissue, or the cells may be transplanted as a partly or fully developed tissue.
  • transplantation of target cells as described herein for delivery of compounds produced natively by the transplanted target cells or transplantation of target cells for delivery of compounds not natively produced by the transplanted target cells is envisaged.
  • Stem cells are known to be able to remain in a given tissue and to produce growth factors, signal molecules etc., which may effect proliferation and differentiation of neighbouring cells. By providing such conditioning factors to a tissue it is believed that the naturally occuring adult stem cells may be stimulated so that new tissue is produced.
  • the transplantation of supraplastic cells or cells derived herefrom is aimed at depositing the cells in a given tissue such as, but not limited to, cardiac muscle or brain tissue in order to attain delivery to the surrounding tissue of compounds such as growth factors, signal molecules etc. produced natively by the supraplastic cells or cells derived herefrom of the present invention.
  • the ability of stem cells to remain in a given tissue may also be used to deliver compounds that are not normally produced by the stem cells by genetically engineering the stem cells prior to transplantation to produce such desired compounds as growth factors, signal molecules, enzymes etc.
  • the supraplastic cells are genetically engineered prior to transplantation to produce compounds not natively produced by the supraplastic cells or cells derived herefrom, and the transplantation of the genetically engineered supraplastic cells or cells derived herefrom is aimed at depositing the cells in a given tissue such as, but not limited to, cardiac muscle or brain tissue in order to attain delivery to the surrounding tissue of compounds such as growth factors, signal molecules etc. produced by the genetically engineered supraplastic cells or cells derived herefrom of the present invention.
  • altering the level of expression of the one or more plasticity modifying factor(s) of covalent modifications of DNA, histones and/or chromatin remodelling covers a situation wherein the level of gene expression is either activated/increased (up-regulated) and/or repressed/decreased (down-regulated).
  • Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product may 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.
  • Gene activation refers to any process which results in an increase in production of a gene product.
  • a gene product may be either RNA (including, but not limited to, mRNA, rRNA, tRNA, and structural RNA) or protein.
  • gene activation includes those processes which increase transcription of a gene and/or translation of a mRNA. Examples of gene activation processes which increase transcription include, but are not limited to, those which facilitate formation of a transcription initiation complex, those which increase transcription initiation rate, those which increase transcription elongation rate, those which increase processivity of transcription and those which relieve transcriptional repression (by, for example, blocking the binding of a transcriptional repressor).
  • Gene activation can constitute, for example, inhibition of repression as well as stimulation of expression above an existing level.
  • Examples of gene activation processes which increase translation include those which increase translational initiation, those which increase translational elongation and those which increase mRNA stability.
  • gene activation comprises any detectable increase in the production of a gene product.
  • the production of a gene product is increased by at least 2-fold, e.g 5-fold, such as 10- fold, e.g 20- fold, such as 30- fold, e.g 40-fold, such as 50- fold, e.g 60-fold, such as 70- fold, e.g 80-fold, such as 90- fold, e.g 100-fold.
  • the term "increased level of expression” refers to a situation wherein the level of expression of a specific gene in a cell is increased when compared to the level of gene expression in a similar cell comprising the same genotype.
  • Determining whether the level of expression of a specific gene in a cell is increased may be measured by the following steps:
  • identifying a cell as having an increased level of expression of a specific gene of interest if the second cell compared to the first cell show an increased level of expression of at least 1%, e.g. 5%, 10%, 20%, 50% or 75%.
  • the level of gene expression may be measured by several different methods all which are known to the skilled person. The methods include e.g. Northern Blotting and quantative RT-PCR. Alternatively, if the level of expression of one or more genes is of interest DNA microarray technology or "tag based" technologies like SAGE or SuperSAGE can provide a relative measure of the cellular concentration of the mRNAs of interest.
  • the term "repressed level of expression” refers to a situation wherein the level of expression of a specific gene in a cell is down- regulated when compared to the level of gene expression in a similar cell comprising the same genotype.
  • Determining whether the level of expression of a specific gene in a cell is repressed may be measured by the following steps:
  • identifying a cell as having an repressed level of expression of the specific gene of interest if the second cell compared to the first cell show an repressed level of expression of at least least 1%, e.g. 5%, 10%, 20%, 50% or 75%.
  • Gene repression refers to any process which results in a decrease in production of a gene product.
  • a gene product may be either RNA (including, but not limited to, mRNA, rRNA, tRNA, and structural RNA) or protein. Accordingly, gene repression includes those processes which decrease transcription of a gene and/or translation of a mRNA. Examples of gene repression processes which decrease transcription include, but are not limited to, those which inhibit formation of a transcription initiation complex, those which decrease transcription initiation rate, those which decrease transcription elongation rate, those which decrease processivity of transcription and those which antagonize transcriptional activation (by, for example, blocking the binding of a transcriptional activator).
  • Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level.
  • Examples of gene repression processes which decrease translation include those which decrease translational initiation, those which decrease translational elongation and those which decrease mRNA stability.
  • Transcriptional repression includes both reversible and irreversible inactivation of gene transcription.
  • gene repression comprises any detectable decrease in the production of a gene product.
  • gene activation comprises any detectable increase in the production of a gene product.
  • the production of a gene product is decreased by at least 2-fold, such as 10- fold, such as 30- fold, such as 50- fold, such as 90- fold.
  • gene repression results in complete inhibition of gene expression, such that no gene product is detectable.
  • the nucleotide sequence encoding MBD2 is selected from the group consisting of a) a nucleotide sequence set forth in SEQ ID NO: 1, and b) nucleotide sequences having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO: 1.
  • nucleotide sequence encoding MBD4 is selected from the group consisting of a) a nucleotide sequence set forth in SEQ ID NO: 4, and b) nucleotide sequences having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO: 4.
  • nucleotide sequence encoding DNMTl is selected from the group consisting of a) a nucleotide sequence set forth in SEQ ID NO: 7, and b) nucleotide sequences having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO: 7.
  • identity is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
  • sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
  • the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
  • nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990).
  • Gapped BLAST may be utilised.
  • PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules.
  • NBLAST NBLAST
  • XBLAST XBLAST
  • Gapped BLAST programs
  • sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
  • sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
  • the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment.
  • the BLASTN and PSI BLAST default settings may be advantageous.
  • the percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • An inherent feature of the supraplastic cells of the present invention is their capacity to renew themselves and to differentiate into a broad spectrum of derivatives of all three embryonic germ layers: ectoderm, mesoderm, and endoderm.
  • Human embryonic stem cell lines have already been isolated, and their ability for multilineage differentiation, including neural lineage, has been demonstrated both in vivo and in vitro. This ability to develop has drawn clinical attention to stem cells as a novel source for new cell-based therapeutic strategies such as tissue regeneration.
  • the present invention relates to a composition for mediating an increase in the plasticity level of a donor cell, said composition comprises one or more of the plasticity modifying factors described herein.
  • said composition for mediating an increase in the plasticity level of a donor cell comprises one or more of the plasticity modifying factors selected from the group consisting of MBD2 factor(s), MBD4 factor(s) and DNMTl factor(s).
  • said composition for mediating an increase in the plasticity level of a donor cell comprises MBD2 factor(s).
  • said composition for mediating an increase in the plasticity level of a donor cell comprises MBD4 factor(s).
  • said composition for mediating an increase in the plasticity level of a donor cell comprises D ⁇ /M ⁇ factor(s).
  • the composition may comprise MBD2 factor(s) in combination with MBD4 factor(s).
  • the composition may be any of the above mentioned compositions further combined with DNMTl factor(s).
  • compositions described herein may further comprising one or more histone acetyltransferases (HATs).
  • HATs histone acetyltransferases
  • the present invention relates to a vector comprising at least one of the genes selected from the group of AOF2, DNMTl, DNMT3A, DNMT3B, MBDl, MBD2, MBD4, MECP2, ZBTB33, ZBTB38, ZBTB4, ATF2, CDYL, CREBBP, ELP3, EP300, GCN5L2, GTF3C1, HATl.
  • HTATIP MYSTl, MYST2, MYST3, MYST4, NCOAl, NCOA2, NCOA3, OGT, PCAF, TAFl, HDACl, HDAC2,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7A, HDAC8, HDAC9, HDAClO, HDACIl, SIRTl, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, DOTlL, EHMTl, EHMT2, EZH2, MLLl, NSDl, PRDM2, PRDM9, PRMTl, PRMT2, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, SETDlA, SETD2, SETD7, SETD8, SETDBl, SMYD3, SUV39H1, SUV39H2, SUV420H1, SUV420H2, AOF2, FBXLIl, JMJD2A, JMJD2B, JMJD2C, PADI4, CHDl, CHD2, CHD3, CHD4, SMARCAl, SMARCA
  • Another embodiment relatest to a vector according to the present invention, wherein said vector comprising at least one of the genes selected from DMNTl, MBDl, ELP3,GCN5L2, HTATIP, MYSTl, HDAC6, SIRT5, SIRT7, PRMT7, SMYD3, SUV39H1, SMARCA2, SMARCA4, DNMT3A, DNMT3B, MBD2, MBD4, ZBTB33, ATF2, CDYL, EP300, NCOAl, NCOA2, OGT, TAFl, HDAC2, HDAC3, HDAC9, SIRTl, SIRT4, DOTlL, EHMTl, PRMTl, PRMT3, SETD2, SETD8 and SUV39H2.
  • said vector comprising at least one of the genes selected from DMNTl, MBDl, ELP3,GCN5L2, HTATIP, MYSTl, HDAC6, SIRT5, SIRT7, PRMT7, SMYD3, SUV39H1, SMARCA2, SMARCA4, DNMT
  • the vector comprises at least one of the genes selected from the group consisting of DMNTl, MBD2 and MBD4.
  • the vector comprises at least one of the genes OCT4, SOX2, C-MYC and KLF4 in combination with MBD2 and/or MBD4.
  • Oocytes were obtained from 3-week-old B6D2F1 mice (Taconic Europe, Ejby, Denmark) and were carefully processed to avoid human contamination. Mice were handled under standard laboratory conditions and experiments were conducted following national guidelines on animal care (Danish Ministry of Justice; approval 2003/561-713).
  • mice were each injected with 15 IL) menotropin (Menopur ® ; Ferring, Copenhagen, Denmark) and killed by cervical dislocation 42 hours later. Ovaries were isolated and antral follicles were punctured by 27-ga needles. Cumulus-enclosed GV oocytes were released into alpha MEM medium (Invitrogen, Carlsbad, CA) supplemented with 4 mmol/l hypoxanthine (Sigma, St. Louis, MO) to inhibit GV breakdown during handling. Only spherical GV oocytes with a distinct GV and attached cumulus cells were collected and immediately underwent mechanical denudation with a fine bore glass pipette.
  • Menotropin Menotropin
  • mice were first primed with 15 IL) menotropin (Menopur ® ; Ferring) followed 44 hr later by injection of 5 IL) of choriongonadotropin (Pregnyl ® ; Organon, Oss, The Netherlands) and were then killed 15 hr later. Collected oviducts were punctured in alpha-MEM to yield the cumulus oocyte complexes (COCs). COCs were briefly exposed to 80 IU/ml hyaluronidase (SynVitro ® Hyadase; MediCult, Jyllinge, Denmark) at 37°C to remove attached cumulus granulosa cells. Only spherical Mil oocytes with a distinct first polar body were selected.
  • Oocyte RNA was obtained using the RNeasy ® Micro Kit (Qiagen, Valencia, CA), which enriches mRNAs from small samples by selectively excluding shorter RNAs such as rRNAs and tRNAs. Briefly, pools of oocytes were lyzed with guanidine- isothiocyanate-containing buffer and were applied to spin columns for adsorption of the RNA. Carrier was included to improve the yield of low-abundance mRNAs further. Pellets were washed with 80% ethanol, centrifuged and air-dried.
  • RNA was eluted in nuclease-free water and immediately stored at -80 0 C until use.
  • RNA 6000 Pico LabChip Agilent Technologies, Palo Alto, CA.
  • the protocol used for microarray chip hybridization required at least 100 ng of total RNA for each biological replicate. Due to limited sample quantities of oocytes, pooling of total RNA from repeated experiments was necessary, so with an expected average amount of 0.3 to 0.5 ng of total RNA in a murine oocyte, each replicate was constructed from pooled RNA of oocytes from approximately 20 mice. Sufficient oocyte RNA was isolated for the generation of three and five biological replicates of GV and Mil oocytes, respectively. Amplification and hybridization to microarray chips
  • RNA transcript labeling assay Due to the small amounts of initial RNA, the process required an amplifying two cycle target labeling assay step to obtain sufficient amounts of labeled cRNA target for analysis with arrays.
  • a minimum of 100 ng of total RNA was used to synthesize double stranded cDNA with the Superscript Choice system (Invitrogen) with an oligo(dT) primer containing a T7 RNA polymerase promoter (GenSet).
  • the cDNA was used as the template for an in vitro transcription reaction to synthesize antisense cRNA (BioArray high yield RNA transcript labeling kit; Enzo, Farmingdale, NY).
  • the signal was then amplified in a second cDNA synthesis step using random primers for first strand synthesis and T/ oligo(dT) primer for the second strand synthesis.
  • the amplified cDNA was used as template for a second in vitro transcription to synthesize biotin labeled antisense cRNA (BioArray high yield RNA transcript labeling kit; Enzo).
  • the labeled cRNA was fragmented at 94°C for 35 min in fragmentation buffer (40 mM Tris, 30 mM magnesium acetate, 10 mM potassium acetate) and was hybridized for 16 hr to the Affymetrix GeneChip ® Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA), which covers transcripts and variants from 34,000 well characterized mouse genes. Probe sets on this array are derived from sequences from GeneBank, dbEST, and RefSeq. The arrays were washed and stained with phycoerythrin streptavidin using a Fluidics Station 450 (Affymetrix), and the arrays were scanned in a GeneArray 3000 scanner (Affymetrix). Data were digitalized and converted into CEL format (v. 3) for data analysis.
  • fragmentation buffer 40 mM Tris, 30 mM magnesium acetate, 10 mM potassium acetate
  • transcripts with an Affymetrix oligonucleotide probe set corresponding to a single gene (“_at” suffices) or with anticipated oocyte specific splice variants (“_a_at” suffices) was retained, thus only allowing less specific probe sets if a unique probe set for a gene was missing on the gene chip at the time of manufacturing.
  • Probe sets were cross examined in the ADAPT database (Leong et al. 2005) to ensure that only probe sets representing mRNAs remained, thus avoiding regions outside exons and 3' untranslated region (L)TR). Eventually, if a gene was represented by more than one unique probe set, the probe set with most present calls or positioned in the most distal 3' end of the transcript was selectd.
  • Hierarchical clustering was performed only on genes showing statistically significant up- or down-regulation as identified by t test and with a P value corrected for multiple testing with the Benjamini-Hochberg method to avoid erroneous result by less variant or absent genes. Significance level was 0.05, distance metric was 1 - r, and linkage method was centroid. To validate the overall quality of the experiment, hierarchical clustering was also performed on sample level, since biological replicates representing the same condition should to be grouped together because of identical global expression pattern. Accordingly, they should be separated from samples representing other conditions.
  • cDNA Complementary DNA
  • Superscript III Invitrogen
  • iScriptTM cDNA Synthesis Kit Bio-Rad Laboratories, Hercules, CA
  • Equal amounts of total RNA 24 ng were converted into cDNA for GV and Mil samples, respectively.
  • the cDNA elute was diluted 1:20 before down-stream application.
  • the gene specific primers were constructed using Primer3 (Rozen & Skaletsky 2000) or PrimerBank (Wang & Seed 2003). Primers were carefully chosen or designed to eliminate the risk of amplifying unspecific targets, such as primer dimers or genomic DNA. Primer sequences, exon complementarity, melting temperature, GenBank accession number, and size of the amplified product are presented in table 1. Expression levels were quantified in real-time on a LightCycler ® Instrument (Roche Diagnostics, Germany) using LightCycler ® FastStart DNA Master PLUS SYBR Green I (Roche Applied Science) or SYBR ® Premix Ex TaqTM kit (Takara, Madison, WI) as detection chemistry.
  • the PCR reaction mixture consisted of 2 ⁇ l of diluted cDNA, 1 ⁇ l of gene specific primers, 2 ⁇ l or 5 ⁇ l of SYBR® Green I master mix (depending on manufacturer), and added water to a total volume of 10 ⁇ l.
  • cDNA was denatured and pre-incubated by heating for 10 sec at 95°C.
  • Template was then amplified by 45 cycles consisting of denaturation for 5 sec at 95°C, annealing 20 sec at 55°C, and extension for 10 sec at 72°C. At least three replicates were used for each reaction, and a minus RT and a minus template served as negative controls.
  • melting point curves were generated following amplification. Samples were heated to 95°C followed by immediate cooling for 15 sec at 65°C and then slowly heating by 0.1°C/sec up to 95°C while monitoring fluorescence continuosly.
  • RNA from GV and Mil oocytes were used for each cDNA reaction.
  • Putative candidate reference genes were amplified and the gene showing the lowest variability was chosen as internal reference using Norm-Finder (Andersen et al. 2004).
  • oocytes such as the zona pellucida glycoproteins (ZPl, ZP2, and ZP3), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), moloney sarcoma oncogene (MOS) 1 cyclin Bl (CCNBl), spindlin (SPIN), MATER (NALP5), zygote arrest 1 (ZARl), STELLA (DPPA3), and OCT4 (POU5F1) were abundantly detected on the chip set (Table 3).
  • oocytes underwent careful denudation, potential contamination of heterologue RNA from cumulus- granulosa cells were investigated. The absence of cumulus transcripts such as hyaluronic synthase 2 (HAS2), pentraxin 3 (PTX3), and gremlin 1 (GREMl) confirmed the oocyte homogeneity of the collected samples.
  • HAS2 hyaluronic synthase 2
  • PTX3
  • Table 3 shows the average expression indices, fold changes and corrected P- values supplemented with values for a number of transcripts preferentially expressed in the oocyte.
  • 57 genes 70 % were detected in GV- and/or MII-oocytes on the chip set.
  • 14 had fold changes from -1 to -2, eight had fold changes from -2 to -5, three had fold changes from -5 to -10, and eight had fold changes less than -10.
  • a total of 24 genes were detected as up-regulated with 16 genes having fold changes from 1 to 2 and eight genes having fold changes ranging from 2 to 5.
  • HATs may be classified into five subgroups according to sequence homology and internal protein domains. All five members of the MYST-subgroup were detected in oocytes. TIP60 (HTATIP), MOF (MYSTl) and MORF (MYST4) showed significant down-regulation, while HBOl (MYST2) and MOZ (MYST3) were unchanged. Of the members belonging to the GNAT-subgroup, elongation protein 3 homolog (ELP3) and GCN5 (GCN5L2) were significantly down-regulated, histone aminotransferase 1 (HATl) was unchanged and p300/CBP-associated factor (PCAF) was absent.
  • TAT elongation protein 3 homolog
  • GCN5L2 histone aminotransferase 1
  • PCAF p300/CBP-associated factor
  • SRC-subgroup SRC-I
  • NCOA2 GRIPl
  • ACTR NCOA3
  • the basal transcription factors TAFII250 (TAFl) and TFIIIC (GTF3C) were also expressed at an unchanged level.
  • HDACs are subdivided into four classes according to phylogenesis.
  • Class I members HDACl, HDAC2, and HDAC3 were expressed in oocytes, whereas HDAC8 was absent.
  • HDACl was down-regulated and HDAC2 and HDAC3 were unchanged.
  • No class II HDAC was detected, except HDAC6 and HDAC9, which were down-regulated and unaffected, respectively.
  • SIRT2 all sirtuins or class III HDACs were expressed in oocytes of which SIRT4 was up-regulated, SIRT3, SIRT5 and SIRT7 were down-regulated, and SIRTl and SIRT6 showed no change.
  • the single class IV member, HDACIl was not detected.
  • Histone methyltransferases and histone demethylases/deiminases HMTs either activate or repress gene function depending on the residue of the modified amino acid. Histone methylation at positions H3K4, H3K36, or H3K79 is associated with gene activation.
  • DotlL (DOTlL) was undifferentially expressed, SMYD3 (SMYD3) was down-regulated, and SETl (SETDlA), MLL (MLLl), SET7/9 (SETD7), and Meisetz (PRDM9) were absent.
  • SUV39hl SUV39H1
  • ESET SETDBl
  • SUV39h2 SUV39H2
  • Eu- HMTasel/GLPl EHMTl
  • ENX-I EZH2
  • PR-SET7/SET8 SETD8
  • SUV4-20hl SUV420H1
  • SUV4-20h2 SUV4-20h2
  • RIZ PRDM2
  • NSDl NSDl
  • arginine HMTs Of the arginine HMTs, one was slightly up-regulated (PRMT3), one was down- regulated (PRMT7), two were undifferentially expressed (PRMTl, PRMT2), and three were absent (PRMT4, PRMT5, PRMT6).
  • HDMs were absent in oocytes, except LSDl (AOF2), which was expressed at an unchanged level.
  • the ChR-ATPases are classified into subfamilies according to the presence of conserved domains outside their catalytic region.
  • the two bromodomain SNF2- subfamily ATPases hBRM (SMARCA2) and BRGl (SMARCA4) were both expressed in oocytes and down-regulated.
  • the SANT domain ISWI-subfamily members SNF2L (SMARCAl) and SNF2H (SMARCA5) were absent or unchanged, respectively.
  • Among the four chromodomain CHD-subfamily ATPases only Mi-2 ⁇ (CHD4) was expressed at unchanged level while CHDl, CHD2 and Mi-2 ⁇ (CHD3) were absent.
  • the four transcripts having statistically significant up-regulation and the 18 transcripts being down-regulated as shown by the present microarray data were selected for further analysis by real-time quantitative RT-PCR.
  • ACTB ⁇ -actin
  • HMBS porphobilinogen deaminase
  • TATA box binding protein TATA box binding protein
  • the RT-PCR expression data for the 22 reprogramming gene transcripts are presented in figure 3.
  • the absence of non-specific amplified products was confirmed by melting curve analysis.
  • some discrepancy regarding direction and amplitude was observed.
  • the RT-PCR using oligo(dT) primers was repeated. In this case, there was a significantly higher correlation between array data and qRT-PCR data regarding direction and amplitude, suggesting the occurrence of selective deadenylation during meiotic maturation.
  • Sequence information for the human MBD2 gene is obtained by use of the Swiss Protein database (www.expasy.org). Swiss-Prot entry Q9UBB5 is used to access DNA sequence information regarding the human MBD2 gene and its splicing isoforms.
  • Human MBD2 is a 411 amino acid protein with a molecular weight of 43255 Da.
  • a synthetic MBD2 gene is purchased from commercial companies like GENEART (www.geneart.com).
  • GENEART www.geneart.com
  • a DNA fragment is manufactured according to the human MBD2 gene sequence as specified by the Swiss-Prot Q9UBB5 entry. Both DNA fragments coding for the standard human MBD2 protein as well as DNA fragments coding for splicing and sequence variants is obtained in this way.
  • Synthetic genes are normally delivered cloned into a standard bacterial vector. This bacterial plasmid with a MBD2 gene insert allows for multiplication of the MBD2 gene material according to the needs for subsequent use.
  • Restriction enzyme sites useful for DNA fragment recloning is inserted before and after the MBD2 gene reading frame. Such restriction enzyme sites is designed into the purchased MBD2 construct, or a PCR reaction with PCR primers containing such restriction enzyme sites is used to amplify a MBD2 gene fragment with these these sites attached at proper positions.
  • the human MBD2 gene fragment is recloned into a commercially available expression system using cloning sites as described by the manufactorer.
  • the RheoSwitch Mammalian Expression System from New England BioLabs (www.neb.com) allows for induction and adjustable control of gene expression for the inserted reading frame.
  • the T-REx System from Invitrogen utilizes a CMW promoter to deliver higher levels of induced expression than other regulated mammalian expression systems.
  • Primary human fibroblast cells are cultivated by use of hospitally derived material from neonatal circumcisions. Alternatively primary human fibroblasts are obtained from commercial suppliers like Cascade Biologies (www.cascadebio.com), a part of Invitrogen (www.invitrogen.com), and primary human fibroblast cells are grown using media and protocols supplied by the cell line manufactorer. Expression system vectors containing a MBD2 gene fragment are transformed into primary human fibroblasts by use of methods described by the expression vector manufactorer. Transformation is performed in the form of transient expression experiments, or with the purpose of generating a stable cell line with a chromosomally integrated MBD2 gene construct.
  • MBD2 expression in transformed primary human fibroblast cells with or without expression vector MBD2 expression is analysed by use of MBD2 specific antibodies in an ELISA assay.
  • Abeam www.abcam.com
  • Abeam is an example of a commercial supplier of MBD2 antibodies
  • the codes ab26848, ab33931, ab38646, ab49136 and abll985 are examples of such Abeam products.
  • a significantly increased MBD2 protein expression level is observed for transformed primary human fibroblast cells relative to control cells without MBD2 overexpression.
  • Human MBD4 is a 580 amino acid protein (unprocessed precursor) with a molecular weight of 66051 Da.
  • a synthetic MBD4 gene is purchased from commercial companies like GENEART (www.geneart.com).
  • a DNA fragment is manufactured according to the human MBD4 gene sequence as specified by the Swiss-Prot 095243 entry. Both DNA fragments coding for the standard human MBD4 protein as well as DNA fragments coding for splicing and sequence variants is obtained in this way.
  • a synthetic gene is either delivered already cloned into a standard bacterial vector or it is cloned into such a vector when received from the commercial supplier.
  • This bacterial plasmid with a MBD4 gene insert allows for multiplication of the MBD4 gene material according to the needs for subsequent use.
  • Restriction enzyme sites useful for DNA fragment recloning is inserted before and after the MBD4 gene reading frame. Such restriction enzyme sites is designed into the purchased MBD4 gene fragment, or a PCR reaction with PCR primers containing such restriction enzyme sites is used to amplify a MBD4 gene fragment with these these sites attached at proper positions.
  • the human MBD4 gene fragment is recloned into a commercially available expression system using cloning sites as described by the manufactorer.
  • the RheoSwitch Mammalian Expression System from New England BioLabs (www.neb.com) allows for induction and adjustable control of gene expression for the inserted reading frame.
  • the T-REx System from Invitrogen utilizes a CMW promoter to deliver higher levels of induced expression than other regulated mammalian expression systems.
  • Primary human fibroblast cells are cultivated by use of hospitally derived material from neonatal circumcisions. Alternatively primary human fibroblasts are obtained from commercial suppliers like Cascade Biologies (www.cascadebio.com), a part of Invitrogen (www.invitrogen.com), and primary human fibroblast cells are grown using media and protocols supplied by the cell line manufactorer. Expression system vectors containing a MBD4 gene fragment are transformed into primary human fibroblasts by use of methods described by the expression vector manufactorer. Transformation is performed in the form of transient expression experiments, or with the purpose of generating a stable cell line with a chromosomally integrated MBD4 gene construct.
  • MBD4 expression in transformed primary human fibroblast cells with or without expression vector MBD4 expression is analysed by use of MBD4 specific antibodies in an ELISA assay.
  • Abeam www.abcam.com
  • Abeam is an example of a commercial supplier of MBD4 antibodies
  • the codes ab3756, abl3793 and abl2187 are examples of such Abeam products.
  • a transient overexpression of both MBD2 and MBD4 is established by co- transfection of the MBD2 expression plasmid described in Example 5 and the MBD4 expression plasmid described in Example 6 into primary human fibroblast cells as previously described.
  • a stable cell line that overexpresses both MBD2 and MBD4 is established by constructing a DNA fragment, where both genes are placed in line behind separate regulated promoters.
  • the resulting dual gene construct inserted into the matching expression plasmid system is transformed into human primary fibroblast cells for stable transformant isolation as previously described in example 5 and 6.
  • the level of MBD2 and MBD4 protein expression is measured using specific antibodies as described in example 5 and 6.
  • Detection of surface markers may be used as a measure for cell plasticity (developmental potential).
  • Primary human fibroblast cells are so-called end-stage- committed cells, and detection of surface markers for non-end-stage-commited cells upon overexpression of MBD2 and/or MBD4 signals a possible induction of plasticity.
  • Primary human fibroblast cells overexpressing MBD2 and/or MBD4 protein are found to display characteristic cell surface proteins, that identifies them as being different from control primary human fibroblast cells without the MBD2 and/or MBD4 overexpression.
  • DNA methylation of cytosine in specific CpG regions is known to be associated with cell differentiation. As induction of plasticity is a process opposite to differentiation, demethylation of CpG sites may also be considered indicative for an increased plasticity, exemplified by the transition from an end-stage committed cell to a non-end-stage commited cell.
  • the methylSEQr Bisulfite Conversion Kit from Applied Biosystems (www.appliedbiosystems.com) is used to determine the methylation status of a CpG motif. Sodium bisulfite deaminates cytosine to produce uracil, and methylated cytosines are protected from this conversion. Hence DNA sequence determination of chromosomal DNA regions with and without the use of bisulfite convertion will identify methylated cytosines.
  • the CpG methylation patterns for primary human fibroblast cells with overexpression of MBD2 and/or MBD4 protein is found to show signs of DNA demethylation compared to primary human fibroblast cells without the MBD2 and/or MBD4 overexpression.
  • DNA methyltransferase 1 is responsible for the methylation of the newly formed DNA strand during DNA replication. Only hemi-methylated CpG sites are recognized, and in this way the pre-existing methylation pattern is copied onto the two daughter cells. Demethylation induced by overexpression of MBD2 and/or MBD4 protein may thus be counterpoised by DNMTl activity, and DNMTl inhibition may be required for efficient plasticity induction.
  • RNAi RNA interference
  • Loss of function for specific genes is induced by way of short RNAi oligonucleotides complementary to the target mRNA transcript.
  • RNAi systems like DNMTl Stealth Select RNAi (RNAi Catalog Nr. HSS102859; HSS102860; HSS102861) from Invitrogen
  • Chromatin structure constitutes another obstacle for an efficient DNA demethylation reaction, where heterochromation is known to be structurally inaccessible for protein factors. Condensation of chromosomal DNA into heterochromatin is associated with histone deacetylation, whereas DNA wrapped around acetylated histones are accessible to protein factors. Histone acetylases (HAT) and histone deacetylases (HDAC) perform opposite reactions, and hyperacetylation is generated by use of histone deacetylase inhibitors.
  • HAT histone acetylases
  • HDAC histone deacetylases
  • MBD2 and/or MBD4 protein Treatment of primary human fibroblast cells with and without overexpression of MBD2 and/or MBD4 protein with known histone deacetylase inhibitors like for example butyrate, valproic acid, oxamflatin and apicidin is used to determine the enhancement of plasticity induction measured as described in example 8.
  • histone deacetylase inhibitors like for example butyrate, valproic acid, oxamflatin and apicidin is used to determine the enhancement of plasticity induction measured as described in example 8.
  • DNMTl inhibition and/or histone hyperacetylation is observed to result in similar or improved plasticity induction, when primary human fibroblast cells overexpressing MBD2 and/or MBD4 protein are analysed as described in example 8.
  • a donor cell that has been de-differentiated and reprogrammed into a higher level of plasticity may be considered pluripotent if it demonstrates capability of being re-differentiated into one or more end-stage committed cell from each the three primary germ layers (ectoderm, mesoderm, and endoderm).
  • Cells are harvested as per any standard procedure, washed with DMEM, and placed in suspension culture in differentiation medium consisting of either MedII/FGF2 medium (DMEM/F12, 1 x N2 [Invitrogen], 20 mM L-glutamine, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 4 ng/ml FGF-2, and 50% serum-free Medll) or DMEM/N2 medium (DMEM, 1 x N2, 20 mM L-glutamine, 0.5 U/ml penicillin, 0.5 U/ml streptomycin). Cultures are differentiated for 2-6 weeks in suspension, and the medium is changed every 5-7 days.
  • Cell aggregates are plated on dishes or Permanox slides coated with 20 ⁇ g/ml polyornithine (Sigma) and 1 ⁇ g/ml laminin (Sigma) in MedII/FGF2 medium or Neurobasal medium (Invitrogen) containing 1 x B27 (Invitrogen), 5% FCS (Hyclone), 2 ng/ml glial-derived neurotrophic factor (GDNF) (R&D Systems), 10 ng/ml brain-derived neurotrophic factor (BDNF) (R&D Systems), 20 mM L- glutamine, 0.5 U/ml penicillin, and 0.5 U/ml streptomycin.
  • tyrosine hydroxylase which is the enzyme responsible for converting L-tyrosine to dihydroxyphenylalanine (DOPA), the precursor for dopamine.
  • de-differentiated donor cells are co-cultured with immortalized fetal human liver clone B (FH-B-hTERT) cells (Wege et al. Gastroenterology 2003; 124:432- 444).
  • CD34+ cells are subsequently purified and subjected to serum-free liquid culture, allowing for the expansion of de-differentiated donor cell-derived hematopoietic stem cells (HSCs).
  • HSCs de-differentiated donor cell-derived hematopoietic stem cells
  • terminal erythroid differentiation is induced via co-culture of putative erythroid progenitors with MS-5 feeder cells, erythropoietin and hemin.
  • upwards of 5x 106 terminally differentiated erythrocytes may be generated from 50 000 dedifferentiated donor cells in less than one month.
  • De-differenatiated donor cells are passaged onto 1% Matrigel (B&D Biosciences)- coated tissue culture dishes. Then, the culture medium is changed to modified CDM: 50% IMDM (Gibco) plus 50% F12 NUT-MIX (Gibco), supplemented with insulin-transferrin-selenium-A (1 : 100, Gibco) and 450 ⁇ M monothioglycerol (Sigma), and 5 mg/ml albumin fraction V (Sigma) or X-Vivol0 (Cambrex) supplemented with 55 ⁇ M 2-Mercaptoethanol (Gibco) and 0.1% albumin fraction V (Sigma).
  • modified CDM 50% IMDM (Gibco) plus 50% F12 NUT-MIX (Gibco), supplemented with insulin-transferrin-selenium-A (1 : 100, Gibco) and 450 ⁇ M monothioglycerol (Sigma), and 5 mg/ml albumin fraction V (
  • the cells Two days later, the cells are induced into definitive endoderm differentiation with CDM containing 50 ng/ml activin A (Sigma) for 4 days, as detected by the expression of the definitive endoderm markers SOXl 7 and Brachyury. After 4 days of activin A induction, the cells are transferred into CDM with 10-6 M all-trans retinoic acid (Sigma) for another 4 days to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors PDXl and HLXB9.
  • CDM containing 50 ng/ml activin A (Sigma) for 4 days, as detected by the expression of the definitive endoderm markers SOXl 7 and Brachyury.
  • the cells are transferred into CDM with 10-6 M all-trans retinoic acid (Sigma) for another 4 days to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors PDXl and HLXB9.
  • Culture medium is then changed from CDM to modified islet maturation medium: DMEM/F12 (Gibco), insulin-transferrin-selenium-A (1 : 100, Gibco) and 2 mg/ml albumin fraction V (Sigma) with 10 ng/ml bFGF (Invitrogen) for the first 3 days and with 10 mM nicotinamide (Sigma) for the next 5 days.
  • the cells are digested by 0.5 mg/ml dispase (Gibco) and transferred into Ultra Low Attachment culture dishes (Costar) for 5 days to achieve islet maturation in suspension culture.
  • the differentiated cells may be evaluated by the expression of islet specific markers such as C-peptide, insulin, glucagon and GLUT2.
  • the level of reprogramming and putative pluripotency is then tested by the concomitant up-regulation of classical stem cell markers (Oct-4, SSEA3, SSEA4, TRA-1-60, TRA-1-81) and down-regulation of epithelia cell markers (e.g. cytokeratins, lamin A/C).
  • classical stem cell markers Oct-4, SSEA3, SSEA4, TRA-1-60, TRA-1-81
  • epithelia cell markers e.g. cytokeratins, lamin A/C
  • Final level of differentiation capacity is assessed by exposing the de-differentiated donor cells to the protocol of erythroid cell differentiation as described in example 15.
  • hFFs Human foetal (foreskin) fibroblast cells
  • Lipofectamine 2000 (Invitrogen) was used for plasmid transfection of hFF cells accordinging to the manufactorers description (use of standard ratio of DNA to Lipofectamine 2000 reagent). Test of the above transfection protocol using a GFP reporter plasmid detected a green fluorescence signal in almost half of the transfected hFF cells.
  • hFFs Human foetal fibroblasts
  • cells were harvested after a period of one, two or three days of culture.
  • cells from the different transfections were also grown in the presence of 5 ⁇ M 5-azacytidine (5aza) in order to inactive the DNMTl enzyme activity.
  • Total RNA from hFFs was obtained using the RNeasy ® Micro Kit (Qiagen, Valencia, CA), which enriches mRNAs from small samples by selectively excluding shorter RNAs such as rRNAs and tRNAs.
  • Cells were deattached from the bottom of the culture dish by trypsination (Trypsin-EDTA treatment followed by resuspension in IMDM medium containing 10% FBS for trypsin inactivation) and isolated by centrifugation. Tubes with cell pellets were rapidly snap frozen by dipping into liquid nitrogen and subsequently stored at -80 0 C.
  • the pelleted cells were lyzed with guanidine-isothiocyanate-containing buffer and were applied to spin columns for adsorption of the RNA.
  • Carrier RNA was included to improve the yield of low- abundance mRNAs further. Pellets were washed with 80% ethanol, centrifuged and air-dried. Subsequently, the RNA was eluted in nuclease-free water and immediately stored at -80 0 C until use.
  • NanoDrop N-1000 device NanoDrop Technologies, Wilmington, DE, USA
  • cDNA quality was evaluated on a NanoDrop N-1000 device (NanoDrop Technologies, Wilmington, DE, USA) and was shown to be of good quality by the outline of the absorbance curve around in relation to 260 and 280 nm.
  • hESCs pluripotent human embryonic stem cells
  • DNMT3B pluripotent human embryonic stem cells
  • GABBR3, GDF3, NANOG pluripotent human embryonic stem cells
  • POU5F1 TDGFl
  • hMSCs multipotent human mesenchymal stem cells
  • Primer sequences were designed using either PrimerBank (Wang & Seed 2003) or PerlPrimer (Marshall O. f 2007).
  • Three house-keeping genes were used as internal reference genes (i.e. ACTB, GAPDH, TBP).
  • melting point curves were generated following amplification. Samples were heated to 95 ° C followed by immediate cooling for 15 s at 65 ° C and then slowly heating by 0.1 ° C/s to 95 ° C, while monitoring fluorescence continuously. All target genes were evaluated against the geometric mean of the three reference house-keeping genes within the log-linear phase of the amplification curve, and ⁇ C T values were assessed using the ⁇ C T method (Pfaffl, 2001).
  • the expression values for a gene across all eight samples are standardized to have mean 0 and S. D. 1 by linear transformation.
  • the distance between two genes is defined as 1 - r, where r is the standard correlation coefficient between the eight standardized values of two genes.
  • Two genes with the closest distance are first merged into a supergene, connected by branches with length representing their distance, and deleted for future merging.
  • the expression level of the newly formed supergene set is the average of standardized expression levels of the two genes (average linkage) for each sample.
  • Expression levels are shown as compared to control population (non-transfected hFFs) at 1, 2 and 3 days post-transfection.
  • Figure 8 Shows a bar chart showing ⁇ Ct changes of 13 genes specific to undifferentiated human embryonic stem cells.
  • Cells have been transfected with MBD2 and MBD4 with the DNA methyltransferase inhibitor (DNMTi) 5-azadeoxycytidine (5aza) present.
  • DNMTi DNA methyltransferase inhibitor 5-azadeoxycytidine
  • Expression levels are shown as compared to control population (non- transfected hFFs) at 2 and 3 days post-transfection.
  • Primer list comprising a group of selected genes important to to epigenetic modifications and chromatin remodelling.
  • the housekeeping gene ⁇ Actb) is used as an internal reference. 5 Gene symbols and descriptions have been adopted from the Mouse Genome Informatics database
  • Primer pairs were carefully designed so that they were separated by at least one intron sequence to reduce risk of co-amplified genomic DNA. Additionally, some primers span intron-exon flanks. No primer pair had inter-primer complementarity thus avoiding formation of primer-dimers. All primer sequences were blasted for potential Ul Ul alternative binding sites.
  • F forward primer
  • R reverse primer
  • T m melting temperature
  • Characteristics for microarray chip samples A high correlation coefficient (r) between biological replicates indicates almost identical global transcription patterns and thus high validity and reliability of the microarray procedure per se performed on 5 minute amounts of RNA.
  • GV germinal vesicle
  • Mil metaphase II
  • GV germinal vesicle
  • Mil metaphase II
  • BH Benjamini
  • MIAME microarray experiment
  • Wilson CL Miller CJ. Simpleaffy: a BioConductor package for affymetrix quality control and data analysis. Bioinformatics 2005; 21 : 3683-3685.

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Abstract

This application relates to reprogramming of cells, as the present invention discloses a method for increasing the plasticity level in a donor cell by introducing one or more plasticity modifying factor(s), it is an aspect of the present invention to provide a composition for mediating an increase in the plasticity level of a donor cell, said composition comprising one or more plasticity modifying factors.

Description

METHOD FOR INCREASING THE PLASTICITY LEVEL OF A CELL
Technical field of the invention
The present invention relates to the reprogramming of partly or fully differentiated mammalian cells, so that they can act like stem cells. The present invention provides a method for such reprogramming by increasing the plasticity level of a cell through changes in the pattern of gene expression in the partly or fully differentiated cell. In particular, the present invention relates to plasticity modifying factor(s), said plasticity modifying factor(s) being selected from the group consisting of plasticity modifying factors increasing DNA demethylation, plasticity modifying factors inhibiting maintenance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones.
Background of the invention
The nature and function of any type of cell depends on the specific set of genes expressed (e.g. transcribed and translated). Accordingly, development of cells exhibiting a high plasticity level (i.e. pluripotent or totipotent cells) into more specialized phenotypes exhibiting a decreased plasticity level (for example end- stage-committed cells) is determined by the type of genes expressed during development. Gene expression is directly mediated by sequence specific binding of gene regulatory proteins that can effect positive or repressive regulation. The ability of any of these regulatory proteins to directly mediate gene expression however, appear to depend on the accessibility of their binding site within the cellular DNA.
In mammals, the culmination of oogenesis is the creation of a mature metaphase II (Mil) oocyte capable of sustaining fertilization, reprogramming of the parental genomes, and supporting early embryonic development. The oocyte cytoplasm rapidly starts to modify the sperm genome upon fertilization resulting in decondensation of paternal chromatin, substitution of protamines with highly acetylated histones, and active demethylation of DNA (Santos et al. 2002; Spinaci et al. 2004). By contrast, the oocyte-derived maternal chromatin seems more protected from this extensive reprogramming activity undergoing DNA demethylation at a more passive level during subsequent cleavages. The observed asymmetry in parental genome reprogramming is believed to result from marked differences in the original nucleosomal structure of the maternal and paternal chromatin, respectively.
Several studies have approached the underlying molecular mechanisms that take place in connection with reprogramming suggesting that the three-dimensional structure of chromatin is highly important in the regulation of gene expression.
Generally, a condensed chromatin structure is associated with an inaccessible and gene repressive state whereas more decondensed and open chromatin is more amenable to gene activation (Turner 2001). Notably, transcription levels may be affected by covalent modifications of the DNA helix and core histones comprising the nucleosome, the core unit of chromatin.
The complex mechanisms involved in the modifications of chromatin are mediated by a group of highly conserved enzymes, including DNA methyltransferases (DNMTs), methyl-CpG-binding proteins (MeCPs), histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), histone demethylases/deiminases (HDMs), and chromatin remodeling complexes (ChRs). Covalent DNA methylation, which is the most abundant form of epigenetic modification, occurs at cytosines in the pyrimidine ring of CpG dinucleotides dispersed throughout the genome and is generally associated with gene silencing.
The repressive effect of DNA methylation is mediated by the MeCPs, which again recruit a wide range of co-repressors, including HDACs. Nucleosome core histones are covalently modified at their N-terminal tails, which are highly charged and tightly associated with the DNA helix. In particular, acetylation and methylation have been associated with regulation of gene expression and it has been suggested that the vast number of combinations may actually form a specific histone code, which could determine the binding patterns of regulatory proteins.
By contrast, ChRs do not interact covalently with DNA in a sequence specific manner but are rather recruited to promoters of target genes by sequence specific transcription factors. Once recruited, the catalytic sub-units of the ChRs (ChR- ATPases) cause an altered conformation and position of nucleosomes by ATP- hydrolysis. This leads to weakened DNA-histone contacts resulting in histone octamers changing positions along the DNA helix, thus either increasing or reducing the binding of transcription factors to promoter regions. The groups of proteins important to epigenetic modifications and chromatin remodeling do not act independently, but are closely coordinated, often by forming large multiprotein complexes.
Remarkably, the highly specialized ability of the Mil oocyte cytoplasm to reprogram a sperm genome extends to a comparable level of reprogramming of a diploid somatic nucleus from adult tissue, i.e. somatic cell nuclear transfer (SCNT) or cloning. By contrast, fully grown germinal vesicle (GV) oocytes are not considered capable of adequate reprogramming. For example, when murine GV oocytes are enucleated and the remaining ooplast subsequently is used for SCNT, nuclear reprogramming is severely reduced without formation of pseudo-pronuclei or cleavage. The factors responsible for gamete and somatic reprogramming are likely to become available during the final stages of oogenesis during the transition from GV to Mil stage, thus highlighting the Mil oocyte cytoplasm as the key player in the reprogramming of a specialized genome into a totipotent state. However, the exact mechanisms by which the Mil oocyte cytoplasm erases the established patterns of gene expression imposed on the sperm genome or a somatic nucleus remain unknown.
Even though successful cloning of frogs and several mammalian species by SCNT has been reported, the therapeutic application of this method has been hindered by technical complications as well as ethical objections. Accordingly, there exists a need to either increase the efficiency of the SCNT method, or alternatively to invent new methods for increasing the level of plasticity of cells.
Takahashi et al. 2006 demonstrated induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four transcription factors Oct3/4, Sox2, c-Myc and Klf4 under ES cell culture conditions. However, the use of retroviral expression systems resulted in only a small fraction of cells expressing the four transcription factors to become induced pluripotent stem cells (iPS). Later Wernig et al. 2007 reported that reprogramming of fibroblasts to a pluripotent state may be induced in vitro through ectopic expression of the four previously mentioned transcription factors (Oct4 (Oct3/4 or Pou5fl), Sox2, c-Myc and Klf4). By employing said method, the authors showed that the DNA methylation, gene expression and chromatin state in induced reprogrammed cells were similar to those of ES-cells.
In the present application, the inventors have investigated further factors involved in early embryonic reprogramming. They discovered and identified a number of genes involved in epigenetic reprogramming.
Accordingly, a method for epigenetic reprogramming is disclosed in the present application.
Summary of the invention The present invention relates to the construction of a cell having an increased level of plasticity. Such a cell is termed a "supraplastic cell" and is capable of differentiating into a variety of end-stage-commited cell types, including the cell types similar to the cell type of the donor cell from which the supraplastic cell was derived and cell types different from the cell type of the donor cell from which the supraplastic cell was derived.
In a first aspect, the present invention describes a method for increasing the plasticity level of a donor cell, said method comprising providing a mammalian, normoploid somatic cell as the donor cell and increasing the plasticity level in said cell by contacting said donor cell with, or introducing into said donor cell, one or more plasticity modifying factor(s), said plasticity modifying factor(s) being selected from the group consisting of plasticity modifying factors increasing DNA demethylation, plasticity modifying factors inhibiting maintenance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones, thereby obtaining a supraplastic cell with the ability to develop into at least two different end-stage-commited cell types.
In another aspect the invention relates to a supraplastic cell obtained by any of the methods described herein. Such supraplastic cell may be used in several different aspects. In one aspect the supraplastic cell may be used for various kinds of cell therapy. One type of cell therapy comprises using the supraplastic cell or cells derived from a supraplastic cell for cell replacement by transplantation, for example replacement of bone marrow in patients suffering from diseases and conditions affecting hematopoietic and mesenchymal stem cells in disorders such as anemia, hypovolemic blood loss, hemoglobinopathies, and platelet disorders. Cell therapy may as described in the present invention also comprise delivery of natively and non-natively produced compounds in situ in patients, said compounds being produced by the transplanted supraplastic cell or cells derived therefrom.
As the present invention discloses a method for increasing the plasticity level in a donor cell by introducing one or more plasticity modifying factor(s), it is an aspect of the present invention to provide a composition for mediating an increase in the plasticity level of a donor cell, said composition comprising one or more plasticity modifying factors.
In a further aspect of the present invention, the supraplastic cell is used for the production of target cells exhibiting the phenotype of end-stage-committed cell types including unipotent pre-cursor cells.
Detailed description of the invention
General
Each of these applications, patents, and each document cited in this text, and each of the documents cited in each of these applications, patents, and documents ("application cited documents"), and each document referenced or cited in the application cited documents, either in the text or during the prosecution of the applications and patents thereof, as well as all arguments in support of patentability advanced during prosecution thereof, are hereby incorporated herein by reference.
It should be understood that any feature and/or aspect discussed above in connection with the methods according to the invention apply by analogy to the uses. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. Thus, preferred features and characteristics of one aspect of the invention may be applicable to other aspects of the invention.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer step, or group of elements, integers or steps.
Increasing the plasticity level
Stem cells are the master cells that replenish all the body's tissues, from skin and blood to the brain and heart. In children and adults, particular types of stem cells are distributed throughout the body, and are specialized to create particular types of tissues. However, in the early embryo, inner cell mass cells can make derivatives of all three primary germ layers including germ cells, a quality known as pluripotency. The "gold standard" test for pluripotency is the ability of a cell to contribute extensively to all adult cell types, including the germ line.
It is an object of the present invention to test the supraplastic cells described herein by injecting these cells into early embryos and observe that these cells can differentiate into all normal adult cell types. The goal for epigenetic reprogramming or production of stem cells or stem-like cells is mainly for regenerative medicine purposes and for understanding the molecular mechanisms behind cellular differentiation and de-differentiation. It is of course preferred to do this in e.g. murine cells for ethical reasons.
The method to assess pluripotency includes as explained injecting these cells into early mouse embryos and observe that these cells differentiate into all normal adult cell types. In human cells, formation of teratomas in severe combined immunodeficient (SCID) mice and/or embryoid body formation in vitro is considered proof of pluripotency (Thomson et al. 1998). In this application, the present inventors describe various plasticity modifying factors that enable reprogramming of partly or fully differentiated cells so they can act like stem cells or stem-like cells. Others have disclosed the principle that pluripotency can be induced in mouse embryonic or adult fibroblasts by introducing four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4, under embryonic stem (ES) cell culture conditions (Takahashi et al. 2006).
The present invention provides an alternative method for such reprogramming by increasing the plasticity level of a cell through changes in the pattern of gene expression by use of new plasticity modifying factors.
The transcriptional profiles, the structure of chromatin (complexes of DNA and histone proteins) and the DNA methylation status of ES cells are very different from those of adult cells, indicating that pluripotency is probably under complex layers of control. It is therefore surprising that contacting partly or fully differentiated cells with, or introducing into partly or fully differentiated cells, a limited number of plasticity modifying factors as described in the present application one can produce stem cell with varying degress of developmental potency.
The present application describes methods for increasing the plasticity level of a donor cell, said method comprising, providing a donor cell and increasing the plasticity level in said cell by contacting said cell with, or introducing into said cell, one or more plasticity modifying factor(s), said plasticity modifying factor(s) being selected from the group consisting of plasticity modifying factors increasing DNA demethylation, plasticity modifying factors inhibiting maintenance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones, thereby obtaining a supraplastic cell.
In one embodiment, the one or more plasticity modifying factor(s) is selected from the group consisting of plasticity modifying factors increasing DNA demethylation and plasticity modifying factors inhibiting maintainance DNA methylation. In another embodiment, the one or more plasticity modifying factor(s) is selected from the group consisting of plasticity modifying factors increasing DNA demethylation and plasticity modifying factors mediating hyperacetylation of histones.
In another embodiment, the one or more plasticity modifying factor(s) is selected from the group consisting of plasticity modifying factors inhibiting maintainance DNA methylation and plasticity modifying factors mediating hyperacetylation of histones.
In one embodiment, the plasticity modifying factor is a factor increasing DNA demethylation.
In one embodiment, the plasticity modifying factor is a factor i inhibiting maintainance DNA methylation.
In one embodiment, the plasticity modifying factor is a factor imediating hyperacetylation of histones.
The cells of the present invention are useful for cell-based therapy including but not limited to autologous treatment schemes. Thus in one embodiment the present invention relates to a method for creating a stem cell comprising, obtaining a partly or fully differentiated donor cell from an individual, inducing in said cell epigenetic reprogramming by altering the level of expression of mediators of covalent modifications of DNA and/or histones and/or mediators of chromatin remodelling, and introducing the resulting stem cell into an individual by transplantation.
Furthermore, the invention thus also relates to use of a transplanted supraplastic cell or a cell derived herefrom for in vivo delivery of a therapeutic compound, said cell either natively producing the compound or being engineered by gene technology to produce the compound. Plasticity
The term "plasticity" refers to the ability of a cell to develop into at least two end- stage-committed cell types. It is the object of the present invention to provide a donor cell and increasing in said donor cell the level of plasticity to obtain a supraplastic cell. Depending on the increase in the level of plasticity, the supraplastic cell may develop into a variety of target cell types.
Accordingly, in one embodiment of the present invention, the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives from one of the three germ layers including germ cells.
Accordingly, in one embodiment of the present invention, the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives from one of the three germ layers.
Accordingly, in one embodiment of the present invention, the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives from one of the representing two germ layers.
In a further embodiment, the supraplastic cell exhibits the ability of differentiating into target cells, said target cells being derivatives of the three or two germs layers.
In the case wherein the target cells represent derivatives of two germ layers, one of said germ layers may be of the same developmental origin as the donor cell from which the supraplastic cell was obtained.
In yet another embodiment of the present invention, the supraplastic cell has the ability of differentiating into target cells derived from one germ layer, said germ layer may be of the same developmental origin as the donor cell from which the supraplastic cell was obtained.
Specifically, as used herein "plasticity" refers to the ability of a cell to differentiate into at least one end-stage-committed cell type different from the cell type from which said cell originated. Thus, in the present context the term "plasticity level" of a cell, refers to the number of end-stage-commited cell types into which said cell is capable of differentiating.
Accordingly, a totipotent stem cell comprises a higher plasticity level than a pluripotent stem cell, as a totipotent stem cell in the present context refers to a cell capable of dividing and producing all types of differentiated cells in an organism, but also the strict extra-embryonic tissue (i.e. outer chorion) whereas a pluripotent stem cell in the present context refers to a cell capable of differentiating into all cell types comprising the adult human body including some tissues of the placenta (inner chorion, yolk sac, amnion, allantois), but not the strict extra-embryonic tissues derived from trophoblast cells (i.e. outer chorion).
The pluripotent stem cell thus comprises a higher plasticity level than a multipotent stem cell, which in the present context refers to a cell capable of dividing and producing only cell types representing the same germ layer, e.g. the hematopoietic stem cell within the mesoderm.
Accordingly, a method for increasing the plasticity level of a donor cell provides the potential of epigenetic reprogramming. Specifically as used herein the term "epigenetic reprogramming" refers to the erasure and remodeling of epigenetic marks, such as, but not limited to, covalent DNA methylation, covalent histone modifications and non-covalent chromatin remodeling. The term "reprogram" refers to the phenomenon, in which a partly or fully differentiated donor cell acquires an increase in the level of plasticity, hence becomes multipotent, pluripotent or totipotent. Donor cells may be reprogrammed to varying degrees as it is possible that an individual donor cell is reprogrammed to be multipotent or pluripotent or even totipotent. Thus, the increased level of plasticity obtained by the method disclosed in the present invention may vary and may thus be increased into different levels.
The term "reprogrammed cell" refers to a cell that had formerly attained a particular degree of differentiation i.e. a particular level of plasticity, but has subsequently regained the ability to differentiate into at least two types of specialized cells (e.g., has become multipotent, pluripotent or totipotent), it has been de-differentiated. It is in general highly unlikely that differentiated cells (i.e cells comprising a decreased level of plasticity) will revert into their precursor cells (i.e., reprogram) in vivo. However, using the method of the present invention, differentiated cells may be reprogrammed or de-differentiated into multipotent, pluripotent or totipotent cells both in vitro and in vivo. The terms "differentiated cell" and "cell comprising a decreased level of plasticity" are used interchangeably to refer to a cell comprising a poorer ability of differentiating into different specialized cell types when compared to its counterparts termed "undifferentiated cell", "supraplastic cell" and "cell exhibiting an increased level of plasticity" (the latter terms also being used herein interchangeably).
In order to determine whether the plasticity level is increased in a supraplastic cell, the plasticity level of said cell is compared to the plasticity level of the donor cell by subjecting said supraplastic and donor cell to one or more differentiation protocols and determining the number and/or type of target cells into which the supraplastic cell and donor cell as well as cells derived herefrom are able to differentiate.
A cell is determined as having an increased level of plasticity (i.e. to be a supraplastic cell), if the number of target cell types into which said cell may develop is larger than the number of target cell types into which the donor cell may develop; or if a supraplastic cell derived from a donor cell is capable of developing into at least one type of target cell into which the donor cell cannot develop.
The term "differentiated cell" refers to a cell that has developed from a relatively unspecialized phenotype (e.g. multipotent stem cell) to a more specialized phenotype (e.g. nerve cell, pancreatic beta-cell, cardimyocyte).
According to the present invention an increase in the plasticity level is desirable, as a cell exhibiting a high level of plasticity has the potential to develop into a number of different cell types, i.e. shows an increased differentiation potential or developmental potential. Plasticity modifying factors
As described herein, the present inventors have investigated several factors involved in early embryonic reprogramming, and thus in one embodiment relates to one or more plasticity modifying factor(s) increasing DNA demethylation. Examples given herein comprises MBD2 factor(s) and MBD4 factor(s)
The MBD2 factor is selected from the group consisting of DNA sequences encoded by the MBD2 gene, proteins encoded by the MBD2 DNA sequence, compounds increasing the transcription of the native MBD2 gene, compounds increasing the translation of MBD2 mRNA species and compounds increasing the activity of the proteins encoded by the MBD2 gene. As will be apparent to the skilled person, the term "MBD2" also covers any well known variants (e.g. MBD2a, MBD2b and MBD2t) as well as any other putative splice variant isoforms hereof.
The MBD4 factor is selected from the group consisting of DNA sequences encoded by the MBD4 gene, proteins encoded by said DNA sequence, compounds increasing the transcription of the native MBD4 gene, compounds increasing the translation of MB42 mRNA species and compounds increasing the activity of the proteins encoded by the MBD4 gene. Again the term "MBD4" covers any well known variants as well as any other putative splice variant isoforms hereof.
In another embodiment, the one or more plasticity modifying factor(s) inhibiting maintenance DNA methylation, comprise DNMTl factor(s).
The DNMTl factor is selected from the group consisting of compounds decreasing the transcription of the native DNMTl gene, compounds decreasing the translation degradation of DNMTl mRNA species, compounds increasing the translation degradation of DNMTl mRNA species and compounds inhibiting the DNA methyltransferase activity of the proteins encoded by DNMTl . As will be apparent to the skilled person, the term "DNMTl" also covers any well known variants (e.g. DMNTIs and DMNTIo) as well as any other putative splice variant isoforms hereof. The compounds decreasing the translational degradation of DNMTl mRNA species does according to the present invention also include siRNA and microRNA. As will be apparent from the literature, DNMTl, DNMT3a, DNMT3b (DNA methyltransferase 1, 3a, and 3b) carry out the process of DNA methylation wherein DNMTl is the proposed maintenance DNA methyltransferase that is responsible for replicating DNA methylation patterns to the daughter strands during DNA replication.
The present invention relates to epigenetic reprogramming or de-differentiation by increasing the plasticity level of a donor cell, such as inducing pluripotency through plasticity modifying factors. Others have shown that the transcription factor(s) selected from the group consisting of Oct4, Sox2, c-Myc, and Klf4 can induce de-differentiation. Thus, in one embodiment, the plasticity modifying factors of the present invention such as but not limited to MBD2 factor(s), MBD4 factor(s) and DNMTl factor(s) is used in combination with any of the factors Oct4, Sox2, c-Myc, and Klf4 for the purpose of de-differenctiation.
In one embodiment, the present invention relates to epigenetic reprogramming by increasing the plasticity level of a donor cell contacting said donor cell with, or introducing into said donor cell, one or more DNMTl factor(s) and/ or MBD2 /actor(s) and/or MBD4 factor(s) combined with the induction of expression of any of the transcription factors selected from the group consisting of OCT4, SOX2, C- MYC and KLF4.
In a further embodiment the one or more plasticity factors mediating hyperacetylation of histones (HATs) is encoded by one or more of the genes selected from the group of HATs consisting of but not limited to ATF2, CDYL,
CREBBP, ELP3, EP300, GCN5L2, GTF3C1, HATl, HTATIP, MYSTl, MYST2, MYST3, MYST4, NCOAl, NCOA2, NCOA3, OGT, PCAF, and TAFl . Like in the above this group also includes splice variants and isoforms, of the genes mentioned herein.
In general the one or more plasticity modifying factor(s) disclosed in the present invention may be selected from the group consisting of small chemical entities, peptides, proteins, RNA species including but not limited to siRNA, microRNA, shRNA, peptide aptamers, modified nucleic acids and DNA species. In order to obtain an decrease in DNA methylation, an inhibition of maintenance DNA methylation or mediate hyperacetylation of histones, the level of gene expression of the genes encoding DNA demethylation, DNA methylation, or hyperacetylation of histones may be altered at the DNA or mRNA level.
In an embodiment of the present invention, the plasticity modifying factor is directed towards the mRNA of the mediators of covalent modifications of DNA and histones and mediators of chromatin remodeling, the plasticity modifying factor being selected from the group consisting of an antisense oligonucleotide, a siRNA, shRNA and microRNA. Thus, instead of interfering directly with the mediators of covalent modifications of DNA and histones and mediators of chromatin remodelling production at the protein level, in this embodiment the production of the mediators of covalent modifications of DNA and histones and mediators of chromatin remodeling is targeted.
Accordingly, said plasticity modifying factor may either increase or decrease the expression of the genes encoding the mediators of covalent modifications of DNA and histones and mediators of chromatin remodelling. It is an object of the present invention to increase the level of gene expression of MBD2 and/or MBD4 whereas it is an object to decrease the level of gene expression of DNMTl .
A preferred plasticity modifying factor is an antisense oligonucleotide that may be specifically directed to the mRNA encoded by the gene of interest by way of complementarity. Preferably, the antisense oligonucleotide is capable of mediating RNase H cleavage of the target mRNA. Thus, the antisense oligonucleotide comprises a stretch of DNA residue and/or DNA analogue residues to make the mRNA/DNA hybrid substrate for RNase H. In a preferred embodiment, the antisense oligonucleotide is of the so-called gapmer structure, which is well known to a person skilled in the art in the field of antisense oligonucleotides.
Another preferred plasticity modifying factor is a siRNA (short interfering RNA). siRNAs are short double stranded RNA complexes of typically 20-22 nucleotides with a overhang of 1-2 nucleotides at the 3'end. siRNAs are capable of activating a sophisticated cellular machinery ultimately leading to degradation or translational inhibition of mRNAs that are complementary to the guide strand of the siRNA complex. siRNAs are typically not produced in the cell, but are introduced into the cell to mediate RNAi (RNA interference).
Yet another preferred plasticity modifying factor is shRNAs (short hairpin RNAs). shRNA may be produced from a gene introduced into a cell and may thus be viewed as vector mediated RNAi.
MicroRNAs (miRNA) are small (typically 21-23 nucleotides in length) and noncoding RNAs which regulates gene expression by mediating sequence-specific repression of mRNA translation. miRNA may be overexpressed in a cell using transfection of synthetic miRNAs or miRNA-expressing plasmids or by down- regulation using transfection of miRNA inhibitors.
In a preferred embodiment, the plasticity modifying factor interferes with the mediators of covalent modifications of DNA and histones and mediators of chromatin remodelling production at the protein level. Accordingly, said plasticity modifying factor is selected from the group consisting of a protein such as but not limited to a monoclonal antibody and a polyclonal antibody, an aptamer, a peptide, and a small chemical entity acting as inhibitor or activator of protein function. It is an object of the present invention to increase the level of activity of products encoded by MBD2 and/or MBD4 whereas it is an object to decrease the level of activity of products encoded of DNMTl .
The mediator of covalent modification of DNA, covalent modification of histones and/or modification of chromatin remodelling may be encoded by the genes selected from the group consisting of DNMTl, DNMT3A, DNMT3B, MBDl, MBD2, MBD4, MECP2, ZBTB33, ZBTB38, ZBTB4, ATF2, CDYL, CREBBP, ELP3, EP300, GCN5L2, GTF3C1, HATl, HTATIP, MYSTl, MYST2, MYST3, MYST4, NCOAl, NCOA2, NCOA3, OGT, PCAF, TAFl, HDACl, HDAC2,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7A, HDAC8, HDAC9, HDAClO, HDACIl, SIRTl, SIRT2, SIRT3,
SIRT4, SIRT5, SIRT6, SIRT7, DOTlL, EHMTl, EHMT2, EZH2, MLLl, NSDl, PRDM2, PRDM9, PRMTl, PRMT2, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, SETDlA, SETD2, SETD7, SETD8, SETDBl, SMYD3, SUV39H1, SUV39H2, SUV420H1, SUV420H2, AOF2, FBXLIl, JMJD2A, JMJD2B, JMJD2C, PADI4, CHDl, CHD2, CHD3, CHD4, SMARCAl, SMARCA2, SMARCA4, SMARCA5. According to the inventors of the present invention, an embodiment of the present invention discloses that the level of expression of at least one of the genes MBD2 and/or MBD4 or any well known variants {MBD2a, MBD2b, MBD2t), as well as putative splice variant isoforms hereof is increased.
In yet another embodiment of the present invention, the level of expression of the genes selected from the group consisting of DN MT3 A, DNMT3B, MBD2, MBD4, ZBTB33, ATF2, CDYL, EP300, NCOAl, NCOA2, OGT, TAFl, HDAC2, HDAC3, HDAC9, SIRTl, SIRT4, DOTlL, EHMTl, PRMTl, PRMT3, SETD2, SETD8 and SUV39H2 are increased.
In a preferred embodiment of the present invention the level of expression of the genes selected from the group consisting of MBD2, MBD4, ZBTB33 and SIRT4 are increased.
In yet another embodiment of the present invention, the level of expression of the gene DNMTl or any well known variants (DNMTIs and DMNTIo), as well as putative splice variant isoforms hereof, is repressed.
In a further embodiment, the level of expression of the genes selected from the group consisting of DMNTl, MBDl, CREBBP, ELP3, GCN5L2, GTF3C1, HATl, HTATIP, MYSTl, MYST2, MYST3, MYST4, NCOA3, HDACl, HDAC6, SIRT3, SIRT5, SIRT6, SIRT7, EZH2, PRDM2, PRMT2, PRMT7, SETDBl, SMYD3, SUV39H1, SUV420H1, SUV420H2, AOF2, CHD4, SMARCA2, SMARCA4 and SMARCA5 are repressed.
In yet an embodiment the level of expression of the genes selected from the group consisting DMNTl, MBDl, ELP3,GCN5L2, HTATIP, MYSTl, HDAC6, SIRT5, SIRT7, PRMT7, SMYD3, SUV39H1, SMARCA2, SMARCA4 are repressed.
Donor cell
In the present context, the term "donor cell" refers to a normoploid somatic cell from the group consisting of end-stage-committed cell types including unipotent pre-cursor cells, and multipotent stem cells, e.g. isolated from adults, newly born and foetuses.
In a preferred embodiment of the present invention, an end-stage-committed somatic cell belonging to any of the three germ layers may be isolated using techniques known in the art, and is selected from the group comprising of keratinizing epithelial cells, wet stratified barrier epithelial cells, exocrine secretory epithelial cells, hormone secreting cells, gut-, exocrine-, glands- and urogenital tract cells, metabolism and storage cells, barrier function cells (lung, gut, exocrine glands and urogenital tract), epithelial cells lining closed internal body cavities, ciliated cells with propulsive function, extracellular matrix secretion cells, contractile cells, blood and immune system cells, sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells and nurse cells. Presently preferred cells are cells readily obtainable i.e. cell which may be obtained without penetration of the body. Such cell may be but is not limited to epithelial cells, e.g. buccal mucosa cells.
An adult stem cell on the other hand refers to a partly differentiated cell characterized by its ability to divide or self-renew indefinitely and generate a number of cell types of the same germ layer from which it originates. Adult stem cells may be found throughout the foetal or adult body and may thus be obtained from already developed tissue using techniques known in the art, said tissues being selected from the group consisting of, but not limited to, brain tissue, heart tissue, adipose tissue, skin, liver and bone marrow tissue. Thus, in a preferred embodiment of the present invention, the adult stem cells of the present invention are obtained from adipose tissue, skin and bone marrow tissue.
The source of the donor cell of the present invention comprises all mammalian species. In one embodiment, the source of the donor cell is human.
It must be emphasized that the isolated donor cell itself may exhibit a range of plasticity levels. Accordingly, the donor cell of the present invention may be selected from the group consisting of end-stage-committed cell types including unipotent pre-cursor cells, and multipotent stem cells, e.g. isolated from adults, newly born and foetuses.
Accordingly the donor cell of the present invention is a cell, in which the plasticity level is to be increased.
Opposite the term "supraplastic cell" which refers to a donor cell in which the plasticity level has been increased.
Target cell
The term "target cell" relates in the present context to a cell developed by differentiation from a supraplastic cell. Target cells may for instance exhibit a phenotype different from the phenotype exhibited by the donor cell from which the supraplastic cell was derived. In an embodiment of the present invention the target cell may be selected from the group consisting of end-stage-committed cells including unipotent pre-cursor cells, oligopotent cells, multipotent cells, and pluripotent cells.
End-stage-committed cell The term "end-stage-committed-cell" refers to all differentiated cell types including tissue-specific unipotent pre-cursor cells with the ability to generate by proliferation and differentiation the specific type of tissue.
Introducing the plasticity modifying factor(s) into a cell In the present application, the introduction of one or more plasticity mediating factor(s) into a cell and the induction of epigenetic reprogramming may be performed employing various methods. Accordingly, the terms introduced herein will be used interchangeably.
Expression of the plasticity modifying factors presented in the present application may be mediated through any means for regulating gene expression, such as, but not limited to, transfection of naked DNA, viral vectors (both episomal and integrative), antisense oligonucleotides, siRNA, shRNA, microRNA, antibodies and aptamers into the cell. Said transfection may either increase or repress the level of selected factors.
In the present invention the viral vector and/or plasmid vector are employed to deliver gene constructs such as a promoter/enhancer, a reporter gene and/or selection marker into the cell.
Transfection may also be performed by various methods all well known to the person skilled in the art. Said methods 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 for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into the cell, which is to be reprogrammed. The transfection may be either transient or stable.
Conventional viral and non-viral based gene transfer methods may be used to introduce the mediators into donor cells. Such methods may be used to administer e.g. nucleic acids encoding reprogramming or plasticity increasing polypeptides. 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 vesicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery into the cell.
Methods of non-viral delivery of nucleic acids include lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, nakes DNA, artificial virolons and agent enhanced uptake of DNA.
The level of reprogramming or increase in plasticity may be assessed by various methods all of which are known to the skilled person. The methods includes but are not limited to the measurement of global DNA methylation patterns (e.g. using methylation chips), down regulation of cell type-specific markers and up- regulation of pluripotency markers. The supraplastic cells of the present invention may e.g. be characterised by biological markers for cellular multipotency, pluripotency or totipotency, e.g any of these pluripotency markers such as, but not limited to, NANOG, Oct-4 (POU5F1), S0X2, or REXl. Thus, cells having an increased OCT4 expression would generally be considered to possess an increased plasticity and this suggests that cells that have been exposed to the gene products of MBD2 and MBD4 could have increased plasticity. Example 17 demonstrates that altering the expression profiles of one or more of the genes (MBD2, MBD4 and DNMTl) may be a valid method of increasing the plasticity of cells through an upragulation of Oct-4 (POU5F1),
The cells and uses thereof
The cells disclosed in the present invention are mammalian cells. However, in a presently preferred embodiment the cells are human cells.
Before the era of human ES-cell-based therapy can be realized, the potential usefulness of these cells must be confirmed in animal models. Nevertheless, once verified, the present invention describes a method for cell therapy based upon transplantation of supraplastic cells or derivatives of the supraplastic cells for cell or tissue replacement or for delivery of compounds produced by the transplanted cells following transplantation into an individual.
It has been contemplated to use differentiated derivatives of human embryonic stem cell lines for cell-based therapy involving transplantation of the cells to an individual with the aim of either replacing lost cells or using the transplanted cells to deliver biologically active compounds to an individual.
The pluripotency of embryonic stem cell represents a risk of the recipient developing tumours originating from the embryonic stem cells, socalled teratomas. It is therefore desireable that the cells to be used in such transplation- based treatment have a desired differentiation potential, which precludes the risk of teratoma formation. Importantly, the present invention allows for therapeutic use of supraplastic cells or derivatives thereof with various degrees of differentiation potential compared to embryonic cells.
The supraplastic cells of the present invention may be used for the production of derivatives of the supraplastic cell, such derivatives comprising a target cell with a desired phenotype as well as a partly differentiated supraplastic cell. Such derivatives of supraplastic cells may be used as described above in transplantation therapy for cell replacement and/or tissue regeneration or for delivery of compounds into an individual of compounds produced by the transplanted cells.
An additional problem in cell-based therapy relates to the need for histocompatibility between the individual receiving a transplantation and the cells being transplanted to the individual. For example, the proposed therapeutic uses of non-histocompatible fetal stem cells in cell replacement treatment of Parkinson's disease or in cell delivery of brain cell growth factors to cure amyotrophic lateral sclerosis is only feasible due to the fact that the target tissue (brain) is immunologically sequestered. Most importantly, the invention allows for therapeutic use of fully histocompatible, i.e. autologous, supraplastic cells and cells derived from the supraplastic cells exhibiting various degrees of differentiation potential compared to embryonic cells thus broadening the range of diseases and conditions amenable to cell-based therapy.
Epigenetic reprogramming of somatic cells into stem cells attracts attention because of the potential for autologous transplantation therapy, as cellular derivatives of the reprogrammed cells will not be rejected by the recipient.
Although autologous stem cells may be isolated from tissues such as bone marrow from an adult individual, the number of such cells is low, and the adult stem cells are difficult to isolate for expansion. Importantly, as described in this application, this quantitative hurdle may be overcome by employing the strategy of epigenetic reprogramming of partly or fully differentiated cells from the individual to be treated.
Another source of autologous stem cells is umbilical cord blood, umbilical cord tissues as well as amniotic fluid, amnion epithelium, and placenta sampled at the time of birth or by means of ante-natal diagnostic invasive procedures and kept in storage under suitable conditions until the individual in question requires cell- based therapy. The use of such cells depends upon their suitability for therapy after prolonged storage, and the use is limited to individuals who have had samples of umbilical blood, umbilical cord tissues or amniotic fluid taken at the time of birth or ante-natal diagnostic procedures. Importantly, as described in this application, these hurdles with regard to stem cell quality and availability may be overcome by employing the strategy of epigenetic reprogramming of partly or fully differentiated cells from any individual to be treated, said reprogramming being carried out at the time that cell-based therapy is desired.
Thus far, in one embodiment the present invention relates to a method for cell therapy in which the transplantation of supraplastic cells or derivatives thereof is done to restore a tissue such as, but not limited to, bone marrow, liver, skin or cartilage. It is contemplated that the cells for replacement therapy may be transplanted as such by intravenous injection or by depositing the cells into a specific tissue, or the cells may be transplanted as a partly or fully developed tissue. Furthermore, transplantation of target cells as described herein for delivery of compounds produced natively by the transplanted target cells or transplantation of target cells for delivery of compounds not natively produced by the transplanted target cells is envisaged.
Stem cells are known to be able to remain in a given tissue and to produce growth factors, signal molecules etc., which may effect proliferation and differentiation of neighbouring cells. By providing such conditioning factors to a tissue it is believed that the naturally occuring adult stem cells may be stimulated so that new tissue is produced. In another embodiment, the transplantation of supraplastic cells or cells derived herefrom is aimed at depositing the cells in a given tissue such as, but not limited to, cardiac muscle or brain tissue in order to attain delivery to the surrounding tissue of compounds such as growth factors, signal molecules etc. produced natively by the supraplastic cells or cells derived herefrom of the present invention.
The possibility of making cell lines with all of the properties of ES cells directly from non-conventional adult sources such as but not limited to the skin holds obvious appeal. It could be a powerful way of making patient specific stem cells to provide tissue-matched cells for therapy, and a source of cells for research into the pathogenesis of complex diseases.
The ability of stem cells to remain in a given tissue may also be used to deliver compounds that are not normally produced by the stem cells by genetically engineering the stem cells prior to transplantation to produce such desired compounds as growth factors, signal molecules, enzymes etc. In yet another embodiment, the supraplastic cells are genetically engineered prior to transplantation to produce compounds not natively produced by the supraplastic cells or cells derived herefrom, and the transplantation of the genetically engineered supraplastic cells or cells derived herefrom is aimed at depositing the cells in a given tissue such as, but not limited to, cardiac muscle or brain tissue in order to attain delivery to the surrounding tissue of compounds such as growth factors, signal molecules etc. produced by the genetically engineered supraplastic cells or cells derived herefrom of the present invention.
Gene expression
In the present context the term "altering the level of expression of the one or more plasticity modifying factor(s) of covalent modifications of DNA, histones and/or chromatin remodelling" covers a situation wherein the level of gene expression is either activated/increased (up-regulated) and/or repressed/decreased (down-regulated).
"Gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product may 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.
"Gene activation" refers to any process which results in an increase in production of a gene product. A gene product may be either RNA (including, but not limited to, mRNA, rRNA, tRNA, and structural RNA) or protein. Accordingly, gene activation includes those processes which increase transcription of a gene and/or translation of a mRNA. Examples of gene activation processes which increase transcription include, but are not limited to, those which facilitate formation of a transcription initiation complex, those which increase transcription initiation rate, those which increase transcription elongation rate, those which increase processivity of transcription and those which relieve transcriptional repression (by, for example, blocking the binding of a transcriptional repressor). Gene activation can constitute, for example, inhibition of repression as well as stimulation of expression above an existing level. Examples of gene activation processes which increase translation include those which increase translational initiation, those which increase translational elongation and those which increase mRNA stability. In general, gene activation comprises any detectable increase in the production of a gene product. In a preferred embodiment of the invention, the production of a gene product is increased by at least 2-fold, e.g 5-fold, such as 10- fold, e.g 20- fold, such as 30- fold, e.g 40-fold, such as 50- fold, e.g 60-fold, such as 70- fold, e.g 80-fold, such as 90- fold, e.g 100-fold.
In the present context the term "increased level of expression" refers to a situation wherein the level of expression of a specific gene in a cell is increased when compared to the level of gene expression in a similar cell comprising the same genotype.
Determining whether the level of expression of a specific gene in a cell is increased may be measured by the following steps:
- providing a first cell having a geneotype which comprises the specific gene of interest
- providing a second cell with the same genoype as the first cell and further adding in said cell at least one modification increasing the level of expression of the specific gene,
- measuring the level of gene expression of the specific gene in both the first and second cell,
- identifying a cell as having an increased level of expression of a specific gene of interest if the second cell compared to the first cell show an increased level of expression of at least 1%, e.g. 5%, 10%, 20%, 50% or 75%. The level of gene expression may be measured by several different methods all which are known to the skilled person. The methods include e.g. Northern Blotting and quantative RT-PCR. Alternatively, if the level of expression of one or more genes is of interest DNA microarray technology or "tag based" technologies like SAGE or SuperSAGE can provide a relative measure of the cellular concentration of the mRNAs of interest.
In the present context the term "repressed level of expression" refers to a situation wherein the level of expression of a specific gene in a cell is down- regulated when compared to the level of gene expression in a similar cell comprising the same genotype.
Determining whether the level of expression of a specific gene in a cell is repressed may be measured by the following steps:
- providing a first cell having a geneotype which comprises the specific gene of interest
- providing a second cell with the same genoype as the first cell and further adding in said cell at least one modification repressing the level of expression of the specific gene,
- measuring the level of gene expression of the specific gene in both the first and second cell,
- identifying a cell as having an repressed level of expression of the specific gene of interest if the second cell compared to the first cell show an repressed level of expression of at least least 1%, e.g. 5%, 10%, 20%, 50% or 75%.
Several methods of modifying gene-expression are known to the skilled person.
"Gene repression" refers to any process which results in a decrease in production of a gene product. A gene product may be either RNA (including, but not limited to, mRNA, rRNA, tRNA, and structural RNA) or protein. Accordingly, gene repression includes those processes which decrease transcription of a gene and/or translation of a mRNA. Examples of gene repression processes which decrease transcription include, but are not limited to, those which inhibit formation of a transcription initiation complex, those which decrease transcription initiation rate, those which decrease transcription elongation rate, those which decrease processivity of transcription and those which antagonize transcriptional activation (by, for example, blocking the binding of a transcriptional activator). Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level. Examples of gene repression processes which decrease translation include those which decrease translational initiation, those which decrease translational elongation and those which decrease mRNA stability. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription. In general, gene repression comprises any detectable decrease in the production of a gene product. In general, gene activation comprises any detectable increase in the production of a gene product. In a preferred embodiment of the invention, the production of a gene product is decreased by at least 2-fold, such as 10- fold, such as 30- fold, such as 50- fold, such as 90- fold. Most preferably, gene repression results in complete inhibition of gene expression, such that no gene product is detectable.
Sequence identity
Whenever mentioned in the present application, the nucleotide sequence encoding MBD2 is selected from the group consisting of a) a nucleotide sequence set forth in SEQ ID NO: 1, and b) nucleotide sequences having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO: 1.
The same applies to the nucleotide sequence encoding MBD4. Said nucleotide sequence is selected from the group consisting of a) a nucleotide sequence set forth in SEQ ID NO: 4, and b) nucleotide sequences having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO: 4.
In accordance with the above the nucleotide sequence encoding DNMTl is selected from the group consisting of a) a nucleotide sequence set forth in SEQ ID NO: 7, and b) nucleotide sequences having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO: 7. As commonly defined "identity" is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. Thus, in the present context "sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment the two sequences are the same length.
One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.
The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
Compositions
An inherent feature of the supraplastic cells of the present invention is their capacity to renew themselves and to differentiate into a broad spectrum of derivatives of all three embryonic germ layers: ectoderm, mesoderm, and endoderm. Human embryonic stem cell lines have already been isolated, and their ability for multilineage differentiation, including neural lineage, has been demonstrated both in vivo and in vitro. This ability to develop has drawn clinical attention to stem cells as a novel source for new cell-based therapeutic strategies such as tissue regeneration.
It would obviously be desirable to generate medicaments that enabled the creation of the supraplastic cells of the present invention by simply adding a composition comprising any of the plasticity modifying factors described herein either in vivo or in vitro to partly or fully differentiated cells. Thus, in one embodiment the present invention relates to a composition for mediating an increase in the plasticity level of a donor cell, said composition comprises one or more of the plasticity modifying factors described herein.
In one embodiment said composition for mediating an increase in the plasticity level of a donor cell comprises one or more of the plasticity modifying factors selected from the group consisting of MBD2 factor(s), MBD4 factor(s) and DNMTl factor(s). In a preferred embodiment said composition for mediating an increase in the plasticity level of a donor cell, said composition comprises MBD2 factor(s).
In a preferred embodiment said composition for mediating an increase in the plasticity level of a donor cell, said composition comprises MBD4 factor(s).
In a preferred embodiment said composition for mediating an increase in the plasticity level of a donor cell, said composition comprises DΛ/MΗfactor(s).
In one embodiment, the composition may comprise MBD2 factor(s) in combination with MBD4 factor(s).
In one embodiment, the composition may be any of the above mentioned compositions further combined with DNMTl factor(s).
In one embodiment the compositions described herein may further comprising one or more histone acetyltransferases (HATs).
Vector Reprogramming of fibroblasts to a pluripotent state can be induced through ectopic expression of transcription factors such as OCT4, SOX2, C-MYC and KLF4. Expression of these four transcription factors proved to be a robust method to induce reprogramming of somatic cells to a pluripotent state. Thus in one embodiment the present invention relates to a vector comprising at least one of the genes selected from the group of AOF2, DNMTl, DNMT3A, DNMT3B, MBDl, MBD2, MBD4, MECP2, ZBTB33, ZBTB38, ZBTB4, ATF2, CDYL, CREBBP, ELP3, EP300, GCN5L2, GTF3C1, HATl. HTATIP, MYSTl, MYST2, MYST3, MYST4, NCOAl, NCOA2, NCOA3, OGT, PCAF, TAFl, HDACl, HDAC2,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7A, HDAC8, HDAC9, HDAClO, HDACIl, SIRTl, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, DOTlL, EHMTl, EHMT2, EZH2, MLLl, NSDl, PRDM2, PRDM9, PRMTl, PRMT2, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, SETDlA, SETD2, SETD7, SETD8, SETDBl, SMYD3, SUV39H1, SUV39H2, SUV420H1, SUV420H2, AOF2, FBXLIl, JMJD2A, JMJD2B, JMJD2C, PADI4, CHDl, CHD2, CHD3, CHD4, SMARCAl, SMARCA2, SMARCA4, SMARCA5, BMPl 5, CCNBl and DPP A3. Another embodiment relatest to a vector according to the present invention, wherein said vector comprising at least one of the genes selected from DMNTl, MBDl, ELP3,GCN5L2, HTATIP, MYSTl, HDAC6, SIRT5, SIRT7, PRMT7, SMYD3, SUV39H1, SMARCA2, SMARCA4, DNMT3A, DNMT3B, MBD2, MBD4, ZBTB33, ATF2, CDYL, EP300, NCOAl, NCOA2, OGT, TAFl, HDAC2, HDAC3, HDAC9, SIRTl, SIRT4, DOTlL, EHMTl, PRMTl, PRMT3, SETD2, SETD8 and SUV39H2.
In one embodiment the vector comprises at least one of the genes selected from the group consisting of DMNTl, MBD2 and MBD4.
In a presently preferred embodiment, the vector comprises at least one of the genes OCT4, SOX2, C-MYC and KLF4 in combination with MBD2 and/or MBD4.
The invention will hereinafter be described by way of the following non-limiting Figures and Examples.
Examples
MATERIALS AND METHODS
Collection of mouse oocytes
Oocytes were obtained from 3-week-old B6D2F1 mice (Taconic Europe, Ejby, Denmark) and were carefully processed to avoid human contamination. Mice were handled under standard laboratory conditions and experiments were conducted following national guidelines on animal care (Danish Ministry of Justice; approval 2003/561-713).
For the collection of GV oocytes, mice were each injected with 15 IL) menotropin (Menopur®; Ferring, Copenhagen, Denmark) and killed by cervical dislocation 42 hours later. Ovaries were isolated and antral follicles were punctured by 27-ga needles. Cumulus-enclosed GV oocytes were released into alpha MEM medium (Invitrogen, Carlsbad, CA) supplemented with 4 mmol/l hypoxanthine (Sigma, St. Louis, MO) to inhibit GV breakdown during handling. Only spherical GV oocytes with a distinct GV and attached cumulus cells were collected and immediately underwent mechanical denudation with a fine bore glass pipette. For the collection of Mil oocytes, mice were first primed with 15 IL) menotropin (Menopur®; Ferring) followed 44 hr later by injection of 5 IL) of choriongonadotropin (Pregnyl®; Organon, Oss, The Netherlands) and were then killed 15 hr later. Collected oviducts were punctured in alpha-MEM to yield the cumulus oocyte complexes (COCs). COCs were briefly exposed to 80 IU/ml hyaluronidase (SynVitro® Hyadase; MediCult, Jyllinge, Denmark) at 37°C to remove attached cumulus granulosa cells. Only spherical Mil oocytes with a distinct first polar body were selected.
After denudation, all oocytes were briefly exposed to 10 mg/ml protease (Sigma) at 37°C to remove the zona pellucida and remaining attached cumulus cell debris. Oocytes were washed several times and collected for immediate RNA extraction.
RNA extraction and quality assessment Oocyte RNA was obtained using the RNeasy® Micro Kit (Qiagen, Valencia, CA), which enriches mRNAs from small samples by selectively excluding shorter RNAs such as rRNAs and tRNAs. Briefly, pools of oocytes were lyzed with guanidine- isothiocyanate-containing buffer and were applied to spin columns for adsorption of the RNA. Carrier was included to improve the yield of low-abundance mRNAs further. Pellets were washed with 80% ethanol, centrifuged and air-dried.
Subsequently, the RNA was eluted in nuclease-free water and immediately stored at -800C until use.
To ensure that high quality RNA was hybridized to the gene expression arrays, isolated RNA was assessed on a 2100 Bioanalyzer using a RNA 6000 Pico LabChip (Agilent Technologies, Palo Alto, CA).
The protocol used for microarray chip hybridization required at least 100 ng of total RNA for each biological replicate. Due to limited sample quantities of oocytes, pooling of total RNA from repeated experiments was necessary, so with an expected average amount of 0.3 to 0.5 ng of total RNA in a murine oocyte, each replicate was constructed from pooled RNA of oocytes from approximately 20 mice. Sufficient oocyte RNA was isolated for the generation of three and five biological replicates of GV and Mil oocytes, respectively. Amplification and hybridization to microarray chips
Due to the small amounts of initial RNA, the process required an amplifying two cycle target labeling assay step to obtain sufficient amounts of labeled cRNA target for analysis with arrays. A minimum of 100 ng of total RNA was used to synthesize double stranded cDNA with the Superscript Choice system (Invitrogen) with an oligo(dT) primer containing a T7 RNA polymerase promoter (GenSet). The cDNA was used as the template for an in vitro transcription reaction to synthesize antisense cRNA (BioArray high yield RNA transcript labeling kit; Enzo, Farmingdale, NY). The signal was then amplified in a second cDNA synthesis step using random primers for first strand synthesis and T/ oligo(dT) primer for the second strand synthesis. The amplified cDNA was used as template for a second in vitro transcription to synthesize biotin labeled antisense cRNA (BioArray high yield RNA transcript labeling kit; Enzo).
The labeled cRNA was fragmented at 94°C for 35 min in fragmentation buffer (40 mM Tris, 30 mM magnesium acetate, 10 mM potassium acetate) and was hybridized for 16 hr to the Affymetrix GeneChip® Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA), which covers transcripts and variants from 34,000 well characterized mouse genes. Probe sets on this array are derived from sequences from GeneBank, dbEST, and RefSeq. The arrays were washed and stained with phycoerythrin streptavidin using a Fluidics Station 450 (Affymetrix), and the arrays were scanned in a GeneArray 3000 scanner (Affymetrix). Data were digitalized and converted into CEL format (v. 3) for data analysis.
Microarray data analyses
To adjust the overall chip brightness of the arrays to a similar level and thus make the individual chips comparable to each other, the quantile normalization method was used (Bolstad et al. 2003). To summarize the probe level data for each probe set, an expression index was calculated by the guanine cytosine corrected robust multi array analysis (GCRMA) method (Irizarry et al. 2003). Expression values were estimated on logarithmic scale (base 2) instead of raw intensities because of the variance-stabilizing effect of this transformation.
To remove the ambiguity of multiple and less specific probe sets, only transcripts with an Affymetrix oligonucleotide probe set corresponding to a single gene ("_at" suffices) or with anticipated oocyte specific splice variants ("_a_at" suffices) was retained, thus only allowing less specific probe sets if a unique probe set for a gene was missing on the gene chip at the time of manufacturing. Probe sets were cross examined in the ADAPT database (Leong et al. 2005) to ensure that only probe sets representing mRNAs remained, thus avoiding regions outside exons and 3' untranslated region (L)TR). Eventually, if a gene was represented by more than one unique probe set, the probe set with most present calls or positioned in the most distal 3' end of the transcript was selectd.
Hierarchical clustering was performed only on genes showing statistically significant up- or down-regulation as identified by t test and with a P value corrected for multiple testing with the Benjamini-Hochberg method to avoid erroneous result by less variant or absent genes. Significance level was 0.05, distance metric was 1 - r, and linkage method was centroid. To validate the overall quality of the experiment, hierarchical clustering was also performed on sample level, since biological replicates representing the same condition should to be grouped together because of identical global expression pattern. Accordingly, they should be separated from samples representing other conditions.
Normalization and calculation of gene expression indices were performed with the gcrma package (Wu et al. 2004), comparisons of samples were performed with the simpleaffy package (Wilson & Miller 2005), and Benjamini-Hochberg corrected P-values were calculated using the multtest package (Pollard et al. 2004). All packages were run in the R statistical software (http://www.r-project.org). Hierarchical clustering was performed in DNA Chip Analyzer (dChip) 2005 (Li & Wong 2003). Microarray analyses are reported according to the MIAME statement (Brazma et al. 2001).
Real-time quantitative RT-PCR Oocytes were collected and total RNA was extracted as described above.
Complementary DNA (cDNA) was synthesized with Superscript III (Invitrogen) or iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) using random hexamers or a random hexamer/oligo(dT) blend as first strand primer. Equal amounts of total RNA (24 ng) were converted into cDNA for GV and Mil samples, respectively. Subsequently, the cDNA elute was diluted 1:20 before down-stream application.
For PCR amplification, the gene specific primers were constructed using Primer3 (Rozen & Skaletsky 2000) or PrimerBank (Wang & Seed 2003). Primers were carefully chosen or designed to eliminate the risk of amplifying unspecific targets, such as primer dimers or genomic DNA. Primer sequences, exon complementarity, melting temperature, GenBank accession number, and size of the amplified product are presented in table 1. Expression levels were quantified in real-time on a LightCycler® Instrument (Roche Diagnostics, Germany) using LightCycler® FastStart DNA Master PLUS SYBR Green I (Roche Applied Science) or SYBR® Premix Ex Taq™ kit (Takara, Madison, WI) as detection chemistry. The PCR reaction mixture consisted of 2 μl of diluted cDNA, 1 μl of gene specific primers, 2 μl or 5 μl of SYBR® Green I master mix (depending on manufacturer), and added water to a total volume of 10 μl. First, cDNA was denatured and pre-incubated by heating for 10 sec at 95°C. Template was then amplified by 45 cycles consisting of denaturation for 5 sec at 95°C, annealing 20 sec at 55°C, and extension for 10 sec at 72°C. At least three replicates were used for each reaction, and a minus RT and a minus template served as negative controls. To verify RT-PCR product identity, melting point curves were generated following amplification. Samples were heated to 95°C followed by immediate cooling for 15 sec at 65°C and then slowly heating by 0.1°C/sec up to 95°C while monitoring fluorescence continuosly.
To determine the effect of meiotic maturation on the expression of a candidate internal reference gene, a range of housekeeping genes was evaluated. Briefly, identical amounts of input RNA from GV and Mil oocytes were used for each cDNA reaction. Putative candidate reference genes were amplified and the gene showing the lowest variability was chosen as internal reference using Norm-Finder (Andersen et al. 2004).
All target genes were normalized against the chosen internal reference gene within the log-linear phase of the amplification curve, and relative quantification was assessed using the 2-ΔΔCT method, where ΔΔCT = (CT-target - CT-reference)Mii - (CT-target - CT-reference)Gv (Pfaffl 2001). All fold changes are presented as means with 95% confidence intervals. EXAMPLE 1
Oocyte collection and validation of RNA
On average, 320 and 421 oocytes were pooled for each biological replicate of GV and Mil oocytes, respectively. An examination of total RNA on a 2100 Bioanalyzer confirmed the extraction of high-quality RNA (data not shown). Correlations between biological replicates were substantially high, showing identical global transcription patterns and thus high reliability of the microarray gene chip procedure performed on minute amounts of oocyte RNA (Figure 1 and Table 2). An unsupervised hierarchical cluster analysis using all detected probe sets showed that biological replicates representing the same developmental stage were independently grouped together as expected. Genes known to be preferentially expressed in oocytes such as the zona pellucida glycoproteins (ZPl, ZP2, and ZP3), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), moloney sarcoma oncogene (MOS)1 cyclin Bl (CCNBl), spindlin (SPIN), MATER (NALP5), zygote arrest 1 (ZARl), STELLA (DPPA3), and OCT4 (POU5F1) were abundantly detected on the chip set (Table 3). Although oocytes underwent careful denudation, potential contamination of heterologue RNA from cumulus- granulosa cells were investigated. The absence of cumulus transcripts such as hyaluronic synthase 2 (HAS2), pentraxin 3 (PTX3), and gremlin 1 (GREMl) confirmed the oocyte homogeneity of the collected samples.
EXAMPLE 2
Global oocyte gene expression and inclusion of genes for further analyses After excluding probe sets that were 100% absent in both groups of replicates, the data were reduced from 45,101 to 24,919 probe sets. To minimize false positives, a probe set was then included for further analysis provided that a 100% present call was found in either one of the two groups of replicates, irrespective of its call percentage in the other replicate group. Accordingly, a total of 14,697 probe sets were expressed exclusively in GV oocytes, in Mil oocytes, or both. A t- test for difference in expression level between GV and Mil oocytes Benjamini- Hochberg corrected for multiple testing yielded that 5,169 (35%) probe sets were significantly down-regulated, 609 (4%) probe sets were up-regulated, and 8,919 (61%) probe sets were equally expressed. A complete list of up- and down- regulated genes will be available at http://www.ebi.ac.uk/arrayexpress/.
EXAMPLE 3
General characteristics of reprogramming genes
Table 3 shows the average expression indices, fold changes and corrected P- values supplemented with values for a number of transcripts preferentially expressed in the oocyte. In total, 57 genes (70 %) were detected in GV- and/or MII-oocytes on the chip set. Within the 33 genes detected as down-regulated, 14 had fold changes from -1 to -2, eight had fold changes from -2 to -5, three had fold changes from -5 to -10, and eight had fold changes less than -10. A total of 24 genes were detected as up-regulated with 16 genes having fold changes from 1 to 2 and eight genes having fold changes ranging from 2 to 5. However, when only statistically significant fold changes were considered, four genes were up- regulated and 18 genes were down-regulated, whereas 35 genes indicated an unchanged level of gene expression. The dendrogram in figure 2 illustrates the final clustering tree, where significantly regulated genes close to each other have high similarity in their standardized expression values across all eight samples.
EXAMPLE 4
DNA methyltransferases and methyl-CpG-binding proteins The gene of the oocyte variant form of the maintenance DNA methyltransferase, Dnmtlo, was abundantly expressed in GV oocytes and then significantly down- regulated in Mil whereas the two de novo DNA methyltransferases DNMT3A and DNMT3B both showed unchanged level of expression. Of the seven known MeCPs that bind to methylated DNA, two were significantly up-regulated (MBD2, MBD4), one was down-regulated (MBDl), one (Kaiso/ZBTB33) showed no change while MECP2, ZBTB4 and ZBTB38 were not detected on the chip. EXAMPLE 5
Histone acetyltransferases
HATs may be classified into five subgroups according to sequence homology and internal protein domains. All five members of the MYST-subgroup were detected in oocytes. TIP60 (HTATIP), MOF (MYSTl) and MORF (MYST4) showed significant down-regulation, while HBOl (MYST2) and MOZ (MYST3) were unchanged. Of the members belonging to the GNAT-subgroup, elongation protein 3 homolog (ELP3) and GCN5 (GCN5L2) were significantly down-regulated, histone aminotransferase 1 (HATl) was unchanged and p300/CBP-associated factor (PCAF) was absent. The two transcriptional 'coactivators' ElA binding protein p300/p300 (EP300) and CREB binding protein/CBP (CREBBP)1 which are almost functionally equivalent (P300/CBP-subgroup), were both expressed oocytes at an unchanged level as were the three nuclear hormone-receptor cofactors (SRC-subgroup) SRC-I (NCOAl), GRIPl (NCOA2), and ACTR (NCOA3). The basal transcription factors TAFII250 (TAFl) and TFIIIC (GTF3C) were also expressed at an unchanged level. The more recent identified HATs, CDYL, ATF2, and OGT, were all expressed in oocytes at an unchanged level.
EXAMPLE 6
Histone deacetylases
HDACs are subdivided into four classes according to phylogenesis. Class I members HDACl, HDAC2, and HDAC3 were expressed in oocytes, whereas HDAC8 was absent. HDACl was down-regulated and HDAC2 and HDAC3 were unchanged. No class II HDAC was detected, except HDAC6 and HDAC9, which were down-regulated and unaffected, respectively. Except SIRT2, all sirtuins or class III HDACs were expressed in oocytes of which SIRT4 was up-regulated, SIRT3, SIRT5 and SIRT7 were down-regulated, and SIRTl and SIRT6 showed no change. The single class IV member, HDACIl, was not detected. EXAMPLE 7
Histone methyltransferases and histone demethylases/deiminases HMTs either activate or repress gene function depending on the residue of the modified amino acid. Histone methylation at positions H3K4, H3K36, or H3K79 is associated with gene activation. Of the activating HMTs, DotlL (DOTlL) was undifferentially expressed, SMYD3 (SMYD3) was down-regulated, and SETl (SETDlA), MLL (MLLl), SET7/9 (SETD7), and Meisetz (PRDM9) were absent. Of the HMTs associated with repression (H3K9, H3K27 and H4K20), SUV39hl (SUV39H1) and ESET (SETDBl) were down-regulated, SUV39h2 (SUV39H2), Eu- HMTasel/GLPl (EHMTl), ENX-I (EZH2), PR-SET7/SET8 (SETD8), SUV4-20hl (SUV420H1), SUV4-20h2 (SUV420H2), and RIZ (PRDM2) were unchanged and G9a (EHMT2) was absent. NSDl (NSDl), which can act both as activator and repressor, was not detected.
Of the arginine HMTs, one was slightly up-regulated (PRMT3), one was down- regulated (PRMT7), two were undifferentially expressed (PRMTl, PRMT2), and three were absent (PRMT4, PRMT5, PRMT6).
All currently known HDMs were absent in oocytes, except LSDl (AOF2), which was expressed at an unchanged level.
EXAMPLE 8
Chromatin remodeling ATPases
The ChR-ATPases are classified into subfamilies according to the presence of conserved domains outside their catalytic region. The two bromodomain SNF2- subfamily ATPases hBRM (SMARCA2) and BRGl (SMARCA4) were both expressed in oocytes and down-regulated. The SANT domain ISWI-subfamily members SNF2L (SMARCAl) and SNF2H (SMARCA5) were absent or unchanged, respectively. Among the four chromodomain CHD-subfamily ATPases, only Mi-2β (CHD4) was expressed at unchanged level while CHDl, CHD2 and Mi-2α (CHD3) were absent. EXAMPLE 9
Real time quantitative RT-PCR
The four transcripts having statistically significant up-regulation and the 18 transcripts being down-regulated as shown by the present microarray data were selected for further analysis by real-time quantitative RT-PCR. To select an appropriate internal reference gene, the expression of the three housekeeping genes β-actin (ACTB)1 porphobilinogen deaminase (HMBS) and TATA box binding protein (TBP) using equivalent amounts of total RNA converted into cDNA were tested. A statistically insignificant relationship existed between oocyte developmental stage and expression of ACTB, which was confirmed by using NormFinder. Accordingly, ACTB was used as internal reference gene for further analyses.
The RT-PCR expression data for the 22 reprogramming gene transcripts are presented in figure 3. The absence of non-specific amplified products was confirmed by melting curve analysis. When comparing RT-PCR fold changes with the microarray data, some discrepancy regarding direction and amplitude was observed. To test whether such differences could be explained by the use of random hexamer primers during cDNA synthesis, the RT-PCR using oligo(dT) primers (Fermentas, Helsingborg, Sweden) was repeated. In this case, there was a significantly higher correlation between array data and qRT-PCR data regarding direction and amplitude, suggesting the occurrence of selective deadenylation during meiotic maturation.
Example 10
Cloning and expression of MBD2
Sequence information for the human MBD2 gene is obtained by use of the Swiss Protein database (www.expasy.org). Swiss-Prot entry Q9UBB5 is used to access DNA sequence information regarding the human MBD2 gene and its splicing isoforms. Human MBD2 is a 411 amino acid protein with a molecular weight of 43255 Da. A synthetic MBD2 gene is purchased from commercial companies like GENEART (www.geneart.com). Here a DNA fragment is manufactured according to the human MBD2 gene sequence as specified by the Swiss-Prot Q9UBB5 entry. Both DNA fragments coding for the standard human MBD2 protein as well as DNA fragments coding for splicing and sequence variants is obtained in this way.
Synthetic genes are normally delivered cloned into a standard bacterial vector. This bacterial plasmid with a MBD2 gene insert allows for multiplication of the MBD2 gene material according to the needs for subsequent use.
Restriction enzyme sites useful for DNA fragment recloning is inserted before and after the MBD2 gene reading frame. Such restriction enzyme sites is designed into the purchased MBD2 construct, or a PCR reaction with PCR primers containing such restriction enzyme sites is used to amplify a MBD2 gene fragment with these these sites attached at proper positions.
The human MBD2 gene fragment is recloned into a commercially available expression system using cloning sites as described by the manufactorer. The RheoSwitch Mammalian Expression System from New England BioLabs (www.neb.com) allows for induction and adjustable control of gene expression for the inserted reading frame. The T-REx System from Invitrogen (www.invitrogen.com) utilizes a CMW promoter to deliver higher levels of induced expression than other regulated mammalian expression systems.
Primary human fibroblast cells are cultivated by use of hospitally derived material from neonatal circumcisions. Alternatively primary human fibroblasts are obtained from commercial suppliers like Cascade Biologies (www.cascadebio.com), a part of Invitrogen (www.invitrogen.com), and primary human fibroblast cells are grown using media and protocols supplied by the cell line manufactorer. Expression system vectors containing a MBD2 gene fragment are transformed into primary human fibroblasts by use of methods described by the expression vector manufactorer. Transformation is performed in the form of transient expression experiments, or with the purpose of generating a stable cell line with a chromosomally integrated MBD2 gene construct. The level of MBD2 expression in transformed primary human fibroblast cells with or without expression vector MBD2 expression is analysed by use of MBD2 specific antibodies in an ELISA assay. Abeam (www.abcam.com) is an example of a commercial supplier of MBD2 antibodies, and the codes ab26848, ab33931, ab38646, ab49136 and abll985 are examples of such Abeam products.
A significantly increased MBD2 protein expression level is observed for transformed primary human fibroblast cells relative to control cells without MBD2 overexpression.
Example 11
Cloning and expression of MBD4
Sequence information for the human MBD4 gene is obtained by use of the Swiss Protein database (www.expasy.org). Swiss-Prot entry 095243 is used to access DNA sequence information regarding the human MBD4 gene and its splicing isoforms. Human MBD4 is a 580 amino acid protein (unprocessed precursor) with a molecular weight of 66051 Da.
A synthetic MBD4 gene is purchased from commercial companies like GENEART (www.geneart.com). Here a DNA fragment is manufactured according to the human MBD4 gene sequence as specified by the Swiss-Prot 095243 entry. Both DNA fragments coding for the standard human MBD4 protein as well as DNA fragments coding for splicing and sequence variants is obtained in this way.
Normally, a synthetic gene is either delivered already cloned into a standard bacterial vector or it is cloned into such a vector when received from the commercial supplier. This bacterial plasmid with a MBD4 gene insert allows for multiplication of the MBD4 gene material according to the needs for subsequent use.
Restriction enzyme sites useful for DNA fragment recloning is inserted before and after the MBD4 gene reading frame. Such restriction enzyme sites is designed into the purchased MBD4 gene fragment, or a PCR reaction with PCR primers containing such restriction enzyme sites is used to amplify a MBD4 gene fragment with these these sites attached at proper positions.
The human MBD4 gene fragment is recloned into a commercially available expression system using cloning sites as described by the manufactorer. The RheoSwitch Mammalian Expression System from New England BioLabs (www.neb.com) allows for induction and adjustable control of gene expression for the inserted reading frame. The T-REx System from Invitrogen (www.invitrogen.com) utilizes a CMW promoter to deliver higher levels of induced expression than other regulated mammalian expression systems.
Primary human fibroblast cells are cultivated by use of hospitally derived material from neonatal circumcisions. Alternatively primary human fibroblasts are obtained from commercial suppliers like Cascade Biologies (www.cascadebio.com), a part of Invitrogen (www.invitrogen.com), and primary human fibroblast cells are grown using media and protocols supplied by the cell line manufactorer. Expression system vectors containing a MBD4 gene fragment are transformed into primary human fibroblasts by use of methods described by the expression vector manufactorer. Transformation is performed in the form of transient expression experiments, or with the purpose of generating a stable cell line with a chromosomally integrated MBD4 gene construct.
The level of MBD4 expression in transformed primary human fibroblast cells with or without expression vector MBD4 expression is analysed by use of MBD4 specific antibodies in an ELISA assay. Abeam (www.abcam.com) is an example of a commercial supplier of MBD4 antibodies, and the codes ab3756, abl3793 and abl2187 are examples of such Abeam products.
A significantly increased MBD4 protein expression level is observed for transformed primary human fibroblast cells relative to control cells without MBD4 overexpression. Example 12
Simultaneous expression of MBD2 and MBD4
A transient overexpression of both MBD2 and MBD4 is established by co- transfection of the MBD2 expression plasmid described in Example 5 and the MBD4 expression plasmid described in Example 6 into primary human fibroblast cells as previously described.
A stable cell line that overexpresses both MBD2 and MBD4 is established by constructing a DNA fragment, where both genes are placed in line behind separate regulated promoters. The resulting dual gene construct inserted into the matching expression plasmid system is transformed into human primary fibroblast cells for stable transformant isolation as previously described in example 5 and 6.
The level of MBD2 and MBD4 protein expression is measured using specific antibodies as described in example 5 and 6.
At significantly increased expression of both MBD2 and MBD4 is observed in dual gene overexpression experiments compared to the background level found for human primary fibroblast cells.
Example 13
Induction of plasticity by MBD2 and/or MBD4 overexpression
Detection of surface markers, that characteristically are associated with stem cell or progenitor cell phenotypes, may be used as a measure for cell plasticity (developmental potential). Primary human fibroblast cells are so-called end-stage- committed cells, and detection of surface markers for non-end-stage-commited cells upon overexpression of MBD2 and/or MBD4 signals a possible induction of plasticity.
Companies like Abeam (www.abcam.com), BD Biosciences (www.bdbiosciences.com) and eBioscience (www.ebioscience.com) market a large number of different antibodies, that are fluorescently labeled, and that each specifically recognize a particular cell surface marker. Primary human fibroblast cells overexpressing MBD2 and/or MBD4 protein is probed with fluorescense labeled antibodies recognizing selected surface marker proteins according to the procedure described by the antibody manufactorer. Fluorescence activated cell sorting equipment like a BD FACSArray from BD Biosciences (www.bdbiosciences.com) is used to count the number of cells expressing a particular cell surface protein marker, and quantitative data for light signal emissions is obtained for characteristic cell type surface markers.
Primary human fibroblast cells overexpressing MBD2 and/or MBD4 protein are found to display characteristic cell surface proteins, that identifies them as being different from control primary human fibroblast cells without the MBD2 and/or MBD4 overexpression.
DNA methylation of cytosine in specific CpG regions is known to be associated with cell differentiation. As induction of plasticity is a process opposite to differentiation, demethylation of CpG sites may also be considered indicative for an increased plasticity, exemplified by the transition from an end-stage committed cell to a non-end-stage commited cell. The methylSEQr Bisulfite Conversion Kit from Applied Biosystems (www.appliedbiosystems.com) is used to determine the methylation status of a CpG motif. Sodium bisulfite deaminates cytosine to produce uracil, and methylated cytosines are protected from this conversion. Hence DNA sequence determination of chromosomal DNA regions with and without the use of bisulfite convertion will identify methylated cytosines.
Primary human fibroblast cells overexpressing MBD2 and/or MBD4 protein are analysed for DNA methylation of particular chromosomal regions using a bisulfite conversion kit as described by the kit manufactorer. DNA sequence determination is performed using an ABI PRISM 310 Genetic Analyzer from Applied Biosystems (www.appliedbiosystems.com) as described by the manufactorer.
The CpG methylation patterns for primary human fibroblast cells with overexpression of MBD2 and/or MBD4 protein is found to show signs of DNA demethylation compared to primary human fibroblast cells without the MBD2 and/or MBD4 overexpression.
Example 14
Enhancement of plasticity induction
DNA methyltransferase 1 (DNMTl) is responsible for the methylation of the newly formed DNA strand during DNA replication. Only hemi-methylated CpG sites are recognized, and in this way the pre-existing methylation pattern is copied onto the two daughter cells. Demethylation induced by overexpression of MBD2 and/or MBD4 protein may thus be counterpoised by DNMTl activity, and DNMTl inhibition may be required for efficient plasticity induction.
A listing of DNA methylation inhibitors is found in an article by CB. Yoo and P. A. Jones (Nature Reviews Drug Discovery, volume 5, January 2006, page 37-50).
Treatment of primary human fibroblast cells with and without overexpression of MBD2 and/or MBD4 protein with known DNA methylation inhibitors like for example 5-azacytidine, hydralazine or procainamide is used to determine the enhancement of plasticity induction measured as described in example 13.
Reduction of cellular DNMTl activity can also be obtained by use of RNA interference (RNAi) technology. Loss of function for specific genes is induced by way of short RNAi oligonucleotides complementary to the target mRNA transcript. Commercially available RNAi systems like DNMTl Stealth Select RNAi (RNAi Catalog Nr. HSS102859; HSS102860; HSS102861) from Invitrogen
(www.invitrogen.com) (www.rnaidesigner.invitrogen.com) is transfected into primary human fibroblast cells as described by the manufactorer and used for plasticity induction analysis as previously described.
Chromatin structure constitutes another obstacle for an efficient DNA demethylation reaction, where heterochromation is known to be structurally inaccessible for protein factors. Condensation of chromosomal DNA into heterochromatin is associated with histone deacetylation, whereas DNA wrapped around acetylated histones are accessible to protein factors. Histone acetylases (HAT) and histone deacetylases (HDAC) perform opposite reactions, and hyperacetylation is generated by use of histone deacetylase inhibitors.
A listing of histone deacetylase inhibitors is found in an article by CB. Yoo and P. A. Jones (Nature Reviews Drug Discovery, volume 5, January 2006, page 37- 50).
Treatment of primary human fibroblast cells with and without overexpression of MBD2 and/or MBD4 protein with known histone deacetylase inhibitors like for example butyrate, valproic acid, oxamflatin and apicidin is used to determine the enhancement of plasticity induction measured as described in example 8.
DNMTl inhibition and/or histone hyperacetylation is observed to result in similar or improved plasticity induction, when primary human fibroblast cells overexpressing MBD2 and/or MBD4 protein are analysed as described in example 8.
Example 15
Re-differentiation of de-differentiated donor cells A number of protocols have been tested for the differentiation of human pluripotent stem cells into a wide range of specialized cells. Thus, a donor cell that has been de-differentiated and reprogrammed into a higher level of plasticity may be considered pluripotent if it demonstrates capability of being re-differentiated into one or more end-stage committed cell from each the three primary germ layers (ectoderm, mesoderm, and endoderm).
Ectoderm
To evaluate the differentiation-potential of de-differentiated donor cells into a target cell of ectodermal origin, a protocol for the derivation of dopaminergic neurons using a serum-free suspension culture system is implemented:
Cells are harvested as per any standard procedure, washed with DMEM, and placed in suspension culture in differentiation medium consisting of either MedII/FGF2 medium (DMEM/F12, 1 x N2 [Invitrogen], 20 mM L-glutamine, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 4 ng/ml FGF-2, and 50% serum-free Medll) or DMEM/N2 medium (DMEM, 1 x N2, 20 mM L-glutamine, 0.5 U/ml penicillin, 0.5 U/ml streptomycin). Cultures are differentiated for 2-6 weeks in suspension, and the medium is changed every 5-7 days. Characteristic folds and rosettes of neural precursors is observed after 5-10 days of culture. Cell aggregates are plated on dishes or Permanox slides coated with 20 μg/ml polyornithine (Sigma) and 1 μg/ml laminin (Sigma) in MedII/FGF2 medium or Neurobasal medium (Invitrogen) containing 1 x B27 (Invitrogen), 5% FCS (Hyclone), 2 ng/ml glial-derived neurotrophic factor (GDNF) (R&D Systems), 10 ng/ml brain-derived neurotrophic factor (BDNF) (R&D Systems), 20 mM L- glutamine, 0.5 U/ml penicillin, and 0.5 U/ml streptomycin. These cultures are highly enriched for nestin÷ neural precursor rosettes and large networks of Bill tubulin+ neurons. In addition, most of these neurons will also express tyrosine hydroxylase, which is the enzyme responsible for converting L-tyrosine to dihydroxyphenylalanine (DOPA), the precursor for dopamine.
Mesoderm
To assess the differentiation-potential of de-differentiated donor cells into a target cell of mesodermal origin, a protocol for the derivation of fully mature primitive erythroid cells is implemented:
First, de-differentiated donor cells are co-cultured with immortalized fetal human liver clone B (FH-B-hTERT) cells (Wege et al. Gastroenterology 2003; 124:432- 444). CD34+ cells are subsequently purified and subjected to serum-free liquid culture, allowing for the expansion of de-differentiated donor cell-derived hematopoietic stem cells (HSCs). These cells are then cultured in the presence of insulin growth factor-1 to induce commitment of de-differentiated donor cell - derived HSCs to the erythroid fate. Finally, terminal erythroid differentiation is induced via co-culture of putative erythroid progenitors with MS-5 feeder cells, erythropoietin and hemin. Using this stepwise protocol, upwards of 5x 106 terminally differentiated erythrocytes may be generated from 50 000 dedifferentiated donor cells in less than one month.
Endoderm To evaluate the differentiation-potential of de-differentiated donor cells into a target cell of endodermal origin, a protocol for the derivation of pancreatic β-cells is implemented:
De-differenatiated donor cells are passaged onto 1% Matrigel (B&D Biosciences)- coated tissue culture dishes. Then, the culture medium is changed to modified CDM: 50% IMDM (Gibco) plus 50% F12 NUT-MIX (Gibco), supplemented with insulin-transferrin-selenium-A (1 : 100, Gibco) and 450 μM monothioglycerol (Sigma), and 5 mg/ml albumin fraction V (Sigma) or X-Vivol0 (Cambrex) supplemented with 55 μM 2-Mercaptoethanol (Gibco) and 0.1% albumin fraction V (Sigma).
Two days later, the cells are induced into definitive endoderm differentiation with CDM containing 50 ng/ml activin A (Sigma) for 4 days, as detected by the expression of the definitive endoderm markers SOXl 7 and Brachyury. After 4 days of activin A induction, the cells are transferred into CDM with 10-6 M all-trans retinoic acid (Sigma) for another 4 days to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors PDXl and HLXB9. Culture medium is then changed from CDM to modified islet maturation medium: DMEM/F12 (Gibco), insulin-transferrin-selenium-A (1 : 100, Gibco) and 2 mg/ml albumin fraction V (Sigma) with 10 ng/ml bFGF (Invitrogen) for the first 3 days and with 10 mM nicotinamide (Sigma) for the next 5 days. The cells are digested by 0.5 mg/ml dispase (Gibco) and transferred into Ultra Low Attachment culture dishes (Costar) for 5 days to achieve islet maturation in suspension culture. The differentiated cells may be evaluated by the expression of islet specific markers such as C-peptide, insulin, glucagon and GLUT2.
Example 16
Reprogramming of human buccal mucosa epithelial cells (ectoderm) into pluripotency followed by re-differentiation into fully mature primitive erythroid cells (mesoderm) Donor cells in the form of human epithelial cells can easily be obtained non- invasively by gentle scraping from the buccal mucosa. Subsequently, the cells are induced to active DNA demethylation by means of increasing activity of DNA demethylating factors and/or inhibition of maintenance methylation in accordance with the principles outlined in examples 10-14.
The level of reprogramming and putative pluripotency is then tested by the concomitant up-regulation of classical stem cell markers (Oct-4, SSEA3, SSEA4, TRA-1-60, TRA-1-81) and down-regulation of epithelia cell markers (e.g. cytokeratins, lamin A/C).
Final level of differentiation capacity is assessed by exposing the de-differentiated donor cells to the protocol of erythroid cell differentiation as described in example 15.
Example 17
Characterisation of stem cell gene expression on mRNA-level in human dermal fibroblasts transfected with MBD2 and/or MBD4 with/without DNA methyltransferase inhibitor (5-aza)
Human foetal (foreskin) fibroblast cells (hFFs) were cultivated in IMDM medium (Invitrogen) containing 10% FBS and 1% pen/strep.
Synthetic DNA fragments with the MBD2 and MBD4 protein reading frames were obtained from GENEART. The DNA fragments were inserted into the pcDNA 3-l(+) expression vektor (Invitrogen). Both the MBD2 and the MBD4 reading frames were sequenced in the final plasmid vectors prior to use for transfection eksperiments.
Lipofectamine 2000 (Invitrogen) was used for plasmid transfection of hFF cells acording to the manufactorers description (use of standard ratio of DNA to Lipofectamine 2000 reagent). Test of the above transfection protocol using a GFP reporter plasmid detected a green fluorescence signal in almost half of the transfected hFF cells.
Human foetal fibroblasts (hFFs) were transfected and grown under the following 3 conditions (plasmid use combinations):
1. hFFs transfected with the MBD2 construct,
2. hFFs transfected with the MBD4 construct, and
3. hFFs transfected with both the MBD2 and MBD4 construct.
For each transfection, cells were harvested after a period of one, two or three days of culture. In addition, cells from the different transfections were also grown in the presence of 5 μM 5-azacytidine (5aza) in order to inactive the DNMTl enzyme activity.
Total RNA from hFFs was obtained using the RNeasy® Micro Kit (Qiagen, Valencia, CA), which enriches mRNAs from small samples by selectively excluding shorter RNAs such as rRNAs and tRNAs. Cells were deattached from the bottom of the culture dish by trypsination (Trypsin-EDTA treatment followed by resuspension in IMDM medium containing 10% FBS for trypsin inactivation) and isolated by centrifugation. Tubes with cell pellets were rapidly snap frozen by dipping into liquid nitrogen and subsequently stored at -800C. The pelleted cells were lyzed with guanidine-isothiocyanate-containing buffer and were applied to spin columns for adsorption of the RNA. Carrier RNA was included to improve the yield of low- abundance mRNAs further. Pellets were washed with 80% ethanol, centrifuged and air-dried. Subsequently, the RNA was eluted in nuclease-free water and immediately stored at -800C until use.
Quantity and integrity were assessed on a NanoDrop N-1000 device (NanoDrop Technologies, Wilmington, DE, USA) and showed to be of good quality according to absorbances at 260 and 280 nm.
RNA was converted into cDNA using Superscript III reverse transcriptase (Invitrogen) with a blend of oligo-d(T) and random hexamer primers (both Fermentas). Briefly, mixtures containing dNTP mix, water, primer blend and RNA template were heated at 700C for 5 min and placed immediately on ice for 3 min to unfold secondary RNA structures. Next, the mixture was added to a blend of first-strand buffer, DTT, RNase inhibitor and Superscript III reverse trancriptase. Samples were incubated in a thermocycler at 55°C for 1 hour followed by inactivation of reverse transcriptase at 700C for 15 min. The synthesized cDNA was stored immediately at -200C.
cDNA quality was evaluated on a NanoDrop N-1000 device (NanoDrop Technologies, Wilmington, DE, USA) and was shown to be of good quality by the outline of the absorbance curve around in relation to 260 and 280 nm.
To assess the level of induced plasticity, six transcripts which has been shown to be characteristic for pluripotent human embryonic stem cells (hESCs) (i.e. DNMT3B, GABBR3, GDF3, NANOG, POU5F1, TDGFl) were designed according to refs. (Adewumi et al., 2007 and Awan et al., 2008). In addition, seven transcripts expressed in multipotent human mesenchymal stem cells (hMSCs) (Abdallah et al., 2008) were designed (i.e. ALCAM, ANPEP, CD44, CD55, DLKl, ENG, ICAMl). Primer sequences were designed using either PrimerBank (Wang & Seed 2003) or PerlPrimer (Marshall O.f 2007). Three house-keeping genes were used as internal reference genes (i.e. ACTB, GAPDH, TBP).
Fold changes in gene expression were evaluated by real-time qPCR using a LightCycler device (Roche). SYBR Green I dye was used as detection chemistry (Takara). Briefly, SYBR Premix Ex Taq mix, template cDNA, gene specific primers, and RNase-free water were mixed inversion and brief spin. Samples (10 μl) were placed in capillaries and rotor (Genaxxon). PCR conditions were as follows: cDNA was denatured and preincubated by heating for 10 s at 95°C. Template was then amplified by 37 cycles consisting of denaturation for 5 s at 95°C, annealing for 20 s at 55°C, and extension for 10 s at 72°C. To verify RT-PCR product identity, melting point curves were generated following amplification. Samples were heated to 95°C followed by immediate cooling for 15 s at 65°C and then slowly heating by 0.1°C/s to 95°C, while monitoring fluorescence continuously. All target genes were evaluated against the geometric mean of the three reference house-keeping genes within the log-linear phase of the amplification curve, and ΔCT values were assessed using the ΔΔCT method (Pfaffl, 2001).
Expression levels of the genes specific to undifferentiated human embryonic stem cells as compared to control population (i.e. non-transfected hFFs) were generally modified only to a modest degree and there were no consistent trend for either up- or down-regulation. However, with one noticeable exception, the classical gene for characterizing undifferentiated hESC is the POU5F1 (OCT4) gene, which consistently in all transfections shows a relatively strong up regulation. The present data actually indicate a 9 to 14 fold up-regulation of the POU5F1 (OCT4) gene.
Figure legends
Figure 1
Shows a correlation plot illustrating the concordance in probe set expression values (n =45,101) between two of the biological replicates. The high correlation coefficient (r) confirms the reliability of Affymetrix gene chip technology performed on minute amounts of mRNA extracted from mammalian oocytes.
Figure 2
Shows a dendrogram illustrating the final hierarchical clustering tree of 22 significantly up- or down-regulated reprogramming genes (for gene symbols, see Table 3) in Mil oocytes when compared with GV oocytes (five and three biological replicates respectively). The expression values for a gene across all eight samples are standardized to have mean 0 and S. D. 1 by linear transformation. The distance between two genes is defined as 1 - r, where r is the standard correlation coefficient between the eight standardized values of two genes. Two genes with the closest distance are first merged into a supergene, connected by branches with length representing their distance, and deleted for future merging. The expression level of the newly formed supergene set is the average of standardized expression levels of the two genes (average linkage) for each sample. Then, the next pair of genes (supergene) with the smallest distance is chosen to merge and the process is repeated until all genes are merged into one final cluster. The genes Prmt3, Mbd4, Mbd2, and Sirt indicate significant down-regulation, whereas the remaining genes indicate down-regulation.
Figure 3
Shows a bar chart showing fold changes of 22 reprogramming genes measured by real-time quantitative RT-PCR. Mean fold changes are expressed on a Iog2 scale as metaphase II oocyte values relative to germinal vesicle stage oocytes. Whiskers represent 95% confidence intervals.
Figure 4
Shows a bar chart showing ΔCt changes of 13 genes specific to undifferentiated human embryonic stem cells. Cells have been transfected with MBD2 in absence of DNA methyltransferase inhibitor (DNMTi) 5-azadeoxycytidine (5aza).
Expression levels are shown as compared to control population (non-transfected hFFs) at 1, 2 and 3 days post-transfection.
Figure 5
Shows a bar chart showing ΔCt changes of 13 genes specific to undifferentiated human embryonic stem cells. Cells have been transfected with MBD4 in absence of DNA methyltransferase inhibitor (DNMTi) 5-azadeoxycytidine (5aza). Expression levels are shown as compared to control population (non-transfected hFFs) at 1, 2 and 3 days post-transfection.
Figure 6
Shows a bar chart showing ΔCt changes of 13 genes specific to undifferentiated human embryonic stem cells. Cells have been transfected with MBD2 with the DNA methyltransferase inhibitor (DNMTi) 5-azadeoxycytidine (5aza) present. Expression levels are shown as compared to control population (non-transfected hFFs) at 2 and 3 days post-transfection. Figure 7
Shows a bar chart showing ΔCt changes of 13 genes specific to undifferentiated human embryonic stem cells. Cells have been transfected with MBD4 with the DNA methyltransferase inhibitor (DNMTi) 5-azadeoxycytidine (5aza) present. Expression levels are shown as compared to control population (non-transfected hFFs) at 2 and 3 days post-transfection.
Figure 8 Shows a bar chart showing ΔCt changes of 13 genes specific to undifferentiated human embryonic stem cells. Cells have been transfected with MBD2 and MBD4 with the DNA methyltransferase inhibitor (DNMTi) 5-azadeoxycytidine (5aza) present. Expression levels are shown as compared to control population (non- transfected hFFs) at 2 and 3 days post-transfection.
Tables
Table 1.
Primer list comprising a group of selected genes important to to epigenetic modifications and chromatin remodelling. The housekeeping gene {Actb) is used as an internal reference. 5 Gene symbols and descriptions have been adopted from the Mouse Genome Informatics database
(http://www.informatics.jax.org/). Primer pairs were carefully designed so that they were separated by at least one intron sequence to reduce risk of co-amplified genomic DNA. Additionally, some primers span intron-exon flanks. No primer pair had inter-primer complementarity thus avoiding formation of primer-dimers. All primer sequences were blasted for potential Ul Ul alternative binding sites.
10
Abbreviations: F, forward primer; R, reverse primer; Tm, melting temperature.
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Table 2
Characteristics for microarray chip samples. A high correlation coefficient (r) between biological replicates indicates almost identical global transcription patterns and thus high validity and reliability of the microarray procedure per se performed on 5 minute amounts of RNA.
Abbreviations: GV , germinal vesicle; Mil, metaphase II.
Developmental stage Biological Median (range) r between P-value replicates (n) biological replicates
GV oocytes 3 0.993 (0.992 - 0.995) < 0.0001
Mil oocytes 5 0.972 (0.955 - 0.992) < 0.0001
Table 3
Expression patterns in murine oocytes of 86 genes involved in epigenetic modifications and chromatin remodelling. Selected transcripts preferentially expressed in oocytes are included for comparison. Fold changes are represented as mean metaphase II stage values relative to germinal vesicle stage. P-values have been corrected for multiple testing with the Benjamini- 5 Hochberg method, and expression indices have been Iog2 transformed. Gene symbols and descriptions are adopted from the Mouse Genome Informatics database (http://www.informatics.jax.org/).
Abbreviations: GV, germinal vesicle; Mil, metaphase II; BH, Benjamini
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Claims

Claims
1. A method for increasing the plasticity level of a donor cell, said method comprising
-providing a mammalian, normoploid somatic cell as the donor cell
-increasing the plasticity level in said cell by contacting said donor cell with, or introducing into said cell, at least one plasticity modifying factor selected from the group consisting of DNMTl factors, MBD2 factors and MBD4 factors,
thereby obtaining a supraplastic cell with the ability to develop into at least two different end-stage-commited cell types.
2. A method according to claim 1, wherein the MDB2 factor is selected from the group consisting of DNA sequences encoded by the MBD2 gene, proteins encoded by said DNA sequence, compounds increasing the transcription of the native MBD2 gene, compounds increasing the translation of MBD2 mRNA species and compounds increasing the activity of the proteins encoded by the MBD2 gene.
3. A method according to claim 1, wherein the MBD4 factor is selected from the group consisting of DNA sequences encoded by the MBD4 gene, proteins encoded by said DNA sequence, compounds increasing the transcription of the native MBD4 gene, compounds increasing the translation of MBD2 mRNA species and compounds increasing the activity of the proteins encoded by the MBD4 gene.
4. A method according to claim 1, wherein the DNMTl factor is selected from the group consisting of compounds decreasing the transcription of the native DNMTl gene, compounds increasing the degradation of DNMTl mRNA species and compounds inhibiting the DNA methyltransferase activity of the proteins encoded by DNMTl .
5. A method according to any of the preceeding claims, wherein the at least one plasticity modifying factor is further combined with at least one plasticity modifying factors mediating hyperacetylation of histones
, wherein said mediator of histone acetylation is selected from the group consisting of ATF2 factor(s), CDYL factor(s), CREBBP factor(s), ELP3 factor(s), EP300 factor(s), GCN 5L2 factor(s), GTF3C1 factor(s), HATl factor(s), HTATIP factor(s), MYSTl factor(s), MYST2 factor(s), MYST3 factor(s), MYST4 factor(s), NCOAl factor(s), NCOA2 factor(s), NCOA3 factor(s), OGT factor(s), PCAF factor(s), and TAFl factor(s).
6. A method according to any of the preceeding claims, wherein the one or more plasticity modifying factor(s) and the mediators of histone acetylation is selected from the group consisting of a small chemical entity, a peptide, a protein, an RNA species including but not limited to SiRNA-, microRNA- and, shRNA-molecules, peptide aptamers, modified nucleic acids and DNA species.
7. A supraplastic cell according to any of claims 1-6.
8. A supraplastic cell according to claim 7, said supraplastic cell being partly differentiated.
9. Use of the supraplastic cell of claims 7-8 for the production of target cells, said target cells being derived from the supraplastic cell and exhibiting an end-stage- committed phenotype including a unipotent pre-cursor target cell.
10. A target cell according to claim 9.
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