WO2007054720A1 - Reprogrammation et modification genetique de cellules - Google Patents

Reprogrammation et modification genetique de cellules Download PDF

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WO2007054720A1
WO2007054720A1 PCT/GB2006/004218 GB2006004218W WO2007054720A1 WO 2007054720 A1 WO2007054720 A1 WO 2007054720A1 GB 2006004218 W GB2006004218 W GB 2006004218W WO 2007054720 A1 WO2007054720 A1 WO 2007054720A1
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cell
cells
nanog
pluripotent
differentiated
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PCT/GB2006/004218
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WO2007054720A8 (fr
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Ian Chambers
Jose Rebero Da Silva
Austin Gerard Smith
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The University Court Of University Of Edinburgh
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Priority claimed from GB0523039A external-priority patent/GB0523039D0/en
Priority claimed from GB0605998A external-priority patent/GB0605998D0/en
Application filed by The University Court Of University Of Edinburgh filed Critical The University Court Of University Of Edinburgh
Priority to AU2006313518A priority Critical patent/AU2006313518A1/en
Priority to JP2008539502A priority patent/JP2009515515A/ja
Priority to EP06808510A priority patent/EP1957643A2/fr
Priority to GB0808244A priority patent/GB2445706A/en
Priority to GB0702261A priority patent/GB2434155A/en
Publication of WO2007054720A1 publication Critical patent/WO2007054720A1/fr
Publication of WO2007054720A8 publication Critical patent/WO2007054720A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/605—Nanog
    • 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
    • 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
    • C12N2501/999—Small molecules 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to reprogramming and genetic modification of cells, in particular cells that are to be reprogrammed.
  • the invention relates especially to reprogramming of cells to a pluripotent state, and to genetic modification of a cell, e.g. to remedy a genetic defect, prior to such reprogramming.
  • pluripotent cells differentiate into many distinct cell types. These are defined by the specific subsets of genes that are either activated or repressed, the epigenome.
  • a differentiated epigenome can be reverted to pluripotency by nuclear transfer into enucleated oocytes and through cell fusion with either ES or EG cells.
  • ES and EG-T cell hybrids it has been shown that the inactive X, silent imprinted genes and a silent Oct4-GFP reporter transgene of T cells are reactivated (Tada et al, 1997 and 2001).
  • ES and EG-T cell hybrids were shown in vivo to contribute to all the three primary germ layers, demonstrating pluripotency properties (Tada et al, 1997; 2001).
  • Other studies have also shown that nuclei of the central nervous system, of bone marrow and of splenocytes exhibit pluripotent potential after fusion with ES cells (Matveeva et al, 1998; Terada et al, 2002; Ying et al, 2002).
  • analysis of the chromatin status of the somatic genome in ES-differentiated cell hybrids showed acquisition of ES cell genome properties (Kimura et al, 2004). Together, these results showed that hybrids do not simply contain the union of the two genomes, the likelihood is that the somatic donor genome has undergone full reprogramming.
  • PPCs Primordial germ cells entering the genital ridges at 10.5dpc-11.5dpc undergo genome wide demethylation, reactivation of the silent X chromosome and erasure of imprints (Hajkova et al, 2002; Sato et al, 2003; Tarn et al, 1994). Recently, it was reported that reprogramming is a feature of early embryos (Mak et al, 2004; Okamoto et al, 2004). In these studies it was shown that imprinted X chromosome inactivation occurs in all the cells of the late morula and blastocyst.
  • the inactive X chromatin showed, along with other epigenetic marks, enrichment of H3-K9 dimethylation and H3-K27 trimethylation. This was a surprising result as the inactive X is a marker of cell differentiation and its state is stable and heritably transmitted, being only reversible in pluripotent cells (Wutz and Jaenisch, 2000). Importantly, the inactive X was shown to erase the epigenetic marks and reactivate, at around 4.5 dpc, in the epiblast (Mak et al, 2004; Okamoto et al, 2004).
  • Nanog is a unique homeodomain-containing protein present in pluripotent mammalian cells and essential for early development.
  • the previously inactivated paternal X chromosome in female XX embryos becomes reactivated. Reactivation is confined to those cells that express Nanog and does not occur in Nano null embryos.
  • Nanog expression in the early embryo marks the future pluripotent epiblast cells and is rapidly downregulated during ES cell differentiation and embryo development (Chambers et al, 2003; Mitsui et al, 2003).
  • Nanog has the unique properties, when overexpressed in ES cells, of allowing self-renewal in the absence of the otherwise essential factors Lif and serum (BMP-4) (Chambers et al, 2003; Ying et al, 2003).
  • BMP-4 otherwise essential factors Lif and serum
  • Nanog is expressed in the pluripotent EG cells and in the PGCs, in which reprogramming is occurring (Chambers et al, 2003; Yamaguchi et al, 2005).
  • ICMs Inner Cell Masses
  • WO 03/064463 describes use of Nanog in combination with LIF to reprogram a cell or nucleus. However, in comparative examples described herein, expression of Nanog was found to be insufficient to induce reprogramming.
  • genetic modification of cells is known using e.g. vectors. Genetic modification of cells produced by SCNT has been used, for example, in the treatment of immunodeficiency (Rideout et al, 2002). But these methods can leave artefacts in the genome.
  • Cell therapy products should be diploid cells, but it is known that fusion of diploid cells yields tetraploid cells or cells in which segregation of chromosomes is uneven, yielding a variety of different aneuploid cell types (Matveeva et al, 1998). This would be unacceptable for regulatory authorities.
  • An object of the present invention is to ameliorate or at least provide an alternative to the above problems.
  • An object of specific embodiments of the invention is to provide improved reprogramming methods and cells obtained thereby.
  • a further object of specific embodiments of the invention is to provide a method for genetic modification of cells, optionally followed by reprogramming of the modified cells and derivation of progeny thereof.
  • Nanog expression and / or inhibition of MEK is used for reprogramming of cells.
  • a method of obtaining a pluripotent genome from a differentiated genome comprising:- fusing a pluripotent cell with a differentiated cell; and overexpressing Nanog in the fused cell.
  • Nanog can be overexpressed by overexpressing Nanog in the pluripotent cell prior to the fusing step, overexpressing Nanog in the differentiated cell prior to the fusing step or overexpressing Nanog in the fused cell, i.e. after the fusing step.
  • Nanog is suitably overexpressed by introducing into a cell a genetic construct which expresses Nanog.
  • Nanog can also be overexpressed by introducing into the cell, or culturing the cell in the presence of, a medium component which increases Nanog expression in the cell.
  • the method comprises:- fusing a pluripotent cell with a differentiated cell to form a fused cell; wherein at least one of the pluripotent cell, the differentiated cell and the fused cell is treated with a MEK inhibitor.
  • Both the pluripotent cell and the differentiated cell can be treated with a MEK inhibitor.
  • Nanog via direct overexpression or via overexpression using e.g. a MEK inhibitor, in this way increases the efficacy of obtaining a cell with pluripotent properties. It is believed that treatment with a MEK inhibitor results in upregulation of expression of Nanog.
  • the inventors have also advantageously found that following further culture a pluripotent, diploid cell can be obtained, containing genetic information from the differentiated cell.
  • a fused cell is maintained in culture and spontaneously loses chromosomes until its chromosome complement reduces and reverts to diploid.
  • at least one or more chromosomes from the differentiated cell contribute to the diploid state of the resultant, pluripotent cell.
  • a pluripotent cell can be obtained and used to derive differentiated progeny thereof including cells and tissue starting from a differentiated cell containing a desired gene or a desired chromosome or other desired genetic material.
  • the pluripotent cell is suitably an embryonic stem (ES) cell, an embryonic carcinoma (EC) cell or an embryonic gonadal (EG) cell, and in specific embodiments of the invention, described in more detail below, cell fusions have successfully resulted in a pluripotent genome by fusing an ES cell with a differentiated cell.
  • the differentiated cell can be a somatic cell and in general it is believed that substantially all somatic cells are suitable for use in application of the invention described herein.
  • the differentiated cell can also be a stem cell and, again, it is believed that the application of the invention is not restricted to any particular stem cells, but extends generally to stem cells, in particular neural stem cells, haematopoietic stem cells etc. In use of a particular example of the invention, reprogramming has successfully been carried out when the differentiated cell is a neural stem cell.
  • Nanog can be provided via various different means. In one embodiment, Nanog is provided by overexpressing Nanog in the pluripotent cell, and in a separate embodiment Nanog is provided by expressing Nanog in the differentiated cell.
  • Nanog is also optional for Nanog to be expressed directly in both of or either of the pluripotent cell and the differentiated cell, conveniently by transfecting one or more of the cells with a vector containing a nucleotide type sequence encoding Nanog together with a suitable promoter.
  • the methods of the present invention result in Nanog being overexpressed in the pluripotent cell, and in practice expression levels of from two times up to fifteen times the endogenous ES cell level are believed suitable, preferably three to ten times, with about five times the normal level being used in one or more of the examples.
  • the invention is believed to be of application generally to mammalian cells, including murine, human, porcine, ovine, bovine and caprine and in all cases it is preferred that the pluripotent cell and the differentiated cell are of the same species. It is further preferred that the pluripotent cell and the differentiated cell are both mouse cells or both human cells. It has hitherto been described that following fusion of ES cells there is unequal segregation of chromosomes (Matveva et al 2005). However, in accordance with the present invention, reprogramming methods comprise culturing the fused cell so as to obtain a diploid cell.
  • chromosome segregation is not, in fact, unequal but can be carried out so as to obtain cells which contain a diploid chromosome complement. Analysis of these cells typically shows that they contain a mixture of chromosomes derived from the pluripotent cell and also chromosomes derived from the differentiated cell, i.e. a mix of chromosomes.
  • fusing of the pluripotent cell and the differentiated cell results in a tetraploid cell and the method comprises obtaining progeny of the tetraploid cell and identifying such progeny which, for example by spontaneous chromosome loss, are or have become diploid.
  • the method comprises obtaining progeny of the tetraploid cell and identifying such progeny which, for example by spontaneous chromosome loss, are or have become diploid.
  • a significant proportion of the cells obtained are diploid. Without wishing to be bound by any theory, it seems both the case that a large proportion of these cells spontaneously revert to diploid status and also that during culture of the cells, preferably continued in the presence of Nanog, there is a survival advantage for truly diploid cells.
  • a diploid cell population can be obtained with chromosomes derived from each of the two starting material cells.
  • the invention thus offers the opportunity to derive a cell, whether a somatic cell or a stem cell or other, from a differentiated cell by carrying out the reprogramming step as described and, thereafter, culturing a diploid, pluripotent cell obtained as progeny of the fused cell, and deriving a differentiated cell from the diploid, pluripotent cell.
  • the invention additionally provides a method of genetically modifying a cell, comprising:- providing a first cell containing chromosomes, providing a second cell containing chromosomes, fusing the first cell and the second cell to form a fused cell, and culturing the fused cell so as to obtain a diploid cell containing at least one chromosome from the first cell and at least one chromosome form the second cell.
  • Nanog is overexpressed in the first cell, the second cell and / or the fused cell.
  • at least one of the first cell, the second cell and the fused cell can be treated with a MEK inhibitor.
  • the first cell and the second cell are preferably both diploid, and can be both mouse cells or both human cells.
  • the thus-obtained cell contains chromosomes derived from each of the first cell and the second cell, and thus the method can be used to combine genetic material from two different sources. After obtaining the resulting diploid cell this can be tested to identify which particular combination of chromosomes have ended up in the diploid cell and, as and when necessary, selection carried out to identify a cell having a particularly desired combination of chromosomes.
  • one of the first cell and the second cell is pluripotent.
  • the other of the first cell and the second cell is optionally pluripotent, and can be a somatic cell, optionally a stem cell.
  • the other cell is a neural stem cell.
  • an embodiment of the invention comprises identifying a first cell having a desired copy of a gene and an undesired copy of the same gene which is to be modified, identifying a second cell having a modified form of the undesired gene, fusing the first cell and second cell, culturing the fused cell, and identifying a progeny of the fused cell containing both the desired copy of the gene and the modified form of the undesired gene.
  • the combination of genes from the first and second cells yields a cell free of a genetic defect, free of the undesired gene.
  • the invention is usefully exploited for curing a genetic defect in the first cell wherein the undesired gene constitutes a gene having a defect and the modified form of the gene in the second cell constitutes the cure.
  • Another embodiment of the invention comprises the fusion of a first cell engineered to alter the expression of a normal endogenous gene with a second cell containing normal copies of the endogenous gene, culturing the fused cell, and identifying a progeny of the fused cell containing both a normal copy of the gene and the engineered form of the gene.
  • This invention is usefully exploited for treating a disease by making a compensatory change in the expression of a gene.
  • a method of the invention in which a suitable second cell is created may comprise genetically modifying the second cell prior to fusion with the first cell so as to introduce into the second cell the modified form of the undesired gene.
  • aneuploid generally tetraploid
  • the resultant cell though initially or potentially aneuploid, generally tetraploid, can undergo chromosomal segregation to become a diploid cell at an acceptable rate, yielding cells useful e.g. for ongoing culture, derivation of progeny and cell therapy.
  • the invention provides a method of cell fusion, comprising:- fusing a first cell and a second cell in the presence of a MEK inhibitor.
  • the first cell is preferably pluripotent, and may be an ES, EC or EG cell.
  • the second cell can also be pluripotent, but is preferably a somatic cell, more preferably a stem cell.
  • Nanog can be provided by expressing Nanog in the first cell and/or second cell, as described elsewhere herein.
  • the first cell, the second cell and / or the fused cell can be cultured in the presence of the MEK inhibitor, again as described elsewhere herein.
  • a method of the invention comprises fusing the first and second cells to yield an aneuploid cell and culturing the fused cell to obtain diploid progeny thereof.
  • Cells are also provided by the invention, being cells obtained according to a method of any aspect of the invention.
  • the invention provides additionally methods of cell therapy using the cells and use of a cell according to the invention in manufacture of a medicament for use in therapy, preferably cell therapy.
  • a method of treatment of the invention comprises administering to a patient an effective amount of cell of the invention.
  • a further method of treatment comprises administering to a patient an effective amount of a genetically modified cell of the invention.
  • Treatment can suitably comprise:- identifying a patient needing a genetically modified cell, identifying the genetic modification required to treat the patient, and preparing a genetically modified cell according to the method of any embodiment of the invention.
  • the invention provides use of Nanog in reprogramming a cell obtained by a fusion of a pluripotent cell and a somatic cell.
  • the pluripotent cell is typically an ES cell
  • the somatic cell is typically a stem cell, and in a specific embodiment is a neural stem cell
  • the invention also provides use of a MEK inhibitor in reprogramming a cell obtained by a fusion of a pluripotent cell and a somatic cell.
  • the pluripotent cell is typically an ES cell
  • the somatic cell is typically a stem cell, and in a specific embodiment is a neural stem cell.
  • the invention also provides use of Nanog in an improved method of somatic cell nuclear transfer to obtain a pluripotent cell.
  • a method of nuclear transfer of the invention comprises transferring a somatic cell nucleus into a recipient cell to form a nuclear transfer cell, and overexpressing Nanog in the nuclear transfer cell. Reprogramming of the donor, somatic cell nucleus is thereby achieved with improved efficiency.
  • the invention also provides use of a MEK inhibitor in an improved method of somatic cell nuclear transfer to obtain a pluripotent cell.
  • a method of nuclear transfer of the invention comprises transferring a somatic cell nucleus into a recipient cell to form a nuclear transfer cell, and treating the nuclear transfer cell with a MEK inhibitor. Reprogramming of the donor, somatic cell nucleus is thereby achieved with improved efficiency.
  • the recipient cell is generally an oocyte, preferably enucleated and also preferably of the same species as the donor.
  • the MEK inhibitor is a MEKl inhibitor.
  • suitable MEK inhibitors include the MEKl inhibitor PD184352, and those discussed in Davies et al (2000).
  • Ultimate progeny of these and other aspects of the invention can include cloned non- human animals (human cloning forms no part of the invention) and pluripotent cell cultures.
  • a method of obtaining a live non-human animal comprising a method of cell fusion or nuclear transfer of the invention and a method of obtaining a culture of pluripotent stem cells, comprising a method of cell fusion or nuclear transfer of the invention.
  • the donor somatic cell is more preferably a stem cell, and in particular a neural stem cell.
  • Nanog is provided as described herein, by expressing it in the donor somatic cell.
  • a MEK inhibitor can be used, again as described elsewhere herein.
  • the MEK inhibitor upregulates expression of Nanog as described herein. It is preferred that Nanog is overexpressed in the donor somatic cell, such as by being expressed at a level greater than the normal, endogenous level of Nanog expression in ES cells, for example at a level that is from 2 to 15 times the level of endogenous expression of Nanog in an ES cell.
  • An embodiment of this invention is to use a somatic donor cell with a gene defect for nuclear transfer, then correct the gene defect in the resulting pluripotent cell by methods known in the art for use in treatment of a disease.
  • This embodiment is believed to be of application generally to mammalian cells, including murine, human, porcine, ovine, bovine and caprine and in all cases it is preferred that the recipient oocyte and the somatic donor cell are of the same species.
  • the cells are pre-treated with a MEK inhibitor.
  • Reference to Nanog herein refers to the family of polypeptides described in WO 03/064463, including the preferred polypeptides described, and hence refers e.g. to a polypeptide which is a pluripotency determining factor having from 200 to 400 amino acids.
  • a mouse pluripotency determining factor is represented by SEQ ID NO 2 therein, a human pluripotency determining factor by SEQ ID NO 4 therein, a rat pluripotency determining factor by SEQ ID NO 6 therein, and a macaque pluripotency determining factor by SEQ ID NO 8 therein, the contents of WO 03/064463 being fully referred to and incorporated herein by reference.
  • Nanog refers also to a factor which maintains a cell in a pluripotent state, acts intracellularly and comprises a homeodomain, in particular a homeodomain that has at least 50% sequence identity with the homeodomain from SEQ ID NO: 2, 4, 6 or 8 therein or, in relation to a factor for cells of a given species, one that has at least 50% sequence identity with the homeodomain of pluripotency determining factor of the same species.
  • a homeodomain is around 60 amino acids in length and the factor comprises a homeodomain in which any 20 amino acid fragment has at least 35% sequence identity with the homeodomain of SEQ ID NO: 2, 4, 6 or 8.
  • Nanog further refers to isolated polypeptides which include (a) polypeptide molecules comprising an amino acid sequence as set out in SEQ ID NO: 2, 4, 6 or 8; (b) naturally occurring variants of (a); (c) orthologues of (a) or (b), and (d) biologically active and diagnostically or therapeutically useful fragments, analogues and derivatives thereof.
  • Nanog additionally includes the polypeptides of SEQ ID NO: 2, 4, 6 and 8 (in particular the mature polypeptide) as well as polypeptides which have at least 50% similarity (preferably at least 50% identity) to the polypeptide of SEQ ID NO: 2, 4, 6 or 8 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO: 2, 4, 6 or 8 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO: 2, 4, 6 or 8 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • Similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Various different approaches are known for the calculation of sequence similarity and identity.
  • a suitable way to perform these calculations is to run database searches using a program such as Smith- Waterman, BLAST or FASTA, and use one or preferably two or even three similarity tables.
  • the Blosum and PAM (Point Accepted Mutation) matrices are suitable amino acids similarity matrices for database searching and sequence alignment.
  • neural stem cells are used as the somatic cell, i.e. as a source of differentiated epigenome.
  • Neural stem (NS) cells were expandable and could be manipulated genetically (Conti et al, 2005). These can be derived from embryonic and adult brain and from ES cells. NS cells form a homogeneous population that expresses morphological and molecular features of radial glial cells in a reproducible manner. In addition, transplantation assays showed that these cells are not transformed, even after prolonged expansion (Conti et al, 2005). Nanog overexpression in ES cells was shown to enhance reprogramming of the NS cell genome after ESxNS cell fusion.
  • Nanog expression is necessary for the reprogramming of NS cells as shown by the failure of Nanog-/- ES cells to generate ES-NS cell hybrids.
  • this property was rescued by the introduction of transgenic expression of Nanog in both Nanog-/- ES cells and, to a lesser extent, in NS cells.
  • Comparison of the ability of ES-ES and ES-NS FACS sorted hybrids to produce ES-like colonies suggested that overexpression of Nanog from ES cells is the only factor required for the reprogramming of the NS cell genome.
  • Nanog in embryonic stem cells increased 200-fold the frequency of pluripotent hybrids obtained after fusion with neural stem (NS) cells. This cannot be explained by effects of Nanog on cell viability or clonogenicity. Forced expression of Nanog in NS cells did not convert them to ES cells indicating that factors additional to Nanog are necessary for reprogramming. However, Nanog overexpressing ES cells formed pluripotent hybrids with NS cells at the same efficiency as ESxES fusions, in which there is no reprogramming. This implied that the level of Nanog is the only limiting factor for reprogramming the NS cell epigenome to pluripotency in hybrids.
  • Nanog null ES cells failed to yield any pluripotent hybrids upon fusion with NS cells. Their capacity was fully restored by introduction of a Nanog transgene.
  • a MEK inhibitor for increasing the efficacy of obtaining a cell with pluripotent properties and upregulating the expression of Nanog in the cells. It has been shown that if cells are treated with a MEK inhibitor then this enhances reprogramming by cell fusion with a similar effect to that of using a Nanog trans gene.
  • the cell is a somatic cell.
  • the cell is reprogrammed in vitro.
  • the cell is not a fused cell.
  • the invention allows for reprogramming a somatic cell back to pluripotency, thus enabling derivation of pluripotent cells without cell fusion or nuclear transfer.
  • MEK inhibitor herein refers to MEK inhibitors in general.
  • MEKl inhibitors for example P184352 (Davies et al, 2000).
  • P184352 has been found to have a high degree of specificity and potency when compared to other known MEK inhibitors.
  • the invention is now illustrated in specific embodiments set out in the following examples and accompanied by drawings in which: -
  • Fig. 1 shows Nanog expression is necessary for X reactivation in vivo
  • Fig. 2 shows Nanog enhances ES cell ability to generate ES-differentiated cell hybrid colonies
  • Fig. 3 shows the effect of PEG in cell viability and differentiation
  • Fig. 4 shows an analysis of NS epigenome in ES-NS cell hybrids
  • Fig. 5 shows FACS analysis of fusion mixtures
  • Fig. 6 shows Nanog is necessary for the generation of ES like ES-NS hybrid colonies
  • Fig. 7 (Supplementary Figure 1) shows a chromosome count of hybrid clones
  • Fig. 8 (Supplementary Figure 2) shows that ES-NS hybrids exhibit pluripotency after deletion of transgenic Nanog; and Fig. 9 shows the effect of PD 184352 in the formation of pluripotent ES-NS hybrid colonies.
  • Figure 1 shows that Nanog expression is necessary for X reactivation in vivo.
  • A-D Immunofluorescence for Eed and Nanog of female (XX) 4.5 dpc and diapause embryos resulted from crosses between Nanog heterozygous mice.
  • Panels (A, B) exhibit complete confocal projections of analysed embryos. Arrows indicate Nanog positive cells.
  • C, D Higher magnification of A and B nanog positive embryos. Note absence of Eed large foci from Nanog positive cells (white arrowheads). Panels exhibit partial confocal projections of analysed embryos. Red arrowheads point to examples of cell nuclei exhibiting large Eed focus (inactive X).
  • E, F Tables summarizing the scores for presence (with Xi) or absence (with no Xi) of Eed large focus in Nanog positive cells of 4.5 (E) and diapause (F) embryos.
  • FIG. 2 shows Nanog enhances ES cell ability to generate ES-differentiated cell hybrid colonies.
  • A-F Illustrated are plates containing hybrid colonies stained with Leishmans. Scored colonies of fusions involving RH and RHN ES cells with ES O4GiP (A), NS 04GiP (embryo) (B), NS TGFP (D), NS 04GiP (Adult) (E) and MEF TGFP (F) exhibited red and green fluorescence (see right column example). Scored EF4 x NS 04GiPGFPIH (C) colonies exhibited green fluorescence only. All scored colonies displayed ES morphology and scores were performed while cells were in culture.
  • Figure 3 shows the effect of PEG in cell viability and differentiation.
  • A Bar chart indicating number of ES cells attached to 100mm plates 24 hours after PEG treatment of RH and RHN cells. 10 6 cells were plated.
  • B - Illustrated are 96- well plates containing RH and RHN colonies stained with Alkaline phosphatase (AP). PEG treated and untreated cells were allowed to sit for 4 hours in culture plates before being tripsinized and single cell FACS sorted into 96 well plates. Cells were then cultured for 12 days.
  • C Table summarizing 96 well plate data.
  • Figure 4 shows an analysis of NS epigenome in ES-NS cell hybrids.
  • A-D Immunofluorescence for trimethyl H3-K27 (me 3 H3-K27) (A) and Ubiquityl H2A (ubH2A) (B) and RNA FISH for Xist (C) and Oct4 (D) in XX NS TGFP, XY RH, XY RHN, XXXY RH-NS TGFP and XXXY RHN-NS TGFP cells.
  • Yellow arrowheads indicate presence of me 3 H3-K27, ubH2A or Xist RNA nuclear body in XX NS TGFP cells.
  • FIG. 1 Illustrated are FACS plots detecting both red and green fluorescence of RHxNS TGFP, RHNxNS TGFP, RHNxES 04GiP, RHNxMEF TGFP and RHNxT TGFP.
  • A mock fusion providing a gate control for the analyses and FACS sorting of the hybrid population;
  • B fusion mixture 24 hours after PEG treatment;
  • C Purity check of FACS sorted hybrids gated in B.
  • D Hybrids sorted in B were plated and the formed colonies were scored as percentage of colonies per plated hybrid. These scores take into account the purity of the FACS sorted cells. Scores from a replicated experiment are indicated in (E).
  • Figure 6 shows Nanog is necessary for the generation of ES like ES-NS hybrid colonies.
  • A,B Illustrated are plates containing hybrid colonies stained for Alkaline phosphatase of fusions between RCxNS O4GiP, RCA-AxNS O4GiP, RCA-/-NZxNS O4GiP, RCA-AxNS O4GiP NP, RCB-AxNS 04GiP, RCB-A NZxNS 04GiP, RCB-A xNS 04GiP NP cells. Scores are indicated under each plate.
  • Figure 7 shows a chromosome count of hybrid clones. Examples show metaphase spreads of RH-NS 04GiP (A), RHN-NS 04GiP (B), RH- NS TGFP (C) and RHN-NS TGFP (C). Metaphase chromosomes were counted in 8 to 11 cells for 3 clones of each hybrid cell line and are indicated under each illustration, cl-clone.
  • Figure 8 shows ES-NS hybrids exhibit pluripotency after deletion of transgenic Nanog.
  • A Schematic representation of EF4 and Neural Stem (NS) O4GiP RB cells.
  • EF4 cells contain a constitutively expressing Nanog transgene flanked by lox P sites, which allows the deletion of Nanog using ere recombinase that will then lead to constitutive expression of GFP.
  • NS 04GiP RB contains a GFP transgene driven by Oct4 promoter sequences and a constitutively expressing RED fluorescent transgene. Grey boxes indicate IRES sequences.
  • C EF4-NS RB 04GiP cells after Cre deletion of Nanog. Note increased GFP expression.
  • E RT-PCR analysis of T-brachyury expression in EF4-NS 04GiP RB cre EBs at 3, 5, 7 and 8 days (d) of differentiated (diff). Undifferentiated hybrids and the parental/donor cell lines were also analysed. Gapdh was used as a ubiquitously expressed control.
  • F Immunoflurescence for ⁇ -actinin (blue) of plated EBs.
  • Figure 9 shows analysis of the effect of PDl 84352 in the formation of pluripotent ES- NS hybrid colonies.
  • A-C - FACS analysis for red and green fluorescence of RHxNS TGFP fusions.
  • A Fusion mixture 24 hours after PEG treatment;
  • B Purity check of FACS sorted hybrids gated in A.
  • C Hybrids sorted in A were plated and the formed colonies were scored as percentage of colonies per plated hybrid. These scores take into account the purity of the FACS sorted cells.
  • D Summary of data.
  • E Examples of hybrid colony morphology.
  • Nanog expression in the early embryo is necessary and correlates with progressive X reactivation
  • X-chromosome reactivation is known to occur in vivo in the female embryo epiblast at around 4.5 dpc (Mak et al, 2004; Okamoto et al, 2004).
  • Eed is a marker of the inactive X chromosome in pre-implantation embryos (Silva et al, 2003; Mak et al, 2004; Okamoto et al, 2004).
  • Nanog negative embryos showed in all the nuclei of the embryo presence of a single large Eed focus, with the exception of apoptotic and rare poorly Eed stained cells (Figure IA).
  • Female Nanog positive embryos exhibited a variable number of Nanog positive ICM cells ( Figure IE). In contrast to the rest of the embryo, a proportion of these cells showed no Eed focus, which is consistent with X reactivation ( Figure 1C,E).
  • Figure 1C,E X reactivation
  • Nanog-/- embryos were smaller. To determine if this would be due to a developmental delay we analysed 6.5 dpc embryos in diapause (Figure IB). Of 31 embryos examined, 8 lacked Nanog staining, of which 3 were female. As with the 4.5 dpc embryos these also exhibited the presence of an Eed focus in all cells. On the contrary, female Nanog positive embryos did not reveal an Eed focus on almost all the cells positive for Nanog ( Figure ID-F).
  • ES cells overexpressing Nanog have enhanced ability to generate ES like ES- differentiated cell hybrid colonies
  • Nanog is involved in reprogramming
  • Pluripotent hybrids have previously been produced using 3 different methods of cell fusion, namely electrofusion, polyethylene glycol (PEG) mediated and spontaneous cell fusion by co-culture (Matveeva et al, 1998; Terada et al, 2002; Ying et al, 2002).
  • PEG polyethylene glycol
  • RH ES cells that express constitutively the red fluorescence and the hygromicin resistance genes, and a RH ES cell derivative, RHN, which overexpresses Nanog. These were fused to both ES and NS cells carrying a transgene containing the regulatory sequences of the mouse Oct4 gene (Yeom et al, 1996) driving GFP and puromycin resistance (04GiP) (Ying et al, 2002). This transgene is expressed exclusively in pluripotent cells, and thus reactivates only if the NS cell genome reprograms, after ES-NS cell fusion.
  • the NS cells were derived from 14.5 dpc foetal forebrains.
  • PEG does not induce increased cell death or differentiation on RH compared to RHnanog ES cells
  • RHN cells were shown to be 2- fold more clonogenic after FACS and PEG treatment, which is insufficient to account for the observed 200 fold increase in hybrids.
  • Hybrid cells segregate chromosomes and revert to diploidy at significant frequencies
  • ES-NS cell hybrids To define the ES-NS cell hybrids we looked at the chromosomal content and to the identity of the NS cell epigenome. To analyse the former we looked at 12 independent ES-NS cell hybrid clones originated from fusions between RH x NS Oct4GiP, RHnanog x NS Oct4GiP, RH x NS TGFP and RHnanog x NS TGFP. After being passaged 5 times, the clones exhibited 80 chromosomes in the majority of metaphases scored (Supplementary Figure 1).
  • NS genome acquires ES cell identity in ES-NS cell hybrids
  • NS cell epigenome inactive X chromosome
  • XX differentiated cells have one inactive X chromosome.
  • the inactive X chromosome is found associated with Xist RNA and is enriched for trimethyl H3-K27 and ubiquitil H2A (Brown et al, 1992; Napoles et al, 2004; Silva et al, 2003).
  • RNA FISH analysis for Oct4 expression of XXXY ES-NS hybrids revealed the presence of 3 or 4 pinpoint signals per cell nucleus demonstrating reactivation of the Oct4 alleles of NS cell origin ( Figure 4D).
  • Further characterization of XXXY ES-NS hybrids included the expression analysis of genes, Olig2 and Blbp, which are expressed in NS cells and silenced in ES cells (Conti et al, 2005). In agreement with the above described results RT-PCR analysis revealed that both Blbp and Olig2 are switched off in ES-NS hybrid cells ( Figure 4E).
  • Nanog overexpressing ES and NS cells were allowed to differentiate, by embryoid body formation, and analysed for expression of the mesoderm marker T-brachyury (supplementary Figure 2).
  • EF4-NS 04GiP RB hybrids were used.
  • EF4 cells contain a floxed Nanog transgene which allows its deletion by the action of ere recombinase, while NS O4GiP RB express constitutively the red fluorescent protein and the blasticidin resistance gene (supplementary Figure 2A).
  • EF4-NS O4GiP RB hybrids exhibited red and green fluorescence from NS origin (supplementary Figure 2B). After ere recombinase treatment, loss of Nanog overexpression was observed as hybrids displayed increased green fluorescence (supplementary Figure 2C). These EF4-NS O4GiP RB ere hybrids were then allowed to differentiate by Lif withdrawal and Embryonic Body (EB) formation and analysed for T-brachyury expression (Supplementary Figure 2D, E). As expected, T-brachyury was not detected in the donor cells or in undifferentiated hybrids. However, and in accordance with pluripotency potential, it was expressed in differentiated hybrids.
  • EB' s were also plated, 1 to 3 per well, and analysed for presence of contracting cells. Approximately 70% (24/35) of wells showed beating cells and stained positively for the specific marker of skeletal and cardiac muscle cells ⁇ -actinin (supplementary Figure 2F).
  • the data shows that for all the analysed markers, the NS genome acquires an ES cell identity in ES-NS cell hybrids. In addition, hybrids retained pluripotent capacity.
  • Nanog overexpression in ES cells is the only factor required for the reprogramming of NS cells after cell fusion
  • Example 7 Neural cells as donor cells in reprogramming
  • somatic cell nuclear transfer the donor cell type used was shown to affect ES cell derivation efficiency from blastocysts (Hoechedlinger and Jaenisch 2002; Blelloch et al, 2004; Eggan et al, 2004; Inoue et al, 2005).
  • ES cells were shown to give the best results suggesting that the differentiation status of the donor cell is an important factor.
  • NS cells are multipotent somatic stem cells (Conti et al, 2005) therefore containing a somewhat plastic epigenome.
  • RHN-NS primary hybrids were shown to produce ES like hybrid colonies at the same efficiency as RHN-ES primary hybrids.
  • Nanog expression in NS cells is not sufficient for reprogramming
  • Nanog expression is sufficient to reprogram the NS genome into that of an ES cell
  • NS cells were transfected with Nanog (NS O4GiP NP). These cells stably express Nanog and the protein levels are similar to those found in
  • Nanog expression is not sufficient to induce reprogramming.
  • Nanog is necessary for the generation of ES like ES-NS cell hybrid colonies
  • RC-/- cells are true ES cells comes from the fact that selection can be made for the activity of endogenous Nanog promoters driving G418 and hygromycin resistant genes, introduced by the targeting vectors. This allowed the selection of a pure ES-like population in which the levels of both Oct4 and Sox2 were similar to other Nanog expressing ES cell populations ( Figure 6D).
  • Parallel fusions of either RC-/- or its parental cell line RC were performed with NSO4GiP cells.
  • RC-/- were also fused with NS 04GiP expressing transgenic Nanog, NS O4GiP NP.
  • Nanog expression is essential for the reprogramming of the NS genome after ESxNS cell fusion.
  • Nanog is increased by a MEK inhibitor
  • PD 184352 an inhibitor of MEK, increases the levels of Nanog in ES cells.
  • the effect of PDl 84352 in reprogramming was also investigated by determining the conversion of NS cells to pluripotency in the context of cell fusion.
  • RH ES cells which express constitutively the dsRed fluorescent protein and hygromycin resistance, were fused to foetal derived Neural Stem cells (NS TGFP) that express the fusion protein TauGFP linked via an IRES to puromycin resistance.
  • NS TGFP foetal derived Neural Stem cells
  • PDl 84352 In one of the fusions RH cells were treated for 3 days prior and after fusion with 3 ⁇ M PDl 84352. In the control no PDl 84352 was added. Treated and untreated primary hybrids were sorted 24 hours after fusion and then plated ( Figure 9A-C). Hygromycin and puromycin selection were added to the ES medium 3 days later. Colonies expressing dsRed2 and GFP fluorescence and exhibiting ES cell morphology were scored ( Figure 9D and E).
  • Results showed that PDl 84352 enhanced ES-NS hybrid colony formation by 45-fold. Interestingly, the percentage of hybrid colonies formed per plated hybrid in PD 184352 treated RH cells was just 2-fold lower compared to Nanog overexpressing ES cells (2.25% vs 4%). This result shows that PD184352 enhances reprogramming in the cell fusion context. This effect is likely to be mediated by the increased levels of Nanog in treated RH cells. Accordingly, if Nanog is endogenously expressed then the MEK inhibitor can be used to upregulate Nanog and achieve associated effects, such as increased reprogramming.
  • the human XIST gene analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell. 1992 Oct 30;71(3):527-42.
  • the invention thus provides methods of reprogramming, genetic modification of cells via cell fusion and combinations, progeny and uses thereof.

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Abstract

L'invention concerne des procédés de reprogrammation et de modification génétique optionnelle de cellules. Un génome multipotent est obtenu à partir d'un génome différencié par fusion d'une cellule multipotente avec une cellule différenciée, en présence de Nanog ou d'un inhibiteur de MEK. Une cellule est génétiquement modifiée en préparant une première et une seconde cellules, chacune d'elles contenant des chromosomes, en fusionnant la première cellule et la seconde cellule, et en cultivant la cellule fusionnée de manière à obtenir une cellule diploïde contenant au moins un chromosome de la première cellule et au moins un chromosome de la seconde cellule. Un procédé de fusion des cellules comprend la fusion d'une première cellule et d'une seconde cellule, en présence de Nanog ou d'un inhibiteur de MEK. L'invention concerne également les cellules ainsi obtenues ainsi que leurs utilisations.
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US8278104B2 (en) 2005-12-13 2012-10-02 Kyoto University Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2
US8129187B2 (en) 2005-12-13 2012-03-06 Kyoto University Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2
US8048999B2 (en) 2005-12-13 2011-11-01 Kyoto University Nuclear reprogramming factor
WO2008087442A1 (fr) * 2007-01-19 2008-07-24 Evocell Limited Matières biologiques et leurs utilisations
WO2008151058A2 (fr) * 2007-05-30 2008-12-11 The General Hospital Corporation Procédés de génération de cellules pluripotentes à partir de cellules somatiques
WO2008151058A3 (fr) * 2007-05-30 2009-01-29 Gen Hospital Corp Procédés de génération de cellules pluripotentes à partir de cellules somatiques
US9714433B2 (en) 2007-06-15 2017-07-25 Kyoto University Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue
US8211697B2 (en) 2007-06-15 2012-07-03 Kyoto University Induced pluripotent stem cells produced using reprogramming factors and a rho kinase inhibitor or a histone deacetylase inhibitor
US8257941B2 (en) 2007-06-15 2012-09-04 Kyoto University Methods and platforms for drug discovery using induced pluripotent stem cells
US9213999B2 (en) 2007-06-15 2015-12-15 Kyoto University Providing iPSCs to a customer
EP2090649A1 (fr) 2008-02-13 2009-08-19 Fondazione Telethon Procédé de reprogrammation de cellules différentiées
AU2019264680B2 (en) * 2008-03-17 2022-03-10 The Scripps Research Institute Combined chemical and genetic approaches for generation of induced pluripotent stem cells
EP3409762A1 (fr) * 2008-03-17 2018-12-05 The Scripps Research Institute Approches chimiques et génétiques combinées pour la génération de cellules souches pluripotentes induites
US9499797B2 (en) 2008-05-02 2016-11-22 Kyoto University Method of making induced pluripotent stem cells
WO2010017562A2 (fr) 2008-08-08 2010-02-11 Mayo Foundation For Medical Education And Research Cellules souches pluripotentes induites
US10047346B2 (en) 2008-08-08 2018-08-14 Mayo Foundation For Medical Education And Research Method of treating heart tissue using induced pluripotent stem cells
WO2010017562A3 (fr) * 2008-08-08 2010-04-22 Mayo Foundation For Medical Education And Research Cellules souches pluripotentes induites
US9932561B2 (en) 2011-07-22 2018-04-03 Mayo Foundation For Medical Education And Research Differentiating induced pluripotent stem cells into glucose-responsive, insulin-secreting progeny
WO2016168658A1 (fr) * 2015-04-15 2016-10-20 University Of Massachusetts Compositions et procédés utilisables en vue de la réactivation de chromosomes xi
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US11268150B2 (en) 2015-04-15 2022-03-08 University Of Massachusetts Compositions and methods for Xi chromosome reactivation

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