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Cell fusion-mediated nuclear reprogramming of somatic cells
RBMOnline - Vol 16 No 1. 2008 51-56 Reproductive BioMedicine Online; www.rbmonline.com/Article/2917 on web 9 August 2007
Symposium: Nuclear reprogramming and the control of differentiation in mammalian embryos Cell fusion-mediated nuclear reprogramming of somatic cells Dr Takashi Tada obtained his PhD from Hokkaido University, Japan. He then became a research associate at the University of Cambridge, UK. Currently Dr Tada is Associate Professor in Kyoto University, Japan.
Dr Takashi Tada Hiroyuki Matsumura1, Takashi Tada1,2,3 1 Laboratory of Stem Cell Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507; 2JST, CREST, 4–1-8 Hon-chou, Kawaguchi, Saitama, 332–0012, Japan 3 Correspondence: Tel: +81 75 7514102, Fax: +81 75 7514102; e-mail; [email protected]
Abstract Pluripotential embryonic stem cells (ESC) possess a unique property of being able to carry out nuclear reprogramming of somatic nuclei, as shown after cell fusion. The nuclear reprogramming activity has been applied for producing pluripotential stem cells from personal somatic cells through several new technologies, including cytoplasmic cell fusion and ES cell factor introduction. Targeted elimination of ESC-derived chromosome(s) following cell fusion-mediated reprogramming of somatic chromosomes is one of the new technologies for producing personalized stem cells. A universal chromosome elimination cassette (CEC) has been developed that confers drug resistance and GFP (green fluorescent protein) fluorescence, flanked by oppositely orientated loxP sites, to induce sister chromatid recombination and targeted chromosome loss. GFPpositive ESC generated with a CEC-integrated chromosome were hybridized with adult thymocytes and then exposed to Cre recombinase. This led to loss of GFP expression and elimination of the CEC-tagged chromosome. Targeted elimination of a pair of ESC-derived chromosome 6s, which are key chromosomes for maintaining pluripotency, demonstrated that the reprogrammed somatic factors are sufficient for the continued pluripotentiality of hybrid cells. Targeted chromosome elimination technology therefore offers a means for developing major histocompatibility complex-personalized or completely personalized pluripotential stem cell populations for use in a range of therapeutic applications. Keywords: cell hybrid, chromosome elimination, epigenetics, ES cells, recombination, transplant rejection
Introduction Regenerative medicine is a therapeutic approach to cure functional damage by replacement with cells, tissues or organs, which are generated by tissue-specific differentiation of plurior multi-potential stem cells in vitro. Candidate sources of stem cells include tissue stem cells, embryonic stem cells (ESC) and reprogrammed stem cells, which are pluripotential stem cells generated from somatic cells through nuclear reprogramming. Tissue stem cells function in maintaining homeostasis through periodic renewal of exhausted cells in various tissues in vivo. Cells derived from tissue stem cells purified from patients are applicable for transplantation of immunogenetically syngeneic
grafts. However, tissue stem cells exist as an extremely small number of cells, and their ability to undergo cell proliferation in vitro in an undifferentiated state is limited. The majority of tissue stem cells have the potential for multilineage differentiation as multipotential but not pluripotential stem cells. It has been thought that multipotential adult progenitor cells (MAPC) isolated from bone marrow (Jiang et al., 2002) and amniotic fluid stem (AFS) cells (De Coppi et al., 2007) are pluripotential stem cells without tumourigenicity. It is, however, unclear whether some type(s) of tissue stem cells isolated from adult mice have the potential to form chimeras by
© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
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Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada microinjection into blastocysts. In ESC, which are derived from the inner cell mass of blastocysts, pluripotency was verified by contribution to all types of tissues, including germ cells, in chimeric mice. Furthermore, ESC have a robust capacity for self-renewable proliferation under culture conditions. ESC and their derivatives are, however, rejected by the immunological response to allogeneic grafts in the majority of recipients. Tumourigenicity of ESC has also been shown by teratoma formation upon intraperitoneal or subcutaneous injection into immunodeficient mice. ESC have been established not only in mice but also in humans (Thomson et al., 1998).
Nuclear reprogramming activity of embryonic stem cells The ability to carry out nuclear reprogramming of a somatic cell nucleus to totipotency was first demonstrated by producing cloned frogs by nuclear transfer from the intestinal endoderm cells of feeding tadpoles into activated eggs (Gurdon, 1962). Similar nuclear reprogramming activity of mammalian oocytes has been shown by the production of cloned animals in several species (Tada and Tada, 2001). Thus, reprogramming-mediated plasticity of the somatic nucleus is detected using some types of somatic cells. Interestingly, nuclear reprogramming activity was found in mouse and human ES and embryonic germ (EG) cells by cell fusion with somatic cells (Tada et al., 1997; Tada and Tada, 2001; Do and Scholer, 2004; Cowan et al., 2005). This finding suggests that key factors involved in nuclear reprogramming could be isolated from the nucleus and/or cytoplasm of ESC. Recently, generation of personalized pluripotential stem cells from individual somatic cells has been realized as an important cell source for applying to regenerative medicine. Selective chromosome elimination from hybrid cells between mouse ES and somatic cells (Figure 1A) (Matsumura et al., 2007), cell fusion of somatic cells with ESCs or ESC cytoplasm in the mouse (Tada et al., 2001; Do and Scholer, 2004) and in humans (Figure 1B) (Cowan et al., 2005; Strelchenko et al., 2006), centrifugation-dependent enucleation of the tetraploid ESC nucleus from a heterokaryon produced by cell fusion with a diploid somatic cell in the mouse (Figure 1C) (Pralong et al., 2005), treatment of 293T epithelial cells with lysates extracted from human NCCIT carcinoma cells (Figure 1D) (Taranger et al., 2005) and induced pluripotential stem (iPS) cells transformed from mouse embryonic and adult fibroblasts by transfection of four key factors, Oct4, Sox2, Klf4 and cMyc (Figure 1E) (Takahashi and Yamanaka, 2006), have been proposed as new technologies applicable for generating tailor-made stem cells. If human iPS cells that closely resemble normal human ES cells in pluripotential stem cell properties could be generated from adult somatic cells, personalized ESlike cells could be widely turned to practical use for generating syngeneic transplantable cells or tissues.
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ES−somatic hybrid cells are one of the strong candidates for pluripotential stem cells applicable for regenerative medicine. Cell fusion with ESC is a reliable approach for inducing nuclear reprogramming of nuclei of specialized somatic cells (Tada et al., 2001; Do and Scholer, 2004; Cowan et al., 2005) through erasure of somatic cell memory and establishment of pluripotential cell memory with epigenetic changes leading to
the formation of globally looser chromatin structure (Kimura et al., 2004). Major architectural proteins are hyperdynamic and bind loosely to chromatin in ES cells (Meshorer et al., 2006), suggesting that a similar architecture becomes established in the reprogrammed somatic chromatin. The detailed pluripotential stem cell-specific properties of the hybrid cells containing reprogrammed somatic nuclei have been demonstrated by reactivation of an inactivated somatic X chromosome and Xist/Tsix, somatic Oct4, Dppa3 (Developmental pluripotency associated-3) (Stella/PGC7) and Nanog (Tada et al., 2001, 2003; Kimura et al., 2002; Do and Scholer, 2004; Hatano et al., 2005; Matsumura et al., 2007), endodermal, mesodermal and ectodermal tissue-specific gene expression from somatic genomes of differentiated hybrid cells in vivo and in vitro (Tada et al., 2003), efficient cell differentiation to neuronal cells in vitro (Tada et al., 2003) promoted by a stromal cell-derived induced activity (SDIA) (Kawasaki et al., 2000) and contribution to chimeric embryo and teratoma formation (Tada et al., 2001, 2003). Cell fusion-mediated reprogrammed somatic nuclei have a gene expression profile similar to that of ESC (Cowan et al., 2005; Ambrosi et al., 2007). These data strongly indicate that ESC possess the activity for nuclear reprogramming of somatic cells, and key factors existing in the nucleus and/or cytoplasm of ESC may function in inducing nuclear reprogramming of mouse and human somatic cell nuclei.
Chromosome elimination from hybrid cell nuclei ES−somatic hybrid cells possess tetraploid nuclei consisting of diploid ES- and diploid somatic cell-derived chromosomes. Thus, elimination of ESC-derived chromosome(s) from hybrid cells once the personal somatic genome has been reprogrammed is a useful biotechnology for generating chromosome-personalized hybrid cells. However, it has been shown that large autosomal deletions and whole autosome loss are deleterious for ESC survival, although genome engineering by Cre-loxP-mediated chromosome rearrangements has facilitated genetic studies such as invivo conditional knockout of a targeted gene (Lewandoski and Martin, 1997; Mills and Bradley, 2001). It was therefore concluded that elimination of autosomes leads to lethality in diploid cells. However, such elimination is apparently not lethal in tetraploid hybrid cells. ES−somatic hybrid cells missing a couple of chromosomes are capable of surviving as pluripotential stem cells. A chromosome elimination cassette (CEC) bearing a fluorescence reporter and drug-resistance gene between oppositely orientated loxP sites was designed for inducing selective elimination of whole chromosome(s) from hybrid cell nuclei. Various types of CEC were produced by construction with different combinations of loxP variants, drug-resistance genes and reporter genes. One type of CEC contains the CAGgfp/IRES.puro-pA gene (CEC-gfp-puro). Site-specific DNA recombinase Cre-dependent homologous recombination between loxP sites occurs in the G1 or G2 phase of the cell cycle in a trans- or cis-targeted recombination manner (Mills and Bradley, 2001). Nulli- or di-centric chromosome(s) generated through unequal recombination are eliminated from a nucleus and disappear during cell division, while ES cells bearing wild-type or Cre-mediation-generated inverse RBMOnline®
Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada
Figure 1. Nuclear reprogramming-mediated pluripotential stem cells. Five approaches are summarized: (A) chromosome elimination from embryonic stem (ES)−somatic hybrid cells, (B) hybridization of somatic cell with enucleated ES cells, (C) selective enucleation of ES-derived nucleus from ES−somatic heterokaryon, (D) direct reprogramming of somatic cell with ES cell extract and (E) direct reprogramming of somatic cell by transfection of reprogramming factors (induced pluripotential stem (iPS) cells). CEC = chromosome elimination cassette.
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Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada CEC-chromosome(s) are fluorescence-activated cell sorted (FACS) out as GFP-positive cells. With the CEC-gfp-puro, chromosome-eliminated ES cells are enriched and collected as GFP-negative cells. To examine whether CEC-tagged chromosome(s) are selectively eliminated from hybrid cell nuclei, CEC-gfp-purointegrated XY ES cell clones were randomly picked up and cell-fused with thymocytes obtained from female adult mice. In the case of ES cells bearing the CEC in the proximal region of chromosome 12, hybrid cells were positive for GFP. GFPnegative hybrid cells 7 days after transient Cre treatment were FACS and karyotyped. In fact, a single chromosome 12 was selectively eliminated from hybrid clones (4n = 79,XXXY,12). Chromosome painting and genomic polymerase chain reaction analyses clearly showed evidence of the targeted loss of ESC-derived chromosome 12 from hybrid cell nuclei (Matsumura et al., 2007). Therefore, desired chromosome(s) can be eliminated from hybrid cell nuclei by the application of the CEC-mediated chromosome elimination system.
Maintenance of pluripotency by reprogrammed somatic Nanog A key pluripotential factor, Nanog, bearing the homeodomain and W-rich domain is expressed in pluripotential early embryonic cells, ES cells, EG cells and EC (embryonal carcinoma) cells (Chambers et al., 2003; Mitsui et al., 2003; Hatano et al., 2005) and essential for normal development of early post-implantation embryos as seen by loss of the epiblast at E5.5 in the absence of Nanog (Mitsui et al., 2003). Nanog
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transcription is activated by the Octamer and Sox element upstream of the transcriptional starting site through binding of Oct4 and Sox2 (Kuroda et al., 2005; Rodda et al., 2005) and also activated by Brachyury and Stat3 (Suzuki et al., 2006) and repressed by p53, germ cell nuclear factor, Brb2/Mek pathway and transcription factor 3 (Lin et al., 2004; Gu et al., 2005; Hamazaki et al., 2006; Pereira and Merrill, 2006). Mouse chromosome 6, which harbours Nanog and genes for other pluripotential factors Dppa3 and Gdf3 (growth and differentiation factor 3) (Clark et al., 2004), functions as a key chromosome in maintaining the undifferentiated state of stem cells. To eliminate the pair of ESC-derived chromosome 6s from ES−somatic hybrid cell nuclei, a CEC containing the Pgkneo/IRES.gfp-pA gene (CEC-neo-gfp) was integrated at the Gt(ROSA)26-Sor locus on chromosome 6 by homologous recombination in ES cells (Srinivas et al., 2001). ES cells having a single CEC-neo-gfp-tagged chromosome 6 (CEC6tg/+ ES cells) were treated with high dose G418 to select ES cells in which all chromosome 6s were CECneo-gfp-tagged (CEC6tg/tg ES cells). Hybrid cells between CEC6tg/tg ES cells and somatic cells positive for GFP were transiently treated with Cre recombinase. GFP-negative hybrid cells missing ESC-derived chromosome 6s maintained the characteristic ESC phenotype and pluripotency (Matsumura et al., 2007), demonstrating that reprogrammed somatic Nanog is sufficient for maintenance of hybrid cell pluripotency with no contribution of ESC-derived Nanog. It seems that the reprogrammed somatic Nanog is functionally equivalent to ESC-derived Nanog.
Figure 2. Induction of immunotolerance to major histocompatibility stem (MHC)-matched embryonic stem (ES)−somatic hybrid cells by chromosome elimination. ES cell-derived MHC glycoprotein (blue dashed lines) and somatic cell-derived MHC glycoprotein (pink dashed lines) are expressed on the surface of ES−somatic hybrid cell. The hybrid cell is specifically recognized and killed as an allogenic cell. Following targeted elimination of ES cell-derived MHC chromosomes, hybrid cells covered only by somatic cell-derived MHC glycoprotein could be recognized as an autogenic cell. RBMOnline®
Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada
Applications of CEC technology for stem cell personalization Mismatch of major histocompatibility complex (MHC) molecules encoding specialized host-cell glycoproteins leads to acute immunological rejection of transplanted grafts or cells by recipients. At least 200 genes for MHC molecules extend for at least 4–7 × 106 bp in chromosome 17 as H-2 genes in the mouse and in chromosome 6 as human leukocyte antigen (HLA) genes in the human. ES−somatic hybrid cells express both ESC- and somatic cell-derived MHC glycoproteins on the cell surface. If ESC-derived MHC genes were selectively eliminated from the hybrid cells, the genetic type of MHC could be completely matched with that of somatic cells and MHCmediated immunorejection of the hybrid cell derivatives could be drastically reduced. The technology for targeted elimination of a pair of ESC-derived chromosomes has been established as described above (Matsumura et al., 2007), indicating that mouse MHC-personalized hybrid cells can be generated through selective elimination of ESC-derived chromosome 17s from hybrid cell nuclei (Figure 2). Further development of chromosome elimination technology for selective elimination of human ESC-derived chromosome 6s bearing MHC genes could make it possible to generate MHC-personalized hybrid cells from an individual’s own somatic cells. This approach could be developed further to directly generate MHC-personalized diploid ES cells by targeted elimination of ES chromosomes harbouring the MHC genes, and replacing both copies with somatic-derived MHC chromosomes using a microcell-mediated chromosome transfer technique. Ultimately, the chromosome elimination technology will allow for the production of personally syngeneic pluripotential stem cells through elimination of all ESC-derived chromosomes. In humans, the exact frequency of aneuploidy at conception is not known, but it has been estimated to be at as low as 8– 10% (Kjii et al., 1978) or 9.3% at 3–4 weeks after gestation (Yamamoto and Watanabe, 1979), suggesting that chromosome abnormalities, especially trisomy, could be found in ES cells established from human blastocysts. Targeted elimination of an extra chromosome from trisomy-containing ES cells could contribute to controlling the quality of human ES cells. Notably, targeted elimination of chromosomes is also applicable to a variety of diverse biomedical purposes. Generating human stem cells in this way by introducing somatic-derived chromosomes with specific mutations from patients should help in elucidating the causes of human diseases as well as for the discovery of appropriate drugs through pharmaceutical evaluation using the differentiated and undifferentiated mutant cells in vitro.
References Ambrosi DJ, Tanasijevic B, Kaur A et al. 2007 Genome-wide reprogramming in hybrids of somatic cells and embryonic stem cells. Stem Cells 25, 1104–1113. Chambers I, Colby D, Robertson M et al. 2003 Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655. Clark AT, Rodriguez RT, Bodnar MS et al. 2004 Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma. Stem Cells 22, 169–179.
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Cowan CA, Atienza J, Melton DA et al. 2005 Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373. De Coppi P, Bartsch G Jr, Siddiqui MM et al. 2007 Isolation of amniotic stem cell lines with potential for therapy. Nature Biotechnology 25, 100–106. Do JT, Scholer HR 2004 Nuclei of embryonic stem cells reprogram somatic cells. Stem Cells 22, 941–949. Gu P, LeMenuet D, Chung AC et al. 2005 Orphan nuclear receptor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation. Molecular and Cellular Biology 25, 8507–8519. Gurdon J 1962 The developmental capacity of nuclei taken from intestinal epithelial cells of feeding tadpoles. Journal of Embryology and Experimental Morphology 10, 622–640. Hamazaki T, Kehoe SM, Nakano T et al. 2006 The Grb2/Mek pathway represses Nanog in murine embryonic stem cells. Molecular and Cellular Biology 26, 7539–7549. Hatano SY, Tada M, Kimura H et al. 2005 Pluripotential competence of cells associated with Nanog activity. Mechanisms of Development 122, 67–79. Jiang Y, Jahagirdar BN, Reinhardt RL et al. 2002 Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49. Kajii T, Ohama K, Mikamo K 1978 Anatomic and chromosomal anomalies in 944 induced abortuses. Human Genetics 43, 247–258. Kawasaki H, Mizuseki K, Nishikawa S et al. 2000 Induction of midbrain dopaminergic neurons from ES cells by stromal cellderived inducing activity. Neuron 28, 31–40. Kimura H, Tada M, Nakatsuji N et al. 2004 Histone code modifications on pluripotential nuclei of reprogrammed somatic cells. Molecular and Cellular Biology 24, 5710–5720. Kimura H, Tada M, Hatano S et al. 2002 Chromatin reprogramming of male somatic cell-derived XIST and TSIX in ES hybrid cells. Cytogenetic and Genome Research 99, 106–114. Kuroda T, Tada M, Kubota H et al. 2005 Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Molecular and Cellular Biology 25, 2475–2485. Lewandoski M, Martin GR 1997 Cre-mediated chromosome loss in mice. Nature Genetics 17, 223–225. Lin T, Chao C, Saito S et al. 2004 p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nature Cell Biology 7, 165–171. Matsumura H, Tada M, Otsuji T et al. 2007 Targeted chromosome elimination from ES−somatic hybrid cells. Nature Methods 4, 23–25. Meshorer E, Yellajoshula D, George E et al. 2006 Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev Cell 10, 105–116. Mills AA, Bradley A 2001 From mouse to man: generating megabase chromosome rearrangements. Trends in Genetics 17, 331–339. Mitsui K, Tokuzawa Y, Itoh H et al. 2003 The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642. Pereira L, Yi F, Merrill BJ 2006 Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Molecular and Cellular Biology 26, 7479–7491. Pralong D, Mrozik K, Occhiodoro et al. 2005 A novel method for somatic cell nuclear transfer to mouse embryonic stem cells. Cloning Stem Cells 7, 265–271. Rodda DJ, Chew JL, Lim LH et al. 2005 Transcriptional regulation of nanog by OCT4 and SOX2. The Journal of Biological Chemistry 280, 24731–24737. Srinivas S, Watanabe T, Lin CS et al. 2001 Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Developmental Biology 1, 4. Strelchenko N, Kukharenko V, Shkumatov A et al. 2006 Reprogramming of human somatic cells by embryonic stem cell cytoplast. Reproductive BioMedicine Online 12, 107–111. Suzuki A, Raya A, Kawakami Y et al. 2006 Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation
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Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada of embryonic stem cells. Proceedings of the National Academy of Science of the United States of America 103, 10294–10299. Tada T, Tada M 2001 Toti-/pluripotential stem cells and epigenetic modifications. Cell Structure and Function 26, 149–160. Tada M, Morizane A, Kimura H et al. 2003 Pluripotency of reprogrammed somatic genomes in embryonic stem hybrid cells. Developmental Dynamics 227, 504–510. Tada M, Takahama Y, Abe K et al. 2001 Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Current Biology 11, 1553–1558. Tada M, Tada T, Lefebvre L et al. 1997 Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO Journal 16, 6510–6520. Takahashi K, Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676.
Taranger CK, Noer A, Sørensen AL et al. 2005 Induction of dedifferentiation, genome-wide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. Molecular Biology of the Cell 16, 5719–5735. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. 1998 Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. Yamamoto M, Watanabe G 1979 Epidemiology of gross chromosomal anomalies at the early embryonic stages of pregnancy In: Klingberg MA and Weatherall JAC (eds) Epidemiologic Methods for Detection of Teratogens, vol.1. Karger, Basel, pp. 101–106. Received 1 May 2007; refereed 23 May 2007; accepted 6 June 2007.
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Symposium: Nuclear reprogramming and the control of differentiation in mammalian embryos Cell fusion-mediated nuclear reprogramming of somatic cells Dr Takashi Tada obtained his PhD from Hokkaido University, Japan. He then became a research associate at the University of Cambridge, UK. Currently Dr Tada is Associate Professor in Kyoto University, Japan.
Dr Takashi Tada Hiroyuki Matsumura1, Takashi Tada1,2,3 1 Laboratory of Stem Cell Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507; 2JST, CREST, 4–1-8 Hon-chou, Kawaguchi, Saitama, 332–0012, Japan 3 Correspondence: Tel: +81 75 7514102, Fax: +81 75 7514102; e-mail; [email protected]
Abstract Pluripotential embryonic stem cells (ESC) possess a unique property of being able to carry out nuclear reprogramming of somatic nuclei, as shown after cell fusion. The nuclear reprogramming activity has been applied for producing pluripotential stem cells from personal somatic cells through several new technologies, including cytoplasmic cell fusion and ES cell factor introduction. Targeted elimination of ESC-derived chromosome(s) following cell fusion-mediated reprogramming of somatic chromosomes is one of the new technologies for producing personalized stem cells. A universal chromosome elimination cassette (CEC) has been developed that confers drug resistance and GFP (green fluorescent protein) fluorescence, flanked by oppositely orientated loxP sites, to induce sister chromatid recombination and targeted chromosome loss. GFPpositive ESC generated with a CEC-integrated chromosome were hybridized with adult thymocytes and then exposed to Cre recombinase. This led to loss of GFP expression and elimination of the CEC-tagged chromosome. Targeted elimination of a pair of ESC-derived chromosome 6s, which are key chromosomes for maintaining pluripotency, demonstrated that the reprogrammed somatic factors are sufficient for the continued pluripotentiality of hybrid cells. Targeted chromosome elimination technology therefore offers a means for developing major histocompatibility complex-personalized or completely personalized pluripotential stem cell populations for use in a range of therapeutic applications. Keywords: cell hybrid, chromosome elimination, epigenetics, ES cells, recombination, transplant rejection
Introduction Regenerative medicine is a therapeutic approach to cure functional damage by replacement with cells, tissues or organs, which are generated by tissue-specific differentiation of plurior multi-potential stem cells in vitro. Candidate sources of stem cells include tissue stem cells, embryonic stem cells (ESC) and reprogrammed stem cells, which are pluripotential stem cells generated from somatic cells through nuclear reprogramming. Tissue stem cells function in maintaining homeostasis through periodic renewal of exhausted cells in various tissues in vivo. Cells derived from tissue stem cells purified from patients are applicable for transplantation of immunogenetically syngeneic
grafts. However, tissue stem cells exist as an extremely small number of cells, and their ability to undergo cell proliferation in vitro in an undifferentiated state is limited. The majority of tissue stem cells have the potential for multilineage differentiation as multipotential but not pluripotential stem cells. It has been thought that multipotential adult progenitor cells (MAPC) isolated from bone marrow (Jiang et al., 2002) and amniotic fluid stem (AFS) cells (De Coppi et al., 2007) are pluripotential stem cells without tumourigenicity. It is, however, unclear whether some type(s) of tissue stem cells isolated from adult mice have the potential to form chimeras by
© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
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Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada microinjection into blastocysts. In ESC, which are derived from the inner cell mass of blastocysts, pluripotency was verified by contribution to all types of tissues, including germ cells, in chimeric mice. Furthermore, ESC have a robust capacity for self-renewable proliferation under culture conditions. ESC and their derivatives are, however, rejected by the immunological response to allogeneic grafts in the majority of recipients. Tumourigenicity of ESC has also been shown by teratoma formation upon intraperitoneal or subcutaneous injection into immunodeficient mice. ESC have been established not only in mice but also in humans (Thomson et al., 1998).
Nuclear reprogramming activity of embryonic stem cells The ability to carry out nuclear reprogramming of a somatic cell nucleus to totipotency was first demonstrated by producing cloned frogs by nuclear transfer from the intestinal endoderm cells of feeding tadpoles into activated eggs (Gurdon, 1962). Similar nuclear reprogramming activity of mammalian oocytes has been shown by the production of cloned animals in several species (Tada and Tada, 2001). Thus, reprogramming-mediated plasticity of the somatic nucleus is detected using some types of somatic cells. Interestingly, nuclear reprogramming activity was found in mouse and human ES and embryonic germ (EG) cells by cell fusion with somatic cells (Tada et al., 1997; Tada and Tada, 2001; Do and Scholer, 2004; Cowan et al., 2005). This finding suggests that key factors involved in nuclear reprogramming could be isolated from the nucleus and/or cytoplasm of ESC. Recently, generation of personalized pluripotential stem cells from individual somatic cells has been realized as an important cell source for applying to regenerative medicine. Selective chromosome elimination from hybrid cells between mouse ES and somatic cells (Figure 1A) (Matsumura et al., 2007), cell fusion of somatic cells with ESCs or ESC cytoplasm in the mouse (Tada et al., 2001; Do and Scholer, 2004) and in humans (Figure 1B) (Cowan et al., 2005; Strelchenko et al., 2006), centrifugation-dependent enucleation of the tetraploid ESC nucleus from a heterokaryon produced by cell fusion with a diploid somatic cell in the mouse (Figure 1C) (Pralong et al., 2005), treatment of 293T epithelial cells with lysates extracted from human NCCIT carcinoma cells (Figure 1D) (Taranger et al., 2005) and induced pluripotential stem (iPS) cells transformed from mouse embryonic and adult fibroblasts by transfection of four key factors, Oct4, Sox2, Klf4 and cMyc (Figure 1E) (Takahashi and Yamanaka, 2006), have been proposed as new technologies applicable for generating tailor-made stem cells. If human iPS cells that closely resemble normal human ES cells in pluripotential stem cell properties could be generated from adult somatic cells, personalized ESlike cells could be widely turned to practical use for generating syngeneic transplantable cells or tissues.
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ES−somatic hybrid cells are one of the strong candidates for pluripotential stem cells applicable for regenerative medicine. Cell fusion with ESC is a reliable approach for inducing nuclear reprogramming of nuclei of specialized somatic cells (Tada et al., 2001; Do and Scholer, 2004; Cowan et al., 2005) through erasure of somatic cell memory and establishment of pluripotential cell memory with epigenetic changes leading to
the formation of globally looser chromatin structure (Kimura et al., 2004). Major architectural proteins are hyperdynamic and bind loosely to chromatin in ES cells (Meshorer et al., 2006), suggesting that a similar architecture becomes established in the reprogrammed somatic chromatin. The detailed pluripotential stem cell-specific properties of the hybrid cells containing reprogrammed somatic nuclei have been demonstrated by reactivation of an inactivated somatic X chromosome and Xist/Tsix, somatic Oct4, Dppa3 (Developmental pluripotency associated-3) (Stella/PGC7) and Nanog (Tada et al., 2001, 2003; Kimura et al., 2002; Do and Scholer, 2004; Hatano et al., 2005; Matsumura et al., 2007), endodermal, mesodermal and ectodermal tissue-specific gene expression from somatic genomes of differentiated hybrid cells in vivo and in vitro (Tada et al., 2003), efficient cell differentiation to neuronal cells in vitro (Tada et al., 2003) promoted by a stromal cell-derived induced activity (SDIA) (Kawasaki et al., 2000) and contribution to chimeric embryo and teratoma formation (Tada et al., 2001, 2003). Cell fusion-mediated reprogrammed somatic nuclei have a gene expression profile similar to that of ESC (Cowan et al., 2005; Ambrosi et al., 2007). These data strongly indicate that ESC possess the activity for nuclear reprogramming of somatic cells, and key factors existing in the nucleus and/or cytoplasm of ESC may function in inducing nuclear reprogramming of mouse and human somatic cell nuclei.
Chromosome elimination from hybrid cell nuclei ES−somatic hybrid cells possess tetraploid nuclei consisting of diploid ES- and diploid somatic cell-derived chromosomes. Thus, elimination of ESC-derived chromosome(s) from hybrid cells once the personal somatic genome has been reprogrammed is a useful biotechnology for generating chromosome-personalized hybrid cells. However, it has been shown that large autosomal deletions and whole autosome loss are deleterious for ESC survival, although genome engineering by Cre-loxP-mediated chromosome rearrangements has facilitated genetic studies such as invivo conditional knockout of a targeted gene (Lewandoski and Martin, 1997; Mills and Bradley, 2001). It was therefore concluded that elimination of autosomes leads to lethality in diploid cells. However, such elimination is apparently not lethal in tetraploid hybrid cells. ES−somatic hybrid cells missing a couple of chromosomes are capable of surviving as pluripotential stem cells. A chromosome elimination cassette (CEC) bearing a fluorescence reporter and drug-resistance gene between oppositely orientated loxP sites was designed for inducing selective elimination of whole chromosome(s) from hybrid cell nuclei. Various types of CEC were produced by construction with different combinations of loxP variants, drug-resistance genes and reporter genes. One type of CEC contains the CAGgfp/IRES.puro-pA gene (CEC-gfp-puro). Site-specific DNA recombinase Cre-dependent homologous recombination between loxP sites occurs in the G1 or G2 phase of the cell cycle in a trans- or cis-targeted recombination manner (Mills and Bradley, 2001). Nulli- or di-centric chromosome(s) generated through unequal recombination are eliminated from a nucleus and disappear during cell division, while ES cells bearing wild-type or Cre-mediation-generated inverse RBMOnline®
Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada
Figure 1. Nuclear reprogramming-mediated pluripotential stem cells. Five approaches are summarized: (A) chromosome elimination from embryonic stem (ES)−somatic hybrid cells, (B) hybridization of somatic cell with enucleated ES cells, (C) selective enucleation of ES-derived nucleus from ES−somatic heterokaryon, (D) direct reprogramming of somatic cell with ES cell extract and (E) direct reprogramming of somatic cell by transfection of reprogramming factors (induced pluripotential stem (iPS) cells). CEC = chromosome elimination cassette.
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Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada CEC-chromosome(s) are fluorescence-activated cell sorted (FACS) out as GFP-positive cells. With the CEC-gfp-puro, chromosome-eliminated ES cells are enriched and collected as GFP-negative cells. To examine whether CEC-tagged chromosome(s) are selectively eliminated from hybrid cell nuclei, CEC-gfp-purointegrated XY ES cell clones were randomly picked up and cell-fused with thymocytes obtained from female adult mice. In the case of ES cells bearing the CEC in the proximal region of chromosome 12, hybrid cells were positive for GFP. GFPnegative hybrid cells 7 days after transient Cre treatment were FACS and karyotyped. In fact, a single chromosome 12 was selectively eliminated from hybrid clones (4n = 79,XXXY,12). Chromosome painting and genomic polymerase chain reaction analyses clearly showed evidence of the targeted loss of ESC-derived chromosome 12 from hybrid cell nuclei (Matsumura et al., 2007). Therefore, desired chromosome(s) can be eliminated from hybrid cell nuclei by the application of the CEC-mediated chromosome elimination system.
Maintenance of pluripotency by reprogrammed somatic Nanog A key pluripotential factor, Nanog, bearing the homeodomain and W-rich domain is expressed in pluripotential early embryonic cells, ES cells, EG cells and EC (embryonal carcinoma) cells (Chambers et al., 2003; Mitsui et al., 2003; Hatano et al., 2005) and essential for normal development of early post-implantation embryos as seen by loss of the epiblast at E5.5 in the absence of Nanog (Mitsui et al., 2003). Nanog
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transcription is activated by the Octamer and Sox element upstream of the transcriptional starting site through binding of Oct4 and Sox2 (Kuroda et al., 2005; Rodda et al., 2005) and also activated by Brachyury and Stat3 (Suzuki et al., 2006) and repressed by p53, germ cell nuclear factor, Brb2/Mek pathway and transcription factor 3 (Lin et al., 2004; Gu et al., 2005; Hamazaki et al., 2006; Pereira and Merrill, 2006). Mouse chromosome 6, which harbours Nanog and genes for other pluripotential factors Dppa3 and Gdf3 (growth and differentiation factor 3) (Clark et al., 2004), functions as a key chromosome in maintaining the undifferentiated state of stem cells. To eliminate the pair of ESC-derived chromosome 6s from ES−somatic hybrid cell nuclei, a CEC containing the Pgkneo/IRES.gfp-pA gene (CEC-neo-gfp) was integrated at the Gt(ROSA)26-Sor locus on chromosome 6 by homologous recombination in ES cells (Srinivas et al., 2001). ES cells having a single CEC-neo-gfp-tagged chromosome 6 (CEC6tg/+ ES cells) were treated with high dose G418 to select ES cells in which all chromosome 6s were CECneo-gfp-tagged (CEC6tg/tg ES cells). Hybrid cells between CEC6tg/tg ES cells and somatic cells positive for GFP were transiently treated with Cre recombinase. GFP-negative hybrid cells missing ESC-derived chromosome 6s maintained the characteristic ESC phenotype and pluripotency (Matsumura et al., 2007), demonstrating that reprogrammed somatic Nanog is sufficient for maintenance of hybrid cell pluripotency with no contribution of ESC-derived Nanog. It seems that the reprogrammed somatic Nanog is functionally equivalent to ESC-derived Nanog.
Figure 2. Induction of immunotolerance to major histocompatibility stem (MHC)-matched embryonic stem (ES)−somatic hybrid cells by chromosome elimination. ES cell-derived MHC glycoprotein (blue dashed lines) and somatic cell-derived MHC glycoprotein (pink dashed lines) are expressed on the surface of ES−somatic hybrid cell. The hybrid cell is specifically recognized and killed as an allogenic cell. Following targeted elimination of ES cell-derived MHC chromosomes, hybrid cells covered only by somatic cell-derived MHC glycoprotein could be recognized as an autogenic cell. RBMOnline®
Symposium - Cell fusion-mediated nuclear reprogramming - H Matsumura & T Tada
Applications of CEC technology for stem cell personalization Mismatch of major histocompatibility complex (MHC) molecules encoding specialized host-cell glycoproteins leads to acute immunological rejection of transplanted grafts or cells by recipients. At least 200 genes for MHC molecules extend for at least 4–7 × 106 bp in chromosome 17 as H-2 genes in the mouse and in chromosome 6 as human leukocyte antigen (HLA) genes in the human. ES−somatic hybrid cells express both ESC- and somatic cell-derived MHC glycoproteins on the cell surface. If ESC-derived MHC genes were selectively eliminated from the hybrid cells, the genetic type of MHC could be completely matched with that of somatic cells and MHCmediated immunorejection of the hybrid cell derivatives could be drastically reduced. The technology for targeted elimination of a pair of ESC-derived chromosomes has been established as described above (Matsumura et al., 2007), indicating that mouse MHC-personalized hybrid cells can be generated through selective elimination of ESC-derived chromosome 17s from hybrid cell nuclei (Figure 2). Further development of chromosome elimination technology for selective elimination of human ESC-derived chromosome 6s bearing MHC genes could make it possible to generate MHC-personalized hybrid cells from an individual’s own somatic cells. This approach could be developed further to directly generate MHC-personalized diploid ES cells by targeted elimination of ES chromosomes harbouring the MHC genes, and replacing both copies with somatic-derived MHC chromosomes using a microcell-mediated chromosome transfer technique. Ultimately, the chromosome elimination technology will allow for the production of personally syngeneic pluripotential stem cells through elimination of all ESC-derived chromosomes. In humans, the exact frequency of aneuploidy at conception is not known, but it has been estimated to be at as low as 8– 10% (Kjii et al., 1978) or 9.3% at 3–4 weeks after gestation (Yamamoto and Watanabe, 1979), suggesting that chromosome abnormalities, especially trisomy, could be found in ES cells established from human blastocysts. Targeted elimination of an extra chromosome from trisomy-containing ES cells could contribute to controlling the quality of human ES cells. Notably, targeted elimination of chromosomes is also applicable to a variety of diverse biomedical purposes. Generating human stem cells in this way by introducing somatic-derived chromosomes with specific mutations from patients should help in elucidating the causes of human diseases as well as for the discovery of appropriate drugs through pharmaceutical evaluation using the differentiated and undifferentiated mutant cells in vitro.
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