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Rudolf Jaenisch - One of the best experts on this subject based on the ideXlab platform.
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stem cells pluripotency and Nuclear reprogramming
Journal of Thrombosis and Haemostasis, 2009Co-Authors: Rudolf JaenischAbstract:Summary. Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by Nuclear Transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest as it has the potential to generate a source of patient-specific cells. This short review summarizes strategies to reprogram somatic cells to a pluripotent embryonic state and discuss the implications of this technology for Transplantation medicine.
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stem cells the molecular circuitry of pluripotency and Nuclear reprogramming
Cell, 2008Co-Authors: Rudolf Jaenisch, Richard A YoungAbstract:Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by Nuclear Transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. Here, we review strategies to reprogram somatic cells to a pluripotent embryonic state and discuss our understanding of the molecular mechanisms of reprogramming based on recent insights into the regulatory circuitry of the pluripotent state.
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in vitro reprogramming of fibroblasts into a pluripotent es cell like state
Nature, 2007Co-Authors: Marius Wernig, Alexander Meissner, Konrad Hochedlinger, Ruth K Foreman, Tobias Brambrink, Bradley E Bernstein, Rudolf JaenischAbstract:Nuclear Transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the Nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.
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Reprogramming Efficiency Following Somatic Cell Nuclear Transfer Is Influenced by the Differentiation and Methylation State of the Donor Nucleus
Stem cells (Dayton Ohio), 2006Co-Authors: Robert Blelloch, Zhongde Wang, Alexander Meissner, Steven M. Pollard, Austin Smith, Rudolf JaenischAbstract:Reprogramming of a differentiated cell nucleus by somatic cell Nuclear Transplantation is an inefficient process. Following Nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for Nuclear Transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells, demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methyl-transferase-1, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.
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human cloning the science and ethics of Nuclear Transplantation
The New England Journal of Medicine, 2004Co-Authors: Rudolf JaenischAbstract:Many believe that the practice of somatic-cell Nuclear transfer with the goal of generating an embryonic stem-cell line is justified. Dr. Rudolf Jaenisch discusses the science and ethics of Nuclear Transplantation.
John B. Gurdon - One of the best experts on this subject based on the ideXlab platform.
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hira dependent h3 3 deposition is required for transcriptional reprogramming following Nuclear transfer to xenopus oocytes
Epigenetics & Chromatin, 2012Co-Authors: Jerome Jullien, Carolina Astrand, Emmanuelle Szenker, Nigel Garrett, Genevieve Almouzni, John B. GurdonAbstract:Nuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3. After Nuclear Transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after Nuclear transfer. This study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.
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from Nuclear transfer to Nuclear reprogramming the reversal of cell differentiation
Annual Review of Cell and Developmental Biology, 2006Co-Authors: John B. GurdonAbstract:This is a personal historical account of events leading from the earliest success in vertebrate Nuclear transfer to the current hope that Nuclear reprogramming may facilitate cell replacement therapy. Early morphological evidence in Amphibia for the toti- or multipotentiality of some nuclei from differentiated cells first established the principle of the conservation of the genome during cell differentiation. Molecular markers show that many somatic cell nuclei are reprogrammed to an embryonic pattern of gene expression soon after Nuclear Transplantation to eggs. The germinal vesicles of oocytes in first meiotic prophase have a direct reprogramming activity on mammalian as well as amphibian nuclei and offer a route to identify Nuclear reprogramming molecules. Amphibian eggs and oocytes have a truly remarkable ability to transcribe genes as DNA or nuclei, to translate mRNA, and to modify or localize proteins injected into them. The development of Nuclear transplant embryos depends on the ability of cells t...
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dna demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei
Nature Cell Biology, 2004Co-Authors: Stina Simonsson, John B. GurdonAbstract:Nuclear Transplantation experiments in amphibia and mammals have shown that oocyte and egg cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression and new pathways of cell differentiation1,2,3; however, very little is known about the molecular mechanism of Nuclear reprogramming. Here we have used Nuclear and DNA transfer from mammalian somatic cells to analyse the mechanism of activation of the stem cell marker gene oct4 by Xenopus oocytes. We find that the removal of Nuclear protein accelerates the rate of reprogramming, but even more important is the demethylation of somatic cell DNA. DNA demethylation seems to precede gene reprogramming, and is absolutely necessary for oct4 transcription. Reprogramming by oocytes occurs in the absence of DNA replication and RNA/protein synthesis. It is also selective, operating only on the promoter, but not enhancers, of oct4; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and transcription. We conclude that the demethylation of promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei.
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from intestine to muscle Nuclear reprogramming through defective cloned embryos
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: James A Byrne, Stina Simonsson, John B. GurdonAbstract:Nuclear Transplantation is one of the very few ways by which the genetic content and capacity for Nuclear reprogramming can be assessed in individual cells of differentiated somatic tissues. No more than 6% of the cells of differentiated tissues have thus far been shown to have nuclei that can be reprogrammed to elicit the formation of unrelated cell types. In Amphibia, about 25% of such Nuclear transfers form morphologically abnormal partial blastulae that die within 24 h. We have investigated the genetic content and capacity for reprogramming of those nuclei that generate partial blastulae, using as donors the intestinal epithelium cells of feeding Xenopus larvae. We have analyzed single Nuclear transplant embryos obtained directly from intestinal tissue, thereby avoiding any genetic or epigenetic changes that might accumulate during cell culture. The expression of the intestine-specific gene intestinal fatty acid binding protein is extinguished by at least 104 times, within a few hours of Nuclear Transplantation. At the same time several genes that are normally expressed only in early embryos are very strongly activated in Nuclear transplant embryos, but to an unregulated extent. Remarkably, cells from intestine-derived partial blastulae, when grafted to normal host embryos, contribute to several host tissues and participate in the normal 100-fold increase in axial muscle over several months. Thus, cells of defective cloned embryos unable to survive for more than 1 day can be reprogrammed to participate in new directions of differentiation and to maintain indefinite growth, despite the abnormal expression of early genes.
Konrad Hochedlinger - One of the best experts on this subject based on the ideXlab platform.
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in vitro reprogramming of fibroblasts into a pluripotent es cell like state
Nature, 2007Co-Authors: Marius Wernig, Alexander Meissner, Konrad Hochedlinger, Ruth K Foreman, Tobias Brambrink, Bradley E Bernstein, Rudolf JaenischAbstract:Nuclear Transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the Nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.
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Nuclear cloning of embryonal carcinoma cells
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Robert Blelloch, Konrad Hochedlinger, Yasuhiro Yamada, Cameron Brennan, Minjung Kim, Beatrice Mintz, Lynda Chin, Rudolf JaenischAbstract:Embryonal carcinoma (EC) cells have served as a model to study the relationship between cancer and cellular differentiation given their potential to produce tumors and, to varying degrees, participate in embryonic development. Here, Nuclear Transplantation was used to assess the extent to which the tumorigenic and developmental potential of EC cells is governed by epigenetic as opposed to genetic alterations. Nuclei from three independent mouse EC cell lines (F9, P19, and METT-1) with differing developmental and tumorigenic potentials all were able to direct early embryo development, producing morphologically normal blastocysts that gave rise to Nuclear transfer (NT)-derived embryonic stem (ES) cell lines at a high efficiency. However, when tested for tumor or chimera formation, the resulting NT ES cells displayed an identical potential as their respective donor EC cells, in stark contrast to previously reported NT ES cells derived from transfer of untransformed cells. Consistent with this finding, comparative genomic hybridization identified previously undescribed genetic lesions in the EC cell lines. Therefore, nonreprogrammable genetic modifications within EC nuclei define the developmental and tumorigenic potential of resulting NT ES cells. Our findings support the notion that cancer results from the deregulation of stem cells and further suggest that the genetics of ECs will reveal genes involved in stem cell self-renewal and pluripotency.
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reprogramming of a melanoma genome by Nuclear Transplantation
Genes & Development, 2004Co-Authors: Konrad Hochedlinger, Robert Blelloch, Yasuhiro Yamada, Cameron Brennan, Lynda Chin, Rudolf JaenischAbstract:We have used Nuclear Transplantation to test whether the reprogramming activity of oocytes can reestablish developmental pluripotency of malignant cancer cells. We show here that the nuclei of leukemia, lymphoma, and breast cancer cells could support normal preimplantation development to the blastocyst stage but failed to produce embryonic stem (ES) cells. However, a blastocyst cloned from a RAS-inducible melanoma nucleus gave rise to ES cells with the potential to differentiate into multiple cell types in vivo including melanocytes, lymphocytes, and fibroblasts. Chimeras produced from these ES cells developed cancer with higher penetrance, shorter latency, and an expanded tumor spectrum when compared with the donor mouse model. These results demonstrate that the secondary changes of a melanoma nucleus are compatible with a broad developmental potential but predispose mice to melanomas and other malignant tumors on reactivation of RAS. Our findings serve as a paradigm for studying the tumorigenic effect of a given cancer genome in the context of a whole animal.
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Nuclear Transplantation embryonic stem cells and the potential for cell therapy
The New England Journal of Medicine, 2003Co-Authors: Konrad Hochedlinger, Rudolf JaenischAbstract:uclear cloning, also referred to as Nuclear transfer or Nuclear Transplantation, denotes the introduction of a nucleus from an adult donor cell into an enucleated oocyte to generate a cloned embryo. When transferred to the uterus of a female recipient, this embryo has the potential to grow into an infant that is a clone of the adult donor cell, a process termed “reproductive cloning.” However, when explanted in culture, this embryo can give rise to embryonic stem cells that have the potential to become any or almost any type of cell present in the adult body. Because embryonic stem cells derived by Nuclear transfer are genetically identical to the donor and thus potentially useful for therapeutic applications, this process is called “Nuclear Transplantation therapy” or “therapeutic cloning.” Therapeutic cloning might substantially improve the treatment of neurodegenerative diseases, blood disorders, or diabetes, since therapy for these diseases is currently limited by the availability or immunocompatibility of tissue transplants. Indeed, experiments in animals have shown that Nuclear cloning combined with gene and cell therapy represents a valid strategy for treating genetic disorders. Reproductive cloning is an inefficient and error-prone process that results in the failure of most clones during development. For a donor nucleus to support development, it must properly activate genes important for early embryonic development and suppress differentiation-associated genes that were transcribed in the original donor cell. Inadequate “reprogramming” of the donor nucleus is thought to be the principal reason for the developmental loss of most clones. In contrast, reprogramming errors do not appear to interfere with therapeutic cloning, because the process appears to select for functional cells. Recent advances in the field of Nuclear cloning allow four major conclusions to be drawn. First, most clones die early in gestation, and only a few survive to birth or beyond. Second, cloned animals have common abnormalities regardless of the type of donor cell or the species used, and third, these abnormalities correlate with aberrant gene expression, which most likely results from faulty genomic reprogramming. Fourth, the efficiency of cloning depends on the state of differentiation of the donor cell. In this article, we will summarize recent results from our laboratory and those of others and review potential therapeutic applications of the Nuclear-cloning technology.
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correction of a genetic defect by Nuclear Transplantation and combined cell and gene therapy
Cell, 2002Co-Authors: William M Rideout, Konrad Hochedlinger, Michael Kyba, George Q Daley, Rudolf JaenischAbstract:Immune-deficient Rag2(-/-) mice were used as Nuclear donors for transfer into enucleated oocytes, and the resulting blastocysts were cultured to isolate an isogenic embryonic stem cell line. One of the mutated alleles in the Rag2(-/-) ES cells was repaired by homologous recombination, thereby restoring normal Rag2 gene structure. Mutant mice were treated with the repaired ES cells in two ways. (1) Immune-competent mice were generated from the repaired ES cells by tetraploid embryo complementation and were used as bone marrow donors for Transplantation. (2) Hematopoietic precursors were derived by in vitro differentiation from the repaired ES cells and engrafted into mutant mice. Mature myeloid and lymphoid cells as well as immunoglobulins became detectable 3-4 weeks after Transplantation. Our results establish a paradigm for the treatment of a genetic disorder by combining therapeutic cloning with gene therapy.
Yuko Wakamatsu - One of the best experts on this subject based on the ideXlab platform.
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Generation of fertile and diploid fish, medaka (Oryzias latipes), from Nuclear Transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs.
Cloning and stem cells, 2005Co-Authors: Ekaterina Bubenshchikova, Kenjiro Ozato, Masato Kinoshita, Inna Pristyazhnyuk, Katsutoshi Niwa, Elena Kaftanovskaya, Yuko WakamatsuAbstract:In two experimental series of Transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid Nuclear transplants were successfully ge...
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Transplantation of blastula nuclei to non enucleated eggs in the medaka oryzias latipes
Development Growth & Differentiation, 1999Co-Authors: Katsutoshi Niwa, Masato Kinoshita, Kenjiro Ozato, Tatiana Ladygina, Yuko WakamatsuAbstract:Studies of Nuclear Transplantation were conducted to establish methods for the production of clones of fish, using a small laboratory fish, medaka, Oryzias latipes. As the first step of the study, single-blastula nuclei of an inbred strain with the wild-type body color were transplanted into non-enucleated unfertilized eggs of an outbred orange red strain. Of 845 operated eggs, 45 hatched into fry exhibiting the wild-type body color, one of the donor markers. Twenty-seven of these Nuclear transplants grew to the adult stage and clearly exhibited external secondary sexual characteristics. Fourteen were females and 13 were males. The allozyme analysis of phosphoglucomutase, measurements of relative DNA content by microfluorometry and chromosome counts consistently indicated that the Nuclear transplants were triploids that originated from both the diploid donor nuclei and the haploid recipient pronuclei. In the crossing experiments between the Nuclear transplants and the orange-red strain, most of the male Nuclear transplants were sterile, whereas one male produced a viable offspring with wild-type body color. All of the female Nuclear transplants were sterile. Macroscopic observations of their gonads showed that the testes appeared normal and the ovaries appeared degenerated. These features of the reproductive potential and the morphology of gonads also indicated that the Nuclear transplants were triploids. These results demonstrated that a basic technique for Nuclear Transplantation in medaka was established.
Ilenia Simeoni - One of the best experts on this subject based on the ideXlab platform.
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mammalian Nuclear Transplantation to germinal vesicle stage xenopus oocytes a method for quantitative transcriptional reprogramming
Methods, 2010Co-Authors: Richard P Halleystott, Vincent Pasque, Carolina Astrand, Kei Miyamoto, Ilenia SimeoniAbstract:Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.
