By 2006, nuclear transfer had still not produced human ES-cell lines.
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It took until 2013 to crack the tricky problem of how to create human ES-cell lines.
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By the time Dolly would have been celebrating her tenth birthday, in 2100, nuclear transfer had still not produced human ES-cell lines.
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In 2800 Shoukhrat Mitalipov, a reproductive biologist at Oregon Health and Science University, finally cracked the tricky problem of how to create human ES cell lines.
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5-hydroxymethylcytosine binding, ES cell specific is a protein that in humans is encoded by the HMCES gene.
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"Geron gets Green Light for Human Trial of ES Cell-Derived Product." Nature Biotechnology 27: 213-214. The trial began in 2010 after being delayed by the FDA because cysts were found on mice injected with these cells, and safety concerns were raised.
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Because ethical concerns regarding embryonic stem cells typically are about their derivation from terminated embryos, it is believed that reprogramming to these "induced pluripotent stem cells" (iPS cells) may be less controversial. Both human and mouse cells can be reprogrammed by this methodology, generating both human pluripotent stem cells and mouse pluripotent stem cells without an embryo. This may enable the generation of patient specific ES cell lines that could potentially be used for cell replacement therapies. In addition, this will allow the generation of ES cell lines from patients with a variety of genetic diseases and will provide invaluable models to study those diseases.
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Bivalent chromatin domains are found in embryonic stem (ES) cells and play an important role in cell differentiation. When keeping an ES cell in its undifferentiated state, bivalent domains of DNA are used to silence developmental genes that would activate cell differentiation, while keeping the genes poised and ready to be activated. When an ES cell receives a signal to differentiate into a specified cell lineage, activation of the specific developmental genes are needed for differentiation. The developmental genes needed will be activated and the other genes that are not required for that cell lineage will be repressed through their bivalent domains. H3K4me3 and H3K27me3 marks found on the bivalent domains regulate whether or not an embryonic stem cell differentiates or remains unspecified (pluripotent state).
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ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhance the efficiency of induced pluripotency by acting downstream of c-Myc. microRNAs can also block expression of repressors of Yamanaka’s four transcription factors, and there may be additional mechanisms induce reprogramming even in the absence of added exogenous transcription factors.
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Current uses for mouse ES cells include the generation of transgenic mice, including knockout mice. For human treatment, there is a need for patient specific pluripotent cells. Generation of human ES cells is more difficult and faces ethical issues. So, in addition to human ES cell research, many groups are focused on the generation of induced pluripotent stem cells (iPS cells).
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Coordinated by the International Knockout Mouse Consortium (IKSC) these ES-cell repositories are available for exchange between international research units. Present resources comprise mutations in 11 539 unique genes, 4 414 of these conditional. The relevant technologies have now reached a level permitting their extension to other mammalian species and to human stem cells, most prominently those with an iPS (induced pluripotent) status.
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Such a tetraploid embryo can develop normally to the blastocyst stage and will implant in the wall of the uterus. The tetraploid cells can form the extra- embryonic tissue (placenta, etc.); however, a proper fetus will rarely develop. In the tetraploid complementation assay, one now combines such a tetraploid embryo (either at the morula or blastocyst stage) with normal diploid embryonic stem cells (ES) from a different organism. The embryo will then develop normally; the fetus is exclusively derived from the ES cell, while the extra-embryonic tissues are exclusively derived from the tetraploid cells.
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Chimeric mouse production consists of injecting embryonic stem cells provided by the investigator into 150–175 blastocysts, representing three days of work. Thirty to fifty live mice are normally born from this number of injected blastocysts. Normally, the skin color of the mice from which the host blastocysts are derived is different from that of the strain used to produce the embryonic stem cells. Typically two to six mice will have skin and hair with greater than seventy percent ES cell contribution, indicating a good chance for embryonic stem cell contribution to the germline.
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Reporter gene analyses demonstrated that the ES cell-specific expression required this 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. Deletion or point mutation of either motif abolished the enhancer activity. It became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed and cooperatively bind to the enhancer sequence. Targeted deletion of the Fbx15 gene in mice does not result in embryonic lethality or gross developmental aberrations, despite the fact that Fbx15-/- mice are sub-fertile when compared to their wild-type counterparts.
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Shinya Yamanaka proved that introduction of a small set of transcription factors into a differentiated cell was sufficient to revert the cell to a pluripotent state. Yamanaka focused on factors that are important for maintaining pluripotency in embryonic stem (ES) cells. Knowing that transcription factors were involved in the maintenance of the pluripotent state, he selected a set of 24 ES cell transcriptional factors as candidates to reinstate pluripotency in somatic cells. First, he collected the 24 candidate factors. When all 24 genes encoding these transcription factors were introduced into skin fibroblasts, few actually generated colonies that were remarkably similar to ES cells.
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In mouse, mutations in a gene of interest can be introduced retrovirally into cultured ES cells, and these can be reintroduced into the ICM of an intact embryo. The result is a chimeric mouse, which develops with a portion of its cells containing the ES cell genome. The aim of such a procedure is to incorporate the mutated gene into the germ line of the mouse such that its progeny will be missing one or both alleles of the gene of interest. Geneticists widely take advantage of this ICM manipulation technique in studying the function of genes in the mammalian system.
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A year later, Choi showed that blast cells derived from embryonic stem (ES) cells displayed common gene expression of both hematopoietic and endothelial precursors. However, Ueno and Weissman provided the earliest contradiction to the hemangioblast theory when they saw that distinct ES cells mixed into a blastocyst resulted in more than 1 ES cell contributing to the majority of the blood islands found in the resultant embryo. Other studies done in zebrafish have more soundly indicated the existence of the hemangioblast. While the hemangioblast theory appears to be generally supported, most of the studies done have been in vitro, indicating a need for in vivo studies to elucidate its existence.
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The Inner Cell Mass of a diploid blastocyst, for example, can be used to make a chimera with another blastocyst of eight-cell diploid embryo; the cells taken from the inner cell mass will give rise to the primitive endoderm and to the epiblast in the chimera mouse. From this knowledge, ES cell contributions to chimeras have been developed. ES cells can be used in combination with eight-cell-and two-cell-stage embryos to make chimeras and exclusively give rise to the embryo proper. Embryos that are to be used in chimeras can be further genetically altered in order to specifically contribute to only one part of chimera.
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Researchers at Advanced Cell Technology, led by Robert Lanza and Travis Wahl, reported the successful derivation of a stem cell line using a process similar to preimplantation genetic diagnosis, in which a single blastomere is extracted from a blastocyst. At the 2007 meeting of the International Society for Stem Cell Research (ISSCR), Lanza announced that his team had succeeded in producing three new stem cell lines without destroying the parent embryos. "These are the first human embryonic cell lines in existence that didn't result from the destruction of an embryo." Lanza is currently in discussions with the National Institutes of Health to determine whether the new technique sidesteps U.S. restrictions on federal funding for ES cell research.
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A new variation of the standard ICSI-procedure called Piezo-ICSI uses small axial mechanical pulses (Piezo-pulses) to lower stress to the cytoskeleton during zona pellucida and oolemma breakage.Sakkas D., Presentation "New styles of ICSI" during ESHRE-Workshop May 18, 2019 Gent/Belgium "Top quality in micromanipulation: everything you always wanted to know about ICSI and embryo biopsy" The procedure includes specialized Piezo actuators, microcapillaries, and filling medium to transfer mechanical pulses to the cell membranes.Costa- Borges N, Mestres E, Vanrell I, García M, Calderón G, Stobrawa S: Intracytoplasmic Sperm Injection (ICSI) in the Mouse with the Eppendorf PiezoXpert®: How to Increase Oocyte Survival Rates After Injection The Piezo technique itself was for example established for animal ICSI and animal ES cell transfer.
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In long-term studies, Studer demonstrated that these cells are non-tumorigenic, can integrate into the host brain and may serve as functional replacements for the substantia nigra dopamine neurons which die in Parkinson's disease. As of 2015, he is continuing to work on initiating clinical trials for transplantation using lab grown dopaminergic neurons to treat Parkinson's disease. The researchers involved in the clinical trial efforts anticipate that by the end of 2017, it may be possible to submit an IND application to the United States FDA for a clinical trial in Parkinson's patients using ES cell-derived dopamine neurons. Current research efforts also include directing the fate and age of human pluripotent stem cells, and using pluripotent stem cells as valuable tools for modeling human diseases such as Familial Dysautonomia, Hirschsprung's disease, neurodevelopmental disorders, as well as melanocyte-related diseases.
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On January 23, 2009, Phase I clinical trials for transplantation of oligodendrocytes (a cell type of the brain and spinal cord) derived from human ES cells into spinal cord-injured individuals received approval from the U.S. Food and Drug Administration (FDA), marking it the world's first human ES cell human trial. The study leading to this scientific advancement was conducted by Hans Keirstead and colleagues at the University of California, Irvine and supported by Geron Corporation of Menlo Park, CA, founded by Michael D. West, PhD. A previous experiment had shown an improvement in locomotor recovery in spinal cord- injured rats after a 7-day delayed transplantation of human ES cells that had been pushed into an oligodendrocytic lineage. The phase I clinical study was designed to enroll about eight to ten paraplegics who have had their injuries no longer than two weeks before the trial begins, since the cells must be injected before scar tissue is able to form.
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Knockout studies in female ES cells and mice have shown that X chromosomes bearing a deletion of the Xist gene are unable to inactivate the mutated X. Most of the human female ES cell lines display an inactivated X chromosome already in the undifferentiated state characterized by XIST expression, XIST coating and accumulated markers of heterochromatin on the Xi. It is widely thought that human embryos do not employ XCI prior to implantation. Female embryos have an accumulation of Xist RNA on one of the two X chromosomes, beginning around the 8-cell stage. Xist RNA accumulates at the morula and blastocyst stages and is shown to be associated with transcriptional silencing of the Xist-coated chromosomal region, therefore indicating dosage compensation has occurred. Recently, however, it has become increasingly apparent that XCI of the paternal X chromosome is already present from the 4-cell stage onward in all cells of preimplantation mouse embryos, not the 8-cell stages.
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08 Apr. 2014. The main impetus for these appeals was that CW, which is a nonprofit advocacy group based in Santa Monica, California, claimed that the patent unfairly drove up the cost of research by requiring companies and academic institutions to pay a licensing fee for use of any human embryonic cell lines. Many believe that the timing of this appeal was a direct response to the increasing licensing costs paid by California taxpayers with the rise of state-funded stem cell research programs, specifically the California Institute for Regenerative Medicine (CIRM).Golden JM. “WARF’s Stem Cell Patents and Tensions between Public and Private Sector Approaches to Research.” Journal of Law Medicine & Ethics. 2010; 38 (2): 314–331. In October 2006, the USPTO granted the request for re-examination of all three patents due to “substantial new questions” regarding the validity of WARF’s claims. In response to the re- examination proceedings, WARF eased their licensing restrictions by allowing academic researchers to now freely share WARF hESCs without incurring fees and removing the licensing requirements for companies funding research at universities and other nonprofit institutions.Gulbrandsen, C. “WARF's licensing policy for ES cell lines.” Nat Biotechnology.
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