78 research outputs found

    Embryonic Stem Cells: New Possible Therapy for Degenerative Diseases That Affect Elderly People

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    The capacity of embryonic stem (ES) cells for virtually unlimited self renewal and differentiation has opened up the prospect of widespread applications in biomedical research and regenerative medicine. The use of these cells would overcome the problems of donor tissue shortage and implant rejection, if the cells are made immunocompatible with the recipient. Since the derivation in 1998 of human ES cell lines from preimplantation embryos, considerable research is centered on their biology, on how differentiation can be encouraged toward particular cell lineages, and also on the means to enrich and purify derivative cell types. In addition, ES cells may be used as an in vitro system not only to study cell differentiation but also to evaluate the effects of new drugs and the identification of genes as potential therapeutic targets. This review will summarize what is known about animal and human ES cells with particular emphasis on their application in four animal models of human diseases. Present studies of mouse ES cell transplantation reveal encouraging results but also technical barriers that have to be overcome before clinical trials can be considere

    Calreticulin reveals a critical Ca2+ checkpoint in cardiac myofibrillogenesis

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    Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca2+-binding protein that regulates diverse vital cell functions, including Ca2+ storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt−/− embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt−/− EBs, despite normal expression levels of cardiac transcription factors. Crt−/− EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt−/− phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca2+ with EGTA or BAPTA, or by inhibiting Ca2+/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca2+ concentration ([Ca2+]c) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt−/− cardiac cells in the absence of ionomycin-triggered [Ca2+]c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca2+-dependent checkpoint critical for early events during cardiac myofibrillogenesis

    Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues

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    Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stemcell-based approaches for liver metabolic diseases

    A teratocarcinoma-like human embryonic stem cell (hESC) line and four hESC lines reveal potentially oncogenic genomic changes

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    The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis

    A teratocarcinoma-like human embryonic stem cell (hESC) line and four hESC lines reveal potentially oncogenic genomic changes

    Get PDF
    The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis

    Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues

    Get PDF
    Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases

    Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells

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    BACKGROUND Feeder cells are frequently used for the early-stage of derivation and culture of human embryonic stem cell (hESC) lines. METHODS We established a conditionally immortalized human foreskin fibroblast line that secreted basic fibroblast growth factor (bFGF). These cells were used as feeder cells for hESC culture and induced pluripotent stem (iPS) cell derivation and expansion. This conditional immortalization was performed using lentiviral vector (LV) mediated transduction of Bmi-1 and human telomerase reverse transcriptase genes and the resulting cell line was further modified by LV-mediated transduction of a secreted form of bFGF gene product. Three different laboratories have tested whether this feeder cell line could support the maintenance of four different hESC lines. RESULTS Immortalized fibroblasts secreting stable amounts of bFGF supported the growth of all hESC lines, which remained pluripotent and had a normal karyotype for at least 10 passages. Even at high passage (p56), these modified cells, when used as feeders, could support iPS derivation and propagation. Derived iPS cells expressed pluripotency markers, had hESC morphology and produced tissue components of the three germ layers when differentiated in vitro. CONCLUSION These modified fibroblasts are useful as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cell

    Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors

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    Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research
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