Takahashi K, Okita K, Nakagawa M et al.Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2:3081-3089
Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-machi, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Nature Protocol
(Impact Factor: 9.67).
02/2007; 2(12):3081-9. DOI: 10.1038/nprot.2007.418
Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.
Available from: Celine de Esch
- "The fibroblasts were reprogrammed to iPSC lines according to established protocols (Takahashi et al., 2007; Warlich et al., 2011). First, four iPSC clones were generated that showed typical characteristics of pluripotent stem cells: morphology similar to that of embryonic stem cells (data not shown), expression of alkaline phosphatase (data not shown), silencing of the multicistronic lentiviral transgene (data not shown), reactivation of genes indicative of pluripotency (data not shown), immunoreactivity for OCT4, NANOG, TRA-1-60, TRA-1-81, and SSEA4 (Figure S3), propagation for a long time in culture (up to passage 30), and maintenance of a normal diploid karyotype (data not "
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ABSTRACT: Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epige-netic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an un-methylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 pro-moter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.
Available from: Andrew M Tidball
- "Fibroblasts and hiPSCs were cultured as described previously (Neely et al., 2012). hiPSC lines CA4, CA6, CB5, SM3, SM4, and SM5 were reprogrammed using viral vectors whereas hiPSC lines CA11, CE6, CF1, SM14, PM12 and PM17 were reprogrammed using an episomal-based method (Okita et al., 2011; Takahashi et al., 2007). Line nomenclature follows an alphanumeric sequence, wherein the first two letters designate the human subject they are derived from. "
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ABSTRACT: Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD.
Available from: Dongwan Don Kang
- "We generated iPS cells as described by the Yamanaka group  with the following modifications: One day before transduction, MEFs were seeded at 4×104 cells per well of a Matrigel coated 12-well plate. On the following day (considered day 0) the concentrated retrovirus were thawed and mixed with fresh MEF media up to 1 ml volume (per well) and 8 ng/ml polyprene, and then exposed to the MEFs at 37°C and 5% CO2. "
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ABSTRACT: PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts.
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