Project description:p53 knockdown by shRNA markedly increased efficiency of human iPS cell generation Gene expression patterns were compared between human ES cells and dermal fibroblasts. Gene expression patterns were also compared between p53 shRNA-treated fibroblasts and control fibroblasts.
Project description:This SuperSeries is composed of the following subset Series: GSE13312: Role of p53 in mouse iPS cell generation GSE13334: Effect of p53 in human iPS cell generation Refer to individual Series
Project description:Human fibroblasts can be induced into pluripotent stem cells (iPS cells), but the reprogramming efficiency is quite low. Here, we screened a panel of candidate factors in the presence of OCT4, SOX2, KLF4 and c-MYC in an effort to improve the reprogramming efficiency from human adult fibroblasts. We found that p53 siRNA and UTF1 enhanced the efficiency of iPS cell generation up to 100-fold, even when the oncogene c-MYC was removed from the combinations. We further demonstrated that by using a novel combination of the four factors OCT4, SOX2, KLF4 and UTF1, iPS cells could be generated at a frequency at least 10 times higher than using the original four reprogramming factors without c-MYC. The iPS cells generated in this work have a similar gene expression profile and differentiation potential as human embryonic stem (hES) cells. In conclusion, two novel supporting factors that increase the efficiency of direct reprogramming have been identified, and a more-efficient method for the generation of human iPS cells has been developed in the absence of the oncogene c-MYC. Keywords: cell type comparison
Project description:Induced pluripotent stem cells (iPSC) are generated from somatic cells by the transgene expression of three transcription factors Oct3/4, Sox2, and Klf4 (OSK), albeit at a low efficiency. The protooncogene c-Myc enhances the efficiency of iPSC generation by OSK, but it also increases the tumorigenicity of the resulting iPSC. In the current study, we found the Gli-like transcription factor Glis1, when expressed together with OSK, to markedly enhance the generation of iPSC from both mouse and human fibroblasts. Mouse iPSC generated by OSK and Glis1 can form germline-competent chimeras. Glis1 is enriched in unfertilized oocytes and one cell-stage embryos. DNA microarray analyses revealed that Glis1 promotes multiple pro-reprogramming pathways, including Myc, Nanog, Lin28, Wnt, mesenchymal-epithelial transition (MET), and Esrrb. These results therefore demonstrated that oocyte transcription factor Glis1 effectively promote direct reprogramming during iPSC generation. Adult human fibroblasts were transduced with OSKM and OSK+Glis1 and were used for microarray analyses.
Project description:Human fibroblasts can be induced into pluripotent stem cells (iPS cells), but the reprogramming efficiency is quite low. Here, we screened a panel of candidate factors in the presence of OCT4, SOX2, KLF4 and c-MYC in an effort to improve the reprogramming efficiency from human adult fibroblasts. We found that p53 siRNA and UTF1 enhanced the efficiency of iPS cell generation up to 100-fold, even when the oncogene c-MYC was removed from the combinations. We further demonstrated that by using a novel combination of the four factors OCT4, SOX2, KLF4 and UTF1, iPS cells could be generated at a frequency at least 10 times higher than using the original four reprogramming factors without c-MYC. The iPS cells generated in this work have a similar gene expression profile and differentiation potential as human embryonic stem (hES) cells. In conclusion, two novel supporting factors that increase the efficiency of direct reprogramming have been identified, and a more-efficient method for the generation of human iPS cells has been developed in the absence of the oncogene c-MYC. Keywords: cell type comparison Total RNA from hFSF, hAFF, hES cells (H1, H7) and 7 established iPS cell lines were labeled with Cy5, hybridized to a human Oligo Microarray (Phalanx Human Whole Genome OneArray™, Phalanx Biotech) according to the manufacturer's protocol. Three technical repetitions were performed. After hybridization, arrays were scanned using GenePix 4000B scanner (Molecular Devices) and processed using the GenePix Pro 6.0 software (Molecular Devices). After removing control probes, a 14/33 presence call (SNR>=5 and foreground-background>0) was used to filter probes for the 33 microarrays, resulting in 12311 probes for further quantile normalization.
Project description:Induced pluripotent stem cells (iPSC) are generated from somatic cells by the transgene expression of three transcription factors Oct3/4, Sox2, and Klf4 (OSK), albeit at a low efficiency. The protooncogene c-Myc enhances the efficiency of iPSC generation by OSK, but it also increases the tumorigenicity of the resulting iPSC. In the current study, we found the Gli-like transcription factor Glis1, when expressed together with OSK, to markedly enhance the generation of iPSC from both mouse and human fibroblasts. Mouse iPSC generated by OSK and Glis1 can form germline-competent chimeras. Glis1 is enriched in unfertilized oocytes and one cell-stage embryos. DNA microarray analyses revealed that Glis1 promotes multiple pro-reprogramming pathways, including Myc, Nanog, Lin28, Wnt, mesenchymal-epithelial transition (MET), and Esrrb. These results therefore demonstrated that oocyte transcription factor Glis1 effectively promote direct reprogramming during iPSC generation.
Project description:Induced pluripotent stem cells (iPSC) are generated from somatic cells by the transgene expression of three transcription factors Oct3/4, Sox2, and Klf4 (OSK), albeit at a low efficiency. The protooncogene c-Myc enhances the efficiency of iPSC generation by OSK, but it also increases the tumorigenicity of the resulting iPSC. In the current study, we found the Gli-like transcription factor Glis1, when expressed together with OSK, to markedly enhance the generation of iPSC from both mouse and human fibroblasts. Mouse iPSC generated by OSK and Glis1 can form germline-competent chimeras. Glis1 is enriched in unfertilized oocytes and one cell-stage embryos. DNA microarray analyses revealed that Glis1 promotes multiple pro-reprogramming pathways, including Myc, Nanog, Lin28, Wnt, mesenchymal-epithelial transition (MET), and Esrrb. These results therefore demonstrated that oocyte transcription factor Glis1 effectively promote direct reprogramming during iPSC generation. Mouse embryonic fibroblasts were transduced with OSKM, OSM+Glis1, OSM+Dmrtb1, and OSM+Pitx2 and were used for microarray analyses.
Project description:We observed a marked increase in efficiency of iPS cell generation when p53 is deleted. Experiment Overall Design: Gene expression patterns were compared between p53 wt MEF and p53-null MEF cells. Experiment Overall Design: Gene expression patterns were also compared between ES cells and MEF.
Project description:E-cadherin upregulation is an early event of reprogramming of fibroblasts to induce pluripotent stem cells (iPS). Knocking down of E-cadherin by shRNA impairs iPS generation, though some colonies with great morphorlogical difference to shRNA control colonies remain. To illustrate the molecular and functional difference between shECAD iPS clones and shRNA control iPS clones, three respective iPS clones (shECAD 4,8,9 and Ctrl 2,3,4) were derived and DNA microarrays were run to analyze the transcriptional profile of these clones.