Project description:We found distinct and self-renewal cell linages that could differentiate into melanocytes, named melanocyte precursor cells(MPCs) in which MPCs revealed drastically differntiation to authentic melanocytes by a small amount of GSK3β inhibitor in about seven days. Comparing MPCs ,iPSCs and melanocytes, we showed wnt sigal systems in differentiation to melanocytes.
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.
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. OG2 MEFs were infected with Sox2, Klf4, Oct4 and c-Myc (SKOM) plus either Luciferase shRNA (shLUC) or E-cadherin shRNA (shECAD) retrovirus. At Day 6 post infection cells were split onto feeder cells. Several colonies from SKOM+shLuc and SKOM+shECAD were picked out at Day 14 post infection respectively and three cell lines were established, namely Ctrl 2,3,4 for SKOM+shLuc iPS and shECAD 4,8,9 for SKOM+shECAD iPS. All clones were maintained on feeder cells in mESC medium. RNA were extracted from these six cell lines and DNA microarrays were run to analyze the transcriptional profile.
Project description:Whole genome sequencing was performed on several murine iPS cell clones (and their parental cells) from each of three independent reprogramming experiments. Hundreds of single nucleotide variants (SNVs) were detected in each clone, with an average of 11 in coding regions. Affymetrix Mouse Exon 1.0ST arrays were used to compare expression patterns in MPSVII iPS lines, and embryo-derived MPSVII ES cells. Unsupervised hierarchal clustering analysis showed that the iPS clones and ES cell lines clustered randomly, suggesting that their global patterns of gene expression are highly similar. Taken together, our data suggest that most of the genetic variation in iPS cell clones is not caused by reprogramming, but is rather a consequence of cloning individual cells, “capturing” random mutations that preexisted in the single cells that were reprogrammed. These mutations can sometimes contribute to reprogramming “fitness”, thus providing a selective advantage for rare cells when they overexpress reprogramming factors.
Project description:We used heterokaryon cell fusion based reprogramming and identified the cytokine IL6 as a potential regulator of reprogramming to pluripotency. We generated iPS clones using the four reprogramming factors (4F) Oct4, Klf4, Sox2, and c-Myc. In addition, iPS clones were generated using only three factors (3F: Oct4, Klf4, amd Sox2) with the addition of the cytokine IL6 to reprogramming culture conditions. Global RNA-Seq of the 3F + IL6 derived iPS clones was done for comparison with 4F-derived iPS clones, mouse embryonic stem cells and mouse embryonic fibroblasts.