Project description:Chromatin state maps (H3K4me3 and H3K27me3) from partially and fully reprogrammed mouse cell lines obtained by ectopic expression of Oct4, Sox2, Klf4 and c-Myc using constitutive retroviral infection of MEFs (MCV6, MCV8, MCV8.1) or induction of lentivirus in secondary B lymphocytes obtained from iPS-derived chimeric mice (BIV8). Keywords: High-throughput ChIP-sequencing, Illumina, cell type comparison
Project description:Chromatin state maps (H3K4me3 and H3K27me3) from partially and fully reprogrammed mouse cell lines obtained by ectopic expression of Oct4, Sox2, Klf4 and c-Myc using constitutive retroviral infection of MEFs (MCV6, MCV8, MCV8.1) or induction of lentivirus in secondary B lymphocytes obtained from iPS-derived chimeric mice (BIV8). Keywords: High-throughput ChIP-sequencing, Illumina, cell type comparison H3K4me3 and H3K27me3 ChIP-Seq in singlicate from three partially reprogrammed cell lines (BIV1, MCV8, MCV6), one iPS cell line (MCV8.1) and MEFs (subsampled from Mikkelsen et al, Nature, 2007)) Raw sequence data files for this study are available for download from the SRA FTP site at ftp://ftp.ncbi.nlm.nih.gov/sra/Studies/SRP000/SRP000215
Project description:Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by Oct4, Sox2, Klf4, plus c-Myc. Recently, Sox2 plus Oct4 were shown to reprogram fibroblasts and Oct4 alone to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here we report that Bmi1 leads to dedifferentiation of mouse fibroblasts into NSC-like cells and, in combination with Oct4, replaces Sox2, Klf4 and c-Myc during reprogramming fibroblasts to iPS cells. Furthermore, activation of sonic hedgehog signalling (by Shh, purmorphamine, or oxysterol) replaces the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One-and two-factor iPS cells are similar to mouse embryonic and adult fibroblasts into iPS cells in global gene expression profile, epigenetic status, in vitro and in bibo differentiation into all three ferm layers, as well as teratoma formation and germline transmission in vivo. These data support that fibroblasts can be reprogrammed into iPS cells by Oct4 alone.
Project description:Induced pluripotent stem (iPS) cells can be obtained from fibroblasts by expression of Oct4, Sox2, Klf4, and c-Myc. To determine how these factors induce this change in cell identity, we carried out genomewide promoter analysis of their binding in iPS and partially reprogrammed cells. Most targets in iPS cells are shared with ES cells and the factors cooperate to activate the ES-like expression program. In partially reprogrammed cells, genes bound by c-Myc have achieved a more ES-like binding and expression pattern. In contrast, genes that are co-bound by Oct4, Sox2, and Klf4 in ES cells and that encode pluripotency regulators show severe lack of both binding and transcriptional activation. Among the factors, c-Myc has a pivotal effect on the initiation of the ES transcription program, including the repression of fibroblast-specific genes. Our analysis begins to unravel how the four factors function together and suggests a temporal and separable order of their effects during reprogramming. For genome wide expression analysis: A. ES/iPS/partial iPS cells were analyzed in duplicates from 2 different cell lines (e.g four samples per cell type) B. tet inducible factors (tetO/tetS/tetC/tetK) were analyzed individually, a control was an uninduced tet line. C. OCK expression was performed in duplicate with 2 different clones. For genome wide location analysis (ChIP-chip): Oct4/Sox2/c-Myc/Klf4 were performed with biological duplicates (2 clonally isolated iPS/partial iPS lines and ES cells were performed with v6.5 and E14 cell lines)
Project description:Induced pluripotent stem (iPS) cells can be obtained from fibroblasts by expression of Oct4, Sox2, Klf4, and c-Myc. To determine how these factors induce this change in cell identity, we carried out genomewide promoter analysis of their binding in iPS and partially reprogrammed cells. Most targets in iPS cells are shared with ES cells and the factors cooperate to activate the ES-like expression program. In partially reprogrammed cells, genes bound by c-Myc have achieved a more ES-like binding and expression pattern. In contrast, genes that are co-bound by Oct4, Sox2, and Klf4 in ES cells and that encode pluripotency regulators show severe lack of both binding and transcriptional activation. Among the factors, c-Myc has a pivotal effect on the initiation of the ES transcription program, including the repression of fibroblast-specific genes. Our analysis begins to unravel how the four factors function together and suggests a temporal and separable order of their effects during reprogramming.
Project description:Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by Oct4, Sox2, Klf4, plus c-Myc. Recently, Sox2 plus Oct4 were shown to reprogram fibroblasts and Oct4 alone to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here we report that Bmi1 leads to dedifferentiation of mouse fibroblasts into NSC-like cells and, in combination with Oct4, replaces Sox2, Klf4 and c-Myc during reprogramming fibroblasts to iPS cells. Furthermore, activation of sonic hedgehog signalling (by Shh, purmorphamine, or oxysterol) replaces the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One-and two-factor iPS cells are similar to mouse embryonic and adult fibroblasts into iPS cells in global gene expression profile, epigenetic status, in vitro and in bibo differentiation into all three ferm layers, as well as teratoma formation and germline transmission in vivo. These data support that fibroblasts can be reprogrammed into iPS cells by Oct4 alone. Total RNAs were isolated from indicated cells and labeled with Cy3. Hybridization was performed once for each sample.
Project description:We tested various architectures of artificial transcription factors (ATFs) in HEK293 cells to test the contribution of the interaction domain, nuclear localization domain, size of DNA-binding domain, and activation domain. Then we made a genome-scale ATF library and tested it in reprogramming mouse embryonic fibroblasts to induced pluripotent stem (iPS) cells. Three combinations of ATFs (C2, C3, and C4) could induce pluripotency when expressed with Sox2, Klf4, and c-Myc. We harvested polyadenylated RNAs from 11 cell types derived with different sets of factors. The transcriptional profiles of ATF-induced iPS cells are similar to that of iPS cells induced with Oct4, Sox2, Klf4, and c-Myc and mouse embryonic stem cells, exhibiting up-regulation of pluripotency markers and down-regulation of fibroblast markers. Comparisons of cells undergoing reprogramming at an intermediate stage before becoming fully reprogrammed suggest that ATFs activate a different set of genes than the set activated by Oct4. This study provides a proof-of-principle that a gene-activating ATF library can be used to identify cell fate-defining transcriptional networks in an unbiased manner.
Project description:Differentiated somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by forced expression of four transcription factors—Oct4, Sox2, Klf4, and c-Myc. However, it remains undetermined whether the reprogrammed iPS cells are fully pluripotent, resembling normal embryonic stem (ES) cells, given that no iPS cell lines have been shown to possess the capability to autonomously generate full-term mice after injection into tetraploid blastocysts. Here, we provide evidence demonstrating that iPS cells induced by the four transcription factors can be fully pluripotent and that full-term mice can be produced from complemented tetraploid blastocysts. This work serves as a proof of principle that iPS cells can generate full term embryos by tetraploid complementation.
Project description:Through a loss-of-function approach, we identified that inhibition of the histone methyltransferase, Dot1L, accelerated somatic cell reprogramming, significantly increased the yield of induced pluripotent stem (iPS) cell colonies, and substituted for Klf4 and c-Myc in the reprogramming cocktail. To understand the mechanism by which Dot1L inhibition results in these phenotypes, we carried out gene expression profiling using Affymetrix microarrays. GSM723207-GSM723224: Embryonic stem cell-derived fibroblasts (dH1fs) were retrovirally transduced in culture with vectors expressing either a control shRNA or an shRNA targeting Dot1L. These cells were then superinfected with either Oct4, Sox2, Klf4 and c-Myc (OSKM) retroviruses or Oct4, Sox2 and c-Myc (OSM) retroviruses. Total RNA was harvested 6 days later. There were three biologic replicates for each condition. GSM880675-GSM880682: dH1fs were treated with 10uM Dot1L inhibitor (EPZ004777) and then superinfected with Oct4, Sox2, Klf4 and c-Myc (OSKM) retroviruses. Total RNA was harvested 6 days later. There were two biologic replicates for each condition.
Project description:Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing heterochromatin marks. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative Electron Spectroscopic Imaging (ESI). In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocenter structures of densely packed 10 nm chromatin fibers. In contrast, chromocenter boundaries are poorly defined in pluripotent ES and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibers in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst prior to differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by Mek/Gsk3 2i inhibitor treatment. Thus, constitutive heterochromatin reorganization serves as a novel biomarker with retroviral silencing for identifying iPS cells in the very late stages of reprogramming. We compared the expression profiles of partially and fully reprogrammed iPS cell lines derived from CD1 mouse embryonic fibroblasts (MEFSs) by retroviral reprogramming (pMX-Oct4, pMX-Klf4 and pMX-Sox2). to the differentiated MEFS and the J1 embryonic stem cell line. We also studied the effect of a 2i cocktail treatment in partially reprogrammed iPS cells.