Project description:To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.
Project description:To investigate functional differences between pluripotent stem cells and somatic cells, we measured protein thermal stability and expression after transitions between cell types using allogenic cell lines. To this purpose, we reprogrammed hFF into human iPSCs and initiated undirected differentiation through formation of EBs during 21 days. We also included human embryonic stem cells (ESC) in order to validate our approach and colon cancer cell line RKO.
Project description:The gene expression profiles of differentiating human pluripotent stem cells (hPSCs) were analyzed by DNA microarray. We identified differences and commonalities among six human pluripotent stem cell lines: the hESCs KhES1, KhES2, KhES3, and H1, and the iPSCs 201B7 and 243G1. Analysis of DNA microarray data suggested that hepatocyte-like differentiation of EBs treated with ammonia in Lanford medium.
Project description:Embryoid bodies, 3D aggregates of spontaneously differentiating iPSCs, have the potential to be a useful model system for genomic studies of diverse cell types. We first collected single-cell RNA-sequencing data from embryoid bodies (EBs) generated from 3 Yoruba individuals (18858, 18511, and 19160) in 3 replicates. We classify functional heterogeneity in EB cells using unsupervised clustering, differential expression analysis, reference annotation, and topic modelling. We then inferred differentiation trajectories that recapitulate known developmental patterns of gene expression. We later collected Day 21 EBs from a single replicate of 5 additional Yoruba iPSC lines (18856, 18912, 19140,19159, and 19210) and use this data to demonstrate robustness of EB cell type composition across iPSC lines. Our results establish EBs as a powerful model system to facilitate discovery of QTLs for cell type composition, as well as eQTLs and dynamic eQTLs across a multitude of human cell types and developmental trajectories.
Project description:Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here, we show that iPSCs obtained from fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies (EBs) and different hematopoietic cells. Importantly, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that low-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and ltered differentiation capacity. These observations might affect ongoing attempts to use iPSCs for disease modeling and also could be exploited for potential therapeutic applications to enhance differentiation into desired cell lineages. Keywords: DNA methylation profiling Direct comparison of DNA methylation in iPS cells derived from different tissues
Project description:Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here, we show that iPSCs obtained from fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies (EBs) and different hematopoietic cells. Importantly, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that low-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and ltered differentiation capacity. These observations might affect ongoing attempts to use iPSCs for disease modeling and also could be exploited for potential therapeutic applications to enhance differentiation into desired cell lineages. Keywords: DNA methylation profiling
Project description:These data include the genome wide location of different histone modifications by ChIP sequencing in mouse ES cells, and RNA Seq data generated from wild type and EED KO mouse ES cells and knocked down for unrelated protein and Setd2 protein. ChIP-Seq: Immuno-precipitation of formaldehyde cross-linked chromatin prepared from wild type mouse E14 ES cells, wild type E36 ES cells, EED KO E36 ES cells, wild type Embryoid bodies (Ebs), EED KO Embryoid bodies (Ebs EED KO) using specific antibody against different histone modifications. RNA-Seq: Total RNA extracted from wild type E36 ES cells, EED KO E36 ES cells, wild type E36 Embryoid bodies (Ebs), EED KO Embryoid bodies (Ebs EED KO), E14 Ctrl KD, E14 Setd2 KD.
Project description:The ability of cells to perceive and translate versatile cues into differential chromatin and transcriptional states is critical for many biological processes1-4. In plants, timely transition to a flowering state is crucial for successful reproduction5-7. EARLY BOLTING IN SHORT DAY (EBS) is a negative transcriptional regulator that prevents premature flowering in Arabidopsis8,9. Here, we revealed that bivalent bromo-adjacent homology (BAH)-plant homeodomain (PHD) reader modules of EBS bind H3K27me3 and H3K4me3, respectively. A subset of EBS-associated genes was co-enriched with H3K4me3, H3K27me3, and the Polycomb repressor complex 2 (PRC2). Interestingly, EBS adopts an auto-inhibition mode to mediate its binding preference switch between H3K27me3 and H3K4me3. This binding balance is critical because disruption of either EBS-H3K27me3 or EBS-H3K4me3 interaction induces EBS-mediated early floral transition. This study identifies a single bivalent chromatin reader capable of recognizing two antagonistic histone marks and reveals a distinct mechanism of interplay between active and repressive chromatin states.The ability of cells to perceive and translate versatile cues into differential chromatin and transcriptional states is critical for many biological processes1-4. In plants, timely transition to a flowering state is crucial for successful reproduction5-7. EARLY BOLTING IN SHORT DAY (EBS) is a negative transcriptional regulator that prevents premature flowering in Arabidopsis8,9. Here, we revealed that bivalent bromo-adjacent homology (BAH)-plant homeodomain (PHD) reader modules of EBS bind H3K27me3 and H3K4me3, respectively. A subset of EBS-associated genes was co-enriched with H3K4me3, H3K27me3, and the Polycomb repressor complex 2 (PRC2). Interestingly, EBS adopts an auto-inhibition mode to mediate its binding preference switch between H3K27me3 and H3K4me3. This binding balance is critical because disruption of either EBS-H3K27me3 or EBS-H3K4me3 interaction induces EBS-mediated early floral transition. This study identifies a single bivalent chromatin reader capable of recognizing two antagonistic histone marks and reveals a distinct mechanism of interplay between active and repressive chromatin states.v
Project description:Recent studies indicated that the differentiation tendency of pluripotent stem cells (PSCs) was affected by a certain small molecule treatment. We found the combination of small molecules that bringed out the differentiation potentials of PSCs, and defined such state of PSC as CTraS. Then, we used microarrays to detail the global programme of gene expression reflecting the effect of CTraS, and carefully compared the characteristics of iPSCs, CTraS-iPSCs, embryoid bodies (EBs), and their derived neurospheres (NSs) based on their expression profiles .
Project description:Keratins 5 and 14 are critical for cytoskeletal integrity, as shown by missense mutations in these genes, which cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS). The complexity of the pathomechanisms in EBS is not fully understood and no effective management exists. In addition to fragility, EBS keratinocytes are characterized by aggregates of misfolded keratin. Here, we tested the chemical chaperone 4-phenylbutyrate (4-PBA) as a putative novel therapy, using keratinocytes from patients with severe generalized EBS due to distinct KRT5 and KRT14 mutations.