Project description:We developed a gradual differentiation protocol to differentiate mESC towards the EpiSC lineage, followed by early mesoderm cells. These were later differentiated to intermediate mesoderm and we found a condition (IM3) that best generates early gonadal progenitor cells which are normally present at the bipotential gonad at E11.5. Forced expression of 2 transcription factors: Nr5A1 (SF1) and Dmrt1 induced a specific-Sertoli marker (TESCO-CFP) and the cells started to self-aggregate and form tubule-like structures, highly resembling the testis cords. There was proper induction of relevant markers at the correct time-points as shown by both qPCR, immuno-staining and RNA-Seq that includes comparison to in-vivo gonads.
Project description:The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is thus extremely valuable for managing the disease progression. Extracellular glucose is known to impact cell identity by impacting the redox balance. Here we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, high exogenous glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex yin-yang action of exogenous glucose on differentiating pancreas progenitors with a distinct proteome signature.
Project description:This SuperSeries is composed of the following subset Series: GSE35379: Genome-wide occupancy map of GATA-1 in proliferating and differentiating murine ES cell derived erythroid progenitors (ES-EP) GSE35384: Transcriptome analysis of differentiating normal and leukemic erythroid progenitors Refer to individual Series
Project description:The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female -induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.
Project description:We find GATA-1 occupies 6,600 sites in proliferating erythroid progenitors and 10,600 sites in differentiating progenitors. 80-90% of GATA-1 binds within intragenic or intergenic regions, while <20% of GATA-1 is found within 2kb of TSS. Assaying GATA-1 occupancy in normal erythroid progenitors in both proliferating and differentiating conditions.
Project description:We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFB3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. RNA fhMSCs of chondrogenically differentiating fhMSCs was isolated at week, 0, 1, 2, 3, 4 and 5. Moreover, RNA of 4 human growth plates from was isolated.
Project description:We find GATA-1 occupies 6,600 sites in proliferating erythroid progenitors and 10,600 sites in differentiating progenitors. 80-90% of GATA-1 binds within intragenic or intergenic regions, while <20% of GATA-1 is found within 2kb of TSS.
Project description:We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFB3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.
Project description:The molecular details for the specification of lung progenitors from human pluripotent stem cells (hPSCs) are unclear. Here, we use single cell RNA-sequencing with high temporal resolution along an optimized differentiation protocol to determine the transcriptional hierarchy of lung specification from human hPSCs. To chart a comprehensive map of the transcriptional states underlying the differentiation of lung progenitors and hepatoblasts in parallel, we performed a 16-day time-series scRNA-seq using the Drop-Seq workflow (Macosko et al., 2015). Single cell suspensions were processed daily, resulting in the analysis of a total of 10,667 cells that were used for downstream analysis.