Project description:Mesenchymal stem/stromal cells (MSCs) are multipotent cells that can differentiate into a variety of cell types forming connective tissue and skeleton, and are essential participants in the development of all organs. However, MSC precursors remain largely unknown. In human embryonic stem cells (hESCs) directed to mesendodermal differentiation through coculture with OP9 stromal cells, we identified a population of mesodermal cells by surface expression of apelin receptor (APLNR1). APLNR+ cells were enriched with precursors generating compact spheroid colonies in semisolid suspension culture. Being formed by single cells, these colonies consisted of a uniform population of mesenchymal cells with a transcriptional profile representative of embryonic mesenchyme originating from lateral plate/extraembryonic mesoderm. Mesenchymal colony formation required serum-free medium and FGF2 as a colony-forming factor, could be significantly enhanced by PDGF-BB, but suppressed by VEGF. When transferred to the adherent cultures in serum-free medium with FGF2, individual colonies gave rise to multipotential mesenchymal cell lines with typical phenotype (CD146+CD105+CD73+CD31-CD43-CD45-), differentiation (chondro-, osteo-, and adipogenesis) and proliferation (>80 doublings) potentials. Consistent with lineage-restricted differentiation pattern, neither endothelial nor hematopoietic cells could be produced from adherent mesenchymal cultures, however endothelial cells could be derived from mesenchymal colonies in the early days of colony-forming culture suggesting that mesenchymal cells arose from cells with primary angiogenic potential (mesangioblasts). Together these studies identified mesangioblasts as the earliest clonogenic mesenchymal precursors at this stage of their specification from mesoderm. This set (11 samples) of expression data is sequential stages of MSC development from hESCs (H1), namely ALPNR+ mesodermal precursors isolated on day 2 and day 3 differentiation, mesangioblast (MB) cores (Day 2 H1-derived cores), hemangioblast (HB) cores (day 3 H1-derived cores), mesangioblast (MB) and hemangioblast (HB) colonies, and colony-derived MSC lines at passage 1 and 5.

Project description:In this work we investigated the molecular mechanisms that sustain the endothelial differentiation of murine embryonic stem cells (ES). When ES cells are co-cultured with the stromal PA6 cells in serum-free medium they differentiate mainly into neurons, thanks to the neural inducing activity exerted by the stroma. The addition of exogenous FGF2 allows also the differentiation of endothelial cells whereas, in presence of exogenous BMP4, ES cells differentiate exclusively into endothelium. The purpose of the gene expression analysis of FGF2 and BMP4 treated co-cultures versus the untreated ones was to profile the transcriptomes of FGF2 and BMP4-driven endothelial differentiation in order to detect molecules and pathways involved upon each of the two exogenous signals. In parallel we also performed transcriptome analysis of single monocultures of stromal PA6 cells to investigate the signals released by the stroma in response to FGF2 and BMP4. We found that TGFb1 is involved in the differentiation of ES cells into endothelium in response to FGF2 while Wnt6 and Wnt pathway sustain the endothelial differentiation of ES cells in response to BMP4. In this work we investigated the molecular mechanisms that sustain the endothelial differentiation of murine embryonic stem cells (ES). When ES cells are co-cultured with the stromal PA6 cells in serum-free medium they differentiate mainly into neurons, thanks to the neural inducing activity exerted by the stroma. The addition of exogenous FGF2 allows also the differentiation of endothelial cells whereas, in presence of exogenous BMP4, ES cells differentiate exclusively into endothelium. The purpose of the gene expression analysis of FGF2 and BMP4 treated co-cultures versus the untreated ones was to profile the transcriptomes of FGF2 and BMP4-driven endothelial differentiation in order to detect molecules and pathways involved upon each of the two exogenous signals. In parallel we also performed transcriptome analysis of single monocultures of stromal PA6 cells to investigate the signals released by the stroma in response to FGF2 and BMP4. We found that TGFb1 is involved in the differentiation of ES cells into endothelium in response to FGF2 while Wnt6 and Wnt pathway sustain the endothelial differentiation of ES cells in response to BMP4.

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FGF2 sustains NANOG and switches the outcome of BMP4 induced human embryonic stem cell differentiation

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