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.

Project description:The identified stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells. Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons are potentially useful for treating Parkinson’s disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factor(s) produced by stromal cells that constitute SDIA is unknown. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons in the human embryonal carcinoma stem cell line, NTera2. Here we further examined the effects of PA6-conditioned medium and found that it can induce DA neuronal differentiation in both the NTera2 cell line and the hESC line, I6. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with proteomic analysis of PA6-conditioned medium. Four candidate factors (hepatocyte growth factor (HGF), stromal cell-derived factor-1 alpha (SDF1alpha), secreted frizzled-related protein 1 (sFRP1) and vascular endothelial growth factor D (VEGFD)) were identified and immunoaffinity capillary electrophoresis (ICE) was used to establish the protein concentration of these factors in conditioned medium. Upon addition of SDF1alpha, sFRP1, and VEGFD, we observed an increase in the number of tyrosine hydroxylase- and TuJ1- positive cells in both the NTera2 and I6 cell lines. These results indicate that SDF1alpha, sFRP1 and VEGF-D are major components of SDIA, and suggest the potential use of these defined factors to elicit dopaminergic differentiation of pluripotent stem cells as a therapeutic intervention in PD. Mouse embryonic fibroblasts were grown in DMEM medium supplemented with 10% FBS; this conditioned media was used as the control. PA6, mouse stromal cells, were grown in MEM-alpha supplemented with 10% FBS; this conditioned media is known to induce differentiation in hES cells.

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Role of TGFb1 and Wnt6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells [3h]

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