Project description:We developed a differentiation protocol to create skeletal myogenic progenitors in vitro by the induction of Mesp1. The goal of this study is to study the heterogenity of Mesp1 derivatives.
Project description:A hESC MESP1-MCHERRY reporter line was used to isolate and study the molecular character of MESP1 expressing pre-cardiac progenitors, derived from hESC. MESP1 is a key-transcription factor for pre-cardiac mesoderm and is marking the progenitor for almost all cells of the heart. This reporter line was used to study cardiac differentiation and the derivation of early cardiac progenitors in vitro. hESCs were differentiated towards the cardiac lineage, expressing MESP1-mCherry at day 3 of differentiation. Total RNA obtained from isolated MESP1-mCherry expressing progenitors was compared to that of non-MESP1-expressing progenitors and undifferentiated hESCs in order to characterize MESP1-specific transcription factors and proteins.
Project description:A hESC MESP1-MCHERRY reporter line was used to isolate and study the molecular character of MESP1 expressing pre-cardiac progenitors, derived from hESC. MESP1 is a key-transcription factor for pre-cardiac mesoderm and is marking the progenitor for almost all cells of the heart. This reporter line was used to study cardiac differentiation and the derivation of early cardiac progenitors in vitro.
Project description:We have developed a protocol to generate cardiopharyngeal mesoderm (CPM) in vitro by Mesp1 induction in ES cells. The goal of this study is to compare the transcriptome of CPM-derived cardiac and skeletal myogenic progenitors to identify novel lineage-specific markers. mRNA profiles of CPM-derived D6 (early) and D12 (late), cardiac (BMP) and skeletal myogenic (control) progenitors were generated
Project description:We explored the role of mammalian ETS1/2 and Mesp homologues of cardiogenic transcription factors of Ciona intestinalis, to convert primary human dermal fibroblasts into cardiac progenitors. ETS1/2 and Mesp homologues of cardiogenic transcription factors of Ciona intestinalis, to convert primary human dermal fibroblasts into cardiac progenitors. Here we show murine Ets2 has an obligatory role for directing cardiac progenitors during cardiopoesis in embryonic stem cells. ETS2 converted fibroblasts into KDR/Flk1+ replicative cells but, like the purported cardiac master regulatory gene Mesp1, could not by itself generate cardiac progenitors de novo from fibroblasts. Co-expression of both Ets2 and Mesp1, however, successfully reprogrammed differentiated fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, gap junction proteins, sarcomeric proteins, electrical activity and contractility. ETS2 and Mesp1 sit at the pinnacle of the cardiopoesis regulatory hierarchy and are well suited for treating human heart disease. Co-expression of both Ets2 and Mesp1, reprogrammed differentiated fibroblasts into cardiac progenitors
Project description:We have developed a protocol to generate cardiopharyngeal mesoderm (CPM) in vitro by Mesp1 induction in ES cells. The goal of this study is to compare the transcriptome of CPM-derived cardiac and skeletal myogenic progenitors to identify novel lineage-specific markers.
Project description:We explored the role of mammalian ETS1/2 and Mesp homologues of cardiogenic transcription factors of Ciona intestinalis, to convert primary human dermal fibroblasts into cardiac progenitors. ETS1/2 and Mesp homologues of cardiogenic transcription factors of Ciona intestinalis, to convert primary human dermal fibroblasts into cardiac progenitors. Here we show murine Ets2 has an obligatory role for directing cardiac progenitors during cardiopoesis in embryonic stem cells. ETS2 converted fibroblasts into KDR/Flk1+ replicative cells but, like the purported cardiac master regulatory gene Mesp1, could not by itself generate cardiac progenitors de novo from fibroblasts. Co-expression of both Ets2 and Mesp1, however, successfully reprogrammed differentiated fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, gap junction proteins, sarcomeric proteins, electrical activity and contractility. ETS2 and Mesp1 sit at the pinnacle of the cardiopoesis regulatory hierarchy and are well suited for treating human heart disease. Co-expression of both Ets2 and Mesp1, reprogrammed differentiated fibroblasts into cardiac progenitors All sample were done in triplicates, controls were NHDF and ETS2 only infected cells. NHDF were first infected with Doxycyline redulated (Doxy-) ETS2 lentivirus and supplemented with doxycycline for 1 week, sequentially cells were infected with Doxy-Mesp1 and treated for 1 more week. Cells were then aggegated to form EB and hangdrop for 1 week, at the end of that period cells were plated and samples were taken every 24 hrs
Project description:A hESC MESP1 reporter line was used to isolate MESP1 expressing pre-cardiac progenitors. These progenitor were replated and fulter differentiated in culture. At four sequential timepoints upon further differentiation, samples were isolated for gene expression analysis, in order to identify key cardiac transcription factors and molecules. hESCs were differentiated towards the cardiac lineage. MESP1-mCherry expressing progenitors were isolated at day 3 of differentiation and replated as aggregates, in presence of Wnt-inhibitor Xav939. Two days after replating (D5), four days (D7), seven days (D10), ad 11 days (D14), total RNA of each sample was isolated for gene expression analysis. MESP1-mCherry positive derivatives were compared to MESP1-mCherry negative derivatives, in order to identify cardiac-specific regulators and cell surface markers.
Project description:Previously, we reported that the transcription factor Mesp1 promotes the cell fates of cardiomyocytes, smooth muscle, and vascular endothelium. Recently, hematopoietic stem cells (HSCs) were shown to derive from hemogenic endothelium. Since Mesp1 regulates development of endothelium, it potentially could influence gene expression related to hematopoietic development. Our present fate mapping study found that Mesp1-cre efficiently labeled hematopoietic lineages in vivo. This result suggested that Mesp1 might be expressed in progenitors of the hematopoietic system, such as hemogenic endothelium. To test this, we purified Flk1+ Tie2+ endothelium derived from differentiating ES cells with or without Mesp1 induction, and used microarray expression analysis to identify genes strongly up-regulated by Mesp1.