MesP1 dependent signalling pathways during the formation of common cardiovascular progenitors in vivo and in vitro

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It is crucial to decipher the biological processes underlying cardiovascular stem cell differentiation in order to preprogram pluripotent cells for cardiovascular cell therapy and tissue engineering. We have shown that the transcription factor MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in stem cells as well as vertebrate embryos. Thereby, we defined the Dkk-1 promoter as a direct target of MesP1 leading to blockage of canonical wnt-signaling. Subsequently, we demonstrated proof of principle for cardiovascular subtype specific programming of ES cells: MesP1 forced the appearance of early/intermediate type cardiomyocytes whereas Nkx2.5 led to preferentially differentiated ventricular cells. Recently, we introduced the MesP1 promoter to isolate common cardiovascular precursors via magnetic cell sorting (MACS). We are now planning to further characterize this MesP1 positive population as well as to decipher MesP1 dependent signaling pathways in an approach comprising systems biology as well as classical cell biology. This will include the characterization of the MesP1 promoter with respect to upstream regulators as well as the identification of co-factors directly interacting with MesP1 at its target promoters. To achieve this we will use our established ES cell lines as well as newly generated ES and iPS cell lines bearing a knocked in Flagtagged MesP1-variant. The Flag-tagged MesP1 will also serve for the identification of novel direct MesP1 target genes via ChiP-Seq. Furthermore, MesP1-MACS purified cells will be analyzed for their mRNA expression patterns via array-based methods and subsequently by deep sequencing of selected samples. Additionally, expression of miRNAs will be investigated based on up-to-date custom-made Agilent miRNA arrays. For all expression analyses, which will be performed after MesP1-MACS, we will use our established MesP1-ΔCD4 lines described above as well as ES cell lines to be newly generated, which will bear a targeted knock-in of ΔCD4. Our MesP1- ΔCD4 lines as well as the lines with a targeted knock-in of ΔCD4 will also be subjected to electrophysiological and pharmacological analyses and used for cell transplantations to see whether MesP1 purified progenitors do recapitulate their cardiovasculogenic multipotency in vivo. Overall we expect to obtain novel important information of yet unknown signaling pathways and factors driving cardiovasculogenic events as well as to come up with novel surface markers possibly allowing purification of cardiovascular precursor cells without genetic modification.