Deep sequencing analysis of MesP1 dependent cardiovascular signalling pathways during stem cell differentiation

干细胞衍生的心肌细胞的增殖潜力是有限的，修复人类梗死心脏的合理产量尚未实现。破译心血管干细胞分化的生物学过程是至关重要的，以便为心血管细胞治疗和组织工程预编程多能细胞。我们已经证明转录因子MesP1是一个主调控因子，足以诱导干细胞和脊椎动物胚胎的心血管发生。因此，我们将Dkk-1启动子定义为MesP1的直接靶点，导致典型wnt信号通路的阻断。最近，我们证明了ES细胞心血管亚型特异性编程的原理：MesP1迫使早期/中期型心肌细胞的出现，而Nkx2.5导致优先分化的心室细胞。依靠稳定和可诱导的MesP1过表达ES细胞系，我们计划使用基于mRNA（转录组）水平深度测序的全局方法分析心血管干细胞分化。因此，MesP1阳性将与MesP1阴性的侧板中胚层细胞进行比较。此外，新的MesP1靶启动子将通过ChIP-Seq鉴定。总的来说，我们期望获得未知的信号通路和驱动这些事件的因素的新的重要信息，并提出新的表面标记物，可能允许在没有基因修饰的情况下纯化心血管前体细胞。

The proliferative potential of stem cell derived cardiomyocytes is limited and reasonable yields to repair a human infarcted heart have yet to be achieved. 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. More recently 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. Relying on stably and inducible MesP1 overexpressing ES cell lines here we plan the analysis of cardiovascular stem cell differentiation using a global approach based on deep sequencing at the mRNA (Transcriptome) level. Thereby MesP1 positive will be compared to MesP1 negative lateral plate mesodermal cells. In addition novel MesP1 target promoters will be identified via ChIP-Seq. Overall we expect to obtain novel important information of yet unknown signalling pathways and factors driving these events as well as to come up with novel surface markers possibly allowing purification of cardiovascular precursor cells without genetic modification.