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

为了为心血管细胞治疗和组织工程预先编程多能细胞，破译心血管干细胞分化的生物学过程至关重要。我们已经证明，转录因子Mesp1代表了一个足以在干细胞和脊椎动物胚胎中诱导心血管生成的主调节因子。因此，我们将DKK-1启动子定义为Mesp1的直接靶点，从而阻断规范的WNT信号转导。随后，我们论证了ES细胞心血管亚型特异性编程的原理证明：Mesp1促使早/中期类型的心肌细胞出现，而NKX2.5导致优先分化的心室细胞。最近，我们引入了Mesp1启动子，通过磁性细胞分选(MACs)分离常见的心血管前体细胞。我们现在正计划用一种包括系统生物学和经典细胞生物学的方法来进一步表征这个Mesp1阳性的群体，以及破译依赖于Mesp1的信号通路。这将包括关于上游调控因子的Mesp1启动子的特征，以及确定在其目标启动子处直接与Mesp1相互作用的辅助因子。为了实现这一点，我们将使用我们建立的ES细胞系以及新产生的带有敲打标记的Mesp1变体的ES和iPS细胞系。FLAG标记的Mesp1还将用于通过CHIP-Seq鉴定新的直接Mesp1靶基因。此外，将通过基于阵列的方法以及随后对所选样本的深度测序来分析Mesp1-MACs纯化细胞的mRNA表达模式。此外，将基于最新的定制Agilent miRNA阵列来研究miRNAs的表达。对于所有将在Mesp1-MACs之后进行的表达分析，我们将使用上面描述的已建立的Mesp1-ΔCD4系以及将新产生的ES细胞系，它们将带有ΔCD4的靶向敲入。我们的Mesp1-ΔCD4系以及靶向敲入ΔCD4系也将接受电生理和药理学分析，并用于细胞移植，看看Mesp1纯化的祖细胞是否在体内重现了它们的心血管生成多能性。总体而言，我们希望获得关于尚不清楚的信号通路和驱动心血管事件的因素的新的重要信息，以及提出新的表面标记，可能允许在不进行遗传修饰的情况下纯化心血管前体细胞。

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.