权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

体内和体外常见心血管祖细胞形成过程中MesP1依赖性信号通路

基本信息

批准号：
35961400
负责人：
Professor Dr. Wolfgang-M. Franz
金额：
--
依托单位：
Klinik und Poliklinik für Herzchirurgie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2007
资助国家：
德国
起止时间：
2006-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/35961400?language=en
关键词：
MesP1 dependent signalling pathways during

项目摘要

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.

因此，研究心血管干细胞分化的生物学机制，对于为心血管细胞治疗和组织工程提供多能干细胞的预编程至关重要。我们已经表明，转录因子MesP 1代表一个主调节足以诱导干细胞以及脊椎动物胚胎心血管发生。因此，我们将Dkk-1启动子定义为MesP 1的直接靶点，导致经典wnt信号传导的阻断。随后，我们证明了ES细胞的心血管亚型特异性编程的原理证明：MesP 1迫使早期/中间型心肌细胞的出现，而Nkx2.5导致优先分化的心室细胞。最近，我们引入了MesP 1启动子，通过磁细胞分选（MACS）分离常见的心血管前体。我们现在正计划进一步表征这种MesP 1阳性人群，并在包括系统生物学和经典细胞生物学的方法中破译MesP 1依赖的信号通路。这将包括MesP 1启动子相对于上游调节器的表征以及在其靶启动子处与MesP 1直接相互作用的辅因子的鉴定。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ES和iPS细胞系，这些细胞系携带敲入的Flagtaged MesP 1变体。Flag标记的MesP 1也将用于通过ChiP-Seq鉴定新的直接MesP 1靶基因。此外，将通过基于阵列的方法分析MesP 1-MACS纯化细胞的mRNA表达模式，随后对选定样品进行深度测序。此外，将基于最新定制的Agilent miRNA阵列研究miRNA的表达。对于将在MesP 1-MACS后进行的所有表达分析，我们将使用上述已建立的MesP 1-Δ CD 4细胞系以及新生成的ES细胞系，这些细胞系将携带Δ CD 4的靶向敲入。我们的MesP 1- Δ CD 4系以及具有Δ CD 4靶向敲入的系也将进行电生理学和药理学分析，并用于细胞移植，以观察MesP 1纯化的祖细胞是否在体内重现其心血管发生多能性。总的来说，我们希望获得新的重要信息，但未知的信号通路和驱动心血管事件的因素，以及提出新的表面标记物，可能允许纯化心血管前体细胞，而无需遗传修饰。