Endoderm & Inflammation-induced Cardiomyogenesis in ESCs

用多能干（PS）细胞治疗性再生患病或受损的心肌取决于在体外有效地将PS细胞预分化为冠状血管和心肌细胞谱系内的专门终点的能力。此外，PS衍生细胞的最佳植入可能需要在这些谱系内的选定发育终点分离细胞。此外，影响因素的内部 必须考虑移植环境对干细胞行为的影响。该子项目解决了这些要求中的每一个，目的是利用多能人类胚胎干细胞（hESC）使受损的心肌重新肌化。目的1检验人定形内胚层（DE）诱导 在多能hESC以及成体（c-kit+）干细胞中的心肌发生。在优化DE诱导的分化后，将对DE分泌的因子进行蛋白质组学表征，以设计生物化学定义的心肌发生混合物，使用实时监测hESC中心肌发生的新型基于内切酶的筛选测定进行评估。目的2将分离Nkx-2.5+/aMHC-细胞，其在免疫反应的早期， 通过FACS选择或直接生物化学诱导，检测与hESC衍生的成熟心肌细胞相比，未成熟的“成心肌细胞”最佳地移植并功能性再生梗死心肌的假设。纯Nkx-2.5+/aMHC-细胞（Aim 2）和成熟心肌细胞（Aim 1）的可用性也将使心肌发生转录组的遗传谱， 将进一步告知心肌发生信号传导和因此生长因子应用以最佳地诱导分化。目的3将评估心肌间质液（MIF）（从缺血心肌分离的非细胞渗出物）中的生长因子/细胞因子对心肌源性谱系中的hESC衍生细胞的行为的影响。这项研究的结果将大大有助于阐明人类如何 心脏病可以通过细胞疗法来改善。

Therapeutic regeneration of diseased or damaged myocardium with pluripotent stem (PS) cells depends on the ability to efficiently pre-differentiate PS cells in vitro to specialized endpoints within the coronary vascular and cardiomyocyte lineages. In addition, optimal engraftment of PS-derived cells will likely require the isolation of cells at selected developmental endpoints within these lineages. In addition, effects of factors within the transplanted environment on stem cell behavior must be considered. This Subproject addresses each of these requirements with the objective of utilizing pluripotent human embryonic stem cells (hESCs) to re-muscularize damaged myocardium. Aim 1 tests the hypothesis that human definitive endoderm (DE) induces cardiomyogenesis in pluripotent hESCs, as well as in adult (c-kit+) stem cells. After optimizing DE-induced differentiation, factors secreted by DE will be proteomically characterized to design a biochemically-defined cardiomyogenic mixture, to be evaluated using a novel luciferase-based screening assay that monitors cardiomyogenesis in hESCs in real-time. Aim 2 will isolate Nkx-2.5+/aMHC- cells, which are early in the cardiomyogenic lineage, via FACS selection or direct biochemical induction to test the hypothesis that immature 'cardiomyoblasts', in comparison with hESC-derived mature cardiomyocytes, optimally engraft and functionally regenerate infarcted myocardium. The availability of pure Nkx-2.5+/aMHC- cells (Aim 2) and mature cardiomyocytes (Aim 1) will also enable the genetic profiling of cardiomyogenic transcriptomes, which will further inform cardiomyogenic signaling and consequently growth factor application to optimally induce differentiation. Aim 3 will assess the effects of growth factors/cytokines in myocardial interstitial fluid (MIF), a non-cellular exudate isolated from ischemic myocardium, on the behavior of hESC-derived cells in the cardiomyogenic lineage. Findings from this study will significantly contribute toward elucidation of how human heart disease may be ameliorated by via cellular therapy.