Embryonic Stem Cell Derived Cardiac Myocytes

该研究领域涉及胚胎干细胞和诱导多能干细胞在分化为心肌细胞之前和期间的研究。在这些研究中，我们使用了胚胎干细胞（ES）（R1，D3，HES 2），胚胎生殖细胞（EG-1），胚胎癌细胞（P19），以及几种小鼠诱导多能干细胞系。过去的成就包括建立了有效的体外系统，从ES细胞体外产生心肌细胞，并分析了ryanodine 2型缺陷的ES细胞及其对心肌细胞的影响。选择协议（最近与心脏限制部分的Na/Ca交换启动子）也允许从这些异质性培养分离纯化的心肌细胞。最近，我们建立了一个体外模型，包括单层培养的高度增殖的胚胎干（ES）细胞来源的CM，可以采用，促进细胞周期控制机制的分析。另外，该研究旨在产生心脏谱系特异性细胞，以了解调节蛋白在体外心肌细胞形成中的作用。这包括BMP/SMAD信号传导和抗坏血酸和suramen的诱导作用的分析。苏拉明对起搏样细胞诱导的作用正在出版中（Weise等人，Int J Cardiol. 2009年9月21日。Epub before the print），关于BMP信号和抗坏血酸的数据正在准备出版。 这一基础研究工作的另一个重点是致力于提高这些细胞在体外培养条件下的活力。为此，我们有项目评估基质胶对心肌细胞分化和稳定性的影响，这导致了对p53和Mdm 2作用的关注。最近，我们与加州大学戴维斯分校/西奈山，纽约的小组合作，研究发育和分化过程中的microRNA表达。 我们已经开始分离（和靶向）细胞，以基于细胞表面标记物选择心脏祖细胞的亚群，这可能更适合于基于细胞的治疗。最近发表了C2成肌细胞的主要研究证据，说明了这些研究中采用的基于蛋白质组学的方法，现在已经扩展到检查未分化的ES细胞。通过研究胚胎干细胞的基本生物学和鉴定分离亚群的方法，我们希望描绘出负责心肌细胞发育和更新的新机制，并将这些结果应用于改善可能适用于人类的基于细胞的疗法。

SUMMARY OF WORK This research area involves the study of embryonic stem and induced pluripotent stem cells prior to and during differentiation to cardiomyocytes. For these studies, we employ embryonic stem (ES) cells (R1, D3, HES2), embryonic germ cells (EG-1) embryonic carcinoma cells (P19), and several lines of induced pluripotent stem cells from mouse. Past accomplishments include establishment of efficient in vitro systems to generation of cardiomyocytes from ES cells in vitro, and the analysis of ryanodine type 2 deficient ES cells and their effects on cardiomyocytes. Selection protocols (most recently with a cardiac-restricted portion of the Na/Ca exchanger promoter) have also permitted the isolation of purified cardiomyocytes from these heterogeneous cultures. Recently, we established an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CMs that can be employed that facilitate the analysis of cell cycle control mechanisms. Separately, the research is aimed at generating cardiac-lineage specific cells to understand the role of regulatory proteins in the formation of cardiomyocytes in vitro. This includes the analysis of BMP/SMAD signalling and the inductive roles of ascorbate and suramen. The effects of suramen on pacemaker-like cell induction are in press (Weise et al, Int J Cardiol. 2009 Sep 21. Epub ahead of print), and the data on BMP signaling and ascorbate are being prepared for publication. An additional focus of this basic research effort is devoted to the improved viability of these cells during in vitro cultivation conditions. For this we have projects evaluating the effects of Matrigel on cardiomyocyte differentiation and stability, which has led to a focus on the roles of p53 and Mdm2. More recently, we have collaborated with groups at UC Davis/Mount Sinai, NY to examine microRNA expression during development and differentiation. We have begun isolating (and targeting) cells to select sub-populations of cardiac progenitor cells based on cell surface markers that may be more appropriate for cellular based therapies. A proof of principal study in C2 myoblasts was recently published that illustrates the proteomic based methodology employed in these studies, which have now expanded to examine undifferentiated ES cells. By studying the basic biology of embryonic stem cells and identifying methods for isolating sub-populations, we hope to delineate novel mechanisms responsible for cardiomyocyte development and renewal, and apply these results to improve cellular based therapies that may be applicable to man.