Embryonic Stem Cell Derived Cardiac Myocytes

本研究领域涉及胚胎干细胞分化为心肌细胞之前和过程中的研究。在这些研究中，我们使用了胚胎干细胞（ES）细胞（R1, D3, HES2），胚胎生殖细胞（EG-1）和胚胎癌细胞（P19）。过去的成就包括建立体外胚胎干细胞生成心肌细胞的高效体外系统，以及分析ryanodine 2型缺陷胚胎干细胞及其对心肌细胞的影响。选择方案（最近使用了Na/Ca交换启动子的心脏限制性部分）也允许从这些异质培养物中分离纯化的心肌细胞。最近，我们建立了一个体外模型，由高增殖性胚胎干（ES）细胞来源的CMs的单层培养物组成，可用于促进细胞周期控制机制的分析。另外，该研究旨在生成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。这包括对BMP/SMAD信号传导的分析以及抗坏血酸和suramen的诱导作用，后者刚刚被接受发表。这项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生存能力。为此，我们重点讨论了p53和Mdm2的作用。最近，我们与加州大学戴维斯分校的研究小组合作，研究了发育和分化过程中microRNA的表达。这项工作已提交出版。我们已经开始分离（和靶向）细胞，以选择基于细胞表面标记的心脏祖细胞亚群，这可能更适合细胞为基础的治疗。最近发表的一项C2成肌细胞的主要研究证明了这些研究中采用的基于蛋白质组学的方法，现在已扩展到检查未分化的胚胎干细胞。通过研究胚胎干细胞的基本生物学和确定分离亚群的方法，我们希望描述心肌细胞发育和更新的新机制，并将这些结果应用于改进可能适用于人类的细胞基础疗法。

SUMMARY OF WORK This research area involves the study of embryonic 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) and embryonic carcinoma cells (P19). 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 latter of which was just accepted 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 focused on the roles of p53 and Mdm2. More recently, we have collaborated with groups at UC Davis to examine microRNA expression during development and differentiation. This work has been submitted for publication. 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.