Embryonic Stem Cell Derived Cardiac Myocytes

这一研究领域涉及胚胎干细胞分化为心肌细胞之前和分化过程中的研究。在这些研究中，我们使用了胚胎干细胞(ES)(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高效的ES细胞体外培养体系，分析Ryanodine 2型缺陷ES细胞及其对心肌细胞的影响。选择方案(最近使用Na/Ca交换启动子的心脏受限部分)也允许从这些异种培养中分离纯化的心肌细胞。最近，我们建立了一个由高增殖胚胎干细胞来源的CMS单层培养而成的体外模型，该模型可用于分析细胞周期调控机制。另外，这项研究的目的是生成心脏谱系特异性细胞，以了解调控蛋白在体外心肌细胞形成中的作用。这包括对BMP/SMAD信号的分析，以及抗坏血酸和腓肠肌的诱导作用，后者刚刚被接受出版。这项基础研究的另一个重点是在体外培养条件下提高这些细胞的生存能力。为此，我们将重点放在p53和MDM2的作用上。最近，我们与加州大学戴维斯分校的研究小组合作，研究了microRNA在发育和分化过程中的表达。这部作品已提交出版。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，这可能更适合于基于细胞的治疗。最近发表了一项关于C2成肌细胞主要研究的证据，说明了在这些研究中采用的基于蛋白质组的方法，这些研究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进可能适用于人类的基于细胞的治疗方法。

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.