Embryonic Stem Cell Derived Cardiac Myocytes

这一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过程中的研究。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。这些细胞是心脏细胞替代治疗的一个很有前途的来源，具有高度的异质性，并显示出各种成熟状态。在最近发表的一项研究中，我们利用含有心脏限制性ncx1启动子驱动的嘌呤霉素抗性盒的ESC克隆株和大规模培养系统来分离表现出非常不成熟的CMS特征的ESC-CMS。细胞表现出增殖、CM限制性标志物、线粒体质量减少和耐缺氧等特性。在移植到啮齿动物心脏后，生物发光成像显示，未成熟的细胞，但不是更成熟的CMS，在注射后至少存活了一个月。这些数据和与更成熟的细胞的比较使我们得出结论，未成熟的耐缺氧的ESC-CMS可以在体外大量分离，并在注入心脏后形成移植物，可能介导梗死后整体和局部心肌收缩功能的长期恢复。 另外，我们合作表明胚胎干细胞来源的心肌细胞(ESCs)经常表现出节律失常的兴奋。利用钙离子成像和膜片钳技术，我们研究了晚期小鼠胚胎干细胞自发节律动作电位(AP)的产生条件。ESCs的肌浆网(SR)产生自发的、节律性的、小波样的局部钙释放(LCRs)(被兰尼定、丁卡因或thapsigargin抑制)。L型钙电流(I(Cal))引起整体钙释放(CICR)，耗尽Ca(2+)含量SR，使LCR振荡器重置相位。在延迟一段时间后，SR在整个细胞内产生高度同步的多个LCR的自发钙释放。LCR产生内向的Na(+)/Ca(2+)交换器(NCX)电流(在无Na(+)的溶液中不存在)，从而点燃下一个AP。干扰SR Ca(2+)循环(ryanodine、咖啡因、thapsigargin、clecopiazonic acid、BAPTA-AM)、NCX(无Na(+)溶液)或I(Cal)(硝苯地平)会导致节律性兴奋或自律性停止。用PKA特异性抑制剂PKI抑制cAMP/PKA信号，可减少SR Ca(2+)负荷，显著减少自发LCR(数目、大小和幅度)和节律性AP放电。相反，cAMP增强PKA信号会增加LCR(数量、大小、持续时间)，并将不规则跳动的ESCs转化为有节奏的“起搏样”细胞。磷蛋白抗体选择性激活肌浆网钙离子泵，也可增加肌浆网钙离子载量和LCR活性。我们认为，肌浆网钙离子负荷和自发节律性LCRs是由固有的cAMP/PKA活性驱动的。I(Cal)使多个LCR振荡器同步，导致强的、部分同步的舒张期Ca(2+)释放和Ncx电流。节律性ESC的自动化可以通过增强“偶联”因子来实现，例如cAMP/PKA信号，从而增强SR和肌膜之间的相互作用。

SUMMARY OF WORK This research area involves the study of embryonic stem and induced pluripotent stem cells prior to and during differentiation to cardiomyocytes. 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. These cells, which represent one promising source for cell replacement therapy in heart, are highly heterogeneous and show a variety of maturation states. In a recently published study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass and hypoxia resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction. Separately, we collaborated to show that embryonic stem cell-derived cardiocytes (ESCs)often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.