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。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。这些细胞是心脏细胞替代疗法的一个有前途的来源，它们是高度异质的，并显示出各种成熟状态。在最近发表的一项研究中，我们采用了一种ESC克隆系，该克隆系含有心脏限制性ncx 1启动子驱动的嘌呤霉素抗性盒，以及大量培养系统，以分离显示非常不成熟CM特征的ESC-CM。细胞显示增殖、CM限制性标志物、减少的线粒体质量和耐缺氧性的特性。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CM，存活至少一个月后注射。这些数据和与更成熟细胞的比较使我们得出结论，未成熟的耐缺氧ESC-CM可以在体外大量分离，并在注射到心脏后形成移植物，其可以介导梗死后全局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衍生的心肌细胞（ESC）经常表现出节律性兴奋。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性动作电位（AP）产生的条件。ESCs的肌浆网（SR）产生自发的、有节奏的、小波样局部Ca（2+）释放（LCR）（受兰尼碱、丁卡因或毒胡萝卜素抑制）。L型Ca（2+）电流（I（CaL））引起整体Ca（2+）释放（CICR），耗尽SR中的Ca（2+）含量，使LCR振荡器的相位复位。在延迟之后，SR然后在整个细胞中产生多个LCR的高度同步的自发Ca（2+）释放。LCR产生向内的Na（+）/Ca（2+）交换器（NCX）电流（在无Na（+）溶液中不存在），其点燃下一个AP。干扰SR Ca（2+）循环（ryanodine、咖啡因、毒胡萝卜素、cyclopiazonic acid、BAPTA-AM）、NCX（无Na（+）溶液）或I（CaL）（硝苯地平）可导致节律性兴奋障碍或自律性停止。通过特异性PKA抑制剂PKI抑制cAMP/PKA信号传导，减少SR Ca（2+）负荷，显著减少自发LCR（数量、大小和幅度）和节律性AP放电。相比之下，通过cAMP增强PKA信号传导增加LCR（数量、大小、持续时间）并将不规则跳动的ESC转化为有节奏的“起搏器样”细胞。受磷蛋白抗体选择性激活SR Ca（2+）泵也可增加SR Ca（2+）负荷和LCR活性。我们的结论是，SR Ca（2+）负荷和自发节律LCR是由固有的cAMP/PKA活性驱动的。I（CaL）是多个LCR振荡器，导致强的、部分同步的舒张期Ca（2+）释放和NCX电流。有节律的ESC自律性可以通过增强“偶联”因子来实现，例如cAMP/PKA信号传导，其增强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.