Embryonic Stem Cell Derived Cardiac Myocytes

本研究领域涉及胚胎干细胞和诱导多能干细胞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。这些细胞是心脏细胞替代疗法的一个有希望的来源，它们是高度异质性的，并表现出各种成熟状态。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耐药盒的ESC克隆系和大众培养系统分离出具有非常不成熟CMs特征的ESC-CMs。细胞表现出增殖、cm限制性标记物、线粒体质量减少和耐缺氧的特性。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，而不是更成熟的CMs，在注射后存活了至少一个月。这些数据以及与更成熟细胞的比较使我们得出结论，未成熟的抗缺氧ESC-CMs可以在体外大量分离，并在注射到心脏后形成移植物，可能介导梗死后整体和局部心肌收缩功能的长期恢复。

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. More recent data have been generated from targeted mouse ESCs which were used to generate inducible knockouts of the ryanodine receptor. These data have been recently published.