Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells
Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells
复制标题
DOI：
10.1089/scd.2012.0490
复制
发表时间：
2013-07-01
影响因子：
4
通讯作者：
Laflamme, Michael A.
中科院分区：
文献类型：
--
作者：
Lundy, Scott D.;Zhu, Wei-Zhong;Laflamme, Michael A.
尽管临床前研究证实了将人多能干细胞来源的心肌细胞(PSC-CMS)移植到受损心肌中的功能益处，但这些未成熟细胞采用更成体样心肌细胞(CM)表型的能力仍然不确定。为了解决这个问题，我们测试了一种假设，即延长人胚胎干细胞(HESC)和人诱导多能干细胞(HiPSC)来源的CMS的体外培养时间将导致它们的结构和收缩特性成熟到更像成人的表型。与早期(体外分化培养20-40天后的PSC-CMS)相比，晚期hESC-CMS和HiPSC-CMS(80-120天)在形态上有很大的不同，包括细胞大小和各向异性增加，肌原纤维密度和排列增加，明场显微镜下可见肌节，多核CMS的比例增加10倍。超微结构分析证实了肌原纤维密度、排列和形态的改善。我们测量了晚期hESC-CMS和HiPSC-CMS的收缩性能，发现与早期细胞相比，收缩幅度增加了一倍，收缩动力学减慢。然后，我们检查了这些成熟的CMS的钙处理特性的变化，发现钙释放和再摄取速率增加，而最大幅度没有变化。最后，我们在hESC-CMS中进行了电生理评估，发现晚期心肌细胞具有超极化的最大舒张期电位，动作电位幅度增加，以及更快的上升速度。为了将这些功能变化与基因表达相关联，我们进行了定量聚合酶链式反应，发现在晚期hESC-CMS和HiPSC-CMS中，关键的心脏结构标志物，包括β-肌球蛋白重链和连接蛋白-43都有很强的诱导作用。这些发现表明，PSC-CMS能够缓慢成熟，更接近于成年CMS的表型，最终可能具有再生丢失心肌的潜力，具有强大的新生力量产生组织。
Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including beta-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.