Engineering Human Heart Tissues with Polyploid Cardiomyocytes

人类诱导多能干细胞(HiPSCs)代表了一种潜在的无限的功能性来源 心肌细胞(hiPSC-CMS)，用于疾病建模、药物开发和再生治疗。在……里面 特别是，在微生理学(芯片上器官)系统中使用HiPSC-CM衍生的微组织成立 Promise是未来药物研究的支柱，也是一个提高我们对 人类心脏单基因和多基因疾病中的基因-表型关系。然而，一个主要的 阻碍HiPSC-CMS在这些应用中广泛使用的障碍是它们的不成熟特性，包括 细胞大小，缺乏T管，主要是糖酵解代谢，以及功能输出减少，仅举几例。 出生后CM成熟的一个重要方面-倍性增加-在很大程度上一直没有得到充分的研究。也就是说， 体外培养的HiPSC-CMS主要为单核和二倍体，而成人心肌 由~90%的多倍体不育系组成。因此，我们建议研究多倍体在HiPSC-CM中的潜在作用 成熟，特别是探索hPSC-CMS的工程多倍体是否可以赋予人类工程化 与对照组织相比，具有更强的功能和成熟度的心脏组织(HECT) 主要是二倍体不育系。我们的初步结果表明，稳定的hiPSC-CM多倍体在遗传或 药物导致HiPSC-CMS的大小和线粒体密度增加，以及收缩 HECTS的强度和传导速度。在建议的研究中，我们会进一步探讨管理人员的角色 HECTs结构、功能和代谢成熟过程中的多倍化及多倍体诱导 HiPSC-CMS的转录和表观遗传学变化。此外，使用一种新型的生物反应器，具有 动态控制将机械预载和后载应用于高强混凝土，我们将研究它们之间的关系 机械负荷的发育模拟机制与CM多倍体之间的关系。我们还将确定 如果多倍体在体外使hPSC-CMS对肥大刺激敏感，并保护hECTs免受氧化应激 体外和体内的缺血损伤。最后，我们将应用CRISPR/Cas9筛查方法来确定 已经多倍体的hiPSC-CMS的末端成熟的遗传诱导剂，并将进行额外的筛选 在二倍体和多倍体HiPSC-CMS中寻找能够促进CM细胞周期活性的候选有丝分裂原。 通过成功地完成这些研究，我们希望提高我们对生理作用的理解 多倍体在心脏发育中的作用并为将来工程化基因的翻译应用奠定基础 心脏组织在疾病建模、药物开发和心脏治疗中的应用。

Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of functional cardiomyocytes (hiPSC-CMs) for use in disease modeling, drug development, and regenerative therapies. In particular, use of hiPSC-CM-derived microtissues in microphysiological (“organ-on-chip”) systems holds promise as the future mainstay of pharmaceutical research and a platform to improve our understanding of genotype-phenotype relationships in mono- and polygenic diseases of the human heart. However, a major obstacle to wide-spread use of hiPSC-CMs in these applications are their immature properties including small cell size, lack of T-tubules, predominantly glycolytic metabolism, and reduced functional output, to name a few. One important aspect of postnatal CM maturation - increased ploidy - has been largely understudied. Namely, in vitro cultured hiPSC-CMs are predominantly mononuclear and diploid, while the adult human myocardium consists of ~90% polyploid CMs. We thus propose to investigate potential roles of polyploidy in hiPSC-CM maturation, and specifically, to explore if engineered polyploidy of hiPSC-CMs can endow human engineered cardiac tissues (hECTs) with increased functionality and maturity compared to control tissues made from primarily diploid CMs. Our preliminary results show that stable hiPSC-CM polyploidy induced genetically or pharmacologically results in increased size and mitochondrial density of hiPSC-CMs, as well as contractile strength and conduction velocity of hECTs. In the proposed studies, we will further examine roles of CM polyploidization in structural, functional, and metabolic maturation of hECTs and investigate polyploidy-induced transcriptomic and epigenetic changes in hiPSC-CMs. Furthermore, using a novel bioreactor with capacity to dynamically control applied mechanical preload and afterload to hECTs, we will investigate the relationships between developmentally-mimetic regimes of mechanical loading and CM polyploidy. We will also determine if polyploidy sensitizes hiPSC-CMs to hypertrophic stimuli in vitro and protects hECTs from oxidative stress in vitro and ischemic damage in vivo. Finally, we will apply CRISPR/Cas9 screening methodologies to identify genetic inducers of terminal maturation in already polyploidy hiPSC-CMs and will perform additional screens in both diploid and polyploid hiPSC-CMs to identify candidate mitogens that can promote CM cell cycle activity. By successfully completing these studies, we expect to improve our understanding of physiological roles of polyploidy in cardiac development and to establish the foundation for the future translational uses of engineered cardiac tissues in disease modeling, drug development, and cardiac therapies.