Maturation of Normal and Disease-specific Human Stem Cell-derived Cardiomyocytes

描述(申请人提供)：心血管疾病仍然是美国的主要死亡原因。干细胞和祖细胞来源的心肌细胞(SPC-CMS)在心肌修复方面有着巨大的前景。最近在将不同类型的体细胞重新编程为诱导多能干细胞(IPSCs)方面的进展为开发针对患者的、基于细胞的治疗方法打开了大门。然而，大多数SPC-CMS表现出异质性和不成熟的电生理(EP)表型，且自动化程度不可控。在进行安全的临床应用之前，需要明确SPC或IPSCs来源的心肌细胞(CM)的分化特征和阶段。此外，IPSC技术使从已知遗传性疾病患者中创建干细胞系成为可能，这已被用于研究疾病的发病机制和设计治疗方法。在这项提议中，我们计划通过与原始CMS共培养内皮细胞(ECs)来创造诱导SPC-或IPSC-CMS成熟的方法。我们已经证实内皮细胞通过内皮素-1途径促进原始CMS的Na+通道表达。此外，我们还建立了3个可分化为CMS的人IPSC系。我们进一步产生了第一个心脏疾病特异性IPSC系，产生了具有致心律失常性右室发育不良(ARVD)病理特征的CMS。此外，我们通过慢病毒载体建立了人类胚胎干细胞(HESC)和具有嘌呤霉素抗性的IPSC系，以便能够快速分离出95%纯度的IPSC-或hESC-CMS，用于遗传和EP分析。最后，为了明确这些正常和患病的IPSC-CMS的性质和命运，我们已经开始利用微阵列技术生成人类胚胎和成人心脏6个区域的广泛遗传图谱。利用来自基因阵列的生物信息学分析，我们将创建一个全面的数据集，称为发育心脏矩阵，以便我们可以将IPSC-或SPC-CMS的特征与人类心脏发育不同阶段的CM亚型的特征进行比较，并确定IPSC-CM的分化阶段。如果该项目完成，将提供遗传特征图谱，以开发诱导原始IPSC-CMS向合适的CM亚型成熟的方法，以实现安全的基于细胞的治疗。最重要的是，IPSC技术可以建立人类ARVD的体外疾病模型，为临床治疗奠定基础。 公共卫生相关性：干细胞和祖细胞来源的心肌细胞(SPC-CMS)对心肌修复具有很大的前景。最近发明的将体细胞重新编程为诱导多能干细胞(IPSCs)的技术使基于患者特定细胞的治疗成为可能。此外，IPSC技术能够创建心脏疾病的特定品系，作为人类体外疾病模型，用于开发治疗干预措施。此外，大多数SPC-CMS是不成熟的。我们将建立体外诱导原始SPC-CMS成熟的方法。此外，我们将通过微阵列技术生成广泛的胚胎和成人心脏发育基因矩阵，以适当地对各种IPSC-或SPC-CMS进行分类，并指导它们的分化和成熟，为心血管疾病的安全细胞治疗奠定基础。

DESCRIPTION (provided by applicant): Cardiovascular diseases remain the major cause of death in the US. Stem and progenitor cell- derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent progress in cellular reprogramming of various somatic cell types into induced pluripotent stem cells (iPSCs) opened the door for developing patient-specific, cell-based therapies. However, most SPC-CMs displayed heterogeneous and immature electrophysiological (EP) phenotypes with uncontrollable automaticity. The characteristics and stages of differentiation of cardiomyocytes (CMs) derived from SPCs or iPSCs need to be clearly defined before a safe clinical application could be performed. Furthermore, iPSC technology enables the creation of stem cell lines from patients with known genetic diseases, which has been used to study disease pathogenesis and to design therapy. In this proposal, we plan to create methods of inducing maturation of SPC- or iPSC-CMs by co-culturing endothelial cells (ECs) with these primitive CMs. We have identified that ECs promote Na+ channel expression of primitive CMs via endothelin-1 pathway. Also, we have created 3 human iPSC lines that could differentiate to CMs. We further generated the first cardiac disease-specific iPSC line that produced CMs with pathological signatures of arrhythmogenic right ventricular dysplasia (ARVD). Furthermore, we created human embryonic stem cell (hESC) and iPSC lines with Puromycin resistance by lentiviral vectors to allow rapid isolation of >95% pure iPSC- or hESC-CMs for genetic and EP analysis. Finally, in order to clearly identify the nature and fate of these normal and diseased iPSC-CMs, we have started to generate an extensive genetic map of 6 regions of embryonic and adult human hearts by micro- array technologies. Using bioinformatic analysis of data from genetic arrays, we will create a comprehensive dataset, termed Developmental HeartMatrix, so that we could compare characteristics of iPSC- or SPC-CMs to those of CM subtypes from various stages of human hearts during development, as well as to determine the stages of iPSC-CM differentiation. This project, if completed, will provide the genetic signature maps to develop methods of inducing maturation of primitive iPSC-CMs toward appropriate CM subtypes for a safe cell-based therapy. Most importantly, a human ARVD in vitro disease model could be generated by iPSC technology as the foundation for developing clinical therapy. PUBLIC HEALTH RELEVANCE: Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repair. Recent invention of technologies in reprogramming somatic cells to induced pluripotent stem cells (iPSCs) make patient-specific cell-based therapy possible. In addition, iPSC technology enables the creation of cardiac disease-specific lines as human in vitro disease models for developing therapeutic interventions. Also, most SPC- CMs are immature. We will create in vitro method to induce maturation of primitive SPC- CMs. Moreover, we will generate an extensive developmental gene matrix of embryonic and adult human hearts by micro-array technologies to properly classify various iPSC- or SPC-CMs as well as to guide their differentiation and maturation for a safe cell-based therapy in cardiovascular diseases.