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

描述（由申请人提供）：心血管疾病仍然是美国的主要死亡原因。干细胞和祖细胞衍生的心肌细胞（SPC-CM）在心肌修复方面具有巨大的潜力。最近在将各种体细胞类型细胞重编程为诱导多能干细胞（iPSC）方面的进展为开发患者特异性的基于细胞的疗法打开了大门。然而，大多数SPC-CM显示异质性和不成熟的电生理（EP）表型与不可控制的自律性。来源于SPC或iPSC的心肌细胞（CM）的分化特征和阶段需要在安全的临床应用之前明确定义。此外，iPSC技术能够从已知遗传疾病的患者中创建干细胞系，该干细胞系已用于研究疾病发病机制和设计治疗方法。在该提议中，我们计划通过将内皮细胞（EC）与这些原始CM共培养来创建诱导SPC-或iPSC-CM成熟的方法。我们已经确定，内皮细胞通过内皮素-1途径促进原始CM的Na+通道表达。此外，我们已经创建了3个可以分化为CM的人iPSC系。我们进一步产生了第一个心脏疾病特异性iPSC系，其产生具有致瘤性右心室发育不良（ARVD）病理特征的CM。此外，我们通过慢病毒载体创建了具有嘌呤霉素抗性的人胚胎干细胞（hESC）和iPSC系，以允许快速分离>95%纯的iPSC-或hESC-CM用于遗传和EP分析。最后，为了清楚地鉴定这些正常和患病iPSC-CM的性质和命运，我们已经开始通过微阵列技术产生胚胎和成人心脏的6个区域的广泛遗传图谱。使用来自基因阵列的数据的生物信息学分析，我们将创建一个称为发育心脏矩阵的综合数据集，以便我们可以将iPSC或SPC-CM的特征与发育过程中人类心脏各个阶段的CM亚型的特征进行比较，以及确定iPSC-CM分化的阶段。该项目如果完成，将提供遗传特征图谱，以开发诱导原始iPSC-CM成熟为适当CM亚型的方法，用于安全的基于细胞的治疗。最重要的是，iPSC技术可以产生人类ARVD体外疾病模型，作为开发临床治疗的基础。 公共卫生相关性：干细胞和祖细胞衍生的心肌细胞（SPC-CM）在心肌修复方面有很大的前景。最近发明的将体细胞重编程为诱导多能干细胞（iPSC）的技术使得基于患者特异性细胞的治疗成为可能。此外，iPSC技术能够创建心脏疾病特异性细胞系，作为开发治疗干预措施的人类体外疾病模型。而且，大多数SPC-CM还不成熟.建立体外诱导原始SPC-CM成熟的方法.此外，我们将通过微阵列技术产生胚胎和成人心脏的广泛发育基因矩阵，以正确分类各种iPSC或SPC-CM，并指导它们的分化和成熟，用于心血管疾病的安全细胞治疗。

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.