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

正常和疾病特异性人类干细胞来源的心肌细胞的成熟

• 批准号: 8118810
• 负责人: HUEI-SHENG Vincent CHEN
• 金额: $ 47.75万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8118810
• 关键词: Aborted Fetus; Accounting; Adult; Affect; Age; Aging; Algorithms; Animal Model; Arrhythmogenic Right Ventricular Dysplasia; Back; Bioinformatics; Biological Models; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cause of Death; Cell Line; Cell Proliferation; Cell Therapy; Cell Transplantation; Cells; Characteristics; Chromosome Mapping; Clinical; Clinical Pathology; Coculture Techniques; Cues; Data Analyses; Data Set; Dermal; Desmosomes; Development; Developmental Gene; Disease; Disease model; Elderly; Electrodes; Embryo; Embryonic Heart; Endothelial Cells; Endothelin A Receptor; Endothelin-1; Engineering; Environment; Fetal Heart; Fibroblasts; Foundations; Future; Genes; Genetic; Genetic Transcription; Germ Layers; Goals; Grouping; Heart; Heart Diseases; Hereditary Disease; Human; Implant; In Vitro; Investigation; Ion Channel; Knowledge; Laboratories; Lentivirus Vector; Maps; Measures; Medical center; Messenger RNA; Methods; MicroRNAs; Microarray Analysis; Molecular; Mutation; Myocardial; Nature; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Population; Pregnancy; Process; Puromycin; Replacement Therapy; Resistance; Sampling; Signal Pathway; Signal Transduction; Sodium; Somatic Cell; Source; Staging; Stem cells; Subfamily lentivirinae; System; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissue Sample; Tissues; Translating; Transplantation; Viral Vector; Western World; Work; base; cell type; clinical application; developmental genetics; fetal; genetic profiling; human disease; human embryonic stem cell; human stem cells; improved; in vitro Model; in vivo; induced pluripotent stem cell; method development; mutant; plakophilin 2; protein protein interaction; public health relevance; repaired; stem; stem cell technology; therapy design; transcription factor

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.

描述（由申请人提供）：心血管疾病仍然是美国人死亡的主要原因。干细胞和祖细胞来源的心肌细胞（SPC-CMs）在心肌修复中具有很大的前景。多种体细胞重编程成诱导多能干细胞（iPSCs）的最新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，具有不可控的自动性。SPCs或iPSCs衍生的心肌细胞（CMs）分化的特征和阶段需要明确界定，才能进行安全的临床应用。此外，iPSC技术能够从患有已知遗传疾病的患者身上创造干细胞系，这已被用于研究疾病发病机制和设计治疗方法。在这项提议中，我们计划通过内皮细胞（ECs）与这些原始CMs共培养来创建诱导SPC-或iPSC-CMs成熟的方法。我们已经发现ECs通过内皮素-1途径促进原始CMs的Na+通道表达。此外，我们已经创建了3个人类iPSC系，可以分化为CMs。我们进一步生成了第一个心脏病特异性iPSC细胞系，该细胞系产生具有心律失常性右心室发育不良（ARVD）病理特征的CMs。此外，我们利用慢病毒载体构建了具有Puromycin抗性的人胚胎干细胞（hESC）和iPSC细胞系，以便快速分离纯度为95%的iPSC-或hESC- cms用于遗传和EP分析。最后，为了清楚地确定这些正常和患病iPSC-CMs的性质和命运，我们已经开始通过微阵列技术生成胚胎和成人心脏6个区域的广泛遗传图谱。利用对基因阵列数据的生物信息学分析，我们将创建一个全面的数据集，称为发育心脏矩阵，这样我们就可以比较iPSC-或SPC-CMs与人类心脏发育过程中不同阶段的CM亚型的特征，并确定iPSC-CM分化的阶段。该项目如果完成，将提供遗传标记图谱，以开发诱导原始iPSC-CMs向适当的CM亚型成熟的方法，用于安全的细胞治疗。最重要的是，iPSC技术可以生成人类ARVD体外疾病模型，为开发临床治疗奠定基础。