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

基本信息

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
项目类别：
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

项目摘要

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死亡的主要原因。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广阔的前景。将各种体细胞类型重新编程为诱导多能干细胞 (iPSC) 的最新进展为开发针对患者的细胞疗法打开了大门。然而，大多数 SPC-CM 表现出异质且不成熟的电生理 (EP) 表型，且具有不可控的自动性。在进行安全的临床应用之前，需要明确定义源自 SPC 或 iPSC 的心肌细胞 (CM) 的特征和分化阶段。此外，iPSC 技术能够从患有已知遗传疾病的患者身上创建干细胞系，该干细胞系已用于研究疾病发病机制和设计治疗方法。在本提案中，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。我们发现 EC 通过内皮素 1 途径促进原始 CM 的 Na+ 通道表达。此外，我们还创建了 3 个可以分化为 CM 的人类 iPSC 系。我们进一步构建了第一个心脏病特异性 iPSC 系，该系产生具有致心律失常性右心室发育不良 (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，以便快速分离 >95% 纯的 iPSC-或 hESC-CM，用于遗传和 EP 分析。最后，为了清楚地识别这些正常和患病 iPSC-CM 的性质和命运，我们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。通过对基因阵列数据进行生物信息分析，我们将创建一个名为“发育心脏矩阵”的综合数据集，以便我们可以将 iPSC 或 SPC-CM 的特征与人类心脏发育过程中各个阶段的 CM 亚型的特征进行比较，以及确定 iPSC-CM 分化的阶段。该项目一旦完成，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，以实现安全的细胞治疗。最重要的是，可以通过 iPSC 技术生成人类 ARVD 体外疾病模型，作为开发临床治疗的基础。公共健康相关性：干细胞和祖细胞来源的心肌细胞 (SPC-CM) 在心肌修复方面具有巨大的前景。最近发明的将体细胞重编程为诱导多能干细胞 (iPSC) 的技术使针对患者的特定细胞治疗成为可能。此外，iPSC 技术还能够创建心脏病特异性细胞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-CM 还不成熟。我们将创建体外方法来诱导原始 SPC-CM 的成熟。此外，我们将通过微阵列技术生成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，以实现心血管疾病的安全细胞治疗。