Multipotent heart cell progenitor lineages in cardiac development and disease

描述(由申请人提供)：确定一种最佳的细胞类型来驱动强大的心肌生成对于改进基于细胞的治疗和开发针对心力衰竭的人类心肌细胞模型至关重要。钱实验室最近的研究允许从小鼠和人类胚胎干细胞(ES)和可诱导的多潜能干细胞(IPS)中鉴定、纯化和更新多潜能的Islet-1心血管祖细胞。利用一种新颖的双色报告系统，从ES细胞中分离出一组完全承诺、自我更新的心肌祖细胞(CMPS)，并将其用于通过组织工程生成心肌薄膜。Melton实验室最近开发了仅使用两个基因和一种化学试剂即可从小鼠和人类皮肤成纤维细胞高效生成iPS细胞的新方案，而Chien/Wu实验室则有效地从iPS细胞来源纯化了心脏祖细胞，证明了从单个患者的iPS来源生成自体人类心血管祖细胞用于基于细胞的治疗和/或生成基于人类心肌细胞的心脏病模型的可行性，这将允许使用大量的遗传工具来确定导致心肌功能障碍的分子途径。这项建议整合了三个领先实验室的独特专业知识、试剂和方案：发育和疾病中的心脏干细胞生物学(Chien)；人类ES和iPS技术(Melton)；以及心肌组织工程(Kit Parker)。因此，这两个拟议项目的具体目标是：项目1：多潜能的胰岛-1/NKX2.5心脏细胞前体细胞和承诺的生心肌祖细胞(CMP)谱系的形成、更新和分化：1)鉴定胰岛-1/NKX2.5心脏祖细胞家族的承诺心肌祖细胞(CMPS)；2)从Islet-1/NKX2.5心脏祖细胞家族的CMPS中产生功能性心肌条；3)鉴定驱动胰岛-1/NKX2.5家族(Wnt/Beta catenin，Creb312等)的致死性心肌祖细胞形成、更新、存活和分化的途径；4)鉴定、纯化和鉴定胰岛-1/NKX2.5心脏祖细胞家族的传导系统祖细胞；项目2：人类心脏疾病的小鼠和人心脏祖细胞模型的建立：1)从人ES和iPS细胞来源的人多能胰岛-1/NKX2.5祖细胞及其心肌细胞系的纯化和鉴定；2)从多能胰岛-1/NKX2.5前体细胞及其来源于ES和iPS细胞的心肌细胞系中培养出具有功能的小鼠和人心肌条；3)从含有心肌病突变的ES细胞和iPS细胞中获得鼠和人心肌条并进行鉴定。。

DESCRIPTION (provided by applicant): The identification of an optimal cell type to drive robust cardiac myogenesis is critical for improving cell-based therapy and to develop human cardiomyocyte models for heart failure. Recent studies in the Chien lab have allowed the identification, purification, and renewal of multipotent Islet-1 cardiovascular progenitors from both murine and human embryonic stem (ES) cells and inducible pluripotent stem cells (iPS). Utilizing a novel two color reporter system, a unique subset of completely committed, self-renewing cardiomyogenic progenitors (CMPs) have been isolated from ES cells, and used to generate thin films of cardiac muscle via tissue engineering. The Melton lab has recently developed novel protocols for the high efficiency generation of iPS cells from murine and human skin fibroblasts using just two genes and a chemical agent, while the Chien/Wu labs have effectively purified heart progenitors from iPS cell sources, documenting the feasibility for generating autologous human cardiovascular progenitors from iPS sources from an individual patient for cell based therapy and/or for the generation of human cardiac muscle cell-based models of cardiac disease, which should allow the use of an arsenal of genetic tools to identify the molecular pathways for cardiac muscle dysfunction. This proposal integrates unique expertise, reagents, and protocols from three leading laboratories: heart stem cell biology in development and disease (Chien); human ES and iPS technology (Melton); and cardiac muscle tissue engineering (Kit Parker). Accordingly, the Specific Aims of the two proposed projects are: Project 1: Multi-potent Islet-1/Nkx2.5 heart cell progenitors and the formation, renewal, and differentiation of committed cardiomyogenic progenitor (CMP) lineages: 1) Characterization of committed cardiomyogenic progenitors(CMPs) of the Islet-1/Nkx2.5 family of heart progenitors; 2) Generation of functional heart muscle strips from CMPs of the Islet-1/Nkx2.5 family of heart progenitors; 3) Identification of Pathways which drive the formation, renewal, survival, and differentiation of committed cardiomyogenic progenitors of the Islet-1/Nkx2.5 family (Wnt/Beta Catenin, Creb312, etc.); 4) Identification, purification, and characterization of the conduction system progenitors of the Islet-1/Nkx2.5 family of heart progenitors; Project 2: Generation of mouse and human heart progenitor derived models of human cardiac diseases: 1) Purification and characterization of human multipotent Islet-1/Nkx2.5 progenitors and their cardiomyogenic lineages derived from human ES and iPS cells; 2) Generation and characterization of functional mouse and human heart muscle strips from multipotent Islet-1/Nkx2.5 progenitors and their cardiomyogenic lineages derived from ES and iPS cells; 3) Generation and characterization of murine and human heart muscle strips from ES and iPS cells that harbor cardiomyopathic mutations. .