Derivation and Functional Characterization of Heart Cells from Human Embryonic an

人胚胎心脏细胞的衍生和功能表征

• 批准号: 7870865
• 负责人: Yibing Qyang
• 金额: $ 9.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7870865
• 关键词: Adult; Autologous; Biomimetics; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Therapy; Cell physiology; Cells; Derivation procedure; Development; ES Cell Line; Embryo; Endothelial Cells; Engineering; Engraftment; Environment; Evaluation; Genes; Goals; Heart; Heart failure; Human; Implant; In Vitro; Injection of therapeutic agent; Laboratories; Lead; Medicine; Modeling; Myosin Light Chains; Organoids; Patients; Pluripotent Stem Cells; Population; Production; Puromycin; Rattus; Reporter; Reporter Genes; Research; Research Institute; Resistance; Smooth Muscle Myocytes; Somatic Cell; Source; Stem cells; Suspension substance; Suspensions; System; Technology; Testing; Tissues; Transplantation; Treatment Efficacy; Ventricular; base; cell type; cellular engineering; embryonic stem cell; enhanced green fluorescent protein; heart cell; human embryonic stem cell; improved; in vivo; in vivo Model; induced pluripotent stem cell; injured; molecular marker; pressure; promoter; public health relevance; repaired; research study; stem

DESCRIPTION (provided by applicant): Induced pluripotent stem (iPS) cells are a type of pluripotent stem cells resembling embryonic stem (ES) cells and can be derived from adult somatic cells by forced expression of certain genes. Both human ES and iPS cells are able to give rise to all the fully differentiated tissues and may provide an ideal source for the production of heart cells for cell-based therapies. Although human iPS cell-derived cardiomyocytes are functionally comparable to those derived from human ES cells in vitro, their functionality has not been compared in a physiologically relevant context. This proposal will test the hypothesis that human iPS cell- derived heart cells are the functional equivalent of their human ES cell-derived counterparts and thus provide an autologous cell source for cardiac repair and cardiovascular research. A mixture of cell types enriched for heart cells can be isolated from human ES and iPS cells in culture, but in order to create functional heart tissue and obtain reproducible results in cell-based therapies, it is critically important to isolate pure populations of cardiac cells that will engraft and promote cardiac repair. Ventricular cardiomyocytes (VCM) are the major cell type injured in patient's hearts, resulting in reduced cardiac function. While implanted VCM may be the optimal mature cell type to readily couple electrically with host VCM, our recent discovery of ISL1+ cardiovascular progenitor cells (ICPC) may represent a better source for cell-based therapies as these are highly proliferative cells that have the capacity to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells, all of which are required for cardiac repair. By combining bacterial artificial chromosomal (BAC) recombineering technology with human ES cell engineering, the Qyang laboratory has established human ES and iPS cell lines that harbor enhanced green fluorescent protein (eGFP) and puromycin-resistance (Puro) dual reporters controlled by the endogenous myosin light chain 2V (MLC2V) promoter, which is VCM specific. Such a strategy has enabled the Qyang laboratory to isolate highly purified VCM based on both GFP expression and Puro-resistance. We will use this same technology to derive pure ICPC by establishing human ES and iPS cell lines that harbor GFP and Puro dual reporters controlled by the endogenous ISL1 promoter. As a proof of principle study to generate patient-specific VCM and ICPC for cell-based therapies, the Qyang laboratory proposes to isolate and characterize VCM and ICPC heart cells generated from human iPS cells using traditional 2D cultures and a recently established, physiologically relevant 3D "cardiac organoid chamber" system in vitro as well as cardiac repair models in vivo. For cardiac repair in vivo, we will employ a recently developed "cell sheet engineering" technology to establish engineered heart tissues. For all experiments, heart cells derived from NIH-approved human ES cells will be used as controls. PUBLIC HEALTH RELEVANCE: This research will lead to the production of pure, well-characterized ISL1+ cardiovascular progenitor cells and ventricular cardiomyocytes as an abundant renewable cell source for cardiac repair. Development of engineered cardiac tissues using human ES and iPS cell cultures will provide exciting opportunities for high- throughput, patient-specific evaluation of cardiac medicines and cell-based therapies for heart failure.

描述（由申请人提供）：诱导多能干细胞（iPS）是一种类似于胚胎干细胞（ES）的多能干细胞，可以通过强制表达某些基因从成体体细胞中衍生出来。人类ES和iPS细胞都能够产生所有完全分化的组织，并可能为基于细胞的治疗提供理想的心脏细胞来源。虽然人iPS细胞衍生的心肌细胞在功能上与体外衍生自人ES细胞的心肌细胞相当，但尚未在生理学相关背景下比较它们的功能。该提案将检验人iPS细胞衍生的心脏细胞是其人ES细胞衍生的对应物的功能等同物的假设，从而为心脏修复和心血管研究提供自体细胞来源。可以从培养的人ES和iPS细胞中分离出富含心脏细胞的细胞类型的混合物，但是为了产生功能性心脏组织并在基于细胞的治疗中获得可重复的结果，分离将移植并促进心脏修复的纯心脏细胞群至关重要。心室心肌细胞（VCM）是患者心脏中受损的主要细胞类型，导致心脏功能降低。虽然植入的VCM可能是容易与宿主VCM电偶联的最佳成熟细胞类型，但我们最近发现的ISL1+心血管祖细胞（ICPC）可能代表了基于细胞的治疗的更好来源，因为这些细胞是高度增殖的细胞，具有分化为心肌细胞，平滑肌细胞和内皮细胞的能力，所有这些细胞都是心脏修复所需的。通过将细菌人工染色体（BAC）重组工程技术与人ES细胞工程相结合，Qyang实验室建立了具有增强的绿色荧光蛋白（eGFP）和嘌呤霉素抗性（Puro）双报告基因的人ES和iPS细胞系，该双报告基因由内源性肌球蛋白轻链2V（MLC2V）启动子控制，该启动子是VCM特异性的。这种策略使Qyang实验室能够基于GFP表达和Puro抗性分离高度纯化的VCM。我们将使用相同的技术，通过建立人ES和iPS细胞系来获得纯的ICPC，这些细胞系含有由内源性ISL 1启动子控制的GFP和Puro双报告基因。 作为为细胞治疗产生患者特异性VCM和ICPC的原理证明研究，Qyang实验室提出使用传统的2D培养物和最近建立的生理相关的3D“心脏类器官室”系统在体外以及体内心脏修复模型分离和表征从人iPS细胞产生的VCM和ICPC心脏细胞。对于体内心脏修复，我们将采用最近开发的“细胞片工程”技术来建立工程心脏组织。对于所有实验，将使用来自NIH批准的人ES细胞的心脏细胞作为对照。 公共卫生关系：这项研究将导致产生纯的，良好表征的ISL1+心血管祖细胞和心室心肌细胞，作为心脏修复的丰富可再生细胞来源。使用人ES和iPS细胞培养物开发工程化心脏组织将为心脏药物和心力衰竭的基于细胞的疗法的高通量、患者特异性评价提供令人兴奋的机会。