Derivation and Functional Characterization of Heart Cells from Human Embryonic an

描述(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细胞)的多能干细胞，可通过某些基因的强制表达从成体细胞分化而来。人类ES细胞和iPS细胞都能分化为完全分化的组织，可能为细胞治疗提供理想的心脏细胞来源。尽管体外培养的人iPS细胞来源的心肌细胞与人ES细胞来源的心肌细胞在功能上具有可比性，但它们的功能尚未在生理学相关的背景下进行比较。这一提议将检验这样一种假设，即人诱导性多能干细胞来源的心脏细胞在功能上等同于人类胚胎干细胞来源的心脏细胞，从而为心脏修复和心血管研究提供自体细胞来源。在培养中可以从人类ES和iPS细胞中分离出富含心脏细胞的多种细胞类型的混合物，但为了创建功能正常的心脏组织并在基于细胞的治疗中获得可重复性的结果，分离将植入并促进心脏修复的纯心肌细胞群至关重要。室性心肌细胞(VCM)是患者心脏损伤的主要细胞类型，导致心功能下降。虽然移植的VCM可能是最容易与宿主VCM电偶联的成熟细胞类型，但我们最近发现的IsL1心血管前体细胞(ICPC)可能是基于细胞治疗的更好来源，因为这些细胞具有高度增殖的能力，具有分化为心肌细胞、平滑肌细胞和内皮细胞的能力，所有这些都是心脏修复所必需的。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，曲阳实验室建立了由内源性肌球蛋白轻链2V(MLC2V)启动子控制的增强型绿色荧光蛋白(EGFP)和嘌呤霉素抗性(Puro)双报告基因的人ES和iPS细胞系。这样的策略使启阳实验室能够根据GFP表达和Puro抗性分离出高纯度的VCM。我们将使用同样的技术，通过建立含有内源性Isl1启动子控制的GFP和Puro双重报告基因的人ES和iPS细胞系来获得纯ICPC。作为为基于细胞的治疗生成患者特定的VCM和ICPC的原理研究的证据，曲阳实验室提议使用传统的2D培养和最近建立的生理相关的体外和体内心脏修复模型分离和鉴定从人类iPS细胞生成的VCM和ICPC心脏细胞。对于活体心脏修复，我们将使用最近开发的“细胞片工程”技术来建立工程化的心脏组织。在所有实验中，从NIH批准的人类ES细胞中提取的心脏细胞将作为对照。 公共卫生相关性：这项研究将导致生产纯净的、特征良好的IsL1心血管前体细胞和心室肌细胞，作为心脏修复的丰富可再生细胞来源。使用人类ES和iPS细胞培养的工程化心脏组织的开发将为高通量、针对患者的心脏药物评估和基于细胞的心力衰竭治疗提供令人兴奋的机会。

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.