Enhanced cardiomyocyte differentiation and maturation using myocardial matrix

描述(申请人提供)：将hESC来源的心肌细胞转化为体外研究成人心脏病和体内治疗人类心肌梗死的一个重要瓶颈是其成熟和/或存活的能力。我们开发了一种将hESC定向到心脏谱系的方法，采用分阶段方案，包括原始条纹样群体的形成、心脏中胚层的诱导和指定以及心血管谱系的扩大，这可用于研究hESC的分化。细胞外基质(ECM)在体外可促进小鼠胚胎干细胞来源的早期心肌细胞分化，因此ECM可能是指导人类心脏干细胞分化的关键决定因素。然而，在体外和体内，有有限的研究评估了心肌基质在指导人类心脏干细胞分化方面的作用。我们最近开发了一种从成年猪心肌中提取心肌基质的方法，它可以在组织培养皿中溶解和吸收，作为体外细胞研究的涂层，并可以作为体内注射生物相容性材料。与传统的明胶基质细胞相比，hESC来源的心肌细胞表现出明显的肌原纤维生长和细胞-细胞连接(桥粒)成熟/分化的增加，从而显示了这种心肌基质的成熟潜力。传统明胶基质上的细胞表现出更明显的桥粒定位的胎儿表达模式。这项为期两年的计划的总体目标是确定心肌基质是否是体外和体内hESC来源的心肌细胞分化/成熟的重要决定因素。这些结果使我们提出假设，需要来自ECM的组织特异性信号来指导hESC来源的心肌细胞在体外和体内的存活、成熟/分化和功能。我们将通过对心肌基质在体外和体内心肌梗死大鼠模型中对hESC来源的心肌细胞的影响进行全面的分子、细胞和功能分析来实现这些目标。具体目标包括：(1)确定心肌基质是否是体外培养人胚胎干细胞来源的心肌细胞成熟、分化和功能的细胞决定因素。(2)将心肌基质联合hESC来源的心肌细胞注入心肌梗死大鼠体内，挽救/改变心肌梗死大鼠心力衰竭的死亡和进展。 与公共卫生相关：我们建议开发新的技术来指导干细胞分化，从而使人们能够更及时地了解干细胞生物学，并推动基于干细胞的心脏病新疗法的发展。干细胞来源的心肌细胞将是最初的焦点；然而，这些技术将适用于其他干细胞领域。

DESCRIPTION (provided by applicant): A significant bottleneck for translation of hESC derived cardiomyocytes to study adult-onset human cardiac diseases in vitro and for treatment of human myocardial infarction in vivo are their abilities to mature and/or survive. We have developed a method to direct hESC to the cardiac lineage, using a staged protocol that involves the formation of a primitive-streak-like population, the induction and specification of cardiac mesoderm and the expansion of the cardiovascular lineages, which can be exploited to study hESC differentiation. The extracellular matrix (ECM) could be a critical determinant of directing human cardiac stem cell differentiation because of its effects in promoting early mouse ESC-derived cardiomyocyte differentiation in vitro. However, there are limited studies, which have assessed the role of the myocardial matrix on directing human cardiac stem cell differentiation in vitro and in vivo. We recently developed a method to extract myocardial matrix from adult porcine cardiac muscle, which can be solublilized and absorbed on tissue culture dishes as a coating for in vitro cell-based studies and can be solubilized as an injectable biocompatible material in vivo. We show the maturation potential of this myocardial matrix by demonstrating that hESC derived cardiomyocytes displayed a significant increase in myofibrillar growth and cell-cell junction (desmosomal) maturation/ differentiation as opposed to cells plated on traditional gelatin substrate, which displayed a more punctate "fetal" expression pattern of desmosomal localization. The overall goal of this two-year proposal is to identify whether the myocardial matrix is an important determinant of hESC derived cardiomyocyte differentiation/maturation in vitro and in vivo. These results led us to the hypotheses that tissue-specific cues from the ECM are required to direct hESC-derived cardiac cell survival, maturation/differentiation and function in vitro and in vivo. We will achieve these goals by comprehensive molecular, cellular and functional analyses of the effects of the myocardial matrix on hESC-derived cardiomyocytes in vitro and in a rat model of myocardial infarction in vivo. Specific Aims include: (1) To determine whether the myocardial matrix is a cellular determinant of hESC derived cardiomyocyte maturation, differentiation and function in vitro. (2) To rescue/alter death and progression of myocardial-infarction induced heart failure in rats by injecting myocardial matrix in combination with hESC-derived cardiomyocytes into the infarcted myocardium in vivo. PUBLIC HEALTH RELEVANCE: We propose to develop new technologies to direct stem cell differentiation, thus enabling the more timely understanding of stem cell biology and the advancement of new stem cell based therapies for heart disease. Stem cell derived cardiomyocytes will be the initial focus; however, these technologies will be applicable to other stem cell fields.