Scalable bioprocess for cardiomyocyte generation from human embryonic stem cells

描述（由申请人提供）：心肌梗死是美国和大多数发达国家发病和死亡的主要原因。心脏移植是重建受损心脏功能的有效治疗手段。然而，由于器官供体的稀缺和与所需免疫抑制相关的并发症，这种方式的广泛应用受到严重限制。旨在替换梗塞心肌的细胞疗法是非常期望的。胚胎干细胞是修复受损心肌的取之不尽的细胞物质来源。人类胚胎干细胞（hESC）已被证明向功能性心肌细胞分化。尽管如此，基于干细胞的心脏修复疗法的临床实现将需要大量产生ESC衍生的心肌细胞。目前的方法需要ESC在静态培养物（例如培养皿）中的增殖和分化，这对于扩大规模具有挑战性。为此，可以在可扩展的搅拌悬浮生物反应器中在严格控制的培养条件下产生大量细胞。我们已经发现，在生物反应器中培养的小鼠ESC可以扩增数倍并分化为多个谱系。我们假设，在搅拌悬浮容器中培养的人胚胎干细胞聚集体也可以繁殖到高浓度。我们建议在搅拌的生物反应器培养系统中培养人胚胎干细胞，并确定有利于人胚胎干细胞生长而不影响其活力的条件。此外，hESC向心肌细胞的分化主要在hESC组织为聚集体或胚状体时进行。因此，在生物反应器中作为聚集体的hESC扩增之后，可以切换到指导细胞采用心肌细胞命运的条件。我们将评估在生物反应器中作为聚集体培养的hESC的心原性潜力。所得细胞将表征心肌细胞相关基因/蛋白的表达，并将进行体外功能测定。这项研究将产生新的信息，有利于开发生物过程，产生大量的hESC衍生的心肌细胞，适用于梗死的心脏治疗。公共卫生相关性心肌梗死引起的心力衰竭是美国的主要死因，缺乏旨在替换或恢复受损心肌的临床治疗。干细胞具有广泛的增殖能力和向功能性心肌细胞分化的能力，可作为再生心脏治疗的可再生细胞来源。该项目旨在进一步了解生物反应器培养对人胚胎干细胞的心源性潜力的影响，并推进用于生产临床相关数量的干细胞衍生心肌细胞的生物工艺技术。

DESCRIPTION (provided by applicant): Myocardial infarction is a major cause of morbidity and mortality in the United States and most developed countries. Heart transplantation is an effective therapeutic modality in reconstituting the function of damaged heart. However, widespread application of this modality is severely limited due to the scarcity of organ donors and complications associated with the required immunosuppression. Cell therapies aiming at replacing infarcted heart muscle are highly desirable. Embryonic stem cells (ESCs) can serve as an inexhaustible source of cellular material for repairing damaged myocardium. Human ESCs (hESCs) have been shown to differentiate towards functional cardiomyocytes. Nonetheless, clinical realization of stem cell-based therapies for heart repair will require the production of ESC-derived cardiomyocytes in large numbers. Current methodologies entail the propagation and differentiation of ESCs in static cultures (e.g. dishes) which are challenging to scale-up. To that end, large cell quantities can be generated under tightly controlled culture conditions in scalable stirred-suspension bioreactors. We have discovered that mouse ESCs cultivated in a bioreactor can be expanded several fold and differentiate to multiple lineages. We hypothesize that hESCs cultured as aggregates in stirred-suspension vessels can also propagate to high concentrations. We propose to culture hESCs in a stirred bioreactor culture system and determine conditions which favor the growth of hESCs without compromising their viability. Furthermore, hESC differentiation to cardiomyocytes is carried out mainly while hESCs are organized as aggregates or embryoid bodies. Therefore, expansion of hESCs as aggregates in the bioreactor may be followed by switching to conditions directing the cells to adopt a cardiomyocyte fate. We will evaluate the cardiogenic potential of hESCs cultured as aggregates in the bioreactor. The resulting cells will be characterized for the expression of cardiomyocyte-associated genes/proteins and will be subjected to functional assays in vitro. This study will yield new information benefiting the development of bioprocesses for the generation of large quantities of hESC-derived cardiomyocytes suitable for infracted heart therapies. PUBLIC HEALTH RELEVANCE Myocardial infarction-induced heart failure is a prevailing cause of death in the United States and clinical therapies aiming at replacing or restoring damaged heart muscle are lacking. Stem cells with their extensive proliferative capacity and their ability to differentiate towards functional cardiomyocytes may serve as a renewable cellular source for regenerative heart therapies. This project seeks to further our understanding of the effects of bioreactor culture on the cardiogenic potential of human embryonic stem cells and to advance the bioprocess technology for the production of stem cell-derived cardiomyocytes in clinically relevant quantities.