Scalable bioprocess for cardiomyocyte generation from human embryonic stem cells

描述(申请人提供)：在美国和大多数发达国家，心肌梗死是发病率和死亡率的主要原因。心脏移植是重建受损心脏功能的有效治疗手段。然而，由于器官捐赠者的稀缺和与所需免疫抑制相关的并发症，这种方法的广泛应用受到严重限制。旨在取代心肌梗死的细胞疗法是非常可取的。胚胎干细胞可以作为修复受损心肌的取之不尽用之不竭的细胞材料。人类胚胎干细胞(HESCs)已被证明分化为有功能的心肌细胞。然而，基于干细胞的心脏修复疗法的临床实现将需要大量干细胞来源的心肌细胞的生产。目前的方法需要在静态培养(例如培养皿)中繁殖和分化ESCs，这对扩大规模具有挑战性。为此，在可扩展的搅拌悬浮生物反应器中，可以在严格控制的培养条件下产生大量细胞。我们发现，在生物反应器中培养的小鼠ESCs可以扩增数倍，并分化为多个谱系。我们假设，在搅拌悬浮容器中作为聚集体培养的hESCs也可以繁殖到高浓度。我们建议在搅拌生物反应器培养系统中培养hESCs，并确定在不影响其生存能力的情况下有利于hESCs生长的条件。此外，人胚胎干细胞主要分化为心肌细胞，而人胚胎干细胞是以聚集体或类胚体的形式组织起来的。因此，在生物反应器中，hESCs作为聚集体扩张之后，可能会切换到指示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hESCs的心脏潜能。由此产生的细胞将被鉴定为心肌细胞相关基因/蛋白的表达，并将在体外接受功能分析。这项研究将产生新的信息，有利于开发生物过程，以产生大量的hESC来源的心肌细胞，适用于心肌梗死的治疗。公共卫生相关性心肌梗死导致的心力衰竭在美国是一种主要的死亡原因，而旨在替换或恢复受损心肌的临床治疗缺乏。干细胞具有广泛的增殖能力和分化为功能性心肌细胞的能力，可作为再生心脏治疗的可再生细胞来源。本项目旨在加深我们对生物反应器培养对人胚胎干细胞生心潜能影响的了解，并推动生物工艺技术在临床上大量生产干细胞来源的心肌细胞。

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.