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方式。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。细胞疗法旨在取代梗死心肌是非常可取的。胚胎干细胞（ESCs）可以作为修复受损心肌的细胞材料的取之不尽的来源。人类ESCs （hESCs）已被证明向功能性心肌细胞分化。尽管如此，临床实现以干细胞为基础的心脏修复疗法将需要大量产生esc来源的心肌细胞。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，在严格控制的培养条件下，可以产生大量细胞。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成多个谱系。我们假设在搅拌悬浮血管中以聚集体形式培养的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，并确定有利于hESCs生长而不影响其生存能力的条件。此外，hESC向心肌细胞的分化主要发生在hESC以聚集体或胚状体组织的过程中。因此，hESCs作为聚集体在生物反应器中扩增之后，可能会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hESCs的心源性潜能。所得到的细胞将被表征为心肌细胞相关基因/蛋白质的表达，并将在体外进行功能测定。这项研究将产生新的信息，有利于生物过程的发展，以产生大量适合梗塞心脏治疗的hesc来源的心肌细胞。心肌梗死引起的心力衰竭是美国主要的死亡原因，目前缺乏旨在替代或恢复受损心肌的临床治疗方法。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类胚胎干细胞致心潜能的影响，并推进生物工艺技术，以生产临床相关数量的干细胞衍生心肌细胞。

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.