Bioprocess for cardiac cell generation from human induced pluripotent 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理想的。最近在患者特异性人类诱导多能干细胞(HiPSCs)的生成方面取得的进展引发了人们的希望，即这些细胞可以作为修复受损心肌的取之不尽的细胞材料。与人类胚胎干细胞(HESCs)一样，HiPSCs已被证明利用类胚体(EB)培养和补充血清的培养液向有功能的心肌细胞分化。然而，基于干细胞的心脏修复疗法的临床实现将需要(I)使用不含动物成分(如血清)的定向分化方法来生产hPSC来源的心肌细胞，以及(Ii)大量生产。我们假设HiPSCs可以定向为具有生理相关因子的心肌细胞样细胞，参与胚胎心脏的发育。为此，我们建议建立一种在静态培养(如培养皿)中将hPSCs定向分化为干细胞的方法。然而，IPSCs在菜肴中的繁殖和分化是规模化的挑战。我们发现，在生物反应器中培养的人胚胎干细胞可以扩增数倍，并分化为多个谱系。然后，我们的第二个假设是，在搅拌悬浮生物反应器中培养在微载体上的HiPSCs也可以培养到高浓度。我们建议在搅拌生物反应器培养系统中培养HiPSCs，并确定在不影响其多能性和活性的情况下有利于HiPSCs生长的条件。此外，干细胞分化为心肌细胞通常是在EB培养中进行的。鉴于并不是EBS内的所有细胞都暴露在心源性因子中，这一过程很难控制，并且具有效率低的特点。因此，在微载体上扩增HiPSCs之后，可能会切换到指示细胞采用心肌细胞命运的条件。我们将评估在微载体生物反应器中培养的HiPSCs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。最后，将使用小鼠梗死心脏模型来评估体内hPSC来源的心脏细胞的功能属性。这项研究将为开发大量适合心脏治疗的HiPSC来源的心肌细胞的生物过程提供新的信息。 公共卫生相关性：在美国，心肌梗死引起的心力衰竭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。患者特异性的人类诱导多能干细胞可以广泛增殖并分化为有功能的心肌细胞，可能成为再生心脏治疗的可再生细胞来源。该项目旨在加深我们对人类诱导的多能干细胞在静态和可扩展培养系统中的生心潜能的理解，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 infracted heart muscle are highly desirable. Recent advances in the generation of patient-specific human induced pluripotent stem cells (hiPSCs) have sparked hopes that these cells can serve as an inexhaustible source of cellular material for repairing damaged myocardium. Like human embryonic stem cells (hESCs), hiPSCs have been shown to differentiate towards functional cardiomyocytes utilizing embryoid body (EB) culture and serum-supplemented media. Nonetheless, clinical realization of stem cell-based therapies for heart repair will require the production of hiPSC-derived cardiomyocytes (i) using directed differentiation methods free of animal components (e.g. serum), and (ii) in large numbers. We hypothesize that hiPSCs can be directed towards cardiomyocyte-like cells with physiologically relevant factors known to participate in embryonic heart development. To that end, we propose to establish a method for the directed differentiation of hiPSCs to stem cardiac cells in static cultures (e.g. dishes). However, the propagation and differentiation of iPSCs in dishes are challenging to scale-up. We have discovered that hESCs cultivated in a bioreactor can be expanded several fold and differentiate to multiple lineages. Then, our second hypothesis is that hiPSCs cultured on microcarriers in stirred-suspension bioreactors can also be propagated to high concentrations. We propose to culture hiPSCs in a stirred bioreactor culture system and determine conditions which favor the growth of hiPSCs without compromising their pluripotency and viability. Furthermore, stem cell differentiation to cardiac cells is typically carried out in EB cultures. Given that not all cells within EBs are exposed to cardiogenic factors, this process is challenging to control and is characterized by poor efficiency. Therefore, expansion of hiPSCs on microcarriers may be followed by switching to conditions directing the cells to adopt a cardiomyocyte fate. We will evaluate the cardiogenic potential of hiPSCs cultured in microcarrier bioreactors. The resulting cells will be characterized for the expression of cardiomyocyte-associated genes/proteins and will be subjected to functional assays in vitro. Lastly, a mouse infarct heart model will be employed to evaluate the functional attributes of hiPSC-derived heart cells in vivo. This study will yield new information benefiting the development of bioprocesses for the generation of large quantities of hiPSC-derived cardiomyocytes suitable for 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. Patient-specific human induced pluripotent stem cells, which proliferate extensively and can differentiate towards functional cardiomyocytes, may serve as a renewable cellular source for regenerative heart therapies. This project seeks to further our understanding of the cardiogenic potential of human induced pluripotent stem cells in static and scalable culture systems and to advance bioprocess technologies for the production of stem cell-derived cardiomyocytes in clinically relevant quantities.