Bioprocess for cardiac cell generation from human induced pluripotent 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。细胞疗法旨在取代心肌梗塞是非常可取的。在患者特异性人类诱导多能干细胞（hipsc）的产生方面的最新进展引发了希望，这些细胞可以作为修复受损心肌的细胞材料的取之不尽的来源。与人类胚胎干细胞（hESCs）一样，hiPSCs在胚胎样体（EB）培养和血清补充培养基中被证明可以分化为功能性心肌细胞。尽管如此，临床实现基于干细胞的心脏修复疗法将需要生产hipsc衍生的心肌细胞(1)使用不含动物成分（如血清）的定向分化方法，以及(2)大量生产。我们假设hipsc可以被导向具有已知参与胚胎心脏发育的生理相关因子的心肌细胞样细胞。为此，我们建议建立一种在静态培养（如培养皿）中将hipsc定向分化为心脏干细胞的方法。然而，多能干细胞在培养皿中的繁殖和分化是一个挑战。我们发现在生物反应器中培养的hESCs可以扩增数倍并分化成多个谱系。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hipsc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hipsc，并确定有利于hipsc生长而不影响其多能性和活力的条件。此外，干细胞向心脏细胞的分化通常在EB培养中进行。考虑到并非所有的细胞都暴露于致心因子，这一过程很难控制，而且效率很低。因此，hipsc在微载体上的扩增可能随后会转向指导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hipsc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。最后，将采用小鼠梗死心脏模型来评估hipsc来源的心脏细胞在体内的功能属性。这项研究将产生新的信息，有利于生物过程的发展，以产生大量适合心脏治疗的hipsc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.