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。旨在替代梗死心肌的细胞疗法是非常可取的。最近在产生患者特异性人类诱导多能干细胞（hiPSC）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细胞材料的无尽来源。与人胚胎干细胞（hESC）一样，hiPSC已显示出利用胚状体（EB）培养物和血清补充培养基向功能性心肌细胞分化。 尽管如此，用于心脏修复的基于干细胞的疗法的临床实现将需要（i）使用不含动物组分（例如血清）的定向分化方法和（ii）大量产生hiPSC衍生的心肌细胞。我们假设hiPSC可以定向于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，我们提出建立一种用于在静态培养物（例如培养皿）中将hiPSC定向分化为心脏干细胞的方法。然而，iPSC在培养皿中的增殖和分化对于扩大规模是具有挑战性的。我们已经发现，在生物反应器中培养的hESC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的微载体上培养的hiPSC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hiPSC，并确定有利于hiPSC生长而不损害其多能性和活力的条件。此外，干细胞分化为心肌细胞通常在EB培养物中进行。鉴于并非EB内的所有细胞都会暴露于心源性因子，因此该过程难以控制，并且效率低下。因此，在微载体上扩增hiPSC之后可以切换到指导细胞采用心肌细胞命运的条件。我们将评估在微载体生物反应器中培养的hiPSC的心源性潜力。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。最后，将采用小鼠梗塞心脏模型来评估体内hiPSC衍生的心脏细胞的功能属性。这项研究将产生新的信息，有利于开发生物过程，以产生大量适用于心脏治疗的hiPSC衍生的心肌细胞。

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.