Growth Factor-Based Manufacturing of Human Pluripotent Stem Cell-Derived Cardiomyocytes Using the Vertical Wheel Bioreactor System

Abstract

BackgroundMultiple protocols have been reported for the large-scale generation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in bioreactors using small molecules; however, no comparable bioreactor-based methods have been established using growth factors. This is despite evidence that differentiation with optimized concentrations of BMP4, Activin A, and bFGF offers finer control of cardiomyocyte phenotype. Here, we develop scalable hPSC expansion and growth factor-based cardiac differentiation protocols using the vertical wheel bioreactor (VWBR) system. Methods and ResultsThe expansion of undifferentiated hPSCs was first optimized in 100 mL VWBRs by varying parameters, including starting cell seeding density, agitation rate, and media exchange schedule. Proliferation, viability, aggregate diameter, media metabolites, and pluripotency were assessed during hPSC expansion. Next, we evaluated the effects of undifferentiated hPSC culture conditions on subsequent cardiomyocyte differentiation potential. We found that hPSCs expanded in static culture or in VWBRs at different densities and agitation rates all differentiated into hPSC-CM populations of similar cardiac purity; however, cardiomyocyte yields were initially lower when VWBR-expanded hPSCs were used. We compared the differentiation kinetics of hPSCs expanded in VWBRs to conventional 2D culture and found that the former had accelerated mesodermal commitment and significantly greater cKit+/CXCR4+/PDGRF- cell formation during differentiation. Modifying our aggregation and mesoderm induction steps improved cell yields and enabled reliable production of >1x106 cells/mL cardiac troponin T+ (cTnT) hPSC-CMs. Highlighting the versatility of our growth factor-based system, variation in the BMP4:Activin A ratio enabled a second heart field-like differentiation and generation of atrial-like cardiomyocytes in VWBRs. We further show that our expansion and differentiation protocols are reproducible and economical in 500 mL VWBRs, yielding on average 1.11x106 hPSC-CMs/mL at a mean purity of 93% cTnT+. Characterization of VWBR produced hPSC-CM force generation, action potentials, and intercellular calcium transients confirmed the expected phenotype of ventricular-like cells. Lastly, VWBR produced hPSC-CMs robustly engrafted in the infarcted guinea pig myocardium, supporting use as a cell therapy product. ConclusionsThis novel bioreactor-based protocol will enable cardiac cell therapy and tissue engineering applications by providing scalable and consistent production of hPSC-derived cardiac cell products.