67 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 Glycogen synthase kinase-3β inhibition and low-density passaging result in massive expansion of beating hiPSC-CMs To determine the maximum extent of hiPSC-CMs expansion through combined GSK-3β inhibition and low-density passaging, we treated hiPSC-CMs continuously with or without 2.0 μM CHIR (CHIR vs. CTR), and serially passaged at low-density (Figure 2A). Remarkably, in the presence of CHIR, the beating hiPSC-CMs continue to divide without notable cell death (Figure S1C) and can be passaged up to 4–5 times over 60+ days (Figure 2B–D). Control dimethyl-sulfoxide (DMSO)-treated hiPSC-CMs cease to proliferate after the first or second passage (Figure 2B–D). Interestingly, despite the capacity for continuous cell division in the presence of CHIR, these hiPSC-CMs remain capable of spontaneous beating (Supplementary Movie 1). Typically, ~2 million hiPSC-CMs (e.g. 1 well in a 6-well plate) at day 12 of differentiation generate 300–900 million hiPSC-CMs (Figure 2E). In addition, the expansion capacity was reproducible in CMs from 4 different iPSC lines (CVI-111, CVI 113, CVI 202 and CVI 273) (Figure 2F). Moreover, no significant difference in expansion capacity was observed among the same batch of day 12 CVI-111 hiPSC-CMs that were either cryopreserved or used directly (Figure S1D). In total, continuous treatment of day 12 hiPSC-CMs with CHIR for >30 days resulted in 100 to 250-fold increase in total CM number when compared to CTR hiPSC-CMs at similar time points (Figure 2G–H). Furthermore, CHIR-treatment appeared to maintain, if not slightly enrich, CM purity as opposed to control DMSO-treated hiPSC-CMs which decreased CM purity with each passage (Figure 2I–J), most likely due to over-growth of non-myocytes. To examine the progression of cell cycle activity of CHIR-treated hiPSC-CMs over multiple passaging, the percentage of troponin T (TnT) positive hiPSC-CMs that express the cell cycle marker Ki67 was assessed at each passage after treatment with CHIR or DMSO (CTR) (Figure S1E). Interestingly, while CTR hiPSC-CMs showed a rapid decline in their expression of Ki67 with each passage where only a small number of proliferative cells can be observed after passage 2 (P2), CHIRtreatment with low-density passaging resulted in significant (P = 4.42E-05) extension (up to 5 passages) of the presence of proliferating cells. Moreover, we found that CHIR-treated hiPSC-CMs indeed proceed through each stage of the cell cycle, undergo cytokinesis and remain more mono-nucleated (Figure 2K–M and S1F–I). Furthermore, our immunostaining data suggests that hiPSC-CMs disassemble their aligned sarcomeres during active mitosis, suggesting the diminution or absence of active hiPSC-CM beating during the mitotic phase of cell division (Figure 2K–M). Collectively, these data support the ability of GSK-3ȕ inhibition and contact removal via serial passaging to maintain hiPSC-CM proliferation.
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