75 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 Long-term GSK-3β inhibition does not preclude terminal differentiation and maturation of expanded hiPSC-CMs and their capacity to form functional cardiac tissue Given the diverse role for GSK-3β signaling to regulate cell lineage commitment and differentiation, a prolonged exposure to CHIR raises concerns regarding possibilities of hiPSCCM phenotype conversion or oncogenic transformation that prevent them from appropriate maturation following expansion, a finding that would significantly hamper their use for tissue engineering applications or regenerative approaches. To address this, we expanded hiPSC-CMs for 4 serial passages in continuous presence of CHIR followed by its withdrawal from the culture media for several weeks. When compared to age-matched controls, previously long-term expanded cells had phenotypically recovered to normal as assessed by immunohistochemistry for α-sarcomeric actinin and real-time PCR for several mature sarcomeric and ion channel genes (Figure 6A–B). Next, as a functional proof-ofprinciple, we created engineered heart tissues from P4 CHIR expanded hiPSC-CMs and agematched non-expanded CMs (Figure 6C). Mass-produced (Figure 6D) engineered heart tissues from expanded hiPSC-CMs exhibited normal sarcomere alignment and fully functional integration when applied in engineered heart tissues (Figure 6E). Moreover, mass produced engineered heart tissues from multiple hiPSC lines (CVI-111 and CVI-273) have comparable or slightly enhanced functional properties when compared to those generated from agematched unexpanded control cells (Figure 6F and Supplementary Movie 5 and 6). The proliferative response of hiPSC-CMs to GSK-3β inhibition raises the possibility that CHIR may act to stimulation cell division in proliferation-competent CMs when applied the late embryonic or postnatal heart in vivo. To investigate this, pregnant mice at E16.5 was treated with a once daily injection of CHIR or DMSO carrier (CTR) for 4 consecutive days, followed by harvesting. Interestingly, at E20.5 CHIR-treated embryos contained 1.8-fold increase in mitotic CMs when compared to the embryos treated with DMSO control (Figure S5A–C). Analysis of individual cell size revealed no difference in the size of single CMs in hearts from CHIR-administered mice compared to those treated with DMSO (Figure S5D–E). These data suggest the potential for residual hyperplasia of CMs during late gestation with GSK-3β inhibition, despite substantially attenuated proliferative effect compared to in vitro CHIRtreated hiPSC-CMs. Furthermore, GSK-3β inhibition with daily CHIR injections for 6 consecutive days in postnatal mice (P10) did not result in increased myocardial growth when compared to controls (Figure S5F–G). These data support the progressive decline in proliferative capacity of embryonic CMs in vivo and the likely inhibitory effects of cell-cell contact that prevents significant CM expansion upon in vivo GSK-3β inhibition.
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