79 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 10 days after birth (Figure S5). This indicates that GSK-3β inhibition alone is not sufficient for cardiac regeneration in the postnatal and adult phases. Nevertheless, these findings point to the potential for residual hyperplasia of CMs during late gestation with Wnt stimulation as a therapy for certain forms of congenital heart disease such as hypoplastic left heart syndrome since a recent in-depth study indicates dysregulation of Wnt signaling as one of the contributors to impaired growth (Liu et al., 2017). SUMMARY We demonstrated a strategy for the massive expansion of beating hiPSC-CMs with simultaneous GSK-3β inhibition and low cell density culture that ultimately generated a 250-fold increase in the number of hiPSC-CMs, which is suitable for multiple translational/ regenerative applications. Furthermore, the in vivo control of late gestational CM proliferation may lead to promising regenerative strategies for treating patients with congenital heart defects. Study Limitations Some limitations should be considered when interpreting the results of this study. First, while we observed a strong expansion capacity of day 12 hiPSC-CMs, this capacity is not unlimited. This suggests that additional factors contribute to the proliferative decline of hiPSCCM with maturation. In line with this finding we found that late gestational but not postnatal administration of CHIR is able to increase in vivo cardiomyocyte proliferation. Second, our mechanics studies focused on the inhibition of GSK-3β signaling to stimulate hiPSC-CM proliferation. While we identified the powerful negative effect of cell-cell contact on hiPSC-CM proliferation, the precise molecular mechanism of this effect remains unknown. Whether the engagement of cell surface molecules between two adjacent cells leads to a change in downstream signaling event that suppresses hiPSC-CM proliferation will require further investigation. Acknowledgements This work was supported by a UMC Utrecht Clinical Fellowship and Netherlands Heart Institute Fellowship (to J.B.); Stanford Child Health Research Institute Postdoctoral Fellowship and NIH NRSA Postdoctoral Fellowship 5F32HL142205 (to S.L.); NIH Pathway to Independence Award 1K99HL127295–01A1 (to V.S.); The Richard and Helen DeVos Foundation (to S.J.); Netherlands Heart Foundation (CVON-Dosis 2014–40), and Netherlands Organization for Sciences (NWO)- ZonMW (VICI 91818602) (to J.V.); R01 HL145676, R01 HL146690 and P01 HL141084 (to J.C.W); NIH (OD004411, HL099776, LM012179), and the Endowed Faculty Scholar Award of Lucile Packard Foundation for Children and Child Health Research Institute at Stanford (to S.M.W.). The sequencing data was generated on an HiSeq purchased with funds from NIH under award
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