58 Chapter 3 have previously demonstrated that it was the most potent CM mitogen in vitro (Mills et al., 2017,2019; Sharma et al., 2018; Titmarsh et al., 2016) . We show that hiPSC-CMs exhibit contactmediated inhibition of proliferation, indicating that the removal of cell-cell contacts is essential to enable the maintenance of their proliferative capacity through continuous passaging. We show that the withdrawal of CHIR or treatment with C59, a small molecule Wnt antagonist, result in rapid cell-cycle exit and restoration of mature contractile, electrophysiological, and cellular characteristics. Further mechanistic studies identified the role of canonical Wnt signal via LEF/TCF to delay hiPSC-CM maturation, and the stimulatory role of AKT T308 and HSP27 phosphorylation in enhancing hiPSC-CM proliferation by increasing cell cycle activity. Lastly, we demonstrate that the expanded hiPSC-CMs exhibit fully differentiated and contractile phenotype as the unexpanded hiPSC-CM and are therefore an excellent cell source for the mass production of engineered heart tissues. Together, these studies reveal a novel and robust method, the mechanistic explanation, and a proof-of-principle demonstration of a translational approach to achieve massive hiPSC-CM expansion (>1 billion) needed for future cardiac regenerative strategies in patients with cardiomyopathy or congenital heart defects. METHODS Cell culture. Four previously established hiPSC lines (SCVI-111, Sendai virus reprogrammed, peripheral blood mononuclear cells (PBMCs), male; SCVI-113, Sendai virus reprogrammed, PBMCs, male; SCVI-202 Sendai virus reprogrammed, PBMCs, female; SCVI-273, Sendai virus reprogrammed, PBMCs, female (Matsa et al., 2016)) were maintained in DMEM/ F12 (ThermoFisher) supplemented with the essential eight (E8) (Thermo Fisher) growth factors in a Matrigel (Corning) coated (1:400 for 24h) polystyrene 2D culture system. Upon 80–90% confluency, cells were dissociated in PBS with 0.5mM EDTA for 5–10 minutes at 37 °C. Dissociation was performed with gentle trituration to obtain small aggregates of undifferentiated hiPSCs. Passaging was performed in 1:15–20 splitting ratios or 10,000 cells per cm2 to achieve low density and reach full confluency within 4–5 days. For the first 24h after replating, 10βM of ROCK inhibitor Y-27632 was included in the hiPSC maintenance media. hiPSC-CM differentiation was performed using the previously described canonical Wnt stimulation and inhibition protocol (Lian et al., 2013) in RPMI 1640-based differentiation media supplemented with B27 minus insulin (Invitrogen). Between day 0–2, a gradient of CHIR99021 (Seleckchem) concentrations (3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μM) was used. Between day 3–5, Wnt-C59 (2.0 μM) (Selleckchem) was added to the differentiation media. At day 7, B27 with insulin was added to the differentiation media. At day 11, wells that contain more than 80% beating cells were treated with TrypLE Select Enzyme 10X (Invitrogen) at 37 °C for 10–40 minutes. Gentle rocking was performed every 10 minutes. Preparations of single dissociated cells were generated with very gentle trituration and transferred to a 15 mL conical tube containing a wash buffer (PBS with 20% FBS). Cells were gently centrifuged at 1000 RPM for 3
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