73 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 To identify the β-catenin-independent process that accounts for the low density plus CHIRmediated hiPSC-CM proliferation, we screened a library of 43 kinases with known functions (Figure 5D and S4A) and found a significant upregulation of AKT1/2/3 phosphorylation at residue T308 (Figure 5D), a residue that is required for AKT activation and cell division (Liu et al., 2014). In addition, we found an increased phosphorylation of HSP27, an AKT binding protein, as well as p70S6K, a downstream AKT target (Figure 5D) (Conejo et al., 2002; Song et al., 2005). Using an antibody directed against the T308 residues of phosphorylated AKT (pAKT), we confirmed by western blotting that the active (i.e. phosphorylated) form of AKT is rapidly increased following CHIR treatment at low cell density (Figure 5E–F). Immunostaining of day 12 hiPSC-CMs revealed that T308 pAKT was abundantly expressed in the cytoplasm of mitotic hiPSC-CMs in the CHIR-treated group (Figure 5G and S4B). Next, we assessed the contribution of T308 pAKT in hiPSC-CM proliferation by using a previously described highly selective AKT phosphorylation inhibitor MK2206 (Lindsley et al., 2007). When co-treated with MK2206, CHIR and low density-induced hiPSC-CM proliferation decreased by ~50% compared with CHIR treatment alone and pAKT expression reduced (Figure 5G–I). Furthermore, we found while MK2206 treatment inhibits AKT T308 phosphorylation (Figure 5I), it does not affect downstream β-catenin signaling, as assessed by the LEF/TCF luciferase reporter (Figure 5J), nor does it alter the expression of genes associated with CM maturation or Wnt signaling (Figure 5K). These data reveal the presence of β-catenin-independent signaling that target downstream effects of GSK-3β inhibition and low cell density via activation of AKT signaling to induce hiPSC-CM proliferation (Figure 5L). (PI3K)-AKT pathway is a well-known regulator of Cyclin D2 protein stabilization and cell-cycle progression (Mirzaa et al., 2014). Interestingly, the addition of MK2206 (AKT inhibitor) or PNU74654 (LEF/TCF inhibitor) reduced amounts of Cyclin D2 positive hiPSC-CMs (Figure 5MN), indicating that Cyclin D2 activity integrates the contribution by β-catenin signaling and AKT phosphorylation to overall hiPSCCM proliferation. Previously, Type D cyclins were reported to assemble with Cyclin-dependentkinases 4/6 (CDK4/6) and form enzymatically active holoenzyme complexes to propel cell cycle progression (Sherr et al., 2016). To address whether CDK4/6 also play an integrative role in CHIR and low cell density-mediate proliferation, CHIR-treated, sparse hiPSC-CMs were treated with palbociclib, a potent small molecular inhibitor of CDK4/6, and shown mitosis was nearly completely abrogated (Figure 5O–P). Taken together, these data demonstrate that GSK-3β inhibition and concomitant cell contact removal induce a molecular interplay to enhance LEF/ TCF activity via canonical β-catenin signaling and activate AKT phosphorylation at T308 to enhance Cyclin D2 activity and promote hiPSC-CM proliferation.
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