69 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 inhibition (Figure 1), prevention of maturation may also in part explain their retained capacity to proliferate. Patterned CHIR-treated single CM that is undergoing mitotic cell division marked by pHH3, exhibited complete sarcomere disassembly (Figure 3A). Moreover, control (CTR) hiPSCCMs demonstrated highly organized and aligned sarcomeres, while CHIR-treated hiPSC-CMs on micropatterns exhibited markedly reduced alignment and organization of sarcomeres (Figure 3A–B). Quantification of z-disc-registered sarcomeric α-actinin confirmed the observed sarcomere disorganization in CHIR-treated hiPSC-CMs (Figure 3C–D). Interestingly, when the Wnt inhibitor C59 was added to cells that had previously expanded with CHIR treatment (CHIR>C59), these cells improved their sarcomeric organization (Figure 3B–D). This supports the reversibility of GSK-3β inhibition signaling effects on hiPSC-CMs. Imaging-based assessment of contractile properties of CTR and CHIR-treated hiPSC-CMs demonstrated a decrease in force generation with CHIR treatment (Figure 3E) that recovered upon replacing CHIR with C59 or after the withdrawal of CHIR (data not shown). Additional single-cell calcium studies of agematched CTR- and CHIR-treated and CHIR withdrawn hiPSC-CMs showed no difference in their spontaneous action potentials, calcium transients, beating frequency nor amplitude (Figure 3F–H). Real-time quantitative PCR analysis revealed CHIR-treated hiPSC-CMs downregulate markers associated with cardiomyocyte maturation (MYL2, TNNI3, MYOM2), excitation (GJA1), contractility (RYR2) and metabolism (COX6A2, CKMT) compared to those treated with DMSO or C59, supporting maturation arrest phenotype at transcriptional level (Figure 3I–K). To further characterize global transcriptional changes in individual hiPSC-CMs in response to GSK-3β inhibition, we performed single-cell RNA sequencing in day 12 hiPSC-CMs treated for 24 hours with either DMSO (CTR), CHIR (4.0 μM), or C59 (4.0 μM) (Figure 3L). We captured a total of 8,381 cells and performed 93,552 mean reads per cell resulting in a median of 1,297 genes analyzed per cell. Unsupervised analysis revealed 5 cell populations characterized as non-proliferative and proliferative ventricular CMs, non-proliferative and proliferative atrial CMs and fibroblast, present in all 3 treatment samples (Figure S2A–D). We found that Wnt target genes such as AXIN2 and LEF1 were upregulated in almost all cells treated with CHIR (Figure 3M). Furthermore, mature cardiac genes were dramatically downregulated in CHIRtreated hiPSC-CMs and conversely correlated with the up-regulation of Wnt signaling target genes (Figure 3N). Interestingly, CHIR treatment resulted in a larger subset of proliferative and immature atrial and ventricular-like hiPSC-CMs (Figure 3O–P and Figure S2E). Importantly, GSK-3β inhibition did not result in increased expression of the cardiac progenitor markers such as CKIT, ISL1 or MESP1/2 (Figure S2F), suggesting that expansion of functional hiPSC-CM with CHIR does not lead to expansion of a rare population of residual cardiac mesoderm or second-heart-field progenitor cells (Wu et al., 2008). Furthermore, expression of key cardiac transcription factors such as GATA4, TBX5, MEF2C and NKX2.5 (Ieda et al., 2010), did not alter significantly in hiPSC-CMs treated with CHIR versus CTR and C59 (Figure S2G). Overall, these data strongly support the inhibition of hiPSC-CM maturation by CHIR/GSK-3β inhibition, a phenomenon that has also been observed in second heart field-derived cardiac progenitor cells and other cell types (Sato et al., 2004; Yin et al., 2014).
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