65 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 RESULTS Glycogen synthase kinase-3β (GSK-3β) inhibition promotes hiPSC-CM proliferation in a cell-density dependent manner through direct cell-cell contact Previous studies have shown CHIR-mediated GSK-3β inhibition leads to cardiomyocyte proliferation in mouse fetal heart, embryonic stem cell-derived cardiomyocytes (Buikema et al., 2013a, 2013b) and hiPSC-CM (Mills et al., 2017; Sharma et al., 2018). Despite the known role of GSK-3β inhibitor as a mitogen, the extent of cardiomyocyte proliferation has been reported to be modest (< 3- to 5-fold increase in total cell number) (Mills et al., 2017; Sharma et al., 2018) and were all performed in densely-plated cell cultures. We investigated whether disruption of cell-cell contact by low-density passaging would further promote hiPSC-CM proliferation. iPSC lines from healthy individuals were differentiated into cardiomyocytes using small molecule-based Wnt-modulated protocol (Figure 1A) (Lian et al., 2012, 2013). The differentiating cells displayed stage-specific gene expression for pluripotent stem cells (NANOG), mesoderm (MESP1) and cardiac progenitor cells (Isl1) before committing to the cardiomyogenic lineage (Figure S1A–B). When hiPSC-CMs were passaged at low-density, the total cell number increased by 8-, 8-, and 4-folds at each passage, resulting overall in a 241-fold increase in hiPSC-CM number (Figure 1B). In contrast, the fold-change in cell number in densely passaged group and w/o passaging group remained comparable, and at the end of three passages or 3 weeks, the number increase was ~10-fold (Figure 1B). The CM number increase with low-density passage was supported by their increase in cell cycle activity (Figure 1C–F) compared with densely passaged or un-passaged CMs assessed by cell cycle marker Ki67 (Figure 1C–D). Moreover, fraction of mitotic CMs was at 1% without passaging and remained at 1% upon passaging at high cell density, but it was only when CMs were passaged at low-density that led to significant increase in mitotic CM fraction up to 10% (Figure 1E–F). This indicates that the maintenance of low cell density is necessary to sustain active cell cycle activity in CMs. Taken together, we demonstrate that massive expansion of hiPSC-CMs can be achieved through sparse passaging in the presence of CHIR. We then asked whether hiPSCCMs at high cell density inhibit CM proliferation and induce cell cycle exit by their secretion of anti-proliferative paracrine factors (Figure 1G). We did not find any significant changes in the proportion of Ki67-expressing hiPSC-CMs between different conditioned media treatment groups (Figure 1H–I). Next, we examined whether the presence of cell-cell contact induces cell cycle exit in hiPSC-CMs (Figure 1J). Interestingly, densely plated hiPSC-CMs significantly down-regulated Ki67 and pHH3, suggesting that cell-cell contact inhibits cell cycle activity in hiPSC-CMs (Figure 1K–N). Taken together, we demonstrate here that cell-cell contact is a major barrier to hiPSC-CM proliferation and that prevention of cell-cell contact is critical to maintain CHIR-induced CM proliferation.
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