IGFBP2 Mediates Human iPSC-Cardiomyocyte Proliferation in a Cellular Contact-Dependent Manner

BACKGROUND: Induction of cardiomyocyte proliferation in situ represents a promising strategy for myocardial regeneration following injury. However, cardiomyocytes possess intrinsic inhibitory mechanisms that attenuate pro-proliferative signaling and constrain their expansion. We hypothesized that cell–cell contact is a key suppressor of cardiomyocyte proliferation. We aimed to delineate the underlying molecular pathways to enable sustained proliferation in 3-dimensional contexts. METHODS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured at varying plating densities to examine the impact of cell-cell contact on cell cycle activity. Phosphoproteomic profiling was performed in sparse versus dense cultures to identify signaling alterations. Conditioned media from sparse cultures were interrogated using a human growth factor array to identify secreted pro-proliferative factors. RESULTS: hiPSC-CM proliferation increased proportionally with plating density until intercellular contacts were established, at which point proliferation was suppressed. Dense cultures exhibited enhanced adherens junction assembly, sarcomeric organization, and contractile function. Increased cell-cell contact in dense conditions attenuated nuclear translocation of β-catenin and reduced TCF/LEF (T cell factor/lymphoid enhancer factor family) transcriptional activity, providing a mechanistic basis for the reduced hiPSC-CM proliferation. Disruption of adherens junctions or sarcomere assembly via siRNA-mediated knockdown of N-cadherin or α-actinin, respectively, resulted in increased cell cycle activation of hiPSC-CMs, but this was not sufficient to drive division of hiPSC-CMs. Additional screening for putative secreted growth factors in the conditioned media from sparsely plated hiPSC-CMs revealed the enrichment of IGFBP2 (insulin-like growth factor-binding protein 2), which was sufficient to drive hiPSC-CM division in the presence of cell-cell contact in 3-dimensional constructs. CONCLUSIONS: Our findings demonstrate that cell-cell contact inhibits hiPSC-CM proliferation through adherens junction formation, sarcomeric assembly, and reduced IGFBP2 secretion. Importantly, exogenous supplementation of IGFBP2 can overcome cell contact-mediated inhibition of hiPSC-CM proliferation and facilitate the growth of 3-dimensional cardiac tissue. These insights provide valuable implications for advancing cardiac tissue engineering and regenerative therapies.