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

Rationale: Inducing cardiomyocyte proliferation in situ is an attractive approach for achieving cardiac regeneration after myocardial injury. However, numerous inhibitory mechanisms are present in cardiomyocytes that silence the pro-proliferative signals for their expansion. We hypothesized that cell-cell contact exerts a major suppressive effect on cardiomyocyte proliferation. Objectives: This study aims to uncover the molecular mechanisms underlying cell contact-mediated inhibition of cardiomyocyte proliferation and leverage this knowledge to sustain proliferation of cardiomyocytes in 3D despite cell-cell contact. Methods and Results: Using human iPSC-derived cardiomyocytes (hiPSC-CMs) as a model system, we found that the proliferative capacity of hiPSC-CMs is initially increased proportional to cell density until cells form intercellular contacts. We found that cell-cell contact exerts a strong inhibitory effect on hiPSC-CM proliferation as cell density increases. Phosphoproteomics analysis and cellular phenotyping revealed that cell-cell contact accompanies adherens junction formation, enhanced sarcomere organization, and increased contractility. Disruption of adherens junctions or sarcomere assembly via siRNA-mediated knockdown of N-cadherin or ɑ-actinin, respectively, resulted in increased cell cycle activation of hiPSC-CMs. Furthermore, disruption of cell-cell contact enhanced nuclear translocation of β-catenin and TCF/LEF transcriptional activity that contributed to hiPSC-CM proliferation. However, 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, which was sufficient to drive hiPSC-CM division in the presence of cell-cell contact in 3D 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 3D cardiac tissue. These insights provide valuable implications for advancing cardiac tissue engineering and regenerative therapies. Human iPSCs (SCVI-111) were directed to differentiate into cardiomyocytes using well-established Wnt modulating protocol. Day 18 hiPSC-CMs were replated at either low (33,000 cells/cm2) or high cell density (181,000 cells/cm2), followed by a two-day culture period to allow for cellular adaptation and transcriptional stabilization. hiPSC-CMs were cultured in cardiomyocyte maintenance media (RPMI supplemented with B27+Insulin and CHI99021). On day 20, cells from both density conditions were dissociated into single-cell suspensions and loaded into the Chromium Controller (10x Genomics) for single-cell barcoded droplet generation, according to the manufacturer's protocol. Gene expression libraries were prepared using the 10x Genomics Single Cell 3' v3 chemistry kit.