Abstract Body: Introduction/Background: Cell-cell contact suppresses cell proliferation, but the mechanisms underlying contact-mediated inhibition in cardiomyocytes remain poorly understood. We previously identified insulin-like growth factor-binding protein 2 (IGFBP2) as a pro-proliferative factor downregulated in the presence of cell-cell contacts, with potential implications for regulating cardiomyocyte proliferation in 3D environments, where physical cell contacts are prevalent.

Hypothesis: We hypothesize that IGFBP2 plays a key role in contact-mediated regulation of human cardiomyocyte proliferation.

Methods/Results: Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we investigated the role of IGFBP2 in cardiomyocyte proliferation under the pro-proliferative condition (Wnt activation via CHIR-99021). We first examined IGFBP2 expression in response to varying degrees of cell-cell contact and found that IGFBP2 secretion decreases as cell-cell contact increases. To determine whether IGFBP2 is necessary for cardiomyocyte proliferation in low-contact conditions, we conducted siRNA-mediated IGFBP2 knockdown (KD) and found a 29.2% reduction in the number of cardiac troponin T+ nuclei. Additionally, conditioned media from IGFBP2 KD cultures confirmed that IGFBP2 secretion is required for cardiomyocyte proliferation. Serial IGFBP2 treatment over 12 days resulted in an 8.12-fold increase in spheroid volume and a 3.84-fold increase in karyokinesis compared to the basal media-treated control. Compared to CHIR treatment alone, IGFBP2 treatment further increased spheroid volume by 1.74-fold and karyokinesis by 1.38-fold, demonstrating its ability to enhance cardiomyocyte proliferation even under high-contact conditions. This IGFBP2-driven 3D expansion was consistently observed across three independent iPSC lines. Finally, IGFBP2-driven cardiomyocyte proliferation was independent of Wnt and Hippo signaling but associated with AKT activation.

Conclusions: Our findings demonstrate that IGFBP2 is a key regulator of hiPSC-CM proliferation. Exogeneous IGFBP2 supplementation enables complete hiPSC-CM division, effectively overcoming the contact-mediated inhibition of proliferation that persists with CHIR treatment alone. These results highlight IGFBP2 as a potential target for enhancing cardiomyocyte proliferation in 3D cardiac tissue engineering applications.