Abstract P1001: IGFBP2 Overcomes Cell Contact-mediated Proliferation Inhibition In Human IPSC-derived Cardiomyocytes

Abstract

Introduction and Hypothesis: Human induced pluripotent stem cells (hiPSCs) and their derivatives provide a promising cellular source for regenerative cardiac therapies. However, generating large-scale hiPSC-derived cardiomyocytes (hiPSC-CMs) remains a major hurdle, caused, in part, by cell contact-driven proliferation inhibition in dense culture conditions. We hypothesize that differentially-secreted factors in sparsely vs densely-cultured hiPSC-CMs are responsible for the increased proliferation rate in sparsely cultured hiPSC-CMs. Methods: We performed a high-throughput screening (HTS) assay to identify growth factors (GFs) released in the supernatant of sparsely- and densely-cultured hiPSC-CMs. Differential expression of candidate GFs in dense vs sparse culture conditions was confirmed by single cell RNA sequencing (scRNAseq), Western Blot (WB), and Immunocytochemistry (ICC) analyses. The confirmed GF hits were further validated by measuring the expression of the cell cycle marker Ki67 in hiPSC-CMs after GF treatment. The GF treatment effects on cell-cell contact was further explored mechanistically using siRNA-based N-cadherin (CDH2) knockdown (KD) model in vitro. Results: Our HTS assay identified IGFBP2, IGFBP6, PDGF-AA as top candidate GFs in the sparse culture supernatant. Subsequent WB, ICC, scRNAseq analyses confirmed 1.5, 3.5, 2.0-fold higher IGFBP2 expression in the sparse condition compared to the dense condition. Supplementation of recombinant IGFBP2 to densely cultured hiPSC-CMs showed a dosage-dependent increase in Ki67 expression level from 13.0 +/- 1.4% in control to 44.1 +/- 8.4% after 3nM IGFBP2 treatment, a level comparable to that achieved in sparse culture. Mechanistically, IGFBP2 treatment resulted in a shift of CDH2 localization from cell-cell junction to the cytoplasm. Reducing CDH2 expression by CDH2-KD to mimic loss of cell-cell contact promoted hiPSC-CM proliferation. Conclusion: We discovered an unexpected role of IGFBP2 to overcome cell contact-mediated inhibition of hiPSC-CM proliferation which provides a promising new approach to engineer in situ large-scale 3D cardiac tissues for regenerative applications.