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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE301852 | GEO | 2025/09/27

REPOSITORIES: GEO

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Project description:Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used to identify potential factors capable of inducing endogenous cardiomyocyte proliferation to regenerate the injured heart. L-type calcium channel blockers have previously been identified as a class of factors capable of inducing matured hiPSC-CMs to proliferate. However, the mechanism by which L-type calcium channel blockers promote hiPSC-CM proliferation remains unclear. Here we provide evidence that matured hiPSC-CMs possess plasticity to undergo dematuration in response to certain pharmacological compounds. Consistent with primary cardiomyocyte maturation during perinatal development, we found that centrosome disassembly occurs in hiPSC-CMs during plate-based, temporal, maturation. A small molecule screen identified Nitrendipine, an L-type calcium channel blocker, and 1-NA-PP1, a Src kinase inhibitor, as factors capable of inducing centrosome reassembly in a subpopulation of hiPSC-CMs. Furthermore, centrosome-positive hiPSC-CMs were more likely to exhibit cell cycle activity than centrosome-negative hiPSC-CMs. In contrast, neither Nitrendipine or 1-NA-PP1 induced centrosome reassembly, or cell cycle activity, in neonatal rat ventricular myocytes (NRVMs). Differential bulk transcriptome analysis indicated that matured hiPSC-CMs, but not NRVMs, treated with Nitrendipine or 1-NA-PP1 undergo dematuration. ScRNA transcriptome analysis supported that matured hiPSC-CMs treated with either Nitrendipine or 1-NA-PP1 undergo dematuration. Collectively, our results indicate that matured hiPSC-CMs, but not primary NRVMs, possess plasticity to undergo dematuration in response to certain pharmacological compounds such as L-type calcium channel blockers and Src-kinase inhibitors. This study shows that once mature, hiPSC-CMs may not maintain their maturity under experimental conditions and thus may have implications for experimental systems where the state of hiPSC-CM maturation is relevant.

Project description:ABSTRACT Background: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing mutant of the coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs. Viral proliferation on hiPSC-CMs was subsequently quantified using bioluminescence imaging. For drug screening, select antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate (PDTC), and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of some of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with the reported drug effects in previous studies. Finally, mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways within these hiPSC-CMs after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to confirm antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that could be used to screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion. For this experiment, human induced pluripotent stem cell derived cardiomyocytes were infected with coxsackievirus at multiplicity of infection (MOI) of 5 for 8 hours. Cells were treated with and without interferon beta 1 in order to determine if treatment activates antiviral response genes and/or viral clearance pathways. 4 total samples (2 for each condition) were analyzed

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

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