Chemical Maturation of human pluripotent stem cell derived-cardiomiocytes

JOAQUIN SMUCLER; JULIA HALEK; GUADALUPE AMIN; SANTIAGO MIRIUKA; CARLOS LUZZANI; ARIEL WAISMAN

Congreso; Reunion anual de la Sociedad Argentina de Investigaciones Clínicas 2022; 2023

Cardiovascular diseases are the main cause of death worldwide. Efforts to model these pathologies are continuously made to improve therapy and drug treatment. In the last years, human induced pluripotent stem cells (hIPSCs) models have yielded a variety of tools to design new clinical approaches. hIPSCs-derived cardiomyocytes (CMs) are an advanced model that has brought the opportunity to study cardiac cells. Nevertheless, hIPSC-CMs lack crucial characteristics present in the adult human heart since they are differentiated in short-term protocols and thus resemble embryonic CMs.In this work, we differentiated hIPSCs into pure immature CMs (day 24) and then applied a maturation cocktail consisting of the hormone T3, dexamethasone, a PPARa small molecule agonist, and palmitic acid in low-glucose DMEM medium until day 38. As a control, we cultured immature CMs in RPMI B27- base medium. We evaluated the expression of cardiac maturation markers by RT-qPCR. These included structural components, metabolic genes, and specific ion channels. We found that the maturation medium significantly upregulated the expression of metabolism (COX6A2, CPT1B) and ion transport (RYR2, ATP2A2, CX43), and enhanced the structural proteins isoform switch from immature to mature (MYL7 to MYL2 and TNNI1 to TNNI3). Using quantitative bioinformatic analysis, we evaluated a key property of mature CMs, which is polynucleation/poliploidy. By DAPI staining of CMs in maturation vs control media, we confirmed that CMs in the mature media have a greater population of 4n nuclei. Finally, we evaluated the proliferative capacity of both conditions, with the control population yielding a near 1.5 fold in the number of cells at the end of the protocol.In conclusion, we show that this protocol robustly generated pure mature like hIPSCs-CMs in 40 days with well-defined sarcomere structures and key maturative traits, generating a unique model to target key questions about cardiac regeneration.