Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes

Abstract

Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of RA during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts and erythroid progenitors. Different concentrations of RA (Control without RA, LowRA with 0.05 μM and HighRA with 0.1 μM) were administered during third to sixth days of the differentiation process. EHTs were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. EHTs were further matured and grown in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPCR and immunofluorescence staining analysis, while RNA sequencing was conducted on EHTs to examine how RA affects gene expression associated with function and structure of EHTs. Results: HighRA-hiPSC-CMs exhibited highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are transcription factors associated with left ventricular development, was also the highest in the HighRA-hiPSC-CM. The expression of TBX5 and NKX2.5 was also found to be highest in the HighRA-EHTs, while expression of right ventricular transcrition factors TBX20 and ISL1 was highest in control-EHTs. RNAseq of EHTs showed augmented extracellular matrix gene expression in HighRA-EHTs. The HighRA-EHTs were functionally most similar to left ventricle with the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paves the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine. German Center for Cardiovascular Research (DZHK81Z0600207 to D.M. and P.S.) China Scholarship Council (CSC202108410141) and Henan Provincial Medical Science and Technology Research Project. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.