Retinoic  acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes
Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart and high concentrations of RA have been shown to steer differentiation towards atrial CMs, whereas lower concentrations of RA promote a more ventricular-like CM profile. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of retinoic acid (RA) during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. The maturation and growth of EHTs were induced in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. The function of CMs and EHTs was analyzed under different conditions. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPRC and immunofluorescence staining analysis. RNA sequencing was conducted on EHTs to examine how RA affects both the function and structure of EHT. Results: In the HRA group, hiPSC-CMs exhibited the first onset of beating and showed the highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are the marker genes for left ventricular CMs, was also the highest in the HRA group. The transcription factor MEF2C associated with RA and ventricular development genes, NPPA and MYH7, were highly expressed in the HRA group, while GATA4 was less expressed in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was more functionally similar to the left ventricle. The expression of TBX5 and NKX2.5 was found to be the highest expression in the HRA group of EHT, while expression of TBX20 and ISL1 was found to be the highest expression in control group. When the electrical stimulation frequency of EHT in the HRA group was raised, it correspondingly increased its contractile force. The heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. 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 paved the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine. Overall design: To investigate molecular changes underlying the improved organisation of the EHTs derived from hiPSC-CMs previously exposed to RA. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the hiPSCs differentiation process. Comparative gene expression profiling analysis of RNA-seq data for EHT from different group.