Biphasic control of retinoic acid signaling promotes left ventricular-like cardiomyocyte differentiation of human pluripotent stem cells

During cardiac development, the left ventricle is predominantly populated by cardiac progenitors from the first heart field. Retinoic acid (RetA) regulates cardiomyocyte subtype differentiation from human pluripotent stem cells through anterior‑posterior axis patterning of the cardiac mesoderm, with high RetA activity in posterior regions promoting atrial and pacemaker cell fates. Although RetA also plays a critical role during early mesoderm induction leading to ventricular differentiation, its precise function and interaction with other cardiogenic cues at this stage remain incompletely understood. In hPSC‑derived cardiac differentiation, cells were treated with a RetA receptor inverse agonist at d0 during mesoderm induction, followed by RetA treatment at d5 at the cardiac progenitor stage. To characterize the differentiated cardiomyocytes (CMs), we examined the expression of first and second heart field marker genes, performed immunostaining for chamber subtype‑specific proteins, and conducted single‑cell patch‑clamp electrophysiology. Contraction/relaxation velocities and contractility were assessed to evaluate functional properties, and hypertrophic responsiveness was tested using endothelin‑1. Finally, bulk RNA sequencing was performed to compare the transcriptomic profiles of the derived CMs with those of fetal human left and right ventricles, including in silico cell‑type deconvolution and CMs‑restricted gene‑set analysis. Biphasic modulation of retinoic acid signaling directs hPSCs toward first heart field (FHF)‑derived ventricular‑like CMs. Early inhibition of RetA signaling followed by subsequent activation promotes differentiation toward ventricular‑like CMs with transcriptomic features aligned with fetal left ventricle‑enriched gene signatures, whereas inhibition alone during mesoderm induction impairs cardiac progenitor formation and activation alone at the progenitor stage favors atrial specification. The resulting cardiomyocytes exhibit molecular signatures and functional properties consistent with ventricular or atrial subtypes, with ventricular‑like cells showing stronger contractility and robust hypertrophic responses to endothelin‑1 stimulation. RetA exerts opposing effects during sequential stages of cardiac differentiation from hPSCs, and biphasic modulation of RetA signaling promotes differentiation toward ventricular‑like CMs with FHF‑associated and fetal left ventricle‑enriched transcriptomic features, providing a simple and broadly applicable approach for chamber‑biased cardiomyocyte generation.