Metabolic Maturation Unveils Left Ventricular Identity in WNT ON/OFF Human Pluripotent Stem Cell-Derived Cardiomyocytes

Deriving high-purity mature left ventricular (LV) cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) is a priority for cardiovascular research and for future therapeutic applications. Small-molecule WNT modulation (WNT ON/OFF) is currently the predominant differentiation method; however, a critical discordance exists regarding its cardiac subtype outcome. While lineage tracing suggests a First Heart Field (FHF) bias, phenotypic characterizations report significant heterogeneity regarding definitive ventricular markers, leading to controversy about the cardiac subtypes generated by this method. Here, we demonstrate that this apparent heterogeneity is a result of CM immaturity. Using single-cell protein analysis, we first show that WNT ON/OFF generates an NKX2.5+ progenitor pool that robustly co-expresses HAND1, confirming uniform FHF specification regardless of differentiation efficiency. We then demonstrate that the CM population negative for the ventricular marker MYL2 observed at early differentiation timepoints mostly represents immature LV cardiomyocytes that have not yet acquired their definitive phenotype. By implementing a targeted metabolic maturation regime, we unlocked this identity, achieving 95% MYL2+/HAND1+/TBX5+ LV CMs by day 38, substantially earlier and with higher chamber-specific purity than previously reported. This phenotypic resolution was accompanied by advanced structural maturation, including sarcomeric protein isoform switching, multinucleation, and notably, the assembly of polarized XIRP2+ intercalated discs, a hallmark of postnatal CM maturation not previously described in 2D differentiations. Validated across three independent hPSC lines, these findings provide the field with a rapid, high-fidelity platform for generating pure mature LV cardiomyocytes for disease modeling and therapeutic research.