Isolation and enrichment of FHF-like CMs using the surface marker CORIN. (A) Heatmap showing co-expression of cardiac contraction and surface marker genes in TBX5⁺ and TBX5⁻ populations at different time points (three biological replicates per group). (B) CORIN and POPDC2 expression assessed using the (top) Kaessmann online database⁴⁷ (one-way ANOVA, P-value (heart vs. other tissues, <0.0001), biological replicates: brain (n = 52), cerebellum (n = 58), heart (n = 44), kidney (n = 36), liver (n = 49), ovary (n = 18), testis (n = 39) for organ specificity, (middle) single-cell RNA sequencing analysis of human foetal heart (10-week gestation) for CM specificity (one biological replicate), and (bottom) Day 20 FHF- and SHF-like CMs for heart field specificity (pool of three biological replicates). (C) qPCR analysis of CORIN expression in (left) FHF and SHF-like CMs (three biological replicates, three technical replicates each) and (right) human foetal LV and RV (18-week gestation, one biological replicate, three technical replicates each). (D) Flow cytometric analysis of CORIN⁺ cells for TNNT2 expression (pool of 3 biological replicates). (E) Flow cytometric analysis of TBX5 and NKX2-5 in CORIN⁺ and CORIN⁻ cells (pool of three biological replicates) using FHF (top) and SHF (bottom) protocols. (F) Quantitative PCR analysis of FHF (TBX5) and SHF (ISL1) in CORIN⁺ and CORIN⁻ Day 10 sorted cells (three biological replicates, three technical replicates each). (G) Immunocytochemistry of Day 10 sorted CORIN⁺ CMs for markers of FHF (TBX5), SHF (ISL1), and nodal cells (SHOX2). Cardiomyocytes are shown by TNNT2 staining and all nuclei were shown by DAPI. Scale bar = 20 μm. (H) Snapshots of optical mapping show spontaneous AP propagation across monolayer of CORIN⁺ CMs. The yellow arrow denotes direction of AP propagation. Numbers denote the area from which the APs were recorded. Red and pink represent depolarized and repolarized phases, respectively (pool of three biological replicates). Statistical analysis is performed by using Student’s t-test, *P < 0.05, **** P < 0.0001, ns = not significant. Error bar = SEM.