Activation of LIMK/Cofilin signaling pathway via extracellular matrix-integrin interaction is critical for generation of mature and vascularized cardiac organoids

Abstract The generation of mature and vascularized human pluripotent stem cell-derived cardiac organoids (hPSC-COs) is necessary to ensure the validity of drug screening and disease modeling. This study investigates the effects of cellular aggregate (CA) stemness and self-organization on the generation of mature and vascularized hPSC-COs and elucidates the mechanisms underlying CO maturation and vascularization. H-COs were generated from early CAs with high stemness, and L-COs were generated from late CAs with low stemness under a self-organized microenvironment by Wnt signaling induction. The study finds that H-COs exhibit ventricular, structural, metabolic, and functional maturation of cardiomyocytes and vessel networks consisting of endothelial cells, smooth muscle cells, pericytes, and basement membranes compared to L-COs. Transcriptional profiling shows the upregulation of genes associated with cardiac maturation and vessel formation in H-COs compared with those in L-COs. Activation of ROCK-LIMK-pCofilin via ECM–integrin interaction leads to cardiomyocyte maturation and vessel formation in H-COs through experiments with LIMK inhibitors. Furthermore, the LIMK/Cofilin signaling pathway induces TGFβ/NODAL and PDGF pathway activation for the maturation and vascularization of H-COs. The study demonstrates for the first time that LIMK/Cofilin axis activation plays an important role in the generation of mature and vascularized COs.