The present study focused on the culture medium of induced pluripotent stem cells (iPSCs) prior to the use of cardiomyocytes differentiation induction medium (pre-culture medium). Seven types (Nos. 1-7) of StemFit AK03 medium (Ajinomoto) for clinical iPSCs with varying compositions were prepared as pre-culture medium. The cardiac muscle troponin T (cTnT) positivity of No. 1 (StemFit AK03 medium) was 84%, No. 3 (similar to E8 medium) was 89%, No. 2 (similar to E8 medium) was 91%, No. 5 (similar to EB Formation medium) was 95%, when using differentiation induction medium prepared with known components available for clinical cell production. The formation of cardiac tissues was assessed by evaluating the expression levels of specific markers, including cTnT, atrial natriuretic peptides (ANP), and pro-B-type natriuretic peptide (proBNP). The results demonstrated that cardiac tissue with high protein expression levels of cTnT and ANP was formed when similar to E8 medium as pre-culture medium.
© 2025. The Author(s).

Reactivating the human epicardium post-cardiac injury holds promise for cardiac tissue regeneration. Despite successful differentiation protocols yielding pure, self-renewing epicardial cells from induced pluripotent stem cells (iPSCs), these cells maintain an embryonic, proliferative state, impeding adult epicardial reactivation investigation. We introduce an optimized method that employs mammalian target of rapamycin (mTOR) signaling inhibition in embryonic epicardium, inducing a quiescent state that enhances multi-step epicardial maturation. This yields functionally mature epicardium, valuable for modeling adult epicardial reactivation. Furthermore, we assess cardiac organoids with cardiomyocytes and mature epicardium, probing molecular mechanisms governing epicardial quiescence during cardiac maturation. Our results highlight iPSC-derived mature epicardium's potential in investigating adult epicardial reactivation, pivotal for effective cardiac regeneration. Additionally, the cardiac organoid model offers insight into intricate cardiomyocyte-epicardium interactions in cardiac development and regeneration.
© 2025. The Author(s).

C-C chemokine receptor type 2 (CCR2-) cardiac-resident macrophages (CCR2- cRMs) are known to promote cardiac repair after myocardial infarction (MI). However, the substantial depletion and slow recovery of CCR2- cRMs pose significant barriers in cardiac recovery. Here, we construct a functional conductive cardiac patch (CCP) that can provide exogenously elastic conductive microenvironment and induce endogenously reparative microenvironment mediated by CCR2- cRMs for MI repair. This CCP exhibits suitable mechanical properties, conductivity, and high water retention, reminiscent of natural myocardium, which can actively engage in modulating CCR2- cRM renewal and their cell crosstalk. The functional CCP can promote the expression of Connexin43 between CCR2- cRMs and cardiomyocytes (CMs) and regulate paracrine signaling to activate epicardial cell epithelial-to-mesenchymal transition (EMT) toward endothelial cells using rat and Wt1CreERT2 transgenic lineage tracing mice. Overall, this study provides a promising strategy to construct a synergistic reparative microenvironment for MI repair.
Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.

Loss of Bcl2-associated athanogene 3 (BAG3) is associated with dilated cardiomyopathy (DCM). BAG3 regulates sarcomere protein turnover in cardiomyocytes; however, the function of BAG3 in other cardiac cell types is understudied. In this study, we used an isogenic pair of BAG3-knockout and wild-type human induced pluripotent stem cells (hiPSCs) to interrogate the role of BAG3 in hiPSC-derived cardiac fibroblasts (CFs). Analysis of cell type-specific conditional knockout engineered heart tissues revealed an essential contribution of CF BAG3 to contractility and cardiac fibrosis, recapitulating the phenotype of DCM. In BAG3-/- CFs, we observed an increased sensitivity to TGF-β signaling and activation of a fibrogenic response when cultured at physiological stiffness (8 kPa). Mechanistically, we showed that loss of BAG3 increased transforming growth factor-β receptor 2 (TGFBR2) levels by directly binding TGFBR2 and mediating its ubiquitination and proteasomal degradation. To further validate these results, we performed single-nucleus RNA sequencing of cardiac tissue from DCM patients carrying pathogenic BAG3 variants. BAG3 pathogenic variants increased fibrotic gene expression in CFs. Together, these results extend our understanding of the roles of BAG3 in heart disease beyond the cardiomyocyte-centric view and highlight the ability of tissue-engineered hiPSC models to elucidate cell type-specific aspects of cardiac disease.

Untethered electrical stimulation or pacing of the heart is of critical importance in addressing the pressing needs of cardiovascular diseases in both clinical therapies and fundamental studies. Among various stimulation methods, light illumination-induced electrical stimulation via photoelectric effect without any genetic modifications to beating cells/tissues or whole heart has profound benefits. However, a critical bottleneck lies in the lack of a suitable material with tissue-like mechanical softness and deformability and sufficient optoelectronic performances toward effective stimulation. Here, we introduce an ultrathin (<500 nm), stretchy, and self-adhesive rubbery bio-optoelectronic stimulator (RBOES) in a bilayer construct of a rubbery semiconducting nanofilm and a transparent, stretchable gold nanomesh conductor. The RBOES could maintain its optoelectronic performance when it was stretched by 20%. The RBOES was validated to effectively accelerate the beating of the human induced pluripotent stem cell-derived cardiomyocytes. Furthermore, acceleration of ex vivo perfused rat hearts by optoelectronic stimulation with the self-adhered RBOES was achieved with repetitive pulsed light illumination.