In Vivo Bioincubation Promotes Maturation of Human iPSC-Derived Cardiomyocytes in Neonatal Rat and Pig Hearts

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac regenerative medicine and disease modeling. However, hiPSC-CMs generated through conventional in vitro differentiation exhibit immature, fetal-like phenotypes. While in vivo bioincubation in neonatal rodent hearts promotes hiPSC-CM maturation toward adult-like phenotypes, studies in large animal models remain limited, particularly with detailed morphological characterization. In this study, we investigated bioincubation of fluorescently labeled hiPSC-CMs in both neonatal rat and pig hearts. Human iPSCs were differentiated into cardiomyocytes expressing GFP or RFP reporters and subsequently injected intramyocardially into neonatal rats (GFP-labeled) and pigs (RFP-labeled). After 4-8 weeks of bioincubation, fluorescent hiPSC-CMs were isolated using large-particle fluorescence-activated cell sorting (COPAS), which preserves cellular morphology of adult-like cardiomyocytes. Immunostaining for cardiac troponin T revealed well-organized sarcomeric structures in multinucleated hiPSC-CMs. Bioincubated hiPSC-CMs displayed rod-shaped morphology with binucleation, characteristic features of mature adult cardiomyocytes. Quantitative analysis demonstrated that bioincubated hiPSC-CMs from rat hearts exhibited sarcomere length and cell circularity comparable to native rat adult cardiomyocytes, though with higher intra-cellular variability in sarcomere organization. Histological examination confirmed successful engraftment of RFP-positive hiPSC-CMs within pig myocardium, with engrafted cells also displaying mature adult-like features. These findings provide critical proof-of-concept data for bioincubation in large animal models and support further investigation for disease modeling, drug screening, and regenerative cell therapies. SIGNIFICANCE STATEMENTHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for cardiac disease modeling and regenerative therapies, but their clinical application is limited by their immature characteristics. Here we show that in vivo bioincubation in neonatal rat hearts enables hiPSC-CMs to achieve structural maturity, exhibiting features of adult cardiomyocytes, including organized sarcomeres, rod-shaped morphology, and multinucleation. We further provided proof-of-concept evidence for engraftment in neonatal pig hearts for maturation, supporting feasibility in large animal models. The use of large-particle cell sorting enables recovery of intact, adult-sized cardiomyocytes for subsequent analysis. These findings establish a practical and scalable platform for generating structurally mature human cardiomyocytes through in vivo bioincubation.