Reprogramming of Human Urine Cells into Cardiomyocytes via a Small Molecule Cocktail in Xeno-Free Conditions

BackgroundCell therapy, particularly using cardiomyocytes, shows significant promise for treating heart failure. Direct reprogramming of somatic cells into cardiomyocytes using small molecules is advantageous due to its efficiency and cost-effectiveness.MethodsThis study demonstrated transdifferentiation of human urine-derived cells (hUCs) into functional cardiomyocyte-like cells (hCiCMs) using a cocktail of 15 small molecules under xeno-free conditions. Various Characterizations were performed, including immunofluorescence, transmission electron microscopy (TEM), qPCR, single-cell RNA sequencing, patch-clamp recordings, and intracellular Ca²⁺ measurements. The therapeutic potential was tested in both mouse and porcine models of myocardial infarction (MI).ResultsReprogramming efficiency achieved 15.08% on day 30, with 96.67% purity on day 60. hCiCMs displayed cardiomyocyte markers, sarcomeric structures, and abundant mitochondria. Electrophysiological analysis confirmed ventricular-like action potentials and regular calcium transients. Single-cell RNA sequencing revealed cardiomyocyte subpopulations resembling 13-week embryonic human heart cells, with gene ontology analysis indicating successful maturation. In the MI model, hCiCM transplantation improved cardiac function, increasing ejection fraction and fractional shortening while reducing fibrosis.ConclusionsThis study demonstrates the successful reprogramming of hUCs into functional hCiCMs using small molecules under xeno-free conditions, offering a scalable, autologous cell source for cardiac repair with significant potential for regenerative therapies.