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

Background Cell 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. Methods This 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). Results Reprogramming 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. Conclusions This 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.背景 细胞疗法，尤其是采用心肌细胞的细胞疗法，在心力衰竭治疗领域展现出显著应用前景。借助小分子将体细胞直接重编程为心肌细胞的策略，凭借高效性与成本效益优势而极具吸引力。 研究方法 本研究在无异种成分的培养条件下，通过15种小分子组合将人尿液来源细胞（human urine-derived cells, hUCs）成功转分化为具有功能活性的心肌细胞样细胞（cardiomyocyte-like cells, hCiCMs）。研究团队开展了多维度表征分析，涵盖免疫荧光染色、透射电子显微镜（transmission electron microscopy, TEM）、实时定量聚合酶链式反应（qPCR）、单细胞RNA测序、膜片钳记录以及细胞内钙离子浓度检测。同时，本研究在小鼠和猪的心肌梗死（myocardial infarction, MI）模型中验证了该细胞的治疗潜力。 研究结果 该重编程策略在第30天时的转化效率达15.08%，第60天时细胞纯度可达96.67%。所获得的hCiCMs表达心肌细胞特异性标志物，具备典型肌节结构与丰富的线粒体。电生理分析证实其具有心室样动作电位与规律的钙瞬变特征。单细胞RNA测序结果显示，该细胞群体存在与人类胚胎心脏13周龄细胞高度相似的心肌细胞亚群，基因本体论（gene ontology, GO）分析表明其已实现有效成熟。在心肌梗死模型中，移植hCiCMs可显著改善心脏功能，提升射血分数与短轴缩短率，同时减轻心肌纤维化程度。 结论 本研究证实，在无异种成分条件下，通过小分子组合可成功将hUCs重编程为功能成熟的hCiCMs，为心脏修复提供了可规模化制备的自体细胞来源，在再生治疗领域具备重要的应用潜力。