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Simple Summary: Simple Summary: Mesenchymal stem cells (MSCs) are adult stem cells capable of differentiating into various cell types, including osteoblasts, chondrocytes, and adipocytes. Renowned for their immunomodulatory and regenerative properties, MSCs hold great promise as tools for tissue repair and therapeutic applications. To facilitate the large-scale production of sheep umbilical cord MSCs (UCMSCs), the telomerase reverse transcriptase (TERT) gene was introduced into UCMSCs to promote immortalization. This study provides valuable insights into (a) the proliferation and anti-aging capabilities of transgenic MSCs compared to primary MSCs; (b) the absence of potential tumorigenicity exhibited in TERT-UCMSCs; and (c) the differentiation potential of TERT-UCMSCs. Immortalized MSCs exhibit enhanced proliferation and anti-aging abilities without tumorigenicity concerns, making them a safe immortalized cell line suitable for medical treatments. Mesenchymal stem cells (MSCs) possess significant differentiation potential, making them highly promising in medicine and immunotherapy due to their regenerative capabilities and exosome secretion. However, challenges such as limited cell divisions and complex testing hinder large-scale MSC production. In this study, we successfully established an immortalized MSC line by transfecting the human telomerase reverse transcriptase (TERT) gene into MSCs isolated from pregnant sheep umbilical cords. This approach effectively inhibits cell senescence and promotes cell proliferation, enabling the generation of umbilical cord mesenchymal stem cells (UCMSCs) on a larger scale. Our findings demonstrate that these transfected TERT-UCMSCs exhibit enhanced proliferative capacity and a reduced aging rate compared to regular UCMSCs while maintaining their stemness without tumorigenicity concerns. Consequently, they hold great potential for medical applications requiring large quantities of functional MSCs. [ABSTRACT FROM AUTHOR]