Establishment and characterization of hTERT-immortalized rat bone marrow mesenchymal stromal cells with preserved
Yuan Chen1, Huimin Xiao1, Weimian Xu1
1State Key Laboratory of Bioreactor Engineering, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, East China University of Science and Technology, Shanghai, 200237 China.
Abstract:
Background The limited proliferative lifespan and progressive senescence of primary mesenchymal stromal cells (MSCs) restrict their long-term expansion and experimental reproducibility. Establishing immortalized MSC lines with preserved biological properties may provide a useful cell resource for basic and preclinical studies. Methods Rat bone marrow-derived MSCs (rBMSCs) were transduced with human telomerase reverse transcriptase (hTERT). The resulting cells were characterized by morphology, hTERT expression, telomerase activity, proliferation, senescence-associated changes, immunophenotype, multilineage differentiation, paracrine-related assays, and tumorigenicity-related assays. Their in vivo activity was further evaluated in an acute dextran sulfate sodium (DSS)-induced colitis model in mice. Results hTERT-rBMSCs maintained a fibroblast-like morphology during long-term culture and showed stable hTERT expression with increased telomerase activity. Compared with late-passage primary rBMSCs, hTERT-rBMSCs exhibited enhanced proliferative capacity, reduced senescence-associated β-galactosidase staining, and lower expression of senescence-related genes. The immortalized cells retained typical MSC surface marker expression and multilineage differentiation potential. Under the tested experimental conditions, hTERT-rBMSCs did not exhibit detectable anchorage-independent growth in soft agar or tumor formation in nude mice. In the DSS-induced colitis model, hTERT-rBMSCs alleviated body weight loss, reduced disease activity, improved colon length and histological appearance, and decreased colonic inflammatory cytokine expression. Their therapeutic effects were broadly comparable to those of primary rBMSCs. Conclusions hTERT-mediated immortalization generated a rat BMSC population with extended proliferative capacity, delayed senescence, and preserved phenotypic and functional characteristics. Under the present experimental conditions, hTERT-rBMSCs retained anti-colitis activity in vivo and may serve as a useful cell resource for studies requiring stable and expandable MSCs.
