Dynamic analysis of metabolic and ultrastructural changes in mesenchymal stem cells at hypoxic preconditioning and post-preconditioning stages: Cobalt chloride on the spotlight

Mesenchymal stem cells (MSCs) have come up as a potential remedy for treatment of various diseases thanks to their regenerative abilities. However, MSC-based therapies face challenges like reduced cell survival and functionality after transplantation. Preconditioning, particularly with hypoxia-mimicking agents like cobalt chloride (CoCl₂), has been explored to enhance MSCs' effectiveness. This study aims to evaluate MSC survival, migration, and therapeutic outcomes at the CoCI₂-preconditioning and post-preconditioning stages. Human umbilical cord-MSCs were treated with 100 µM CoCI₂ with/out serum for 24-hours, and then passaged and planted in corresponding culture conditions without CoCI₂, these two consecutive passages were named as the preconditioning and post-preconditioning stages, respectively. In each stage, total protein concentrations, total antioxidant and total oxidant status (TAS/TOS) of the conditioned media derived from the cells were investigated with bicinchoninic acid assay and TAS/TOS kits, respectively. The proliferation rates, migratory capacities, cellular senescence, expression levels of hypoxia-inducible factor1-α (HIF1-α), Ki-67, active caspase-3 and beclin-1 proteins and ultrastructures of the cells were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide test, wound healing assay, β-galactosidase-activity assessment, immunocytochemistry and transmission electron microscopy, respectively. Our results indicated that preconditioning MSCs with CoCl₂ did not significantly enhance their proliferation, migration, or secretory abilities. However, it increased antioxidant capacity and along with normalization of senescence-status post-preconditioning, possibly by shifting energy metabolism from oxidative-phosphorylation to glycolysis through the upregulation of the HIF1-α signalling pathway. These findings indicate that CoCl₂ preconditioning could be an effective approach to boost the therapeutic potential of MSCs, especially in enhancing their survival and functionality after transplantation.