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

Abstract

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 cordMSCs were treated with 100 mu M 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-alpha (HIF1-alpha), 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, beta-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-alpha 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.