Preconditioning mesenchymal stromal cells with physioxia and resveratrol enhances mitochondrial biogenesis

Mesenchymal stromal cells (MSCs) are adult progenitor cells displaying multi-lineage differentiation potential. They can be isolated from adult tissues through minimally invasive procedures and are promising for regenerative medicine due to their immunomodulatory properties and paracrine effects. Mitochondria are increasingly recognized as a cornerstone of MSC regenerative mechanisms. Therefore, stimulation of mitochondrial biogenesis and function could promote MSC potency. Standard in vitro expansion conditions using atmospheric oxygen (~21% O₂) fail to replicate the physiological microenvironments MSCs naturally reside in, characterized by oxygen tensions between 2–7% O₂. Physioxic culture conditions or treatment with resveratrol, a natural polyphenol, are known to modulate mitochondrial function. In our experiments, we evaluated effects of resveratrol concentration, treatment duration, and oxygen tension on mitochondrial density and function. Among different concentrations tested (1-20 μM), treatment with both 2.5 and 5 μM resveratrol was sufficient to increase mitochondrial biogenesis, as evidenced by increased organelle density assessed by MitoTracker staining and confocal microscopy. In further experiments, both physioxia (5% O2) and resveratrol (5μM) treatment for 48h independently enhanced mitochondrial biogenesis in MSCs from multiple donors. Consistently, these findings correlated with elevated mitochondrial respiration as detected by Seahorse Mito Stress test. Complementary analyses, including mtDNA copy number quantification and Western blotting for mitochondrial proteins may further substantiate these observations. In addition, data from quantitative metabolomic profiling of the same samples characterize the metabolic differences in stimulated versus non-stimulated MSCs. This mitochondrial enrichment may result in improved metabolic fitness and energy efficiency within the cells, which potentially enhances their therapeutic performance in regenerative applications. These findings suggest that simple modifications to cell culture protocols, such as oxygen level adjustment and bioactive compound supplementation, can fundamentally tune MSC function.

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