Metabolic profiling of licensed mesenchymal stromal cells reveals a disconnect between glycolytic fitness and immunomodulatory responsiveness

Mesenchymal stromal cells (MSCs) hold potential as cell-based treatments, but their efficacy is compromised by cryopreservation. Inflammatory licensing can boost MSC functionality, yet protocols are inconsistent and their impact on cellular metabolism is unclear. Our research evaluated mitochondrial and glycolytic activity in MSCs derived from umbilical cord (UC) and bone marrow (BM) tissues following licensing with interferon-gamma (IFN-γ), tumor necrosis factor- (TNF-), or both.

Short exposure to either cytokine altered MSC metabolism towards glycolysis and decreased mitochondrial respiration, while prolonged exposure produced varied outcomes. In contrast, temporary exposure to both IFN-γ and TNF- heightened glycolytic flux and respiratory capacity in most donors, detectable within 2 hours and at concentrations lower than typical use.

Cryopreservation disrupted aerobic metabolism, followed by donor-specific recovery. Brief preconditioning with combined IFN-γ and TNF- partially enhanced post-thaw metabolism, particularly in BM MSCs, and bolstered early inflammatory responsiveness, particularly in UC MSCs, as indicated by IDO and candidate biomarkers CTSS and C1S.

However, donors exhibiting improved post-thaw metabolism did not invariably display heightened biomarker responses. These results underscore that metabolic fitness and induced biomarkers provide distinct insights into post-thaw MSC quality, potentially guiding preconditioning methods for MSC products derived from cryopreserved cells.