Dose-Dependent Effects of Metformin Preconditioning on Metabolic, Clonogenic, and Differentiation-Associated Responses in Human Umbilical Cord-Derived Mesenchymal Stromal Cells: An Exploratory In Vitro Study

Background: Human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) are used in regenerative research, but culture performance varies across donors. Metformin may modulate cellular metabolism and lineage-associated responses, although its effects are dose- and context-dependent. Therefore, this study aimed to evaluate the effects of metformin on metabolic, clonogenic, expansion, and differentiation responses in UC-MSC cultures. Methods: Wharton’s jelly cultures were established from individual umbilical cords without pooling. Qualitative immunofluorescence supported a stromal phenotype. 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) metabolic activity was assessed at 0–1000 µM metformin (n = 10 donors); colony-forming unit–fibroblast (CFU-F)/plating efficiency at 0, 100, 500, and 1000 µM (n = 9); interval population doubling (PD) and descriptive population doubling time (PDT) across P3–P6 (n = 6 complete donors); exploratory osteogenic and neurogenic induction-associated morphology after 100 µM preconditioning (n = 9). Within-donor comparisons used Friedman tests and exact paired Wilcoxon tests with Holm adjustment. Results: XTT signal varied across concentrations (χ2 (6) = 57.88, p < 0.001; Kendall’s W = 0.965), peaking at a median of 106.39% of control at 100 µM and declining to 95.51% at 1000 µM. CFU-F/plating efficiency also differed (χ2 (3) = 19.99, p < 0.001; W = 0.740): medians were 5.23% in controls, 6.60% at 100 µM (Holm p = 0.039), 4.57% at 500 µM (p = 0.039), and 3.13% at 1000 µM (p = 0.035). PD showed ordered dose patterns at each passage (Holm-adjusted omnibus p < 0.001), but no dose-versus-control contrast remained significant after the 12-comparison Holm adjustment. Osteogenic scores did not differ (p = 0.445); the neurogenic-associated score was higher after 100 µM (p = 0.008) but remained exploratory. Conclusions: Metformin was associated with concentration-dependent phenotypic responses in UC-MSCs. The 100 µM condition produced the highest XTT signal and CFU-F/plating efficiency, whereas higher concentrations were associated with lower clonogenic and expansion measures. These findings do not establish mature neuronal differentiation, a molecular mechanism, or therapeutic efficacy and require confirmation in rigorously characterized models.