Figure 5.

Intracellular GSH Levels Modulate the Self-Renewal and Migration Activities of Mesenchymal Stem Cells

(A) Sorting of hES-MSCs according to the F₅₁₀/F₅₈₀ ratio (FR) into three populations: FR^High, FR^Mid, and FR^Low cells. Cells were characterized as described below, following the removal of FreSHtracer.

(B and C) Luminescence-based quantification of GSH in cell lysates (n = 2 independent biological replicates; B) and comparison of FR changes following treatment with 100 μM H₂O₂ (n = 3 cells; C) in FR^High, FR^Mid, and FR^Low hES-MSCs.

(D–I) Analyses of cell proliferation (n = 6 independent biological replicates; D), colony-forming unit fibroblasts (CFU-F; n = 15 independent biological replicates; E), limiting dilution by replating primary CFU colonies (n = 6 independent biological replicates; F), chemotaxis to stromal derived factor-1α (SDF1α; 150 ng/mL, n = 8 independent biological replicates; G), chemotaxis to 10 ng/mL platelet-derived growth factor (PDGF)-AA in the absence or presence of STI571 (0.5 μg/mL), a PDGFR inhibitor (n = 8 independent biological replicates; H), and qPCR of OCT4, SOX2, CXCR4, cMET, PDGFRA, PDGFRB, VEGFR1, and VEGFR2 (n = 8 independent biological replicates; I) in hES-MSCs sorted based on the FR and in unsorted control (naive) cells.

(J–L) Functional role of high GSH levels in hES-MSCs. CFU-F (n = 10 independent biological replicates; J), chemotaxis to 10 ng/mL PDGF-AA (n = 8 independent biological replicates; K), and qPCR assays of stemness and migration-related genes (n = 4 independent biological replicates; L) in control and BSO-treated FR^High hES-MSCs or control and GSH-EE supplemented FR^Low cells.

For all bar graphs, values represent mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; n.s., not significant. Scale bars, 200 μm (G and H). See also Figures S5 and S6.