Inhibition of GSK-3β Restores Differentiation Potential of Late-Passage Mesenchymal Stem Cells

<b>Background/Objectives:</b> Mesenchymal stem cells (MSCs) are regarded as a promising cell type with significant therapeutic benefits owing to their ease of isolation, maintenance, and characterisation. However, repeated passages during cultural maintenance frequently result in cellular senescence, limiting their utility in regenerative medicine. <b>Methods:</b> We investigated the differentiation capability between early- (P3) and late-passage MSCs (>P15) and tested the potential of Wnt agonist 99021 to reverse MSCs using standard cell culture protocols that define minimal criteria for MSCs, primarily tri-lineage differentiation assays, biochemical staining gene expression analysis, and senescence assays. <b>Results:</b> We initially noticed distinct signs of morphological aging between early- (P3) and late-passage MSCs (>P15) and further examined the differentiation capability between early- (P3) and late-passage MSCs (>P15). We found a diminished differentiation potential in late-passage MSCs. Our senescence assay also revealed >P15 cells were able to absorb the senescence dye, indicating that >P15 MSCs underwent senescence. We further demonstrated that CHIR 99021 reversed the differentiation inhibitory potential-mediated impasse of late-passage MSCs by employing tri-lineage specific differentiation assays, biochemical labelling, and gene expression analysis. Senescence assays after CHIR 99021 treatment also revealed no senescence dye uptake at all. <b>Conclusions:</b> Our findings demonstrated that CHIR 99021 Wnt agonist maybe aids in the reversal of MSC aging-related differentiation inhibition glitches and offers a proven demonstrated protocol for rejuvenating late-passage MSCs. Thus, CHIR99021 treatment inherently reverts the tri-lineage potency in late-passage MSCs, and this method could be further employed to ensure a plentiful MSC source for clinical purposes.