Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.