Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages

Mesenchymal stem cells (MSCs) have been extensively studied for therapeutic potential, due to their regenerative and immunomodulatory properties. Serial passage and stress factors may affect the biological characteristics of MSCs, but the details of these effects have not been recognized yet. To investigate the effects of stress factors (high glucose and severe hypoxia) on the biological characteristics of MSCs at different passages, in order to optimize the therapeutic applications of MSCs. In this study, we investigated the impact of two stress conditions; severe hypoxia and high glucose on human adipose-tissue derived MSCs (hAD-MSCs) at passages 6 (P6), P8, and P10. Proliferation, senescence and apoptosis were evaluated measuring WST-1, senescence-associated beta-galactosidase, and annexin V, respectively. Cells at P6 showed decreased proliferation and increased apoptosis under conditions of high glucose and hypoxia compared to control, while the extent of senescence did not change significantly under stress conditions. At P8 hAD-MSCs cultured in stress conditions had a significant decrease in proliferation and apoptosis and a significant increase in senescence compared to counterpart cells at P6. Cells cultured in high glucose at P10 had lower proliferation and higher senescence than their counterparts in the previous passage, while no change in apoptosis was observed. On the other hand, MSCs cultured under hypoxia showed decreased senescence, increased apoptosis and no significant change in proliferation when compared to the same conditions at P8. These results indicate that stress factors had distinct effects on the biological processes of MSCs at different passages, and suggest that senescence may be a protective mechanism for MSCs to survive under stress conditions at higher passage numbers.