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

BACKGROUND 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.AIM 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.METHODS 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.RESULTS 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.CONCLUSION 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.

Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment

BACKGROUND Mesenchymal stem cells(MSCs)are a type of stem cells that possess relevant regenerative abilities and can be used to treat many chronic diseases.Diabetes mellitus(DM)is a frequently diagnosed chronic disease characterized by hyperglycemia which initiates many multisystem complications in the long-run.DM patients can benefit from MSCs transplantation to curb down the pathological consequences associated with hyperglycemia persistence and restore the function of damaged tissues.MSCs therapeutic outcomes are found to last for short period of time and ultimately these regenerative cells are eradicated and died in DM disease model.AIM To investigate the impact of high glucose or hyperglycemia on the cellular and molecular characteristics of MSCs.METHODS Human adipose tissue-derived MSCs(hAD-MSCs)were seeded in low(5.6 mmol/L of glucose)and high glucose(25 mmol/L of glucose)for 7 d.Cytotoxicity,viability,mitochondrial dynamics,and apoptosis were deplored using specific kits.Western blotting was performed to measure the protein expression of phosphatidylinositol 3-kinase(PI3K),TSC1,and mammalian target of rapamycin(mTOR)in these cells.RESULTS hAD-MSCs cultured in high glucose for 7 d demonstrated marked decrease in their viability,as shown by a significant increase in lactate dehydrogenase(P<0.01)and a significant decrease in Trypan blue(P<0.05)in these cells compared to low glucose control.Mitochondrial membrane potential,indicated by tetramethylrhodamine ethyl ester(TMRE)fluorescence intensity,and nicotinamide adenine dinucleotide(NAD+)/NADH ratio were significantly dropped(P<0.05 for TMRE and P<0.01 for NAD+/NADH)in high glucose exposed hAD-MSCs,indicating disturbed mitochondrial function.PI3K protein expression significantly decreased in high glucose culture MSCs(P<0.05 compared to low glucose)and it was coupled with significant upregulation in TSC1(P<0.05)and downregulation in mTOR protein expression(P<0.05).Mitochondrial complexes I,IV,and V were downregulated profoundly in high glucose(P<0.05 compared to low glucose).Apoptosis was induced as a result of mitochondrial impairment and explained the poor survival of MSCs in high glucose.CONCLUSION High glucose impaired the mitochondrial dynamics and regulatory proteins in hAD-MSCs ensuing their poor survival and high apoptosis rate in hyperglycemic microenvironment.