Effect of Smoking and Culture Rejuvenation on Mesenchymal Stem Cell Quality From Veterans with Peripheral Artery Disease and Diabetes
Carson Hoffmann1, Maiko Teichmann1, Feifei Li2, Luke Brewster3
1Atlanta VA Medical Center; Emory University Hospital, Atlanta, GA; 2Emory University Hospital, Atlanta, GA; 31582 Mason Mill Road Northeast, Atlanta, GA
Introduction: Peripheral artery disease (PAD) is a significant age-related disease that occurs earlier in persons with diabetes. Diabetes increases a patientsírisk of major amputation 8 fold, and diabetic patients who smoke have 15-20 fold risk of major amputation. Together, this landscape suggests a defect in reparative function in patients with diabetes. Mesenchymal stem cells (MSC) are a robust reparative cell that may be useful in regenerative therapies. Mesenchymal stem cell quality can be measured in vitro by their proliferation rate (doubling time [DT]) and colony forming units (CFU). The objective of this work was to discover the impact of smoking on MSC quality, and to determine if human platelet lysate (PL) is effective in rejuvenating MSCs over time compared to fetal bovine serum (FBS).Methods: MSC from 11 consecutive diabetic male Veterans (5 active smokers) with PAD were isolated from the tibia during lower extremity amputation under an IRB approved protocol. MSCs were confirmed by immunophenotyping and expanded for each Veteran in 10% FBS or 5% PL. CFU were performed by serial dilution. DT was calculated over passages 2-5 in parallel culture experiments, and MSCs were passaged while subconfluent. Paired students t-test was used to compare differences between FBS and PL culture supplementation. Unpaired students t-test were used to compare active smokers and non-smokers. Results are presented as mean average (standard deviation).Results: Smoking significantly retarded DT (3.5[0.8] versus 1.8[0.6]; P=.01. For non-smokers, PL conferred a faster DT versus FBS (1.3 [1.2] versus 2.0 [1.3] days; P=.0003), but PL was not significantly faster in active smokers (2.0 [1.5] versus 3.3 [2.2]; P=.07). When comparing the effects of smoking on doubling time, there were no significant differences in PL or FBS groups (PL: 1.3 non-smokers versus 2.0 smokers; P=.15. FBS: 2.0 non-smokers versus 3.3 smokers; P=.053). There was no difference between PL and FBS in CFUs in non-smokers (87.7 [32.8] vs. 57.1 [13.9], p = 0.14) or smokers (74.7 [56.9] vs. 75.5 [18.1], p = 0.97).Conclusion: In Veterans with PAD and diabetes, smoking impairs MSC DT but not CFU. PL improves MSC DT in non-smokers, but not in active smokers. Culture rejuvenation techniques may be limited by active smoker status, and it is likely that active smoking status confers additional disruption to cell proliferation pathways already dysregulated by diabetes. Potentially, these results may also support a functional connection between MSC senescence and increased risk of major amputation in diabetic smokers.
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