Diabetes is associated with impaired wound healing. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment and p53 gene silencing prevents cellular senescence and helps vascular regeneration. Previously we have shown that p53 silenced progenitor cell therapy helped tissue regeneration. Our goal is to test whether conditioned media obtained from p53 silenced human stem cells can also improve cellular regeneration.
Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control in bone marrow derived mesenchymal stromal cells. To collect conditioned mediaBM-MSCs were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% exosome free FBS for only 48h and the collected supernatant was concentrated 10-fold to obtain the conditioned media (CM). Endothelial regeneration was tested using endothelial wound healing assay kit ( from Cell Biolabs # CBA-120-T).
Results: We compared CM obtained from null and p53sh in adipose and bone marrow derived mesenchymal stem cells on human endothelial cells in normal and high glucose conditions. Proliferation capacity of the p53sh CM was 2-3 fold more compared to null and non-transduced CM. The CM obtained from the untreated and null samples did not show increased proliferation in any of the cells tested. We are currently analyzing the secretome by mass spectroscopy.
Conclusion: P53 silencing appears to improve CM obtained from p53 silenced cells and may have therapeutic role in treating diabetes complications such as diabetic wound healing. [NSR1]is [NSR2]delete
S. Nandula: None. A. Argaw: None. S. Sen: None.
