DNA Damage Levels and DNA Repair Capability of Bone Marrow‐Derived Mesenchymal Stem Cells from Sprague Dawley® SPF rats as a Quality Parameter Before Surgical Transfection for the Treatment of Gestational Diabetic Myopathy

Abstract

Mesenchymal stem cells (MSCs) have an important role for regenerative medicine through their self‐renewal and differentiation capability; besides its involvement in tissue development, maintenance, and repair, being useful for skeletal muscle regeneration by the production of secretory factors with trophic and engraftment functions. We propose to use the alkaline comet assay to evaluate the DNA damage levels and DNA repair capability according to cell culture passages as one of the parameters to test the quality of MSCs before surgical transplantation to the periurethral and rectus abdominis muscle (RAM) for the treatment of gestational diabetic myopathy in female rats at postpartum. MSCs were collected from bone marrow of female Sprague Dawley® rats. The characterization of these cells was done according to the International Society for Stem Cell. After isolation using Ficoll‐Paque and washing in low glucose Dulbecco's Modified Eagle Medium (DMEM) enriched with L‐glutamine, the cells were seeded in 25 cm2 plastic flasks containing high glucose DMEM enriched with L‐glutamine plus 20% bovine fetal serum, penicillin, streptomycin and amphotericin B. The cells were harvested at 90% of confluence and subjected to comet assay analysis. The slides were incubated in alkaline electrophoresis solution for 20 minutes for DNA unwinding and the run was conducted at 25v, 0.3A and 8W for 30 minutes, at 4 °C. The slides were stained with SYBR GOLD® (1:10.000 dilution) and analyzed in a fluorescent microscope using COMET ASSAY VI software. The DNA damage levels were ranked according to the percentage of damage as follows: 1) 0 % – 5% of damage; 2) 6% – 20% of damage; 3) 21% – 40% of damage; 4) 41% –95% of damage; 5) > 95% of damage. The percentage of nucleoids with such levels of damage was calculated and analyzed using the Chi‐Square test. The analysis was conducted using SPSS (21.0) and p < 0.05 was considered significant. This study was approved by the local Animal Ethics Committee (1234/2017 – CEUA). The partial results demonstrated that the 5th passage has a high percentage (70.04%) of cells with high levels of DNA damage (41% – 95%) assessed by tail intensity. The proportion of cells with high percentage of DNA damage (41% – 95%) is statistically significant from the proportion of cells with low DNA damage levels (0% – 5%, 6% –20%, and 21% –40%; p < 0.001). The preliminary results demonstrated that the 5th passage is not useful for surgical transplantation because of high DNA damage levels. Future experiments will be conducted to test the DNA damage of the other passages and also evaluated the effect of cell culture on DNA repair capability. Furthermore, such results will be compared with the functional evaluations of periurethral and RAM after the surgical transplantation of MSC.Support or Funding InformationSupported by São Paulo Research Foundation (2016/01743‐5)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.