Evaluation of pH effects on genomic integrity in adipose-derived mesenchymal stem cells using the comet assay

Abstract

ABSTRACT. The use of mesenchymal stem cells (MSCs) in experimental, clinical, and therapeutic trials has grown in recent years. However, the issue remains of whether these procedures are completely safe for transplant patients. Therefore, this study was designed and carried out with the aim of evaluating two different comet assay protocols for genomic damage pattern analysis in MSCs derived from adipose tissue. The analyzed and interpreted results suggest that genetic testing is needed to support clonal expansion safety in cell therapy procedures with MSCs. Furthermore, they also suggest that if the comet assay technique would be used as a genomic integrity screening assay, the protocol performed at pH = 12 (that yielded a frequency of damaged cells: tail intensity = 9.50 ± 0.60, tail moment = 0.0122 ± 0.0007; results are reported as means ± standard deviation) would be indicated as genomic damage, and that subsequent single-strand breaks occur at pH > 13 (frequency of damaged cells: tail intensity = 30.71 ± 4.23, tail moment = 0.0447 ± 0.0073). Our study demonstrates that, in the era of regenerative medicine, it is necessary to standardize and establish a battery of tests in order to identify genomic damage prior to MSC transplantation.

Mesenchymal stem cell therapy promotes the improvement and recovery of renal function in a preclinical model

“…The MSCs used in the present study were characterized by adipogenic, osteogenic and chondrogenic differentiation. These results are consistent with previous studies (Bagnaninchi et al , 2011; Pendleton et al , 2013; Vieira et al , 2014 ; Hermeto et al , 2015 ), as was the efficiency of the selection and expansion methods used here.…”

“…At the end of the respective culture periods, media were removed and cells were fixed in paraformaldehyde for 1 h. Adipogenic and osteogenic differentiation were assessed by staining with Oil Red O and Alizarin Red S, respectively. Chondrogenic differentiation was assessed by submitting the pellet to conventional histological processing and staining with toluidine blue ( Hermeto et al , 2015 ).…”

Cardanol: toxicogenetic assessment and its effects when combined with cyclophosphamide

“…This result indicated that genotoxic lesions observed in the comet assay were not fixed in the DNA as a permanent DNA damage. Rather, lesions detected by this comet assay, including single- and double-strand breaks, alkaline-labile sites, crosslinks, excision repair sites, methylation damage and adducts ( Singh, 2000 ; Tice et al , 2000 ; Hermeto et al , 2014 ), may be repaired without becoming mutations ( Oliveira et al , 2007 ). In contrast, the micronucleus assay evaluates aneugenic and clastogenic activities that are not prone to repair.…”

Comet Assay on Toxicogenetics; Several Studies in Recent Years on Several Genotoxicological Agents

“…Hermeto et al, have evaluated pH effects on genomic integrity in adipose-derived mesenchymal stem cells (MSCs); with the aim of evaluating two different comet assay protocols for genomic damage pattern analysis in MSCs derived from adipose tissue [50]. They found that in the pH>13 alkaline version detects damage in the form of single-strand breaks, double alkali-labile sites, cross-linking, and loss of excision.…”

“…They found that in the pH>13 alkaline version detects damage in the form of single-strand breaks, double alkali-labile sites, cross-linking, and loss of excision. The other version, pH=12 detects mainly single-and double-strand breaks [50].…”