Document Type : Original Article
Graduate Program in Veterinary Medicine, Federal University of Santa Maria, Santa Maria, Brazil
Biotecnos Research Center, Santa Maria, Rio Grande do Sul, Brazil; Catholic University of Uruguay, Montevideo, Uruguay
Morphology Department, Federal University of Santa Maria, Santa Maria, Brazil
The aim of this study was to evaluate the potential use of a DNA comet assay, DNA fragmentation fluorimetric assay and reactive oxygen species levels as potential biomarkers of genome conditions of dental pulp stem cells (DPSCs) isolated from dog canine teeth. Mesenchymal stem cells were isolated from the dental pulp collected from dog teeth. The results obtained suggest the ideal moment for clinical application of cellular therapy for this type of cell. The cell culture was maintained with Dulbecco’s modified Eagle’s medium supplemented with 10.00% fetal bovine serum for eight passages. During each passage, cell proliferation, oxidative stress and level of DNA fragmentation were assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, testing 2,7 dichlorodihydro-fluorescein-diacetate and PicoGreen®, respectively. There were important differences among the first three DPSC passages compared to passages 4–8 and a large number of nuclei with some levels of DNA damage (30.00 to 40.00% in initial DPSC passages and > 50.00% in late passages), indicating in vitro DPSC genomic fragility. Within the limitations of this study, the results suggest these relatively simple and inexpensive approaches - comet and DNA fragmentation assays - could help sort stem cells with less DNA damage for use in research or therapies.
- Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000; 97(25): 13625-13630.
- Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004; 364(9429): 149-155.
- Nakamura S, Yamada Y, Katagiri W, et al. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod 2009; 35(11): 1536-1542.
- Park JY, Jeon SH, Choung PH. Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis. Cell Transplant 2011;20(2):271-285.
- Kawashima N. Characterisation of dental pulp stem cells: A new horizon for tissue regeneration? Arch Oral Biol 2012; 57(11): 1439-1458.
- Cavazzana-Calvo M, Fischer A, Bushman FD, et al. Is normal hematopoiesis maintained solely by long-term multipotent stem cells? Blood 2011; 117(17): 4420-4424.
- Baum C, Modlich U, Göhring G, et al. Concise review: Managing genotoxicity in the therapeutic modification of stem cells. Stem Cells 2011; 29(10): 1479-1484.
- Roschke AV, Stover K, Tonon G, et al. Stable karyotypes in epithelial cancer cell lines despite high rates of ongoing structural and numerical chromosomal instability. Neoplasia 2002; 4(1): 19-31.
- Draper BW, McCallum CM, Stout JL, et al. A high-throughput method for identifying N-ethyl-N-nitrosourea (ENU)-induced point mutations in zebrafish. Methods Cell Biol 2004; 77: 91-112.
- Buzzard JJ, Gough NM, Crook JM, et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004; 22(4): 381-382.
- Hoffman LM, Carpenter MK. Human embryonic stem cell stability. Stem Cell Rev 2005; 1(2): 139-144.
- Rebuzzini P, Neri T, Zuccotti M, et al. Chromosome number variation in three mouse embryonic stem cell lines during culture. Cytotechnology 2008; 58(1): 17-23.
- Lefort N, Perrier AL, Laâbi Y, et al. Human embryonic stem cells and genomic instability. Regen Med 2009; 4(6): 899-909.
- Lund RJ, Närvä E, Lahesmaa R. Genetic and epigenetic stability of human pluripotent stem cells. Nat rev Genet 2012; 13(10): 732-744.
- Yang M, Büsche G, Ganser A, et al. Cytological characterization of murine bone marrow and spleen hematopoietic compartments for improved assessment of toxicity in preclinical gene marking models. Ann Hematol 2013; 92(5): 595-604.
- Duailibi MT, Kulikowski LD, Duailibi SE, et al. Cytogenetic instability of dental pulp stem cell lines. J Mol Histol 2012; 43(1): 89-94.
- Ribitsch I, Burk J, Delling U, et al. Basic science and clinical application of stem cells in veterinary medicine. Adv BiochemEngBiotechnol 2010; 123: 219-263.
- Bernardi L, Luisi SB, Fernandes R, et al. The isolation of stem cells from human deciduous teeth pulp is related to the physiological process of resorption. J Endodont 2011; 37(7): 973-979.
- Rao MS, Mattson MP. Stem cells and aging: Expanding the possibilities. Mech Ageing Dev 2001; 122(7): 713-734.
- Wallace DR, Dodson SL, Nath A, et al. Delta opioid agonists attenuate TAT(1–72)-induced oxidative stress in SK-N-SH cells. Neurotoxicology 2006; 27(1): 101-106.
- Batel R, Jaksić Z, Bihari N, et al. A microplate assay for DNA damage determination (fast micromethod). BAnal Biochem 1999; 270(2): 195-200.
- Georgiou CD, Papapostolou I, Grintzalis K. Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks). Nat Protoc 2009; 4(2): 125-131.
- Singh N, McCoy M, Tice R, et al. A simple technique for quantification of low levels of DNA damage in individuals cells. Exp Cell Res 1995; 175(1): 184-191.
- Tice RR, Agurell D, Anderson D, et al. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000; 35(3): 206-221.
- Hartmann A, Agurell E, Beevers C, et al. Recommendations for conducting the in vivo alkaline comet assay. 4th International comet assay workshop. Mutagenesis 2003; 18(1): 45-51.
- Nadin S, Vargas-Roig L, Ciocca D. A silver staining method for single-cell gel assay. J Histochem Cytochem 2001; 49(9): 1183-1186.
- Rocha CR, Lerner LK, Okamoto OK, et al. The role of DNA repair in the pluripotency and differentiation of human stem cells. Mutation Res 2013; 752(1): 25-35.
- Fung H, Weinstock DM. Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells. PLoS One 2011; doi.org/10.1371/journal. pone.0020514.
- Impens S, Chen Y, Mullens S, et al. Controlled cell-seeding methodologies: A first step toward clinically relevant bone tissue engineering strategies. Tissue Eng Part C Methods 2010; 16(6): 1575-1583.
- Chen Y, Sonnaert M, Roberts SJ, et al. Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures. Tissue Eng Part C Methods 2012; 18(6): 444-452.
- Nikitina VA, Chausheva AI, Zhanataev AK, et al. Assessment of DNA damage in human bone marrow cells and multipotent mesenchymal stromal cells. Bull Exp Biol Med 2011; 151(4): 550-552.
- Nagaria P, Robert C, Rassool FV. DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity. Biochim Biophys Acta 2013; 1830(2): 2345-2353.