A Study of Cytogenetic Stability of Induced Pluripotent Stem Cells Using Karyotyping and Comet Assay Techniques

Abstract

Background & Aims: Induced pluripotent stem cells (iPSCs) have the capability to undergo unlimited selfrenewal and differentiation into all cell types in the body. These cells are artificially derived from a nonpluripotent cell, typically human dermal fibroblasts (HDFs). The study of cytogenetic stability of these cells, in order to use iPS cells and apply studies in therapeutic applications, is essential. Methods: In the present experimental study, HDFs were isolated and cultured from human foreskin samples. The cytogenetic stability of these cells was evaluated in early passages (1-3) of HDFs using karyotype test and alkaline comet assay technique. The HDF cells treatment with hydrogen peroxide (H2O2) was used as a positive control for alkaline comet assay. The iPS cells with low passage (4-7) derived from reprogrammed HDFs were cultured on mouse embryonic fibroblast (MEF) feeder layer and cytogenetic stability of these cells were evaluated in early passages using karyotype test and alkaline comet assay technique. Results: The iPS cells in early passages (4-7) had normal karyotype (46, XY) and DNA damage and comet were not observed in these cells. In addition, HDF cells showed normal karyotype in early passages (1-3), but using comet assay, abnormality and DNA damages were observed in positive control (HDFs treated with H2O2). The comparison of alkaline comet assay parameters of iPS and HDF cells with positive control group showed statistically significant differences (P < 0.05). Conclusion: Since the comet assay is a sensitive technique for finding DNA damage, it is best if cytogenetic stability of these cells were evaluated before performing functional experiments on iPS cells. Therefore, for the precise evaluation of DNA damage and cytogenetic stability of iPS cells, the two techniques could complement each other