Background aimsSpontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC) are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa.MethodsThe immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR) analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells.ResultsThe immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12%) are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described.ConclusionsThe spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before use in medicine.

Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion.

Mechanisms underlying the effects of low-dose ionizing radiation (IR) exposure (10-100 mGy) remain unknown. Here we present a comparative study of early (less than 24h) and delayed (up to 11 post-irradiation passages) radiation effects caused by low (80 mGy) vs intermediate (1000 mGy) dose X-ray exposure in cultured human bone marrow mesenchymal stem cells (MSCs). We show that γН2АХ foci induced by an intermediate dose returned back to the control value by 24 h post-irradiation. In contrast, low-dose irradiation resulted in residual γН2АХ foci still present at 24 h. Notably, these low dose induced residual γН2АХ foci were not co-localized with рАТМ foci and were observed predominantly in the proliferating Кi67 positive (Кi67+) cells. The number of γН2АХ foci and the fraction of nonproliferating (Кi67-) and senescent (SA-β-gal+) cells measured at passage 11 were increased in cultures exposed to an intermediate dose compared to unirradiated controls. These delayed effects were not seen in the progeny of cells that were irradiated with low-dose X-rays, although such exposure resulted in residual γН2АХ foci in directly irradiated cells. Taken together, our results support the hypothesis that the low-dose IR induced residual γH2AХ foci do not play a role in delayed irradiation consequences, associated with cellular senescence in cultured MSCs.

The long-term in vivo cytogenetic effects of high-dose radiation exposure can be traced in accidentally irradiated persons, and particularly useful for developing strategies of monitoring and therapy of such patients, as well as for elucidating the fundamental aspects of hematopoiesis and radiobiology. Using 24-color fluorescent in situ hybridization (mFISH), we analysed the frequency and the spectrum of chromosomal aberrations (CA) in peripheral blood lymphocytes of the Chernobyl Nuclear Power Plant (NPP) accident victim 30, 31, 32 and 33 years after acute accidental exposure to high-dose gamma radiation of the whole body. Totally, 993 metaphase cells were analyzed (or 219, 272, 258, 244 cells each year), of which 297 were aberrant. Our study demonstrated a constant aberrant cell frequency at 28% in 2016–2018 years, while in 2019, a significant increase up to 35% occurred due to contribution of significantly elevated frequency of simple aberrations in the absence of evident recent genotoxic factors. Four clonal aberrations were detected, three of which persisted for more than one year at a frequency up to 2.5% of analyzed cells. The distribution of 731 breakpoints per individual chromosomes was nearly proportional to their physical length, excepting Chromosomes 13 and 20, which were significantly breakpoint-deficient compared to the genome median rate. Monitoring of the long-term effects on chromosomal instability caused by radiation exposure is important for understanding and predicting the long-term effects of ionizing radiation.