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.

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.

Background: The search for an effective therapy for local radiation injuries (LRI) is urgent; one option is mesenchymal stem cells (MSC) derived from the placenta and their conditioned medium for the regenerative processes of the skin. Methods: We used 80 animals, randomly assigned to four groups: control (C) animals that did not receive therapy; control with the introduction of culture medium concentrate (CM); introduction of MSCs (PL); introduction of CMPL. LRI modeling was performed on an X-ray machine at a dose of 110 Gy. Histological and immunohistochemical tests were performed. Results: On the 112th day, the area of the open wound surface in the CMPL group was 6.7 times less than in the control group. Complete healing of the open wound surface of the skin in the CM group was observed in 40%, in CMPL 60%, in the PL group 20%, and in the C group there were no animals with a prolonged wound defect. A decrease in inflammatory processes was observed in the CMPL group. Conclusions: the use of a concentrate of conditioned MSCs (CMPL group) in severe LRI in laboratory animals accelerates the transition of the wound process to the stage of regeneration and epithelization.

Purpose: To study the regeneration processes in the treatment of radiation skin lesions with the mesenchymal stem cells (MSC) derived from human gingiva and their conditional medium concentrate (CCM) during animal studies. Material and methods: The study includes 80 white male Wistar rats weighing 210 ± 30 g at the age of 8–12 weeks, randomized into 4 groups (20 animals in each): control group (C), animal did not receive treatment; control with the introduction of the conditional medium concentrate (CCM) three times on days 1, 14 and 21; the introduction of MSC in a dose of 2 million cells per 1 kg three times on days 1, 14 and 21; the introduction of CCM in the estimated dose of 2 million cells per 1 kg three times on days 1, 14 and 21. Radiation burn simulation was performed (using on an X-ray unit at a dose of 110 Gy) and each animal was observed 17 times: at days 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105 and 112. Histological (stained with hematoxylin-eosin) and immunohistochemical (CD31, CD68, and VEGF) studies were performed. MSC was cultivated according to the standard procedure up to passages 3–5, the conditioned medium was collected and concentrated 10 times. The MSC immunophenotype (CD34, CD45, CD90, CD105, CD73, HLA-DR) and viability (7-ADD) were determined using flow cytometry. Results: Under the assessment of the animal skin on the day 7 in the CCM group, the area was significantly larger compared to the C, MSC, CM groups (р ≤ 0.05). In the CM group on the day 14 the area of the open wound surface and ulcers from day 28 to day 42 was significantly less, compared with the C, MSC and CCM groups (р ≤ 0.05). In group C, from 42 to 77 days of observation, an increase in the area of skin ulcers was observed compared with the CM and CCM groups (р ≤ 0.05). On the day 112, healing of skin ulcers in the CM group was observed in 40 %, in the MSC group in 60 %, and only in 20 % of animals in the CCM group, and in the C group it was not registered. Expression of VEGF marker on endothelial cells and stromal cells was observed in groups C and CM on day 28 and in groups MSCs and CCM on day 112. On the 28th day in the MSC group, the average number of vessels (CD31) in the field of view was 6.0, and on day 112 it was 12.75, р ≤ 0.05, in the CCM group – 19.10 and 28.6, respectively, р ≤ 0.05. An increase in the number of macrophages (CD68) was found in group C from 28 to 112 days (11.6 and 24.73, р ≤ 0.05), and in the CM group the decrease was 22.1 and 13.07, respectively, р ≤ 0.05. Conclusion: Thus, all used treatment modes of radiation skin lesions, including 3-fold administration of CM, MSC and CCM at a dose of 2 million cells per 1 kg, were effective and resulted in a reduction in the damage area, accelerated ulcer healing, and improvement of the regenerative processes. In addition, the use of MSCs led to the improvement of inflammatory processes’ vascularization and reduction in the radiation skin lesions.

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Each person is inevitably exposed to low doses of ionizing radiation (LDIR) throughout their life. The research results of LDIR effects are ambiguous and an accurate assessment of the risks associated with the influence of LDIR is an important task. Mesenchymal stromal cells (MSCs) are the regenerative reserve of an adult organism; because of this, they are a promising model for studying the effects of LDIR. The qualitative and quantitative changes in their characteristics can also be considered promising criteria for assessing the risks of LDIR exposure. The MSCs from human connective gingiva tissue (hG-MSCs) were irradiated at doses of 50, 100, 250, and 1000 mGy by the X-ray unit RUST-M1 (Russia). The cells were cultured continuously for 64 days after irradiation. During the study, we evaluated the secretory profile of hG-MSCs (IL-10, IDO, IL-6, IL-8, VEGF-A) using an ELISA test, the immunophenotype (CD45, CD34, CD90, CD105, CD73, HLA-DR, CD44) using flow cytometry, and the proliferative activity using the xCelligence RTCA cell analyzer at the chosen time points. The results of study have indicated the development of stimulating effects in the early stages of cultivation after irradiation using low doses of X-ray radiation. On the contrary, the effects of the low doses were comparable with the effects of medium doses of X-ray radiation in the long-term periods of cultivation after irradiation and have indicated the inhibition of the functional activity of MSCs.

Purpose: To conduct a comparative assessment of the effect of irradiation of human mesenchymal stem cells (MSC)at ultrahigh doses at liquid nitrogen temperature (–196 °C) and room temperature (+22 °C) on the yield of “residual” DNA double-strand breaks (DSB) and proliferative activity of thawed MSC. Material and methods: Isolation and cultivation of MSC was carried out according to standard methods. Dimethyl sulfoxide (DMSO) at a final concentration of 10 % was used for cells cryopreservation. The cells were irradiated with bremsstrahlung photon radiation with photon nominal energy 5 MeV, the accelerator UELR-10-100-T-100 (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of 22 °C and –196 °C. The yield of “residual” DNA DSB was assessed using an immunocytochemical analysis of the foci of the protein-marker DSB – γH2AX. To assess the proliferative activity, the number of Ki67 (protein marker of cell proliferation) of positive cells was analyzed. Results: The results of γH2AX foci assessment in MSCs after 48 hours irradiation at a dose of 50 Gy showed that the number of “residual” γH2AX foci in the MSC nuclei irradiated at 22 °C is about 3.2 times (p = 0.0002) higher than in the MSC nuclei irradiated at –196 °C. Analysis of the proliferative activity of cells using the molecular marker of cell proliferation of the Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of 22 °C, completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, is significantly reduced, but some of the cells (3.5 ± 1.1 %) still retain the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lose their ability to proliferate, but partially retain the ability to adhere. The integral fluorescence conjugated with the flurochrome of antibodies to γH2AX of MSC nuclei irradiated at a dose of 500 Gy at a temperature of –196 °C is 1.8 times lower than that of nuclei irradiated at a temperature of 22 °C. Conclusion: The results of the studies indicate that cryopreserved MSCs irradiated at liquid nitrogen temperature (–196 °C) in a preservation medium containing 10 % DMSO can tolerate the effects of ionizing radiation in large doses (up to 50 Gy). However, there is a rather high yield of “residual” DSB DNA and a very low proliferative activity, which makes them unsuitable in clinical practice. It seems promising to use a quantitative analysis of γH2AX foci to assess genome damage and the functional state of cells irradiated in a cryopreserved state.