The Use of Neural Stem Cells-Derived Exosomes to Prevent Late Radiation-Induced Cognitive Impairments in Mice.

We studied the effect of intranasal administration of neural stem cell (NSC)-derived exosomes on behavior and cognitive functions of mice in the late period after head irradiation in a dose of 8 Gy. The used exosomes had specific markers (CD9+/CD63+, 99.5%; TSG101+, 98.4%) and mean size 105.7±8.8 nm according to dynamic light scattering data and 119.0±12.4 nm according to nanoparticle tracking analysis (NTA). Exosome suspension (2×1012 particles/ml according to NTA measurements) was administered intranasally for 4 weeks starting from 48 h after irradiation in a volume of 5 µl/nostril (2×1010 exosomes/mouse). It was shown that intranasal administration of mouse NSC-derived exosomes prevented delayed radiation-induced behavioral changes and recognition memory impairments in mice after head irradiation.

Stem Cell Exosomes Improve Survival of Neural Stem Cells after Radiation Exposure.

The death of neural stem cells in the hippocampus during radiation therapy of brain tumors leads to neurogenesis impairment and the development of cognitive dysfunctions at delayed terms after irradiation. Exosomes secreted by stem cells can provide a protective effect on neural stem cells. We isolated and characterized exosomes from the medium conditioned by neural stem cells and mesenchymal stem cells from mouse adipose tissue and studied their efficiency in protecting irradiated neural stem cells. According to dynamic light scattering data, the exosome size varied from 44 to 68 nm for neural stem cells, and from 80 to 130 nm for mesenchymal stem cells. All exosomes carried markers CD9, CD63, and TSG101. The survival rate and clonogenic activity of neural stem cells irradiated in a dose of 1 Gy was found to increase after culturing in the presence of stem cell exosome preparations.

Improved Survival and Regeneration of Irradiated Mouse Neural Stem Cells after Co-Culturing with Non-Irradiated Mouse Neural Stem Cells or Mesenchymal Stem Cells from the Adipose Tissue.

We studied the effect of neural stem cells (NSC) and mesenchymal stem cells (MSC) from mouse adipose tissue on survival, clonogenic activity, and senescence of NSC after exposure to γ-radiation. It was found that survival and clonogenic activity of NSC irradiated in doses of 1 and 2 Gy was enhanced when irradiated cells were co-cultured with non-irradiated NSC and MSC in permeable Transwell inserts. The proportion of senescent NSC (cells with high ß-galactosidase activity) increased with increasing irradiation dose. Co-culturing with non-irradiated NSC in 3 days after irradiation in a dose of 1 Gy led to a decrease in the proportion of senescent cells among irradiated NSC. Factors secreted by NSC and MSC can become the basis for the development of means for prevention and treatment of damage to brain cells resulting from radiation therapy of head and neck cancer.

Mechanisms of Stimulation of the Growth of Mouse Mammary Adenocarcinoma by Mesenchymal Stem Cells.

Mesenchymal stem cells from the adipose tissue (AT MSC) and the bone marrow (BM MSC) stimulated migration of melanoma B16 cells, while mammary adenocarcinoma Ca755 cells stimulated migration of mesenchymal stem cells. Mesenchymal stem cells retained these properties at late terms after γ-irradiation in vitro. Tumors that developed after injection of Ca755 cells alone and in combinations with BM MSC or AT MSC had similar histological structure corresponding to breast adenocarcinoma. Only AT MSC stimulated tumor growth, which was determined by more intensive secretion of factors stimulating proliferation of tumor cells, including chemokine CCL2. The use of AT MSC in regenerative medicine requires careful monitoring of the absence of tumors in patients.

[Effectiveness of the DNA double-strand breaks repair system in lymphocytes of patients with cognitive impairments and healthy volunteers]. / Aktivnost' sistemy reparatsii dvunitevykh razryvov DNK v limfotsitakh patsientov s kognitivnym snizheniem v sravnenii so zdorovymi dobrovol'tsami.

The individual differences in the efficiency of DNA DSB repair were estimated by the level of residual γH2AX foci after γ-irradiation at a dose of 2 Gy, in lymphocytes of patients with amnestic mild cognitive impairment (AMCI) and Alzheimer's disease (AD) and of healthy volunteers. Lymphocytes were isolated from the peripheral blood of the examined patients and were frozen in a medium for freezing cells. Before the study, the lymphocytes were thawed, suspended in RPMI 1640 culture medium supplemented with 10% inactivated fetal bovine serum, and half of the cells were γ-irradiated at 4°C from a 60Co source on a GUT-200M facility at a dose of 2 Gy (a dose rate of 0.75 Gy/min). Control and irradiated lymphocytes were cultured for 24 h, collected, fixed, and stored until the study of the number of spontaneous and residual foci of γH2AX using fluorescent microscopy after staining with fluorescent labeled antibodies. In lymphocytes of patients with AMCI and AD a higher number of residual γH2AX foci in lymphocytes and the higher number of lymphocytes with foci were found compared with healthy volunteers. This indicates a decrease in the ability to repair DNA DSB in these patients. Indicators of cellular immunity and the concentration of TNF-α in the blood serum in the group of examined patients were normal. In the group of patients with the cognitive impairments (AMCI+AD), a correlation was found between the number of residual foci of γH2AX and the number of CD3+CD4+ lymphocytes and the concentration of proinflammatory cytokine TNF-α in the blood serum. This suggests the development of stronger neuroinflammation in patients with reduced ability to repair DNA DSB in this pathology.

[Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells]. / Aktivnost' telomerazy, ékspressiia gena mTert i dlina telomer v otdalennyi period posle γ- i γ,n-oblucheniia v mezenkhimal'nykh stvolovykh kletkakh myshi i v opukholiakh, obrazovavshikhsia iz étikh kletok.

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

Mesenchymal Stem Cells from Mouse Adipose Tissue Stimulate Tumor Growth.

We studied the effect of mesenchymal stem cells from the bone marrow and adipose tissue on the growth rate of melanoma B16 and mammary adenocarcinoma Ca755 tumors after their co-administration with tumor cells to syngeneic mice. Stimulation of tumor growth and formation of melanoma metastases in the lungs was found under the influence of adipose tissue-derived, but not bone marrow-derived stem cells. At delayed terms after irradiation in sublethal doses, the adipose tissue-derived mesenchymal stem cells also stimulated the tumor growth. Stimulation of the tumor growth by adipose tissue-derived mesenchymal stem cells was caused by factors secreted by these cells. Transplantation of mesenchymal stem cells to humans is possible only after accurate exclusion of malignant tumors.

CHARACTERISTICS OF TUMORS THAT DEVELOPED IN MICE AFTER TREATMENT WITH IRRADIATED SYNGENEIC MESENCHYMAL STEM CELLS OF BONE MARROW.

Mesenchymal stem cells (MSCs) are present in almost all organs and tissues of the organism. It is believed, that MSCs could be transformed into cancer stem cells spontaneously or under influence of genotoxic factors and trigger the growth of tumors. The aim of this work was to study the possibility of malignant transformation of cultured MSCs from murine bone marrow (MSCs-BM) after g-irradiation in vitro and characterize of biochemical and histological features of the tumors that developed after transplantation of MSCs-BM into syngeneic mice. Tumors were observed in 3­4 months after MSCs-BM transplantation. After administration of MSCs-BM irradiated at a dose of 1 Gy, tumors were seen in 2 of 5 mice. After transplantation of MSCs-BM irradiated at a dose of 6 Gy, tumors were found in all 5 of 5 mice. In the case of control MSCs-BM, only one tumor appeared in 6 months after transplantation. The telomerase activity was two times higher in the tumor developed from 6 Gy irradiated MSCs-BM than from 1 Gy irradiated MSCs-BM. The tumors developed from control and irradiated MSCs-BM were classified as multicomponent mesenchymomas («mixture of sarcomas¼). Histological examination showed that tumors contained tissue areas of different histogenesis. Thus, MSCs-BM g-irradiated at doses of 1 and 6 Gy and, much less frequently, control MSCs-BM can transform into tumor cells and induce development of multicomponent mesenchymomas.

Radiation Stress Changes the Size of Side Population of Human Epithelial Cells.

Stem cell cultures are heterogeneous and include true stem cells and progenitor cells. True stem cells are identified by flow cytofluorometry as a cell subset characterized by low accumulation of fluorescent dye rhodamin-123 and forming a side population. Low-dose γ-irradiation (10-200 mGy) of human skin epithelial stem cells and epithelial H69 tumor cells to was followed by an increase in cell counts by day 7 after the exposure. In parallel, reduction of the side population to 4-30% from the control for epithelial stem cells on the next day after exposure and to 22-36% from the control for H69 cells in 3 days after exposure. The size of the side population remained reduced to 8-37% of that in the control cultures of epithelial stem cells and H69 cells for at least 7 days after exposure. The decrease of the side population fraction of cells was not caused by cell death, but could be due to radiation-induced activation of the signal pathways, regulating the velocity of autoregeneration of the pool of true stem cells and acceleration of their transition to the pool of rapidly proliferating progenitor cells, this leading to an increase in the total cell count in the studied cultures under the effect of low-dose γ-radiation.

[Effects of Low and Sublethal Doses of γ-Radiation on Mesenchymal and Neural Stem Cells from Mouse Brain].

Mesenchymal stem cells (MSC) exist in the brain in addition to the neural stem cells (NSC). The aim of this work was to investigate the sensitivity of mouse brain MSC (MSC(BR)) to sublethal doses of γ-radiation in comparison with the sensitivity of bone marrow MSC (MSC(BM)) and NSC and to study the effects of γ-irradiation at low doses on these cells. Cells were exposed to γ-radiation (137Cs) at the doses of 10 to 200 mGy at a dose rate of 10 mGy/min; higher doses were achieved at the dose rates of 200 and 500 mGy/min (60Co). The survival of cells was assessed by counting living cells after staining with trypan blue in the Goryaev's chamber or using the MTT test for NSC growing as neurospheres. SP fraction was measured using flow cytometry after incubation with rhodamine-123. Exposure to the doses in the range of 10 to 500 mGy stimulated cell proliferation. The maximum decrease in the cells number was seen on the seventh day after irradiation and it was practically the same for the MSC(BR) and MSC(BM). NCS were more radiosensitive than MSC. Exposure to the doses of 100 to 500 mGy stimulated cells proliferation of all SCs except of MSC(BM). It was shown that the size of SP fraction of MSC(BR) was diminished after γ-irradiation at low doses. Thus, the stimulation of cell proliferation after γ-irradiation at low doses is accompanied by the redistribution of distinct cell subpopulations: the decrease in the SP fraction and the increase in the general population of cells were observed.