Воздействие цисплатина на мультипотентные мезенхимные стромальные клетки жировой ткани человека при различном уровне кислорода

Objective: To evaluate the damaging effects of cisplatin on MMSCs from adipose tissue in a phase of active proliferation and the state of the monolayer, which was exposed at standard (20%) and reduced to 1% and 5% level of oxygen.Methods: The incubation MMSC with cisplatin was performed on cultures of 2 passage in a state in monolayer and cultures in the active growth phase. Profile surface markers of MMSC determined by flow cytometry. MMSCs viability after incubation with cisplatin was detected by the number of apoptotic and necrotic cells using ANNEXIN V-FITC - PI Kit (Immunotech, France). Standard culture conditions (~ 20% O2) created in a CO2 incubator (Sanyo, Japan), 5% O2 created using multigas incubator (Sanyo, Japan), 1% O2 - using an airtight chamber (Stemcell Technologies, USA).Results: Incubation of monolayer MMSC with cisplatin at a concentration of 10 ug/ml for 72 hours leads to death of half of the cells in culture under 20% O2, 5% O2 and 1% O2. Cisplatin increased the fracture of PI+-cell, and PI+/Ann+-cells under all culture conditions. The short-term exposure with cisplatin (24 and 48 hours) did not cause the damaging effect. Effects of cisplatin on the MMSC in the growth phase for 48 hours led to accumulation of Ann+-cells and PI+/Ann +-cells under all culture conditions. However least damaging effect of cisplatin was observed in culture under hypoxic conditions (1% O2).Conclusion: These data suggest that monolayer MMSCs are dying primarily through necrosis, whereas MMSC in the growth phase in response to cisplatin treatment are dying by apoptosis, regardless the oxygen tension.Цель исследования: оценить повреждающее воздействие цисплатина на ММСК жировой ткани, находящиеся в фазе активной пролиферации и в состоянии монослоя, при стандартном (20%) и сниженном до 1% и 5% уровне кислорода в среде культивирования.Методы: Оценку влияния цисплатина на ММСК проводили на культурах 2 пассажа в состоянии монослоя и в культурах в активной фазе роста. Профиль поверхностных маркеров ММСК определяли с помощью проточной цитофлуориметрии. Жизнеспособность ММСК после инкубации с цисплатином оценивали по количеству апоптотических и некротических клеток с помощью набора ANNEXIN V-FITC – PI (Immunotech, Франция). Стандартные условия культивирования (~20% О2) создавали в СО2-инкубаторе (Sanyo, Япония), 5% О2 создавали используя мультигазовый инкубатор (Sanyo, Япония), 1% О2 – используя герметичную камеру (Stemcell Technologies, США).Результаты: Инкубация ММСК в состоянии монослоя с цисплатином в концентрации 10 мкг/мл в течение 72 часов вызывала гибель практически половины клеток в культуре как при 20% О2, так и при 5% и 1% О2 в среде. Во всех условиях культивирования увеличивалась доля PI+-клеток и PI+/Ann+-клеток. Более кратковременные экспозиции с цисплатином (24 и 48 часов) не оказывали выраженного повреждающего эффекта. Воздействие цисплатина на ММСК в активной фазе роста в течение 48 часов сопровождалось накоплением количества Ann+-клеток и PI+/Ann+-клеток. При этом наименьшее повреждающее воздействие цисплатин оказал на клетки, культивируемые в условиях гипоксии (1% О2).Заключение: Полученные данные свидетельствуют, что ММСК в состоянии монослоя погибают преимущественно путем некроза, тогда как для культур ММСК в фазе роста в ответ на воздействие было характерно накопление клеток, погибающих путем апоптоза, вне зависимости от уровня содержания кислорода в среде

Osteogenic Potential Reduction in Mesenchymal Stem Cells under Prolonged Simulated Microgravity

Increasing the duration of orbital space flights up to 6–12 months and planning interplanetary missions actualizes the need for a better understanding of the mechanisms of osteopenia caused by microgravity. Investigation of mesenchymal stem cells (MSCs) that support the tissue homeostasis under microgravity conditions allows a deeper insight into the processes underlying bone loss. The purpose of this study was to investigate the osteogenic potential of MSCs under prolonged simulated microgravity by clinorotation. Using the method of mineralized matrix detection, it has been found that MSCs osteogenic potential decreased after long-term clinorotation. The investigation of major osteogenic gene expression has showed decreased trans­criptional activity in RUNX2, ALPL-1, Col-1, but increased expression of PPARγ. One of the reasons for the decreased osteogenic potential of MSCs may be an increased level of reactive oxygen species (ROS) after 30 days of clinorotation. ROS may affect cellular signaling cascades, such as Wnt, Hedgehog and FOXO pathways, thereby leading to a shift of the differentiation potential to adipogenesis

BONE LOSS RECOVERY IN MICE FOLLOWING MICROGRAVITY WITH CONCURRENT BONE-COMPARTMENT-SPECIFIC OSTEOCYTE CHARACTERISTICS

Space missions provide the opportunity to investigate the influence of gravity on the dynamic remodelling processes in bone. Mice were examined following space flight and subsequent recovery to determine the effects on bone compartment-specific microstructure and composition. The resulting bone loss following microgravity recovered only in trabecular bone, while in cortical bone the tissue mineral density was restored after only one week on Earth. Detection of TRAP-positive bone surface cells in the trabecular compartment indicated increased resorption following space flight. In cortical bone, a persistent reduced viability of osteocytes suggested an impaired sensitivity to mechanical stresses. A compartment-dependent structural recovery from microgravity-induced bone loss was shown, with a direct osteocytic contribution to persistent low bone volume in the cortical region even after a recovery period. Trabecular recovery was not accompanied by changes in osteocyte characteristics. These post-space-flight findings will contribute to the understanding of compositional changes that compromise bone quality caused by unloading, immobilisation, or disuse