Cell and Organ Transplantology. 2018; 6(2):170-175.
The effect of transplantation of bone marrow cells induced by the contact with thymus-derived multipotent stromal cells on the immune system of mice, regenerating after cyclophosphamide treatment
Demchenko D. L.
State Institute of Genetic and Regenerative Medicine National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
The effect of transplantation of syngeneic bone marrow cells (BMCs) after their contact in vitro with thymus-derived multipotent stromal cells (MSCs) for regeneration of damaged by cyclophosphamide immune system of mice was studied.
Materials and methods. MSCs were obtained from C57BL/6 mice’s thymus by explants method. BMCs were obtained by flushing the femurs. BMCs were induced for 2 hours on the monolayer of thymus-derived MSCs. The immune deficiency of mice was modelled using cyclophosphamide injection. After that, cell transplantation was performed and the state of the immune system was assessed. The number of erythrocytes, hematocrit, hemoglobin concentration in the peripheral blood; the phases of the cell cycle and apoptosis of mesenteric lymph node cells were determined. The amount of antibody-producing cells in the spleen and the delayed hypersensitivity response was determined. The study of proliferative and cytotoxic activity of natural killer lymphocytes, the analysis of phagocytosis, spontaneous and induced bactericidal activity of peritoneal macrophages were performed.
Results. It was shown that unlike intact bone marrow cells, BMCs induced by thymus-derived MSCs provided increased spontaneous proliferative activity of lymphocytes with a decrease in the number of lymph node cells in G0/G1 phase by 6.2 % and an increase the number of lymphocytes in S+G2/M phase by 28 % in comparison with the group of mice treated with cyclophosphamide, as well as the recovery of cellularity of the bone marrow, lymph nodes and spleen. At the same time in the lymph nodes, the number of cells in the apoptosis increased. BMCs induced by MSCs showed a pronounced negative effect on natural cytotoxicity, reducing its rates by 3 times compared with the group of cyclophosphamide-treated mice, and on adaptive immunity: the rates of delayed hypersensitivity response decreased by 1.7 times, the number of antibody-producing cells by 1.8 times. Red blood cell regeneration was stimulated by intact BMCs, which was manifested by the normalization of hematocrit and hemoglobin and an increase in the number of reticulocytes in the blood by 2.2 times compared with the group of mice treated with cyclophosphamide.
Conclusion. Transplanted BMCs improve erythropoiesis in mice after cyclophosphamide treatment, and BMCs, previously induced by thymusderived MSCs, lose this ability. BMCs after co-culture are strongly activated to impact on the immune system, which is most likely due to the effect of contact interaction with thymus-derived MSCs, effectively impact on hematopoietic cells and possess immunomodulatory properties.
Key words: hematopoietic bone marrow cells; thymus-derived multipotent stromal cells; regeneration of the immune system; cyclophosphamide-induced immunodeficiencyFull Text PDF (eng) Full Text PDF (ua)
|1. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature. 2014; 505(7483):327-34. DOI: 10.1038/nature12984.
|2. Méndez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature. 2010; 466(7308):829-34. DOI: 10.1038/nature09262.
|3. Block GJ, Ohkouchi S, Fung F, et al. Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin‐1. Stem Cells. 2009; 27(3):670-681. DOI: 10.1002/stem.20080742.
|4. Nikolskaya EI, Butenko GM. Structural-functional organisation of the bone marrow hematopoietic stem cells niches. Cell and organ transplantology. 2016; 4(1):82-100.|
|5. Prockop DJ, Phinney DG, Bunnell BA. Mesenchymal stem cells: methods and protocols. Humana Press. 2008. 192 p.
|6. Gregory CA., Gunn WG, Peister A., et. al. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem. 2004; 329(1):77-84. DOI: 10.1016/j.ab.2004.02.002.
|7. Kim WK, Jung H, Kim DH, et al. Regulation of adipogenic differentiation by LAR tyrosine phosphatase in human mesenchymal stem cells and 3T3-L1 preadipocytes. J Cell Sci. 2009; 122(22):4160-7. DOI: 10.1242/jcs.053009.
|8. Nikolskiy IS, Nikolskaya VV, Demchenko DL, Zubov DO. Potentiation of directed osteogenic differentiation of thymic multipotent stromal cells by prior co-cultivation with thymocytes. Cell and Organ Transplantology 2016; 4(2):220-223. DOI:10.22494/cot.v4i2.59.
|9. Said R, Abdel-Rehim M, Sadeghi B, et al. cyclophosphamide Pharmacokinetics in Mice: A Comparison Between Retro Orbital Sampling Versus Serial Tail Vein Bleeding. The Open Pharmacology Journal. 2007; 1:30-35. DOI: 10.2174/1874143600701010030.
|10. Frimmel G. Immunological methods edited by Frimel G. M: The world. 1987. 472 p.|
|11. Nikolsky IS. Dynamic features of hemo-immune deficiency induced by cyclophosphamide Proceedings of the international forum “Clinical immunology and allergology”. 2014:195-196.|
|12. Khaitov RM, Pinegin BV, Yarilin AA. Guide to Clinical Immunology. Diagnosis of immune system diseases: a guide for doctors. M: GEOTAR-Media. 2002. 352 p.|
|13. Mossman T. Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assay. J Immunol Methods. 1983; 65(1-2):55-63.
|14. Sugiura H, Nishida H, Inaba R, et al. Effects of different durations of exercise on macrophage functions in mice. J Appl Physiol. 2001; 90(3):789-94. DOI: 10.1152/jappl.2001.90.3.789.
|15. Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008; 14(1). DOI: 10.1002/0471142735.im1401s83.
|16. Acar M, Kocherlakota K., Murphy S., et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature. 2015. 526, № 7571. P. 126-30. DOI: 10.1038/nature15250.
|17. Abbuehl J-P, Tatarova Z, Held W, et al. Long-Term Engraftment of Primary Bone Marrow Stromal Cells Repairs Niche Damage and Improves Hematopoietic Stem Cell Transplantation. Cell Stem Cell. 2017. 21, № 2. P. 241-255. DOI: 10.1016/j.stem.2017.07.004. doi: 10.1016/j.stem.2017.07.004
|18. Frasca D, Guidi F, Arbitrio M, et al. Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells. Bone Marrow Transplant. 2000; 25(4):427-33. DOI: 10.1038/sj.bmt.1702169.
|19. Jing D, Fonseca AV, Alakel N, et al. Hematopoietic stem cells in co-culture with mesenchymal stromal cells–modeling the niche compartments in vitro. Haematologica. 2010; 95(4):542-50.
|20. Patel DM, Shah J, Srivastava AS. Therapeutic potential of mesenchymal stem cells in regenerative medicine. Stem Cells Int. 2013; 2013:496218. DOI: 10.1155/2013/496218.
|21. Rota C, Morigi M, Cerullo D, et. al. Therapeutic potential of stromal cells of non-renal or renal origin in experimental chronic kidney disease. Stem Cell Res Ther. 2018; 9(1):220. DOI: 10.1186/s13287-018-0960-8.
|22. Bryniarski K, Szczepanik M, Ptak M, et. al. Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice. Pharmacol Rep. 2009; 3:550-7.
|23. Teriukova NP, Pogodina ON, Blinova GI, Ivanov VA. Immunomodulating effect of cyclophosphamide on cytotoxic activity of rats and mice splenocytes. Tsitologiia. 2011; 53(10):800-7.
|24. Tanner A, Hallam SJ, Nielsen SJ, et al. Development of human B cells and antibodies following human hematopoietic stem cell transplantation to Rag2(-/-)γc(-/-) mice. Transpl Immunol. 2015; 32(3):144-50. DOI: 10.1016/j.trim.2015.03.002.
Demchenko D. The effect of transplantation of bone marrow cells induced by the contact with thymus-derived multipotent stromal cells on the immune system of mice, regenerating after cyclophosphamide treatment. Cell and Organ Transplantology. 2018; 6(2):170-175. doi:10.22494/cot.v6i2.89