CULTIVATION AND CHARACTERISTICS OF MESENCHyMAL STEM CELLS FROM THE BONE MARROW OF THE PATIENTS WITH ORTHOPEDIC PATHOLOGY

The article presents data on the cultivation and characteristics of mesenchymal stem cells (MSC) isolated from the bone marrow of patients with dysplastic coxarthrosis. Several morphological phenotypes were found in the fraction of adhesive MSC: spindle-shaped elongated cells, large flattened cells, and thin stellate cells in both samples of bone marrow. Immunophenotypic analysis showed that cells express surface antigens (CD90, CD73, CD105, CD45 and CD34), which are characteristic for typical stem cells. It was shown that the use of a new growth medium containing no components of animal origin for the cultivation of human MSC allowed to achieve confluence of the cell culture on the 16th-8th day of incubation without delaying the proliferative activity of the cells and without loss of ability to differentiate into chondro- and osteogenic types of tissues. Multipotency of MSC was confirmed by osteogenic and chondrogenic differentiation of cells, during prolonged cultivation of MSCs in induction media in vitro . The differentiation of MSC into osteoblasts was confirmed by immunocytochemical staining for alkaline phosphatase and alizarin red S. Specific differentiation of MSC in chondrogenic type was revealed by staining of cartilage deposits with alcian blue. For the first time, such characteristics of human MSC as: mitotic index, trajectory of cells migration and average speed of migration on culture plastics were determined. The mitotic index of actively proliferating MSC was from 2.7 to 3.4 % of the total cell number. The moving activity (speed of cell migration) was 38-42 μm/h. Thus, bone marrow aspirate from patients with orthopedic pathology is the source of stem cells that meet all the criteria for MSC as determined by the International Society of Cellular Therapy and can be used in regenerative therapy of bone and cartilage.

мезенхимальные стромальные клетки, костный мозг, диспластический коксоартроз, митотический индекс, скорость миграции, дифференцировка, регенерация кости и хряща

1. Андреева Н.В., Бонарцев А.П., Жаркова И.И., Махина Т.К., Мышкина В.Л., Харитонова Е.П., Воинова В.В., Бонарцева Г.А., Шайтан К.В., Белявский А.В. Культивирование мезенхимных стволовых клеток мыши на матриксах из поли-3-оксибутирата // Клеточные технологии в биологии и медицине. 2015. (2). 114-119.

2. Кожевникова М.Н., Микаелян А.С., Старостин В.И. Молекулярно-генетический и иммунофенотипический анализ антигенного профиля, остеогенных и адипогенных потенций мезенхимальных стромальных клеток из печени зародышей и костного мозга половозрелых крыс // Цитология. 2009. 51. (6). 526-538.

3. Banfi A., Muraglia A., Dozin B., Mastrogiacomo M., Cancedda R., Quarto R. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy // Exp. Hematol. 2000. 28. (6). 707-715.

4. Bara J., Richards R.G., Alini M., Stoddart M.J. Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic // Stem Cells. 2014. 32. 1713-1723.

5. Beyer Nardi N., da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization // Handb. Exp. Pharmacol. 2006. (174). 249-282.

6. Bianco P., Riminucci M., Gronthos S., Robey P.G. Bone marrow stromal stem cells: nature, biology, and potential applications // Stem Cells. 2001. 19. (3). 180-192.

7. Charbord P. Bone marrow mesenchymal stem cells: historical overview and concepts // Hum. Gene Ther. 2010. 21. 1045-1056.

8. Da Silva Meirelles L., Caplan A.I., Nardi N.B. In search of the in vivo identity of mesenchymal stem cells // Stem Cells. 2008. 26. (9). 2287-2299.

9. Dominici M., Le Blanc K., Mueller I., Slaper-Cortenbach I., Marini F., Krause D., Deans R., Keating A., Prockop D., Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement // Cytotherapy. 2006. 8. (4). 315-317.

10. Dong Y., Chen X., Hong Y. Tissue-engineered bone formation in vivo for artificial laminae of the vertebral arch using beta-tricalcium phosphate bioceramics seeded with mesenchymal stem cells // Spine. 2013. 38. (21). 1300-1306.

11. Gimble J.M., Grayson W., Guilak F., Lopez M.J., Vunjak-Novakovic G. Adipose tissue as a stem cell source for musculoskeletal regeneration // Front. Biosci. 2011. 3. 69-81.

12. Horner C.B., Hirota K., Liu J., Maldonado M., Hyle Park B., Nam J. Magnitude-dependent and inversely-related osteogenic/chondrogenic differentiation of human mesenchymal stem cells under dynamic compressive strain // J. Tissue Eng. Regen. Med. 2016. doi: 10.1002/term.2332.

13. Horwitz E.M., Le Blanc K., Dominici M., Mueller I., Slaper-Cortenbach I., Marini F.C., Deans R.J., Krause D.S., Keating A. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement // Cytotherapy. 2005. 7. (5). 393-395.

14. Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review // J. Nippon. Med. Sch. 2009. 76. (2). 56-66.

15. Mizuno H. Adipose-derived stem and stromal cells for cell-based therapy: current status of preclinical studies and clinical trials // Curr. Opin. Mol. Ther. 2010. 12. (4). 442-449.

16. Mosna F., Sensebe L., Krampera M. Human bone marrow and adipose tissue mesenchymal stem cells: a user’s guide // Stem Cells Develop. 2010. 19. (10) 1449-1470.

17. Di Maggio N. Bone marrow mesenchymal stem cell niches and regenerative medicine. Basel, 2010. 142 p.

18. Wu Y., Yang Z., Law J.B., He A.Y., Abbas A.A., Denslin V., Kamarul T., Hui J.H., Lee E.H. The combined effect of substrate stiffness and surface topography on chondrogenic differentiation of mesenchymal stem cells // Tissue Eng. Part A. 2017. 23. (1-2). 43-54.