The cell cycle features of canine bone mesenchymal stem cells on different passages of cultivation

Authors

  • L. V. Kladnytska National University of Life and Environmental Sciences of Ukraine image/svg+xml

Abstract

The features of the cell cycle of culture of dog bone marrow mesenchymal stem cells inthe  different cultivating passages were studied.

Mesenchymal stem cells were obtained from the bone marrow of the dog under a laminar flow hood. Cell cultivation was carried out at 37 ° C, 100 % moisture and 5 % CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10-15 % fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The culture medium was changed 2-3 times per week and the cells were selected by their capacity to attach to the flask surface. When culture flasks became 80 % confluence, cells were detached with 0.25 % trypsin containing 1 mmol/L EDTA and subsequently replayed at a concentration of 104 cells/cm2 for next passaging. A cells culture of bone marrow mesenchymal stem cells was obtained on the 2nd, 7th and 12th passages. The method of flow cytometry determined the level of diploid, aneuploid cells and the distribution in the cell cycle phases. The morphology of cells of different passages was studied using an inverted microscope Axiovert 40.

It was investigated that the culture of mesenchymal stem cells from  dog bone marrow in the 2nd passage contain a significant number of the proliferative pool cells (S+G2/M)  and it was 29.33 + 0.19 % of the total number of diploid cells. The number of diploid cells was 100%, and the aneuploids, respectively, 0 %. All cells had fibroblast-like morphology. 

  It has been established that in the middle passages (7th) in the culture of mesenchymal stem cells from bone marrow, significant changes in the distribution of cells in the cell cycle phases were found. The number of diploid cells was 100%. The number of cells of the proliferative pool G2 / M + S significantly decreased to 20.33 + 1.27 %**. The presynthetic period G0 / G1 was characterized by a significant increase in the number of cells to 79.67 + 0.84 %*. Morphologically, the cells appeared on the appendages. The indicated changes characterize cell culture with signs of aging.

In the 12th passage of cultivation of MSCs from bone marrow, a significant decrease in the number of cells of a proliferative pool (S + G2 / M), which is 13.90 + 1.40 %*** of the total number of diploid cells compared with the 2nd passage The presynthetic  period G0 / G1 was characterized by a significant increase in the number of cells to 86.10 + 2.29 %**.

Consequently, the first characteristic signs of aging of the culture of stem cells from bone marrow appear on the 7th cultivation passage, which is characterized by significant changes in the distribution of cells in the cell cycle phases.

Key words: mesenchymal stem cells, bone marrow, dog, cultivation, cell cycle, phases, diploid, aneuploids, proliferative pool

References

Achille, V. (2011). Cell-cycle phases and genetic profile of bone marrow-derived mesenchymal stromal cells expanded in vitro from healthy donors. J Cell Biochem, 112(7), 1817-21. doi: 10.1002/jcb.23100.

Boward, B. (2016). Control of cell fate through cell cycle and pluripotency networks. Stem Cells, 34(6), 1427–1436. doi:10.1002/stem.2345.

Kladnytska, L., Nikulina, V., Garmanchuk, L., Mazurkevych, A., Kovpak, V., Nikolaienko, Т., Shelest, D., Dasyukevich, O. (2014). Influence allogeneic mesenchymal stem cells on the tumour growth parameters and metastatic potential in the transplantable carcinoma lung Lewis. Journal of Animal and Veterinary Sciences. 1(1). 1-5.

Soufi, A. (2016). Cycling through developmental decisions: how cell cycle dynamics control pluripotency, differentiation and reprogramming. Development. 143(23). 4301-4311.

Mazurkevy`ch, A. J., Kladny`cz`ka, L. V., Kovpak, V. V. (2013). Opty`mal`ni umovy` vy`dilennya ta kul`ty`vuvannya adgezy`vnoyi frakciyi mononuklearny`x klity`n kistkovogo mozku my`shi [Optimal conditions for the selection and cultivation of the adhesive fraction of mononuclear cells of the bone marrow of the mouse]. Visny`k Sums`kogo nacional`nogo agrarnogo universy`tetu. Seriya «Vetery`narna medy`cy`na». 9 (33).142-145.

Mazurkevy`ch, A. J., Malyuk, M. O., Tkachenko, S. M., Xarkevy`ch, Yu. O. (2014). Do metody`ky` otry`mannya kistkovogo mozku u sobak [To the method of obtaining bone marrow in dogs]. Biologiya tvary`nT. 16. № 2. 66–70.

Nicoletti, I., Migliorati, G., Pagliacci C.M. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods. №139( 2). 271-280.

Volk S., Theoret, C. (2013). Translating stem cell therapies: the role of companion animals in regenerative medicine. Wound Repair Regen. № 3. 382–394.

Zhang Z.X., Wang S.,Cao P.C., Wang Y.H., Wang Z., Dai L.J (2007). Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro. Cell Biol. Int. № 31(6):64. 5-8.

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Published

2017-11-25

Issue

No. 273 (2017)

Section

Article

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