Veterynarna biotehnologija – Veterinary biotechnology, 2016, 28, 165-172 [in Ukrainian].
National University of Life and Environmental Sciences of Ukraine
Institute of Veterinary Medicineof NAAS
CYTOGENETIC ANALYSIS OF MESENCHYMAL STEM CELLS OF RATS AT DIFFERENT PASSAGES
Introduction. Mesenchymal stem cells (MSC) represent a pluripotent stem cell pool of adult organism and possess a range of positive characteristics that open infinite possibilities of their utilization in case of degenerative processes. In order to use MSC for clinical purposes the significant quantity of cells is needed, that can be achieved only by long-standing in vitro cultivation. Literature data analysis doesn't give an unambiguous answer regarding genetic stability of MSC during their in vitro cultivation, which necessitated further research.
Goal of the work. Performing of cytogenetic analysis of mesenchymal stem cells of rats at early passages.
Materials and methods. Mesenchymal stem cells of rats of the first to the sixth passages were used in this research. The cytogenetic analysis was performed on 30 metaphase plates of stem cells of rats from every passage. Slides were obtained through modification of standard cytogenetic method. In the course of the research we considered: quantitative abnormalities of chromosomes – aneuploidy, polyploidy. The same preparations were used to calculate the quantity of binuclear cells, cells with micronuclei, mitotic index, apoptotic cells (frequency was calculated for 500 cells).
Results of research and discussion. Presented the results of cytogenetic analysis of mesenchymal stem cells of rats during their in vitro culture. We found alterations in genetic apparatus of cells, that occurred in the form of aneuploidy, polyploidy as well as micronuclei, the amount of which varied depending on the passage. However, the variability of karyotype of the mentioned cells didn't exceed self-existing level of mutations, specific to this animal species.
Conclusion and prospects for further research:
- The karyotype analysis of mesenchymal stem cells culture of rats showed presence of cases of aneuploidy (8.9–18.9%) and polyploidy (1.1–4.4%), number of which changed with every passage but didn't exceed the limits of self-existing mutagenesis specific to mammals.
- According to the results of cytogenetic assessment of culture it was determined that quantity of cells with micronuclei (0.2–1.9%), binucleated cell (0.5–2.0%) and cells in apoptotic (0.1–0.7%) state lied within normal limits.
- Mitotic index decreased from 4.1% to 2.7% from the first to the fourth passage, respectively, and then gradually increased to 3.5% (sixth passage).
- Dynamics of the indicators listed in paragraphs 1–3 findings suggests that the culture of stem cells of rat during the first 6 passages is genetically stable, which is important for the use of such cells in regenerative medicine.
Keywords: cytogeneticanalysis, micronucleustest, mesenchymalstemcells, passage, mutations.
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