Dynamics properties of two lines of mesenchymal stem cells, derived from the Wharton’s jelly of the human umbilical cord, during long-term cultivation
The comparative analysis of characteristics of two lines MSC (MSCWJ-1 and MSCWJ-2), generated from Wharton’s Jelly of human umbilical cord was done at 6, 13 and 28 passages. The next results were obtained. 1. Morphological analysis showed, that during long-term cultivation morphological changes took place in both lines, but in line MSCWJ-2 the cells were larger and the greater number of debris was observed compared with MSCWJ-1. 2. The analysis of growth characteristics showed, that during long-term cultivation significantly increases of the average population doubling time in both cell lines, which correlates with morphological changes of cells, and with the gradual increase of activity P-galactosidase, indicating the occurrence of replicative senescence. The interline changes are associated with the level of proliferative activity, with the dynamics of the index of proliferation, with the time of intering in phase of active senescence, corresponding to 30 population doublings in line MSCWJ-2 and to 60 population doublings in line MSCWJ-1 with a higher percentage of senescent cells (80 %) in line MSCWJ-2 compared with MSCWJ-1 (58 %). 3. Numerical karyotypic analysis showed that the both lines at early and late passages have normal karyotype of 46, XX and 46, XY, respectively. The structural karyotypic analysis showed cytogenetic heterogeneity, associated in both lines with the emergence with low frequency of random clonal and non-clonal chromosomal rearrangements, disappearing during long-term cultivation. Unlike line MSCWJ-1 in MSCWJ-2 at 6th passage one detected clonal chromosomal rearrangement of the short arm of chromosome 7, occurring with high frequency at 39 % of the investigated cells, but disappearing on the 13th passage. 4. To determine status of these cell lines at 6, 13 and 28th passages, comparative analysis of the surface markers was performed. By this analysis, all the lines expressed the surface antigenes characteristic human MSC: CD44, Cd73, CD90, CD105, HLA-ABC, vimentin and did not express CD34, and HLA-DR. 5. The analysis of expression of markers of non-differentiated hESC in both lines showed a decrease in expression level surface marker SSEA-4 and the lack of expression of the transcription factor SOX-2 in line MSCWJ-2 at 13th passage compared with MSCWJ-1 at 28 passage. 6. Induction of osteogenic and adipogenic differentiation in both lines at later passages showed disruption of adhesion to the cells compared to the 6 passage. But the ability to differentiate in these directions at later passages is confirmed. The lack of chondrogenic differentiation in line MSCWJ-2 are showed at late passage in comparison with line MSCWJ-1. Thus, in line MSCWJ-2 discovered the correlation between frequently occurring clonal chromosomal rearrangement identified at an early passage, early replicative senescence with a large proportion of senescent cells, a significant decrease index proliferation during long-term cultivation, decrease or disappearance of the expression of markers non-differentiated ESCs, decreased differentiation potential. Given the metabolic cooperation between the cells, we can cautiously assume that is detected at an early passage clonal chromosomal rearrangement of the short arm of chromosome 7, occurring with high frequency, is associated with activation of cellular processes which proceed in the absence of this chromosomal aberration.