The Dynamics of Cell Properties during Long-Term Cultivation of Two Lines of Mesenchymal Stem Cells Derived from Wharton’s Jelly of Human Umbilical Cord

Abstract

The cell properties of two MSC lines (MSCWJ-1 and MSCWJ-2) established from Wharton’s jelly of human umbilical cord were analyzed at the 6th, 13th, and 28th passages. The following were discovered. (1) The cell morphology during long-term cultivation was changed in both lines, with the cells being larger and having more debris in the MSCWJ-2 line than in the MSCWJ-1 line. (2) As shown by analysis of growth characteristics, long-term cultivation was accompanied with a significant increase of the average population-doubling time in both cell lines. This was correlated with morphological changes and a gradual increase in β-galactosidase activity, indicating the occurrence of replicative senescence. The interline changes concerned the level of proliferative activity, the index of proliferation, the time of the entry into the phase of replicative senescence (30 population doublings in the MSCWJ-2 line and 60 population doublings in the MSCWJ-1 line), and the percentage of senescent cells (80% in MSCWJ-2 line compared to 58% in the MSCWJ-1 line). (3) Numerical karyotypic analysis showed that both cell lines at early and late passages had normal karyotype 46, XX (MSCWJ-1 cells), and 46, XY (MSCWJ-2 cells). Structural karyotypic analysis showed a cytogenetic heterogeneity in both lines: low frequency of random clonal and nonclonal chromosomal rearrangements disappeared during long-term cultivation. Unlike in the case of the MSCWJ-1 line, the clonal chromosomal rearrangement of the short arm of chromosome 7 with a frequency of 39% was detected at the sixth passage in the MSCWJ-2 line but disappeared at the 13th passage. (4) Surface marker analysis of these cell lines at the 6th, 13th, and 28th passages revealed that these cells expressed surface antigens characteristic of human MSC (CD44, CD73, CD90, CD105, HLA-ABC), as well as vimentin, and did not express CD34 and HLA-DR. (5) Analysis of nondifferentiated hESC markers in both lines showed a decrease in the level of expression of the surface marker SSEA-4 and a lack of expression of the transcription factor SOX-2 at the 13th passage in the MSCWJ-2 and at the 28th passage in MSCWJ-1 cells. (6) Cell adhesion in both lines was lower in late passages compared to the cells at the sixth passage. The cells retained an osteogenic and adipogenic differentiation potential at late passages. A lack of chondrogenic differentiation in MSCWJ-2 cells, but not in MSCWJ-1 cells, was registered at late passages. The MSCWJ-2 cell line exhibited a frequently occurring clonal chromosomal rearrangement at an early passage, early replicative senescence with a large proportion of senescent cells, a significantly decreased index of proliferation during long-term cultivation, a decrease or disappearance of the expression of nondifferentiated ESCs of markers, and decreased differentiation potential. Given the metabolic cooperation between the cells, we can cautiously suppose that the clonal chromosomal rearrangement of the short arm of chromosome 7 detected at an early passage with high frequency is coupled with activation of cellular processes that proceed in the absence of this chromosomal aberration.