Characterization of a Nonimmortalized Mesenchymal Stem Cell Line Isolated from Human Epicardial Adipose Tissue

Abstract

A new nonimmortalized mesenchymal stem cell (MSC) line from human epicardial adipose tissue (ADH–MSC) of a 50-year-old donor in the process of coronary artery bypass has been characterized. In the process of long-term cultivation (passages 8-16), the proportion of senescent cells gradually increases. According to the β-galactosidase activity, the number of senescent cells by passage 16 reached 66%. By this time, other signs indicate the onset of the active phase of replicative senescence: increased cell size and spreading, declined cloning efficiency and proliferation rate, increased the cell population doubling time. These results confirm the limited lifespan of ADH–MSC cells that is common for nonimmortalized cell populations. The karyotypic analysis showed, that at the eighth passage, the cells had normal human karyotype. By passage 12, the karyotypic heterogeneity became substantially increased, and by passage 16 it exceeded the permissible level of chromosomal disorders in normal MSCs. The formation of novel structural karyotype variants (SVK) was shown. The predominant participation of the short arm of one of the homologues of chromosome 21 in clonal and nonclonal rearrangements, as well as in dicentrics by the type of telomeric associations, was found. At passages 8 and 16, the cells exhibited high expression of surface antigens characteristic for human MSCs (CD44, CD73, CD90, CD105, HLA–ABC) and an absence of CD34, CD45, and HLA–DR expression. At the eighth passage, there was a significant expression of SSEA-4, a marker of early ESC differentiation. Its expression significantly reduced at passage 16. At passages 8–16, ADH–MSC cells were able to differentiate equally in the osteogenic and chondrogenic directions. The cell capacity for adipogenic differentiation decreases by passage 16. Collectively, our results show that the MSC cell line demonstrated significant changes in the process of early replicative senescence, possibly related to the altered microenvironment for the cells of the donor with heart disease. Early senescence and karyotypic instability may be associated with more significant impairment of the DNA repair system of ADH–MSC cells compared to other lines.