Date Published: February 12, 2018
Publisher: Public Library of Science
Author(s): Victoria Nikitina, Tatiana Astrelina, Vladimir Nugis, Aleksandr Ostashkin, Tatiana Karaseva, Ekaterina Dobrovolskaya, Dariya Usupzhanova, Yulia Suchkova, Elena Lomonosova, Sergey Rodin, Vitaliy Brunchukov, Stanislav Lauk-Dubitskiy, Valentin Brumberg, Anastasia Machova, Irina Kobzeva, Andrey Bushmanov, Aleksandr Samoilov, Daniela Cimini.
Spontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC) are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa.
The immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR) analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells.
The immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12%) are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described.
The spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before use in medicine.
Quality control and standardization of cellular biomedical products are important for favourable outcomes of cell therapies. Even with optimal conditions of cultivation in vitro, there can be a change of properties and loss of functional characteristics of the tissue in vivo. This can be attributed to a lack of regulatory humoral factors, loss of intracellular and intertissue interactions, genetic variability and spontaneous mutagenesis. Mutation variability is a genetic variation among members of the same species, which leads to the emergence of genotypic and phenotypic differences from parent forms. Genetically modified cells that may occur as a result of adaptation to culture conditions give rise to new cell clones. If a chromosome aberration increases the rate of proliferation or leads to other changes, which are useful for cells, then the faster growing cells could rapidly displace the slower cells with normal karyotype. In the cytogenetics of continuous cell lines, this process is called “karyotypic evolution” and is subdivided into two stages, namely establishment and stabilization. The stages are different in karyotypic variability of the cell populations and in peculiarities of clone selection. “Establishment” is characterized by genetic heterogeneity of the cell population and selection of cell clones that are best adapted to the existence in vitro and may last for a prolonged period of time before emergence of a stable population. During “stabilization” the cell population of lines usually consists of one predominant cell clone and some minor subclones [1, 2].
A previously studied, genetically stable (up to passage 5) MSC culture, with a normal karyotype, was defrosted and cultivated until the mitotic activity significantly decreased (passage 26). Despite the significant differences in the number of chromosome breaks at some passages (Table 2) and although previous studies have indeed shown increased genomic instability in stem cell cultures that have been expanded for a prolonged period of time, the current study did not find a clear relationship between passage number and genomic instability. Breakpoints were heterogeneous from one batch to another (see Fig 2); they were probably random and characterized by the spontaneous level of chromosome aberrations in MSC. The level of spontaneous random chromosome aberrations estimated using mFISH was approximately 3 times higher than in previously studied MSC, obtained from adipose tissue analysed at early culture passages . This can be attributed to both the duration of the cultivation and the difference in staining methods, since routine staining of chromosomes does not allow for the evaluation of the spectrum of chromosomal rearrangements as well as mFISH. The chromosome was counted according to the modal class principle (Fig 4), and the spread of values was discovered (45–99 chromosomes). Metaphases with more than 50 chromosomes are characterized by ploidy– 3n, 4n. The number of polyploid cells was 3.1% by passage 12 (Fig 4, Table 3) and stays almost invariable. In this study, polyploid cells were detected at all cultivation passages. The polyploidization (multiple sets of homologous chromosomes), including cells with clonal aberrations, is one of the most common ways of immortalized cell lines establishment. The rising amount of polyploid cells in the MSC line is an unfavourable sign for the cellular biomedical products.