Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture.

BACKGROUND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells.

Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γaH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion.