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

Abstract

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.

Keywords: DNA double-strand breaks; cellular senescence; genome instability; long-term cultivation; mesenchymal stromal cells; replicative senescence; γH2AX foci.

Figure 1. Immunocytochemical analysis of γH2AX and pATM foci

(A) Changes in γH2AX, pATM foci and their co‐localization depending on the passage number in MSCs. (B) Comparative analysis of γH2AX, pATM foci and their co‐localization in early (3‐8) vs. late (18‐22) passages of MSCs. (C) Fraction of γH2AX foci that co‐localize with pATM at early (3‐8) vs. late (18‐22) passages of MSCs. (D) Representative immunofluorescent microphotographs of MSC showing γH2AX (green), pATM (red) foci and their co‐localization (yellow) at passage 5 and 20. Nuclei were counterstained with DAPI.

Figure 2. Differential immunocytochemical analysis of γH2AX foci in proliferating (Ki67(+)) and resting (Ki67(−)) cells

(A) Changes in the γH2AX number in Ki67(+) and Ki67(−) cells on 3-22 passages (B) Comparative analysis of γH2AX in Ki67(+) and Ki67(−) cells on early (3-8) vs. late (18-22) passages; (C) Representative immunofluorescent microphotographs of MSC showing Ki67 (green), γH2AX (red) foci and their co-localization (yellow) at passage 5 and 20. Nuclei were counterstained with DAPI.