Long-term culture of mesenchymal stem cells impairs ATM-dependent recognition of DNA breaks and increases genetic instability

Abstract

Background Mesenchymal stem cells (MSCs) are attracting increasing interest for cell-based therapies, making use of both their immuno-modulating and regenerative potential. For such therapeutic applications, a massive in vitro expansion of donor cells is usually necessary to furnish sufficient material for transplantation. It is not established to what extent the long-term genomic stability and potency of MSCs can be compromised as a result of this rapid ex vivo expansion. In this study, we investigated the DNA damage response and chromosomal stability (indicated by micronuclei induction) after sub-lethal doses of gamma irradiation in murine MSCs at different stages of their in vitro expansion. Methods Bone-marrow-derived tri-potent MSCs were explanted from 3-month-old female FVB/N mice and expanded in vitro for up to 12 weeks. DNA damage response and repair kinetics after gamma irradiation were quantified by the induction of γH2AX/53BP1 DSB repair foci. Micronuclei were counted in post-mitotic, binucleated cells using an automated image analyzer Metafer4. Involvement of DNA damage response pathways was tested using chemical ATM and DNA-PK inhibitors. Results Murine bone-marrow-derived MSCs in long-term expansion culture gradually lose their ability to recognize endogenous and radiation-induced DNA double-strand breaks. This impaired DNA damage response, indicated by a decrease in the number of γH2AX/53BP1 DSB repair foci, was associated with reduced ATM dependency of foci formation, a slower DNA repair kinetics, and an increased number of residual DNA double-strand breaks 7 h post irradiation. In parallel with this impaired efficiency of DNA break recognition and repair in older MSCs, chromosomal instability after mitosis increased significantly as shown by a higher number of micronuclei, both spontaneously and induced by γ-irradiation. Multifactorial regression analysis demonstrates that in vitro aging reduced DNA damage recognition in MSCs after irradiation by a multiplicative interaction with dose ( p < 0.0001), whereas the increased frequency of micronuclei was caused by an additive interaction between in vitro aging and radiation dose. Conclusion The detrimental impact of long-term in vitro expansion on DNA damage response of MSCs warrants a regular monitoring of this process during the ex vivo growth of these cells to improve therapeutic safety and efficiency. Electronic supplementary material The online version of this article (10.1186/s13287-019-1334-6) contains supplementary material, which is available to authorized users.

Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

“…This disparity is hypothesized, in part, to be directly related to how these cells are delivered. There is wide debate on the effects of prolonged ex vivo cell culture of MSCs 39 , whether their potency changes with cryopreservation [40][41][42] , and if infused systemically they get trapped in the lungs 13 or induce an innate immune attack 18 . Our ex vivo MSC technology was designed to overcome these limitations by housing MSCs in an ex vivo blood-contacting device that significantly extends the duration of cellular therapeutic activity compared to injection.…”

Revealing Cellular Heterogeneity andIn VitroDifferentiation Trajectory of Cultured Human Endometrial Mesenchymal-like Stem Cells Using Single-cell RNA Sequencing

“…To obtain large amount MSCs for clinical use requires in vitro expansion because of limited supply of primary MSCs. However, in vitro expansion has large inter-sample variations and causes functional loss of the cells [43][44][45][46]. Thus, understanding of in vitro differentiation is critical.…”

Molecular Mechanisms Contributing to Mesenchymal Stromal Cell Aging

“…Signals generated downstream DNA damage are mostly addressed to counteract transformation but can also promote tissue dysfunction. A variety of mechanisms can contribute to DNA damage accumulation in MSC aging: telomere shortening, replicative stress and reduced DNA repair system efficiency [18,68]. Accumulation of DNA damage can lead to genomic instability and ultimately affect differentiation ability, self-renewal ability or induce cell transformation [2].…”

“…While the ATM pathway is mostly involved in double strand break (DSB) repair [69], the ATR one is prevalently involved in single strand break (SSB) and replicative stress-derived damage repair [70]. Murine bone marrow MSCs (BM-MSCs) in long-term expansion progressively lose their ability to recognize DSBs (as detected by phosphorylated histone H2AX-γH2AX-and 53BP1 foci); they show a slower repair kinetics and a consequent increased number of residual DSBs, due to an impaired ATM-mediated DNA damage response [68].…”