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

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.

The circRNA interactome-innovative hallmarks of the intra- and extracellular radiation response.

Generated by Quaking (QKI), circular RNAs (circRNAs) are newly recognised non-coding RNA (ncRNA) members characterised by tissue specificity, increased stability and enrichment within exosomes. Studies have shown that ionizing radiation (IR) can influence ncRNA transcription. However, it is unknown whether circRNAs or indeed QKI are regulated by IR. Microarray circRNA profiling and next generation sequencing revealed that circRNA expression was altered by low and medium dose exposure sourced predominantly from genes influencing the p53 pathway. CircRNAs KIRKOS-71 and KIRKOS-73 transcribed from the WWOX (WW Domain Containing Oxidoreductase) tumor suppressor (a p53 regulator) responded within hours to IR. KIRKOS-71 and KIRKOS-73 were present in exosomes yet exhibited differential transcript clearance between irradiated cell lines. Dual-quasar labelled probes and in-situ hybridization demonstrated the intercellular distribution of KIRKOS-71 and KIRKOS-73 predominantly within the perinucleus. QKI knockdown removed nuclear expression of these circRNAs with no significant effect on cytosolic KIRKOS-71 and KIRKOS-73. Distinct QKI transcription between cell lines and its augmented interaction with KIRKOS-71 and KIRKOS-73 was noted post IR. This foremost study provides evidence that QKI and circRNAs partake in the cellular irradiation response. KIRKOS-71 and KIRKOS-73 as stable secreted circRNAs may afford vital characteristics worth syphoning as promising diagnostic radiotherapy biomarkers.