Dataset Information

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 ?H2AX/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 ?H2AX/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 ?-irradiation. Multifactorial regression analysis demonstrates that in vitro aging reduced DNA damage recognition in MSCs after irradiation by a multiplicative interaction with dose (p ConclusionThe 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.

SUBMITTER: Hladik D

PROVIDER: S-EPMC6664790 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Publications

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

Hladik Daniela D Höfig Ines I Oestreicher Ursula U Beckers Johannes J Matjanovski Martina M Bao Xuanwen X Scherthan Harry H Atkinson Michael J MJ Rosemann Michael M

Stem cell research & therapy 20190729 1

<h4>Background</h4>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, ...[more]

PMID: 31358047

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Project description:BACKGROUND:We studied a primary culture developed from a biopsy of a clear cell carcinoma of the ovary (O-CCC) by (a) assessing its capacity to retain in vitro pathological features of the tumor of origin; (b) characterizing the main cells released from the complex mass without forced purification of any particular cellular entity; and (c) investigating its long-term proliferative capacity. METHODS:A primary cell culture was developed from a pelvic mass diagnosed as an O-CCC. The morphological analysis of the cell culture was carried out by phase contrast microscopy. Markers of epithelial, mesenchymal, and tumor initiating cells were evaluated by immunocytochemistry. Cell proliferation was studied by detection of bromodeoxyuridine (BrdU) incorporated into newly synthesized DNA. As a biomarker of O-CCC, we assessed the expression of hepatocyte nuclear factor (HNF) 1?. RESULTS:We show that cells with epithelial morphological features express E-cadherin and expand with time in culture, a fact that the incorporation of BrdU confirms. Cells with mesenchymal-like characteristics that express the mesenchymal marker vimentin, however, allocate to the edges of the epithelial compartment. Moreover, we found that some cells with epithelial features also expressed vimentin. At the beginning of incubation, over 60 % of primary cells expressed the O-CCC marker HNF1?; such percentage declined upon passaging. We show that epithelial not mesenchymal cells undergo DNA replication, and that few cells in both epithelial and mesenchymal compartments express the stem-like tumor antigen CD133. CONCLUSIONS:We provide proof-of-principle that cells separated in bulk from a biopsy of an O-CCC can be maintained in culture for several months, and that two consistent cellular compartments-one epithelial that retains the O-CCC marker HNF1?, and another mesenchymal-persist, and seem to have a cooperative interaction leading to the multiplication of epithelial cells within a mesenchymal cellular environment.

Dataset Information

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

Background

Methods

Results

Publications

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

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