Exposure of Cultured Human Mesenchymal Stem Cells to Chemotherapy Induces An Early and Permanent Telomere Loss: Biological and Clinical Implications
Authors: Buttiglieri Stefano , Spatola Tiziana , Ruella Marco , Risso Alessandra , Montanini Melissa , Tracchi Silvia , Silengo Lorenzo , Avvedimanto Enrico , Tarella Corrado 
Published: October 2019

Abstract
Abstract 4776

Introduction.
A good indicator of the rate of cell ageing is the length of telomeres, the long tandem repeat sequences at the end of chromosomes. Telomere length (TL) in humans decreases at each somatic cell division, and undergoes progressive shortening with increasing age. Indeed, cell proliferation has been considered the primary factor responsible of TL shortening and ultimately of the cell ageing process. Exposure to DNA-damaging substances has been proposed as an alternative and relevant factor involved in the cell ageing process. In spite of the number of studies in both human and animal models, few information is available on the role of both cell proliferation and DNA damage in normal human stem cells. The present study addressed this issue by investigating the ageing process in cultured Bone-Marrow (BM) human Mesenchymal Stem Cells (MSCs). Aim of the study has been to investigate whether and how exposure to toxic agents may affect the rate of TL shortening in cultured MSCs.

Methods.
1. MSCs were cultured from human mononuclear BM cells, obtained from normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium containing heparin and 10% platelet lysates and fed at 3–4 day intervals, until they reached confluence; they were then detached with trypsin-EDTA solution and reseeded for the experimental tests. Cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and negativity for CD14, CD34 and CD45 by flow cytometry; 2. cultured MSC were assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed to induce DNA damage: Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times; 3. telomere was evaluate in MSCs before (day 0 or T0) and after exposure to chemotherapeutic drugs, i.e. at 7 – 14 –21 and 28 days of culture since drug exposure. TL was determined by the cytofluorimetric Flow-fish assay and by southern-blot analysis; 4. MSC were also cultured in differentiating medium in order to induce osteogenic and adipogenic differentiation, evaluated by Alizarin red and Oil red staining, respectively; 5. lastly, both ATM phosphorilation and telomerase activation were assessed, by Western blot analysis and the TRAP assay, respectively.

Results.
1. as expected, TL progressively decreased after 7 days in culture in untreated (NT) MSC (88,8% compared to T0); TL was further shortened as long as the cells were maintained in culture; 2. a marked TL shortening occurred in MSC already at day 7 after exposure to sub-lethal doses of Doxo and Eto, with a mean of 81,7% and 79,6% compared to T0, respectively. Indeed, a minimum of 5 days in culture was required to identify the earliest signs of telomere loss. Again, further TL shortening was observed later on and the difference in TL between NT and chemotherapy-exposed cells progressively increased: compared to T0, TL resulted of 82 % and 74% in NT cells, whereas TL was 68% and 54% in Doxo-treated, and 70% and 62% in Eto-treated, at week 3 and 4, respectively; 3. a single 2-hr exposure to Doxo or Eto induced a TL loss compared to NT cells, that was maintained unchanged at long-term in culture; 4. when MSC were assayed in differentiation inducing medium, cells exposed to chemotherapy displayed enhanced adipogenic and osteogenic differentiation compared to NT cells; 5. ATM was strongly phosphorilated following exposure to Doxo and Eto compared to NT MSC; ATM phosphorilation preceded TL loss; by contrast, telomerase was unaffected by chemotherapy exposure.

Conclusions.
i. human cultured MSCs exposed to chemotherapeutic drugs offer a simple and reliable means to investigate the correlation between DNA damage and cell ageing; ii. telomere loss is clearly detectable in MSCs already after 7 days following exposure to Doxo and Eto and remains detectable at long-term in culture as a permanent signature of the previous DNA damage; iii. the in vitro observation suggests that not only hematopoietic cells but also stromal cells, including MSCs, may experience relevant DNA damages following in vivo treatments with chemotherapeutic drugs.

Disclosures:
No relevant conflicts of interest to declare.