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Exposure to 20 mGy radiation decreases HSC functions
HSC pool, demonstrating that preserving genome integrity is crucial for HSC long-term maintenance (reviewed by Biechonski and Milyavsky).16 For instance, ku80, lig4 and atm-deficient mice exhibit defects in HSC maintenance and self-renewal.10-12 Atm-deficient HSC harbor increased levels of reactive oxygen species (ROS) responsible for their loss of hematopoietic reconstitution capacity10 that can be res- cued by treatment with the antioxidant N-acetylcystein (NAC). Indeed, increased ROS in HSC induce their differ- entiation and their exhaustion17,18 and quiescent HSC with the lowest level of ROS have the highest hematopoietic reconstitution potential compared to 'activated' HSC har- boring higher ROS levels.17 Interestingly, in mouse and human, ROS and DNA damage accumulate in HSC upon serial transplantation resulting in decreased self-renewal capacities. NAC-treated HSC are protected against the accumulation of oxidative DNA damage.18,19
Ionizing radiations (IR) represent the main source of DNA damage and ROS. Importantly, the human popula- tion is increasingly exposed to low doses of IR (LDIR, <100 mGy) due to the recurrent use of medical imaging.20 Studies have shown that combinations of several comput- ed tomography (CT) scans (thoracic or cranial) can increase the risk of developing cancer.21 Indeed, having more than five CT scans (corresponding to a cumulative dose of 30 mGy) can lead to a 3-fold increase in the risk of developing pediatric leukemia. Moreover, a recent study showed that 20 mGy LDIR affects the fundamental prop- erties of HSC in mouse.22 In this context, it is crucial to study the consequences of LDIR exposure in human cells, in particular in human HSC. Here we show through com- bining in vitro and in vivo studies that a single acute 20 mGy LDIR decreases human HSPC serial clonogenic and recon- stitution potentials, and that these effects are mediated through a ROS/p38MAPK-dependent signaling pathway.
Methods
Primary cells
Cord blood (CB) samples were collected from healthy infants with the informed written consent of the mothers according to the Declaration of Helsinki. Samples were obtained in collaboration with the Clinique des Noriets, Vitry-sur-Seine, and with the Cell Therapy Department of Hôpital Saint-Louis, Paris, France. Samplings and experiments were approved by the Institutional Review Board of INSERM (Opinion n. 13-105-1, IRB00003888). CD34+ cells were purified by immuno-magnetic selection using a CD34 MicroBeads kit (Miltenyi Biotec, Paris, France). For each experiment, we used a pool of CD34+ cells from different healthy infants to diminish individual variability.
Low dose of ionizing radiations
20 mGy LDIR was delivered with a dose rate of 20 mGy/minute (min) using a Cobalt 60 Irradiator (Alcyon). 2.5 Gy was delivered with a dose rate of 1 Gy/min.
Flow cytometry and cell sorting
CD34+CD38low cells and CD34+CD38lowCD45RA–CD90+ HSPC were isolated after labeling with human specific monoclonal anti- bodies (MoAbs, see Online Supplementary Table S1 for details). Cell sorting was performed using either a Becton Dickinson (BD)- FACS-ARIA3 SORP or a BD-FACS-Influx (laser 488, 405, 355, 561 and 633, BD Bioscience). Flow cytometry experiments are described in the Online Supplementary Methods.
Transplantation assays
NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice (Jackson Laboratory, Bar Harbor, ME, USA) were housed in the pathogen- free animal facility of IRCM, CEA, Fontenay-aux-Roses, France. Adult 8-12-week old NSG mice received a 3 Gy sublethal irradia- tion using a GSRD1 γ-irradiator (source Cesium137, GSM) and were anesthetized with isoflurane before intravenous retro-orbital injection (i.v.) of human cells as described in the Online Supplementary Methods. All experimental procedures were carried out in compliance with French Ministry of Agriculture regulations (animal facility registration n.: A9203202, Supervisor: Michel Bedoucha, APAFIS #9458-2017033110277117v2) for animal exper- imentation and in accordance with a local ethical committee (#44).
Immunofluorescence
Immunofluorescence was performed on cell-sorted HSPC irra- diated and incubated 30 min, 1 hour (h), or 3 h at 37°C in MyeloCult medium, as previously described.22-24 Details of the methods used are available in the Online Supplementary Methods.
Drug treatments
CD34+ cells or CD34+ CD38lowCD45RA–CD90+ HSPC were treated with several drugs as described in the Online Supplementary Methods.
Colony forming unit-cell assay
Colony forming unit-cell assay (CFU-C) and serial platings were performed as previously described;6 see Online Supplementary Methods for details. Depending on CB pool samples, 60-80 colonies were generated from 500 HSPC non-irradiated or irradi- ated at 20 mGy.
Primary and extended long-term culture initiating cell assays
Long-term culture initiating cell assay was performed as previ- ously described6 and is described in detail in the Online Supplementary Methods.
Intracellular flow cytometry
Ki67, cleaved caspase 3, phospho-p38MAPK (P-p38, phospho- rylation on Thr180/Tyr182), phospho-ATM, p53 and phospho- p53 staining were performed as previously described4 (Ki67) and according to the manufacturer's instructions, respectively. More details can be found in the Online Supplementary Methods.
Carboxyfluorescein diacetate succinimidyl ester staining
3x105 CD34+ cells were labeled with carboxyfluorescein diac- etate succinimidyl ester (CFSE) (2.5 mM, Sigma, France) and cul- tured in StemSpan medium supplemented with cytokines (Stem Cell Technologies), as described in the Online Supplementary Methods.
Reactive oxygen species quantification and mitochondria activity assay
Reactive oxygen species quantification was performed with fresh CD34+ cells using CellRox Orange reagent following the manufacturer's instructions (ref. C10443, Molecular Probes, ThermoFisher Scientific). Mitochondrial activity assay was per- formed using mitotracker green (MTG) and TMRE products, according to the manufacturer's instructions (Molecular Probes, ThermoFisher Scientific).
Statistical analysis
Mann and Whitney (M&W) and Kruskal and Wallis (K&W)
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