Thawing and shipping of human bone marrow-derived HSC: Frozen human CD34+ cells isolated from healthy adult donors of typical astronaut age (30–55 years) were purchased from AllCells, LLC or Stem Cell Technologies, Inc. and used for these experiments. Cells received from both commercial sources were of similar purity (>90% CD34+ cells) and viability (>90% viable). On the day prior to the scheduled irradiations at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), human CD34+ cells were thawed using procedures to maximize recovery and viability. In brief, thawing medium consisting of RPMI (Gibco™, ThermoFisher Scientific, Grand Island, NY) containing 30% fetal bovine serum (FBS; Lonza, Walkersville, MD), and supplemented with 20 U/mL heparin, penicillin/streptomycin (100 U/ml), and L-glutamine (all from Gibco™, ThermoFisher Scientific, Grand Island, NY), was prepared fresh on the morning the cells were to be thawed. One hundred microliters of 1 mg/mL DNase (SigmaAldrich, St. Louis, MO) was then added to each 10 mL of media, just prior to use to prevent cell clumping. Each 2 mL cryovial of cells was then quickly thawed in a 37°C water bath, and immediately diluted with 10 mL of thawing medium. The cells were then incubated in a 37°C water bath for one hour, spun down at 300xg for 10 minutes, washed once with 10 mL of thawing medium, and spun down again at 300xg for 10 minutes. Cells were then resuspended in QBSF-60 serum-free media (Quality Biologicals, Inc., Gaithersburg, MD) containing 100 ng/mL recombinant human stem cell factor (SCF; PeproTech, Rocky Hill, NJ), and transferred to sterile 1.8 mL cryovials (Nunc, ThermoFisher Scientific, Grand Island, NY), which were closed tightly and sealed with Parafilm (SigmaAldrich, St. Louis, MO), carefully packed in an insulated box, and shipped via FedEx First Overnight® for delivery to BNL early the following morning.
Exposure of human HSC to simulated SEP and GCR radiation:
Upon receipt at BNL, the HSC cryovials were maintained in a sterile 370C incubator in the BNL Medical building and transferred to the NSRL cell culture facility approximately one hour before the scheduled irradiation time. After the NSRL physicists completed calibration and dosimetry of the NSRL proton and 56Fe ion beams, the HSC cryovials were placed upright in custom foam holders and irradiated at room temperature with 1 Gy of 50 MeV protons (LET = 1.26 keV/μm) at a dose rate of 50–100 cGy/min. Doses were measured by the NSRL physicists at the approximate cell position within the flasks using a NIST-traceable tissue-equivalent ion chamber (EG&G model IC-17) used to calibrate a series of custom parallel-plate beamline ionization chambers to control beam delivery. The NSRL ion source was then immediately switched to deliver iron ions, tuning/dosimetry was again completed, and 15 minutes following the completion of the proton irradiations, additional HSC cryovials were irradiated with 20 cGy 56Fe ions (LET = 151.4 keV/µm) at a dose rate of 20–40 cGy/min. Some samples received sequential doses of protons and 56Fe ions (dual ion exposures) to better simulate a more typical deep space mixed-field exposure scenario. After irradiation, cells were immediately returned to a 37°C incubator. Another group of samples from each donor was irradiated with 1 Gy of 137Cs γ-irradiation (LET = 0.91 keV/μm) at a dose rate of 125 cGy/min using a J.L. Shepherd and Associates Mark I Model 68A located in the BNL Biomedical building. A final group of samples from each donor was also transported to the NSRL and the gamma source to serve as unirradiated (sham-irradiated) controls. At the completion of all irradiations, the cryovials were securely packaged up for overnight return shipping back to Wake Forest Institute for Regenerative Medicine (WFIRM) via FedEx First Overnight® service.
Upon receipt at WFIRM, cells in each cryovial were washedwith 10 mL of fresh QBSF-60 serum-free media (Quality Biologicals, Inc., Gaithersburg, MD), collected by centrifugation at 300xg for 10 minutes, counted with a hemocytometer, and their viability assessed by Trypan Blue exclusion according to standard methods. To assess the in vitro functionality/colony-forming potential of HSC following exposure to each irradiation scheme, we performed HALO assays, as detailed in Supplementary Materials and Methods. a highly sensitive, high-throughput ATP bioluminescence proliferation assay for lympho-hematopoietic stem and progenitor cells (Hematopoietic/Hemotoxicity Assay via Luminescence Output; HALO) 1 was performed according to the manufacturer's instructions (HemoGenix, Inc., Colorado Springs, CO). Briefly, to a HALO master mix tube containing 0.8mL of the appropriate growth factor combination (using manufacturer-provided premade master mixes with optimized cytokines/concentrations for each specific colony type to be assayed), approximately 200μL of CD34+ cell suspension was added for a final cell concentration of 10,000cells/mL. The contents of each tube were mixed by vortexing, and the tube was then allowed to settle for a minute. 100μL of the master mix containing cells was dispensed into individual wells of 96-well culture plates in 6–8 replicates such that ~1000cells were deposited into each well. The culture plates were placed in sterile 37°C incubators supplied with atmosphere of 95% air/5% CO2 mixture. After 6–7days of culture, 100μL of the ATP Enumeration Reagent (prepared fresh according to the manufacturer’s instructions [HemoGenix, Inc., Colorado Springs, CO]) was added to each well using a multichannel pipette, and the contents of each well were mixed well by repeated pipetting. The plate was then allowed to incubate at room temperature in the dark for 15 minutes, after which the bioluminescence of each well was measured with a Veritas microplate luminometer (Turner BioSystems, Sunnyvale, CA) and the data exported directly into an Excel spreadsheet (Microsoft Corp., Seattle, WA).
Transplantation of immunodeficient mice to assess human HSC functionality in vivo:
To test their in vivo potential/functionality, human HSC exposed to each irradiation scheme were used to reconstitute human hematopoiesis in immunodeficient NOD/LtSz-ScidIL2Rgamma-/- (NSG) mice (The Jackson Laboratory, Bar Harbor, ME), using previously published methods 2, in accordance with a Wake Forest University Health Science IACUC-approved protocol. In brief, on each of the two days prior to HSC transplant, the mice were given an IP injection of a sublethal-conditioning dose of busulfan (20 mg/kg). This dosing regimen has been shown to result in highly efficient engraftment of human HSC, yet is mild enough that, if left untransplanted, the endogenous murine hematopoiesis recovers with sufficient rapidity to ensure all animals survive 2. To perform the transplant, mice were warmed under a heat lamp for several minutes to dilate the tail vein and were briefly restrained (~1 minute) while human HSC (~2x105 cells in 0.1–0.3 mL) were injected into the tail vein, using a 30-g needle. The behavior of the mice was carefully monitored during the procedure for any signs of stress/overheating.
While there are several inherent drawbacks to using xenogeneic transplant systems to study the in vivo behavior/functionality of human HSC, they provide a very valuable avatar model to evaluate immediate and long-term radiation effects 3. One issue is the wide range of variability that is often seen in engraftment levels between individual mice in different treatment groups. However, the variation in engraftment levels we observed within animals receiving the same cell source in these studies was more tightly clustered than other recent studies employing NSG mice 4.
Histopathology/Immunohistochemistry on enlarged spleens:
To define the cause of the marked splenomegaly in the mice transplanted with human HSC exposed to 56Fe ions, the spleen was collected from all animals in the second transplant cohort at the time of euthanasia. The spleen was “bread-loafed” with a scalpel, and the resulted tissue pieces were then fixed with 4% PFA overnight, and preserved in paraffin blocks using standard methodology. 6-µm tissue sections were prepared from paraffin blocks and immunostained with various human-specific antibodies to confirm the human origin of the cells within the enlarged spleens and to establish their immunophenotype. Briefly, following tissue deparaffinization and rehydration, slides were washed with PBS and stained with Hematoxylin-Eosin for histopathology, using standard procedures, or stained with human-specific antibodies to various antigens in a Leica Bond 3 Autostainer (Leica Biosystems, Buffalo Grove, IL).Specifically, all sections were subjected to antigen retrieval using Epitope Retrieval solution #2 for 20 minutes and were then stained with anti-CD7 (Clone LP15 – 1:100), anti-TdT (Clone SEN28 – predilute/ready-to-use), and CD3 (Clone LN10 – 1:100). Detection was then performed using a DAB Polymer Refine detection kit according to manufacturer’s instructions.All reagents employed were from Leica Biosystems. Sections were mounted with MM24 mounting media (Leica Biosystems), visualized and images acquired using a Leica DM4000B microscope, stored as TIFF files, and subjected to minimal global processing using Adobe Photoshop CS5 (Adobe Systems, San Jose, CA).
FACS analysis of human hematopoietic engraftment and differentiation:
At euthanasia, all long bones and the spleen were harvested from each animal and processed with a glass homogenizer to obtain a single cell suspension of bone marrow cells or splenocytes, respectively. The cells were then incubated overnight at 4°C in PBS containing 5% FBS (Lonza, Walkersville, MD) to block non-specific binding. On the following morning, the cells were subjected to red blood cell lysis using a commercially available solution according to manufacturer’s instructions (Becton Dickinson Immunosystems [BDIS], San Jose, CA). Cells were then spun down at 300xg for 10 minutes, resuspended in ice-cold PBS, and counted using a hemocytometer. Cells were then stained with the following fluorophore-conjugated antibodies to the following human markers/antigens to identify the various human hematopoietic populations: CD59 (labels all human cells in NSG mice with high specificity 5), CD34, CD38, CD4, CD7, CD8, CD14, CD19, CD33, CD45, and Glycophorin A. All tubes were co-stained with antibodies to mouse CD45 and Ter119 to exclude endogenous mouse cells from the analyses. All antibodies used for flow cytometric analysis were obtained from BDIS (San Jose, CA), except anti-Glycophorin A, which was purchased from Abcam (Cambridge, MA). Following a 15-minute incubation in the dark at 4°C, cells were washed with 3 mL of PBS containing 0.1% NaN3, and spun down as before. The wash solution was decanted, and the cell pellet resuspended in 350 μL of PBS containing 1% formaldehyde. Cells were then analyzed on a FACScaliber (BDIS, San Jose, CA) flow cytometer. Data were then analyzed in FlowJo Data Analysis Software (FlowJo, LLC, Ashland, OR), and statistical analyses performed using Prism 6 (GraphPad, Inc. Software, La Jolla, CA). Engraftment levels within specific lineages were compared between mice receiving sham-irradiated human HSC and those receiving human HSC exposed to each irradiation scheme, by performing one-way ANOVA, Brown-Forsythe test, and Bartlett's test. For statistical tests, data outliers with Q = 2% were removed by Prism 6.
Whole exome sequencing of DNA from human HSC exposed to SEP and GCR:
Immediately upon receipt at WFIRM (~18 h post-irradiation), aliquots of CD34+ cells exposed to each irradiation scheme were washed once with QBSF-60 serum-free media (Quality Biologicals, Inc., Gaithersburg, MD), and collected by centrifugation at 300xg for 10 minutes. Total genomic DNA was then extracted from each HSC aliquot using the Quick-gDNA™ MicroPrep kit according to the manufacturer’s instructions (Zymo Research, Irvine, CA). DNA quality/purity was assessed with a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE), and all samples passing this initial quality check were then used for unbiased whole genome amplification using the REPLI-g kit according to manufacturer’s instructions (Qiagen, Valencia, CA). Samples were then packed on dry ice and shipped to BGI Tech Solutions Co., LTD (Cambridge, MA), where they underwent an additional quality check, and subjected to whole exome sequencing (WES) using the new Ion Torrent™ Next-Generation Sequencing platform, which enables highly accurate sequencing, with 100X target coverage.
To identify genetic variants induced by the specific irradiation schemes, control and irradiated exome sequencing data from each individual donor were called independently, followed by simple subtraction. Variants annotation was performed using the software Annovar6.To find regions in the genome that were potentially enriched in mutations in each irradiated sample, we performed the following steps: 1) first the genome was divided into intervals (windows) of 50 kb; 2) for each interval, we counted the number of variants that were induced by the treatment; 3) we extracted the top 0.1% of those intervals from step 2. These lists of variant-containing regions were limited further by only considering the variants that fell within coding regions.
References:
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