FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2012156551> ?p ?o ?g. }

• W2012156551 endingPage "956" @default.
• W2012156551 startingPage "944" @default.
• W2012156551 abstract "Hematopoiesis is regulated by the bone marrow (BM) niche microenvironment. We recently found that posttransplant administration of AMD3100 (a specific and reversible CXCR4 antagonist) enhanced donor cell engraftment and promoted recovery of all donor cell lineages in a congeneic mouse transplant model. We hypothesized that AMD3100 enhances donor cell reconstitution in part by modulating the levels and constitution of soluble factors in the niche microenvironment. In the current study, the effects of the BM extracellular fluid (supernatant) from AMD3100-treated transplant recipient mice on colony-forming units (CFUs) were examined. A semiquantitative, mass spectrometry–based proteomics approach was used to screen for differentially expressed proteins between the BM supernatants of PBS-treated transplant mice and AMD3100-treated transplant mice. A total of 178 proteins were identified in the BM supernatants. Thioredoxin was among the 32 proteins that displayed greater than a twofold increase in spectral counts in the BM supernatant of AMD3100-treated transplant mice. We found that thioredoxin increased CFUs in a dose-dependent manner. Thioredoxin improved hematopoiesis in irradiated mice and protected mice from radiation-related death. Furthermore, ex vivo exposure to thioredoxin for 24 hours enhanced the long-term repopulation of hematopoietic stem cells. Additionally, combined posttransplant administration of thioredoxin and AMD3100 improved hematologic recovery in primary and secondary transplant recipient mice. Our studies demonstrated that factors in the BM niche microenvironment play a critical role in hematopoiesis. Identifying these factors provides clues on potential novel targets that can be used to enhance hematologic recovery in hematopoietic stem cell transplan`tation. Hematopoiesis is regulated by the bone marrow (BM) niche microenvironment. We recently found that posttransplant administration of AMD3100 (a specific and reversible CXCR4 antagonist) enhanced donor cell engraftment and promoted recovery of all donor cell lineages in a congeneic mouse transplant model. We hypothesized that AMD3100 enhances donor cell reconstitution in part by modulating the levels and constitution of soluble factors in the niche microenvironment. In the current study, the effects of the BM extracellular fluid (supernatant) from AMD3100-treated transplant recipient mice on colony-forming units (CFUs) were examined. A semiquantitative, mass spectrometry–based proteomics approach was used to screen for differentially expressed proteins between the BM supernatants of PBS-treated transplant mice and AMD3100-treated transplant mice. A total of 178 proteins were identified in the BM supernatants. Thioredoxin was among the 32 proteins that displayed greater than a twofold increase in spectral counts in the BM supernatant of AMD3100-treated transplant mice. We found that thioredoxin increased CFUs in a dose-dependent manner. Thioredoxin improved hematopoiesis in irradiated mice and protected mice from radiation-related death. Furthermore, ex vivo exposure to thioredoxin for 24 hours enhanced the long-term repopulation of hematopoietic stem cells. Additionally, combined posttransplant administration of thioredoxin and AMD3100 improved hematologic recovery in primary and secondary transplant recipient mice. Our studies demonstrated that factors in the BM niche microenvironment play a critical role in hematopoiesis. Identifying these factors provides clues on potential novel targets that can be used to enhance hematologic recovery in hematopoietic stem cell transplan`tation. Hematopoietic stem cell transplantation (HSCT) provides a potentially curative treatment approach for a wide variety of diseases. HSCT, however, is associated with a high incidence of morbidity and mortality. Approximately 25% of patients die within 1 year of transplant from transplant-related complications. The speed of donor cell engraftment and hematopoietic recovery has been correlated with patients' overall survival and transplant outcomes. Recombinant colony-stimulating factor granulocytes are commonly used in HSCT to enhance neutrophil recovery [1Dekker A. Bulley S. Beyene J. Dupuis L.L. Doyle J.J. Sung L. Meta-analysis of randomized controlled trials of prophylactic granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor after autologous and allogeneic stem cell transplantation.J Clin Oncol. 2006; 24: 5207-5215Crossref PubMed Scopus (94) Google Scholar]; however, the effects are limited to myeloid cell lineage [2Battiwalla M. McCarthy P.L. Filgrastim support in allogeneic HSCT for myeloid malignancies: a review of the role of G-CSF and the implications for current practice.Bone Marrow Transplant. 2009; 43: 351-356Crossref PubMed Scopus (27) Google Scholar]. Furthermore, over the last few decades we have seen advances in identifying molecules that regulate hematopoietic stem cell (HSC) expansion and reconstitution [3Almeida-Porada G.D. Stem cell gene manipulation and delivery as systemic therapeutics.Adv Drug Deliv Rev. 2010; 62: 1139-1140Crossref PubMed Scopus (3) Google Scholar, 4Yu X. Zou J. Ye Z. et al.Notch signaling activation in human embryonic stem cells is required for embryonic, but not trophoblastic, lineage commitment.Cell Stem Cell. 2008; 2: 461-471Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Crispino J.D. GATA1 in normal and malignant hematopoiesis.Semin Cell Dev Biol. 2005; 16: 137-147Crossref PubMed Scopus (159) Google Scholar, 6Jude C.D. Gaudet J.J. Speck N.A. Ernst P. Leukemia and hematopoietic stem cells: balancing proliferation and quiescence.Cell Cycle. 2008; 7: 586-591Crossref PubMed Scopus (40) Google Scholar, 7Goldman D.C. Bailey A.S. Pfaffle D.L. Al Masri A. Christian J.L. Fleming W.H. BMP4 regulates the hematopoietic stem cell niche.Blood. 2009; 114: 4393-4401Crossref PubMed Scopus (91) Google Scholar]; however, genetically modifying HSCs with these molecules has several limitations and is not readily translatable into the clinic. There is an unmet medical need to develop novel approaches for enhancing hematologic recovery in HSCT.After HSCT, donor HSCs home to the bone marrow (BM), engraft, and eventually reconstitute the entire hematologic and immunologic repertoire of the recipient [8Chavakis E. Urbich C. Dimmeler S. Homing and engraftment of progenitor cells: A prerequisite for cell therapy.J Mol Cell Cardiol. 2008; 45: 514-522Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 9Voermans C. van Hennik P.B. van der Schoot C.E. Homing of human hematopoietic stem and progenitor cells: New insights, new challenges?.J Hematother Stem Cell Res. 2001; 10: 725-738Crossref PubMed Scopus (40) Google Scholar]. HSC homing is not a random event, and transplanted HSCs are not evenly distributed in the BM space. BM space is compartmentalized as individual specialized microenvironments, termed niches [10Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell.Blood Cells. 1978; 4: 7-25PubMed Google Scholar], that provide cellular and molecular support for HSCs to reside, proliferate, and differentiate [11Li L. Xie T. Stem cell niche: structure and function.Annu Rev Cell Dev Biol. 2005; 21: 605-631Crossref PubMed Scopus (934) Google Scholar]. HSCs in the niches are regulated by: (1) cytokines and chemokines [12Wilson A. Trumpp A. Bone-marrow haematopoietic-stem-cell niches.Nat Rev Immunol. 2006; 6: 93-106Crossref PubMed Scopus (1044) Google Scholar, 13Yin T. Li L. The stem cell niches in bone.J Clin Invest. 2006; 116: 1195-1201Crossref PubMed Scopus (636) Google Scholar, 14Eliasson P. Jönsson J.-I. The hematopoietic stem cell niche: low in oxygen but a nice place to be.J Cell Physiol. 2010; 221: 17-22Crossref Scopus (337) Google Scholar], (2) supportive cellular elements such as mesenchymal stem cells and endothelial cells and their secreted, soluble molecules, or membrane-bound factors [11Li L. Xie T. Stem cell niche: structure and function.Annu Rev Cell Dev Biol. 2005; 21: 605-631Crossref PubMed Scopus (934) Google Scholar], (3) the nervous system and neurotransmitters [15Spiegel A. Kalinkovich A. Shivtiel S. Kollet O. Lapidot T. Stem cell regulation via dynamic interactions of the nervous and immune systems with the microenvironment.Cell Stem Cell. 2008; 3: 484-492Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Spiegel A. Shivtiel S. Kalinkovich A. et al.Catecholaminergic neurotransmitters regulate migration and repopulation of immature human CD34+ cells through Wnt signaling.Nat Immunol. 2007; 8: 1123-1131Crossref PubMed Scopus (266) Google Scholar], and (4) extracellular matrix.The interaction between stromal-derived factor-1 (SDF-1, now termed CXCL12) and the CXCR4 chemokine receptor plays a critical role in hematopoiesis after HSCT. The SDF-1/CXCR4 interaction is important in attracting HSCs into the niches, anchoring HSCs in the niches, and maintaining HSCs in a quiescent state [17Peled A. Petit I. Kollet O. et al.Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.Science. 1999; 283: 845-848Crossref PubMed Scopus (1452) Google Scholar, 18Bowie M.B. McKnight K.D. Kent D.G. McCaffrey L. Hoodless P.A. Eaves C.J. Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect.J Clin Invest. 2006; 116: 2808-2816Crossref PubMed Scopus (268) Google Scholar, 19Cashman J. Clark-Lewis I. Eaves A. Eaves C. Stromal-derived factor 1 inhibits the cycling of very primitive human hematopoietic cells in vitro and in NOD/SCID mice.Blood. 2002; 99: 792-799Crossref PubMed Scopus (111) Google Scholar, 20Cashman J. Dykstra B. Clark-Lewis I. Eaves A. Eaves C. Changes in the proliferative activity of human hematopoietic stem cells in NOD/SCID mice and enhancement of their transplantability after in vivo treatment with cell cycle inhibitors.J Exp Med. 2002; 196: 1141-1150Crossref PubMed Scopus (49) Google Scholar]. Plerixafor (AMD3100) is a specific and reversible CXCR4 antagonist and is currently approved by the U.S. Food and Drug Administration to be used in pretransplant setting for stem cell collection. We recently found that giving AMD3100 subcutaneously every other day beginning at day 2 after transplant selectively enhanced donor cell engraftment and promoted recovery of all donor cell lineages in a congeneic mouse transplant model [21Kang Y. Chen B.J. Deoliveira D. Mito J. Chao N.J. Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.PLoS One. 2010; 5: e11316Crossref PubMed Scopus (39) Google Scholar]. In addition, we found that posttransplant administration of AMD3100 significantly improved animal survival, likely resulting from a significant reduction in the levels of proinflammatory cytokines and chemokines [21Kang Y. Chen B.J. Deoliveira D. Mito J. Chao N.J. Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.PLoS One. 2010; 5: e11316Crossref PubMed Scopus (39) Google Scholar]. Enhancing donor cell engraftment and reducing transplant-related mortality are important goals in HSCT, and our approach offers a potentially effective, novel approach to improve the care and outcome of HSCT patients. We have recently initiated a two-center (Duke University and Medical University of South Carolina), phase I/II clinical trial to test the safety and efficacy of posttransplant administration of plerixafor in myeloablative allogeneic HSCT patients (ClinicalTrials.gov: NCT01280955).The detailed mechanisms underlying the enhanced donor cell engraftment with posttransplant administration of AMD3100 are not completely understood. CXCR4 is expressed not only on HSCs, but also on mesenchymal stem cells, endothelial cells, T cells, dendritic cells, neurons, and other cells. All these cells constitute BM niche microenvironments and regulate hematopoiesis through cell-cell interaction or secretion of soluble factors, including cytokines and chemokines. In this study, we aimed to understand the effects of posttransplant administration of AMD3100 on the BM niche microenvironement and to identify the soluble factors that can potentially be used to enhance hematologic recovery in HSCT.MethodsAnimalsEight to twelve-week-old C57BL/6 CD45.2 mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Breeding pairs of C57BL/6 CD45.1 (BSJL) mice were purchased from the Jackson Laboratories and their offspring produced and maintained in our pathogen-free animal facility at the Medical University of South Carolina (MUSC). Studies were performed in accordance with procedures approved by the MUSC Institutional Animal Care and Use Committee.Antibodies and reagentsAMD3100 and thioredoxin-1 (Trx) were purchased from Sigma-Aldrich (St. Louis, MO). Biotin-conjugated anti-mouse CD3e (145-2C11), CD4 (RM4-5), CD5 (53-7.3), CD8a (53-6.7), CD11b (M1/70), B220 (RA3-6B2), Gr-1 (RB6-8C5), TER-119 (TER119); allophycocyanin (APC)-conjugated anti-mouse CD117 (c-Kit, 2B8); phycoerythrin (PE)-conjugated anti-mouse Sca-1 (D7); APC-conjugated anti-mouse CD3 (145-2C11); APC-Cy7–conjugated anti-mouse Gr-1 (RB6-8C5); fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD45.2 (104); PE-conjugated anti-mouse CD45.1 (A20); and APC-conjugated anti-mouse CD45.1-PE (A20) were purchased from BD Pharmingen (San Diego, CA). PE-Cy5.5–conjugated anti-mouse B220 (RA3-6B2) was purchased from eBioscience (San Diego, CA). The secondary antibodies were FITC-labeled streptavidin (BD Pharmingen) and Brilliant Violet 605-labeled streptavidin (BioLegend, San Diego, CA). Anti-mouse thioredoxin antibody was purchased from Cell Signaling (Danvers, MA).Preparation of BM supernatants from PBS- or AMD3100-treated transplant recipient miceRed blood cell (RBC)-depleted BM cells were obtained from C57BL/6 CD45.1 mice and enriched for Lineage negative (Lin–) cells using murine lineage cell depletion kit (Miltenyi Biotec, Auburn, CA). Lin– BM cells were subsequently stained with a cocktail of primary monoclonal antibodies consisting of biotin-conjugated anti-mouse CD3, CD4, CD5, CD8a, CD11b, B220, Gr-1, anti-TER-119, as well as APC-conjugated anti-mouse c-Kit and PE-conjugated anti-mouse Sca-1 antibodies, followed by secondary staining with FITC-labeled streptavidin. Propidium iodide (1 μg/ml) was added to the cell suspension to exclude dead cells. The stained BM cells were sorted for Lin–/lowSca-1+c-Kit+ (LSK) HSCs using MoFlo cell sorter (Beckman Coulter, Brea, CA). Sorted LSK cells were then injected via the tail vein to lethally irradiated (10.5 Gy) C57BL/6 CD45.2 mice within 4 hours after irradiation (250 LSK cells per recipient mouse).The transplant recipient mice were injected subcutaneously with PBS buffer or AMD3100 at 5 mg/kg body weight in a volume of 100 μL every other day beginning at day 2 after transplant for a total of three doses. The mice were then sacrificed at day 7 after transplant, and the two femurs and two tibias of each mouse were harvested. We have performed three separate sets of experiments with 10 mice in each group of each experiment. In our first set of experiments, all femurs and tibias from the 10 mice of each treatment group were combined and flushed with 500 μL of PBS. In our second and third sets of experiments, the mice were divided into four groups, and five femurs and five tibias were combined and flushed with 500 μL of PBS. The flushed BM suspension was then centrifuged at 15,000 rpm for 5 min, and the BM supernatants were collected for colony-forming unit (CFU) assay and for proteomics analysis. The BM cell pellets were used for real-time polymerase chain reaction (RT-PCR) measurement of thioredoxin as described below.CFU assaysThe effects of BM supernatants and thioredoxin on CFUs were performed similarly. Briefly, BM cells were isolated from sublethally irradiated (5.5 Gy) C57BL/6 mice and plated (5 × 105 cells/dish) in Methocult GF 3434 (StemCell Technology, Vancouver, Canada) that contained 30 μL/dish of RPMI 1640 medium, RPMI 1640 medium with 0.78 μg/mL AMD3100, BM supernatant from PBS-treated transplant recipient mice, BM supernatant from AMD3100- treated transplant recipient mice, or various concentrations of thioredoxin. CFUs were performed in triplicate. BM CFUs from primary and secondary transplant recipient mice were also performed at 5 × 104 cells/dish. CFU–granulocyte and monocyte (GM), burst-forming unit erythrocytes (BFU-Es) and CFUs-granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) were counted at days 7, 9, and 12, respectively.Proteomic analysisProtein concentration in the supernatant was determined using the Pierce 660-nm protein assay (Thermo Scientific, Waltham, MA). A total of 20 μg protein from each pooled sample was resuspended in 150 μL ammonium bicarbonate and 0.12% Rapigest (Waters) for a final protein concentration of 0.13 μg/μL. The samples were reduced in 5 mM Dithiothreitol at 60°C for 30 min and alkylated in 15 mM iodoacetamide for 30 min. Trypsin (Promega) was added to the sample at 1:20 and incubated overnight at 37°C. Tryptic digestion was halted by acidifying the sample by adding 1.8 mL 0.1% formic acid. Peptides were isolated using solid-phase extraction cartridges (Phenomenex Strata-X, 60 mg). Cartridges were conditioned with methanol and 0.1% formic acid, and the sample was passed through the cartridge, washed twice with 0.1% formic acid, and once with 5% acetonitrile. Peptides were eluted stepwise in 0.1% formic acid solution containing 20%, 25%, 30%, 35%, 40%, and 60% acetonitrile. Each fraction was dried using a vacuum concentrator and resuspended in 50 μL mobile phase A (MPA; 98% water, 2% acetonitrile, 0.1% formic acid). Peptide concentration was estimated by absorbance at 280 nM to ensure that no more than 2 μg peptide was injected per fraction. Five microliters of each fraction was injected by autosampler and loaded onto an Acclaim PepMap 100 c18 trap column (100 μm ID × 2 cm, C18, 5 μm, 100 Å; Thermo Scientific). Trapped peptides were washed with 15 μL MPA before separation on an Acclaim PepMap100 analytical column (75 μm ID × 15 cm, C18, 3 μm, 100 Å; Thermo Scientific). Peptides were separated using a three-step gradient at 300 nL/min: (1) mobile phase B (MPB; 2% water, 98% acetonitrile, 0.1% formic acid) was increased from 5% to 35% over 30 minutes; (2) MPB was increased from 35% to 60% over 12 minutes; (3) MPB was increased from 60% to 80% over 1 min and held at 80% MPB for 4 min. Peptides were eluted into the mass spectrometer source using 10 μm nanospray tips (New Objective, Wobum, MA), and tandem mass spectrometry was performed using an AB SCIEX Triple TOF 5600 mass spectrometer. Parent ion TOF scans were conducted across 400–1800 m/z with an accumulation time of 0.25 sec. Product ion scans of parent ions between 400 and 1250 m/z were conducted using the information-dependent acquisition mode. Briefly, 20 candidate parent ions were monitored per cycle in high-sensitivity mode, with exclusion of former target ions for 10 sec after one occurrence. Product ion data were collected in high sensitivity mode between 75 and 2000 m/z, with an accumulation time of 0.05 sec. Mass tolerance was set to 50 mDa, and isotopes were excluded within 4 Da. Rolling collision energy was selected. Total cycle time was 1.3 sec.Data files for each run were converted to Mascot generic format (MGF) using a conversion tool provided by AB SCIEX version 1.1 beta. MGF files were merged and searched using Mascot (version 2.3.02) against the mouse protein database (UniprotKB/Swiss-Prot version 2012_03, 24,410 entries), with the addition of common contaminants. Digestion enzyme was set to trypsin allowing for two missed cleavages, parent ion tolerance was set to 10 ppm, and fragment tolerance was set to 0.25 Da. Fixed modification was +57 on C (carbamidomethyl) and variable modifications included: +16 on M (oxidation), +1 on N/Q (deamidation), and −17 on Q n-terminal (pyro-Glu). Mascot search results were uploaded to SCAFFOLD (version 3.5.1) for viewing. Spectral count data was normalized using the quantitative value tool. Protein identifications were included for analysis at two levels of false discovery. A conservative list was generated for proteins identified with three unique peptides, with a threshold of 80%, and with a protein threshold of 99% to ensure a conservative false discovery rate (<0.1%). Normalized spectral count fold change was calculated using normalized spectral counts, and proteins that displayed a twofold change in spectral count data were listed.The data associated with this manuscript can be downloaded from the ProteomeCommons.org Tranche network using the following hash:XselMgl3o2tPEVec4osCyCwmg4zwjJNJ0vscXO79Tim16UtLenDsxm3JS9OJxarK1Y8js41dmfs7Wl79SyRWMnz5tgwAAAAAAYm8AA==.Thioredoxin quantitative RT-PCRBM cells were harvested from PBS- or AMD3100-treated transplant recipient mice and total RNA extracted. Quantitative RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The primers to amplify thioredoxin were 5′-GCCAAAATGGTGAAGCTGAT and 5′-TGATCATTTTGCAAGGTCCA. The actb was used as reference gene and amplified with the primers, 5′-GATCTGGCACCACACCTTCT and 5′-GGGGTGTTGAAGGTCTCAAA.Thioredoxin for radioprotectionC57BL/6 mice were given 9.5 Gy total body irradiation using a cesium irradiator. Two hours after the irradiation, the mice were injected intraperitoneally with PBS buffer or thioredoxin at 32 μg/mouse in a volume of 100 μL and then every other day for a total of five doses. Animal survival was monitored daily. Blood samples were collected at day 7 after irradiation, and peripheral blood cell counts were measured using Beckman automatic cell counter.BM transplantation with thioredoxin-pretreated HSCsLSK cells were obtained from C57BL/6 CD45.1 mice and cultured in StemPro-34 SFM media (Invitrogen, Carlsbad, CA) containing stem cell factor (100 ng/mL) and thrombopoietin (TPO-1, 100 ng/mL) with or without thioredoxin (10 μg/mL) for 24 hours. Cells were then harvested, washed, counted, and injected via the tail vein to lethally irradiated (9.5 Gy) C57BL/6 CD45.2 mice (2000 LSK cells per recipient mouse). Peripheral blood hematologic recovery and donor cell engraftment were followed. At 11 weeks after transplant, the mice were sacrificed and BM was harvested for CFU assay, flow cytometric analysis, and secondary transplantation.Bone marrow transplantation with posttransplant administration of thioredoxin or AMD3100LSK cells were isolated from C57BL/6 CD45.1 mice and cultured in StemPro-34 SFM media with stem cell factor (100 ng/mL) and TPO-1 (100 ng/mL) for 24 hours. The cells were then injected intravenously to lethally irradiated (9.5 Gy) C57BL/6 CD45.2 mice (2000 cells/recipient). At day 2 after transplant, the recipient mice were injected with: (1) PBS buffer subcutaneously (100 μL/mouse every other day for 8 weeks); (2) AMD3100 subcutaneously (5 mg/kg body weight in a volume of 100 μL every other day for 8 weeks); (3) thioredoxin intraperitoneally (32 μg/mouse in a volume of 100 μL every other day for a total of six doses); or (4) AMD3100 plus thioredoxin (i.e., AMD3100 subcutaneously at 5 mg/kg body weight every other day for 8 weeks plus thioredoxin intraperitoneally at 32 μg/mouse every other day for a total of six doses).Secondary BM transplantationThe primary transplanted recipient mice were sacrificed at 11 weeks after transplant, and BM cells were collected. The BM cells were injected into lethally irradiated C57BL/6 CD45.2 mice (6 × 106 BM cells/recipient, 8 mice/group, one primary to one secondary matched transplant).Measurement of donor cell engraftment and hematological recoveryPeripheral blood donor cell engraftment was measured as described previously [21Kang Y. Chen B.J. Deoliveira D. Mito J. Chao N.J. Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.PLoS One. 2010; 5: e11316Crossref PubMed Scopus (39) Google Scholar]. Whole blood (50 μL) was collected at various times following transplant and was stained with a combination of cell subset-specific antibodies for 15 min at room temperature. The stained blood samples were then processed in BD FACS lysing solution. Flow-Count Fluorospheres (50 μL; Beckman-Coulter) were added into the samples and the samples were analyzed using BD Fortessa analyzer. Whole blood cell counts including white blood cell count, RBC count, hemoglobin count, and platelet count were measured using a Scil Plus hematology analyzer (Gurnee, IL) per the manufacturer's instructions.Bone marrow donor cell engraftment was determined in primary and secondary transplanted recipient mice at 11 and 12 weeks after transplant, respectively. RBC-depleted BM cells from one tibia and one femur were counted, and 2 × 106 cells were stained with indicated antibodies and analyzed for donor CD45.1+ LSK cells. The absolute number of donor LSK cells in two femurs and two tibias was calculated and shown.Statistical analysisThe values were reported as mean ± SEM of combined data of multiple experiments or as mean ± SD from a representative experiment. All experiments were performed at least two times. Differences were analyzed using Student t test; p < 0.05 was regarded as significant and is indicated in the text or figure legends.ResultsBM supernatant of AMD3100-treated transplant recipient mice contains factors that promote the growth of hematopoietic progenitor cellsTo test whether posttransplant administration of AMD3100 affected the BM niche microenvironment, we collected the BM extracellular fluids (also named BM supernatants) at day 7 after transplant by flushing the bones with PBS and centrifuging the bone marrow suspension. We chose day 7 because our previous studies indicated that at this time, the plasma cytokine profile was clearly affected by AMD3100 treatment [21Kang Y. Chen B.J. Deoliveira D. Mito J. Chao N.J. Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.PLoS One. 2010; 5: e11316Crossref PubMed Scopus (39) Google Scholar]. The BM supernatants were then tested for their effects on hematopoietic progenitors by adding them to the CFU assay using marrow cells isolated from sublethally (5.5 Gy) irradiated mice. We used BM cells harvested from irradiated mice for the CFU assay to evaluate the influence of the BM microenvironment on HSCs that have been radiated, thus mimicking the setting of radiation preconditioning.As shown in Figure 1, compared to those that had no BM supernatants added (i.e., RPMI 1640 medium only), the number of CFUs was significantly reduced when BM supernatants from PBS-treated transplant recipient mice were added, suggesting that the BM microenvironment in the lethally irradiated transplant mice contains factors that inhibit hematopoiesis. In contrast, the BM supernatants from AMD3100-treated transplant recipient mice significantly increased CFUs. Because the last dose of AMD3100 was given 24 hours before the mice were sacrificed, we estimated that the concentration of AMD3100 remaining in the BM supernatant was approximately 0.78 μg/mL (5 mg/kg of AMD3100 was injected in a 20 gram mouse that had ∼2 ml blood distribution and the half-life of AMD3100 was 4 hours; 0.1 mg / 2 mL / 26 = 0.78 μg/mL). AMD3100 at 0.78 μg/mL did not affect CFUs; results were similar in CFUs when medium only or medium containing 0.78 μg/mL AMD3100 was added (Fig. 1). These data suggest that in the early phase of transplant following lethal total-body irradiation, the BM microenvironment of transplant recipient mice contains soluble factors that inhibit hematopoiesis. Posttransplant administration of AMD3100 promotes hematopoiesis likely by reducing these inhibitors or increasing factors that enhance hematopoiesis.Proteomic analysis screens for proteins that are differentially expressed between AMD3100-treated BM supernatant and PBS-treated BM supernatantIdentifying the proteins in the BM supernatants that were affected by AMD3100 treatment will provide important insights into the regulation of hematopoietic reconstitution following HSCT. To this end, we used a liquid chromatography tandem mass spectrometry (MS) approach [22Liu H. Sadygov R.G. Yates 3rd, J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2055) Google Scholar] to screen for proteins that were expressed differentially in the BM supernatants at day 7 after transplant between PBS-injected transplant recipient mice and AMD3100-treated transplant recipient mice. A conservative list was generated for proteins identified with three unique peptides, with a threshold of 80%, and with a protein threshold of 99% to ensure a conservative false discovery rate (<0.1%). Proteins that displayed a ≥ twofold change between PBS-treated transplant recipient mice and AMD3100-treated transplant recipient mice were considered to be potential candidate proteins affected by AMD3100 treatment.Using this conservative proteomics approach, we identified a total of 178 proteins in the marrow supernatants of lethally irradiated transplant recipient mice. Gene ontology analysis of these 178 proteins suggested that the top three categories of the molecular functions were molecular function (169), binding (133), and catalytic activity (79). The biological processes with the three largest numbers of identified proteins were cellular process (138), metabolic process (107), and biological regulation (94). The majority of the proteins present in the BM supernatants were from cytoplasm (122), intracellular organelle (96), and extracellular regio" @default.
• W2012156551 created "2016-06-24" @default.
• W2012156551 creator A5008453908 @default.
• W2012156551 creator A5019007603 @default.
• W2012156551 creator A5025064016 @default.
• W2012156551 creator A5042789216 @default.
• W2012156551 creator A5080929469 @default.
• W2012156551 creator A5081728414 @default.
• W2012156551 date "2013-11-01" @default.
• W2012156551 modified "2023-10-18" @default.
• W2012156551 title "Proteomic analysis of murine bone marrow niche microenvironment identifies thioredoxin as a novel agent for radioprotection and for enhancing donor cell reconstitution" @default.
• W2012156551 cites W1523887533 @default.
• W2012156551 cites W1537465102 @default.
• W2012156551 cites W1550909330 @default.
• W2012156551 cites W1551392207 @default.
• W2012156551 cites W1555938743 @default.
• W2012156551 cites W1565745037 @default.
• W2012156551 cites W1567647424 @default.
• W2012156551 cites W1572899018 @default.
• W2012156551 cites W1969268163 @default.
• W2012156551 cites W1970909450 @default.
• W2012156551 cites W1974389004 @default.
• W2012156551 cites W1980156876 @default.
• W2012156551 cites W1989776946 @default.
• W2012156551 cites W1991387227 @default.
• W2012156551 cites W1991962444 @default.
• W2012156551 cites W1992049458 @default.
• W2012156551 cites W1997382890 @default.
• W2012156551 cites W1997405293 @default.
• W2012156551 cites W1997504014 @default.
• W2012156551 cites W1999178985 @default.
• W2012156551 cites W2001173092 @default.
• W2012156551 cites W2004949125 @default.
• W2012156551 cites W2008484243 @default.
• W2012156551 cites W2014655794 @default.
• W2012156551 cites W2016907191 @default.
• W2012156551 cites W2021741522 @default.
• W2012156551 cites W2030526926 @default.
• W2012156551 cites W2039311043 @default.
• W2012156551 cites W2039748263 @default.
• W2012156551 cites W2040937069 @default.
• W2012156551 cites W2045222138 @default.
• W2012156551 cites W2045907249 @default.
• W2012156551 cites W2052350491 @default.
• W2012156551 cites W2059315336 @default.
• W2012156551 cites W2062504428 @default.
• W2012156551 cites W2064386642 @default.
• W2012156551 cites W2067373124 @default.
• W2012156551 cites W2073692681 @default.
• W2012156551 cites W2074355200 @default.
• W2012156551 cites W2076680341 @default.
• W2012156551 cites W2078295067 @default.
• W2012156551 cites W2084136532 @default.
• W2012156551 cites W2084874441 @default.
• W2012156551 cites W2090036710 @default.
• W2012156551 cites W2097312865 @default.
• W2012156551 cites W2125326158 @default.
• W2012156551 cites W2135470678 @default.
• W2012156551 cites W2141837666 @default.
• W2012156551 cites W2146864852 @default.
• W2012156551 cites W2148625728 @default.
• W2012156551 cites W2155338282 @default.
• W2012156551 cites W2162371077 @default.
• W2012156551 doi "https://doi.org/10.1016/j.exphem.2013.08.004" @default.
• W2012156551 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3955273" @default.
• W2012156551 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23994289" @default.
• W2012156551 hasPublicationYear "2013" @default.
• W2012156551 type Work @default.
• W2012156551 sameAs 2012156551 @default.
• W2012156551 citedByCount "6" @default.
• W2012156551 countsByYear W20121565512014 @default.
• W2012156551 countsByYear W20121565512016 @default.
• W2012156551 countsByYear W20121565512017 @default.
• W2012156551 countsByYear W20121565512022 @default.
• W2012156551 crossrefType "journal-article" @default.
• W2012156551 hasAuthorship W2012156551A5008453908 @default.
• W2012156551 hasAuthorship W2012156551A5019007603 @default.
• W2012156551 hasAuthorship W2012156551A5025064016 @default.
• W2012156551 hasAuthorship W2012156551A5042789216 @default.
• W2012156551 hasAuthorship W2012156551A5080929469 @default.
• W2012156551 hasAuthorship W2012156551A5081728414 @default.
• W2012156551 hasBestOaLocation W20121565511 @default.
• W2012156551 hasConcept C153991713 @default.
• W2012156551 hasConcept C203014093 @default.
• W2012156551 hasConcept C2780007613 @default.
• W2012156551 hasConcept C55493867 @default.
• W2012156551 hasConcept C70721500 @default.
• W2012156551 hasConcept C86803240 @default.
• W2012156551 hasConcept C95444343 @default.
• W2012156551 hasConceptScore W2012156551C153991713 @default.
• W2012156551 hasConceptScore W2012156551C203014093 @default.
• W2012156551 hasConceptScore W2012156551C2780007613 @default.
• W2012156551 hasConceptScore W2012156551C55493867 @default.
• W2012156551 hasConceptScore W2012156551C70721500 @default.
• W2012156551 hasConceptScore W2012156551C86803240 @default.
• W2012156551 hasConceptScore W2012156551C95444343 @default.
• W2012156551 hasIssue "11" @default.
• W2012156551 hasLocation W20121565511 @default.

• next