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The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.
HSC quiescence is increased in E-selectin knockout (Sele−/−) mice. (a–c) HSC turnover measured by BrdU incorporation in mice of the indicated genotypes continuously administered BrdU. At set periods of time, the percentage of BrdU incorporation in phenotypic HSCs was measured. (a) Dot plot showing the LKS+ gate within Lin− bone marrow cells (left). BrdU after 3 d of incorporation and CD34 expression on gated LKS+ cells from representative mice (right). Inserted quadrants show percentages of cells in each quadrant of the dot plots. (b) Cytospins of sorted HSCs (LKS+CD34−) stained for BrdU incorporation after 5 d of BrdU administration. Scale bar, 50 μm. (c) Percentage of bone marrow LKS+CD34− HSCs that incorporated BrdU over time. Data are pooled from four separate experiments. ***P < 0.001. (d,e) Cell-cycle analysis of LKS+CD34− HSCs. (d) Typical dot plots of DNA content (Hoechst 33342) plotted versus Ki-67 nuclear antigen staining. The cell-cycle phases were defined as G0 (Ki-67− and 2n DNA), G1 (Ki-67+ and 2n DNA) and S-G2-M (Ki-67+ and DNA >2n). (e) Percentage of LKS+CD34− HSC in phase G0 (quiescence). (f) HSC quiescence in Sele−/− and WT mice as measured by hydroxyurea (HU) in vivo suicide assay. Data shows the number of surviving reconstituting units (RU) per femur after injection with HU or no HU control. (g,h) Rhodamine efflux by HSCs. (g) Contour plot showing Rho efflux and CD48 staining on gated bone marrow LKS+ cells from representative mice. (h) Percentage of bone marrow LKS+ cell able to efflux Rho. Data are pooled from three separate experiments. Each symbol in c,e,f and h represents data for one mouse. All statistical significance was calculated by nonparametric Mann-Whitney test. All data are shown as the mean ± s.d.
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PSGL-1 and CD44 receptors do not mediate the effects of E-selectin on HSC cycling. (a) Static adhesion assay of sorted bone marrow LKS+ cells on immobilized recombinant selectins. Shown are the percentages of adherent cells compared to input cells. In all cases, 97–100% of the input cells adhered to immobilized recombinant mouse VCAM-1 in parallel wells. Data are the mean ± s.d. for triplicate wells. (b) E-selectin (Esel)-IgM binding assay. A suspension of bone marrow cells was stained for the indicated cell-surface antigens and then incubated with a preformed complex of E-selectin–IgM and CY5-labeled antibody to IgM. Shown are overlays of E-selectin binding to CD11b+Gr1+/low neutrophils and monocytes (top left, Gr1+/low) and to gated LKS+ cells (top right). Black line, WT; bold gray line, Selplg−/−Cd44−/− (KO); light gray filled line, EDTA negative control. Also shown is a histogram (bottom) of the percentage of gated cells that bound E-selectin–IgM. Data are the mean ± s.d. n = 6 mice per group. ***P < 0.001. (c) Three-day BrdU incorporation plotted relative to CD34 expression in gated LKS+ cells in WT and Selplg−/−Cd44−/− mice. Inserted quadrants show percentages of cells in each quadrant of the dot plots. (d) Timecourse of BrdU incorporation in gated bone marrow LKS+CD34− cells. Bars are the mean ± s.d. NS, not significant. (e) Rho efflux by HSCs. Contour plot showing Rho efflux plotted against CD48 staining on gated bone marrow LKS+ cells from representative WT and Selplg−/−Cd44−/− mice. (f) Percentage of bone marrow LKS+ cells able to efflux Rho. (g) E-selectin pulldown experiments. Lysates of Lin−Kit+ cells from Selplg−/−Cd44−/− mice were incubated with recombinant mouse E-selectin–huIgG1–Fc (Esel-IgG) or control (IgG)-coated beads, pulled down, eluted with EDTA and western blotted with a rabbit antibody to ESL-1. MW, molecular weight. (h) Binding of LKS+ cells from WT and Selplg−/−Cd44−/− mice to recombinant human E-selectin–IgM (top histograms) and P-selectin–IgM (bottom histograms) after an overnight incubation with or without PPMP, as measured by flow cytometry. Data are the mean ± s.d. (triplicate wells, three repeats). Each symbol in d and f represents data from a single mouse. All statistical significance was determined by nonparametric Mann-Whitney test.
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... However, BMM or "niche"-mediated resistance may be associated with extrinsic cytokines or chemokines, such as CXC chemokine receptor 4 (CXCR4)/CXC motif ligand 12 (CXCL12) and endothelial (E)-selectin (CD62E)/E-selectin ligands (E-selectin-L). Both CXCR4 and E-selectin ligands are crucial for directing leukocyte or hematopoietic stem cell (HSC) homing to BMM 10,11 . In addition, the hypoxic condition (1-3% O 2 ) in BMM can trigger the upregulation of genes glycosylating E-selectin-L such as fucosyltransferase VII (FUT7) and sialyltransferase ST3Gal-I (ST3O) through the hypoxiainducible factor (HIF)1-α---a key transcriptional factor induced by hypoxia, known to be involved in the synthesis of the carbohydrate ligands for E-selectin and associated with resistance to therapy in AML 12,13 . ...
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Citation: Faisal, M.; Hassan, M.; Kumar, A.; Zubair, M.; Jamal, M.; Menghwar, H.; Saad, M.; Kloczkowski, A. Hematopoietic Stem and Progenitor Cells (HSPCs) and Hematopoietic Microenvironment: Molecular and Bioinformatic Studies of the Zebrafish Models. Int. J. Mol. Sci. 2022, 23, 7285.
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Chapter
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Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has been assessed for its potential in vivo to protect hematopoietic progenitor cells from the cytotoxic effects of a cycle-specific drug--in this case hydroxyurea (HU). Two doses of HU, 7 hours apart, were administered to mice to induce spleen colony-forming unit (CFU-S) cycling and then to kill them during DNA-synthesis. MIP-1 alpha, in a variety of dose and time combinations, was injected before the second dose of HU in an attempt to prevent recruitment or maintain CFU-S quiescence, and thus protect them from the second dose of HU. Without MIP-1 alpha, recovery of the CFU-S population was complete in 7 days. In a dose-dependent manner, MIP-1 alpha either reduced the initial kill and accelerated recovery, or completely protected the CFU-S population. We conclude that MIP-1 alpha does protect multipotent progenitor cells in vivo and that these observations provide a base from which to build practical clinical applications.