![]() The day before blood harvest, recipient congenic B6.SJL CD45.1 + mice were lethally irradiated with 11.0 Gy in 2 split doses 3 hours apart. The content of mobilized blood samples in competitive repopulating HSCs was determined in competitive repopulation assays. Probe(94f)FAM-5′-CACTGACCGGCCTGTATGCTATCCA-3′-BHQ1 Probe(1039b)FAM-5′-TCCCAGCACCCAGTGTTGGC-3′-BHQ1 Reverse(1090b) 5′-GGATGATCCACTTCTTGTGC-3′ Probe(949b)FAM-5′-CCCTAAATCACTGAGGCGATCAGAGAA-3′-BHQ1 Reverse (995b) 5′-ATACTGGGATGAGGAATGCG-3′ Reverse (288b) 5′-CAGTCCCAGATCATGGAGTC-3′įorward (904f) 5′-CCAAGAAGGCACAGACAGAA-3′ ![]() Reverse (902b) 5′-TGGGTCCACAACAGTCAGAA-3′ Reverse (1806b) 5′-ATGGTGGTGGAACGTAAGGA-3′įorward (722f) 5′-CGTGGTGGAAACACAAGATG-3′ Reverse (298b) 5′-CGGGTCAATGCACACTTGTC-3′įorward (1645f) 5′-CAAATGCGCTCTCATGCTAA-3′ Reverse (245b) 5′-GCGTATGGCTTCTTTGTGCCT-3′įorward (198f) 5′-GAGCCAACGTCAAGCATCTG-3′ Reverse (137b) 5′-CCCTCCTGCTTGGACATGAA-3′įorward (126f) 5′-AGCTCACTATGGCTCCAGTCC-3′ 11 Thus, it appears that the endosteal region and osteoblast-lineage cells provide a unique environment necessary for HSC survival, quiescence, self-renewal, and contribution to long-term hematopoiesis. 10 Finally, targeted deletion in osteoprogenitors of Dicer-1, necessary for the maturation and processing of all microRNA, results in pancytopenia, exaggerated HSC proliferation, ineffective hematopoiesis, and myelodysplasia. Transgenic mice with increased osteoblast function have more HSCs in the BM, 8, 9 whereas targeted ablation of osteoblasts in vivo results in a progressive loss of HSCs from the BM. 7 Functional studies support a role for osteoblast-lineage cells to maintain HSCs in the BM in vivo. 6 Similarly, Lin −CD41 −Sca1 +KIT +CD48 −CD150 + HSCs isolated from the femoral endosteal regions have higher long-term reconstitution potential than phenotypically identical HSCs isolated from the central BM. Recent microscopy studies have shown that quiescent, long-term, reconstituting HSCs preferentially reside within 20 μm of the interface between compact bone and BM (endosteum) and 10 μm (or 2 cell diameters) from osteoblasts, 2-5 in poorly perfused regions of the BM. While HSC niches were conceptualized in the 1970s, 1 it is only in recent years, with the advent of mouse genetic models and live microscopy, that the precise locations and nature of HSC niches have been proposed. These fates are largely controlled by specific local microenvironments (ie, “niches”), in which HSCs and HPCs reside. Individual HSCs undergo one of several fates: quiescence, apoptosis, cycling either to self-renew, or to generate a more committed hematopoietic progenitor cell (HPC), or migration. Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and produce all cells required to replenish the blood and immune systems this process is tightly regulated to maintain a steady number of leukocytes, platelets, and red cells in the blood. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum.
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