The mechanism(s) underlying the release of stem/progenitor cells from bone marrow into the circulation is poorly understood. We hypothesized that matrix metalloproteinases (MMPs), especially gelatinases, which are believed to participate in the proteolysis of basement membranes and in the migration of leukocytes, may facilitate this process. First, we investigated whether CD34+stem/progenitor cells express gelatinases A (MMP-2) and/or B (MMP-9) and whether growth factors and cytokines (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], stem cell factor [SCF], macrophage colony-stimulating factor [M-CSF], interleukin-3 [IL-3], IL-6, IL-8, and tumor necrosis factor- [TNF-]) are able to modulate their expression. Next, we examined the transmigration of these stem/progenitor cells through reconstituted basement membrane (Matrigel) and its modulation by growth factors and cytokines. CD34+ cells were obtained from steady-state bone marrow and peripheral blood (from leukapheresis products collected either in steady-state hematopoiesis or after mobilization with G-CSF plus chemotherapy or G-CSF alone). We found that peripheral blood CD34+ cells, regardless of whether they were mobilized or not, strongly expressed both gelatinases (MMP-2 and MMP-9) in contrast to steady-state bone marrow CD34+ cells, which did not. However, all the growth factors and cytokines tested could induce MMP-2 and MMP-9 secretion by the latter cells. Moreover, the stimulatory effects of G-CSF and SCF on both MMP-2 and MMP-9 secretion were found to be significantly higher in CD34+ cells isolated from bone marrow than in those from peripheral blood. In addition TNF-, GM-CSF, and IL-6 increased the secretion of a partially active form of MMP-2. Basal transmigration of bone marrow CD34+ cells through Matrigel was lower than that of peripheral blood CD34+ cells (P < .0001), but growth factors and cytokines increased it by 50% to 150%. Positive correlations were established between expression of gelatinases and CD34+cell migration (r > .9). The stimulatory effect of G-CSF was significantly greater on the migration of CD34+ cells from bone marrow than on those from peripheral blood (P = .004). Moreover, CD34+ cell migration was reduced to approximately 50% by antibodies to MMP-2 and MMP-9, tissue inhibitors of metalloproteinases (rhTIMP-1 and -2), ando-phenanthroline. TNF-–induced gelatinase secretion and migration of CD34+ cells and of clonogenic progenitors (colony-forming unit–granulocyte-macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], and colony-forming unit-megakaryocyte [CFU-MK]) were dose-dependent. Therefore, this study demonstrated that CD34+ cells that are circulating in peripheral blood express both MMP-2 and MMP-9 and transmigrate through Matrigel. In contrast, CD34+ cells from steady-state bone marrow acquire similar properties after exposure to growth factors and cytokines, which upregulate expression of gelatinases and transmigration of these cells when they enter the bloodstream. Hence, we suggest that growth factors and cytokines induce release of stem/progenitor cells from bone marrow into peripheral blood during mobilization, as well as during steady-state hematopoiesis, by signaling through gelatinase pathways.
Summary:In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34 þ cells, and granulocytemacrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34 þ cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P ¼ 0.136 and 0.417, respectively) or platelets (P ¼ 0.88 and 0.126, respectively). However, the reinfused viable CD34 þ cells/ kg of patient weight pre-or post-cryopreservation showed significant correlation to engraftment of neutrophils (P ¼ 0.0001 and 0.001, respectively) and platelets (P ¼ 0.023 and 0.010, respectively), whereas CFU-GM pre-or post-cryopreservation was significantly correlated to neutrophils (P ¼ 0.011 and 0.007, respectively) but not to platelets (P ¼ 0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34 þ cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34 þ cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.
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