Migration Of CD34 Research Articles

Organ-Specific Migration of CD133+ Stem Cells Triggered by CpG Motifs From DSS-Induced Colitis Mouse Model

Organ-Specific Migration of CD133+ Stem Cells Triggered by CpG Motifs From DSS-Induced Colitis Mouse Model

Differential chemokine receptor expression regulates functional specialization of endothelial progenitor cell subpopulations

Postnatal vasculogenesis is mediated by endothelial progenitor cells (EPCs) which consist of subpopulations with different functional capacities. Our goal was to profile chemokine receptor expression on relevant subsets of EPCs and to characterize their role for effector functions. CD34(+)/CD133(+)/VEGFR2(+) EPCs were characterized by high expression of chemokine receptors CXCR4, CX3CR1, BLT1, and low level expression of CXCR2 and CCR2, while primordial CD34(-)/CD133(+)/VEGFR2(+) EPCs express these chemokine receptors at comparably low levels. Migration assays revealed that SDF-1, fractalkine, and LTB4 significantly increase migration of CD34(-)/CD133(+)/VEGFR2(+) EPCs, while SDF-1 was the only potent agonist of migration of CD34(+)/CD133(+)/VEGFR2(+) EPCs. SDF-1, fractalkine, and LTB4 trigger significant increase adhesion of CD34(+)/CD133(+)/VEGFR2(+) EPCs, while in CD34(-)/CD133(+)/VEGFR2(+) EPCs SDF-1 and fractalkine are equipotent agonists and LTB4 triggers a smaller though still significant increase in adhesion. Differential expression of specific chemokine receptors is an important regulator in terms of migration and adhesion of biologically relevant EPC-subpopulations, which may have implications for cell therapeutic strategies for treatment of ischemic vascular disease.

Lenalidomide Upregulates CXCR4 on CD34+ Hematopoietic Cells Resulting In Increased Binding to Bone Marrow Niche and Inhibiting Mobilization Into Peripheral Blood In Multiple Myeloma Patients

AbstractAbstract 4079 Background:In multiple myeloma (MM), lenalidomide has impressive clinical activity in patients with both relapsed/refractory and newly diagnosed disease. Nevertheless hematopoietic stem cell transplantation (HSCT) is still the “backbone” in the treatment of newly diagnosed MM patients and very often lenalidomide treatment (as induction therapy) is combined with HSCT in order to achieve high response rates. Clinical data have shown that in up to 43% of those patients, standard mobilization of CD34+ cells with G-CSF alone failed to mobilize significant numbers of hematopoietic progenitors into peripheral blood, raising concerns about potential stem cell toxicity of lenalidomide (Mazumder et al, Leukemia 2008). Interestingly mobilization of hematopoietic progenitors with AMD-3100 (Plerixafor) overcomes mobilization failures in almost all patients previously treated with lenalidomide. The fact that AMD-3100 antagonizes the binding of chemokine stromal- cell–derived factor-1α (SDF-1α) to CXC chemokine receptor 4 (CXCR4) suggests a potential role of the CXCR4/SDF-1α axis in mediating mobilization failure after lenalidomide treatment. Subsequently, the findings noted above raised questions on: 1) the stem cell toxicity of lenalidomide; 2) the underlying mechanism that mediates the development of G-CSF resistance; and 3) the mechanism of the modulation of the CXCR4/SDF-1α axis by lenalidomide. In our previous work we showed the following. 1) Lenalidomide neither inhibited colony formation in standard colony assays nor the development of cobble stone area forming cells (CAFC) in LTC-IC assays, suggesting that lenalidomide is not stem cell toxic (Koh et al, Blood 2005). 2) We further showed that lenalidomide significantly upregulated G-CSF secretion of CD34+ cells (600%), suggesting that the high levels of G-CSF may mediate a relative resistance towards G-CSF-induced mobilization (Pal et al, Blood, 2010). Results and Methods:We analyzed why blocking CXCR4 by AMD-3100 overcomes mobilization failure to G-CSF. We first examined the CXCR4 expression profile of CD34+ cells treated with lenalidomide. Lenalidomide treatment significantly upregulated the expression of CXCR4 on cell surface after 48h treatment measured by flow cytometry. Increased expression of CXCR4 onCD34+ cells remained high with continuous treatment. Western blot assay of the hydrophobic (membrane) and the hydrophilic (cytosol) cell fraction confirmed our data showing that lenalidomide increases the expression of CXCR4 on CD34+ cell surface. Confocal microscopy showed that lenalidomide inhibited SDF-1α induced CXCR4 internalization. In accordance with the increased CXCR4 surface expression, transwell migration assay revealed that the SDF-1α-induced migration of CD34+ cells in the presence of lenalidomide significantly increased by 52% in comparison to control. Quantitation of SDF-1α showed that CXCR4 had no significant effect on the secretion of SDF-1α by CD34+ cells and in human stromal cells. Result:In conclusion, our data show that lenalidomide is not toxic to hematopoietic progenitors. The strong increase of G-CSF secretion by CD34+ cells might contribute to a desensitization of CD34+ cells to G-CSF mobilization. Primarily our data indicate that increased expression of CXCR4 followed by blocked internalization, increases binding to SDF-1α secreted by the bone marrow niche. This subsequently prohibits the mobilization of CD34+ cells. These data suggest that blocking the CXCR4 receptor by AMD-3100 disrupts this circle and finally permits the mobilization of hematopoietic cells from the bone marrow niche into peripheral blood. This study provides novel insights into the effects of lenalidomide on CD34+ cells relevant for HSCT in MM. Disclosures:Roodman:Amgen: Consultancy; Celgene Corp: Consultancy; Acceleron: Consultancy; Millennium: Consultancy. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

MicroRNA Mir-23a Regulates SDF-1alpha Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells

MicroRNAs (miRNAs) can regulate hematopoietic stem/progenitor cells (HSPC) by modulation of intrinsic cell components such as transcription factors and receptors. In addition, miRNAs could play a role in the microenvironment were HSPC host and consequently affect HSPC via extrinsic factors, which to our knowledge is an hypothesis that has not been tested. Since the chemokine stromal derived factor 1alpha (SDF-1alpha) is essential for both homing and retention of HSPC in the bone marrow, we tested if miRNAs expressed by human bone marrow-derived mesenchymal stem cells (MSC; a key source of SDF-1alpha), could potentially inhibit SDF-1alpha expression. In deed, using luciferase reporter-systems we show specific binding of miR-23a to the 3′UTR of SDF-1alpha. Consequently, transfection of MSCs with a precursor miR-23a (pre-miR-23a) leads to a 30% reduction of SDF-1alpha at both mRNA and protein levels. In contrast, inhibition of endogenous miR-23a with anti-sense oligonucleotides (anti-miR-23a), leads to a significant increase of SDF-1alpha also at both mRNA (30%) and protein (10%) levels as compared to controls (scramble pre-/anti-miR). As a result, migration of CD34+ HSPC in transwell assays is strongly affected upon overexpression or inhibition of miR-23a in MSCs (with pre-miR-23a 35% less migration; with anti-miR-23a 20% more migration, as compared to respective controls). Interestingly, transforming growth factor beta 1 (TGF-beta1) inhibits SDF-1alpha expression in MSCs in a concentration dependent manner, while miR-23a is increased under same experimental settings. Even more, silencing endogenous miR-23a significantly reduces the effect of TGF-beta1 on SDF-1alpha mRNA and protein levels, suggesting that at least in part TGF-beta1 inhibits SDF-1alpha expression via increasing miR-23a levels. This is to our knowledge the first established connection between miRNA biology and HSPC-niche related factors.

Thymodepressin inhibits migration of CD34+ cells from bone marrow in normal and granulocyte CSF-stimulated hemopoiesis

We studied the effect of thymodepressin on migration and adhesion of mouse hemopoietic CD34+ cells under normal conditions and under the effect of granulocytic CSF. It was found that the peptide reduced the absolute number of CD34+ hemopoietic cells in the peripheral blood, increased the percent of cells bound to fibronectin and expressing receptor for integrin beta1 (CD29+) in the bone marrow of mice under normal conditions and after stimulation with granulocytic CSF, and reduced the relative number of cells carrying CXCR4 receptor for stromal factor-1 (CD184+) in the bone marrow (CD34+CD184+) and blood (CD184+) of mice stimulated with granulocytic CSF. The results suggest that thymodepressin can inhibit migration of CD34+ cells from bone marrow into peripheral blood under conditions of normal and granulocytic CSF-stimulated hemopoiesis.

The Value of Bone Marrow Biopsy in the Mobilization of Hematopoietic Stem Cells into the Peripheral Blood in Hematologic Malignancies.

Background: The mobilization the Peripheral Blood Stem Cells (PBSC) from bone marrow (BM) to peripheral blood (PB) is a complex process yet. This process is related to mobilization growth factor and/or chemotherapy protocol and disease status. CXCR4, receptor of SDF-1 and VLA-4 searched in primitive CD34+ and CD34+Thy+ cells have shown some functions in migration of CD34+ cells. Marrow disease infiltration and grade of fibrosis seem to be important features related to CD34+ cells trafficking from BM to PB. The objective of this study was to compare the cellularities, degrees of disease infiltration, and grades of fibrosis of hematopoietic stem cells (HSCs) in BM biopsies before and after mobilization and to correlate the results with the success of the mobilization in patients with hematologic malignancies and in donors.Patients and Methods: Fifty-nine patients and 21 PB stem cell (PBSC) donors participated in this prospective study between January/2003 and May/2006. The patients included 21 with non-Hodgkin lymphoma (NHL), 21 with multiple myeloma (MM), 10 with Hodgkin lymphoma (HL), and 7 with leukemia (5 AML, 1 ALL, and 1 CLL). The mobilization regimen consisted of 5 g/m2 of cyclophosphamide in 29 patients (21 MM, 4 NHL, 3 HL, and 1 CLL), DHAP in 8 (7 NHL and 1 NHL), ICE in 14 (9 NHL and 5 HL), high-dose Ara-C in 5 (all with AML), and G-CSF in 24 (21 donors, 1 NHL, 1 HL, and 1 ALL). The mean numbesr for previous chemotherapy cycles were 11, 10, 6, and 5 for NHL, HL, MM, and leukemia, respectively. We collected PBSC using a Spectra-Cobe device (Cobe, Lakewood, CO, USA). Large-volume leukoapheresis (LVL) was used in all cases. Mobilization success was defined as > 8 × 106 CD34+ cells/L in PB before collection, and adequate LVL yield was defined as 2 × 106 CD34+ cells/kg in LVL products. Overall BM and WBC precursor cellularities were visually assessed as normal (20–50%), decreased (<20%), and increased (> 50%). Fibrosis was graded as absent, slight (grades 1/2), or severe (grades 3/4). The clonal patterns of infiltration were confirmed by immunohistochemicaly analyses using monoclonal antibodies.Results: Eighty proposals (59 patients and 21 donors) were presented for mobilization. There were 49 men and 31 women; the median age and mean duration of disease at time of mobilization were 40.3 years (range 25–68 years) and 15.9 months (range 10.3–20.1 months). Eighteen patients were complete responders, 37 were partial responders, and 4 had refractory disease. The median duration of G-CSF usage was 5 days. Mobilization failed in 17 patients (2 MM, 8 NHL, 3 HL, and 4 leukemia) and 1 donor. The mean numbers of CD+34 cells/μL in the PB at time of LVL were 110 in MM (1.1–647), 64.5 in NHL (0–211), 29.3 in HL (2–80), and 16.2 in leukemia (1.3–65). The mean yield of PBSC × 106/Kg in LVL were 15.3 in MM (1.1–85.5), 9.4 in NHL (2.4–26.6), 6.3 in HL (1.3–14.1), 6.1 in donors (1.9–11.9), and 5.0 in leukemia (2.9–7.3). There were correlations between successful mobilization and high overall cellularity pre- and post-mobilization (p=0.015 and p=0.002, respectfully) and successful mobilization and high WBC precursors post-mobilization (p=0.009).Conclusion: BM biopsy is a method important to re-statement at pre transplant, should be continue searched by overall and WBS precursor's celularities, fibrosis and clonal infiltration before mobilization.

The Neurotransmitter GABA Is a Potent Inhibitor of the Stromal Cell-Derived Factor-1αInduced Migration of Adult CD133+Hematopoietic Stem and Progenitor Cells

The ability of hematopoietic stem and progenitor cells (HSPCs) to migrate is a prerequisite for bone marrow homing and tissue regeneration processes. Induction of HSPC migration is chiefly directed by stromal cell-derived factor-1alpha (SDF-1alpha). Considerably less is known about factors that terminate HSPC migration. Adult CD133(+) HSPCs were isolated from mobilized peripheral blood by immunomagnetic separation. Cell migration was assessed using the three-dimensional collagen matrix migration assay, which allows detailed migration analysis on a cell population and single-cell level. The SDF-1alpha-induced locomotory activity of CD133(+) cells was efficiently blocked by the neurotransmitter gamma-aminobutyric acid (GABA). GABA signaling was effected via the GABA(B)-receptor. This was verified by flow cytometry and cell migration studies using the specific GABA(A)-receptor and GABA(B)-receptor agonists isoguvacine and baclofen, respectively. Baclofen blocked SDF-1alpha-induced migration of CD133(+) cells. Flow cytometry-based calcium measurements revealed that GABA inhibits the SDF-1alpha-induced migration of CD133(+) cells by blocking the SDF-1alpha-induced calcium influx. Similar results were obtained with the specific calcium-release-activated calcium (CRAC) channel inhibitor BTP-2, which both blocked the SDF-1alpha-induced calcium influx and migration of CD133(+) cells. These results suggest that GABA(B)-receptor signaling modulates the activity of CRAC channels, whereby the mechanism in detail remains unclear. In summary, the neurotransmitter GABA is a potent blocker of the SDF-1alpha-induced migration of CD133(+) HSPCs from mobilized peripheral blood.

Identification of Putative Stem Cell Markers, CD133 and CXCR4, in hTERT–Immortalized Primary Nonmalignant and Malignant Tumor-Derived Human Prostate Epithelial Cell Lines and in Prostate Cancer Specimens

Understanding normal and cancer stem cells may provide insight into the origin of and new therapeutics for prostate cancer. Normal and cancer stem cells in prostate have recently been identified with a CD44(+)/alpha(2)beta(1)(high)/CD133(+) phenotype. Stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, have multiple essential functions, including homing of stem cells and metastasis of cancer cells. We show here that human telomerase reverse transcriptase (hTERT)-immortalized primary nonmalignant (RC-165N/hTERT) and malignant (RC-92a/hTERT) tumor-derived human prostate epithelial cell lines retain stem cell properties with a CD133(+)/CD44(+)/alpha(2)beta(1)(+)/34betaE12(+)/CK18(+)/p63(-)/androgen receptor (AR)(-)/PSA(-) phenotype. Higher CD133 expression was detected in the hTERT-immortalized cells than in primary prostate cells. These immortalized cells exhibited "prostaspheres" in nonadherent culture systems and also maintained higher CD133 expression. The CD133(+) cells from these immortalized cell lines had high proliferative potential and were able to differentiate into AR(+) phenotype. In three-dimensional culture, the CD133(+) cells from RC-165N/hTERT cells produced branched structures, whereas the CD133(+) cells from RC-92a/hTERT cells produced large irregular spheroids with less branched structures. SDF-1 induced, but anti-CXCR4 antibody inhibited, migration of CD133(+) cells from RC-92a/hTERT cells, which coexpressed CXCR4. CXCR4/SDF-1 may sustain tumor chemotaxis in cancer stem cells. Furthermore, immunostaining of clinical prostate specimens showed that CD133 expression was detected in a subpopulation of prostate cancer cells and corresponded to the loss of AR. Expression of CXCR4 was also detected in CD133(+) cancer cells. These novel in vitro models may offer useful tools for the study of the biological features and functional integration of normal and cancer stem cells in prostate.

The Many Facets of SDF-1α, CXCR4 Agonists and Antagonists on Hematopoietic Progenitor Cells

Stromal cell-derived factor-1alpha (SDF-1α) has pleiotropic effects on hematopoietic progenitor cells (HPCs). We have monitored podia formation, migration, proliferation, and cell-cell adhesion of human HPC under the influence of SDF-1α, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Whereas SDF-1α induced migration of CD34+ cells in a dose-dependent manner, CTCE-0214, CTCE-9908, and AMD3100 did not induce chemotaxis in this concentration range albeit the peptides CTCE-0214 and CTCE-9908 increased podia formation. Cell-cell adhesion of HPC to human mesenchymal stromal cells was impaired by the addition of SDF-1α, CTCE-0214, and AMD3100. Proliferation was not affected by SDF-1α or its analogs. Surface antigen detection of CXCR4 was reduced upon treatment with SDF-1α or AMD3100 and it was enhanced by CTCE-9908. Despite the fact that all these molecules target the same CXCR4 receptor, CXCR4 agonists and antagonists have selective effects on different functions of the natural molecule.

Protein Phosphatase 2A Plays an Essential Role in Migration of CD34+ Cells to SDF-1/CXCL12.

Although cord blood contains significant numbers of hematopoietic stem and progenitor cells (HSPC), its applicability has remained largely in children (1Broxmeyer and Smith, 2004). Homing of hematopoietic stem progenitor cells (HSPC) to bone marrow is a critical determinant for success of transplantation. Enhancement of homing of HSPC from cord blood has the potential to increase the applicability of cord blood transplantation in adults. Therefore, it is important to understand the molecules that underlie directional movement of HSPC. Stromal derived factor-1 (SDF-1)/CXCL12 is the most potent chemoattractant for both mouse and human HSPC and has been shown to play an important role in the homing and retention of HSPC in bone marrow. Earlier studies have shown that PI-3 kinase plays a critical role in chemotaxis by attracting various pleckstin homology (PH) containing domain proteins at the leading edge and spatial localization of PI3Kinase and PTEN is critical for maintaining the leading edge. Controlled activation of Akt/PKB, one of the various PH containg domains, has been described to be required for efficient chemotaxis. In this study we have demonstrated that protein phosphatase 2A (PP2A), a serine-threonine phosphatase, plays an important role in chemotaxis of cord blood CD34+ cells towards SDF-1, primarily by modulating Akt activity. Inhibition of PP2A by okadaic acid (OA) or siRNA impairs chemotaxis; this involved impairment in the ability of CD34+ cells to polarize and reduced speed of movement. This was associated with robust and prolonged Akt phosphorylation. Indeed, over expression of constitutive active Akt in CD34+ cells impaired SDF-1 directed chemotaxis. Co-immunoprecipitation experiments demonstrated increased association of Akt with PP2A following SDF-1 stimulation and this increased association of Akt and PP2A-catalytic subunit was observed at the plasma membrane of SDF-1 stimulated CD34+ cells by confocal microscopy. The importance of PP2A in maintaining a critical level and duration of activated Akt was also supported by our finding that inhibition of PI-3kinase by low dose of LY294002, partially recovered chemotactic activity in CD34+ cells pretreated with OA. Interestingly, glycogen synthase kinase-3 (GSK-3) associated with Akt following SDF-1 stimulation, although it was found to be constitutively associated with PP2A-C. Inhibition of GSK-3 using GSK-3 IX inhibitor impaired chemotaxis, raising the possibility that GSK-3 is a downstream target for Akt in SDF-1 directed chemotaxis. The physiological relevance of our in vitro findings is established from the observation that OA pretreatment impaired engraftment potential of CD34+ cells in NOD-SCID mice. Our findings contribute to the growing understanding of molecules that affect directional movement and may have the potential implications for homing and engraftment of HSPC.

G-CSF-Stimulation of Human Marrow Stromal Cells Induces In Vitro Migration of Hematopoietic Progenitor Cells Involving MMP-2 and MMP-9 but Not MMP-1.

The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitro migration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+ cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+ cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitro trans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+ across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.

Growth Factors and Cytokines Upregulate Gelatinase Expression in Bone Marrow CD34+ Cells and Their Transmigration Through Reconstituted Basement Membrane

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-alpha [TNF-alpha]) 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-alpha, 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), and o-phenanthroline. TNF-alpha-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.