Comparison between Progenitor Cells from Mobilized Peripheral Blood (PB) in Healthy Donors and Non-Hodgkin’s Lymphoma (NHL) Patients.

Abstract INTRODUCTION: Peripheral blood stem cell (PBSC) mobilization is impaired in patients receiving chemotherapy but, as far as we know there is no data about the impact of chemotherapy on different PB progenitor cell subpopulations. AIM: to ascertain whether or not immature or committed progenitor cell are affected by chemotherapy prior PBSC mobilization in NHL patients. MATERIAL AND METHODS: a total of 27 PB samples from NHL patients and 36 PB samples from healthy donors were studied. Immunophenotypic analysis of CD34+ cell subpopulations was performed using the following four colour combinations of monoclonal antibodies (FITC/PE/PC5/APC): CD90/CD133/CD38/CD34 and CD71/CD13/CD45/CD34. In order to study committed progenitor cells “in vitro”, standard colony-forming assays were used and, in order to investigate the behaviour of the uncommitted progenitors Delta Assays of plastic adherent progenitor cells (PΔ) were performed. RESULTS: The comparison between NHL patients and healthy donors is shown in Table 1. The relationship between data obtained by flow cytometry and cultures was statistically significant (p<0.05, r>0.568) for all the progenitors analysed. Table 1: Results of Immunophenotypic and Functional Assays LNH patients Healthy donors p Data expressed as median (range). 1. Percentage among CD34+ cells. 2. Number of CFU/10 5 planted cells. 3. Number of CFU/10 6 planted cells % CD34 0.16(0.04–3.65) 0.57(0.11–1.81) 0.013 Immunophenotypic Data Erithroid 1 0.05(0.01–0.60) 0.14(0.02–0.42) 0.098 Myelo–monocytic 1 0.11(0.02–2.41) 0.37(0.07–1.18) 0.014 Immature 1 0.01(0.00–0.63) 0.05(0.01–0.19) 0.014 CFU-GM 2 70(4–440) 90(0–904) 0.327 Clonogenic and Delta Assays data BFU-E 2 62(6–172) 85(0–240) 0.046 CFU-Mix 2 18(0–124) 42(0–140) 0.018 CFU Δ3 356(0–3509) 953(90–8320) 0.033 CONCLUSIONS: We can conclude that in NHL, mobilized committed and above all immature progenitors are impaired when compared with healthy subjects, both analysed by immunological and functional assays. Only granulomonocytic progenitors analysed by a functional approach seemed to be preserved.

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In Vitro Manipulation of Peripheral Blood Progenitor Cell Collections

The International Journal of Artificial Organs ◽

10.1177/039139889802106s01 ◽

1998 ◽

Vol 21 (6_suppl) ◽

pp. 1-10

Author(s):

C. Carlo-Stella ◽

V. Rizzoli

Keyword(s):

Progenitor Cells ◽

Peripheral Blood ◽

Progenitor Cell ◽

High Dose ◽

Hematopoietic Stem ◽

Peripheral Blood Progenitor Cells ◽

Stem And Progenitor Cells ◽

Therapy Strategies ◽

Mobilized Peripheral Blood

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used to reconstitute hematopoiesis in patients undergoing high-dose chemoradiotherapy. PBPC collections comprise a heterogeneous population containing both committed progenitors and pluripotent stem cells and can be harvested (i) in steady state, (ii) after chemotherapeutic conditioning, (iii) growth factor priming, or (iv) both. The use of PBPC has opened new therapeutic perspectives mainly related to the availability of large amounts of mobilized hematopoietic stem and progenitor cells. Extensive manipulation of the grafts, including the possibility of exploiting these cells as vehicles for gene therapy strategies, are now possible and will be reviewed.

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Cell Cycle Progression and Self-Renewal Divisions of Cord Blood Derived CD34+ Cells Treated with the Polyamine Copper-Chelator Tetraethylenepentamine.

Blood ◽

10.1182/blood.v104.11.4237.4237 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4237-4237

Author(s):

Toni Peled ◽

Noga R. Goudsmid ◽

Frida Grynspan ◽

Sophie Adi ◽

Efrat Landau ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Fluorescence Intensity ◽

Cell Expansion ◽

Progenitor Cell ◽

Self Renewal ◽

Cell Cycle Profile ◽

Cd34 Cells ◽

Copper Chelator

Abstract In vitro cell expansion is constrained by default pathways of commitment and differentiation resulting in limited expansion of hematopoietic stem-progenitor cells (HSPCs). Still, several ex vivo manipulations have been reported to achieve expansion of HSPCs by altering cell cycle kinetics and enhancing progression through the G1-S barrier. We have previously shown that addition of tetraethylenepentamine (TEPA), a polyamine copper chelator, to cytokine-supplemented CD34+ cell cultures modulates cytokine-driven hematopoietic cell fate in vitro, resulting in remarkable expansion of a cell population that displays phenotypic and functional characteristics of HSPCs (Exp Hematol.2004;32 (6):547–55). The objective of the present study was to evaluate the mechanism leading to expansion of early progenitor cells following short-term exposure to TEPA. To this end, cell cycle profile, tracking of proliferation history, as well as determination of actual numbers of progenitor subsets were studied. In order to follow the extent of proliferation by tracking the number of cellular divisions, freshly isolated CD34+ cells were labeled with PKH2, a membrane dye that is sequentially diluted during every cell division. Fluorescence intensities of CD34+ and that of a more immature CD34+CD38− cell subset were determined immediately after staining. The cells were then cultured in serum-containing medium and a cocktail of cytokines (SCF, TPO, IL-6, Flt3-ligand, at 50 ng/ml each and IL-3 at 20 ng/ml), with and without TEPA. Total nucleated cells (TNC), purified CD34+ cells and CD34+CD38− cells were analyzed for PKH2 fluorescence intensity during the first two weeks of culture. Cell cycle profile was detected with the DNA intercalating agent propidium iodide, which determines cellular DNA content. FACS analysis of the cultured cells as well as progenitor cell quantification by immuno-affinity purification revealed comparable expansion levels of TNC and CD34+ cells in both TEPA-treated and control cultures during the first two weeks, as previously published. Although similar CD34+ cell numbers were observed, the mean frequency of CD34+CD38− and CD34+CD38-Lin- cells within the CD34+ cell population was significantly higher in TEPA-treated cultures over the control (0.2 vs. 0.04 and 0.07 vs. 0.01, respectively; n=6, p<0.05). Median PKH2 fluorescence intensity of CD34+CD38− subset was two fold higher in TEPA than in control cultures, demonstrating that early progenitor cells derived from TEPA-treated cultures consistently accomplished less proliferation cycles as compared to early progenitor cells derived from control cultures. This effect was not mirrored by a significant alteration of the cell cycle profile (Control (%): G1=26±14, S=2.6±0.1, G2=0.7±0.4; TEPA(%): G1=29±12, S=1.7±0.9, G2=0.4±0.2). Taken together, the data suggest that during cycling, the CD34+CD38− phenotype is preserved more successfully in TEPA-treated than in control cultures, suggesting retention of self-renewing potential of early progenitor cells under these culture conditions. This mechanism also supports a role for TEPA in inhibition of early progenitor cell differentiation. Ongoing work is aimed at further defining whether phenotype reversion or self-renewal (or both) lie at the foundation of TEPA-mediated progenitor cell expansion.

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MT1-MMP and RECK Inversely Regulate Hematopoietic Progenitor Cell Egress.

Blood ◽

10.1182/blood.v110.11.1259.1259 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1259-1259

Author(s):

Abraham Avigdor ◽

Yaron Vagima ◽

Polina Goichberg ◽

Shoham Shivtiel ◽

Melania Tesio ◽

...

Keyword(s):

Steady State ◽

Progenitor Cell ◽

Scid Mice ◽

Hematopoietic Progenitor Cell ◽

Chimeric Mice ◽

Hematopoietic Progenitor ◽

Human Cd34 ◽

Healthy Donors ◽

Cd34 Cells

Abstract Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

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Mobilization of Allogeneic Peripheral Blood Stem Cells in Family Donors with Single-Dose Pegfilgrastim.

Blood ◽

10.1182/blood.v114.22.2148.2148 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2148-2148

Author(s):

Vladan Vucinic ◽

Nadezda Basara ◽

Runa Stiegler ◽

Kristina Bartsch ◽

Constanze Kliem ◽

...

Keyword(s):

Stem Cells ◽

Body Weight ◽

Single Dose ◽

Peripheral Blood ◽

Peripheral Blood Stem Cells ◽

Weight Range ◽

Healthy Donors ◽

Cd34 Cells ◽

Blood Stem Cells ◽

Pbsc Mobilization

Abstract Abstract 2148 Poster Board II-125 Introduction: The standard procedure for obtaining peripheral blood stem cells (PBSC) is donor mobilization with G-CSF. Pegfilgrastim is a covalently bound conjugate of filgrastim and monomethoxypolyethylene glycol with longer half-life elimination due to decreased plasma clearance and could represent an alternative approach for PBSC mobilization in healthy donors. Design and Methods: From July 2006 till August 2009 28 related healthy donors (50% male, 50% female) were treated with single dose of 12 mg pegfilgrastim for mobilization of allogeneic PBSC. The harvests were performed as large-volume, continuous-flow collections using a Cobe Spectra blood cell separator on day 4 and if necessary on day 5 of the mobilization regimen. In case of inadequate CD34+ counts (less than 4×106/kg body weight of recipient on day 5), stimulation was continued with filgrastim. In addition, the serum level of filgrastim was determined twice daily. Results: We present the results of 27 donors (the results of the 28th donor are still pending). In all 27 cases the harvests were successful. In 22 out of 27 donors (82%) only a single apheresis was needed to reach the target. Two of the donors required additional treatment with non-pegylated filgrastim. The maximal concentration of circulating CD34+ cells was achieved on day 4 (median 74.3/μl; range 24.6-136.6). The median yield of CD34+ cells was 5.9×106/kg of the recipients body weight (range 3-14.5), and the median CD3+ count was 9.1×108/kg of the recipient body weight (range 1.4-6.2). Serum filgrastim level peak was on day 2 of the mobilization regimen with a median level of 226 ng/ml (range 35 to 1123 ng/ml), thus preceding the increase of CD34+ cells in blood. The main adverse events were WHO grade 1 and included headaches, bone pain and transient elevations of alkaline phosphatase and lactate dehydrogenase. Conclusion: PBSC mobilization with a single dose of pegfilgrastim is feasible for healthy donors. The graft composition was comparable to that obtained with the conventional regimen of short-term G-CSF. Long-term follow-up of healthy donors treated with pegfilgrastim should be further investigated. Disclosures: No relevant conflicts of interest to declare.

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The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.739.739 ◽

2010 ◽

Vol 116 (21) ◽

pp. 739-739

Author(s):

Vijay P. S. Rawat ◽

Natalia Arseni ◽

Farid Ahmed ◽

Medhanie A. Mulaw ◽

Silvia Thoene ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Cell Lines ◽

Myeloid Cells ◽

Lymphoid Cells ◽

Hematopoietic Development ◽

Cd34 Cells ◽

The Impact

Abstract Abstract 739 Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Impact of Constitutional Polymorphisms in VCAM1, CD44, CXCL12, CXCR4 and CSF3R in Progenitor Cell Mobilization in Patients with Hematological Malignancies

Blood ◽

10.1182/blood.v118.21.2997.2997 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2997-2997

Author(s):

Maria L. Lozano ◽

Cristina Castilla-Llorente ◽

Elkin A. Niño ◽

Ana I. Antón ◽

Jose Padilla ◽

...

Keyword(s):

Peripheral Blood ◽

Genetic Variants ◽

Acute Leukaemia ◽

Progenitor Cell ◽

Hematological Malignancies ◽

Cell Transplant ◽

Healthy Donors ◽

Cd34 Cells ◽

Cell Mobilization ◽

Poor Mobilizers

Abstract Abstract 2997 Introduction. The identification of genetic variants predictive of response to G-CSF mobilization might be useful in deciding the best strategy to obtain haematopoietic progenitors (HP) in patients scheduled for autologous peripheral blood progenitor cell transplant. Recently, one study has demonstrated the relationship among polymorphisms in genes implicated in trafficking and homing of CD34+ cells and the degree of mobilization after G-CSF therapy among healthy donors (Haematologica 2011; 96: 102–109). Aims. To evaluate if polymorphisms in five genes (CD44 rs13347 C>T, CSF3R rs3917924 A>G, CXCR4 rs2680880 A>T, CXCL12 rs1801157 G>A, and VCAM1 rs1041163 T>C) previously associated with the number of G-CSF mobilized CD34+ cells in healthy donors, can also predict the mobilization efficacy in a group of patients with hematological malignancies. Patients and Methods. We retrospectively evaluated 183 patients who were treated with s.c. G-CSF at 10 mcg/kg during 4 days. HP collection was initiated or not at day 5 according to the CD34+ number in peripheral blood (PB). Patients were selected among two groups: (1) poor mobilizers (n=109), who failed a mobilization attempt, presenting with <10 CD34+cells/mcl of PB, and (2) good mobilizers (n=74), those achieving >2 ×106CD34+ cells/kg in a first and only apheresis. The genetic variants were genotyped by allelic discrimination polymerase chain reaction (PCR) assays using TaqMan®Genotyping Assays (Applied Biosystems). Results. Patients diagnosed with lymphoma, myeloma and acute leukaemia were 40%, 38% and 21% of poor mobilizers, and 38%, 46% and 16% of good mobilizers, respectively. On the overall group, the genetic variant TT rs1801157 in CXCL12 was significantly associated with poor mobilization (p=0.040). Among lymphoma patients, the presence of the C allele in VCAM1 was significantly associated with mobilization rate (49% vs 19% among poor and good mobilizers respectively, p=0.011). In this lymphoma group, a trend towards poor mobilization was also observed in relation with homocygosis for the T allele of CXCL12 (12% vs 0% in poor and good mobilizers, respectively, p=0.066). The analyzed variables had no impact on the mobilization capacity in patients with myeloma or acute leukaemia. Discussion. Genetic variants in VCAM and CXCL12 seem to be related with the mobilization yield after G-CSF, particularly in lymphoma patients. Other polymorphisms in adhesion molecules related to the degree of CD34+ cell mobilization in healthy donors have not shown a relevant role in patients with hematological malignancies, probably reflecting the predominant effect of disease biology and/or of previous treatments. Funding. This study was supported in part by a research grants 04515/GERM '2f 06; RECAVA RD06/0014/0039, and FIS 10/02594 Disclosures: No relevant conflicts of interest to declare.

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The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽

10.1182/blood.v126.23.1169.1169 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1169-1169

Author(s):

Kam Tong Leung ◽

Karen Li ◽

Yorky Tsin Sik Wong ◽

Kathy Yuen Yee Chan ◽

Xiao-Bing Zhang ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Scid Mouse ◽

Conflicts Of Interest ◽

Chimeric Mice ◽

Hematopoietic Stem ◽

Cell Engraftment ◽

Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

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Transendothelial Migration of CD34+ and Mature Hematopoietic Cells: An In Vitro Study Using a Human Bone Marrow Endothelial Cell Line

Blood ◽

10.1182/blood.v89.1.72.72_72_80 ◽

1997 ◽

Vol 89 (1) ◽

pp. 72-80 ◽

Cited By ~ 2

Author(s):

Robert Möhle ◽

Malcolm A.S. Moore ◽

Ralph L. Nachman ◽

Shahin Rafii

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Transendothelial Migration ◽

Plating Efficiency ◽

Cd34 Cells ◽

Upper Chamber

To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

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Membrane Type 1-Matrix Metalloproteinase Is Directly Involved in G-CSF Induced Human Hematopoietic Stem and Progenitor Cell Mobilization.

Blood ◽

10.1182/blood.v104.11.2675.2675 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2675-2675

Author(s):

Abraham Avigdor ◽

Shimon Schwartz ◽

Polina Goichberg ◽

Isabelle Petit ◽

Izhar Hardan ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Peripheral Blood ◽

Progenitor Cell ◽

Human Cells ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Cd34 Cells ◽

Membrane Type

Abstract G-CSF induced hematopoietic stem/progenitor cell (HSC/HPC) mobilization is regulated by a complex interplay between extracellular matrix (ECM), cytokines/chemokines, adhesion molecules, and proteases, which affect stem cell retention and proliferation in the bone marrow microenvironment. Several studies suggest that membrane type 1-matrix metalloproteinase (MT1-MMP), expressed on the surface of various cell types, is a key enzyme for normal cell motility and tumor cell migration and invasion. We found that enriched human CD34+ cells express various surface MT1-MMP levels, depending on the cell source and G-CSF treatment. CD34+ cells obtained from BM of healthy donors treated with G-CSF were found to have the highest mean fluorescence intensity (>900 arbitrary units), while level of expression was lower in CD34+ cells derived from G-CSF mobilized peripheral blood from healthy donors (159±40), human steady-state (SS) BM (80±19), and human cord blood (41±4). Following 48 hr incubation of human SS-BM CD34+ cells with G-CSF, the expression of MT1-MMP increased 2-fold compared to untreated cells, whereas treatment with other cytokines, such as SCF, SDF-1 and IL-6, had only a minimal impact. Immunocytochemical analysis of human cord blood CD34+ enriched cells plated on fibronectin or hyaluronate-coated cover slips revealed that in response to SDF-1, MT1-MMP changes its localization in the polarized and spreading cells, suggesting a role in the process of HPCs directional migration. Indeed, incubation with neutralizing antihuman MT1-MMP Ab, targeting its catalytic site, significantly reduced human progenitor migration towards a gradient of SDF-1 in transwells. Interestingly, low concentrations of tissue inhibitor of the metalloproteinase-2 (TIMP-2) enhanced SDF-1 induced transwell migration, while higher concentrations hampered this process. More importantly, 5 daily injections of G-CSF to NOD/SCID mice previously engrafted with human cells, up-regulated MT1-MMP expression on CD45+ and CD34+ human cells in the BM and peripheral blood of the mobilized chimeric mice compared to untreated, control chimeras. Treatment of chimera mice with antihuman MT1-MMP Ab on days 3–5 of G-CSF induced mobilization, significantly reduced the number of CD45+ and CD34+ human cells in peripheral blood. In summary, based on our data we suggest that following G-CSF treatment, increased levels of MT1-MMP on the surface of human progenitors in the BM facilitates their mobilization most probably due to pericellular ECM degradation and/or activation of other regulatory molecules, pointing to the essential role of MT1-MMP in G-CSF induced mobilization.

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Probing the Impact of AMD3100/GCSF Mobilization on the Peripheral Blood T Cell Content Using a Rhesus Macaque Model: Increased Proportions of FoxP3+/CD4+ T Cells Occur After Mobilization and Leukapheresis

Blood ◽

10.1182/blood.v116.21.350.350 ◽

2010 ◽

Vol 116 (21) ◽

pp. 350-350

Author(s):

Leslie Kean ◽

Sharon Sen ◽

Mark E Metzger ◽

Aylin Bonifacino ◽

Karnail Singh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Cell Populations ◽

Macaque Model ◽

Cd34 Cells ◽

Cell Subpopulations ◽

T Cell Subpopulations ◽

The Impact

Abstract Abstract 350 Introduction: Leukapheresis is a widely utilized modality for collecting hematopoietic stem cells (HSCs). While collection of CD34+ cells with stem-cell activity is the primary goal of most mobilization and leukapheresis procedures, these cells only represent ∼1% of most leukapheresis products. The profile of the non-CD34+ cells is likely influenced by the choice of mobilization strategy, and has the potential to profoundly impact the post-transplant immune milieu of the transplant recipient. Two of the most critical of the CD34-negative cell populations that are collected during leukapheresis include effector and regulatory T cells. Thus, in evaluating mobilization regimens, the impact on these regimens on the mobilization of each of these T cell populations into the peripheral blood should be rigorously evaluated. Methods: We used a rhesus macaque model to determine the impact that mobilization with AMD3100 (a.k.a., Plerixafor or Mozobil®)+ G-CSF (“A+G”) had on peripheral blood CD4+ and CD8+ effector T cell populations as well as on FoxP3+/CD4+ T cells. Three rhesus macaques were mobilized with 10ug/kg SQ of G-CSF for five consecutive days prior to leukapheresis. AMD3100 was administered at 1mg/kg SQ in combination with the last dose of G-CSF two hours prior to leukapheresis. Leukapheresis procedures were performed for two hours using a modified CS3000 Plus cell separator. A peripheral blood sample was taken before cytokine therapy, just prior to leukapheresis following mobilization, one hour during leukapheresis, and at the end of the procedure. These samples were analyzed by multicolor flow cytometry using a BD LSRII flow cytometer. Results: Bulk, effector, and regulatory T cell subpopulations were analyzed flow cytometrically. The proportion of total CD3+ T cells remained stable during mobilization and apheresis: Thus, CD3+ T cells represented 77% of peripheral blood lymphocytes prior to mobilization, and 69% post-apheresis). The balance of CD4+ to CD8+ T cells was also relatively stable. Thus, for one of the three animals tested, the CD4+ and CD8+ proportions remained unchanged after apheresis. For two animals, the average CD4+ % decreased from 67% prior to mobilization to 52% post-apheresis. In these two animals, there was a reciprocal increase in the % of CD3+ T cells that were CD8+ (28% pre-G+A to 40% post-apheresis). The CD28+/CD95- naïve (Tn), CD28+/CD95+ central memory (Tcm) and CD28-/CD95+ effector memory (Tem) subpopulation balance of CD4+ and CD8+ T cells was also determined, by comparing the relative percentages of each subpopulation post-apheresis with their relative percentages prior to mobilization. Compared to their pre-G+A percentages, the post-apheresis CD4+ percentages of Tn, Tcm and Tem were 92%, 93% and 160%, respectively. Thus, the relative proportions of Tn and Tcm CD4+ cells decreased post-apheresis, while the relative proportion of CD4+ Tem increased compared to cytokine administration. For CD8+ T cell subpopulations, the post-apheresis proportions of Tn, Tcm, and Tem compared to their pre-G-CSF proportions were 99%, 70% and 130%, respectively–thus demonstrating the same direction of change as observed for CD4+ T cells. The most striking change in T cell subpopulations occurred in the CD4+/FoxP3+ compartment. The proportion of CD4+ T cells expressing FoxP3 increased by an average of 600% when post-apheresis samples were compared to pre-mobilization samples (FoxP3+ cells were 9.6% of CD4+ T cells post-apheresis versus 1.5% pre-GCSF). An average of 32% of these FoxP3+ CD4+ T cells expressed high levels of CXCR4. CXCR4 expression has been previously documented on human FoxP3+ T cells (Zou et al., Cancer Res, 2004), but this is the first observation of high level expression of CXCR4 on macaque FoxP3+ CD4 T cells, or of their ability to be efficiently mobilized with AMD3100. Discussion: These results suggest that treatment with AMD3100 and G-CSF may mobilize T cell subsets into the peripheral blood that could have beneficial effects during allo-transplantation. The combination of an increase in Tem cells, which have been observed to have decreased ability to cause GvHD (Zheng et al., Blood 2008), along with FoxP3+/CD4+ T cells, which may have regulatory functions, suggests that A+G mobilization could produce an apheresis product with a beneficial CD34-negative cell profile for allogeneic transplantation. Disclosures: No relevant conflicts of interest to declare.

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