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Hyaluronan-Derived Oligosaccharides Enhance SDF-1-Dependent Chemotactic Effect on Peripheral Blood Hematopoietic CD34⁺ Cells

^a Groupe de Recherche sur le Micro-Environnement et le Renouvellement Cellulaire Intégré (M.E.R.C.I.);
^b Laboratoire d’Oncologie Moléculaire, Centre Henri-Becquerel;
^c Hôpital Charles Nicolle, Hématologie-Oncologie Pédiatrique, Rouen, France

Key Words. CD34⁺ • SDF-1 • Hyaluronan-derived oligosaccharides

Elhem Sbaa-Ketata, Ph.D., Groupe M.E.R.C.I., Faculté De Médecine-Pharmacie, 22, Boulevard Gambetta, 76 183 Rouen Cedex , France. Telephone: 33-2-35-14-83-50 or 49; Fax: 33-2-35-14-83-40; e-mail: Elhem.Ketata{at}Free.Fr

Hyaluronan (HA), a high-molecular-mass glycosaminoglycan that plays a key role in structuring tissue architecture, is an important component of the extracellular matrix that promotes processes such as migration of normal and malignant hematopoietic cells. CD44, a receptor for HA, has been shown to participate in the adhesion of normal and malignant stem cells to extracellular matrix components and to stromal elements [1]. Chemoattractant factors, such as the stromal cell-derived factor-1 (SDF-1), play essential roles in directing the migration of hematopoietic stem cells to bone marrow. SDF-1 is an -chemokine that binds to the CXCR-4 receptor. CXCR-4 is expressed on many cell types, including hematopoietic stem cells and progenitor cells. It has been shown that SDF-1/CXCR-4-mediated migration is essential for the homing and engraftment of human stem cells into nonobese diabetic/severe combined immunodeficient mice [2].

To assess the effects of HA and its fragments on hematopoiesis, an in vitro colony assay of hematopoietic stem cells was performed after migration in transwell. This technical approach, to our knowledge, has never been reported in the literature. No study has been reported on the effect of HA fragments on the SDF-1-dependent migration of steady-state CD34⁺ peripheral blood (PB) stem cells collected from human healthy donors.

Normal PB cells were obtained from 12 healthy adult volunteers after informed consent. Mononuclear cells were separated on a Ficoll-Hypaque density gradient centrifugation, and adherent cells were depleted. The isolation of hematopoietic progenitor cells was performed through positive selection of CD34-expressing cells, using anti-CD34 antibody coupled to microbeads (Miltenyi Biotech, Paris, France; http://www.miltenyibiotech.com). To evaluate the purity of the isolated CD34⁺ cells, direct immunofluorescence staining was used to examine the expression of CD34 on the surface of hematopoietic progenitor cells. Cells were incubated with anti-CD34 monoclonal antibody conjugated to fluorescein isothiocyanate (clone 581; Beckman Coulter; Paris, France; http://www.beckman.com) and analyzed by flow cytometry (EPICS XL-MCL; Coulter; Paris, France; http://www.coulter.com). In our hands, the purity of peripheral blood CD34⁺ cells was between 85%-96%. HA from Streptococcus was purchased from Sigma (Saint Quentin Fallavier, France; http://www.sigmaaldrich.com). HA-derived oligosaccharides were prepared as described [3].

For all experiments, CD34⁺ cells were incubated in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO/ BRL; Life Technologies; Cergy Pontoise, France; http://www.invitrogen.com) and supplemented with 0.5% bovine serum albumin (BSA) (Sigma) for chemotactic assay.

The in vitro chemotactic system used in this study was derived from the Boyden’s chamber using transwell inserts (6.5-mm diameter, 3-µm pore size, polycarbonate membrane, Dutscher; Brumath, France) to define the upper and the lower chambers, separated by an insert. In all experiments of migration, except in the control well, the chemokine SDF-1 (100 ng/ml) was added to the lower chamber at a final volume of 600 µl of IMDM 0.5% BSA. Then, 15 x 10³ CD34⁺ cells were placed into the upper chamber at a final volume of 100 µl of IMDM 0.5% BSA. To test the influence of HA or its fragments (HA₁₂ and HA₆) on the chemotactic effect of SDF-1, the upper and/or lower chambers were loaded with these components diluted in IMDM 0.5% BSA, at a concentration of 100 µg/ml.

After 18 hours at 37°C in a fully humidified atmosphere flushed with a combination of 5% CO₂ in air, the inserts were taken off, the 24-well plates were centrifuged, and supernatant was removed. Serum-free collagen medium (MegaCult; StemCell Technologies; Meylan, France; http://www.stemcell.com) containing growth factor was added directly to migrated cells remaining in the lower chamber of the plates. All growth factors were used at predetermined optimal concentrations: recombinant human (rh) interleukin-3 (IL-3), 10 ng/ml; rhG-CSF, 5 ng/ml; rh stem cell factor, 50 ng/ml; rhIL-6, 10 ng/ml (all growth factors from R&D Systems; Abingdon, United Kingdom; http://www.rndsystems.com); rh erythropoietin, 1 U/ml (MABIO-International; Tourcoing, France; http://www.mabio.net). After collagen polymerization, cultures were incubated at 37°C in a 5% CO₂ humidified incubator for 10 days and scored for colony formations of colony-forming unit (CFU) granulocyte-macrophage and burst-forming unit-erythroid. Assays were performed in duplicate on 12 different PB mononuclear cell CD34⁺ preparations.

Except for the control, results were expressed as the percentage of migrating cells (number of migrated cells/total number of input cells x 100), and as mean ± standard error (SE). Student’s t-test was used to compare paired experiments. Differences were considered significant at p 0.05.

In the absence of SDF-1, due to the small size of pores, no cells were recovered in the lower chamber and there was no spontaneous migration of stem cells (<1 CFU/well). However, addition of SDF-1 to the lower chamber resulted in important migration of CD34⁺ cells, as demonstrated by the number of colonies obtained (mean: 26 ± 7 CFU corresponding to 100% of migration).

The effect of HA and HA fragments on SDF-1-dependent chemotaxis was tested in different compartments. HA had no significant effect on migration of CD34⁺ cells in the presence of SDF-1 (n = 12, data not shown). Figures 1A and 1B showed that HA₁₂ and HA₆ significantly enhanced the chemotaxis effect of SDF-1 when added to the lower chamber (153% ± 18% and 167% ± 17% for HA₁₂ and HA₆, respectively), to the upper chamber (183% ± 21% and 153% ± 23%, respectively), or to both chambers (171% ± 24% and 149% ± 22%, respectively). However, HA or HA-derived oligosaccharides alone had no chemotactic effect (<1 CFU/well, data not shown) on CD34⁺. As a control, nonmigrating CD34⁺ cells were removed from the upper chamber and cultured in collagen assay for the hematopoietic stem cells. In all experiments, numerous colonies were observed (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 1. HA12 (A) and HA6 (B) enhance SDF-1-dependent migration of peripheral blood hematopoietic CD34+ cells. CD34+ cells (15 x 103) were added to the upper chambers of transwell. Lower chambers did or did not contain SDF-1 at 100 ng/ml. Chemotactic effect was tested for 18 hours. HA12 or HA6 (100 µg/ml) was loaded in the upper and/or lower chamber. Results represent the percentage of migrated cells to the lower chambers (mean ± SE) in five separate experiments performed in duplicate. *0.01 < p 0.05; **p < 0.01 compared with migration in presence of only SDF-1 .

The effect on migration may implicate the receptors of HA, CD44, or hyaluronan-mediated motility receptors (RHAMM). Trochon et al. have shown that HA₁₂ stimulated migration of endothelial cells, while HA₆ had no effect. They suggest that the action of HA₁₂ might be mediated by CD44 [4]. Pilarski et al. demonstrated that RHAMM interactions with HA might facilitate migratory behavior of HPCs, whereas CD44 interactions with HA may facilitate anchoring [5]. These results suggest that the mechanisms by which HA fragments stimulate the migration have not yet been determined.

ACKNOWLEDGMENT

We thank Richard Medeiros for his advice in editing the manuscript. This work was supported by grants from Association Vie et Espoir.

REFERENCES

1. Lundell BI, McCarthy JB, Kovach NL et al. Activation of b1 integrins on CML progenitors reveals cooperation between b1 integrin and CD44 in the regulation of adhesion and proliferation. Leukemia 1997;11:822–829.[CrossRef][Medline]

2. Peled A, Petit I, and Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.[Abstract/Free Full Text]

3. Courel MN, Maingonnat C, Tranchepain F et al. Importance of hyaluronan length in a hyaladherin-based assay for hyaluronan. Anal Biochem 2002;302:285–290.[CrossRef][Medline]

4. Trochon V, Mabilat C, Bertrand P et al. Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro. Int J Cancer 1996;66:664–668.[CrossRef][Medline]

5. Pilarski LM, Pruski E, Wizniak J et al. Potential role for hyaluronan and hyaluronan receptor RHAMM in mobilization and trafficking of hematopoietic progenitor cells. Blood 1999;93:2918–2927.[Abstract/Free Full Text]

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