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Cell Adhesion Molecule Expression in Cord Blood CD34⁺ Cells

Department of Pediatric Hematology-Oncology, University of Torino, Torino, Italy

Key Words. Cell adhesion molecules • L-selectin • Cord blood • CD34⁺ cells • Hematopoietic progenitors • Cytokines • Transplantation

Correspondence: Dr. Fabio Timeus, Pediatric Department, University of Torino, Piazza Polonia, 94, 10126 Torino, Italy.

	Abstract

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Self-renewal, proliferation, differentiation, homing, and mobilization of hematopoietic progenitor cells (HPCs) are regulated by a complex mechanism that involves the bone marrow (BM) microenvironment. Cell adhesion molecules (CAMs) expressed on HPCs and on endothelial and stromal cells play a pivotal role in this process. In this study, we have used three-color cytofluorometric analysis to compare CAM expression in the subsets of cord blood (CB) and BM HPCs and examined the effect of a short exposure to various cytokines on L-selectin expression. The study was carried out on unseparated samples to avoid any possible bias from positive CD34 selection. CAMs were highly expressed in both CB and BM CD34⁺CD38⁺ cells. In this population, L-selectin, H-CAM, and LFA-1 were significantly more expressed in BM than in CB.

With regard to the more immature progenitors, the subsets of CD34⁺/CD38^–/L-selectin⁺ and CD34⁺/CD38^–/LFA1⁺ cells were significantly larger in CB than in BM.

Since the expression of such CAMs has been related to the repopulating capacity of HPCs, our results suggest a possible advantage in homing and engraftment of more undifferentiated CB as opposed to BM HPCs. A 4/24-h exposure to various cytokines significantly increased the percentage of CB CD34⁺/CD38⁺/L-selectin⁺ cells, while HPCs were differentiated since the percentage of CD34⁺/CD38^–/L-selectin⁺ cells was reduced. These data show that a short exposure to cytokines increases L-selectin expression in the more differentiated CB HPCs. This could improve their homing in a transplant setting.

	Introduction

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During ontogeny, the site of hematopoiesis moves from the yolk sac to the liver, the spleen, and finally to the marrow. This process is characterized by the migration and selective homing of hematopoietic progenitor cells (HPCs). At birth, cord blood (CB) contains a large number of HPCs with extensive proliferative potential and capable of sustaining early and late engraftment in a transplant setting [1-4]. After birth, peripheral blood HPCs fall rapidly within a few days and colonize the bone marrow (BM) [1].

Self-renewal, proliferation, and differentiation of HPCs are regulated by a complex mechanism which involves the BM microenvironment, where stimulating and inhibiting cytokines as well as cell-to-cell and cell-to-extracellular matrix (ECM) interactions play a pivotal role [5]. These interactions are mediated by cellular adhesion molecules (CAMs) expressed on HPCs and on endothelial and stromal cells. CAMs are also involved in homing and mobilization of HPCs. In particular, HPCs express beta 1 and beta 2 integrins, the homing-associated cell adhesion molecule (H-CAM/CD44), the platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), L-selectin, and sialyl Lewis^x [6-8]. Monoclonal antibodies (mAbs) against beta 1 integrin VLA-4 (CD49d) promote HPC mobilization in primates [9], and antibodies against the ligand of VLA-4, VCAM-1 cause the disruption of the aggregates of hematopoietic cells with stromal cells [10]. Blocking mAbs against CD44 and CD49d inhibits hematopoiesis in long-term bone marrow cultures [11], and mAbs against alpha 4 or beta 1 integrins inhibit bone marrow colony-forming cell proliferation evaluated by the ³H-thymidine suicide assay [12].

The CD34⁺/CD38^– immunophenotype defines a rare subpopulation of HPCs enriched for CFU-blast and long-term culture-initiating cells (LTC-ICs) [13, 14] and responsible for late engraftment in BM transplantation.

There is evidence that L-selectin is involved in the homing of hematopoietic progenitors in the transplant setting: CD34⁺/L-selectin⁺ cells are predictive of rapid platelet recovery after peripheral blood stem cell transplantation [15].

CB has so far been successfully utilized for transplants in various BM failures, malignancies, and congenital immunologic and metabolic diseases. It contains a higher percentage of early progenitors than BM, and this could explain the achievement of a good engraftment with a relatively small number of nucleated cells [16, 17]. In consideration of the possible role in homing mechanisms, we have compared the expression of adhesion molecules in the subsets of CB and BM progenitors and analyzed the effect of a short exposure to various cytokines on L-selectin expression. We have studied the expression of VLA-4 (CD49d), VLA-5 (CD49e), H-CAM (CD44), LFA-1 (CD11a), and L-selectin (CD62L) in CB and BM CD34⁺/CD38⁺ and CD34⁺/CD38^– cells. Subsequently, L-selectin expression was evaluated after 4-24 h exposure to different combinations of stem cell factor (SCF), interleukin 3 (IL-3), flt-3 ligand (FL), and leukemia inhibiting factor (LIF).

	Materials and Methods

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Samples
Ten CB samples obtained during normal full-term deliveries by venipuncture of the placental end of transected umbilical cord were collected in sterile bags containing CPD 21 ml. Ten BM samples obtained after informed consent from aspirates taken in subjects with suspected hematologic disease not confirmed by subsequent analysis were collected in sterile tubes containing preservative-free heparin 100 U/ml. The number of nucleated cells in each sample was assessed with an automatic counter (Sysmex K-800, Toa Medical Electronics; Kobe, Japan).

Antibodies
The following mAbs were utilized:

• anti-CD34 PE or FITC-conjugated (8G12, Becton-Dickinson; San Jose, CA);
  
• anti-CD38 Cy-5-conjugated (HIT 2, Caltag; South San Francisco, CA.), and
  
• mAbs against cell adhesion molecules are shown in Table 1.
  
  

  View this table: [in this window] [in a new window]   Table 1. mAbs against CAMs utilized in the study

   
  

Flow Cytometry
Whole CB and BM were analyzed without cell separation. The percentage of cells expressing CD34 and/or CD38 and/or CAM was assessed by three-color cytofluorometric analysis as follows: 10⁶ nucleated cells were incubated for 20' at 4°C with a sufficient quantity of anti-CD38 Cy5-conjugated mAbs, anti-CD34 and anti-CAM PE or FITC-conjugated mAbs, according to their commercial availability. So, while an anti-CD34 PE-conjugated mAb was utilized in association with the anti-CD49d and CD49e FITC-conjugated mAbs, an FITC-conjugated mAb was utilized in association with the anti-CD62L, CD44, and CD11a mAbs ( Table 1). Isotype control was performed. After incubation and red cell lysis by ammonium chloride, cells were analyzed with a flow cytometer XL2 EPICS COULTER equipped with an argon laser. The analysis was performed as shown in Figure 1. A gate on mononuclear cells and CD34⁺ cells was first established. Then mononuclear cells and CD34⁺ cells were gated on plot 7 (CD38/CAM). CD34⁺ cells were first assessed in each CB and BM sample by the anti-CD34 PE-conjugated mAb, which allows more accurate evaluation, and then anti-CD34 FITC-conjugated was utilized. A mean of 177 (100-459) CD34⁺ cells was analyzed in CB and 259 (100-569) in BM samples.

View larger version (44K): [in this window] [in a new window]   Figure 1. Example of three-color cytofluorometric analysis of CB (A) and BM (B) cells. The study was performed on whole CB and BM, without cell separation. Cells were stained with the following mAbs: anti-CD34 8G12 FITC, anti-CD38 HIT 2 Cy-5 and anti-CD62L PE-conjugated. Gates were established on mononuclear and CD34+ cells. Almost 100 CD34+ cells were analyzed in each sample.

Pre-Exposure Experiments in Liquid Cultures
Whole BM or CB was incubated (1 x 10⁶ nucleated cells/ml) for 4 h and 24 h at 37°C in a humidified atmosphere of 5% CO₂ in air in Iscove's modified Dulbecco's medium (IMDM, GIBCO; Grand Island, NY) with different combinations of cytokines: A) SCF 50 ng/ml; B) SCF 50 ng/ml, IL-3 5 ng/ml; C) FL 50 ng/ml; D) SCF 50 ng/ml, FL 50 ng/ml; E) SCF 50 ng/ml, IL-3 5 ng/ml, FL 50 ng/ml, and F) SCF 50 ng/ml, FL 50 ng/ml, LIF 50 ng/ml. Cells were then stained with anti-CD34, CD38, and CD62L mAbs and analyzed.

Statistical Analysis
Statistics were obtained with Student's t-test.

	Results

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No significant difference in the percentage of CD34⁺ cells was found between the analyses performed with anti-CD34 PE or FITC-conjugated, even though CD34 fluorescence intensity was lower with FITC mAbs.

The number of CD 34⁺/CD38^– cells was significantly higher in CB than in BM (16% ± 8.8% and 4.7% ± 3% of total CD34⁺ cells respectively, p = 0.002). The CD34⁺/CD38⁺ subset was composed of two populations expressing CD38 with high and low fluorescence intensity. CD34⁺/CD38⁺ low intensity was significantly more expressed in CB than in BM (60.5% ± 9.9% and 32.8% ± 5.9% of total CD34⁺/CD38⁺, p = 0.008).

The percentage of BM and CB CD34⁺ cells expressing CAM and the mean fluorescence intensity of each molecule are shown in Table 2 and Figures 2 -4.

View larger version (23K): [in this window] [in a new window]   Figure 2. CD38 (y axis) and CAM (x axis) expression of CB (left) and BM (right) CD34+ cells. The percentage of CD34+/CD38– and CD34+/CD38+ low cells is higher in CB than in BM. CAMs are highly expressed in both CB and BM CD34+ cells.

View larger version (24K): [in this window] [in a new window]   Figure 3. Upper panel: CAM expression in CB and BM CD34+/CD38+ cells. L-selectin, H-CAM, and LFA-1 are significantly more expressed in BM. Lower panel: mean fluorescence intensity for CAM in CB and BM CD34+/CD38+ cells. No significant difference between CB and BM was found.

View larger version (21K): [in this window] [in a new window]   Figure 4. Upper panel: CAM expression in CB and BM CD34+/CD38– cells. L-selectin and LFA-1 are significantly more expressed in CB. Lower panel: mean fluorescence intensity for CAM in CB and BM CD34+/CD38– cells. No significant difference between CB and BM was found.

With regard to the more immature progenitors, the subsets of CD34⁺/CD38^–/CD62L⁺ and CD34⁺/CD38^–/CD11a⁺ cells were significantly larger in CB than in BM (10.9 ± 7 and 7.4 ± 4.4 versus 4 ± 2.2, and 3.2 + 1.8, p = 0.01 and 0.05, respectively).

In the subsets of CD34⁺/CD38^– and CD34⁺/38⁺ cells, no significant difference between CB and BM in mean fluorescence intensity of each CAM was found.

The effect of exposure to different cytokines on CD62L expression is summarized in Table 3 and Figure 5.

View larger version (26K): [in this window] [in a new window]   Figure 5. Upper panel: L-selectin expression in CB and BM CD34+/CD38+ cells before (basal) and after 24-h exposure to cytokines. 1 = SCF; 2 = SCF + IL-3; 3 = FL; 4 = SCF + FL; 5 = SCF + FL + IL-3; 6 = SCF + FL + LIF. Exposure to FL significantly enhanced L-selectin expression in CB. Lower panel: mean fluorescence intensity for L-selectin in CB and BM CD34+/CD38+ cells before (basal) and after 24-h exposure to cytokines. No significant difference was found in both CB and BM between basal and 24-h exposure.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several hundred CB transplants have been performed in various congenital and acquired diseases. Their engraftment is delayed in comparison with that of BM transplants [16-18].

We have observed a high level of early progenitors in CB; CD34⁺/38^– and CD34⁺/CD38⁺ low-intensity cells were significantly more expressed in CB than in BM. This matches previous studies demonstrating the more differentiated phenotype of BM CD34⁺ cells [19].

We have observed that the majority of CD34⁺/CD38⁺ in both CB and BM express CAMs. In BM, the number of CD34⁺/CD38⁺ cells expressing L-selectin, H-CAM, and LFA-1 was significantly higher than in CB. This could suggest an easier homing of more differentiated BM progenitors. However, the delayed engraftment observed in CB transplants seems more likely to be related to the fewer late progenitors in a CB collection.

With regard to the earlier progenitor subset, CD34⁺/CD38^– cells expressing L-selectin and LFA-1 were significantly higher in CB than in BM. Since the expression of these adhesion molecules has been related to the HPCs' repopulating capacity, our results suggest a possible advantage in homing and engraftment of more undifferentiated CB as opposed to BM progenitors.

With respect to the design of the study, while a positive selection of CD34⁺ cells could have allowed us to analyze a higher number of rare events, the experiments were carried out on unseparated CB and BM to avoid any possible bias from cell manipulation (Ficoll gradient, monoclonal antibodies, beads) that might modify the expression of CAMs and result in the possible selective loss of some CD34⁺ cell subsets. We also evaluated CD38 and L-selectin expression of CB and BM CD34⁺ cells after a short exposure to various cytokines to investigate their direct effect on CAM expression and their minimizing effect on cell proliferation.

Cytokine exposure was performed on unseparated samples to maintain the same experimental conditions as in the basal analysis. This approach could be of interest, since unseparated cord blood units are usually kept in cord blood banks, and the possibility of progenitor cell expansion by cytokine exposure could be considered in selected cases.

A short exposure to cytokines significantly enhanced L-selectin expression in CB CD34⁺/CD38⁺ cells and stimulated progenitor differentiation as evidenced by the decrease in the percentage of CD34⁺/CD38^– cells.

In conclusion, these data show that a short exposure to cytokines increases L-selectin expression in the more differentiated hematopoietic progenitors. This could improve their homing in a transplant setting. Moreover, our data support the view that exposure of a small volume of CB to cytokines could facilitate engraftment, as previously suggested [20].

	Acknowledgments

 
This work was partially supported by an A.I.R.C. grant. We thank Angelo Rossi for his technical assistance.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gabutti V, Foa R, Mussa F et al. Behaviour of human haemopoietic stem cells in cord and neonatal blood. Haematologica 1975;60:427.

2. Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as a potential source of transplantable hematopoieic stem/progenitors cells. Proc Natl Acad Sci USA 1989;86:3828-3832.[Abstract/Free Full Text]

3. Gluckman E, Broxmeyer HE, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med 1989;321:1174-1178.[Medline]

4. Lu L, Xiao M, Shen RN et al. Enrichment, characterisation and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 1993;81:41-48.[Abstract/Free Full Text]

5. Dexter TM. Hemopoiesis in long term bone marrow cultures. A review. Acta Haematol 1979;62:299-305.[Medline]

6. Saeland S, Duvert V, Caux C et al. Distribution of surface-membrane molecules on bone marrow and cord blood CD34⁺ hematopoietic cells. Exp Hematol 1992;20:24-33.[Medline]

7. Verfaille CM, Benis A, Iida J et al. Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alfa 4 beta 1 and the CD44 adhesion receptor. Blood 1994;84:1802-1811.[Abstract/Free Full Text]

8. Teixidò J, Hemier ME, Greenberger JS et al. Role of beta 1 and beta 2 integrins in the adhesion of human CD34^hi stem cells to bone marrow stroma. J Clin Invest 1992;90:358-367.

9. Papayannopoulou T, Nakamoto B. Peripheralization of hematopoietic progenitors in primates treated with anti-VLA-4 integrin. Proc Natl Acad Sci USA 1993;90:9374-9378.[Abstract/Free Full Text]

10. Funk PE, Kincade PW, Witte PL. Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates. Blood 1994;83:361-369.[Abstract/Free Full Text]

11. Miyake K, Medina KL, Hayashi S et al. Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures. J Exp Med 1990;171:477-488.[Abstract/Free Full Text]

12. Hurley RW, McCarty JB, Wayner EA et al. Monoclonal antibody crosslinking of the alfa 4 or beta 1 integrin inhibits committed clonogenic hematopoietic progenitor proliferation. Exp Hematol 1997;25:321-328.[Medline]

13. Terstappen LWMM, Huang S, Safford M et al. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34⁺ CD38^– progenitor cells. Blood 1991;77:1218-1227.[Abstract/Free Full Text]

14. Hao QL, Saha AJ, Thiemann FT et al. A functional comparison of CD34⁺CD38^– cells in cord blood and bone marrow. Blood 1995;86:3745-3753.[Abstract/Free Full Text]

15. Dercksen MW, Gerritsen WR, Rodenhuis S et al. Expression of adhesion molecules on CD34⁺ cells: CD34⁺ L-selectin⁺ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation. Blood 1995;85:3313-3319.[Abstract/Free Full Text]

16. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157-166.[Abstract/Free Full Text]

17. Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373-381.[Abstract/Free Full Text]

18. Arnold R, Schmeiser T, Heit W et al. Hemopoietic reconstitution after bone marrow transplantation. Exp Hematol 1986;14:271-277.[Medline]

19. Fritsch G, Stimpfl M, Kurz M et al. The composition of CD34 subpopulations differs between bone marrow, blood and cord blood. Bone Marrow Transplant 1996;17:169-178.[Medline]

20. Gabutti V, Timeus F, Ramenghi U et al. Expansion of cord blood progenitors and use for hemopoietic reconstitution. STEM CELLS 1993;11(suppl 2):105-112.[Abstract]

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