Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of alpha4 integrin- and CXCR4-mediated engraftment in NOD/SCID beta2-microglobulin-null mice

Abstract

Background: Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of alpha4 integrin, alpha5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34(+) cells.

Design and methods: Uncultured or expanded CD34(+) cells were infused in NOD/SCID-beta(2)microglobulin-null mice. The function of alpha4, and alpha5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of alpha4 integrin was further tested by adhesion and migration assays.

Results: Neutralization of either alpha4 integrin or CXCR4 abolished engraftment of uncultured CD34(+) cells at 6 week spost-transplant, while alpha5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking alpha4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of alpha5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that alpha4 integrin affinity in fresh CD34(+) cells was low and susceptible to stimulation while in cultured CD34(+) cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34(+) cells but not in cultured CD34(+) cells.

Conclusions: Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both alpha4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of alpha4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34(+) cells.

Figure 1.

α4 and α5 function in freshly isolated or ex vivo expanded SRC. The percentage of human CD45⁺ cells was measured in bone marrow cells of recipient mice transplanted 6 weeks previously with 100–150×10³ uncultured cord blood CD34⁺ cells (A) or their expansion product (B) after short-term culture in stem cell factor, thrombopoietin, Fit-3 ligand, interleukin-6 and granulocyte colony-stimulating factor. The contribution of α4 and α5 integrins to bone marrow repopulation was determined by incubating cells with neutralizing monoclonal antibodies P4C2 (anti-α4, triangles) or P1D6 (anti-α5, squares) prior to transplantation. Control cells were incubated with anti-CD34 (circles). Solid symbols indicate the mean ± SEM engraftment levels in each experimental condition (n=3 independent experiments of one to three mice per group). *p<0.05 compared to transplantation of control cells.

Figure 2.

Role of α4 and α5 in bone marrow homing of uncultured or ex vivo-expanded cord blood CD34⁺ cells. The presence of human CD34⁺ cells was determined in bone marrow (BM) harvested from NOD/SCID β₂m-null mice transplanted 16–24 hours previously with 500×10³ cord blood CD34⁺ cells before (A) or after (B) short-term culture. The function of α4 and α5 was assessed by incubating cells with P4C2 and P1D6 monoclonal prior to infusion in recipient mice antibodies, respectively. Control cells were treated with anti-CD34. p<0.05 compared to transplantation of control cells (n=3 experiments of two or three mice per group).

Figure 3.

Activation state of α4 and β1 integrins on fresh or expanded CD34⁺ cells. (A) Fresh or (B) expanded CD34⁺ cells were resuspended in Tyrode’s buffer 1% BSA (shaded histogram) or Tyrode’s buffer 1% BSA supplemented with 5 mmol/L EDTA (gray line), with 1 mmol/L MnCl₂ (black line), or with 1 mmol/L MnCl₂ together with function-blocking anti-α4 monoclonal antibody P4C2 (dotted line). Soluble VCAM-1 was added and cells were incubated for 3 hours at 37°C. Binding was measured by cell staining with phycoerythrin (PE)-conjugated anti-VCAM-1 (anti-CD106) monoclonal antibody. Results of one representative experiment out of four are shown. As compared to IgG-PE, binding of CD106 was undetectable in fresh (C) or cultured CD34⁺ cells (D) which had not been previously exposed to soluble VCAM-1, either in Tyrode’s buffer (TB) 0.1% BSA or in TB supplemented with 1 mmol/L MnCL₂. (E) The activation index of α4 integrin on fresh and expanded CD34⁺ cells was calculated as 100 × [(Fo−Fr)/(Fmax−Fr)], where Fo is the mean fluorescence intensity of soluble VCAM-1 binding in Tyrode’s buffer 1% BSA, Fr is background fluorescence in the presence of 5 mmol/L EDTA, and Fmax is the fluorescence intensity in the presence of 1 mmol/L MnCl₂. *p<0.05, n=4. (F) Fresh or expanded CD34⁺ cells were stained with HUTS-21 monoclonal antibody which is directed against an activation epitope of β1 integrin. HUTS-21 expression was measured in cells incubated in Tyrode’s buffer (−), in Tyrode’s buffer supplemented with 5 mmol/L EDTA or with β1 activating monoclonal antibody TS2/16 (n=3).

Figure 4.

Role of CXCR-4 in repopulating activity and homing of fresh or expanded CD34⁺ cells in NOD/SCID β₂m-null mice. (A) Mice were transplanted with 100–150×10³ CD34⁺ cells or their expansion equivalent after a 3-day expansion culture. Prior to infusion, cells were incubated with function-blocking anti-CXCR4 12G5 monoclonal antibody or anti-CD34 as control. Bone marrow chimerism was determined after 6 weeks by staining with human-specific anti-CD45. Solid symbols indicate the mean ± SEM engraftment levels in each experimental condition. (B) Mice were infused with 500×10³ fresh or 500×10³ expanded CD34⁺ cells. Homed CD34⁺ cells were measured in bone marrow of recipient mice 16–20 hours later. *p<0.05 compared to transplantation of control cells (n=3 experiments with two to four mice in each condition).

Figure 5.

Effect of SDF-1 in mediating actin polymerization, adhesion to VCAM and VCAM-supported transmigration of fresh and cultured CD34⁺ cells. (A) Fresh or cultured CD34⁺ cells were stimulated with 100 ng/mL SDF-1 for the indicated times and stained for filamentous actin with phalloidin-fluorescein isothiocyanate. Mean channel fluorescence (MCF) is shown as a function of time (n=3). (B) Adhesion assays were carried out on BSA- and VCAM-coated plates. The effect of SDF-1 and anti-α4 antibody was studied. Adhesion of fresh and cultured CD34⁺ cells is shown (n=3). (C) Transmigration assays were set up in response to 100 ng/mL SDF-1 across VCAM-coated Transwells or across BSA as a control. The effect of α4 neutralization is depicted. Migration of fresh and cultured CD34⁺ cells is shown (n=4).