Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of  4 integrin- and CXCR4-mediated engraftment in NOD/SCID  2-microglobulin-null mice

Abstract

BackgroundSeveral studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of α4 integrin, α5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34 + cells. Design and MethodsUncultured or expanded CD34 + cells were infused in NOD/SCID-β2microglobulin-null mice. The function of α4, and α5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of α4 integrin was further tested by adhesion and migration assays. ResultsNeutralization of either α4 integrin or CXCR4 abolished engraftment of uncultured CD34 + cells at 6 weeks post-transplant, while α5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking α4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of α5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that α4 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 cellderived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34 + cells but not in cultured CD34 + cells. ConclusionsOur data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both α4 integrin-and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of α4 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.Key words: stem cells, hematopoiesis, cell trafficking. -and CXCR4-mediated engraftment in NOD/SCID β2-microglobulin-null mice. Haematologica 2009; 94:185-194. doi: 10.3324/haematol.13206 ©2009 Ferrata Storti Foundation. This is an open-access paper. Citation: Foguenne J, Di Stefano I, Giet O, Beguin Y, and Gothot A. Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of α4 integrin Ex vivo

Co-culture of hematopoietic stem cells with mesenchymal stem cells increases VCAM-1-dependent migration of primitive hematopoietic stem cells

“…3.1 VCAM-1-dependent migration was enhanced in expanded HSC co-cultured with MSC It has been shown that short-term HSC expansion using early-acting cytokines, such as SCF, TPO and Flt3L, reduced HSC in vitro ability to migrate in VCAM-1-covered inserts [5,7]. This finding has been correlated with reduced in vivo HSC homing and engraftment [6,7].…”

“…This finding has been correlated with reduced in vivo HSC homing and engraftment [6,7]. We first assessed whether short-term HSC expansion with SCF, TPO and Flt3L in the presence of MSC would improve HSC migration, since ex vivo HSC co-culturing with MSC led to enhanced HSC engraftment [13,15,16].…”

“…Key HSC homing regulators of mobilization from and to the bone marrow (BM) include the stromal derived factor (SDF-1) and its CXCR4 receptor, very late antigens VLA-4 (CD49d/CD29) and VLA-5 (CD49e/ CD29), and the lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) [9][10][11][12]. Specific alterations in VLA-4-mediated and CXCR4-induced HSC adhesion and migration, appearing after cytokine expansion, may limit HSC homing and engraftment [5][6][7]. For instance, targeting VCAM-1/VLA-4 interaction by CD49d deletion, CD49d or VCAM-1 antibody blockade has induced a sustained increase of circulating CD34 ?…”

“…Many studies have shown that HSC cytokine expansion induces changes in homing-related molecule expression and function [4][5][6][7][8]. Key HSC homing regulators of mobilization from and to the bone marrow (BM) include the stromal derived factor (SDF-1) and its CXCR4 receptor, very late antigens VLA-4 (CD49d/CD29) and VLA-5 (CD49e/ CD29), and the lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) [9][10][11][12].…”

Impaired CXCR4 expression and cell engraftment of bone marrow-derived cells from aged atherogenic mice

“…11 Inactivation of CXCR4 by neutralizing antibodies has been shown to impair stem/progenitor cell engraftment in the bone marrow. 12 Accumulating evidence indicates that the level of CXCR4 surface expression on BMCs determines the efficiency of homing and the subsequent angiogenic response of target tissues. 13, 14 Over-expression of CXCR4 in mesenchymal stem cells (MSCs) or CD34+ cells enhances the migration of these cells towards SDF-1 in vitro 15 and increases the efficiency of bone marrow transplant in vivo 16 .…”