Modulation of homing properties of primitive progenitor cells generated by ex vivo expansion.
Foguenne, Jacques ; ; Greimers, Roland et al
in Haematologica (2005), 90(4), 445-51
BACKGROUND AND OBJECTIVES: The maintenance of adequate interactions with the bone marrow (BM) microenvironment is critical to ensure efficient homing of ex vivo-expanded hematopoietic cells. This study ... [more ▼]
BACKGROUND AND OBJECTIVES: The maintenance of adequate interactions with the bone marrow (BM) microenvironment is critical to ensure efficient homing of ex vivo-expanded hematopoietic cells. This study was intended to assess adhesion and migration properties of long-term culture-initiating cells (LTC-IC) harvested after self-renewal division in ex vivo culture and to determine their susceptibility to growth-inhibitory signals mediated by adhesion to BM stromal ligands. DESIGN AND METHODS: We used cell tracking to isolate primitive LTC-IC that had accomplished 1 or 2 divisions ex vivo. Adhesion, migration and growth inhibition of divided LTC-IC were determined in the presence of purified BM ligands, and compared to the properties of uncultured LTC-IC. RESULTS: As compared to undivided LTC-IC, adhesion and migration mediated by very late antigen (VLA)-4 integrin across both vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (Fn) were downregulated in post-mitotic LTC-IC. Conversely, binding and motility via VLA-5 across Fn were stimulated. No changes occurred in LTC-IC interactions with intercellular adhesion molecule-1 (ICAM-1) or with E- or P-selectin. Proliferation of uncultured LTC-IC was inhibited by VLA-4-mediated binding to VCAM-1 and the CS-1 domain of Fn, as well as binding to P-selectin. Growth of ex vivo-generated LTC-IC became unresponsive to these 3 ligands but was suppressed through VLA-5 engagement by the cell binding domain of Fn. INTERPRETATION AND CONCLUSIONS: The generation of LTC-IC in expansion culture is associated with functional alterations of adhesion receptors, modulating not only binding and migration in the BM but also responsiveness to adhesion-mediated growth inhibitory signals. Such changes may limit homing and engraftment of expanded primitive stem/progenitor cells. [less ▲]Detailed reference viewed: 28 (5 ULg)
Binding and migration across fibronectin and VCAM-1 of cycling hematopoietic progenitor cells.
Gothot, André ; Giet, Olivier ; et al
in Leukemia & Lymphoma (2003), 44(8), 1379-83
Using different experimental approaches, it has been established that transplantability of hematopoietic/stem progenitor cells is ineffective during transit through the cell cycle. Although primitive stem ... [more ▼]
Using different experimental approaches, it has been established that transplantability of hematopoietic/stem progenitor cells is ineffective during transit through the cell cycle. Although primitive stem cells are responsive to mitogenic stimulation in optimized ex vivo conditions, defective engraftment of generated cells may limit their detection in standard transplantation models as well as their use in clinical cell therapy. The activation level of adhesion receptors is modulated by stimulation of cytokine receptors via "inside-out" signaling. This prompted us to study the interactions of progenitor cells with fibronectin (Fn) in different phases of the cell cycle. We first demonstrated that adhesion to Fn was stimulated in S/G2 + M as compared to G0/G1, in ex vivo cultured CD34+ cells, with a predominant usage of very late antigen (VLA)-5 over that of VLA-4. We next determined that maximal Fn binding in active phases of the cell cycle limited cell motility toward stromal cell-conditioned medium. It was also observed that VLA-4 and VLA-5 ability to mediate adhesion or migration varied independently during cell cycle transit. Finally, in synchronized progenitor cells executing a first cell cycle ex vivo, a reversible increase in Fn binding was associated with a reversible decrease in adhesion to vascular cell-adhesion molecule (VCAM)-1. Overall, these observations suggest that defective engraftment of cycling stem/progenitor cells may result, at least in part, from abnormal trafficking related to changes in the activation level of adhesion receptors. [less ▲]Detailed reference viewed: 38 (3 ULg)
Increased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells.
Giet, Olivier ; ; et al
in Blood (2002), 99(6), 2023-31
Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood ... [more ▼]
Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood CD34+ cells. Migration toward medium conditioned by the stromal-derived factor-1-producing cell line MS-5 was studied in bovine serum albumin- and fibronectin (Fn)-coated transwells. Migration was reduced in cycling CD34+ cells and long-term culture-initiating cells (LTC-ICs) compared with their noncycling counterparts across Fn but not across bovine serum albumin. Conversely, Fn binding was higher in cycling CD34+ cells and LTC-ICs compared with noncycling progenitor cells, while adhesion of both subsets to bovine serum albumin was undetectable. The contribution of alpha4 and alpha5 integrins in mediating adhesion and migration of activated CD34+ cells onto Fn was analyzed by neutralization experiments. While alpha4-mediated Fn binding decreased during G(2)/M, alpha5 integrin-mediated adhesion increased during transit from G(0)/G(1) to S and G(2)/M phases. As for migration, the contribution of alpha4 integrin was similar in all phases, whereas alpha5-directed migration was lower in G(2)/M compared with G(0)/G(1) and S phases. Defective migration of cycling CD34+ cells was not due to differences in alpha5 integrin expression. In conclusion, chemotaxis across Fn is less efficient in cycling progenitor cells in correlation with an increased Fn binding capacity. In addition, alpha4 and alpha5 integrin functions are independently modulated during cell cycle transit. [less ▲]Detailed reference viewed: 109 (3 ULg)
Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture.
; Giet, Olivier ; et al
in Blood (2002), 100(8), 2744-52
Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto ... [more ▼]
Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of alpha4 and alpha5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34(+) cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-alpha4 but not by anti-alpha5 antibodies. In mitotically activated CD34(+) cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on alpha5 integrin and to a lesser extent on alpha4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant alpha4 to a preferential alpha5 integrin-dependent interaction with Fn. [less ▲]Detailed reference viewed: 80 (3 ULg)
Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5-mediated adhesion to fibronectin.
Giet, Olivier ; ; Beguin, Yves et al
in Experimental hematology (2001), 29(4), 515-24
Recent studies suggested that trafficking of hematopoietic progenitor cells is related to cell cycle status. We studied whether adhesion of progenitor cells to extracellular matrix proteins was modulated ... [more ▼]
Recent studies suggested that trafficking of hematopoietic progenitor cells is related to cell cycle status. We studied whether adhesion of progenitor cells to extracellular matrix proteins was modulated by cell cycle transit.Mobilized peripheral blood CD34+ cells were stimulated ex vivo for 48 hours with stem cell factor, flt-3 ligand, and thrombopoietin and fractionated by adhesion to fibronectin or vascular cell adhesion molecule-1 (VCAM-1). Adherent and nonadherent cells were assayed for cell cycle status, long-term culture-initiating cell frequency, and integrin function. Binding to fibronectin, but not to VCAM-1, displayed a cell cycle selectivity as the adherent fraction to fibronectin was enriched in cycling CD34+ cells and in cycling long-term culture-initiating cells compared to the nonadherent fraction. Combined cell cycle and phenotypic analysis showed that the expression of VLA-5 was upregulated during S/G2+M but that of VLA-4 remained constant. The selective binding of cycling CD34+ cells to fibronectin was reverted by anti-VLA-5 but not by anti-VLA-4 blocking antibodies. Also, cycling CD34+ cells preferentially adhered to the VLA-5 binding domain but not to the VLA-4 binding domain of fibronectin. Adhesion of cycling CD34+ cells to fibronectin was a reversible process modulated by cell cycle progression, because adherent cells could exit the cell cycle and return to a nonadhesive state within an additional 48-hour culture period.The results indicate that the enhanced binding capacity of cycling progenitor cells to fibronectin is mediated by VLA-5. [less ▲]Detailed reference viewed: 36 (4 ULg)