Sandra Huygen

Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5–mediated adhesion to fibronectin

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.

Upregulation of VLA-5 during cell cycle transit prevents effective migration of CD34+ cells through fibronectin-coated filters

Abstract Several reports have suggested that engraftment of stem/progenitor cells is decreased during cell cycle transit. This prompted us to study cell cycle-associated changes in adhesion and migration after a 2-day ex vivo culture in SCF, FL and TPO. We previously demonstrated that VLA-5 expression of CD34+ cells was enhanced during cell cycle transit and mediated increased adhesion to fibronectin (Fn). Here, we examined whether the increase in VLA-5 mediated adhesion to Fn had any impact on transmigration of cord blood CD34+ cells. Migration was assayed on 5-μm pore Transwells during 3 hours. Migration towards control medium through BSA-coated filters was observed in 5% of the cells. An additional 15% of input cells migrated when the filter was coated with Fn. Migration across Fn-coated filters towards conditioned medium (CM) from the stromal-derived factor (SDF)-1 producing cell line MS-5 rose to 42%. Migration was dependent on the presence of CXCR4, as 48% of migrating (Mg) cells were CXCR4+ versus 20% of non migrating (NMg) cells (P

Regulation of adhesion and migration of human long-term culture-initiating cells during cell cycle synchronization

Abstract Changes in adhesion molecule expression and/or function during cell cycle transit may be critical for engraftment of ex vivo expanded stem/progenitor cells. Here, we studied modulation of adhesion and migration of cell-cycle synchronized LTC-IC. Freshly isolated CB CD34+ cells, which reside in G0/G1, were stimulated ex vivo with SCF, FL and TPO. Aphidicolin was then added to reversibly block cells at the G1/S transition. Upon removal of aphidicolin, cells entered S phase synchronously after 3 hours and G2/M after 9–12 hours. At different time points, cells were assayed for adhesion onto fibronectin (Fn)-coated plates and replated in secondary LTC-IC assays (n=4). When freshly isolated or blocked at the G1/S transition, 15% of LTC-IC were adherent to Fn. When released from the aphidicolin block, LTC-IC adhesion was transiently increased during S-phase (up to 24%, P