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<.05), while VLA-5 expression was similar in both fractions. When the Transwell filter was coated with BSA, the percentage of CD34+ cells in S/G2+M was similar in Mg and NMg fractions towards MS-5 CM. In contrast, when the Transwell filter was coated with Fn, cycling cells were predominantly collected in the NMg fraction (33% of Mg cells in S/G2+M versus 41% of NMg cells in S/G2+M; P<.05). The NMg fraction was further fractionated by collecting separately non adherent (NAd) and adherent (Ad) cells to Fn-coated filters after migration. VLA-5 expression and cell cycle activity (%S/G2+M) were higher in the Ad fraction, as compared to the NAd (P<.05) or to the Mg (P<.05) fractions. Importantly, CXCR4 expression was similar in Ad and Mg cells. Collectively, these results suggest that SDF-1-induced chemotaxis is selective for non cycling cells when migration is assayed across a Fn-coated barrier. In this case, maximal VLA-5 expression in cycling cells confers a full adhesive state and prevents or delays effective transmigration.