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

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<.05) before returning to baseline (10%, P<.05) after cell cycle completion. Using the same synchronization model, LTC-IC migration towards MS-5 conditioned medium was assayed in Fn-coated Transwells (n=4). The proportion of migrating LTC-IC was 68% in freshly isolated cells, 53% at the G1/S transition and 43% in S-phase. Dependence of LTC-IC adhesion and migration on VLA-4 and/or VLA-5 was assessed by blocking antibodies (n=4). In freshly isolated cells, both adhesion and migration of LTC-IC were inhibited by anti-VLA-4 but not by anti-VLA-5. At the G1/S transition and during S-phase transit, adhesion was inhibited mainly by anti-VLA-5 and to a lesser extent by anti-VLA-4 while migration was blocked only by anti-VLA-5. We conclude that S-phase transit during a single cell cycle is associated with a transient increase in adhesion of LTC-IC to Fn and a reciprocal decrease in migration through Fn-coated filters. In addition, our data show that unstimulated LTC-IC interact with Fn via VLA-4 while cytokine-stimulated LTC-IC use mainly VLA-5 to adhere and migrate on Fn. The impact of such changes on the homing capacity of ex vivo stimulated progenitor cells warrants further investigation.