Microanatomic distribution of transplanted α4^+/+ and α^−/− cells. (A) Flow cytometric analysis of donor cell populations. Lineage-depleted (using antibodies, magnetic beads, and column) cells were labeled with anti–c-kit (top 2 panels) and with anti-α4 Ab (bottom panels). Kit positivity was 82.8% in α4⁺ donor cells and 77.3% in α4^−/− donor cells. Shaded areas represent isotype control. α4 integrin was expressed on > 90% of normal donor cells and virtually absent on α4-ablated donor cells. (B) Relative distribution of transplanted cells from normal or α4 integrin-deficient donors to nonirradiated or irradiated normal hosts to endosteal regions of trabecular bone. The relative seeding to endosteal regions of trabecular bone was not affected by irradiation, and did not differ significantly between normal and α4 integrin-deficient donor cells. (□, normal donor cells; ■, α4^−/− donor cells; mean ± standard error of the mean; see Table 1 for values.) (C) Relative microanatomic distribution of transplanted cells from normal or α4 integrin–deficient donors to nonirradiated or irradiated normal hosts to endosteal, subendosteal, and central regions of compact bone. ▨ in each graph represent the relative surface area estimated by image analysis of each of the 3 zones (Figure 1A). Relative distribution frequencies equivalent to these values would thus indicate random distribution of donor cells throughout the marrow mass. Such random distribution was observed in nonirradiated hosts (□), irrespective of donor cell type, and in irradiated recipients (■) given α4^−/− cells. In contrast, normal cells transplanted into irradiated hosts distributed preferentially to the endosteal region (top panel). Asterisks indicate statistically significant differences between normal and α4^−/− cells and between no-irradiation (□) and irradiation (■) conditioning.