Differentiation of PU.1(−/−) ES cells. ES cells were cultivated under conditions optimized for production of macrophages as described in Materials and Methods. (A) Comparison of the development of embryoid bodies at day 8 of differentiation. Note that the two PU.1(−/−) ES cells have generated smaller embryoid bodies than either the (+/+) or (+/−) lines at this stage of differentiation. (B) After 31 days of differentiation, each of the lines of ES cells produced adherent cells with morphology resembling macrophages. The yield of macrophage-like cells was considerably lower at this time point in the (−/−) cells. The cells were harvested, replated, and incubated with latex beads as described in Materials and Methods. Panel shows adherent cells from each culture as indicated; black staining represents the uptake of latex beads. (C) An independent experiment to (B). In this case, adherent cells generated from differentiated ES cells were incubated first with latex beads, then fixed, and stained using an indirect immunoperoxidase method for expression of F4/80 antigen. In the (+/+) and (+/−) cultures, the brown reaction product demonstrating positive staining for F4/80 antigen is clearly evident on all phagocytic cells. In the two (−/−) lines, staining can be seen particularly in larger cells (eg, in the panel showing D3.15 cells), but was generally not sufficiently strong to permit photographic reproduction. Original magnification for (A) was 4× and for (B) and (C), 20×.