Fig. 4. Fetal spleen stroma drives HSCs to the sole macrophage lineage. (A) HSCs were sorted from E14 FL and cultured for 8-10 days onto three different FSS lines (n=5). (B) To understand the lineage relationship between these two cell subsets, sorted CD45^lo F4/80^- cells were cultured for 7 days on FSS cells. Only CD45⁺ F4/80⁺ macrophages were obtained. Both F4/80^- and F4/80⁺ populations were sorted and scored for the expression of myeloid/macrophage-related genes by RT-PCR. (C) For the RT-PCR, we used as positive control, two populations: E15 FL cells and macrophages from the peritoneal cavity (PeC). (D) Analysis of cell surface proteins by cytometry showing that macrophages are highly auto-fluorescent. The expression of indicated markers (gray line) was compared to a negative control (dashed line) that corresponds to the same unstained population (respectively, CD45^lo F4/80^- and CD45⁺ F4/80⁺).