M-CSF induces the expression of mature endothelial cell markers in PCLP-1⁺CD45⁻ cells.

(A) Expression of the M-CSF receptor in adherent cells in AGM culture. Adherent cells in AGM culture were harvested on the eighth day and were analyzed by FACS using anti–M-CSF receptor antibody with FITC-conjugated anti-rat IgG. Approximately 48% of cells in AGM culture expressed the M-CSF receptor. (B) After 10 days of incubation, adherent cells in AGM culture were harvested and sorted using PCLP-1 and CD45 antibodies. A set of specific primers for c-fms,M-CSF, and hprt were used for amplification of the DNA fragments. The expression of c-fms was detected in PCLP-1⁺CD45⁻ cells, and the expression of M-CSF was detected in the AGM region at 11.5 dpc. (C) Immunostaining of the M-CSF receptor. Positive cells were detected within and around the clusters of endothelial cells (arrowhead). (D) PCLP-1⁺CD45⁻ cells were isolated from the AGM region of the GFP transgenic mouse at E11.5 and were cocultured with OP9 stromal cells in the presence (OFVM) and the absence (OFV) of M-CSF. After 8 days of incubation, adherent cells were harvested and analyzed by FACS using antibodies against PECAM-1, VCAM-1, and E-selectin. Numbers in the figure indicate the percentage of cells positive (upper) and negative (lower) for GFP-positive cells. Results were summarized in the bottom panel. This experiment was performed 3 times, and generally the same results were obtained. P < .05 in PECAM-1, VCAM-1, and E-selectin. Culture conditions: OFV—OSM, bFGF, and VEGF; OFVM—OSM, bFGF, VEGF, and M-CSF.