Hematopoietic and endothelial-like potential of type I yolk sac precursor cell lines.
(A) Single type I yolk sac precursor cell–derived colony following limiting dilution. (B) Representative type I yolk sac precursor cells stained with the Hema 3 Stain Set (Biochemical Sciences, Swedesboro, NJ). (C) Cells were allowed to differentiate in hematopoietic growth factor–containing medium. Compact colonies derived were harvested and cytospin preparations were stained with the Hema 3 Stain Set. M indicates macrophage; E, definitive erythroid cells. (D) Compact colonies were harvested and subjected to FACS analyses with the indicated antibodies. (E) Cells from compact colonies were replated into the second-stage culture containing the same hematopoietic growth factors as the first stage. Secondary compact colonies and hematopoietic colonies generated from 7.5 × 103 primary compact colony cells were examined and scored after 7 to 10 days of incubation. Mix indicates mixed colony; Ery-D, definitive erythroid colony; M, macrophage colony; Mk, megakaryocyte colony; Compact, secondary compact colony. (F) Type I precursor cell lines were allowed to differentiate into confluent adherent cells in tissue culture plates. Suspension cells were washed off, and 10 μg/mL Ac-LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL; Biomedical Technologies, Stoughton, MA) was added to the cultures. Four hours later, medium was removed and the plates were washed 3 times with prewarmed phosphate-buffered saline. Cells were visualized under a fluorescence microscope. Fi, NIH3T3 cells (negative control); Fii, bone marrow–derived adherent (endothelial) cells (positive control); Fiii, adherent endothelial-like cells generated from type I precursor cells. (G) Matrigel assay: Gi, type I precursor cells; Gii, type II (hematopoietic) cells. (H) FACS analyses of type I precursor cell–derived adherent cells with the indicated antibodies. Shown in this figure are representatives of 2 (panels D,E, and H) to 4 (panels A-C, F, and G) type I precursor cell lines and 10 type II hematopoietic cell lines (panel G). Original magnifications: ×100 (A, F-G); × 200 (B-C).
