WNT-5a stimulates colony formation of flASK cells. (A) Freshly sorted flASK cells or Lin^lo Sca⁺ bone marrow cells were plated into myeloid methylcellulose with control or WNT-5a CM. Colony number was from quadruplicate plates evaluated after day 12 of culture and repeated in three independent experiments. (B) flASK cells expanded for 11 days on stromal layers (fetal liver cell line 10-6) with control or WNT-5a CM and the resultant cells were replated into myeloid or lymphoid methylcellulose and scored for colony growth as described above. (C) flASK cells expanded for 11 days in suspension culture containing c-kit ligand (KL) with control or Wnt-5A CM or KL, IL-3, and IL-6 with control or Wnt-5A CM were replated into myeloid or lymphoid methylcellulose. KL was supplied at 25 ng/mL. IL-3 and IL-6 were supplied at 2.0 ng/mL each. Colony growth was scored as described above. For methylcellulose assays, myeloid cell growth was obtained using medium-containing sreum, GM-CSF, IL-3, IL-6, erythropoietin, and KL and lymphoid cell growth was obtained using medium-containing serum, KL, and IL-7.