Expansion of CAFCs.
Lin−Sca-1+c-kit+ cells were
purified from BM of the chimeric receptor transgenic mice by FACS
sorting. Without preincubation, 100
Lin−Sca-1+c-kit+
cells were incubated on an OP9 stromal cell layer without adding
cytokines (░⃞). On the other hand, 100
Lin−Sca-1+c-kit+
cells were incubated with SCF (□), hGM-CSF and SCF (▪), IL-6 and
SCF (▧), or IL-11 and SCF (▨) in serum-free suspension culture for
4 d and then washed well with α-medium and incubated on an OP9
stromal cell layer without addition of cytokines. (A) On day 7 of
coculture, numbers of cobblestone area were counted. (B) To evaluate
the hematopoietic capacity of the cobblestone area, on days 12, 20, and
28, clonogenic cells produced from the cobblestone area during the
coculture were analyzed by in vitro colony assay. Assays were performed
in triplicate cultures and repeated in two independent experiments.