Hh pathway antagonists impair maintenance of pro-B cells ex vivo. (A) Expansion of pro-B cells ex vivo. Flow cytometric analysis of pro-B cell (B220⁺CD43⁺) expansion from murine BM cells maintained for 5 days in the presence of autologous stromal cells and IL-7 (left). Presence of Smo, Ptch, and Gli1 transcripts in pro-B cells maintained ex vivo as assayed by RT-PCR (right). (B-C) Effects of Hh antagonists on pro-B cell proliferation. Pro-B cells were maintained on PA6 stromal cells with IL-7 in the absence or presence of Hh antagonist for 48 hours and proliferation was measured by ³H thymidine incorporation (mean and SD, n = 3, *P < .01, **P < .005). (B) Cyclopamine was omitted (control) or added at increasing concentration as indicated. (C) Pro-B cells were treated with isotype control Ab (control), anti–Hh Ab 5e1, or Ab 5e1 supplemented with recombinant sonic Hh (r-shh) as indicated.