Hh pathway activity in stromal cells promotes B lymphopoiesis. (A) Expression of Hh pathway components in stromal cells. Detection of transcripts encoding Hh signaling components (top) and ligands (bottom) in primary BM-derived stromal cells (lane 1), stromal cell lines PA6 (lane 2), or OP9 (lane 3), and mouse brain (lane 4). (B) Characterization of Smo-depleted OP9 derivatives. OP9 cells were transduced with lentivirus particles expressing Smo-specific (Smo^kd) or control (NT) shRNA. Smo (left) and Ptch (right) transcripts were quantitated by RT-PCR. (C-D) Impaired B lymphopoiesis from LSK progenitors maintained on Smo-depleted OP9 cells. Purified LSK progenitors were maintained on control (NT) or Smo-deficient (Smo^kd) OP9 cells under conditions that promote B lymphopoiesis. Lymphoid (CD19⁺Mac1⁻) and myeloid (CD19-Mac1⁺) differentiation were assessed by flow cytometry. (C) Representative plots of LSK cultures at 8 days on control (NT) or Smo-deficient (Smo kd) OP9 cells. (D) Percentages of lymphoid (CD19⁺Mac1⁻), myeloid (CD19⁻Mac1⁺), and nonlineage (CD19⁻Mac1⁻, CD19⁺Mac1⁺) progeny (n = 3, P < .01). (E-F) Impaired B lymphopoiesis in the presence of neutralizing Ab to Hh. LSK progenitors were cultured on OP9 cells in the presence of anti–Hh Ab 5e1 or an isotype control under B-lymphopoietic conditions. B-lymphoid and myeloid progeny were detected by flow cytometry. (E) Representative plots of control and Ab-treated (5e1) LSK cultures at 8 days. (F) Percentages of B-lymphoid and myeloid cells (n = 3, P < .005). A kinetic analysis of LSK differentiation from days 3-15 of culture is presented in the supplemental materials. (G) Undetectable DJ_H rearrangement in 5e1-treated LSK-OP9 cultures. DFL16-J_H (left) and DSP2-J_H (right) rearrangements were assayed in control (lane 1) and 5e1-treated (lane 2) LSK-OP9 cultures at 12 days or in spleen (lane 3). Expected positions of rearrangements to J_H1-4 are indicated. Amounts of template DNA were monitored by amplification of Rag1 (bottom left).