Primary osteoblasts stimulate BM B lymphopoiesis in vitro. (A) Hematopoietic proliferation during hematopoietic-osteoblast coculture is dependent on OB contact. (A, left-hand panel) Lin⁻Sca⁺ BM cells (CD45.1⁺) were isolated by FACS and cultured either directly on (CD45.2⁺) osteoblast monolayers (OB) or separated from osteoblast monolayers by a Transwell membrane (OB + Trans). (A, right-hand panel) Hematopoietic proliferation was nearly completely abrogated in the Transwell cultures, and no B220⁺ cells were produced. In the LS BM-OB contact cultures, 4% to 8% of all CD45.1⁺ cells produced were B220⁺, and these B220⁺ cells were found approximately equally portioned between cells adherent to the OB monolayer (OB-Ad) and nonadherent cells readily decanted from the suspension culture (OB-Susp). Data are shown as means ± SD; n = 4. (B) Flow cytometric analyses of B-lymphocyte precursors generated from Lin⁻Sca⁺ BM-osteoblast (non-Transwell) cocultures, recovered from both OB-adherent (OB-adh) and suspension cell (OB-susp) fractions. Nearly 80% of B220⁺ cells found adherent to the OB monolayer expressed CD24, 60% expressed CD43, and 44% were CD24⁺CD43⁺, but none expressed surface IgM. B220⁺ cells recovered from the culture supernatants expressed lower levels of CD43, and included (9%) B cells expressing surface IgM. (C) PTH stimulation increases the survival and proliferation of both B220⁺CD19⁻ and B220⁺CD19⁺ cells in osteoblast cocultures. IgM⁻Mac1⁻NK1.1⁻ (CD45.1⁺) BM cells were further FACS purified to isolate B220⁺CD19⁻ and CD19⁻B220⁺CD19⁺ cells, which were then cultured with (CD45.2⁺) osteoblasts for 6 days in the presence or absence of PTH. At the end of the cultures, the cells were collected and cells with the phenotype of the input cells (B220⁺CD19⁻ or B220⁺CD19⁺) were measured by flow cytometry. Data shown are expressed as the ratio of output/input B220⁺CD19⁻ cells in the culture initiated with B220⁺CD19⁻ cells (C, bars on left), or the ratio of output/input B220⁺CD19⁺ cells in the culture initiated with B220⁺CD19⁺ cells (C, bars on right). (D) Osteoblasts support B-lymphoid transitions from pre-pro B cells, pro-B cells, and B cells. Primary BM B220⁺CD19⁻ pre-pro-B cells, B220⁺CD19⁺ pro-B cells, and B220⁺⁺CD19⁺ pre-B cells were identified by flow cytometry and isolated by FACS, and each fraction was separately cultured with osteoblasts. Flow cytometric analysis after 5 days demonstrates further continued maturation in each of these compartments along B lymphopoiesis, including the acquisition of cell surface CD19 and/or IgM.