Osteoblasts support the differentiation of RAG2⁻ LSK cells to RAG2⁺ cells, and to B-lymphoid precursors. (A) BM LSK cells from RAG2 GFP NG BAC mice were analyzed for GFP expression by FACS, and sorted into GFP⁻ and GFP⁺ populations. GFP⁻ LSKs were cultured for 14 days on either primary osteoblasts, MS-1 endothelial cells, or S17 stromal cells, after which the hematopoietic cells were recovered, stained with nonfluorescein dyes for B220 and CD19, and analyzed for Rag2⁻GFP, CD19, and B220 expression by flow cytometry. (A) Rag-2⁻GFP⁻ BM cells generate EGFP-bright cells after 14-day coculture with osteoblasts, and S-17 cells, but not MS-1 endothelial cells. Each coculture was initiated with 3000 LSK Rag2⁻ cells. After 14 days, 1.09 × 10⁵ total hematopoietic cells were recovered from the OB coculture, of which 1.64 × 10⁴ were EGFP bright, whereas none of the 1.2 × 10⁵ cells recovered from the MS-1 coculture was EGFP bright. S17 cocultures generated 7.6 × 10⁴ total cells, of which 1.67 × 10⁴ were EGFP bright. Percent of output cells with green fluorescence higher than control (nontransgenic and transgenic, sorted, Rag-2 GFP⁻) cells is shown in the figure. (B) GFP⁺B220⁺CD19⁺ B lymphocytes are generated from both GFP⁻ LSK cells and GFP⁺ LSK cells isolated from RAG2 GFP NG BAC mice, following 14-day cultures on osteoblast monolayers. (C) Absolute numbers of B-lymphocyte precursors produced from cocultures initiated with 10⁵ LSK cells.