CD31⁺ c-kit⁺Ly6C⁻ adult BM cells (2000) were cocultured on a confluent plate of irradiated stromal cells. All cells in the coculture were harvested at 1 or 4 weeks and injected into 4 irradiated adult recipient mice. Thus, each recipient received the equivalent of 500 sorted CD31⁺c-kit⁺Ly6C⁻ adult BM cells (1.6 HSCs). The number of HSCs in the CD31⁺c-kit⁺Ly6C⁻ adult BM cells was calculated by L-Calc software (StemCell Technologies) to be 1:300 by direct injection of the sorted cells into irradiated recipients. The results of these transplantations were: 100 cells injected per mouse resulted in 4 positive mice out of 10 having transplantation (4 of 10), 300 cells injected per mouse gave 8 of 12, and 500 cells injected per mouse gave 7 of 10. Mice were tested for repopulation at more than 4 months after transplantation, and only those mice with more than 10% of peripheral blood cells donor derived were considered positive. Three or more separate coculture and transplantation experiments were performed for most of the stromal clones and the no stroma (none) control. For stromal clones AM30-3F4 and UG15-1B7, only 2 experiments were performed. Cell lines derived from both LacZ and tsA58 embryos showed good HSC support. LacZ transgenic–derived stromal clones are EL08-1D2, AM14-1C4, and UG15-1B7; tsA58 transgenic–derived stromal clones are AM30-3A3, AM30-3F4, UG26-1B6, UG26-2D3, and GI29-2B4.

BM indicates bone marrow; HSCs, hematopoietic stem cells; other abbreviations are explained in Table 1.