V⁺P⁻ stromal cells do not support maintenance of long-term repopulating HSCs. (A) Outline of the long-term HSC repopulating assay. Bone marrow–derived CD45.1⁺ SLAM cells cultured in low-cytokine medium onto stromal layers were transplanted into F1 double congenic CD45.1⁺/CD45.2⁺ irradiated mice. Bone marrow cells from primary recipient mice were transplanted into F1 secondary recipient mice. (B) Percentage of donor cells in the bone marrow of secondary recipient mice 18 weeks posttransplant are presented (# indicates groups for which all recipient animals displayed multilineage engraftment). Values represent mean ± standard deviation (SD) from 2 independent experiments. (C) Representative images of the cocultures after 14 days. Arrows point to putative MKs. Scale bars represent 80 μm. CK, cytokine.