Endosteal populations maintain the LTR activity of HSCs. (A and B) LSK cells were cultured on a feeder layer of ALCAM⁺Sca-1⁻, ALCAM⁻Sca-1⁻, or ALCAM⁻Sca-1⁺ cells. After 5 days of culture, cells were harvested and cultured in methylcellulose medium. (A) Number of CFU-C. (B) Number of CFU-Mix. Data represent means ± SD (*P < .01). (C and D) LSK cells (Ly5.1⁺) were cultured on feeder layers of ALCAM⁺Sca-1⁻, ALCAM⁻Sca-1⁻, or ALCAM⁻Sca-1⁺ cells for 2 days. Then, CD45.1⁺ cells were sorted, and 200 cells were transplanted into recipient mice (Ly5.2⁺) along with 2 × 10⁵ competitor cells. (C) Percentages of donor-derived (Ly5.1⁺) cells in recipient mice 1 to 4 months after BM transplantation. Approximately 200 or 300 LSK cells without coculture were transplanted into recipient mice as a control. Data represent means ± SD (n = 5, *P < .01). (D) Representative FACS profiles of donor-derived (Ly5.1⁺) myeloid, B, T, erythroid, and megakaryocytic lineages in recipient mice. (E) LSK cells were cultured on feeder layers of ALCAM⁺Sca-1⁻, ALCAM⁻Sca-1⁻, or ALCAM⁻Sca-1⁺ cells. After 2 days of coculture, CD45⁺ cells were sorted, and the expression of Cxcr4, Itg2b (Cd41), Itgb2, Cd44, Cdh2, Vcam1, Gfi1, Hoxb4, Tel, Cdkn1c, Foxo3, and Sox2 was analyzed by Q-PCR array. Data represent means ± SD (*P < .05). Hprt1 was used for an endogenous control.