All functional HSCs in adult BM express EMCN. (A) Sorting strategy for the purification of 7AAD⁻L⁻EMCN⁺ (L⁻E⁺) and 7AAD⁻Lin-EMCN⁻ (L⁻E⁻) cells from human BM and representative plot demonstrating a significant enrichment of immunophenotypic HSCs in L⁻E⁺ as compared with L⁻E⁻ populations. (B) Transplantation of 50 000 L⁻E⁻ and 2000 L⁻E⁺ highly purified BM cells into sublethally irradiated NOG mice and assessment of their capability for human long-term multilineage engraftment in BM and spleen 20 weeks after transplantation. (C-D) NOG mice transplanted with 2000 L⁻E⁺ (red squares) cells demonstrated marked human multilineage engraftment after 20 weeks (2 independent experiments; 3-4 replicates each, error bars shown as standard error of the mean), whereas mice transplanted with 50 000 L⁻E⁻ (blue squares) cells demonstrated human engraftment at very low levels without detection of multilineage engraftment. (E) Representative flow cytometric analysis of human multilineage engraftment in the BM of NOG mice including human CD45⁺CD19⁺ B cells and human CD45⁺CD33⁺ myeloid cells. FACS, fluorescence-activated cell sorting.