GPR56 expression identifies the engrafting compartment in CD34⁺ AML. (A) Experimental setup of in vitro and in vivo experiments. (B, left) Sorting strategy for sample 05H163. (Right) Engraftment levels (percentage of human CD33⁺CD45⁺ cells in mouse bone marrow) in 3 to 4 recipient mice 14 weeks posttransplantation of decreasing cell doses (cell doses are cell equivalents, eg, 5 × 10³ bulk cells were equivalent to 800 CD34⁺GPR56⁺ (16%) cells). Bars indicate mean engraftment levels. (C) Absolute LSC frequencies of bulk cells and sorted fractions in AML 05H163 estimated by extreme limiting dilution analysis. **P < .005, ***P < .0005, χ² test. (D) In vivo and in vitro analysis of sorted GPR56 subsets. (Top row) FACS profiles for CD34 and GPR56 and initial percentages of sorted fractions in sorted bulk cells. (Center row) Engraftment levels (% human CD45⁺CD33⁺ cells shown for each recipient, bars indicate mean engraftment levels) yielded by each sorted fraction after 14 weeks in NSG mice (see supplemental Figure 6C for 8 and 12 weeks). Fractions were injected at cell doses respecting the percentages in total cells. Mice with <0.1% human engraftment were positioned at 0.1% and considered negative. (Bottom row) The number of CD34^+/− and GPR56^+/− cells generated by the sorted fractions in 7 days in optimized in vitro conditions. Bars are stacked (ie, the sum of stacked bars indicates total cell number). Equal numbers (5000 cells/well) of the 3 predominant fractions were seeded per well; bars and error bars represent means and standard deviations of 3 to 6 replicate wells.