In vivo leukemia initiation by CD34⁺CD38⁺CD19⁺ and CD34⁻CD19⁺ cells with MLL translocations. (A-C) Representative flow cytometry plots showing identification of LIC populations in MLL-AF4 case 3. (A) Using sort gates indicated by rectangles, 1000 CD34⁺CD38⁺CD19⁺, CD34⁻CD19⁺CD33⁺, and CD34⁻CD19⁺CD33⁻ cells were FACS purified and transplanted into newborn NSG recipients. (B) Leukemia initiation was confirmed by the presence of engrafted human CD45⁺ cells in the BM of recipients. Expression of CD34, CD38, CD19, and CD33 was analyzed in the engrafted human CD45⁺ cells. Five cell fractions (indicated by rectangles labeled Fr. 1-Fr. 5) were isolated and transplanted into secondary recipients. (C) Engraftment of human CD45⁺ cells and expression patterns of CD34 and CD38 in the secondary NSG recipients. (D) Representative flow cytometry plots showing identification of LIC populations in 2 MLL-ENL cases: one with dominant CD34⁺ cell population (case 18) and another with dominant CD34⁻ cell population (case 20). Using sort gates indicated by rectangles, CD34⁻CD19⁺ or CD34⁺CD38⁺CD19⁺ cells were FACS purified and transplanted into NSG recipients. (E) Leukemia initiation was confirmed by the presence of engrafted human CD45⁺ cells. Expression of CD34, CD38, CD19, and CD33 in engrafted human CD45⁺ cells are shown. In A-E, frequencies of each population is indicated and the number of MLL translocation-positive cells out of total number of cells examined are shown as fractions beside sort gates. (F,G) Engraftment levels of human CD45⁺ cells in (F) primary and (G) secondary recipient BM. Primary transplantation: MLL-AF4 CD34⁺CD38⁺ recipients, n = 54; CD34-CD19⁺ recipients, n = 60, from 11 patients; MLL-AF9 CD34⁻19⁺ recipients, n = 35 from 6 patients; MLL-ENL CD34⁺CD38⁺ recipients, n = 11; CD34⁻CD19⁺ recipients, n = 15, from 4 patients. Secondary transplantation: MLL-AF4 CD34⁺CD38⁺ recipients, n = 38; CD34⁻CD19⁺ recipients, n = 14, from 6 patients; MLL-AF9 CD34⁻19⁺ recipients, n = 12, from 4 patients; MLL-ENL CD34⁺CD38⁺ recipients, n = 6, from 2 patients; CD34⁻CD19⁺ recipients, n = 4, from 2 patients.