CD34⁺CD38⁻CD19⁻CD33⁻ cells in MLL ALL are enriched for normal hematopoietic stem/progenitor cells. (A-E) Representative flow cytometry plots showing the identification of HSC/HPC population in MLL-AF4 case 3. (A) Within the CD34⁺CD38⁻ population, CD34⁺CD38⁻CD19⁻CD33⁻, CD34⁺CD38⁻CD19⁺CD33⁻, and CD34⁺CD38⁻CD19⁻CD33⁺ cells were sorted according to rectangular gates indicated and analyzed for MLL rearrangement by FISH. Numbers of MLL translocation-positive cells in total number of cells examined are shown as fractions. Sorted CD34⁺CD38⁻CD19⁻CD33⁻ cells were transplanted into NSG recipients. In normal human BM and CB almost all CD34⁺CD38⁻ cells do not express CD19. (B) Differentiation and maturation of human CD45⁺CD19⁺ B cells engrafted in recipient spleen examined through the expression of human CD10, CD20, IgM, and IgD. (C) BM and spleen of CD34⁺CD38⁻CD19⁻CD33⁺ cells were analyzed for human CD45⁺ cell engraftment and expression of CD34, CD38, CD19, CD33, CD10, CD20, IgD, and IgM. (D) Human CD10 and CD20 were not expressed by human CD19⁺ leukemia cells in the spleen of recipients transplanted with CD34⁺CD38⁺CD19⁺CD33⁻ and CD34⁻CD19⁺CD33⁻ cells. (E) Thymic repopulation by the CD34⁺CD38⁻CD19⁻CD33⁻ cells indicated by the engraftment of CD4⁺CD8⁺ DP cells, CD4 SP cells, and CD8 SP cells. (F,G) B-cell maturation in the BM and spleen of recipients transplanted with (F) CD34⁺CD38⁻CD19⁻CD33⁻ cells (MLL-AF4, n = 5; MLL-AF9, n = 7; MLL-ENL, n = 2) and those transplanted with (G) CD34⁺CD38⁺CD19⁺ or CD34⁻CD19⁺ LICs (MLL-AF4, n = 39; MLL-AF9, n = 16; MLL-ENL, n = 7) is summarized. (H) Frequencies of human CD3⁺ T cells within human CD45⁺ cells and the frequencies of double negative, DP, and single positive cells within human CD45⁺CD3⁺ cells in CD34⁺CD38⁻CD19⁻CD33⁻ recipient thymus are summarized (MLL-AF4, n = 3; MLL-AF9, n = 2; MLL-ENL, n = 1).