Myeloid immortalization potential of LT-HSCs, ST-HSCs, MPP2 cells, and MPP3/4 cells with conditional expression of MLL-ENL. (A) Experimental strategy for myeloid immortalization assays using serial replating. LT-HSCs, ST-HSCs, MPP2 cells, and MPP3/4 cells were purified from BM cells derived from C/Tg mice conditionally expressing CAG promoter-driven MLL-ENL by inducible Cre-estrogen receptor α chain (CreER) fusion protein expressed from the Rosa26 locus by sorting with CD150/CD48 expression in KSL-gated cells. Initially, 100 cells from sorted cells were seeded with 4-hydroxytamoxifen (4OHT) or EtOH, followed by serial replating of 10⁴ cells without drug. In cases with >5 colonies at the end of the third round (3R) of plating, 5 × 10³ cells were replated for 4R plating. ^†RNA was extracted only from initially 4OHT-treated cells. (B) Myeloid immortalization assays. (C) Reverse transcription (RT)-qPCR of MLL-ENL in colony-forming cells at the end of initial plating (1R) and third plating (3R) in myeloid immortalization assays. (D) Typical morphology of immortalized LT-HSCs constituting the colonies. Cells with Wright-Giemsa staining were viewed with an Olympus CKX41 microscope using a ×4/0.13 objective lens, and an Olympus BX41 microscope using a ×20/0.5 objective lens. Images were acquired with Olympus DP21 software. Original magnification, ×200; bar, 50 μm. (E) RT-qPCR of Hoxa9, Meis1, and Evi1 in colony-forming cells at the end of initial plating (with treatment of EtOH or 4OHT) and third plating (only cells derived from initially 4OHT-treated LT-HSCs, ST-HSCs, and MPP2 cells) in myeloid immortalization assays. In several samples from MPP3/4 cells, expression levels of Evi1 were below the limit of detection. Colors and patterns of bars are the same as shown in panel B. (F) Representative FACS plots of immortalized LT-HSCs, ST-HSCs, and MPP2 cells. Bar graphs show the mean with standard deviation (SD) of at least 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant (analysis of variance [ANOVA] followed by Tukey-Kramer multiple comparisons for panels C and E and unpaired Welch t tests for panel E).