Role of FL in THPO-independent maintenance of GM but not MkE progenitors. (A) Representative FACS profile showing sorting gates for THPOR^hi and THPOR⁻ LMPPs cells (top panels) and purity reanalysis (bottom panels). LSK cells were gated on the 25% highest FLT3 expressing cells to define LMPPs and further based on THPOR-high and -negative subpopulations. (B) THPOR^hi and THPOR⁻ LMPPs were sorted and cultured at 1 cell per well (manually plated) in Terasaki plates with indicated cytokines and clones were scored after 7 to 8 days. Data represent mean (SD) values from 2 to 3 experiments, with 120 cells being evaluated per population and experiment. (C) The preGMPs were sorted and cultured at 1 cell per well (directly deposited from the sorter) in Terasaki plates with indicated cytokines and clones were scored after 7 to 8 days. Data represent mean (SD) values from 2 experiments, with 120 cells being evaluated per population and experiment. (D) Single-cell RT-PCR analysis of transcriptional expression of Flt3, Thpor, and coexpression of Flt3 and Thpor in preGMPs. Only cells expressing Kit were included for further analysis (> 98% of cells analyzed). Data are means (SD) from 2 experiments, in which 88 cells were analyzed per experiment. (E) Representative FACS profiles in WT and Thpo^−/− and Fl^−/− × Thpo^−/− mice, showing frequencies of preGMPs (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁻CD105^low) and GMPs (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁺). Numbers represent frequencies of gated populations relative to total BM cells, and are mean values from more than 3 experiments and 10 to 13 mice per genotype. (F) Number of total BM cells, preGMPs (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁻CD105^low), GMPs (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁺), MkP (Lin⁻Kit⁺Sca-1⁻CD41⁺), and CFU-Es (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁻CD105^high) in WT, Thpo^−/−, and Fl^−/− × Thpo^−/− mice. Each dot represents an individual mouse. Data are from 3 experiments. Statistical significance was tested between Thpo^−/− and Fl^−/− × Thpo^−/− mice. ns indicates not significant; **P < .01, ***P < .001. (G) CFU-GM cultures; 10 000 unfractionated BM cells from WT and Thpo^−/− and Fl^−/− × Thpo^−/− mice, respectively, were added to methylcellulose supplemented with FL, IL-3, GM-CSF, and G-CSF. Clones were scored after 7 to 8 days. Each dot represents data from 1 mouse (means of 2 replicates). Data are from 3 experiments. Statistical significance was tested between Thpo^−/− and Fl^−/− × Thpo^−/− mice. ***P < 0.001.