FLT3-KO in normal human HSCs does not prevent engraftment and self-renewal. (A) FLT3 cell surface expression on HSCs (CD34+CD38–CD45RA–CD90+CD49f+), primitive progenitors (CD34+CD38–), and maturing progenitors (CD34+CD38+) from human FL, CB, and adult BM, n = 4 to 5 for each tissue. (B) Overview of the in vivo experiments of FLT3-KO in normal FL, CB, and BM. (C) KO percentages of CRISPR/Cas9-mediated FLT3-KO and control gene-KO in human HSCs from FL (4 samples) and CB (3 samples) and in HSPCs (HSCs and multipotent progenitors defined as CD34+38−45RA−90−49f−) from BM (2 samples), determined by Sanger sequencing and indel analysis. (D) Levels of human engraftment in mice injected with FLT3-KO and control gene-KO HSCs from FL and CB and HSPCs from BM, at 20 weeks (FL, CB) and 12 weeks (BM); recipients were sublethally irradiated female NSG mice; percentages of engraftment in injected femurs; 3 human samples of each tissue were used in 3 independent experiments (15 mice per condition). (E) FLT3 and control gene-KO percentages in human cells engrafted in each mouse from panel D; KO percentages were determined independently in the injected femurs; 2 mice transplanted with FLT3-KO FL HSCs were not engrafted by human cells and human DNA was not detectable, so genotyping was not performed on those. (F) FLT3 cell surface expression (by flow cytometry) in CD34+CD38– human cells engrafted in mice from panel D injected with FL and CB cells. (G) NSG-repopulating cell frequencies in FLT3-KO or control gene-KO HSCs from FL and CB, determined in primary recipients using limiting-dilution assays; 3 to 5 mice per cell dose. (H) Hematopoietic lineage distribution based on cell surface markers expressed in human cells engrafted in mice from panel D, determined by flow cytometry. (I) NSG-repopulating cell frequencies in FLT3-KO or control gene-KO HSCs from FL and CB, in secondary recipients (sublethally irradiated sex-matched NSG-SGM3 mice), using limiting-dilution assays; 3 to 5 mice per cell dose; human CD45+ cells collected from primary recipients described in panel D. Positive engraftment was considered if ≥0.1% human cells; lineage characterization was performed only on grafts with ≥1% of human cells. Mice engrafted with cells with less than 60% KO were excluded from the lineage output analysis. Mice with 0% of human engraftment where no human DNA was detectable were excluded from the genotyping analysis. Unpaired Student t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; mean ± standard deviation values are reported in the graphs. ns, nonsignificant.

FLT3-KO in normal human HSCs does not prevent engraftment and self-renewal. (A) FLT3 cell surface expression on HSCs (CD34+CD38–CD45RA–CD90+CD49f+), primitive progenitors (CD34+CD38–), and maturing progenitors (CD34+CD38+) from human FL, CB, and adult BM, n = 4 to 5 for each tissue. (B) Overview of the in vivo experiments of FLT3-KO in normal FL, CB, and BM. (C) KO percentages of CRISPR/Cas9-mediated FLT3-KO and control gene-KO in human HSCs from FL (4 samples) and CB (3 samples) and in HSPCs (HSCs and multipotent progenitors defined as CD34+38−45RA−90−49f−) from BM (2 samples), determined by Sanger sequencing and indel analysis. (D) Levels of human engraftment in mice injected with FLT3-KO and control gene-KO HSCs from FL and CB and HSPCs from BM, at 20 weeks (FL, CB) and 12 weeks (BM); recipients were sublethally irradiated female NSG mice; percentages of engraftment in injected femurs; 3 human samples of each tissue were used in 3 independent experiments (15 mice per condition). (E) FLT3 and control gene-KO percentages in human cells engrafted in each mouse from panel D; KO percentages were determined independently in the injected femurs; 2 mice transplanted with FLT3-KO FL HSCs were not engrafted by human cells and human DNA was not detectable, so genotyping was not performed on those. (F) FLT3 cell surface expression (by flow cytometry) in CD34+CD38– human cells engrafted in mice from panel D injected with FL and CB cells. (G) NSG-repopulating cell frequencies in FLT3-KO or control gene-KO HSCs from FL and CB, determined in primary recipients using limiting-dilution assays; 3 to 5 mice per cell dose. (H) Hematopoietic lineage distribution based on cell surface markers expressed in human cells engrafted in mice from panel D, determined by flow cytometry. (I) NSG-repopulating cell frequencies in FLT3-KO or control gene-KO HSCs from FL and CB, in secondary recipients (sublethally irradiated sex-matched NSG-SGM3 mice), using limiting-dilution assays; 3 to 5 mice per cell dose; human CD45+ cells collected from primary recipients described in panel D. Positive engraftment was considered if ≥0.1% human cells; lineage characterization was performed only on grafts with ≥1% of human cells. Mice engrafted with cells with less than 60% KO were excluded from the lineage output analysis. Mice with 0% of human engraftment where no human DNA was detectable were excluded from the genotyping analysis. Unpaired Student t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; mean ± standard deviation values are reported in the graphs. ns, nonsignificant.

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