PLAG1 is enriched and essential in human HSCs. (A) Schematic of Lin⁻CD34⁺ CB HSPC in vitro and in vivo functional assay timelines and lentivectors used for PLAG1 knockdown. (B) Primary CFU output by Lin⁻CD34⁺ HSPCs expressing control or (1sh) PLAG1-targeting hairpins. (C) Cumulative in vitro CD34⁺ cell fold change of cultured of Lin⁻CD34⁺ HSPCs expressing 1shPLAG1 or control hairpins. (D) GFP⁺ engraftment in the uninjected femur of primary NSG mice 6 weeks after xenotransplantation of Lin⁻CD34⁺ cells expressing either 1shPLAG1 (n = 4) or control (n = 6) hairpins normalized to input % GFP⁺ levels. (E) GFP⁺ engraftment in the injected femur and bone marrow of primary NSG mice 16 weeks after xenotransplantation of Lin⁻CD34⁺ cells expressing either 1shPLAG1 (n = 4) or control (n = 5) hairpins normalized to input % GFP⁺ levels. (F) Primary CFU output by Lin⁻CD34⁺ HSPCs expressing control or a second (2sh) PLAG1-targeting hairpin. (G) GFP⁺ engraftment in the injected femur and bone marrow of primary NSG mice 16 weeks after xenotransplantation of Lin⁻CD34⁺ cells expressing either 2shPLAG1 (n = 3) or control (n = 6) hairpins normalized to input % GFP⁺ levels. Data are presented as average ± SEM unless otherwise indicated. Each point represents one mouse or an individual CB unit. ***P < .005, **P < .01, *P < .05. n.s., not significant. See also supplemental Figure 1.