PLAG1-S is a positive regulator of human HSPC fitness. (A) PLAG1 and MSI2 transcript expression in human bone marrow cell populations determined by single-cell RNA-seq.⁵⁴ (B) MSI2 protein expression measured by immunofluorescence microscopy in PLAG1-S overexpressing Lin⁻CD34⁺ cells. (C) Change in variance-stabilizing transformed (vst) PLAG1 transcript expression in Lin⁻ cord blood cells cultured for 2 or 4 days showing the P value from 1-tailed Student t-test³⁹ and in 72-hour cultured long-term (Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺) CB HSCs showing the P value from 1-tailed Student t-test and differential expression from DEseq analysis.⁵⁶ (D) Schematic of Lin⁻CD34⁺ CB HSPC in vitro and in vivo functional assay timelines and lentivectors used for overexpression of PLAG1 protein isoforms. (E) Primary CFU output by BFP⁺ Lin⁻CD34⁺ cells overexpressing PLAG1-A, B, or S, or Luciferase control (n = 3 per experiment). (F) Secondary CFU replating efficiency (for each condition, 12 GEMMs from 3 distinct CB units were replated into new wells. Negative indicates no secondary colonies were derived from the primary GEMM, Positive indicates at least 1 secondary colony was derived from the primary GEMM) and the total number of secondary colonies on positive plates with images of representative primary GEMM colonies used. (Square data points are from experiment 1 and circle data points are from experiment 2, n=3 per experiment.) (G) Cumulative in vitro total nucleated cell (TNC) and (H) CD34⁺ cell fold change of cultured of Lin⁻CD34⁺ cells overexpressing PLAG1-A (n = 3), B, or S, or Luciferase control (n = 6). (I) Frequency of CD34 positivity in PLAG1-A (n = 3), B, or S, or Luciferase control (n = 6) overexpressing cultures after 4 and 7 days ex vivo. (J) Representative flow plots and quantification relative to input proportions of BFP representation in CD45⁺ human grafts in bone marrow of primary NSG mice 16 weeks after receiving Lin⁻CD34⁺ cells overexpressing either PLAG1-S or Luciferase control (n = 6). (K) Representative flow plots of input and output BFP fluorescence intensity and quantification of output/input BFP median fluorescence intensity in bone marrow of primary NSG mice 16 weeks after receiving Lin⁻CD34⁺ cells overexpressing either PLAG1-S or Luciferase control (n = 6). Data are presented as average ± SEM unless otherwise indicated. Each point represents 1 mouse or an individual CB unit. ***P < .005, **P < .01, *P < .05. n.s., not significant. See also supplemental Figure 2.