Selective HLF reporter expression in immunophenotypic human LT-HSC populations. CB-derived CD34⁺ cells were processed as in Figure 3D, with the addition of siTP53 and transduction of rAAV6 HLF-ZsG P2A tEGFR at multiplicity of infection 400. One representative of 4 independent experiments covering 4 biological replicates is shown. (A) HR allele frequencies in CD201⁺/⁻ presorted fractions as determined by ddPCR. Gated on FAM⁺ (common probe) droplets, HEX⁺ droplets (red) identified HR allele amplicons. (B-C) Reporter expression in CD201⁺/⁻presorted fractions. Aggregated fluorescence-activated cell sorting (FACS) analysis (B) and summary by repeat (n = 4 for CD201⁻ and n = 5 for CD201⁺ sorted; unpaired, 2-sided P value by Student t test is indicated) (C). (D-E) Immunophenotypes of ex vivo–expanded (UM171⁺) HLF-targeted HSPCs. FACS analysis of total (black) vs reporter-expressing (green) populations at day 7. Percentages of increasingly restricted HSC gates are provided for each population. Aggregated FACS data (D) and summary (E). (F) Dimensional reduction based on FACS analysis. UMAP reduction using CD34, CD45RA, CD201, CD90, ITGA3, and ZsG FACS intensities from panel D were calculated and are represented as a 2-dimensional density plot of all cells (gray, n = 306 797). Cells falling into surface immunophenotypic HSC or ZsG−gates are overlaid and color coded as in panel D. (G-H) HLF reporter expression within immunophenotypic HSC gates. Reverse gating of the same data as above showing reporter expression in increasingly restricted HSC gates. Aggregated FACS data from all repeats (G) and summary (H). FSC, forward scatter; PE, phycoerythrin.