Mutated COPZ1 suppressed granulocytic differentiation of human HSPCs in vitro. (A) Schematic representation of the experimental protocol for gene editing and granulocytic differentiation of iPSCs. Created with BioRender.com. (B) Flow cytometry analysis at day 28 of iPSC myeloid differentiation. The percentage of iPSC-derived CD15⁺CD16⁺CD45⁺ neutrophils is shown. Means from 3 independent experiments (large symbols) performed in technical duplicates (small symbols) are plotted (pink, first experiment; gray, second experiment; green, third experiment). Statistical significance, ∗∗P < .01. (C) Representative cytospin images of iPSC-derived hematopoietic cells at day 28 of differentiation stained with May-Grünwald-Giemsa stain (×60 original magnification) for the indicated iPSC lines. (D) CFU assay of iPSC-derived CD34⁺CD45⁺ cells isolated at day 14 of iPSC differentiation. CFU counts are shown for the indicated iPSC lines. Data are represented as mean from 3 independent experiments (large symbols) performed in technical duplicates (small symbols). Pink represents the first experiment, gray is the second, and green is the third. CFU types included granulocytes, erythrocytes, monocytes, megakaryocytes (GEMM); granulocytes, monocytes (GM); granulocytes (G); monocytes (M). Statistical significance, ∗∗P < .01; ∗∗∗∗P < .0001. (E) Representative western blot images of lysates from iPSC clones C2 (lines 3 to 5) and G2 (lines 6 to 8) demonstrate a truncated (TR) COPZ1 protein at day 1, 14, and 28 of myeloid differentiation. The first lane on the left shows the control cell line THP1 (WT COPZ1) for comparison. (F) Schematic representation of gene editing and granulocytic differentiation of CD34⁺ CB-HSPCs after introducing TR- or MS COPZ1. Created with BioRender.com. (G) Percentage of neutrophilic CD45⁺CD15⁺CD66b⁺CD11b⁺CD16⁺ population at day 14 of LCD of COPZ1-TR or AAVS1 edited CD34⁺ CB-HSPCs from healthy donors. Data from 4 independent experiments are presented (pink, first experiment; gray, second experiment; green, third experiment; blue, fourth experiment). Large symbols represent the average value for each experiment, and small symbols represent technical replicates. Statistical significance, ∗∗P < .01. (H) Representative images of Wright-Giemsa–stained cytospin preparations of differentiated cells on day 14 of LCD of 2 healthy donors (×60 original magnification). (I) Percentage of different cell populations on cytospin slides. 100 cells are counted per slide. Data are plotted as means from 4 independent experiments (color coded per experiment). Statistical significance, ∗∗P < .01. (J) Percentage of CD45⁺CD15⁺CD66b⁺CD11b⁺CD16⁺ neutrophils at day 14 of LCD of COPZ1-MS and WT control-edited CD34⁺ CB-HSPCs from healthy donors. Data from 4 independent experiments are presented (pink, first experiment; gray, second experiment; green, third experiment; blue, fourth experiment). Large symbols represent the average value for each experiment, and small symbols represent technical replicates. Statistical significance: ∗∗∗P < .001. (K) Representative images of Wright-Giemsa–stained cytospin preparations of cells on day 14 of LCD of 2 healthy donors (×60 original magnification). (L) Percentage of different cell populations on cytospin slides of in COPZ1-MS group compared with WT control-edited cells. Data are plotted as means from 4 independent experiments (large symbols: pink, gray, green, and blue), performed in 2 to 4 technical replicates (small symbols). Statistical significance, ∗P < .05; ∗∗P < .01. BC, band cell; EB, embryoid based; Mϕ, macrophage; MB, myeloblast; MM, metamyelocyte; MY, myelocyte; ns, not significant; PM, promyelocyte; PMN, polymorphonuclear cell.