PIWIL4 is dispensable for normal human hematopoiesis in vivo. (A) CFU assay showing number of CFU-GEMM, myeloid (CFU-GM), and the erythroid (BFU-E blast forming unit erythroid, CFU-E) colonies at day 14, formed by CB-derived CD34⁺ HSPCs transduced with scrambled control or shPIWIL4. Bars indicate mean colony number and SEM. “n” indicates number of independent experiments. (B) Representative image of the morphology of colonies observed in CFU assay of PIWIL4-depleted healthy human HSPCs vs control. (C) Total percentage of human engraftment in BM of NSG mice at 12 weeks after transplantation with scrambled or shRNA-transduced healthy CD34⁺ HSPCs. Engraftment was determined by GFP positivity. Dots indicate engraftment levels in individual mice. (D) Flow cytometry analysis of immunophenotypic subpopulations, stem cell–enriched (CD34⁺), myeloid (CD33⁺), or B-cell (CD19⁺), in BM of NSG mice that underwent transplantation with scrambled or shRNA-transduced healthy CD34⁺ HSPCs. (E) Apoptosis assay of PIWIL4-depleted CD34⁺ HSPCs vs scrambled control. “n” indicates the number of biological replicates.