Figure 2.

Characteristic features of iPSC-derived neutrophils. (a) Detached iPSCs were co-cultured on a layer of C3H10T1/2 cells and induced to undergo hematopoietic differentiation. One week later, HPCs could be harvested from sac-like structures and then further induced to undergo neutrophil differentiation. (b) Histological appearance of differentiated neutrophils Wright Giemsa (WG) staining, myeloperoxidase (MPO) (black dots) and alkaline phosphatase (ALP) (purple dots). (c) NBT-coated yeast were added to a suspension of differentiated cells for 1 hour at 37 °C and then stained with 1% safranin-O. Phagocytosed yeast is black in color due to the reduction of NBT to formazan by ROS (black arrows). (d) Differentiated cells were seeded onto Poly-l-lysine coated cover slips and stimulated with PMA to induce the formation of neutrophil extracellular traps (white arrows). After permeabilization and fixation, cells were stained for SYTO 13 (green) and MPO (red). (e) Recapitulation of the XCGD phenotype. Neutrophils were differentiated from either healthy (Healthy) or XCGD iPSCs. Nonstimulated (gray) and stimulated cells with PMA (black line) were assessed for ROS production through the DHR assay. Bars = 200 μm. DHR, dihydrorhodamine123; PMA, phorbol myristate acetate.