Journal: Stem Cells Translational Medicine

Article Title: Neutrophils Derived from Genetically Modified Human Induced Pluripotent Stem Cells Circulate and Phagocytose Bacteria In Vivo

doi: 10.1002/sctm.18-0255

Figure Lengend Snippet: Analyses of reactive oxygen species (ROS), phagocytosis, and neutrophil extracellular traps (NET) formation of human PB and induced pluripotent stem cells (iPSC) derived neutrophils. (A): FACS histogram plots showing fluorescence emission resulting from the oxidation of cell permeant reagent 2′,7′‐dichlorofluorescin diacetate (DCFDA) by intracellular ROS to 2′,7′‐dichlorofluorescin (DCF) in healthy donor PB and iPSC‐derived neutrophils. (B): Quantification of ROS levels in PB and iPSC‐derived neutrophils as measured by mean fluorescence intensity (MFI) of DCF. (C): FACS histogram plots showing the phagocytosis of pH sensitive fluorescent Escherichia coli (pHrodo‐ E. coli ) by PB and iPSC‐derived neutrophils. (D): Quantification of E. coli phagocytosis as measured by the percentage of E. coli + neutrophils. (E): Quantification of E. coli phagocytosis as measured by ratio of the MFI of control (DMSO) and E. coli treated neutrophils. (F): Confocal microscopic images of PB and iPSC‐derived neutrophils treated with phorbol 12‐myristate 13‐acetate (PMA) to induce NET formation. (G): Quantification of chromatin spreading (maximum length) during NET formation as measured by confocal microscopy. (H): FACS histogram plots showing G‐CSF induced activation of AKT (phospho‐AKT‐S473) in PB and iPSC‐derived neutrophils. (I): Quantification of AKT activation as measured by the ratio of the MFI of Alexa Fluor 647‐anti‐pAKT and Alexa Fluor 647‐control IgG. Scale bars = 20 μm data are presented as mean ± SD of a minimum of two independent experiments. *, p < .05; **, p < .01; ***, p < .001.

Article Snippet: After 4 days of culture, 15–20 EBs (starting from 1 to 2 × 10 6 undifferentiated iPSC) were transferred to each well of a 6‐well culture dish and cultured in StemDiff APEL 2 culture media (Stem Cell Technologies) supplemented with 25 ng/ml IL‐3 (Peprotech) and 50 ng/ml G‐CSF (Peprotech).

Techniques: Derivative Assay, Fluorescence, Confocal Microscopy, Activation Assay