Fig. 6

Patient-specific mutations in iPSC-derived neutrophils recapitulate the patient’s phenotype.

A Microscopy image of the patient’s bone marrow aspiration. Myelocyte, arrow. B Number of floating cells released from 6 iPSC colonies per well at indicated days of differentiation from indicated cell lines. n = 3 technical replicates per cell lines (1 VPS18^+/+, 2 VPS18^+/-, 2 VPS18^Arg234*, 1 VPS18^-/- cell lines). Mean ± SEM. ****P < 0.0001 compared to VPS18^+/+. C Quantification of viable, preapoptotic, early and late apoptotic cells of indicated genotypes at day 28 of differentiation in % (100%, sum of all detected single cells) analyzed by flow cytometry. n ≥ 1. Data presented as mean. D Quantification of neutrophil-lineage directed cells (CD45⁺Siglec⁻) from indicated cell lines at day 28 analyzed by flow cytometry. n ≥ 6. Mean ± SEM. *P < 0.05, ***P < 0.001 compared to VPS18^+/+. E Annexin V⁺ cells within populations of proNeu1 (CD11b^-, CD49d⁺, SSC-A^low), proNeu2 (CD11b^-, CD49d⁺, SSC-A^high), preNeu (CD49d⁺, CD101^-), immature (CD35⁺, CD16⁻) and mature (CD35⁺, CD16⁺) neutrophils from indicated cell lines at day 28 in % (100%, sum of all single cells) analyzed by flow cytometry. n ≥ 6. Mean ± SEM. *P < 0.05, **P < 0.01 compared to VPS18^+/+. F Representative Western blots of VPS11, VPS16 and VPS33A (upper panel) & LC3B expression (lower panel) in cell lysates from indicated cell lines at day 28. β-actin was used as loading control. n = 2. B–E Two-way ANOVA, Tukey’s multiple comparisons test.