Generation and phenotypic characterization of iPSC-derived BRAF^V600E/WT cells differentiating toward the monocytic lineage. (A) PBMCs of a patient with Langerhans cell histiocytosis (LCH) were reprogrammed into induced pluripotent stem cells (iPSCs) and then edited via CRISPR-associated protein 9 (Cas9) to introduce the V600E mutation in 1 endogenous BRAF allele as shown by Sanger sequencing. A synonymous mutation was introduced as part of the editing to prevent recutting by Cas9. A second iPSC line (ShiPS-miFF3) derived from a healthy infant was edited in similar way with Cas9 to introduce the V600E mutation in 1 endogenous BRAF allele. (B) Schematic of iPSCs differentiation toward CD14⁺ iMonocytes using STEMdiff Monocyte kit: a monolayer of iPSCs colonies is first differentiated toward mesoderm, then toward iHPCs, and finally toward CD14⁺ iMonocytes. First microscopy image on the left was acquired with a 5× air objective at room temperature on a Zeiss Axio Vert.A1 microscope; the remaining microscopy images were acquired with 20× objective with the same setup; scale bar, 100 μm. Images were processed with ImageJ. (C) Density plots of iPSCs at day 13 to 14 of monocytic differentiation showing smaller CD33⁻/CD34⁺ and CD33⁻/CD34⁻ fractions for mutated than WT cells (n = 9). (D) BRAF^V600E/WT iPSCs express CD14 at day 10 of differentiation, as shown by flow cytometry analysis (n = 5). (E) More BRAF^V600E/WT iPSCs-derived CD14⁺ cells coexpressed CD11c than their WT counterpart as shown by flow cytometry analysis at day 13 of iMonocyte differentiation (n = 6). (F-G) BRAF^V600E/WT develop unique CD14^low/CD1c⁺ (n = 7) and CD1a⁺ (n = 8) populations that are missing in the WT isogenic control while they differentiate into iMonocytes as shown by flow cytometry density plots. (H) Schematic of developmental trajectory experiment. BRAF^V600E/WT iPSCs differentiating toward iMonocytes were sorted in 4 fractions via fluorescence-activated cell sorting (FACS): (1) CD14⁻/CD1c⁻, (2) CD14^low/CD1c⁻, (3) CD14^high/CD1c^int, and (4) CD14^low/CD1c⁺. Each fraction was then cultured separately from the others for 4 days after which cells were restained and analyzed as shown in panel I and J. (I) Density plot showing the 4 fractions sorted in panel H analyzed after 4 days of culture: CD14⁻/CD1c⁻ can reconstitute all other fractions but developmental potential decreases as cells differentiate toward CD14^low/CD1c⁺ cells. (J) Same cells shown in panel I were also stained and gated for CD1a and CD207 expression to demonstrate that CD14^low/CD1c⁺ fraction is enriched of CD1a⁺/CD207⁺ iLCH cells that derive from a CD14⁺ precursor and have a similar surface marker expression to lesional LCH cells. (K) Representative FACS plot of BRAF^V600E/WT CD14^low/CD1c⁺, sorted cells as in panel I and cultured for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF-α, and transforming growth factor β1 (TGF-β1). (L) Quantification of CD1a⁺/CD207⁺ iLCH cells (n = 4) obtained from cells sorted as in panel I and cultured for 7 days with GM-CSF, TNF-α, and TGF-β1. For statistical significance of panels C-G unpaired parametric Student t test was used with Welch correction when variance was significant between groups, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. All histograms except in panel L include replicates from patient-derived iPSCs as well as ShiPS-MIFF3 iPSCs cell line. iPSC, induced pluripotent stem cells; LCH, Langerhans cell histiocytosis.