Transcriptomic characterization of iPSC-derived CD1a⁺/CD207⁺ iLCH cells using scRNA-seq reveals similarity to LCH subsets found in lesions. (A) BRAF^V600E/WT CD14^low/CD1c⁺ were cultured with GM-CSF, TNF-α, and TGF-β1 to generate iLCH cells. Living CD1a⁺/CD207⁻ and CD1a⁺/CD207⁺ cells were sorted and used for scRNA-seq. (B) UMAP of 8483 sequenced cells from 2 independent experiments. (C) Feature plot showing the expression of CD1A and CD207 in the data set. (D) Violin plots showing that iLCH cells are heterogeneous and express markers defining different subsets of lesional cells in LCH biopsies. Cluster iLCH_5 expresses proliferative markers (HMMR, CDK1, and MKI67) similar to high-entropy LCH subsets, whereas cluster iLCH_7 expresses IFI6, IFIT3, and ISG15, similar to low-entropy LCH subsets.⁶⁵ (E) UCell⁶⁶ scoring was created using gene lists from 3 different data sets: (1) GSE173923, top100 makers (using FindMarkers function in Seurat) of LCH cell cluster, of Kvedaraite et al¹⁹; (2) GSE133706, 77 marker genes distinguishing LCH from non-LCH immune cells described in Table S2 of Halbritter et al⁶⁵; and (3) GSE35340 884 genes that are differentially expressed in LCH vs LC described in Hutter et al²⁰ (and listed as supplemental File 1 in Schwentner et al¹⁸). The results show that iLCH cells are much closer to lesional LCH cells than to physiological LCs (E-MTAB-8142). (F) Projection of iLCH on a scRNA-seq data set composed of healthy LCs (E-MTAB-8142) and tumoral LCH cells from biopsies.⁶⁵ Overall, 99% of iLCH cells project on the LCH data set and only 1% on the subset of healthy epidermal LCs. (G) Dot plot of several transcripts expressed in LCH vs LC cells showing similar expression profiles between iLCH and LCH cells.