CerS4 regulates HFSC differentiation in a stem cell–autonomous manner. (A) Outline of HFCS organoid generation. (B) qRT-PCR analyses of Cers4 expression of FACS purified CD34⁺ α6⁺ integrin HFSCs and CD34⁻ integrin α6⁺ non-HFSCs from organoids (n = 5 independent experiments). (C) Representative brightfield images and quantification of the size and loss of cohesion of control (Ctr) and CerS4^epi−/− organoids cultured for 5 days. Scale bar 50 μm. (D) FACS-based quantification CD34⁺ integrin α6⁺ HFSCs from organoids cultured for time points indicated show reduced HFSCs in CerS4^epi−/− organoids (n = 4 mice/genotype for 14 days; six mice/genotype all other time points; mean ± SD; unpaired t test). (E) Quantification of EdU-positive cells after a 24-h chase of control and CerS4^epi−/− organoids cultured for indicated time points (n = 6 mice/genotype; mean ± SD; unpaired t test). (F) Volcano plot of differentially expressed proteins in control and CerS4^epi−/− organoids (n = 4 mice/genotype). (G) GO-term enrichment analyses of differentially expressed proteins in CerS4^epi−/− organoids. (H) GSEA of the differential protein expression of Ctr and CerS4^epi−/− organoids indicate underrepresentation of proteins from gene expression signatures from OB and overrepresentation of proteins from uHF signatures (from Joost et al., 2016) in CerS4^epi−/− organoids. (I) Representative images and quantification of control and CerS4^epi−/− organoids stained for keratin-6 (grey) and keratin-14 (magenta) show increased keratin-6 and abundance of loosely attached, differentiating cells in CerS4^epi−/− (scale bar 20 µm; n = 8 organoids/genotype; mean ± SD; unpaired t test). NES, normalized enrichment score.