Fig. 3: Distinct fibroblast populations show differences in morphogenetic and adipogenic capacity.

a UMAP representations and violin plots showing the expression of Entpd1 (CD39 gene), Ly6a (SCA1 gene) and Dpp4 in mammary fibroblasts. b Representative images showing the expression of CD39 and SCA1, alongside αSMA and COL1A1, in mammary gland regions containing ducts, TEBs or distal fat pad. The insets show details on fibroblasts. The dashed yellow line demarcates the epithelial compartment, which is indicated by *. Scale bars: 100 µm, 10 µm in detail. c Quantification of marker positive stromal cells in different regions of the mammary gland, shown as box plots; the dots indicate single fields of view (FOV), n = 3 biological replicates, N = 68 FOVs for CD39 and 55 FOVs for SCA1. Statistical analysis: Wilcoxon test, two-sided. d Representative FACS plots separating mammary fibroblasts into CD39+; SCA1+ DPP4^neg and SCA1 + DPP4+ populations. e–g Co-culture of sorted fibroblasts with mammary epithelial organoids. e Representative images of organoids after 5 days of co-culture with FACS-sorted fibroblasts (organoids in cyan, fibroblasts in red). Scale bars: 100 µm. f Quantification of the percentage of budding organoids, shown as box plots. Each dot represents a biological replicate, lines connect paired experiments; n = 3 independent experiments. Statistical analysis: Student’s t-test, two-sided. g Quantification of the number of buds formed per organoid, where each dot represents one organoid, and the dot colours code paired experiments; n = 3 independent experiments, N = 91, 40 and 44 organoids for CD39+, DPP4^neg and DPP4+ population, respectively. Statistical analysis: Student’s t-test, two-sided. h–j Adipogenesis assay on sorted fibroblasts. h Representative images of FACS-sorted fibroblasts after 11 days of culture in minimal or complete adipogenic medium in 2D (on plastic) or 3D (in Matrigel). Scale bars: 50 µm. i, j Quantification of adipogenic differentiation in 2D (i) and 3D (j) cultures, shown as box plots. Each dot represents a biological replicate; n = 3 independent experiments. Statistical analysis: Student’s t-test, two-sided.