Figure 1. Single fallopian epithelial cells give rise to organoids that can be maintained long term.

(a) Representative confocal image of fallopian tube tissue labelled for acetylated tubulin, a marker of ciliated cells (red), the proliferation marker Ki67 (green) and DNA (DRAQ5, blue). Ki67-positive nuclei are dispersed along epithelial folds, with occasional clustering (asterisk). (b) Phase contrast images of spheroid formation and growth. Small spheres are already visible 1 day after seeding and expand to reach a diameter of over 100 μm within 7 days. The same culture shows no apparent morphological differences after 2 weeks and after 10 months in vitro. (c) Phase contrast microscopy images of focal planes inside mature organoids showing the epithelial invaginations and folding (arrows), which are routinely present but not visible in most images focused on the organoid surface. (d) Representative image of organoids generated with independently labelled GFP and mCherry epithelial isolates showing almost exclusively single-colour organoids. Quantification of the organoids from two independent experiments and donors (table) reveals that the percentage of hybrids is on average below 1%. (e) Time course of a monoclonal organoid generated from a single EpCAM+ cell sorted and embedded in Matrigel in a 96-well format. Monoclonal organoid cultures were successfully generated from four different donor isolates, confirming the presence of stem cells in the fallopian tube epithelium. (f) Confocal microscopy image of a monoclonal organoid generated from a single fallopian tube stem cell after 2 months of 3D culture (P3), labelled for b-Cat (red) and tubulin, showing ciliated and non-ciliated cells. (g) Confocal images of early organoid cultures at different time points labelled for b-Cat (red) and ac tubulin (green), revealing that cilia develop after 2 weeks in 3D culture.