Extended Data Fig. 3: Wnt signaling, TGFβ inhibition, and feeders are required to maintain 2D culture.

(a) Representative phase contrast image of p5 corpus and antrum 2D culture using recombinant Wnt3a and Rspo1 in lieu of conditioned media, scale bars = 50 μm. (b) Representative immunofluorescence images of corpus and antrum 2D culture stained with Epcam following the iterative removal of Wnt signaling from the culture, scale bars = 50 μm. (c) Quantification of corpus 2D culture following iterative removal of Wnt agonists from the culture, staining for Epcam, and measuring colony size. 5 total fields quantified for each replicate, n = 3 biologically independent experiments. *** = p > 0.001 using students 2-sided t-test. (d) 2D culture in the presence or absence of recombinant Jagged1 and HGF were stained for Epcam and the number of Epcam^pos cells, as well as colony size per field, were quantified. 8 total fields quantified, n = 3 biologically independent experiments. *** = p > 0.001 using students 2-sided t-test. (e) A8301 was included or removed from 2D media conditions and proliferation was measured in epithelial cells by Epcam and Mki67 immunofluorescence staining. Representative colony from each condition is shown. (f) 2D culture was moved onto various extracellular matrices and the ability of the culture to expand was monitored. Representative phase contrast images are shown, scale bars, 50 μm. (g) 2D complete media preconditioned on feeders for 72 hours was used to expand 2D culture without the use of feeders. Representative phase contrast images are shown for each passage, scale bars, 50 μm. (h) 2D culture was serially passaged at a 1:2 ratio in the absence of feeders. Representative phase contrast images are shown for each passage, scale bars, 50 μm.