Fig 1: Pre-pubertal Lgr5high uterus cells behave as Wnt-regulated stem/progenitor cells in ex vivo organoid culture assays. a Representative image of a developing uterine organoid generated from a single EPCAM+GFPhigh cell isolated from a P14 Lgr5-2A-EGFP uterus. b H&E and IHC for K8 on a Lgr5high cell-derived organoid. c Endogenous EGFP fluorescence on a Lgr5high cell-derived organoid. Yellow arrowheads highlight the heterogenous expression of Lgr5-EGFP within individual organoids. Scale bars, 50 µm. d Organoids generated from single EPCAM+GFPhigh cells and EPCAM+GFPneg cells (image representative of five replicates). Scale bars, 1000 µm. e Quantification of organoid formation efficacy from single EPCAM+GFPhigh and EPCAM+GFPneg cells. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test. *** P < 0.001. f Passage frequency of organoids derived from single EPCAM+GFPhigh and EPCAM+GFPneg cells. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (P = 7E-07). g Experimental strategy to ablate Lgr5+ cells in vitro. h Organoid cultures derived from Lgr5-DTR-EGFP and wild-type (control) EPCAM+ uterus cells following Lgr5+ cell ablation by DT treatment in vitro (image representative of five replicates). Scale bars, 200 µm. i Outgrowth efficiency of organoids derived from Lgr5-DTR-EGFP and wild-type mouse EPCAM+ uterus cells following DT treatment in vitro. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (P = 4E-06). j Organoids derived from wild-type EPCAM+ uterus cells cultured under low Wnt3a and low Wnt3a + CHIR conditions (image representative of five replicates). Scale bars, 200 µm. k The average proportions of organoid types under low Wnt3a, low Wnt3a + CHIR and high Wnt3a conditions. Data from five independent experiments are presented. Data were tested for significance by a chi-square test (Low Wnt3a vs. Low Wnt3a + CHIR: P = 2E-17, Low Wnt3a vs. High Wnt3a: P = 2E-08). l QPCR analysis of Lgr5 and Fzd10 expression on organoids generated under each culture condition. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (Lgr5: Low Wnt3a vs. Low Wnt3a + CHIR: P = 0.002, Low Wnt3a vs. High Wnt3a: P = 0.012, Fzd10: Low Wnt3a vs. Low Wnt3a + CHIR: P = 0.018, Low Wnt3a vs. High Wnt3a: P = 0.027). m QPCR analysis of GE markers (Foxa2, Lif, Prss28) and LE markers (Wnt7a, Scnn1a, Irg1) expression on organoids generated under each culture condition Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (Foxa2: P = 9E-04, Lif: P = 0.006, Prss28: P = 0.54, Wnt7a: P = 0.46, Scnn1a: P = 0.49, Irg1: P = 5E-06). n, o Immunostaining for Foxa2, Lif, and Ki67 on round and vacuolated-type organoids. Scale bars, 50 µm. Red arrows indicate Foxa2-positive cells (image representative of three independent mice). ***P < 0.001, **P < 0.01, *P < 0.05, N.S., not significant.