Figure 7.

Spheroids predominantly express Bmi1, but express Lgr5 during crypt-bud formation. (A) Experimental paradigm for mAb-mediated (E5D10^lo/F5C12^neg) isolation of Bmi1-enriched intestinal epithelial populations from either Lgr5-GFP, Bmi1-GFP, or Bmi1-CreERT/R-TdT mice. Representative brightfield (left) and corresponding GFP fluorescence images (right) of spheroids generated from FACS-isolated Bmi1-enriched E5D10^lo/F5C12^neg cell population from (B) Lgr5-GFP or (C) Bmi1-GFP reporter mice after 12 and 14 days in culture. White dashed lines represent structure outline. White arrowheads denote regions of crypt-bud restricted GFP expression. Boxed region of day 14 culture corresponds to higher-magnification image of GFP channel. Asterisk denotes autofluorescent cells within the lumen. Data are representative of N = 3 independent experiments, N = 6 mice per genotype. (D) Experimental paradigm (left) and example brightfield (inset) and corresponding TdT fluorescence images of spheroids derived from Bmi1-CreERT/R-TdT (Bmi1-Cre/R-TdT) mice using mAbs E5D10/F5C12 in combination induced on day 2 with 100 nmol/L 4-OHT (left) or vehicle control (right) after 6 days in culture. N = 2 independent experiments with technical replicates, N = 4 mice. FACS analyses were performed on a BD Influx cell sorter at the OHSU Flow Cytometry Core Facility. Culture images were captured on an inverted Leica DMIRB microscope. Scale bar: 200 μm.