Figure 5

Olfm4/eGFP+ adult mouse prostate cells formed colonies, spheres, or organoids in culture. (a) Representative graph from FACS-based cell-sorting analysis of Olfm4/eGFP+ cells isolated from whole adult prostate from Olfm4^eGFP mice. Data represent mean ± SD of 3 independent experiments. (b) Representative graph from FACS analysis of Olfm4/eGFP/CD44+ and Olfm4/eGFP/CD49F+ cells isolated from whole adult prostate from Olfm4^eGFP mice. Data represent mean ± SD of 3 independent experiments. (c) Representative cell-growth images of Olfm4/eGFP+ or Olfm4/eGFP- cells sorted following single-cell preparation of whole adult Olfm4^eGFP mouse prostate. After sorting, cells were grown in 2D culture for 7 days. Experiments were repeated 3 times. Scale bars: 200 μm. (d) Representative cell-growth images of Olfm4/eGFP+ and Olfm4/eGFP- cells sorted following single-cell preparation of whole adult Olfm4^eGFP mouse prostate. After sorting, cells were subjected to prostate sphere-formation assays for 5 and 14 days. Experiments were repeated 3 times. GFP, GFP filter; Light, light field. Scale bar: 20 μm. (e) Representative cell-growth images of Olfm4/eGFP+ cells sorted following single-cell preparation of whole adult Olfm4^eGFP mouse prostate. After sorting, cells were subjected to prostatic-organoid culture for 4 and 7 days. Experiments were repeated 3 times. GFP, GFP filter; Light, light field. Scale bar: 10 μm. (f) Representative cell-growth images of Olfm4/eGFP+ and Olfm4/eGFP- cells sorted following single-cell preparation of whole adult Olfm4^eGFP mouse prostate. After sorting, cells were subjected to prostatic-organoid culture for 2 and 7 days. Experiments were repeated 3 times. Scale bars: 20 and 200 μm. (g) Olfm4/eGFP+ and Olfm4/eGFP- cells were sorted following single-cell preparation of whole adult Olfm4^eGFP mouse prostate. After sorting, cells were subjected to prostatic-organoid culture for 7 days. Left, representative images of H&E staining in Olfm4/eGFP+ cell- and Olfm4/eGFP- cell–formed organoids. Right, representative merged images from double-color or triple-color immunofluorescent staining in Olfm4/eGFP+ cell- and Olfm4/eGFP- cell–formed organoids with Olfm4, Ck8, and Ck5 antibodies, with Ck19, p63, and Ck5 antibodies, or with androgen receptor and Ck5 antibodies. Experiments were repeated 3 times. Ar, androgen receptor. Scale bars: 20 μm. Blue represents DAPI nuclear staining. (h) Quantitation of the p63+ cells in Olfm4/eGFP+ cell- and Olfm4/eGFP- cell–formed large organoids. Data were obtained from analysis of single-color images from immunofluorescent staining with P63 antibody using the 3D objects counter function (Image J) for each individual organoid. Olfm4/eGFP+ cell- (n = 4); Olfm4/eGFP- cell–(n = 6). P < 0.01 (two-tail student T test).