Figure 4.

Clonal expansion of single Lgr5 cells derived from Lgr5-LacZ pancreatic organoids. (A–E) Lgr5⁺ cells were sorted from Lgr5^LacZ-derived pancreas organoids cultured for 20–25 days in our defined medium. Organoid formation efficiency was analysed 12 days after seeding. (A) Representative image of an FACS plot of wild-type (left) and Lgr5^LacZ (right) pancreas organoids stained with Detectagene Green CMFDG (for detecting beta-galactosidase expression) and EpCAM (for selecting epithelial cells). (B) Representative image of cultures derived from 500 sorted cells from the high (Lgr5^hi) or 500 sorted cells derived from the negative fraction (Lgr5^neg) 12 days after seeding. (C) Graph showing the % of colony formation efficiency of the Lgr5^hi and Lgr5^neg fractions. (D) Representative serial DIC images showing the outgrowth of pancreatic organoids originated from a single Lgr5^LacZ+cell. Magnifications: × 40 (days 1–2), × 20 (day 7), × 10 (days 12–20), × 4 (2 months). (E) Representative DIC image of XGAL staining in a 12-day-old clonal culture derived from Lgr5^hi fraction. Magnification: × 10.