Figure 3.

The ductal CD133^highCD71^lowFSC^mid-high fraction self-renews robustly in vitro. (a) Schematic of serial replating strategy to assess self-renewal capacity. (b–c) Cell growth curves from the CD133^highCD71^lowFSC^low (FSC^low) or the CD133^highCD71^lowFSC^mid-high (FSC^mid-high) fraction over time. n = 2 (b) or 3 (c) independent experiments consisting of 4 technical replicates each. Error bars represent SEM. (d) Average number of cells per well over serial replating. (e) Average number of colonies per well over serial replating. (f) Photomicrographs of representative 3-week-old Cystic colonies from the 1st and the 4th generations. (g) Individual colonies were micro-manipulated and gene expression analyzed using microfluidic qRT-PCR for ductal (Ca2, Prom1, Krt19), acinar (Amy2a, Cpa1, Cela1), endocrine (Ppy, Gcg, Ins2, Ngn3), and other markers (Hnf6, Notch2, Pax4, Unc3). Data represent fold-change calculation of gene expression in colonies of the 4th generation compared to the 1st generation. Each data point is a colony. n = 22 individual colonies from each group. Error bars represent SEM. Statistics were performed comparing FSC^mid-high vs. FSC^low using two-tailed Student’s t-test.