Figure 3.

Isolation and in vitro expansion of single, endocrine-depleted pancreatic epithelial cells. (A) Representative fluorescence-activated cell sorting (FACS) plot illustrating the distribution of EpCAM⁺ and EpCAM⁻ cells from dissociated adult mouse pancreas, following epithelial cell enrichment by magnetic beads as described in Materials and methods. (B) Representative FACS plot showing the distribution of EpCAM⁺ non-granulated TSQ⁻ epithelial cells and EpCAM⁺ granulated TSQ⁺ endocrine cells. (C) EpCAM⁺TSQ⁺ and EpCAM⁺TSQ⁻ sorted fractions were cytocentrifuged and immunostained (red) for Synaptophysin (SYP), Amylase (AMY) and pancytokeratin (CK); nuclei were counterstained with Hoechst 33342 (blue). Magnification: × 40. (D) Representative FACS analysis purity of sorted EpCAM⁺TSQ⁻ cells indicating that this population is isolated with high purity (>99.6%). (E) EpCAM⁺TSQ⁻-sorted single cells were assessed for their growth potential in 3D expansion culture conditions: this population gave rise to organoids that could be expanded for many passages (>5 months). (F) EpCAM⁺TSQ⁺-sorted single cells were assessed for their growth potential under the same conditions: endocrine TSQ⁺ cells survived in culture but did not proliferate. Scale bars: 30 μm.