FIG. 5.

Whole-mount immunostaining and electron microscopy analyses of individual colonies. Dissociated 7-day-old pancreatic cells were plated in the presence of Matrigel, aECM-scr, or aECM-lam. After 7 days, colonies were collected, fixed, and co-stained with antibodies against markers for (A) ductal (Mucin1) and β-cells (C-peptide), (B) acinar (Amylase) and β-cells (Insulin), and (C) α- (Glucagon) and β-cells (C-peptide). Nuclei were stained with DAPI (blue). The white box in (A) was enlarged to show a C-peptide⁺Mucin1⁻ cell and several C-peptide⁻Mucin1⁺ cells within a cystic colony supported by Matrigel. The green and red boxes in (B) were enlarged to show Insulin⁻Amylase⁺ cells and an Insulin⁺Amylase⁻ cell in the cystic colonies supported by Matrigel. (C) C-peptide⁺Glucagon⁻ and C-peptide⁻Glucagon⁺ cells were detected in colonies supported by aECM-scr or aECM-lam. (D) Ultrastructural analysis by transmission electronmicroscopy of a colony supported by aECM-lam. Dashed lines represent cell membranes. Highlighted area is enlarged on the right, showing dense core, insulin-like granules in one cell and multilobed nucleus in a neighboring cell, indicating a ductal phenotype.