Epidermal Growth Factor Increases Undifferentiated Pancreatic Embryonic Cells In Vitro
A Balance Between Proliferation and Differentiation
Article Figures & Tables
Figures
- FIG. 1.
Effect of the inhibitor of the MAP kinase pathway PD98059 on cell proliferation and differentiation. Pancreatic epithelia were grown in culture for 3 days in the absence or presence of PD98059 (50 μmol/l). A: Immunostaining for BrdU (red) and insulin plus glucagon (green) in controls (DMSO) and PD98059-treated rudiments. B: Quantification of the size and number of BrdU-positive cells and the cell surface area occupied by insulin/glucagon−positive cells that developed during 3 days in the presence of PD98059 when compared with control rudiments grown for the same period in the absence of PD98059. The sizes of the epithelia and total surface of insulin/glucagon−positive cells were also determined before culture. Three rudiments were analyzed for each condition. Data are means ± SE. *P < 0.05.
- FIG. 2.
Effect of EGF on the size of pancreatic epithelia in culture. e13.5 pancreatic epithelial rudiments grown in culture for 7 days with or without EGF. A: Photographs of the epithelia before and after 7 days of culture. The pictures were taken at the same magnification. Left panel: an epithelium before culture; middle panel: an epithelium grown for 7 days in control conditions, with endocrine budding visible (arrows); right panel: an epithelium grown for 7 days with EGF. B: Quantification of the total size of the epithelial rudiments before and after 7 days in culture in the absence or in the presence of EGF. The surface of all sections prepared for immunohistochemistry were measured and areas of all sections were added. Seven rudiments were analyzed for each condition. Mean values in μm2 ± SE are shown on the graph. **P < 0.005.
- FIG. 3.
Immunohistological analysis of pancreatic epithelium grown in vitro during 7 days with or without EGF. Pancreatic epithelia were grown for 7 days without or with EGF. They were sectioned and consecutive sections were stained for amylase or insulin and glucagon. A: Superimposition of staining from adjacent sections for amylase (green), insulin (red), and glucagon (blue, artificial color) in epithelia developed without (control) or with EGF. B: Absolute surface areas in μm2 occupied by insulin- (left) and glucagon-expressing (right) cells in rudiments grown for 7 days with or without EGF. Six to seven rudiments were analyzed for each condition. **P < 0.005.
- FIG. 4.
Characterization of the cells developed in the presence of EGF that stain negative for endocrine and acinar markers. A and B: Pancreatic epithelia were grown for 7 days in the absence (A) or presence of EGF (B). Consecutive sections were analyzed by immunohistochemistry for amylase (green), cytokeratin (red), insulin plus glucagon (blue, artificial color), and vimentin (yellow, artificial color). The images were next superimposed. Note that in EGF-treated rudiments, nearly all cells that stained negative for endocrine and acinar markers expressed high levels of cytokeratin. Moreover, very few cells stained positive for vimentin, either in the presence or absence of EGF. C−E: Pancreatic epithelia were grown for 7 days without (C) or with (D and E) EGF. Double-staining for cytokeratin (red) and BrdU (green). The picture presented in (E) was obtained after confocal microscopy.
- FIG. 5.
Epithelial cells that developed during the first week of culture in the presence of EGF differentiated into insulin-expressing cells when EGF was removed. A: Pancreatic epithelia were grown for 7 days with (a and b) or without EGF (c and d). The rudiments were either fixed (a and c) or grown for an additional week in the absence of EGF (b and d). The rudiments were sectioned and analyzed by immunohistochemistry for insulin. B: Quantification of insulin-positive cell mass that developed in each condition. An 8.93-fold (P < 0.005) increase in the insulin-positive cell mass occurred during the second week of culture when EGF was removed at day 7. Moreover, at day 14, more insulin-expressing cells developed (P < 0.05) when the rudiments were grown during the first week with EGF and during the second week in the absence of EGF when compared with rudiments grown 2 weeks in the absence of EGF. Six to seven rudiments were analyzed in each condition. *P < 0.05; **P < 0.005; NS, not significantly different.
- FIG. 6.
Insulin-expressing cells that developed after EGF removal derived from cells that proliferated when EGF was present. Pancreatic epithelia were grown for 7 days with EGF and pulsed with BrdU (10 μmol/l) during the last 6 h. They were either fixed (A) or kept in culture without BrdU during an additional week in the absence of EGF (B) and finally fixed. They were sectioned and immunohistochemistry for insulin (green) and BrdU (red) was performed. Although insulin-expressing cells present at day 7 stained negative for BrdU, some insulin-expressing cells found at day 14 stained positive for BrdU (white arrows), indicating that such insulin-expressing cells differentiated during the second week of culture in the absence of EGF from cells that were proliferating when EGF was present.
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