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Abstract
Background
Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. This has led to a search for alternative sources of transplantable insulin-producing cells including pancreatic duct cells. We aimed to establish in vitro culture of pancreatic duct cells from a partial pancreas tissue in human, which could be harnessed to differentiate into pancreatic β cells.
Methods
We isolated pancreatic duct cells from small pieces of pancreas tissue (1~3 g) derived from non-diabetic humans (n = 8) undergoing pancreatic surgery due to cancer. Pancreas tissue was finely minced after injection of collagenase P into the parenchyma. The mince was incubated in a shaking water bath at 37℃ for 25 min and passed through a 150 µm mesh. The released cells were recovered, washed, and plated in a dish containing CMRL culture medium with serum.
Results
Isolated pancreatic cells grew in monolayer and became confluent in 1~2 wks showing typical epithelial cobblestone morphology. Immunochemistry demonstrated that ~90% of the cultured cells were cytokeratin7-positive duct cells. To induce β cell differentiation, the cells were incubated in DMEM/F12 culture medium without serum. In addition, treatment with Matrigel overlay, exendin-4, cholera toxin or forskolin was done. Though β cell differentiation was found by immunostaining and RT-PCR, the differentiation efficiency was very low. Over-expression of neurogenin-3 by recombinant adenovirus did not increase β cell differentiation of the cultured duct cells significantly.
Figures and Tables
Fig. 3
Cytokeratin 7 (CK7) immunostaining. A, human pancreas (×100); B, human pancreas (×200); C, Isolated pancreatic cells; D, Cultured pancreatic cells 4 days after isolation.
Fig. 4
Double immunostaining of CK7 and insulin in pancreatic duct cells cultured in differentiation medium for 1 week.
Fig. 5
Matrigel overlay on pancreatic duct cells cultured in differentiation medium for 4 week. A, B, morphology under inverted microscope; C, CK7 staining; D, insulin staining.
Fig. 6
RT-PCR of insulin mRNA from pancreatic duct cells 3 days after treatment of forskolin (FK), exendin-4 (Ex4) or cholera toxin (CT).
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