J Korean Diabetes Assoc.  2007 Jan;31(1):44-50. 10.4093/jkda.2007.31.1.44.

Activin A and Glucose Derived Human Pancreatic Ductal Cells into Insulin-producing Cells

• Hong SH
• Han C
• Kim HS
• Park MK
• Lee YJ
• Jeong JH
• Min YK
• Lee MS
• Kim KW
• Lee MK

• KMID: 2008095
• DOI: http://doi.org/10.4093/jkda.2007.31.1.44

Abstract

BACKGROUND: Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Human postnatal pancreatic ductal cells are a potential source of new beta cells. Therefore, we investigated the potential of human pancreatic ductal cells could be differentiated into endocrine cells that would be capable of secreting insulin in response to glucose.
METHODS
Cell fractions enriched with pancreatic ductal cells after human islet isolation were treated with streptozotocin to remove residual beta cells, grown in monolayer culture, changed the media for differentiation in the presence of activin A and glucose, supplemented with 10% FCS. The differentiation markers, insulin secretion and cell proliferation were examined. RESULT: No insulin was detectable in cell preparations after 5 days of treatment with streptozotocin. In monolayer culture, 80% of the streptozotocin-treated pancreatic ductal cells expressed cytokeratin-19. Cell cultures with a high proportion of cytokeratin-19 cells had greater plasticity for differentiation into cells with phenotypic and functional markers of beta cells. This property were significantly enhanced by treatment of activin A and glucose. The differentiated human pancreatic ductal cells secreted insulin sensitively responded with high glucose.
CONCLUSION
Human pancreatic ductal cells are a potential source of new glucose -induced insulin producing cells that may be developed further for clinical use. Therefore, the present data support a possible role for human adult pancreatic ductal cells, following expansion and differentiation, as a source of insulin by transplantation cells to type I diabetes patients.

Keyword

Activin A; Ductal cell

MeSH Terms

Activins*
Adult
Antigens, Differentiation
Cell Culture Techniques
Cell Proliferation
Diabetes Mellitus
Endocrine Cells
Glucose*
Humans*
Insulin
Keratin-19
Pancreatic Ducts*
Plastics
Streptozocin
Tissue Donors
Activins
Antigens, Differentiation
Glucose
Insulin
Keratin-19
Plastics
Streptozocin

Figure

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  Fig. 1 Removal of beta cell from pancreatic ductal cell. A, Reduction of insulin mRNA during STZ treatment (n = 4). Variation of insulin mRNA expression of STZ-treated ductal cell compaired with STZ-nontreated ductal cell; B, RT-PCR showing preproinsulin and CK19 mRNA expression in the cells following STZ treatment; C, Cell constitution after STZ treatment. *P < 0.05 vs. STZ nontreated ducatal cell.

• 

  Fig. 2 Morphological charateristic of pancreatic ductal cell. A, Complete Monolayer after STZ treatment for 5 days; B, Cluster formation after activin A and glucose treatment for 30 days.

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  Fig. 3 Effect of activin A on the differentiation of ductal cell. A, The mRNA expression of differentiation marker of activin A and glucose treated or nontreated ductal cells for 30 days; B, To characterize the cells of dispersed duct cell, triple immuno-staining with CK19, inslulin and DAPI was performed. Duct cells stained with CK19 (red). Beta cells stained with insulin (green) b, STZ treated ductal cell for 5 days. c, Activin A and glucose treated ductal cell for 15 days after STZ treatment. d, Activin A and glucose treated ductal cell for 30 days after STZ treatment; C, The mRNA expression of insulin and preproinsluin during differentiation.

• 

  Fig. 4 Variation of glucose-induced insulin secretion during differentiation. *P < 0.05 vs. glucose 3 mM.

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  Fig. 5 Variation of cell growth during differentiation. A, Doubling times were calculated using the Schwartz equation; B, The proliferation rates were assayed by the rate of BrdU incorporation.

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