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¹ Research Division, Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215, USA² Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA³ Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

(Correspondence should be addressed to G C Weir; Email: gordon.weir{at}joslin.harvard.edu)

Transdifferentiation of cells from a patient's own liver into pancreatic β-cells could be useful for β-cell replacement. We hypothesized that intrahepatic biliary epithelial cells (IHBECs) could become a new source of insulin-producing cells. IHBECs isolated from adult mice were expanded using our novel culture method termed, collagen-embedded floating culture method (CEFCM). With CEFCM, IHBECs formed three-dimensional ductal cysts and rapidly expanded their number by about 15-fold within 2 weeks. Over 90% of cells were positive for cytokeratin 7 and 19. At day 14, IHBECs were transfected with adenoviral (Ad)- pancreas duodenum homeobox 1 (Pdx-1), NeuroD or Pdx-1/VP16. After 7 additional days in serum- and insulin-free differentiation medium (DM), cell phenotypes were determined by RT-PCR, immunostaining and ELISA for insulin. In DM control IHBECs started to express some endocrine progenitor genes (Neurog3, NeuroD, Nkx6.1, and Pdx-1) but lacked insulin gene (Ins) mRNA. Transduced expression of PDX-1, NEUROD or PDX-1/VP16 led to expression of not only INS but also GLUT2 and prohormone convertase 1 and 2. About 3% of 4000 cells counted in PDX-1/VP16 transduced cultures stained strongly for C-peptide suggesting that a subpopulation may have the capacity for differentiation. Transduced cells released insulin (Ad-PDX-1 0.08±0.05, Ad-NEUROD 0.33±0.09, Ad-PDX-1/VP16 0.37±0.14 ng/1x10⁵ cells after 48 h in culture). IHBECs can be markedly expanded, and then with molecular manipulation a subpopulation of these cells can differentiate towards a β-cell phenotype. This approach may lead to a new source of β-cells that can be used for transplantation in diabetes.

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