Adult mouse intrahepatic biliary epithelial cells induced in vitro to become insulin-producing cells.
Transdifferentiation of cells from a patient's own liver into pancreatic beta-cells could be useful for beta-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/1x10(5) 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 beta-cell phenotype. This approach may lead to a new source of beta-cells that can be used for transplantation in diabetes.
Nagaya
Masaki
M
Research Division, Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215, USA.
Katsuta
Hitoshi
H
Kaneto
Hideaki
H
Bonner-Weir
Susan
S
Weir
Gordon C
GC
D000256:Adenoviridae / Q000235:genetics; D000367:Age Factors; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001653:Bile Ducts, Intrahepatic / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D054338:Cell Transdifferentiation / Q000235:genetics*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D020869:Gene Expression Profiling; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
D051792:Basic Helix-Loop-Helix Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D018398:Homeodomain Proteins; D007328:Insulin; D009419:Nerve Tissue Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; C093564:Neurogenic differentiation factor 1