Adult mouse intrahepatic biliary epithelial cells induced in vitro to become insulin-producing cells

Abstract

Transdifferentiation of cells from a patient's own liver into pancreatic b-cells could be useful for b-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 . 08G0 . 05, Ad-NEUROD 0 . 33G0 . 09, Ad-PDX-1/VP16 0 . 37G 0 . 14 ng/1!10 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 b-cell phenotype. This approach may lead to a new source of b-cells that can be used for transplantation in diabetes.

Nkx2.2 repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming

“…In addition, recent studies have exploited the known islet transcriptional networks to transdifferentiate nonpancreatic or exocrine tissue into endocrine cells. Ectopic expression of different combinations of pancreatic transcription factors-including Pdx1, Ngn3, NeuroD, MafA, and Nkx6.1-promotes liver-to-pancreatic b-cell reprogramming Kojima et al 2003;Song et al 2007;Delisle et al 2009;Nagaya et al 2009;Gefen-Halevi et al 2010). Similarly, ectopic expression of a defined set of transcription factors in adult pancreatic acinar cells results in an acinar-to-b-cell conversion (Zhou et al 2008).…”

Pancreatic progenitors: The shortest route to restore islet cell mass

“…41 Interestingly, nestin positive postnatal mesenchymal stem cells of non-pancreatic origin also have the potential to generate functional islets which could reverse experimental diabetes in mice upon transplantation. 15 Nagaya et al 42 demonstrated for the first time that a sub-population of intrahepatic biliary epithelial cells (IHBECs) can be induced to a β-cell like phenotype. Differentiation of dental pulp stromal cells to islet-like cell aggregates (ICAs) has been alternate strategy…”

Perspectives of Cell Therapy in Type 1 Diabetes

“…It has been recently shown that adult human pancreas contains rare multipotent stem cells that express insulin ). Several studies demonstrate that the  cell phenotype can be induced both in vitro and in vivo by transfection of pancreatic transcription factors into the liver that develops from the same embryological origin of pancreas (Kojima et al, 2003;Lemaigre & Zaret, 2004;Nagaya et al, 2009). The potential of bone marrow derived MSC has also been explored.…”