Pdx-1 or Pdx-1-VP16 protein transduction induces β-cell gene expression in liver-stem WB cells

WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. β-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into β-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and β-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein.These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for β-cell engineering for diabetes cell therapy.The difficulties encountered in obtaining sufficient supply of transplantable β-cells is a major problem in cell therapy of type I diabetes. Liver may be a potential source of cells for β-cell engineering. Indeed, liver and pancreas derive from the same endodermal region during embryogenesis [1] and hepatocytes and β-cells share similar built-in glucose-sensing systems.Among transcription factors involved in pancreatic β-cell specification, Pdx-1 plays a central role. All progenitors of the endocrine as well as the exocrine pancreas express Pdx-1 [2,3]. In the adult, Pdx-1 expression is mainly restrained to β-cells where it regulates important β-cell functions like insulin transcription. Several in vitro studies, using viral or stable plasmid gene transfer, show that Pdx-1 expression in hepatic cells results in reprogrammation into insulin producing cells [4-7]. Fusion of Pdx-1 to the VP16 activation domain from Herpes simplex virus (Pdx-1-VP16) leads to more efficient liver-to-pancreas conversion than Pdx-1 alone [8-12]. Stable mouse Pdx-1 or Pdx-1-VP16 gene transfection initiates conversion of rat epithelial liver stem-like WB cell line into pancreatic endocrine precursor cells [13,14]. In these cell