Abstract
Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells.
Highlights
In the last years, several attempts to reprogram liver cells into pancreatic endocrine cells have been proposed [1,2,3]
The main purpose of our study was to evaluate the capability of recombinant Pdx1 to induce the differentiation of a population of normal human Biliary Tree Stem/progenitor Cells (hBTSCs) toward β-pancreatic cells [6,7,8,9,10,11]
Demonstration of equal quantities of Pdx1 uptake and appropriate localization of Pdx1 in hBTSCs and HepG2 cells have been obtained by both WB and IF. This excludes the possibility that failure of Pdx1 to induce HepG2 cell pancreatic differentiation was caused by its impaired uptake
Summary
Several attempts to reprogram liver cells into pancreatic endocrine cells have been proposed [1,2,3]. In a recent study by Banga et al [5] a strategy to drive liver cell toward pancreatic endocrine cells through genetic reprogramming of Pdx expression, culminated in the expression of pancreatic islet markers within glandular Sox positive elements of the bile ducts. We have recently identified a heterogeneous stem/progenitor cell population within the peribiliary glands (PBGs) of the human biliary tree [6,7,8,9,10,11] These cells, referred to as human Biliary Tree Stem/progenitor Cells (hBTSCs), express a broad panel of endoderm stem cell markers, display in vitro long-term persistence and self-renewal, and are able to give rise to a more restricted progeny of different mature lineages (hepatocytes, cholangiocytes and β-pancreatic cells) [6,7,8,9,10,11]. We sought to induce the differentiation of hBTSCs to functional insulin-producing cells trough an innovative protein-based strategy
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