Abstract
The rising prevalence of chronic liver disease, coupled with a permanent shortage of organs for liver transplantation, has sparked enormous interest in alternative treatment strategies. Previous protocols to generate hepatocyte-like cells (HLCs) via pancreas-to-liver transdifferentiation have utilised fetal bovine serum, introducing unknown variables and severely limiting study reproducibility. Therefore, the main goal of this study was to develop a protocol for transdifferentiation of pancreatic progenitor cells to HLCs in a chemically defined, serum-free culture medium. The clonal pancreatic progenitor cell line AR42J-B13 was cultured in basal growth medium on uncoated plastic culture dishes in the absence or presence of Dexamethasone on uncoated, laminin- or fibronectin-coated culture substrata, with or without serum supplementation. The hepatocytic differentiation potential was evaluated: (i) morphologically through bright-field and scanning electron microscopy, (ii) by assessing pancreatic and hepatic marker expression and (iii) by determining the function of HLCs through their ability to synthesise glycogen or take up and release indocyanine green. Here we demonstrate for the first time that transdifferentiation of pancreatic cells to HLCs is not dependent on serum. These results will assist in converting current differentiation protocols into procedures that are compliant with clinical use in future cell-based therapies to treat liver-related metabolic disorders.
Highlights
Irrespective of the underlying aetiology, chronic liver diseases such as alcoholic liver disease, non-alcoholic steatohepatitis or viral hepatitis infection may progress to cirrhosis and eventually hepatocellular carcinoma
Possible changes in AR42J-B13 cell morphology were investigated under the following conditions: (1) undifferentiated cells grown under basal culture conditions; all subsequent conditions used differentiation medium and were: (2) cells grown on tissue culture plastic without extracellular matrix (ECM) coating in fetal bovine serum (FBS)-containing medium, cells grown on fibronectin with (3), and without FBS (4), and cells grown on laminin with (5), and without FBS (6)
As a progression from earlier reports using the AR42J-B13 cell line as a pancreas-to-liver transdifferentiation model[5,6,7,14], we demonstrate in this study that these pancreatic progenitor cells can reliably be induced to transdifferentiate into functional hepatocyte-like cells (HLCs)
Summary
Irrespective of the underlying aetiology, chronic liver diseases such as alcoholic liver disease, non-alcoholic steatohepatitis or viral hepatitis infection may progress to cirrhosis and eventually hepatocellular carcinoma. Treatment of the subclone AR42J-B13 with the synthetic glucocorticoid Dexamethasone induces these cells to convert to a functional hepatocytic phenotype, as determined by downregulation of amylase with concomitant induction of liver-enriched transcription factors and the expression and activity of hepatocyte-specific proteins[5,14]. These previous studies have been performed in culture medium containing fetal bovine serum (FBS) to supply a cocktail of the nutrients, growth factors, hormones, carrier proteins for lipoid substances and trace elements that are necessary for substratum attachment, cell viability and proliferation. To the best of our knowledge, this is the first report of successful hepatocytic transdifferentiation of pancreatic cells in serum-free conditions, facilitated by ECM proteins
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