In vitro, pancreas-to-liver transdifferentiation can be successfully recapitulated with the amphicrine cell line AR42J, which was derived from azaserine-treated rats 13. Pancreas-derived HLCs can be induced in vivo by subjecting rats to a copper depletion-repletion protocol 11 or by transgenically overexpressing keratinocyte growth factor in pancreatic β-cells 12. Pancreas-to-liver transdifferentiation reflects the close developmental relationship of the two tissues, both of which arise from the same endodermal region during embryogenesis 10. A natural case of metaplasia is the development of Barrett’s metaplasia in the context of severe gastroesophageal reflux disease, where normal stratified squamous epithelial cells in the distal oesophagus are replaced by a simple columnar epithelium that includes acid mucin-containing goblet cells - a cell type normally found in the gastrointestinal tract 9. Transdifferentiation belongs to a wider class of cell transformations termed metaplasias and refers to the phenomenon of one differentiated cell type irreversibly converting to another 9. In particular the pancreatic progenitor cell line AR42J-B13 has been used as a pancreas-to-liver transdifferentiation model 5, 6, 7, 8. Pancreatic progenitor cells have also been studied as hepatocyte precursors. As an alternative, hepatocyte-like cells (HLCs) have been generated, with variable rates of efficacy, from a variety of cell sources including embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells and human amniotic stem cells 4. For these therapies, hepatocytes can be isolated from surplus or rejected donor livers, however availabilities are restricted and the viability of the generated hepatocyte population is often compromised if harvested from livers obtained from non-heart beating cadavers 3. Extracorporeal or bioartificial liver devices and hepatocyte transplantation represent two promising strategies to support the failing liver, and may either buy time for the native liver to recover through repair and regeneration, or may prolong a patient’s life until liver transplantation.
However, the demand for donor livers drastically exceeds their availability and an increasing number of patients with end-stage liver disease die on the waiting list for transplantation, highlighting the need for alternative treatment approaches. Currently, tumour resection, ablation or orthotopic liver transplantation are the primary treatment options. Despite a recent decline in death rates from other cancers, the prevalence and disease burden of hepatocellular carcinoma continue to rise due to an increase in risk factors such as diabetes, obesity or dietary aflotoxin B1 exposure 1, 2. 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. 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. Here we demonstrate for the first time that transdifferentiation of pancreatic cells to HLCs is not dependent on serum. 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. 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. 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.
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. 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.
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