Molecular reprogramming of hepatocytes into beta-like cells

The conversion of one differentiated cell-type into another, challenges our long-held view of differentiated cells being in a fixed terminal stage of differentiation, incapable of reversal or change. Such conversions are most favourable between developmentally related cell-types such as hepatocytes from the liver and cells of the pancreas. The main drawback of culturing hepatocytes in vitro has been their limited life span and their rapid dedifferentiation in culture. However, the de-differentiation of hepatocytes, and the progressive diminution in the transcription levels of liver-specific genes, is in fact associated with a concomitant induction of a pancreatic gene expression profile. In this ‘primed’ state, it is possible to convert these dedifferentiated hepatocytes more efficiently into beta-like cells by the transduction of the pancreatic master switch gene, Pdx1, in rat, mouse and human hepatocytes. An extensive screen of several other key-regulatory pancreatic transcription factors has yielded that along with Pdx1, the transcription factors Neurogenin3, NeuroD, Pax4 and MafA are crucial for achieving efficient reprogramming. The ‘beta-phenotype’ of the cells generated can be further enhanced by using the extrinsic factors betacellulin, Activin A and nicotinamide. Many of these factors, most notably betacellulin can promote endocrine differentiation in favour of beta cells, as was demonstrated using an explant model of pancreatic development. The betalike cells generated by the molecular reprogramming of hepatocytes in addition to expressing a wide array of beta cell-specific genes, also exhibit many beta cell functions. The beta-like cells can process pro-insulin into the mature insulin protein and secrete insulin in a regulated manner in response to glucose stimulation. These cells therefore could serve as an attractive alternative for generating autologous beta cells for transplantation as a means of a cell-based therapy for type 1 diabetes mellitus. LIST OF ABBREVIATIONS AFP α-foetoprotein kDa kilo Daltons β-gal β-galactosidase KGF keratinocyte growth factor BMP bone morphogenic protein Kir K + channel subunit C/EBP CCAAT/Enhancer binding protein LAP liver activation protein CAR coxsackievirus B and adenovirus receptor LIP liver inhibitory protein Cdx caudal type homeo box genes LIF leukaemia inhibitory factor ChrA chromogranin A M.O. I multiplicity of infection CK cytokeratins Maf musculoaponeurotic fibrosarcoma oncogene homolog A genes CMV cytomegalovirus (promoter) MODY maturity onset diabetes of the young CPE cytopathic effect NeuroD neuronal determination factor CpE carboxypeptidase E Ngn3 Neurogenin3 CPS carbamoyl phosphate synthetase Nic nicotinamide Dex dexamethasone dpc days post coitus Nkx natural killer cell transcription factor genes dpp days post partum NOD non-obese diabetic (mice) E embryonic stage/day Pax paired box genes EGF(R) epidermal growth factor (receptor) Pbx1 pre-B cell leukaemia transcription factor genes ES embryonic stem (cells) PC prohormone convertases Exe4 exendin4 Pdx1 pancreatic and duodenal transcription factor 1 (a.k.a Ipf1) FGF fibroblast growth factor PH3 phospho-histone 3 Fox forkhead box containing genes RT-PCR reverse transcriptase polymerase chain reaction GCK glucokinase SCID severe combined immunodeficient (mice) GFP green fluorescent protein Shh sonic hedgehog GLP1 glucagon-like peptide 1 Sox SRY(sex-determining region Y)-box containing genes GLUT glucose transporter STZ streptozotocin GS glutamate synthetase SUR sulphonylurea receptor subunit GSIS glucose-stimulated insulin secretion TFN transferrin HA haemagglutinin TGF transforming growth factors HGF hepatocyte growth factor TTR transthyretin HNF hepatic nuclear factor v/v volume by volume IAPP islet amyloid polypeptide w/v weight by volume IH(C)C immunohisto(cyto)chemistry (e)YFP (enhanced) yellow fluorescent protein iPS induced pluripotent stem (cells) Zebu zebularine Isl islet LIM/homeodomain 1 genes -/double knockout