Reprogramming Of Hepatocyte Towards Pancreatic Stem/progenitor-like Cells Mediated By Oct4and Small Molecular Compounds

Objective1. To observe the morphological changes and gene expression trends of mouseprimary hepatocyte after Oct4-lentivirus transduction and investigate the right time toinduce the dedifferentiated hepatocytes towards pancreatic progenitor/stem-like cells.2. To induce dedifferentiated hepatocytes towards pancreatic progenitor/stem-likecells using small molecular compounds.Materials and MethodsPart1: Mouse primary hepatocytes were isolated and cultured. PlasmidpWPT-Oct4was constructed and lentivirus expressing Oct4were packaged.Hepatocytes were transduced with Oct4-lentivirus and the morphological changes wereobserved. Expressions of nestin, ALB, AFP, and Foxa2were monitored andquantitative analysis was made. The expression trends of “intermediate cells” markers:SOX9, SOX17, HNF1β and HNF6were monitored by RT-PCR. The morphologicalchanges were taken into account to investigate the right time to induce thededifferentiated hepatocytes towards pancreatic progenitor/stem like cells.Part2: According to the expression trends of SOX9, a combination of smallmolecular compounds including EGF, bFGF and exendin-4were used to induce thededifferentiated hepatocytes. The morphological changes were observed. Expressionsof pancreatic stem cell markers CK19, ABCG2were identified using RT-PCR andimmunofluorescence staining.ResultsPart1: After Oct4-lentivirus transduction, the intracellular particles weregradually released from the primary hepatocytes and cell proliferation increased from the7thday. According to the RT-PCR analysis, the expression of mature hepatocytemarkers ALB weakened and immature hepatocyte markers AFP, Foxa2increased,indicating that hepatocytes dedifferentiated and gradually lost the mature phenotypesafter Oct4-lentivirus transduction. During this process, the expression of multilineageprogenitor cell marker nestin increased, lasting from the3rdday to the9thday, andweakened from the12thday. HNF1β and HNF6expressions were observed on the2ndday while SOX9expressions were observed on the6thday.Part2: From the6thday on which expression of SOX9was observed, smallmolecular compounds were utilized to induce the dedifferentiated hepatocytes towardspancreatic progenitor/stem-like cells. Four days after induction, cells grew to formclusters. They were positive for pancreatic stem cell markers CK19and ABCG2.ConclusionMediated by gene Oct4and small molecular compounds, primary hepatocytes candedifferentiate and then be induced towards pancreatic cell lineage. We explored a newavenue to generate pancreatic stem cell in vitro and provided experimental basis forexpanding the pool of pancreas transplantation as well as for realization of β cellreplacement therapy in diabetes.