Study On The Differentiation Of Rat Liver Stem-like Cells Into Ancreatic Endocrine Precursor Cells Using Small Molecules

Objective：To establish an in vitro method of differentiation of pancreatic endocrineprogenitor from WB cells, we induced the expressing of pancreatic endocrineprogenitor gene PDX1with small molecules in rat hepatic stem-like cells. The newmethod will be used for transdifferentiation of pancreatic cells from hepatic stem-likecells, and lay the foundation for further study to differentiate the liver stem-like cellsto pancreatic cells.Methods：Using small molecules as5-AZA TSA, RA, and ITS, to directly inducedifferentiation of WB cells into PDX1-expressed pancreatic endocrine progenitors bytwo steps. The different concentrations of5-AZA are used in culture of WB celldifferentiation, and the optimal concentration will be analyzed. The changes of cellshape were observed by phase contrast microscope. The interim cell and progenitorare identified by RT-PCR, immunofluorescence, and glucose stimulationResults：1. Phase contrast microscope observation: after treated with5-AZA at5uM for2days, TSA for1day, about20%the WB cells changed to spindle-liked WB-1cell.With combination of RA and ITS for7days, about50%spindle-liked WB-A cells.2. The results of semi-quantitative PCR of WB-1cells: no expression of PDX1and other development-related genes of pancreas in WB-1cells; the expression ofliver cell gene (ALB, AFP), and differentiation related gene (C/EBP) in WB cells isdown-regulated.3. The results of quantitative PCR and the semi-quantitative PCR of the WB-Acells: The highest expression of PDX1was observed in WB-A cell treated with5-AZA at5uM. The downregulation of live stem cell differentiation related gene,C/EBP, was also observed. Meanwhile, WB-A cell expressed pancreatic endocrineprogenitor related gene of NGN3, Neurod, and NKX2.2. The sustained expression of Nestin and upregulation of Insulin1were also observed.4. Immunofluorescence results of the WB-A cells: the PDX1-positive WB-A cellwas about5%, and very few WB-A cells showed insulin positive.5. Glucose stimulation test: WB-A cells showed no response to glucose.Conclusion:Small molecules, such as5-AZA, TSA, and RA, have ability to induce PDX1expression in liver stem-like cells, and differentiated cells (WB-A cells) showedcharacteristics of pancreatic endocrine progenitor cells. We established a new methodof transformation of pancreatic cell from liver stem cell by using small molecules.Lay the foundation for further study to differentiate the liver stem cells to pancreaticβcells.