Overexpression Of Transcription Factor HNF1A And FOXA2 Induced Rat Bone Mesenchymal Stem Cells Into Hepatocytes

In recent years,researchers have found that mesenchymal stem cells(MSCs)can be induced to a variety of cells across germ layer.The induction of MSCs to hepatocyte-like has been received special attention by researchers,what's more,this research provides a new kind of seed cells for the treatment of liver disease and drug screening.Especially,bone marrow mesenchymal stem cell(BMSCs)have characteristics of self-renewal,highly malleable and immunomodulatory.In addition,the isolation and culture of BMSCs in vitro is simple,exogenous gene can be transferred and express in BMSCs easily.It also has the potential capability to be induced to a variety of cells cross germ layer.FOXA2 and HNF1 A are important members of the hepatocyte nuclear factor(HNF)family,they are abundantly expressed in the liver,and play an important role in the differentiation and function of the liver cells.Based on these studies,we hypothesized that FOXA2 and HNF1 A could directly induce BMSCs to functional hepatocytes.Five days after the two factors were transduced to cells,we could observe some changes in cell morphology,pick out the cells with red fluorescent protein and the cell colonies with changed shape.RT-PCR,western blotting and immunofluorescence were used to verify whether the FOXA2+ and HNF1A+ BMSCs expressed the markers of hepatocytes.HNF4?,FOXA3,ALB,AFP,G6 P,CK18,TAT and TTR were chose as the marker genes of hepatocytes,and the results revealed that these converted epithelial-like cells had the same characteristics with hepatocytes.Meanwhile,the proteins encoded by these marker genes were detected by western blotting,and the corresponding expression at the transcriptional level had been in accordance with the above results.Furthermore,the hepatic proteins such as ALB,G6 P,TAT,TTR,CK18 and AFP were assayed by immunofluorescence,the result indicated all or part of the infected cells expressed those hepatic proteins.The results suggested that the transduced cells had the characteristics of hepatocytes.To further verify the function of induced cells,we chosed the Periodic Acid-Schiff(PAS)stain to detect the capacity of carbohydrate storage,and the results showed that the red deposited was found in the cytoplasm,which indicated these cells could produce and store glycogen.Indocyanine Green(ICG)stain was used to examine the drug absorption capacity of the cells,the result showed the most number of cells had the ability of ICG uptake at 37oC after being incubated for 1 h in the medium with 1 mg/mL ICG.Additionally,the oil red-O staining was ultilized to test the lipid accumulated,and the results revealed lipid was accumulated in some induced cells.However,we used BMSCs as negative control and there were no signs of glycogen storage,ICG uptake and lipid accumulated in the cytoplasm compared with the infected cells.All above results show that rat hepatocyte-like cells are successfully induced from BMSCs.This is a novel and efficient method to prepare HLCs with a short timeline.