Tet-on Regulated Hes1 Expression Induces Cholangiocyte Differentiation Of Liver Epithelial Progenitor Cells

Objective The Hes1 gene plays an important role in differentiation of liver stem cells, however, the underlying mechanism is still unclear . Therefore ,we tried to analyze the relationship between Hes1 gene expression and lineage differentiation of mouse liver progenitor cells(LEPCs, also known as liver stem cells) by a Tet-on regulated System. Our data showed that over-expression of Hes1 influences LEPCs differentiation.Methods Plasmids pTRE2hyg-Hesl and pTRE2hyg-EGFP-Hesl were constructed so that Hes1 is under the transcriptional regulation of Dox(Doxycycline)in LEPCs. LEPCs were co-transfected with pTet-on and the the above Hes1 plasmids, and stable transfectants were obtained. The dose-dependent induction of Hes1 in cell lines induced by DOX at different concentrations ( 0,0.1,1,5,10,50,100,500μg/ml) was established. We detected them by RT-PCR,Real-Time PCR and imunocytochemistry (ICC). Some differentiation factors, including the hepatocyte markers(Gss) and cholangiocyte markers (Krt19,Krt7,Foxa1) were assessed upon Hes1 induction.Results RT-PCR validated DOX -controlled expression of Hes1 in stably transfected LEPCs . The highest level of induction was achieved by 10μg/ml DOX treatment for five days, (approximately 11.21-fold more than untreated LEPCs ). Hes1 expression increased with treatment of DOX and maintained the peak expression from day 5 to day 7. Western blot analysis showed that expression of HES1 protein levels increased with the treatment of increasing concentration of DOX has been increased, compared with HES1 expression by untreated LEPCs. Immunocytochemistry also showed that 98% of the cells were positive to HES1 protein after transfection. In LEPCs cells , with the increase of Hes1 expression, the hepatocyte markers Gss decreased ,but the expression of cholangiocytes molecular markers Krt19 was up-regulated.Conclusions In presence studies, we successfully constructed Tet-inducible plasmids pTRE2hyg-Hesl and pTRE2hyg-EGFP-Hesl ,and established inducible expression of Hes1 in LEPC cells. It was found that Hes1 appears to be able to induce LEPCs differentiation to cholangiocytes in vitro, which provide a firm basis for the future investigation of roles of Hes1 in the regulation and differentiation of liver stem cells.