The Induction Of Differentiation From BMSCs To Hepatocytes & The Expression And Modulation Of Hepatocyte Polarity Molecules During Hepatocyte Differentiation

Background and ObjectiveAfter 20 years of development, hepatocyte transplantation as a replacement therapy for liver transplantation has become an effective treatment for acute and chronic liver failure and genetic liver-related metabolic diseases. However, hepatocyte transplantation is still facing many problems such as hepatocyte in short supply, limit the number of transplanted cells, cell proliferation after transplantation, replacement of damaged hepatocytes and the immune rejection, and so on. It is reported that as early as 2002, BMSCs can be induced into hepatocyte in vitro. The BMSC is expected to have its own unique advantages of cell transplantation therapy in liver diseases and is very prominent in clinical application.Our laboratory has succeeded in hepatocyte induced BMSC in various methods. In order to provide easier, controlled, components defined cell culture method for clinical application, we use human serum albumin instead of FBS, using serum-free culture method to obtain better hepatocyte transplantation cell source in clinical.Physiological functions of the liver depend on the formation and maintenance of hepatocyte polarization. However, the hepatocyte polarization we know is very limited. Hepatocyte polarization is limited by the lack of ideal hepatocyte polarization models in vitro. Therefore, we applied the bone marrow mesenchymal stem cells derived hepatocyte to study the hepatocyte polarization for establishing a good model for hepatocyte polarization in vitro.Ursodeoxycholic acid (UDCA) has choleretic, anti-apoptosis and antagonized the hydrophobic bile acid toxicity and other effects, used to treat a variety of hepatobiliary diseases in the clinical. But we have limited knowledge of intracellular mechanisms of UDCA. It is still lack of study of UDCA on the hepatocyte polarization. In this study, SR-B1, MRP2 and NTCP were detected to study the mRNA levels during hepatocyte differentiation and the role of UDCA in hepatocyte polarization.Methods1. Adult bone marrow mesenchymal stem cells were isolated and induced to hepatocyte in serum-free conditions. Cell morphology of 0 days,7 days,14 days,21 days cells was observed by using optical microscopy, a serum-induced differentiation group and non-induced differentiation group as controls. Hepatocyte specific marker ALB was detected by using albumin biochemical assay kit. After immuno-fluorescence staining, fluorescence microscopy was used to observe albumin synthesis of hepatocyte. It is verified if serum-free culture hepatocytes had the characteristics of mature hepatocyte, as well as cells of serum-induced.2. The mRNA levels of MRP2, NTCP, SR-B1 were compared between induced and non-induced cultures at the time 7 days,14 days,21 days and the last 7 days plus 50μmol /L UDCA (21+U groups) using real-time quantitative PCR. The distribution patterns of MRP2 and SR-B1 were observed by confocal microscopy.Results1. After 14 days induced, adult bone marrow mesenchymal stem cells in serum-free conditions perform a mature hepatocyte morphology (group induced in serum-free condition, A group), were significantly different with non-induced group (C group), but similar with serum-induced group (B group). Three groups of cells were measured albumin expression, the differentiation 7,14,21 days of A group and B group albumin gradually increased, after 14 days on a higher level, the three time points A group and B group albumin expression was significantly higher than the C group (p<0.05). A group and B cells 14 days after induction, cells significantly positive compared with their control.2. The mRNA expression levels of MRP2, SR-B1 and NTCP time-dependently increased during the hepatocyte differentiation. The expression level of 14 days,21 days were significantly higher than that of 7 days groups (p<0.05). The MRP2, SR-B1 and NTCP mRNA of 21+U group was significantly higher than that of 21 days group (p <0.05). The location of MRP2 and SR-B1 on the cell membrane was characteristically distributed. Immunofluorescence staining showed that cells induced 14 days after, the MRP2, SR-B1 are localized in different parts of the hepatocyte membrane, showing significant polarization characteristics.Conclusions1. In serum-free condition, bone marrow mesenchymal stem cells can differentiate into mature hepatocyte when in the presence of epithelial cell growth factor (EGF), basic fibroblast growth (bFGF), hepatocyte growth factor (HGF), and is similar to the cell differentiated in serum condition. Such cells are a good source of hepatocytes which used to clinical transplantation. 2. Adult human bone marrow mesenchymal stem cells have the potential to differentiate into polarized hepatocytes. Hepatocytes derived from bone marrow mesenchymal stem cells can be a good model which is used to study the formation and the regulatory mechanism of hepatocyte polarization.