Differentiated Hepatocyte Derived From Bone Mesenchymal Stem Cells In3D Culture And Its Polarity Characteristic

Background and objective:End-stage liver disease, especially acute liver failure, remains associated with significant morbidity and mortality. Liver transplantation has become the effective treatment for the patients with end-stage chronic liver disease. Due to the scarcity of donor organs,<30%of the patients on waiting list receive a transplant. It has been suggested that hepatocyte-based therapy is an alternative to whole organ transplantation. However, one major limitation of cell-based therapy for liver disease is the availability of human hepatocyte. Bone mesenchymal stem cells (BMSCs) can differentiate into hepatocyte with the hepatic function to synthesis and secrete albumin and other hepatic metabolin induced by hepatocyte growth factor. The differentiated hepatocyte derived from BMSCs can be a new cell source for hepatocyte transplantation. Previous researches of the hepatocyte differentiation from BMSC mainly rely on flat2-dimentional (2D) cell culture system. It is significant different with the3-dimentional (3D) surrounding in vivo. Recently,3D cell culture system has been used on the regeneration of tissue and tumor progression. Type I rat collage can mimic the extracellular matrix necessary to the growth of cell, and the collagen gen Ⅰ3D cell culture system is the most common system. It has been reported that embrayonic stem cell can differentiate into other tissues on3D culture. Hepatocytes are highly polarity cells in vivo, and its polarity characteristic is vital to the normal function of hepatocyte. It remains unknown whether BMSC can differentiate into hepatocyte and establish its polarity structure. The main purpose of this research is to further characterize the phenotype of differentiated hepatocytes derived from BMSC using3D culture in vitro, and to understand the cell polarization, the gerneration and maintenance factors impacting on the polarization. This study may provide fundamental basement for understanding clinical diseases due to the disturbance of hepatocyte polarity.Methods:1. BMSC isolated from adult bone marrow was differentiated into hepatocyte using flat2D culture and3D culture in vitro. The morphology of cell in flat2D culture was observed using inverted microscope. The structure and morphology of cell in3D culture was observed using HE staining. The expression of ALB in hepatocyte differentiated in flat2D culture was measured using immunofluorescence. The expression of ALB and AFP in hepatocyte differentiated in3D culture was measured using immunohistochemistry.2. The expression of polarity protein BSEP and SRBI and the polarity characteristic in differentiated hepatocyte in flat2D culture and3D culture was all measured using immunofluorescence and observed by Confocal. The expression of polarity protein BSEP and SRBI in mouse hepatocyte by sandwich culture was considered as the control.Results:1. Adult BMSC can differentiate into hepatocyte with ALB expression in2D culture as we observed before. BMSC can differentiate into hepatocyte with ALB and AFP expression in3D culture. HE staining results showed that hepatocyte in3D culture can connect to each other and form tubular or spherical structures in3D culture.2. Differentiated hepatocyte derived from BMSC in3D culture can express the polarity protein BSEP and SRBI as the hepatocyte differentiated in2D culture. The differentiated hepatocyte in3D culture can also form the polarity and similar bile canaliculus structures, which was not found in 2D culture. SRBI mainly located at the basal membrane of hepatocyte, however, BSEP mainly located at the apical membrane of hepatocyte. The polarity characteristic was similar to the mouse hepatocyte.Conclusions:1. BMSC can differentiate into hepatocyte in3D culture, and hepatocyte in3D culture can form special tubular or spherical structure with the positive expression of hepatocyte protein.2. Differentiated hepatocyte derived from BMSC in3D culture can form polarity and bile canaliculus structure. This3D culture system can provide significant model for the research of mechanisms of hepatic differentiation and polarity.