Human Mesenchymal Stem Cells Are Directionally Differentiated To Self-assemble The Liver Bud-like Tissue

Background & aims: Liver disease is the major cause leading to death in the world.At present,the treatment methods for the end-stage liver disease mainly include cell transplantation and liver orthotopic transplantation.However,stem cell transplantation for clinical application is still at a very early stage and needs further study.Allograft transplantation is the most direct and effective treatment,but lacking of donor liver organ,expensive cost,postoperative immune rejection and other reasons limit its clinical application.So it is very urgent to look for alternative portable liver.Studies have shown that human mesenchymal stem cells are suitable for autologous transplantation with the advantages of simple selection,non-ethical controversy,rapid proliferation in vitro,and low immunogenicity.They can be differentiated into various types of tissue cells under specific induction conditions in vivo and in vitro.The cell transplant cells are even used as seed cells for liver tissue engineering.Up to now,most studies are directed to the use of induced pluripotent stem cells to construct 3D liver buds in vitro.However,few studies have used mesenchymal stem cells to assemble liver buds.Hepatic stellate cells and liver sinusoidal endothelial cells are important members of liver cells,because they play vital roles in hepatic fibrosis.But,there have no researches involved in the differentiation of human mesenchymal stem cells from the same source into hepatocytes,hepatic stellate cells,and hepatic endothelial cells to assemble them.Here,we focus on inducing mesenchymal stem cells to differentiate directly into three types of liver cells,and assemble them in vitro into 3D human liver buds in a certain proportion.Moreover,it is preliminarily explored for the possibility of its application to the treatment of acute liver injury.Methods: The purified human mesenchymal stem cells were cultured and identified.Human mesenchymal stem cells were induced to differentiate into hepatocyte-like cells,liver endothelium-like cells,and hepatic stellate cells by adding differentiation factors such as Wnt3 a,FGF4,and BMP4 to the culture medium.These cells were separated by flow cytometry for further detection by immunofluorescence staining and RT-PCR,and mixed at the ratio of 10:3:3:1 on Matrigel,and the gene expression profile between 3D liver bud and early liver tissue of mice was compared by microarray analysis.Then,a ganciclovir-induced mouse acute liver injury model was established,and the assembled liver buds were transplanted into the mesentery of mice with liver damage.The survival time of mice was observed,and hematoxylin-eosin staining was used to detect the pathological changes of damaged liver tissues.At the same time,the expression profiles of marked genes from the new liver bud and the liver bud after transplanted were analyzed by microarray.The levels of AFP,ALB,CYP3A4,CYP2C9,CYP2D6,G6 PC,GLUT2,HNF4α,and UGT2B7 genes were detected by RT-qPCR.The secretion of ALB and AAT in the serum of the treated mice was detected by ELISA,and the changes of functional proteins ALB and CK8/18 in the damaged liver tissue and the transplanted liver bud were observed by in situ immunofluorescence staining.Results: The results from morphology,gene expression and flow cytometric sorting showed that human umbilical cord blood stem cells were induced to differentiate into hepatocyte-like cells,hepatic stellate-like cells,and hepatic endothelium-like cells with differential efficiency of 74 %,21 %,and 19 %,respectively.Each cell series could be self-assembled into 3D liver buds after in vitro mixed culture within 72 h,and its gene expression profile is similar to that of the liver of the first 10 d of mouse embryo.Eighteen days after treated by using 3D liver bud transplantation,the survival rate(90%)of the mice with the acute liver injury was significantly higher than that of the sham operation group(50%),and the degree of liver tissue injury was significantly improved.The serum levels of AAT and ALB in mice transplanted with liver buds increased to 400 ng/m L and 800 ng/m L,respectively.ALB and CK8/18 were significantly expressed in orthotopically transplanted liver buds,and the expression level of ALB,CYP3A4,CYP2C9,CYP2D6,G6 PC,GLUT2,HNF4α,UGT2B7,and other mature liver genes in the transplanted liver buds were significantly increased,showing similar gene expression characteristics to adult liver.Conclusion: Human umbilical cord blood stem cells are successfully induced to differentiate into hepatocyte-like cells,hepatic stellate-like cells,and hepatic endothelium-like cells.These differentiated cells can be mixed and cultured in a proportioned ratio and self-assembled into liver buds.Liver bud transplantation can significantly improve the degree of acute liver injury and reduce the mortality of the mice with acute liver injury.This study provides a novel method for the differentiation of mesenchymal stem cells into liver cells and self-assembling functional liver buds in vitro,which lays the foundation for the establishment of drug screening models.