Human Hepatocyte Propagation System in the Mouse Livers: Functional Maintenance of the Production of Coagulation and Anticoagulation Factors

Abstract

We previously reported that cell-based therapies using isolated hepatocytes including hepatocyte transplantation and liver tissue engineering approaches provide therapeutic benefits to hemophilia. For clinical application of these approaches, it is important to establish an active hepatocyte proliferation system that enables providing a sufficient number of hepatocytes. We also reported that human hepatocytes, which were transplanted into the liver of urokinase-type plasminogen activator transgenic severe combined immunodeficiency (uPA/SCID) mice, were able to proliferate while retaining their ability to produce coagulation factor IX. The objective of this study was to explore the functionalities of other coagulation and anticoagulation factors of the propagated human hepatocytes in uPA/SCID mice. Human hepatocytes were transplanted into the liver of uPA/SCID mice, and the propagation status of human hepatocytes in the mice was monitored by the increase in serum human albumin levels and immunohistochemical evaluation on the liver sections. Using uPA/SCID livers with various stages of human hepatocyte propagation, we analyzed the gene expression levels of coagulation factors (prothrombin, factor VII, factor X, and factor VIII) and anticoagulation factors (protein C and protein S) by real-time polymerase chain reaction (PCR) using human-specific primers. As a result, the total amount of raw messenger RNA expression levels increased in all genes analyzed according to the progress of hepatocyte propagation and proliferation. Except for factor VIII, the gene expression levels of the highly repopulated uPA/SCID mouse livers with human hepatocyte showed higher levels than those of normal human livers, indicating that propagated human hepatocytes in the uPA/SCID system possess full functions to produce most of the coagulation-related factors. The current work demonstrated that human hepatocytes can be propagated in experimental animals while maintaining normal gene expression levels of coagulation-related factors. It could be speculated that the propagated cells serve as a cell source for the treatment of various types of coagulation factor deficiencies.