1. ¹Department of Surgery, Nara Medical University, Kashihara, Nara, Japan
2. ²Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
3. ³Institute of Advanced Biomedical Engineering, Tokyo Women's Medical University, Shinjyuku, Tokyo, Japan

Abstract

Background: Recent success in clinical liver transplantation toward hemophilia patients has encouraged further investigation into bioengineering hepatic tissues as a cell-based therapy. Since factors VIII and IX are synthesized primarily by hepatocytes, it would be reasonable to hypothesize that engineering a functional hepatic tissue in vivo using isolated hepatocytes would provide potential impact as a new therapy toward hemophilia. This study describes several approaches to engineer hepatic tissues that express factors VIII and IX gene expressions.

Methods and Results: [1] Engineering hepatic tissue under the kidney capsule. Hepatocytes were isolated and purified from adult mice using two-step collagenase perfusion method and were transplanted under the kidney capsule space of isogenic mice. In order to increase initial cell engraftment rate, EHS extracellular matrix components were provided to the hepatocytes at the time of transplantation. With this procedure, the transplanted hepatocytes demonstrated persistent survival resulting in engineering stable hepatic tissues composing of differentiated hepatocytes. When chronic liver regeneration stimulus were introduced to the recipient mice, the engineered hepatic tissues under the kidney capsule demonstrated repeated proliferation leading to a formation of large hepatic tissue mass. mRNA analyses of the engineered hepatic tissues demonstrated factors VIII and IX expressions at similar levels to the na|[iuml]|ve livers. When we engineered hepatic tissues in hemophilia A mice, therapeutic levels of clotting activities were obtained, demonstrating a therapeutic efficacy of this tissue engineering approach. [2] Engineering hepatic tissue into subcutaneous space. Transplantation of the isolated hepatocytes into the subcutaneous space with EHS extracellular matrix components failed to provide persistent hepatocyte survival. However, preparing the local transplantation sites in advance with FGF-releasing device in order to create functional vascular network followed by hepatocyte transplantation allowed persistent hepatocyte survival and functional hepatic tissue engineering. mRNA analyses of the engineered hepatic tissues that were precisely obtained by laser capture microdissection method confirmed similar expression levels of factors VIII and IX to the host na|[iuml]|ve livers.

Conclusions: This study demonstrates several novel approaches for engineering hepatic tissues and their ability for clotting factor gene expressions as a potential cell-based therapy toward hemophilia.