Takashi Yokoyama

Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets

Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds.

Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole‐organ transplantation or liver‐directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth‐Holm‐Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth‐Holm‐Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naïve liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naïve liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders. (HEPATOLOGY 2005;41:132–140.)

In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity.

Recent success in clinical hepatocyte transplantation therapy has encouraged further investigation into bioengineering hepatic tissues in vivo. Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF‐releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver‐specific mRNAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug‐metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte‐based therapies.

Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice.

Liver tissue engineering using hepatocyte transplantation has been proposed as a therapeutic alternative to liver transplantation toward several liver diseases. We have previously reported that stable liver tissue with the potential for liver regeneration can be engineered at extrahepatic sites by transplanting mature hepatocytes into an extracellular matrix. The present study was aimed at assessing the liver tissue persistence after induced regeneration by hepatectomy and repeat regeneration potential induced by repeat hepatectomy. Mouse isolated hepatocytes mixed in EHS extracellular matrix gel were transplanted under both kidney capsules of isogenic mice. The hepatocyte survival persisted for over 25 weeks. In some of the mice, we confirmed that the grafted hepatocytes developed a thin layer of liver tissues under the kidney capsule, determined by specific characteristics of differentiated hepatocytes in cord structures between the capillaries. We then assessed the regenerative potential and persistence of the exogenous liver tissue. To induce liver regeneration, we performed a two-thirds hepatectomy at 70 days after hepatocyte transplantation. Three weeks after this procedure, the engineered liver tissues showed active regeneration, reaching serum marker protein levels of 261 ± 42% of the prehepatectomy level. We found that the regenerated liver tissue was stably maintained for 100 days (length of the experiment). Repeat regeneration potential was established by performing a repeat hepatectomy (that had been two-thirds hepatectomized at day 70) 60 days after the initial hepatectomy. Again, the regenerated engineered liver tissues showed active regeneration as there was an approximately twofold increase in the serum marker protein levels. The present studies demonstrate that liver tissue, which was recognized as a part of the host naive liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.

597. Liver Tissue Engineering at Extra-Hepatic Site: A Novel Therapeutic Approach toward Hemophilia

Introduction: In response to the demand to establish new-generation treatment approaches for the inherited liver diseases including hemophilia, cell-based therapies using hepatocyte transplantation and liver tissue engineering have been highlighted. We have recently established technique to engineer stable liver tissues at extra-hepatic site (under the kidney capsule) with the potential for liver regeneration by transplanting mature hepatocytes with extracellular matrix components. Since the liver is a major contributory organ for clotting factor VIII production, we hypothesized that engineering a liver tissue in vivo would be a therapeutic option for hemophilia. The present study was aimed at assessing the therapeutic efficacy of the liver tissue engineering on the mouse model of hemophilia A.

876. Towards establishing a heterologous liver in rodents

Top of pageAbstract nLiver tissue engineering has been proposed as an approach toward treating several liver diseases as a means to avoid organ transplantation. We have previously reported that stable liver tissues can be engineered and maintained under the kidney capsule by transplanting mature hepatocytes together with extracellular matrix. Because the liver has robust regeneration potential, we investigated if liver tissue engineered at extra-hepatic sites (without portal circulation) would actively regenerate. The present study was aimed to assess graft regenerative activity, and persistence after the induction of active liver regeneration. METHODS: Hepatocytes were isolated from transgenic mice that express a serum marker protein, human alpha1-antitrypsin (hAAT). Two million hepatocytes were then transplanted with EHS gel matrix component under the kidney capsule of isogenic mice. The survival of the transplanted hepatocytes was monitored by histology and periodic measurement of serum hAAT. RESULTS: The hepatocytes were engrafted and maintained for over 20 weeks in all treated mice (n=28). In some of the mice, we confirmed that the grafted hepatocytes developed small pockets of liver tissue because they showed specific characteristics of differentiated hepatocytes composed of hepatocytes in cord structures between the capillaries. We then assessed if the engineered liver tissues maintain their regeneration potential in vivo. We induced an intrinsic mode of liver regeneration (compensatory regeneration) by performing a 2/3 hepatectomy of the naive liver 70 days after hepatocyte transplantation. Two weeks after this procedure, the serum hAAT levels in mice reached 237 ± 18% of the pre-hepatectomy level, establishing similar regeneration activity as that occurred in the naive host livers during this period. The daily increase in liver tissue regeneration reached a peak at day 3 in the engineered liver tissues, showing a similar trend to the naive livers. The regeneration of the engineered livers resolved with restoration of the naive liver to its pre-surgical mass. We also followed the liver tissues long-term after the regeneration, and found that the regenerated liver tissues were stably maintained for over 80 days (length of the experiment). CONCLUSION: The present studies demonstrate that liver tissue, which was recognized as a part of the host naiuml]ve liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.