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

Abstract

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.)

Stability and Repeat Regeneration Potential of the Engineered Liver Tissues under the Kidney Capsule in Mice

“…The method for hepatocyte isolation and purification is based on a modification of procedures reported by active regeneration potential (18). We also demonstrated these tissues were able to support human-specific hepatiBerry and Friend (2) and reported previously (3, 16,18).…”

“…including hepatocyte transplantation and liver tissue engineering, have been highlighted (12,13 the area of the body other than the liver, such as under MATERIALS AND METHODS the kidney capsule space or mesenteric leave) can also Experimental Animals have therapeutic efficacy (6,10,13,17). However, this approach results in short-term survival of the transAll animal studies used the institutional guidelines set forth by Stanford University and Nara Medical Univerplanted hepatocytes (13,17,18). A number of modifications in the transplantation setting, such as genetic transsity Animal Care Committees.…”

“…However, this approach results in short-term survival of the transAll animal studies used the institutional guidelines set forth by Stanford University and Nara Medical Univerplanted hepatocytes (13,17,18). A number of modifications in the transplantation setting, such as genetic transsity Animal Care Committees.…”

Heterotopically Transplanted Hepatocyte Survival Depends on Extracellular Matrix Components

“…Since the marker protein of the transplanted hepatocytes, hAAT, demonstrates a short half-life (less than 2 hours) in the mouse 7 and is produced only from the hepatocytes, the viability and survival of the transplanted hepatocytes in vivo can reasonably be assessed by periodic mouse serum measurement of the hAAT reporter protein, as described previously. [3][4][5][6][7] Mice were sacrificed at day 140; the grafts were excised and processed for histological examination.…”

“…1,2 We recently reported that the inefficient hepatocyte survival may be overcome by providing ECMs extracted from murine Engelbreth-Holm-Swarm tumor cell lines (EHS-ECMs). [3][4][5][6] The EHS-ECMs are rich in ECM components, predominantly laminin and type IV collagen, but also contain a small amount of growth factors, such as epidermal growth factor. This study was performed to determine the effects of EHS-ECMs on hepatocyte survival and whether it was solely due to ECMs or to a combination of ECMs and growth factors in EHS-ECMs.…”