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

Abstract

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.Key words: Tissue engineering; Hepatocyte transplantation; Liver regeneration; Liver failure; Regenerative medicine INTRODUCTIONin the clinic with more than 50 cases treated for several types of liver diseases (13). Recent reports of CriglarNajjar syndrome and a glycogen storage disease have Organ replacement therapies, including liver transplantation and living related partial liver transplantation, demonstrated the therapeutic efficacy clearly as an alternative to liver transplantation (5,9). However, there is have become a standard treatment modality for many forms of end-stage liver diseases. However, the potential one limiting factor in transplanting hepatocytes into the liver: the number of cells that can be grafted is limited worldwide shortage of organ donors greatly limits the number of patients able to receive this treatment. In rebecause of the potential complications related to the large number of cells infused and the risk of portal vein sponse to the demand to establish alternative treatment approaches for the liver diseases, ...

Heterotopically Transplanted Hepatocyte Survival Depends on Extracellular Matrix Components

“…In our previous reports, significantly higher hepatocyte survival was achieved when human or mouse hepatocytes were cotransplanted with EHS-ECMs at heterotopic sites as compared to hepatocytes without ECMs. 1,4,5 In line with these data, significantly higher and sustained survivals were achieved in group 2 (hepatocytes with EHS-ECMs) compared with the survival of hepatocytes alone (group 1). When hepatocytes were transplanted with growth factor-reduced EHS-ECMs (group 3), sustained survival was also achieved with no difference in survival compared to group 2.…”

“…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.…”

Therapeutic Effects of Hepatocyte Transplantation on Hemophilia B

“…Using primary hepatocytes, our group has developed several innovative approaches to create a functional liver system under the kidney capsule or in subcutaneous locations (13,15,16,24,25), and we have clearly demonstrated that these ectopically engrafted hepatocytes also possess the ability for proliferation (13,16,26). This would be a significant benefit in the use of these hepatocytes, because most of the adult hemophilia B patients presented with chronic hepatitis B and/or C viral infection as a result of treatments with blood-borne contaminated plasma-derived FIX concentrates.…”

“…Hepatocytes were isolated from C57Bl/6 wild-type mice using a collagenase perfusion method as previously described (13)(14)(15)(16). The recipient FIX knock-out (FIX-KO) mice, syngeneic to donor mice (17), were transplanted with the isolated hepatocytes (1.5ϫ10 6 cells in 200 L) into the liver through the inferior pole of the spleen (nϭ25).…”