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Liver transplantation (LTx) is the standard therapy for patients with terminal liver failure. Nonetheless long-term survival is still affected by serious side effects of immunosuppression. Hepatocyte transplantation (HcTx), a possible alternative to LTx until now has not shown long-lasting clinical success.
The aim of this study was to create a novel rat model for investigating the feasibility of combining an orthotopic LTx and transplantation of autologous liver cells isolated from the patient’s own diseased liver. Therefore, we performed allogeneic LTx with Retrorsin-pretreated liver grafts from female donors (Dark Agouti) into female recipients (Lewis). Following this procedure syngeneic hepatocytes and hepatic progenitor cells (hPC) were isolated from 2-AAF/PH -preconditioned male Lewis rats and these combined liver cells were transplanted into the recipient’s spleen (cLCTx).
Once these cells have engrafted, it is postulated that these syngenic cells will repopulate the allogeneic liver graft as they have a selective advantage over the donor tissue. It is hypothesized that this will lead to a neo-hybrid liver graft (NHL), reducing the necessity of immunosuppression and possibly inducing tolerance to both the entire engrafted tissue and the allogeneic donor matrix.
Aim of this study was to investigate the feasibility of the NHL concept in the Dark Agouti/Lewis rat model under stable immunosuppression (cyclosporin A). Animals were sacrificed at four different time-points (day 8, 15, 30, 90) after LTx.
In order to analyse the degree of cell engraftment and repopulation, Y-chromosome detection of transplanted male cells was performed by Fluorescence in situ Hybridisation (FisH). Immunohistochemical analyses of hPC specific markers (OV-6, Thy-1 and EpCam) in combination with proliferation specific staining (PCNA) were used to identify transplanted cells. Correlating immunohistochemical results of hPC specific markers and proliferation stains with FisH demonstrated efficient engraftment of transplanted cells.
The results of FisH-based detection of the transplanted liver cells were as follows: day 8 with 7.75% +/- 0.79% SEM, day 15 7.97% +/- 0.85% SEM, day 30 10.20% +/- 0.91% SEM and day 90 with 19.61% +/- 0.78% SEM after combined cLCTx and LTx. The percentage of Y-chromosome-positive cell nuclei obtained on day 8, 15 and 30 each demonstrated significant difference (p < 0,001) in comparison to day 90.
The increasing quantity of Y-chromosome-positive cell nuclei detected during follow-up confirmed successful repopulation of the liver graft and thus leading to the establishment of a NHL. Animals surviving the first 3 days after combined liver transplantation and liver cell transplantation (cLTx) showed stable liver function until they were sacrificed. Survival rate after cLTx (n= 29) was 75.86% thus underlining the feasibility of this newly established rat model. The Neo-Hybrid Livergraft therefore provides a valuable foundation for future experimental research models regarding the induction of operational tolerance.