Neohybrid Liver Graft - a Novel Concept of in Vivo Tissue-Engineering

Abstract

Objectives: Liver transplantation (LTx) is the standard therapy for end-stage liver disease, even though long-term survival is still affected by serious side-effects of immunosuppression. Liver cell transplantation (LCT), a possible alternative to LTx, still has not shown long-lasting clinical success. We developed a novel concept for in vivo tissue-engineering based on the combination of orthotopic LTx and transplantation of autologous liver cells isolated from the patient's own diseased liver: the Neohybrid Liver Graft. Autologous hepatocytes and hepatic progenitor cells (hPC) may repopulate the allogeneic liver using the graft as biological matrix. This could eventually lead to an entirely syngeneic liver and operational tolerance. Materials and methods: Aim of this study was to investigate the feasibility of the Neohybrid Liver Graft concept in the Dark Agouti/Lewis rat model under stable immunosuppression. Therefore, we performed orthotopic LTx with Retrorsin-pretreated liver grafts from female donors (Dark Agouti) into female recipients (Lewis). Afterwards, syngeneic hepatocytes and hPCs were isolated from 2AAF/PH -preconditioned male Lewis rats and combined liver cells (cLCT) were transplanted into the recipient's spleen. 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 quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) for the SRY gene and by Fluorescence in-situ Hybridisation (FisH-typing). Immunohistochemical analyses of hPC specific markers (OV6, CK18, AFP and EpCam) in correlation with proliferation stains (PCNA, Ki67, BrdU) were used to identify transplanted cells. Results: Correlating immunohistochemical results, hPC specific markers and proliferation stains demonstrated engraftment of transplanted cells with increasing quantity during follow-up. FisH-typing confirmed successful repopulation of the liver graft at day 8 with 8.1% ± 4.0% SEM and day 90 with 23.0% ± 3.8% SEM after combined cLCT and LTx. Validating these results by qRT-PCR using a standard curve, proportion of SRY-positive DNA and absolute weight of each sample were calculated. Collected data for 3 animals showed 0% SRY-positive DNA at day 8, but 16.4% ± 7.9% SD SRY-positive DNA at day 90. Conclusion: We could proof successful engraftment of transplanted cells as well as repopulation of the allograft after combined LTx and cLCT with growing efficiency during follow-up. Histological analyses indicated a continuous cell proliferation activity and FisH-typing results were partly confirmed by qRT-PCR calculating absolute weight of SRY-positive DNA of liver samples. Influence of hPCs on engraftment and proliferation of transplanted hepatocytes are under evaluation, especially with respect to minimization of immunosuppression and induction of tolerance. This new concept for in vivo tissue-engineering could enable repopulation of liver allografts with autologous cells from the patient's own liver to generate a Neohybrid Liver Graft.