Improved Hepatocyte Engraftment After Portal Vein Occlusion in LDL Receptor-Deficient WHHL Rabbits and Lentiviral-Mediated Phenotypic Correction In Vitro

Abstract

Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hypercholesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65-70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL ligands. In conclusion, our results have applications for the treatment of inherited metabolic liver diseases, such as FH, by transplantation of lentivirally transduced hepatocytes.

Keywords: Familial hypercholesterolemia; Hepatocyte transplantation; Lentiviral vector; Liver; Portal vein embolization; Rabbit.

Figure 1

Effects of left lobectomy and portal vein embolization (PVE) on rabbit liver. Successive schematic anatomy of the liver (left) and portograms (right) are shown: (A) before surgery (double arrows indicate lobe to be resected), (B) immediately after left lobectomy, and (C) after embolization of the median lobe (gray) of the liver. The right portal branch of the liver remained patent. Rabbit liver lobes: ML, median lobe; LLL, left lateral lobe; SRL, right lobe divided into superior right lobe; IRL, inferior right lobe; CL, caudate lobe.

Figure 2

Transduction of rabbit hepatocytes. (A) Dose–response of rabbit hepatocytes to lentiviral transduction. Hepatocytes isolated from New Zealand White (NZW) rabbits were transduced in suspension with increasing amounts of mouse transthyretin–green fluorescent protein–woodchuck hepatitis virus posttranscriptional regulatory element (mTTR-GFP-WPRE) lentiviral vectors. The percentages of GFP-positive and viable cells were determined by fluorescent-activated cell sorting (FACS) at day 5 posttransduction. The data (mean±SEM) are representative of three independent experiments. (B) Representative FACS analyses showing mock-transduced Watanabe heritable hyperlipidemic (WHHL) hepatocytes (left) and WHHL hepatocytes transduced at an multiplicity of infection (MOI) of 30 (right). FL2-H and FL1-H represent unspecific fluorescence and specific GFP fluorescence, respectively. (C) Fluorescent microscopy of GFP-expressing hepatocytes in WHHL rabbits. Arrow indicates a GFP-expressing hepatocyte with two nuclei. (D) Morphology of the transduced hepatocytes in WHHL rabbits under phase contrast microscopy. Original magnification: 20×.

Figure 3

Tolerance of the surgical procedures and histological analysis of transplanted hepatocytes. (A) Portal pressure values before left lobectomy and PVE (pre-LB/Pre-PVE), after left lobectomy (LB), before cell transplantation (before Cell T), at the end of cell transplantation (End Cell T), and 2 h after the end of cell transplantation (2 h after cell T) in NZW rabbits. (B) Mean values of liver function tests before and after surgical procedures and cell transplantation. D, days; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; GGT, gamma glutamyl transpeptidase; Bilirubin, total bilirubin in WHHL rabbits. (C) Hematoxylin–eosin staining of a representative cryostat section of the embolized lobe showing the presence of Histoacryl^® in the lumen of a portal vessel in WHHL rabbits. (D, E, F) Histological analysis of WHHL rabbit liver tissue showing transplanted hepatocytes trapped within the portal tract (D, arrows), located within the sinusoidal lumen (E, arrows), and in the process of being integrated and surrounded by an inflammatory reaction (F, arrows). (E) is a magnification of the area in the square in (D). Original magnification: 10× (C, D), 40× (E), 20× (F).

Figure 4

Engraftment of transplanted transduced hepatocytes. (A, B) Representative images of Hoechst-labeled hepatocytes in NZW liver sections 7 days after transplantation. (C, D) Immunohistochemical staining of representative sections of transduced hepatocytes expressing GFP at day 7 after transplantation in a WHHL rabbit. (E, F) Immunohistochemical staining of representative sections of GFP-positive hepatocytes 56 days after transplantation in a NZW rabbit. Original magnification: 20×.

Figure 5

Phenotypic correction of WHHL rabbit hepatocytes. Hepatocytes isolated from WHHL rabbits were transduced with a lentiviral vector encoding human low-density lipoprotein receptor (LDLR). (A) Western blot analysis of cell extracts from transduced (LV) and mock-transduced (NT) hepatocytes using antibodies against actin and human LDLR. Binding and uptake of LDL cholesterol by transduced hepatocytes (LV) was evaluated by FACS using bodipy-LDL (B) and by fluorescent microscopy using Dil-LDL (C). Representative image of fluorescent hepatocytes demonstrating the phenotypic correction of WHHL hepatocytes (C, right). Mock-transduced cells were used as negative control (NT).