Treatment of acute liver failure in mice by hepatocyte xenotransplantation

Abstract

Liver diseases still have a high mortality even though liver transplantation has become a standard treatment. Currently, hepatocyte transplantation has been proposed as another promising strategy. One limitation is the availability of human livers as a source of hepatocytes. Because of an unlimited supply, the use of porcine hepatocytes might address this problem. Regardless of the source, once isolated hepatocytes lose specific functionality due to the loss of the natural microenvironment. For this reason, we tested the ability of a self-assembling peptide nanofiber (SAPNF) to provide a provisional three-dimensional (3D) support to interact with cells to control their function in vivo. Isolated porcine hepatocytes were embedded in SAPNF, or collagen type I and transplanted by direct injection into the splenic pulp of SCID mice suffering from acute liver failure (ALF) by 90% hepatectomy. SAPNF porcine hepatocyte transplantation produced engraftment that was far superior to that obtained using collagen and prolonged the survival of mice with ALF, in contrast with controls. An ultrastructural evaluation using transmission electron microscopy indicated extensive cell-cell communication and preservation of hepatocyte architecture. The transplanted SAPNF hepatocytes showed higher expression of albumin and PAS and lower apoptotic events assessed by TUNEL staining. Hepatocytes culture in a truly 3D network allows in vivo maintaining of differentiated functions, and once transplanted between widely divergent species can function to correct acute liver failure in mice and prolong their survival.

Fig. 1. Schematic representation of the present study

Porcine hepatocytes were isolated and embedded in Self-assembling peptide nanofiber (SAPNF) or Collagen Type I (Coll I). Such porcine hepatocytes were transplanted in the spleen of 90 % hepatectomized-mice by direct injection into the splenic pulp. The animals were followed for 30 days to evaluate their survival.

Fig. 2. Survival of hepatectomized mice after Hepatocyte transplantation and histological findings of ALF mice

We evaluated the effect of SAPNF transplantation on the surface of the spleen containing porcine hepatocytes on SCID mice that were hepatectomized (90% liver removal). (A) survival was determined for 30 days. Seventy percent survival rate at 30 days was achieved in the mice with SAPNF-porcine hepatocyte transplantation (G1, n=10). On the other hand 50%, and 40% survival rate was achieved with Collagen-porcine hepatocytes (G2, n=10), and hepatocytes alone (G3, n=10), respectively. In contrast, control mice with transplantation of cellular homogenate (G4, n=10) and no hepatocytes (G5, n=10) died within 4 days due to liver failure. * indicates p < 0.05 for G1 vs G2, G3, G4 and G5. (B) The liver specimen obtained from control mice that died of ALF demonstrated massive liver necrosis and hemorrhage (original magnification; x100). (C) The liver of the mice regenerated and showed the normal structure when sacrificed on the 30th day post-transplant (original magnification; x100). Bar= 100 micrometer. (D) TEM showed that SAPNF-hepatocytes demonstrated well-preserved nuclei (N), active mitochondria (M). (E) Gap-junction (Gj) and Tight Junction (Tj) between the cells, well-developed bile caniculi (Bc) and glycogen rosettes (Gly); (original magnification; x 15, 000).

Fig. 3. Histological findings of the spleen after porcine hepatocyte transplantation

(A) In the spleen removed 7 days after porcine hepatocyte transplantation, a considerable number of porcine hepatocytes were detected when SAPNF was used for transplantation, they had a trabecular arrangement, and demonstrated capacity for glycogen storage as demonstrated by PAS staining (D). (B, E) small clusters of porcine hepatocytes, weakly stained for PAS where observed when collagen was used. (C, F) Few hepatocytes were detected when transplanted alone (original magnification; x 40). Bars=50μm

Fig. 4. Immunohistological, apoptotic and gene expression analysis in porcine hepatocytes after transplantation

(A) Immunofluorescent study to detect albumin expression indicated by green signal indicated strong expression of albumin in hepatocytes transplanted with SAPNF, (B) collagen-hepatocyte transplantation, (C) hepatocytes alone. (D), less apoptotic events indicated by red signal in SAPNF hepatocytes, (E) collagen hepatocytes with more apoptotic cells and (F) hepatocytes alone. (Original magnification; x 20). Bars=50μm (G) Albumin gene expression in the spleen containing porcine hepatocytes. Note the higher expression when SAPNF was used as a substrate. (negative control = spleen without hepatocyte transplantation, positive control= porcine liver).