Figure 1.
hHGF-modified HOC differentiation potential.
LIF was withdrawn from culture medium to induce cell differentiation. (A) Intracellular hHGF protein expression was determined by Western blot analysis. hHGF was used as positive control and β-actin used as an internal control. (B) Undifferentiated or differentiated normal HOCs or hHGF-modified HOCs were monitored under phase contrast microscopy. Arrows indicated long-spindle like bile duct epithelial cells and arrowheads indicat the large, round-shaped hepatocytes. (C) Immunostaining of differentiated cells with specific antibodies for ALB or CK19. Arrows indicated immuno-positive cells (dark brown cells). Sections were double-stained with H&E. Magnification ×200.
Table 1.
Quantification of ALB and CK19 mRNA levels using quantitative RT-PCR.
Figure 2.
Experimental design outline.
Figure 3.
In situ hybridization of SRY-positive donor HOCs.
In situ hybridization was performed 21 days post transplantation. Ovarian tissue was used as a negative control and renal tissues from male Lewis rats used as positive controls. SRY-positive cells exhibited dark brown staining of their nuclei and/or cytoplasm. Following HOC administration to liver transplant recipients, SRY-positive cells were found in liver tissues and hepatic sinusoids. Additionally, SRY-positive hHGF-HOCs were observed in the surrounding portal area, central vein and bile duct, and were diffusely distributed in hepatic lobules. Magnification is indicated on each image.
Table 2.
Measurement of parameters associated with HOC administration and liver transplantation.
Figure 4.
Analysis of serum cytokine levels post transplantation.
Serum IL-2, IL-10, TNF-α, TGF-β1, and IFN-γ levels were examined at the indicated time points post transplantation by ELISA. Differences were analyzed using an analysis of variance or an independent sample t-test. *P<0.05, liver transplantation only vs. hHGF-HOC+liver transplantation or HOC+liver transplantation vs. hHGF-HOC+liver transplantation; #P<0.05, liver transplantation vs. HOC+liver transplantation.
Figure 5.
Survival curves of recipients.
Figure 6.
Cell proliferation and apoptosis.
The beneficial effects of hHGF-HOCs on liver transplantation were evaluated by measuring cell proliferation and apoptosis. (A) Fourteen days post-transplantation, tissue sections were stained with specific antibodies to PCNA. Arrows indicated immuno-positive cells. Magnification ×100. (B) The protein expression of NFκB was examined by Western blot analysis.
Figure 7.
Histological examination of liver allograft rejection.
(A) Sections obtained from experimental groups were staining with H&E, magnification ×100. (B) Liver allograft rejection was assessed using the rejection activity index according to the Banff classification of hepatic allograft rejection described in the Materials and Methods. Data were compared with analysis of variance or an independent sample t-test. *P<0.05 liver transplantation vs. hHGF-HOC+liver transplantation or HOC+liver transplantation vs. hHGF-HOC+liver transplantation; #P<0.05, liver transplantation vs. HOC+liver transplantation.
Figure 8.
Immunostaining for ICAM-1, Fas, CD44 and CD40 28 days post transplantation.
Immunopositive cells exhibited dark-brown staining. Sections were double stained with H&E (blue). Magnification is indicated on each image.