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Therapeutic efficacy of human hepatocyte transplantation in a SCID/uPA mouse model with inducible liver disease.

Douglas DN, Kawahara T, Sis B, Bond D, Fischer KP, Tyrrell DL, Lewis JT, Kneteman NM - PLoS ONE (2010)

Bottom Line: In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment.Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Alberta, Edmonton, Alberta, Canada. donnad@ualberta.ca

ABSTRACT

Background: Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice.

Methodology/principal findings: In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.

Conclusions/significance: Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

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Ultrastructural changes after GCV treatment.A–B. Chimeric vTK+ SCID/uPA livers after GCV treatment (100 mg/kg) showing swollen electron lucent mitochondria (A) with aberrant cristae and matrices (B). C–D. vTK+ SCID/uPA livers with no GCV showing normal cellular (C) and mitochondrial (D) ultrastructure. E–F. vTK− SCID/uPA livers showing normal cellular (E) and mitochondrial (F) ultrastructure (Uranyl acetate-lead citrate, original magnification, A,C,E: ×1500; B,D,F: ×3600). These micrographs represent ultrastructural features that are present in 10 biopsies with a minimum of 5 random fields examined per mouse.
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pone-0009209-g011: Ultrastructural changes after GCV treatment.A–B. Chimeric vTK+ SCID/uPA livers after GCV treatment (100 mg/kg) showing swollen electron lucent mitochondria (A) with aberrant cristae and matrices (B). C–D. vTK+ SCID/uPA livers with no GCV showing normal cellular (C) and mitochondrial (D) ultrastructure. E–F. vTK− SCID/uPA livers showing normal cellular (E) and mitochondrial (F) ultrastructure (Uranyl acetate-lead citrate, original magnification, A,C,E: ×1500; B,D,F: ×3600). These micrographs represent ultrastructural features that are present in 10 biopsies with a minimum of 5 random fields examined per mouse.

Mentions: Ultrastructural analysis was performed by TEM (Fig. 11). GCV induced swollen electron lucent mitochondria with aberrant cristae and matrices (Fig. 11A,B). We did not observe other ultrastructural changes (Figure 11A). In contrast, livers from vTK+ SCID/uPA mice with no GCV (Fig. 11C,D) or vTK− SCID/uPA mice (Fig. 11E,F) had normal ultrastructural morphology with unremarkable mitochondrial matrix and cristae. Ultrastructural analysis performed with biopsies obtained from experimental chimeric vTK+ and vTK− SCID/uPA mice (having RI>60%) demonstrate aberrant mitochondrial features in hepatocytes from chimeric vTK+ SCID/uPA mice that were administered GCV (not shown).


Therapeutic efficacy of human hepatocyte transplantation in a SCID/uPA mouse model with inducible liver disease.

Douglas DN, Kawahara T, Sis B, Bond D, Fischer KP, Tyrrell DL, Lewis JT, Kneteman NM - PLoS ONE (2010)

Ultrastructural changes after GCV treatment.A–B. Chimeric vTK+ SCID/uPA livers after GCV treatment (100 mg/kg) showing swollen electron lucent mitochondria (A) with aberrant cristae and matrices (B). C–D. vTK+ SCID/uPA livers with no GCV showing normal cellular (C) and mitochondrial (D) ultrastructure. E–F. vTK− SCID/uPA livers showing normal cellular (E) and mitochondrial (F) ultrastructure (Uranyl acetate-lead citrate, original magnification, A,C,E: ×1500; B,D,F: ×3600). These micrographs represent ultrastructural features that are present in 10 biopsies with a minimum of 5 random fields examined per mouse.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823785&req=5

pone-0009209-g011: Ultrastructural changes after GCV treatment.A–B. Chimeric vTK+ SCID/uPA livers after GCV treatment (100 mg/kg) showing swollen electron lucent mitochondria (A) with aberrant cristae and matrices (B). C–D. vTK+ SCID/uPA livers with no GCV showing normal cellular (C) and mitochondrial (D) ultrastructure. E–F. vTK− SCID/uPA livers showing normal cellular (E) and mitochondrial (F) ultrastructure (Uranyl acetate-lead citrate, original magnification, A,C,E: ×1500; B,D,F: ×3600). These micrographs represent ultrastructural features that are present in 10 biopsies with a minimum of 5 random fields examined per mouse.
Mentions: Ultrastructural analysis was performed by TEM (Fig. 11). GCV induced swollen electron lucent mitochondria with aberrant cristae and matrices (Fig. 11A,B). We did not observe other ultrastructural changes (Figure 11A). In contrast, livers from vTK+ SCID/uPA mice with no GCV (Fig. 11C,D) or vTK− SCID/uPA mice (Fig. 11E,F) had normal ultrastructural morphology with unremarkable mitochondrial matrix and cristae. Ultrastructural analysis performed with biopsies obtained from experimental chimeric vTK+ and vTK− SCID/uPA mice (having RI>60%) demonstrate aberrant mitochondrial features in hepatocytes from chimeric vTK+ SCID/uPA mice that were administered GCV (not shown).

Bottom Line: In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment.Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Alberta, Edmonton, Alberta, Canada. donnad@ualberta.ca

ABSTRACT

Background: Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice.

Methodology/principal findings: In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.

Conclusions/significance: Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

Show MeSH
Related in: MedlinePlus