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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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Functional expression of vTK in Huh7 cells.A. Huh7 cells were transfected with pCI-vTK and G418-resistant Huh cellular clones (cl) (1–6) were evaluated for vTK expression (∼43 KDa, arrowheads) by immunoblot analysis. Lmw, low molecular weight markers; 293TvTK, 293T cells transiently transfected with pCI-vTK. B. In vitro cytotoxicity of GCV in vTK expressing cells. HuhvTKcl1 and HuhvTKcl6 cells were incubated with various concentrations of GCV for 72 hours, followed by cell survival quantitation by MTT assay. Data represent the Mean +/− S.D from quadruplicate cell cultures for each GCV dose. Mock, Huh cells that underwent stable selection after transfection with empty pCIneo vector.
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pone-0009209-g002: Functional expression of vTK in Huh7 cells.A. Huh7 cells were transfected with pCI-vTK and G418-resistant Huh cellular clones (cl) (1–6) were evaluated for vTK expression (∼43 KDa, arrowheads) by immunoblot analysis. Lmw, low molecular weight markers; 293TvTK, 293T cells transiently transfected with pCI-vTK. B. In vitro cytotoxicity of GCV in vTK expressing cells. HuhvTKcl1 and HuhvTKcl6 cells were incubated with various concentrations of GCV for 72 hours, followed by cell survival quantitation by MTT assay. Data represent the Mean +/− S.D from quadruplicate cell cultures for each GCV dose. Mock, Huh cells that underwent stable selection after transfection with empty pCIneo vector.

Mentions: Immunoblot analysis demonstrated Huh7 cellular clones (1 and 6) stably transfected with pCI-vTK expressed vTK protein (Fig. 2A). These vTK expressing cellular clones started to detach 48 h post exposure to a single dose (50 mmolL−1) GCV whereas the mock transfected cells grew and proliferated normally. Increased cell death was also assessed by MTT assay (Fig. 2B). Stable expression of vTK in Huh7 cells significantly reduced their survival rate in the presence of the toxic pro-drug GCV.


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)

Functional expression of vTK in Huh7 cells.A. Huh7 cells were transfected with pCI-vTK and G418-resistant Huh cellular clones (cl) (1–6) were evaluated for vTK expression (∼43 KDa, arrowheads) by immunoblot analysis. Lmw, low molecular weight markers; 293TvTK, 293T cells transiently transfected with pCI-vTK. B. In vitro cytotoxicity of GCV in vTK expressing cells. HuhvTKcl1 and HuhvTKcl6 cells were incubated with various concentrations of GCV for 72 hours, followed by cell survival quantitation by MTT assay. Data represent the Mean +/− S.D from quadruplicate cell cultures for each GCV dose. Mock, Huh cells that underwent stable selection after transfection with empty pCIneo vector.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009209-g002: Functional expression of vTK in Huh7 cells.A. Huh7 cells were transfected with pCI-vTK and G418-resistant Huh cellular clones (cl) (1–6) were evaluated for vTK expression (∼43 KDa, arrowheads) by immunoblot analysis. Lmw, low molecular weight markers; 293TvTK, 293T cells transiently transfected with pCI-vTK. B. In vitro cytotoxicity of GCV in vTK expressing cells. HuhvTKcl1 and HuhvTKcl6 cells were incubated with various concentrations of GCV for 72 hours, followed by cell survival quantitation by MTT assay. Data represent the Mean +/− S.D from quadruplicate cell cultures for each GCV dose. Mock, Huh cells that underwent stable selection after transfection with empty pCIneo vector.
Mentions: Immunoblot analysis demonstrated Huh7 cellular clones (1 and 6) stably transfected with pCI-vTK expressed vTK protein (Fig. 2A). These vTK expressing cellular clones started to detach 48 h post exposure to a single dose (50 mmolL−1) GCV whereas the mock transfected cells grew and proliferated normally. Increased cell death was also assessed by MTT assay (Fig. 2B). Stable expression of vTK in Huh7 cells significantly reduced their survival rate in the presence of the toxic pro-drug GCV.

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