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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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Impact of vTK/GCV on hAAT production and human hepatocyte engraftement in chimeric SCID/uPA mice.A. Baseline blood samples were obtained from experimental mice (Table 2) 1 h prior to the commencement of GCV dosing (t0) for hAAT analysis. Subsequent samples were obtained from all chimeric mice for hAAT analysis once weekly for 2 weeks with the exception of v241 which had to be euthanized after 10 days of GCV dosing. B. A correlation plot of final hAAT concentration vs. replacement index (RI) was constructed for those experimental mice (Table 2) that were severely impacted by GCV (left) and those that remained healthy during the course of dosing period (right). RI was determined as the ratio of the area occupied by Alu-positive HH relative to the entire area examined in the in situ hybridization sections expressed as percent. Data represent the mean RI +/− SEM of at least 3 separate lobes).
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pone-0009209-g007: Impact of vTK/GCV on hAAT production and human hepatocyte engraftement in chimeric SCID/uPA mice.A. Baseline blood samples were obtained from experimental mice (Table 2) 1 h prior to the commencement of GCV dosing (t0) for hAAT analysis. Subsequent samples were obtained from all chimeric mice for hAAT analysis once weekly for 2 weeks with the exception of v241 which had to be euthanized after 10 days of GCV dosing. B. A correlation plot of final hAAT concentration vs. replacement index (RI) was constructed for those experimental mice (Table 2) that were severely impacted by GCV (left) and those that remained healthy during the course of dosing period (right). RI was determined as the ratio of the area occupied by Alu-positive HH relative to the entire area examined in the in situ hybridization sections expressed as percent. Data represent the mean RI +/− SEM of at least 3 separate lobes).

Mentions: Serum hAAT levels for the majority of transplanted animals (13 of 16 mice) were higher at the end of the GCV treatment period relative to baseline (Fig. 7A). There was a good correlation between hAAT levels and replacement index (RI) for histologically integrated HH (Fig. 7B) and this did not appear to be impacted by health status since correlations were similar between chimeric mice that remained healthy and thrived during the course of GCV dosing (left) and those that were severely impacted and required euthanasia (right). These results suggest continued expansion of HH in chimeric mice during the course of GCV administration but do not exclude the involvement of GCV in a more subtle effect on engrafted HH. Therefore, increased serum levels of hAAT, ALT, and AST may reflect bystander killing of vTK-negative HH.


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)

Impact of vTK/GCV on hAAT production and human hepatocyte engraftement in chimeric SCID/uPA mice.A. Baseline blood samples were obtained from experimental mice (Table 2) 1 h prior to the commencement of GCV dosing (t0) for hAAT analysis. Subsequent samples were obtained from all chimeric mice for hAAT analysis once weekly for 2 weeks with the exception of v241 which had to be euthanized after 10 days of GCV dosing. B. A correlation plot of final hAAT concentration vs. replacement index (RI) was constructed for those experimental mice (Table 2) that were severely impacted by GCV (left) and those that remained healthy during the course of dosing period (right). RI was determined as the ratio of the area occupied by Alu-positive HH relative to the entire area examined in the in situ hybridization sections expressed as percent. Data represent the mean RI +/− SEM of at least 3 separate lobes).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009209-g007: Impact of vTK/GCV on hAAT production and human hepatocyte engraftement in chimeric SCID/uPA mice.A. Baseline blood samples were obtained from experimental mice (Table 2) 1 h prior to the commencement of GCV dosing (t0) for hAAT analysis. Subsequent samples were obtained from all chimeric mice for hAAT analysis once weekly for 2 weeks with the exception of v241 which had to be euthanized after 10 days of GCV dosing. B. A correlation plot of final hAAT concentration vs. replacement index (RI) was constructed for those experimental mice (Table 2) that were severely impacted by GCV (left) and those that remained healthy during the course of dosing period (right). RI was determined as the ratio of the area occupied by Alu-positive HH relative to the entire area examined in the in situ hybridization sections expressed as percent. Data represent the mean RI +/− SEM of at least 3 separate lobes).
Mentions: Serum hAAT levels for the majority of transplanted animals (13 of 16 mice) were higher at the end of the GCV treatment period relative to baseline (Fig. 7A). There was a good correlation between hAAT levels and replacement index (RI) for histologically integrated HH (Fig. 7B) and this did not appear to be impacted by health status since correlations were similar between chimeric mice that remained healthy and thrived during the course of GCV dosing (left) and those that were severely impacted and required euthanasia (right). These results suggest continued expansion of HH in chimeric mice during the course of GCV administration but do not exclude the involvement of GCV in a more subtle effect on engrafted HH. Therefore, increased serum levels of hAAT, ALT, and AST may reflect bystander killing of vTK-negative HH.

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