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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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Tissue distribution of vTK in transgenic mice.A. Expression of vTK protein (47 kDa) in various tissues from experimental FVB/N mice (listed in Table 1) were examined by immunoblot analysis (as shown for vTK+ mice 3.3, 3.21 and 3.11 and vTK− mice 3.18, 3.12, and 3.12, left panel). A smaller vTK immunoreactive protein (∼39 kDa) was identified in the testis of vTK+ mice. (right panel). B. Expression of vTK mRNA was evaluated in several tissues obtained from vTK+ mice (as shown for 3.3, left). Expression of delta-vTK mRNA was evaluated in the liver and testis of vTK+ (as shown fo 3.3, right) and vTK− (as shown fo 3.2, right) mice.
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pone-0009209-g003: Tissue distribution of vTK in transgenic mice.A. Expression of vTK protein (47 kDa) in various tissues from experimental FVB/N mice (listed in Table 1) were examined by immunoblot analysis (as shown for vTK+ mice 3.3, 3.21 and 3.11 and vTK− mice 3.18, 3.12, and 3.12, left panel). A smaller vTK immunoreactive protein (∼39 kDa) was identified in the testis of vTK+ mice. (right panel). B. Expression of vTK mRNA was evaluated in several tissues obtained from vTK+ mice (as shown for 3.3, left). Expression of delta-vTK mRNA was evaluated in the liver and testis of vTK+ (as shown fo 3.3, right) and vTK− (as shown fo 3.2, right) mice.

Mentions: Various tissues from vTK+FVB/N mice underwent immunoblot and RT-PCR analysis for vTK expression (Fig. 3). Full-length vTK protein (47 kDa) was detected exclusively in the livers of vTK+ mice and was absent from vTK- mice (Fig. 3A, left). A smaller immunoreactive protein was detected in the testis of vTK+ mice (Fig. 3A, right) but was absent from testis from vTK- mice (not shown). RT-PCR for vTK (Fig. 3B, left) and delta-vTK (Fig. 3B, right) confirmed the presence of full length vTK mRNA in the liver and delta-vTK mRNA in the testis from vTK+ mice (as shown for mouse vTK+ mouse 3.3). The detection of a similar amplicon in heart was found to be due to genomic DNA contamination of the total RNA prepared from heart tissue since this amplicon disappeared when the total RNA was treated with DNase (not shown). An amplicon corresponding to delta-vTK was also detected in the liver of vTK+ mice and was expected since the primers for delta-vTK RT-PCR are expected to amplify cDNA generated from full length vTK transcripts (refer to Materials and Methods section). Transcripts for delta-vTK were absent in the testis and liver from vTK- mice (B, right for vTK- mouse 3.2).


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)

Tissue distribution of vTK in transgenic mice.A. Expression of vTK protein (47 kDa) in various tissues from experimental FVB/N mice (listed in Table 1) were examined by immunoblot analysis (as shown for vTK+ mice 3.3, 3.21 and 3.11 and vTK− mice 3.18, 3.12, and 3.12, left panel). A smaller vTK immunoreactive protein (∼39 kDa) was identified in the testis of vTK+ mice. (right panel). B. Expression of vTK mRNA was evaluated in several tissues obtained from vTK+ mice (as shown for 3.3, left). Expression of delta-vTK mRNA was evaluated in the liver and testis of vTK+ (as shown fo 3.3, right) and vTK− (as shown fo 3.2, right) mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009209-g003: Tissue distribution of vTK in transgenic mice.A. Expression of vTK protein (47 kDa) in various tissues from experimental FVB/N mice (listed in Table 1) were examined by immunoblot analysis (as shown for vTK+ mice 3.3, 3.21 and 3.11 and vTK− mice 3.18, 3.12, and 3.12, left panel). A smaller vTK immunoreactive protein (∼39 kDa) was identified in the testis of vTK+ mice. (right panel). B. Expression of vTK mRNA was evaluated in several tissues obtained from vTK+ mice (as shown for 3.3, left). Expression of delta-vTK mRNA was evaluated in the liver and testis of vTK+ (as shown fo 3.3, right) and vTK− (as shown fo 3.2, right) mice.
Mentions: Various tissues from vTK+FVB/N mice underwent immunoblot and RT-PCR analysis for vTK expression (Fig. 3). Full-length vTK protein (47 kDa) was detected exclusively in the livers of vTK+ mice and was absent from vTK- mice (Fig. 3A, left). A smaller immunoreactive protein was detected in the testis of vTK+ mice (Fig. 3A, right) but was absent from testis from vTK- mice (not shown). RT-PCR for vTK (Fig. 3B, left) and delta-vTK (Fig. 3B, right) confirmed the presence of full length vTK mRNA in the liver and delta-vTK mRNA in the testis from vTK+ mice (as shown for mouse vTK+ mouse 3.3). The detection of a similar amplicon in heart was found to be due to genomic DNA contamination of the total RNA prepared from heart tissue since this amplicon disappeared when the total RNA was treated with DNase (not shown). An amplicon corresponding to delta-vTK was also detected in the liver of vTK+ mice and was expected since the primers for delta-vTK RT-PCR are expected to amplify cDNA generated from full length vTK transcripts (refer to Materials and Methods section). Transcripts for delta-vTK were absent in the testis and liver from vTK- mice (B, right for vTK- mouse 3.2).

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