Correction of copper metabolism is not sustained long term in Wilson's disease mice post bone marrow transplantation.

PURPOSE: Alternative cell sources have been sought for the treatment of liver diseases, since liver cells are in short supply for cell transplantation. Although bone marrow-derived cells have been investigated as an alternative cell source, few studies have demonstrated long-term disease correction. Here we examined bone marrow stem cell transplantation into the toxic milk (tx) mouse model for Wilson's disease, a mild liver disease characterized by hepatic copper accumulation. The aim of this study was to determine whether bone marrow cells engrafted in the liver could sustain correction of abnormal copper metabolism in the tx mouse. METHODS: Bone marrow cells were isolated from congenic normal mice, transduced to express enhanced green fluorescent protein, sorted for stem cell (Sca-1) and lineage cell (Lin) surface markers, and then transplanted into sub-lethally irradiated normal or tx mice. Analysis for donor cell activity and distribution was undertaken 5 and 9 months post-transplant to allow for sufficient time to repopulate the liver and demonstrate disease correction. RESULTS: Donor bone marrow cells engrafted in both normal and tx mouse liver within 5 months. Significantly reduced liver copper was found in tx mice with donor cell liver engraftment at 5 months post-transplant compared to controls, demonstrating partial correction of abnormal copper metabolism in the short term. However, disease correction was not maintained 9 months post-transplantation. Lin(-)Sca-1(+) cells were more likely to partially correct disease than Lin(+)Sca-1(-) cells in the short term. CONCLUSION: These results demonstrate that bone marrow transplants cannot maintain disease correction in a mouse model of mild hepatic damage, although initial copper metabolism correction was observed.

Heterozygous tx mice have an increased sensitivity to copper loading: implications for Wilson's disease carriers.

Wilson's disease carriers constitute 1% of the human population. It is unknown whether Wilson's disease carriers are at increased susceptibility to copper overload when exposed to chronically high levels of ingested copper. This study investigated the effect of chronic excess copper in drinking water on the heterozygous form of the Wilson's disease mouse model--the toxic milk (tx) mouse. Mice were provided with drinking water containing 300 mg/l copper for 4-7, 8-11, 12-15 or 16-20 months. At the completion of the study liver, spleen, kidney and brain tissue were analyzed by atomic absorption spectroscopy to determine copper concentration. Plasma ceruloplasmin oxidase activity and liver histology were also assessed. Chronic copper loading resulted in significantly increased liver copper in both tx heterozygous and tx homozygous mice, while wild type mice were resistant to the effects of copper loading. Copper loading effects were greatest in tx homozygous mice, with increased extrahepatic copper deposition in spleen and kidney - an effect absent in heterozygote and wild type mice. Although liver histology in homozygous mice was markedly abnormal, no histological differences were noted between heterozygous and wild type mice with copper loading. Tx heterozygous mice have a reduced ability to excrete excess copper, indicating that half of the normal liver Atp7b copper transporter activity is insufficient to deal with large copper intakes. Our results suggest that Wilson's disease carriers in the human population may be at increased risk of copper loading if chronically exposed to elevated copper in food or drinking water.

Chronological changes in tissue copper, zinc and iron in the toxic milk mouse and effects of copper loading.

The toxic milk (tx) mouse is a rodent model for Wilson disease, an inherited disorder of copper overload. Here we assessed the effect of copper accumulation in the tx mouse on zinc and iron metabolism. Copper, zinc and iron concentrations were determined in the liver, kidney, spleen and brain of control and copper-loaded animals by atomic absorption spectroscopy. Copper concentration increased dramatically in the liver, and was also significantly higher in the spleen, kidney and brain of control tx mice in the first few months of life compared with normal DL mice. Hepatic zinc was increased with age in the tx mouse, but zinc concentrations in the other organs were normal. Liver and kidney iron concentrations were significantly lower at birth in tx mice, but increased quickly to be comparable with control mice by 2 months of age. Iron concentration in the spleen was significantly higher in tx mice, but was lower in 5 day old tx pups. Copper-loading studies showed that normal DL mice ingesting 300 mg/l copper in their diet for 3 months maintained normal liver, kidney and brain copper, zinc and iron levels. Copper-loading of tx mice did not increase the already high liver copper concentrations, but spleen and brain copper concentrations were increased. Despite a significant elevation of copper in the brain of the copper-loaded tx mice no behavioural changes were observed. The livers of copper-loaded tx mice had a lower zinc concentration than control tx mice, whilst the kidney had double the concentration of iron suggesting that there was increased erythrocyte hemolysis in the copper-loaded mutants.

The potential of bone marrow stem cells to correct liver dysfunction in a mouse model of Wilson's disease.

Metabolic liver diseases are excellent targets for correction using novel stem cell, hepatocyte, and gene therapies. In this study, the use of bone marrow stem cell transplantation to correct liver disease in the toxic milk (tx) mouse, a murine model for Wilson's disease, was evaluated. Preconditioning with sublethal irradiation, dietary copper loading, and the influence of cell transplantation sites were assessed. Recipient tx mice were sublethally irradiated (4 Gy) prior to transplantation with bone marrow stem cells harvested from normal congenic (DL) littermates. Of 46 transplanted tx mice, 11 demonstrated genotypic repopulation in the liver. Sublethal irradiation was found to be essential for donor cell engraftment and liver repopulation. Dietary copper loading did not improve cell engraftment and repopulation results. Both intravenously and intrasplenically transplanted cells produced similar repopulation successes. Direct evidence of functionality and disease correction following liver repopulation was observed in the 11 mice where liver copper levels were significantly reduced when compared with mice with no liver repopulation. The reversal of copper loading with bone marrow cells is similar to the level of correction seen when normal congenic liver cells are used. Transplantation of bone marrow cells partially corrects the metabolic phenotype in a mouse model for Wilson's disease.