Muhsen Al-Dhalimy

Purified hematopoietic stem cells can differentiate into hepatocytes in vivo

The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH−/− mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.

Cell fusion is the principal source of bone-marrow-derived hepatocytes.

Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.

Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I

Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer

Robust expansion of human hepatocytes in Fah −/− / Rag2 −/− / Il2rg −/− mice

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.

Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2–(2–nitro–4–trifluoro–methylbenzyol)–1,3–cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH−/− mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

Kinetics of Liver Repopulation after Bone Marrow Transplantation

Recent work has convincingly demonstrated that adult bone marrow contains cells capable of differentiating into liver epithelial cells in vivo. However, the frequency and time course with which fully functional hepatocytes emerge after bone marrow transplantation remained controversial. Here, we used the fumarylacetoacetate hydrolase knockout mouse to determine the kinetics of hepatocyte replacement after complete hematopoietic reconstitution. Single donor-derived hepatocytes were first detected 7 weeks after lethal irradiation and bone marrow transplantation. Liver disease was not required for this transdifferentiation. In the presence of selective pressure the single cells evolved into hepatocyte nodules by 11 weeks after transplantation and resulted in >30% overall liver repopulation by 22 weeks. The frequency with which hepatocytes were produced was between 10(-4) and 10(-6), resulting in only 50 to 500 repopulation events per liver. Hepatic engraftment was not observed without previous hematopoietic reconstitution even in the presence of liver injury. In addition, significant liver repopulation was completely dependent on hepatocyte growth selection. We conclude that hepatocyte replacement by bone marrow cells is a slow and rare event. Significant improvements in the efficiency of this process will be needed before clinical success can be expected.

The Repopulation Potential of Hepatocyte Populations Differing in Size and Prior Mitotic Expansion

Recently the stem cell-like regenerative potential of adult liver cells was demonstrated by serial transplantation. This repopulation capacity could be useful for the treatment of genetic liver diseases by cell transplantation and/or expansion of genetically manipulated cells. However, previous experiments used unfractionated populations of liver cells, and therefore it remained undetermined whether all hepatocytes or only a subpopulation (stem cells) possessed this high regenerative ability. To address this question we used centrifugal elutriation to separate hepatocytes by cell density. Unexpectedly, small hepatocytes (16 microm) had lower repopulation capacity during the first round of transplantation when compared with both the medium-sized (21 microm) and large (27 microm) cells. We also compared the repopulation capacity of hepatocytes that had undergone different degrees of in vivo expansion. Previous cell division neither reduced nor increased the repopulation capacity of transplanted liver cells. Finally, retroviral tagging experiments demonstrated that liver-repopulating cells occur at a frequency of >1:10,000. We conclude that short-term therapeutic liver repopulation does not require progenitor or stem cells.

A Single Mutation of the Fumarylacetoacetate Hydrolase Gene in French Canadians with Hereditary Tyrosinemia Type I

BACKGROUND Hereditary tyrosinemia type I is an autosomal recessive inborn error of metabolism caused by a deficiency of the enzyme fumarylacetoacetate hydrolase. The disorder clusters in the Saguenay-Lac-St.-Jean area of Quebec. In this region, 1 of 1846 newborns is affected and 1 of every 22 persons is thought to be a carrier. Recently, we identified a splice mutation and two nonsense mutations in the fumarylacetoacetate hydrolase gene in two patients from Quebec with tyrosinemia type I. METHODS We used allele-specific-oligonucleotide hybridization to examine the frequency of these three candidate mutations in patients with tyrosinemia type I and in the population of Quebec. RESULTS The splice mutation was found in 100 percent of patients from the Saguenay-Lac-St.-Jean area and in 28 percent of patients from other regions of the world. Of 25 patients from the Saguenay-Lac-St.-Jean region, 20 (80 percent) were homozygous for this mutation, a guanine-to-adenine change in the splice-donor sequence in intron 12 of the gene, indicating that it causes most cases of tyrosinemia type I in the region. The frequency of carrier status, based on screening of blood spots from newborns, was about 1 per 25 in the Saguenay-Lac-St.-Jean population and about 1 per 66 overall in Quebec. CONCLUSIONS This study identified the most prevalent mutation causing hereditary tyrosinemia in French Canada; it also showed the feasibility of DNA-based testing for carriers in the population at risk.

Liver Repopulation and Correction of Metabolic Liver Disease by Transplanted Adult Mouse Pancreatic Cells

The emergence of cells with hepatocellular properties in the adult pancreas has been described in several experimental models. To determine whether adult pancreas contains cells that can give rise to therapeutically useful and biochemically normal hepatocytes, we transplanted suspensions of wild-type mouse pancreatic cells into syngeneic recipients deficient in fumarylacetoacetate hydrolase and manifesting tyrosinemia. Four of 34 (12%) mutant mice analyzed were fully rescued by donor-derived cells and had normal liver function. Ten additional mice (29%) showed histological evidence of donor-derived hepatocytes in the liver. Previous work has suggested that pancreatic liver precursors reside within or close to pancreatic ducts. We therefore performed additional transplantations using either primary cell suspensions enriched for ducts or cultured ducts. Forty-four mutant mice were transplanted with cells enriched for pancreatic duct cells, but only three of the 34 (9%) recipients analyzed displayed donor-derived hepatocytes. In addition, 28 of the fumarylacetoacetate hydrolase-deficient mice were transplanted with cultured pancreatic duct cells, but no donor-derived hepatocytes were observed. Our results demonstrate for the first time that adult mouse pancreas contains hepatocyte progenitor cells capable of significant therapeutic liver reconstitution. However, contrary to previous reports, we were unable to detect these cells within the duct compartment.

Chronic liver disease in murine hereditary tyrosinemia type 1 induces resistance to cell death

The murine model of hereditary tyrosinemia type 1 (HT1) was used to analyze the relationship between chronic liver disease and programmed cell death in vivo. In healthy fumarylacetoacetate hydrolase deficient mice (Fah‐/‐), protected from liver injury by the drug 2‐(2‐ nitro‐4‐trifluoromethylbenzoyl)‐1,3‐cyclohexanedione (NTBC), the tyrosine metabolite homogentisic acid (HGA) caused rapid hepatocyte death. In contrast, all mice survived the same otherwise lethal dose of HGA if they had preexisting liver damage induced by NTBC withdrawal. Similarly, Fah‐/‐ animals with liver injury were also resistant to apoptosis induced by the Fas ligand Jo‐2 and to necrosis‐like cell death induced by acetaminophen (APAP). Molecular studies revealed a marked up‐regulation of the antiapoptotic heat shock proteins (Hsp) 27, 32, and 70 and of c‐Jun in hepatocytes of stressed mice. In addition, the p38 and Jun N‐terminal kinase (JNK) stress‐activated kinase pathways were markedly impaired in the cell‐death resistant liver. In conclusion, these results provide evidence that chronic liver disease can paradoxically result in cell death resistance in vivo. Stress‐induced failure of cell death programs may lead to an accumulation of damaged cells and therefore enhance the risk for cancer as observed in HT1 and other chronic liver diseases. (HEPATOLOGY 2004;39:433–443.)