Extensive double humanization of both liver and hematopoiesis in FRGN mice.

Preclinical research in animals often fails to adequately predict the outcomes observed in human patients. Chimeric animals bearing individual human tissues have been developed to provide improved models of human-specific cellular processes. Mice transplanted with human hematopoietic stem cells can be used to study human immune responses, infections of blood cells and processes of hematopoiesis. Animals with humanized livers are useful for modeling hepatotropic infections as well as drug metabolism and hepatotoxicity. However, many pathophysiologic processes involve both the liver and the hematolymphoid system. Examples include hepatitis C/HIV co-infection, immune mediated liver diseases, liver injuries with inflammation such as steatohepatitis and alcoholic liver disease. We developed a robust protocol enabling the concurrent double-humanization of mice with mature hepatocytes and human blood. Immune-deficient, fumarylacetoacetate hydrolase (Fah(-/-)), Rag2(-/-) and Il2rg(-/-) deficient animals on the NOD-strain background (FRGN) were simultaneously co-transplanted with adult human hepatocytes and hematopoietic stem cells after busulfan and Ad:uPA pre-conditioning. Four months after transplantation the average human liver repopulation exceeded 80% and hematopoietic chimerism also was high (40-80% in bone marrow). Importantly, human macrophages (Kupffer cells) were present in the chimeric livers. Double-chimeric FRGN mice will serve as a new model for disease processes that involve interactions between hepatocytes and hematolymphoid cells.

Transcriptomes of the major human pancreatic cell types.

AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

The clinical picture of the Börjeson-Forssman-Lehmann syndrome in males and heterozygous females with PHF6 mutations.

The usual description of the Börjeson-Forssman-Lehmann syndrome (BFLS) is that of a rare, X-linked, partially dominant condition with severe intellectual disability, epilepsy, microcephaly, coarse facial features, long ears, short stature, obesity, gynecomastia, tapering fingers, and shortened toes. Recently, mutations have been identified in the PHF6 gene in nine families with this syndrome. The clinical history and physical findings in the affected males reveal that the phenotype is milder and more variable than previously described and evolves with age. Generally, in the first year, the babies are floppy, with failure to thrive, big ears, and small external genitalia. As schoolboys, the picture is one of learning problems, moderate short stature, with emerging truncal obesity and gynecomastia. Head circumferences are usually normal, and macrocephaly may be seen. Big ears and small genitalia remain. The toes are short and fingers tapered and malleable. In late adolescence and adult life, the classically described heavy facial appearance emerges. Some heterozygous females show milder clinical features such as tapering fingers and shortened toes. Twenty percent have significant learning problems, and 95% have skewed X inactivation. We conclude that this syndrome may be underdiagnosed in males in their early years and missed altogether in isolated heterozygous females.

Long-term therapy with NTBC and tyrosine-restricted diet in a murine model of hereditary tyrosinemia type I.

In human patients with hereditary tyrosinemia type I (HT1) a combination therapy of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexane dione (NTBC) and dietary restriction of phenylalanine and tyrosine is currently widely used. We previously reported that the use of NTBC in a murine model of HT1 abolished acute liver failure but did not prevent the development of hepatocellular carcinoma (HCC) in the setting of nonrestricted protein intake. Here we present the results obtained with higher doses of NTBC plus dietary tyrosine restriction on long-term follow up (>2 years). Liver function tests and succinylacetone levels were completely corrected with this regimen and cancer-free survival was improved when compared to historical controls. However, while no HT1 animals had HCC at age 13 months, the incidence was 2/16 (13%) at age 18 months and 1/6 (17%) after 24 months. Thus, even the most stringent therapy could not prevent the emergence of HCC in the mouse model of HT1, even when initiated prenatally.

Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells.

OBJECTIVE: Fanconi anemia (FA) is a human autosomal-recessive cancer susceptibility disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to mitomycin C (MMC). FA has at least eight complementation groups (A, B, C, D1, D2, E, F, G), and six of the FA genes have been cloned. Several FA proteins, including FANCA, FANCC, FANCF, and FANCG, interact in a nuclear complex, and this complex is required for the activation (monoubiquitination) of the downstream FANCD2 protein. Activation of FANCD2 results in the assembly of FANCD2/BRCA1 foci. The aim of this study was to analyze the FA pathway in several FA patient-derived cell lines. MATERIALS AND METHODS: We generated an antibody to FANCF and analyzed FANCF expression in human lymphoblasts corresponding to all known FA subtypes. We systematically analyzed the FA pathway (FANCD2 monoubiquitination and assembly of FANCD2 nuclear foci) in patient-derived FA-F and FA-D1 cell lines. RESULTS: FANCF protein expression is normal in cells derived from all FA complementation groups except FA-F and does not vary during cell cycle progression. FANCF, but not FANCD2, is a component of the nuclear FA protein complex and appears to stabilize other subunits of the complex. FANCF is required for the monoubiquitination of the FANCD2 protein following ionizing radiation. FANCD2 is monoubiquitinated in FA-D1 cells, even though these cells are highly sensitive to MMC. CONCLUSIONS: The recently cloned FANCF protein is required for FANCD2 activation, and the yet uncloned FANCD1 protein functions further downstream or independently of the FA pathway.

The pathophysiology and treatment of hereditary tyrosinemia type 1.

The topic of this review is hepatorenal tyrosinemia (hereditary tyrosinemia type 1 [HT1], or fumarylacetoacetate hydrolase deficiency; OMIM# 276700). HT1 is the most serious and common of the genetic defects in tyrosine degradation. In addition, this disorder has importance as a model of spontaneous self-correction of liver disease, as a model of liver repopulation by transplanted cells and gene therapy, and as a genetic cause of hepatocarcinoma. However, other forms of hypertyrosinemia exist; hence, the differential diagnosis also will be described briefly. Recent years have seen much progress in our understanding of the molecular basis, the pathophysiology, and especially the treatment of HT1. The current intervention with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) therapy has improved the outcome of this once devastating disorder. The successful repopulation of the HT1 liver with transplanted cells and positive results in the use of gene therapy in animal models may someday lead to therapy in humans that will obviate the need for life-long dietary and pharmacological therapy.

Fanconi anemia and DNA repair.

Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects. The principal cellular phenotype is hypersensitivity to DNA damage, particularly interstrand DNA crosslinks. The FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. Five of the FA proteins (FANCA, C, E, F and G) interact in a nuclear complex upstream of FANCD2. FANCB and FANCD1 have not yet been cloned, but it is likely that FANCB is part of the nuclear complex and that FANCD1 acts downstream of FANCD2. The FA nuclear complex regulates the mono-ubiquitination of FANCD2 in response to DNA damage, resulting in targeting of this protein into nuclear foci. These foci also contain BRCA1 and other DNA damage response proteins. In male meiosis, FANCD2 also co-localizes with BRCA1 at synaptonemal complexes. Together, these data suggest that the FA pathway functions primarily as a DNA damage response system, although its exact role (direct involvement in DNA repair versus indirect, facilitating role) has not yet been defined.

Loss of p27(Kip1) enhances the transplantation efficiency of hepatocytes transferred into diseased livers.

p27(Kip1) is an important regulator of cyclin-dependent kinases. Studies with p27 knockout mice have revealed abnormalities in proliferation and differentiation of multiple cell types. Here we show that primary hepatocytes isolated from livers of adult p27 knockout mice exhibit higher levels of DNA synthesis activity in culture than do wild-type cells. Interestingly, we found that, compared with control hepatocytes, p27 knockout hepatocytes proliferate better after transplantation into diseased livers to reverse liver failure. These results reveal an aspect of p27 that could be used to benefit cell-based therapy.

Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9.

OBJECTIVE: Fanconi anemia (FA) is an autosomal-recessive cancer susceptibility syndrome with seven complementation groups. Six of the FA genes have been cloned (corresponding to subtypes A, C, D2, E, F, and G) and the encoded proteins interact in a common pathway. Patient-derived mutations in FA genes have been helpful in delineating functional domains of FA proteins. The purpose of this work was to subtype FA patient-derived cell lines in our repository and to identify FA gene mutations. METHODS: We subtyped 62 FA patients as type A, G, C, or non-ACG by using a combination of retroviral gene transfer and immunoblot analysis. Among these FA patients, we identified six FA-G patients for further analysis. We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients. We functionally analyzed FANCG mutant alleles by transducing the corresponding cDNAs into a known FA-G indicator cell line and scoring correction of MMC sensitivity. RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations). Based on this mutational screen, a carboxy terminal functional domain of the FANCG protein appears to be required for complementation of FA-G cells and for normal assembly of the FANCA/FANCG/FANCC protein complex. CONCLUSION: The identification of patient-derived mutant alleles of FA genes can provide important insights to the function of FA proteins. FA subtyping is also a necessary precondition for gene therapy.

Liver repopulation for the treatment of metabolic diseases.

Orthotopic liver transplantation is the treatment of choice for several inborn errors of metabolism. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte transplantation could potentially overcome the shortage in donor livers by use of cells from a single donor for multiple recipients. In classic hepatocyte transplantation, however, only 1% of the liver mass or less can be replaced by donor cells. Recently, though, it has been shown in animal models that >90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term 'therapeutic liver repopulation'. This phenomenon is analogous to repopulation of the haematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. It has been discovered that transplanted cells from extrahepatic sources such as the adult pancreas or bone marrow can also be used for liver repopulation. Because bone marrow donors are widely available, this finding raises the hope of therapeutic application of these cells in the future. Here, the current knowledge regarding therapeutic liver repopulation and the hopeful implications for treatment of liver diseases will be discussed.

Positional cloning of a novel Fanconi anemia gene, FANCD2.

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway.

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiquitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation-induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.

Preclinical protocol for in vivo selection of hematopoietic stem cells corrected by gene therapy in Fanconi anemia group C.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, progressive bone marrow failure, and cellular hypersensitivity to DNA cross-linking agents. Bone marrow transplantation is therapeutic and therefore FA is a candidate disease for hematopoietic gene therapy. We have previously used mitomycin C (MMC) to achieve in vivo selection of wild-type hematopoietic stem cells (HSC) transplanted into FANCC knockout mice. However, clinical application of MMC in human FA gene therapy is unlikely because of its unknown toxicity profile in human FA patients. In contrast, cyclophosphamide (CPA) and gamma-irradiation (IR) are already in use with human FA patients and we therefore tested these regimens for their ability to achieve selection of genetically corrected HSCs in vivo. We found that nonmyeloablative doses of CPA or IR or combinations of CPA + IR were highly efficient at achieving in vivo selection of transplanted wild-type HSC. Furthermore, this nontoxic regimen also selected FANCC-mutant HSC corrected by ex vivo retroviral gene therapy. We suggest those nontoxic doses of CPA and/or IR could also be used to enhance gene therapy in human FA patients.

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.

Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6.

Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3.

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.

Phenotypic correction of Fanconi anemia group C knockout mice.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc -/- nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity of fancc -/- hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc -/- bone marrow cells were transduced with the use of retrovirus carrying the human fancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment of fancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells. (Blood. 2000;95:700-704)

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.

In vivo suppressor mutations correct a murine model of hereditary tyrosinemia type I.

Hereditary tyrosinemia type I and alkaptonuria are disorders of tyrosine catabolism caused by deficiency of fumarylacetoacetate hydrolase (FAH) and homogentisic acid dioxygenase (HGD), respectively. Tyrosinemia is a severe childhood disease that affects the liver and kidneys, but alkaptonuria is a more benign adult disorder in comparison. Because HGD is upstream of FAH in the tyrosine pathway, mice doubly mutant in both enzymes were found to be protected from the liver and renal damage of tyrosinemia as hypothesized. Mice mutant at the tyrosinemic locus but heterozygous for alkaptonuria spontaneously developed clonal nodules of functionally normal hepatocytes that were able to rescue the livers of some mice with this genotype. This phenotypic rescue was a result of an inactivating mutation of the wild-type homogentisic acid dioxygenase gene, thus presenting an example of an in vivo suppressor mutation in a mammalian model.