Artigo Acesso aberto Revisado por pares

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

2001; Elsevier BV; Volume: 158; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63999-5

ISSN

1525-2191

Autores

Xin Wang, Muhsen Al-Dhalimy, Eric Lagasse, Milton J. Finegold, Markus Grompe,

Tópico(s)

Liver physiology and pathology

Resumo

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. 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. In embryonic development the liver and pancreas both originate from the same location in the ventral foregut.1Zaret KS Molecular genetics of early liver development.Annu Rev Physiol. 1996; 58: 231-251Crossref PubMed Scopus (98) Google Scholar, 2Zaret KS Liver specification and early morphogenesis.Mech Dev. 2000; 92: 83-88Crossref PubMed Scopus (151) Google Scholar, 3Gualdi R Bossard P Zheng M Hamada Y Coleman JR Zaret KS Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control.Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (474) Google Scholar, 4Spooner BS Walther BT Rutter WJ The development of the dorsal and ventral mammalian pancreas in vivo and in vitro.J Cell Biol. 1970; 47: 235-246Crossref PubMed Scopus (97) Google Scholar, 5Rutter WJ The development of the endocrine and exocrine pancreas.Monogr Pathol. 1980; 21: 30-38PubMed Google Scholar The liver bud develops anteriorly toward the cardiac mesenchyme beginning at embryonic day 8.5 in the mouse. The pancreas has dorsal and ventral lobes with the dorsal bud growing in a posterior direction. The ventral lobe, however, develops more anteriorly and shares part of its ductal system with the liver. Because hepatocytes, bile ducts, pancreatic ducts, and exocrine and endocrine pancreatic cells are all endodermal in origin and because of the anatomical proximity of the developing liver and ventral pancreas, it is possible that all of these cell types are the offspring of a common stem cell. If this hypothesis is correct, adult animals might also have such hepatopancreatic stem cells with a broad differentiation potential for endodermal lineages, including hepatocytes and β-cells. Several experimental models have indeed supported the hypothesis that multipotent stem cells persist in the adult pancreas and can give rise to a variety of differentiated offspring. In transgenic mice expressing interferon-γ under the transcriptional control of the insulin promoter, new β cells are generated de novo throughout the life of the animal from cells that reside within or close to the pancreatic ducts.6Gu D Sarvetnick N Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice.Development. 1993; 118: 33-46PubMed Google Scholar, 7Gu D Arnush M Sawyer SP Sarvetnick N Transgenic mice expressing IFN-gamma in pancreatic beta-cells are resistant to streptozotocin-induced diabetes.Am J Physiol. 1995; 269: E1089-E1094PubMed Google Scholar Another indication of the existence of pancreatic endodermal stem cells is the emergence of hepatocytes in the adult. The best known example is the appearance of hepatocytes in copper-depleted rats after re-feeding of copper.8Rao MS Subbarao V Reddy JK Induction of hepatocytes in the pancreas of copper-depleted rats following copper repletion.Cell Differ. 1986; 18: 109-117Crossref PubMed Scopus (99) Google Scholar, 9Rao MS Dwivedi RS Subbarao V Usman MI Scarpelli DG Nemali MR Yeldandi A Thangada S Kumar S Reddy JK Almost total conversion of pancreas to liver in the adult rat: a reliable model to study transdifferentiation.Biochem Biophys Res Commun. 1988; 156: 131-136Crossref PubMed Scopus (92) Google Scholar In this system, weanling rats are fed a copper-free diet for 8 weeks that leads to complete acinar atrophy. When they are re-fed copper, cells with multiple hepatocellular characteristics emerge from the remaining pancreatic ducts within weeks. These cells have hepatocyte morphology and express a variety of hepatocyte markers, such as albumin. This work therefore suggested the presence of a pancreatic liver progenitor cell in or close to the pancreatic duct.10Rao MS Yeldandi AV Reddy JK Stem cell potential of ductular and periductular cells in the adult rat pancreas.Cell Differ Dev. 1990; 29: 155-163Crossref PubMed Scopus (36) Google Scholar Copper depletion alone, without re-feeding, results in acinar atrophy and the proliferation of cells very similar to hepatic oval cells.11Dabeva MD Hwang SG Vasa SR Hurston E Novikoff PM Hixson DC Gupta S Shafritz DA Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver.Proc Natl Acad Sci USA. 1997; 94: 7356-7361Crossref PubMed Scopus (180) Google Scholar On transplantation into the liver, these pancreatic oval cells can differentiate and display multiple characteristics of hepatocytes.11Dabeva MD Hwang SG Vasa SR Hurston E Novikoff PM Hixson DC Gupta S Shafritz DA Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver.Proc Natl Acad Sci USA. 1997; 94: 7356-7361Crossref PubMed Scopus (180) Google Scholar More recently, specific cytokines have been identified as candidates to be involved in this process. Transgenic mice in which the keratinocyte growth factor gene is driven by insulin promoter consistently develop pancreatic hepatocytes.12Krakowski ML Kritzik MR Jones EM Krahl T Lee J Arnush M Gu D Sarvetnick N Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells.Am J Pathol. 1999; 154: 683-691Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar The anatomical location of pancreatic stem cells has been determined to be in or near the ducts in both β-cell neogenesis and hepatocyte generation6Gu D Sarvetnick N Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice.Development. 1993; 118: 33-46PubMed Google Scholar, 10Rao MS Yeldandi AV Reddy JK Stem cell potential of ductular and periductular cells in the adult rat pancreas.Cell Differ Dev. 1990; 29: 155-163Crossref PubMed Scopus (36) Google Scholar Other work has also supported a direct role of pancreatic ducts in these processes. Cultured pancreatic duct cells were transplanted subcutaneously in the rat and then displayed hepatocyte markers such as albumin and α-fetoprotein.13Chen JR Tsao MS Duguid WP Hepatocytic differentiation of cultured rat pancreatic ductal epithelial cells after in vivo implantation.Am J Pathol. 1995; 147: 707-717PubMed Google Scholar Thus, pancreatic duct epithelium itself has been considered to represent a facultative stem cell by some investigators.10Rao MS Yeldandi AV Reddy JK Stem cell potential of ductular and periductular cells in the adult rat pancreas.Cell Differ Dev. 1990; 29: 155-163Crossref PubMed Scopus (36) Google Scholar, 14Ramiya VK Maraist M Arfors KE Schatz DA Peck AB Cornelius JG Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.Nat Med. 2000; 6: 278-282Crossref PubMed Scopus (676) Google Scholar Although the earlier work described above had shown the existence of pancreas-derived cells expressing hepatocyte markers, it remains unknown whether these cells are fully functional and therefore therapeutically useful. Although pancreatic liver precursors seemed to be associated with the ducts, it remains unclear whether the ducts themselves contained the hepatocyte precursors or whether they are only anatomically close to the ducts (periductular). To address these questions, we tested adult murine pancreatic cells in the fumarylacetoacetate hydrolase (FAH) knockout liver repopulation model.15Overturf K Al-Dhalimy M Tanguay R Brantly M Ou CN Finegold M Grompe M Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1996; 12: 266-273Crossref PubMed Scopus (500) Google Scholar, 16Overturf K Al-Dhalimy M Ou CN Finegold M Grompe M Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.Am J Pathol. 1997; 151: 1273-1280PubMed Google Scholar, 17Overturf K Al-Dhalimy M Finegold M Grompe M The repopulation potential of hepatocyte populations differing in size and prior mitotic expansion.Am J Pathol. 1999; 155: 2135-2143Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar Here we report that pancreatic cells from adult mice contain hepatocyte progenitor cells that can significantly repopulate the livers in fumarylacetoacetate hydrolase-deficient (FAH−) mice15Overturf K Al-Dhalimy M Tanguay R Brantly M Ou CN Finegold M Grompe M Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1996; 12: 266-273Crossref PubMed Scopus (500) Google Scholar, 18Grompe M Al-Dhalimy M Finegold M Ou CN Burlingame T Kennaway NG Soriano P Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice.Genes Dev. 1993; 7: 2298-2307Crossref PubMed Scopus (303) Google Scholar, 19Grompe M Laconi E Shafritz DA Principles of therapeutic liver repopulation.Semin Liver Dis. 1999; 19: 7-14Crossref PubMed Scopus (83) Google Scholar and reconstitute normal liver function. Furthermore, we provide evidence that these pancreatic liver progenitor cells are not the ducts themselves. As transplant recipients we used the FAHΔexon 5 strain mice previously described by this lab.18Grompe M Al-Dhalimy M Finegold M Ou CN Burlingame T Kennaway NG Soriano P Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice.Genes Dev. 1993; 7: 2298-2307Crossref PubMed Scopus (303) Google Scholar Transplant donors were transgenic ROSA-26 mice, a gift from P. Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA).20Friedrich G Soriano P Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.Genes Dev. 1991; 5: 1513-1523Crossref PubMed Scopus (1195) Google Scholar All transplantation experiments were performed with congenic mice of the 129SvJ background. All FAH mutant animals were treated with NTBC-containing drinking water at a concentration of 7.5 mg/L (a gift from S. Lindstedt, Gøtheborg, Sweden).21Lindstedt S Holme E Lock EA Hjalmarson O Strandvik B Treatment of hereditary tyrosinaemia type I by inhibition of 4-hydroxyphenylpyruvate dioxygenase.Lancet. 1992; 340: 813-817Abstract PubMed Scopus (544) Google Scholar, 22Grompe M Lindstedt S Al-Dhalimy M Kennaway NG Papaconstantinou J Torres-Ramos CA Ou CN Finegold M Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1995; 10: 453-460Crossref PubMed Scopus (269) Google Scholar This provides an approximate dose of 1 mg kg−1 body weight per day. For genotyping, polymerase chain reaction (PCR) was performed with a 3 primer PCR on 200-ng tail-cut DNA as previously described. Animal care and experiments were all in accordance with the Guidelines of the Department of Animal Care at Oregon Health Sciences University. Whole pancreatic cells were isolated from adult (>3 months) male wild-type mice transgenic for Escherichia colilacZ with a two step protease digestion. Briefly, the pancreas was dissected from sacrificed mice carefully avoiding injury of the liver. The harvested pancreas was immediately cut into small pieces and digested by collagenase D (Roche, Indianapolis, IN) (2.5 mg/ml, dissolved in Earle's basic salt solution) for 25 minutes at 37°C with agitation by a magnetic stir bar. The cells were then pelleted by centrifugation, washed once with calcium-free phosphate-buffered saline (PBS), and then further digested in trypsin ethylenediaminetetraacetic acid (0.05% w/v) (Life Technologies, Inc., Rockville, MD) for 3 minutes. The proteases were neutralized by addition of 3 volumes of Dulbecco's minimal essential medium (DMEM) with 10% bovine serum. Next, the digested cell mix was filtered through an 85-μm nylon mesh twice and the cells that came through the filter were collected. Cell number and viability were determined by Trypan blue exclusion staining under a hemocytometer. For pancreatic duct cell enrichment, a different protease digestion protocol was used. The chopped pancreas was first partially digested by collagenase D (Roche) (2.5 mg/ml, dissolved in Earle's basic salt solution) for 20 minutes. The digestion was then filtered through a 85-μm nylon mesh. The cells retained on the mesh, enriched for ducts, were collected and then further digested under the same conditions for another 20 minutes. Mouse pancreatic ducts were cultured by a published method23Githens S Schexnayder JA Moses RL Denning GM Smith JJ Frazier ML Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures.In Vitro Cell Dev Biol Anim. 1994; 30: 622-635Crossref Scopus (40) Google Scholar with some modifications. Briefly, the harvested pancreas was dissected from the animal and immediately chopped into small pieces. The tissue was then partially digested by collagenase D (0.7 mg/ml, Roche) for 20 minutes. The digestion mix was sieved through a nylon mesh attached to a glass funnel to remove the predominant acinar tissue. The nylon mesh with attached duct tissue was then cut from the funnel using sterile procedures and embedded in rat-tail collagen gel. The preparation of rat collagen was modified from a previous report.24Richards J Larson L Yang J Guzman R Tomooka Y Osborn R Nandis WI Method for culturing mammary epithelial cells in a rat tail collagen gel matrix.J Tissue Culture Methods. 1983; 8: 31-36Crossref Scopus (106) Google Scholar Briefly, the rat tail was harvested and sterilized in 70% ethanol. Then, the collagenous fibers of the tail were collected, weighed, and dissolved in 0.1% acetic acid (4.8 g/L) by stirring at 4°C for 3 to 5 days. The dissolved collagen solution was centrifuged at 10,000 × g at 4°C to remove the debris and sterilized by γ-irradiation (150 Gy). Before use, the acidic collagen stock solution was tested for its ability to form a gel at neutral pH. Eight times DMEM/F12 (Life Technologies, Inc.) medium was prepared, and two parts of 8× medium was combined with one part of serial NaOH stock solutions from 0.30 mol/L to 0.42 mol/L with 0.01 mol/L increments to make the serial neutralizing solutions. Then, one part of each of neutralizing solutions was mixed with 4.25 parts of acidic rat collagen stock solution. Each mixture was plated on each well of a 24-well culture plate. The plate was transferred to a tissue culture incubator with 5% CO2 and to equilibrate overnight. The desired result was neutral pH (based on the medium color) and a solid gel. The cultures were fed with a DMEM/F12 (1:1) medium supplemented with 5% Nu-serum V (Collaborative Biomed, Bedford, MA), gentamicin (5 mg/L), and soybean trypsin inhibitor (100 mg/L; Sigma Chemical Co., St. Louis, MO), and a variety of factors including insulin (2.6 mg/L, Life Technologies, Inc.), murine epithelial growth factor (10 μg/L, Life Technologies, Inc.), dexamethasone (1 μmol/L, Sigma), and cholera toxin (100 μg/L, Life Technologies, Inc.). Half of the medium was exchanged every 3 days. After reaching confluency duct epithelial cells growing in the mesh were harvested by collagenase D (2.5 mg/ml, Roche) for 1 hour. The digestion was sieved by 85-μm size nylon mesh to collect single cells. The number and viability of harvested whole pancreatic cells, enriched duct cells, and cultured duct cells were determined by Trypan blue exclusion in a hemocytometer. The appropriate number of donor cells were resuspended in 100 μl of Dulbecco's minimal essential media (Life Technologies, Inc.) with 10% fetal bovine serum and injected intrasplenically or directly into the portal vein of FAH mutant female recipient animals. All FAH mutant transplant recipients were kept on NTBC until the time of transplantation. One day after transplantation they were switched to regular drinking water, not containing NTBC. The weight of experimental animals was measured weekly. Samples from animals were obtained as follows. Animals were sacrificed by decapitation and blood collected by dabbing the wound onto parafilm (American National Can, Menasha, WI). For anticoagulation, the blood was immediately mixed with 10 μl of Na-heparin (Becton-Dickinson, Franklin Lakes, NJ) using a Pipetman. The red blood cells were removed by a brief centrifugation and the plasma was frozen at −80°C. Twenty μl of plasma were mixed with 80 μl of a solution of 7% bovine serum albumin and assayed for aspartate serine aminotransferase, bilirubin, and creatinine levels with a Kodak Ektachem 700 chemistry analyzer (Eastman Kodak, Rochester, NY). Quantitative serum amino acid analyses were performed on a Beckman 6300 amino acid analyzer using published methodology.25Sturman JA Applegarth DA Automated amino acid analysis.in: Boulton AA Baker GB Wood JD Neuromethods. vol. 3. Humana Press Inc., Totowa, NJ1985Google Scholar Plasma succinylacetone levels in plasma were measured by a δ-aminolevulinate dehydratase inhibition assay as described.26Grenier A Lescault A Succinylacetone.in: Bergmeyer HU Methods of Enzymatic Analysis. VCH Verlagsgesellschaft, Weinheim1985: 79Google Scholar FAH enzyme assays were performed at 30°C on a cytosolic fraction of homogenized liver as described previously.27Knox WE Edwards SW Enzymes involved in conversion of tyrosine to acetoacetate.Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar Fumarylacetoacetate, the substrate for the assay, is not commercially available and was prepared enzymatically from homogentisic acid as described in the same reference. Protein concentrations were measured with a Bio-Rad kit (Bio-Rad, Richmond, CA).28Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (219357) Google Scholar Liver tissues fixed in 10% phosphate-buffered formalin, pH 7.4, were dehydrated in 100% ethanol and embedded in paraffin wax at 58°C. Four-μm sections were rehydrated and stained with hematoxylin and eosin and with a polyclonal rabbit antibody to rat FAH (graciously provided by Robert Tanguay, University of Laval, Quebec, Canada) or glutamine synthetase.29Smith DDJ Campbell JW Distribution of glutamine synthetase and carbamoyl-phosphate synthetase I in vertebrate liver.Proc Natl Acad Sci USA. 1988; 85: 160-164Crossref PubMed Scopus (75) Google Scholar The antibody was diluted in PBS, pH 7.4, and applied at concentrations of 1:300,000 at 37°C for 30 minutes. The glutamine synthetase antibody was used at a dilution of 1:10,000. Endogenous peroxidase activity was blocked with 3% H2O2 and methanol. Avidin and biotin pretreatment was used to prevent endogenous staining. The secondary antibody was biotinylated goat anti-rabbit IgG used at 1:250 dilution (BA-1000; Vector Laboratories, Burlingame, CA). Color development was performed with the AEC detection kit (catalogue no. 250–020; Ventana Medical Systems, Tucson, AZ). β-galactosidase staining on the harvested liver tissues was performed. The liver tissues were fixed in 2% formaldehyde and 0.2% glutaraldehyde in PBS for 30 minutes. Then the tissues were washed with PBS for two times and moved to staining medium that contained 1 mg X-gal (Research Products International, Prospect, IL) in buffer of 5 mmol/L K ferricyanide, 5 mmol/L K ferrocyanide, 2 mmol/L MgCl2, 0.02% Nonidet P-40 (all Sigma), and 40 mmol/L Hepes (pH 8.0; Life Technologies). The staining incubation was at 37°C overnight. β-galactosidase histochemistry in frozen sections was performed as previously described.16Overturf K Al-Dhalimy M Ou CN Finegold M Grompe M Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.Am J Pathol. 1997; 151: 1273-1280PubMed Google Scholar The 8- to 10-μm-thick sections of OCT-embedded liver were fixed with 1.25% glutaraldehyde in ice-cold PBS for 10 minutes and stained overnight. For detection of male cells, in situ hybridization was performed with a digoxigenin-labeled high-copy number Y chromosome-specific repeat DNA probe as previously described.30Eckert JW Buerkle CJ Major AM Finegold MJ Brandt ML In situ hybridization utilizing a Y chromosome DNA probe. Use as a cell marker for hepatocellular transplantation.Transplantation. 1995; 59: 109-111Crossref PubMed Scopus (9) Google Scholar For electron microscopy, the cultured duct cells were harvested by collagenase D digestion. The cells were fixed in 3% glutaraldehyde buffered in 0.1 mol/L Na-cacodylate (pH 7.4). The cell samples were then postosmicated, embedded in araldite, sectioned on an AO-Reichert ultracut E microtome, and stained with uranyl acetate and lead citrate. Sections were examined in a Joel 100-CX microscope at magnifications ranging from ×800 to ×12,000. Total cellular RNA of pancreatic duct cells were isolated from harvested pancreatic tissues and pancreatic duct cell cultures.31Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar RT-PCR and Northern blots were performed according to standard protocols.31Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar For the detection of the pancreatic duct markers carbonic anhydrase type II (CAII) and cystic fibrosis transmembrane regulator (CFTR), specific primers for both cDNA fragments were generated based on the published CAII (GenBank accession, K00811) and CFTR (GenBank accession, M69289) sequences. The primer pairs for CAII were: forward 5′-GGAGACCGGCAGTCCCCTGT-3′ and reverse 5′-AGAGAGGCGGTCACACTTGT-3′. The primer pairs for CFTR were: forward 5′ACCCTTGTGGATGGGGGTTATGTGC-3′ and reverse 5′-CATGGGTTCTGGGAATGGACTC-3′. RT was performed by using 2 μg of total RNA samples with random hexamers and M-MLV reverse transcriptase (Life Technologies, Inc.). The PCR conditions were: 94°C for 5 minutes; 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, for 35 cycles; 72°C for 10 minutes for ending. After RT-PCR, the DNA fragments were resolved on a 0.8% agarose gel. To further estimate the relative quantities of CFRT mRNA in different cells we used a 4-primer RT-PCR with the housekeeping gene APRT (adenine phosphoribosyltransferase) as an internal control. The primer pairs for APRT were: forward 5′-AGCGTGCTGATACCTACCTC-3′ and reverse 5′-AAGCAGTTCCTAGTGCTGCT-3′. The PCR conditions were: four primers added in a single reaction, including 40 ng of each CFTR primers and 200 ng of APRT primers in 25 μl reaction; 94°C for 5 minutes for starting; 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, for 28 cycles; 72°C for 10 minutes for ending. After RT-PCR, the DNA fragments were resolved on a 1.5% agarose gel. Northern blots were probed with both RT-PCR generated cDNA fragments as probes. Fifteen μg of total RNA was separated on the formaldehyde denature gel and transferred onto Hybond-N nylon membrane. The hybridization was performed in 50% formamide hybridization solution the α-32P-dCTP labeled probes (5 × 106 cpm/ml) at 42°C for 18 hours. The radioactive signals were shown by the exposure on the X-ray film. To test the hypothesis that pancreas contains a population of liver progenitor cells, we harvested cells from whole pancreas of adult (>3 months) male, FAH wild-type ROSA-26 mice transgenic for an ubiquitously expressed E. coli lacZ gene.20Friedrich G Soriano P Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.Genes Dev. 1991; 5: 1513-1523Crossref PubMed Scopus (1195) Google Scholar A single cell suspension was transplanted into 10-week-old female FAH− mutant congenic recipients. Three to five × 105 crude pancreatic cells were injected into the spleens of the FAH− recipients and NTBC treatment was discontinued immediately after transplantation to permit selection of FAH+ hepatocytes.15Overturf K Al-Dhalimy M Tanguay R Brantly M Ou CN Finegold M Grompe M Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1996; 12: 266-273Crossref PubMed Scopus (500) Google Scholar Our previous work with hepatocyte transplantation has shown that liver repopulation is only partial at 4 weeks of selection and is near-complete after 8 weeks.15Overturf K Al-Dhalimy M Tanguay R Brantly M Ou CN Finegold M Grompe M Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1996; 12: 266-273Crossref PubMed Scopus (500) Google Scholar Even nontransplanted FAH mutants typically survive 6 to 8 weeks off NTBC.22Grompe M Lindstedt S Al-Dhalimy M Kennaway NG Papaconstantinou J Torres-Ramos CA Ou CN Finegold M Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1995; 10: 453-460Crossref PubMed Scopus (269) Google Scholar Therefore, transplanted recipients were divided into two groups for repopulation analysis: one group was harvested at 4 weeks after transplantation, whereas the other group was harvested after 8 weeks. In total, 68 FAH mutants were transplanted in 12 independent experiments. During the selection period, weights of the transplant recipients were followed weekly and all recipients lost weight during the first 4 weeks of transplantation. In the early harvesting group with a total of 28 recipients, nodules of FAH+ hepatocytes were found in 10 (29%) of the transplanted FAH− recipients. In these animals, only a single 4- to 5-μm liver section representing <0.1% of the total liver was analyzed. The FAH+ nodules were small with 6 to 20 cells visible in cross-section, indicating that three-dimensionally each clone contained ∼10 to 50 cells (Figure 1A). In the 8-week-time (late) observation group of 40 recipients, most of transplanted FAH− recipients died with tyrosinemic symptoms for liver failure before 6 weeks, ie, before the scheduled harvesting time. However, six of 40 transplanted mice regained the originally lost weight and completely recovered at 6 to 8 weeks after transplantation. Of these harvested at 8 weeks, four recipients (generated in three independent transplantation experiments) had from 50 to 90% FAH+ hepatocyte repopulation as shown by the immunohistochemistry (Figure 1B). The other two recipients had developed hepatocarcinoma thus making evaluation of liver function impossible. The carcinomas originated in recipient tissue, not transplanted donor cells, as has been reported previously reported in the FAH knockout model.32Overturf K Al-Dhalimy M Ou CN Finegold M Tanguay R Lieber A Kay M Grompe M Adenovirus-mediated gene therapy in a mouse model of hereditary tyrosinemia type I.Hum Gene Ther. 1997; 8: 513-521Crossref PubMed Scopus (62) Google Scholar, 33Grompe M Overturf K Al-Dhalimy M Finegold M Therapeutic trials in the murine model of hereditary tyrosinaemia t

Referência(s)