WO1996039810A1 - Human hepatocellular tissue in chimeric immunocompromised animals - Google Patents

Human hepatocellular tissue in chimeric immunocompromised animals Download PDF

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Publication number
WO1996039810A1
WO1996039810A1 PCT/EP1996/002456 EP9602456W WO9639810A1 WO 1996039810 A1 WO1996039810 A1 WO 1996039810A1 EP 9602456 W EP9602456 W EP 9602456W WO 9639810 A1 WO9639810 A1 WO 9639810A1
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host
hepatocytes
human
cells
organ
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PCT/EP1996/002456
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French (fr)
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David E. B. Knudsen
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Novartis Ag
Systemix, Inc.
Novartis-Erfindungen Verwaltungsgesellschaft Mbh
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Priority to AU62229/96A priority Critical patent/AU6222996A/en
Publication of WO1996039810A1 publication Critical patent/WO1996039810A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the field of this invention is immunocompromised mammals comprising xenogeneic hepatocellular tissue.
  • the liver is a critically important organ for monitoring and adjusting plasma constituents.
  • Hepatocytes are active in controlling levels of blood glucose, lipids and cholesterol, and a number of plasma proteins.
  • the plasma proteins include albumin, fibrinogen and prothrombin, and several complement factors.
  • the structure of a liver lobule is that of a hexagon with portal triads at each corner, where each triad contains branches of the hepatic portal vein, hepatic artery and bile duct, so that each hepatocyte is in a close association with the vascular system.
  • Hepatocytes synthesize triglycerides, cholesterol and phospholipids. Much of the lipid synthesized is then packaged with proteins and released into the circulation as VLDLs, providing a source of fatty acids for all cells. Hepatocytes also synthesize the enzyme essential for formation of cholesteryl esters in HDL, remove chylomicron fragments from the circulation, and are an indirect source of LDLs, which are formed in plasma from VLDLs depleted of fatty acids. Balancing the lipoprotein levels and cholesterol content in the circulation has proven to be a critical factor in vascular disease.
  • Glucose from the blood is stored by hepatocytes in the form of glycogen. This is a major source of glucose for other cells in the body. During meals with high glucose, insulin increases the ability of hepatocytes to synthesize glycogen. As blood glucose drops, glucagon and epinephrine increase the ability of hepatocytes to degrade glycogen. Enzyme deficiencies associated with glycogen deficiencies can result in storage diseases.
  • liver has a unique regenerative capacity. After partial hepatectomy, the liver mass is restored by division of fully differentiated hepatocytes. Even in adults, these cells have a tremendous replicative ability. The existence of liver stem cells remains controversial, but such cells may be active in liver growth after severe injury.
  • hepatocytes The response of hepatocytes to tissue damage is mediated by several cytokines.
  • cytokines such as epidermal growth factor (EGF), tumor growth factor (TGFa), and hepatocyte growth factor (HGF).
  • EGF epidermal growth factor
  • TGFa tumor growth factor
  • HGF hepatocyte growth factor
  • HBV human hepatitis B virus
  • the only animals that can be infected with human hepatitis B virus (HBV) are humans and chimpanzees, and the major tissue that is productively infected is the liver, althou there have been reports of infected stromal cells.
  • HBV human hepatitis B virus
  • Primary hepatocyte cultures are susceptible to infection for only a few days, if at all, and do not produce the characteristic infectious particles.
  • Human hepatitis D virus (HDV) requires envelope proteins produced by HBV, and therefore can only infect cells susceptible to HBV. The need for a good experimental system having cells that are susceptible to productive infection by viruses such as the hepatitis viruses, and other hepatic pathogens, remains.
  • the field of medicine relies heavily on animal models. These models provide a means of analyzing the effect of viruses and other pathogens, cytokines, environmental factors, hormones, diet, and the like. Without animal models, it is extremely difficult to perform controlled experiments. Having viable human tissue in an animal model provides numerous advantages. One can investigate the effect of agents on the tissue at various stages in the development of the disease. The interactions of cells, secreted agents and tissue can also be analyzed. A xenogeneic animal model further provides a means of testing the effect of factors and other agents on cells that are difficult to maintain in culture. Short-lived lymphocyte subsets, neural cells, complex tissues, neutrophils, etc. that cannot easily be grown in culture for extended periods of time may be examined.
  • hepatocyte stimulatory effect of FK506 is described in Sakata et al. (1994) Int. Hepatol Commun. 2:67-69.
  • Hepatic cell transplantation in the mouse is disclosed in Rhim et al. (1994) Science 263:1149-1152.
  • the in vivo response of hepatocytes to growth factors is discussed in Webber et al. (1994) Hepatol 19:489-497.
  • Engraftment of hepatocytes after intra-splenic injection is described in Gupta et al. (1991) Hepatologv 14:144-149.
  • the growth of liver cells in the pancreas after intra-splenic transplantation is discussed in Jaffe et al. (1991) Int.
  • Immunocompromised non-human mammalian hosts comprising functional human hepatocytes.
  • Isolated human hepatocytes or fragments of human hepatic tissue are introduced into the xenogeneic host at a site that permits survival of the cells, in conjunction with hepatocyte growth factors.
  • the hepatocytes are able to survive and function in the host animal.
  • the chimeric animal has broad applicability in the study of human hepatic diseases.
  • the chimeric animal has broad applicability in the study of human infectious diseases with hepatocellular tropism, degenerative and metabolic diseases of the human liver, and toxic or carcinogenic agents that target the human liver.
  • Methods and compositions are provided for the growth of hepatocytes in an immunocompromised heterologous mammalian host, particularly a mouse, for extended periods of time.
  • the method comprises implanting isolated human hepatocytes in an appropriate site in an immunocompromised host, in conjunction with growth factors.
  • the chimeric animal provides an easily manipulated experimental model that is useful for studying human hepatic diseases.
  • Hepatocytes are isolated from human liver tissue by any convenient method, as known in the art.
  • the liver tissue is dissociated mechanically or enzymatically to provide a suspension of single cells.
  • fragments of intact human hepatic tissue may be used.
  • the suspension may be enriched for hepatic precursors by Ficoll-hypaque density gradient centrifugation, fluorescence activated cell sorting, panning, magnetic bead separation, elutriation within a centrifugal field, or rosetting.
  • a suspension of partially purified hepatocytes usually at least about 50% hepatocytes by number, more usually at least about 80% hepatocytes by number, is used for implantation.
  • the ability of hepatocytes to engraft is increased by providing growth factors involved in liver regeneration.
  • the factors may be administered from an exogenous source, or expression may be induced in the animal host.
  • Factors that increase proliferation of hepatocytes include insulin, epidermal growth factor (EGF), tumor growth factor a (TGFa) and hepatocyte growth factor (HGF), as well as a wide variety of factors produced by the gut, pancreas and liver.
  • the factors may be administered singly, particularly HGF, or as a cocktail.
  • Preferably human factors will be used, although cross- reactive factors from other species may be used in place of human. Administration may be through any convenient method, e.g.
  • the animal host may be stimulated to produce growth factors by a partial hepatectomy. Removal of one-third to two-thirds of the host liver provides adequate hepatocyte growth factors for engraftment. These factors may be those previously characterized, as well as chalones, a class of molecules believed to be responsible for liver regeneration.
  • the hepatocyte growth factors produced by partial hepatectomy of the animal host are sufficiently active on human hepatocytes to provide for engraftment.
  • the site of implantation is important for successful engraftment of hepatocytes, such that the liver cells will be placed in contact with the host growth factors, nutrients, and other factors required by the human hepatocytes. For the most part, factors regulating the total functional liver mass are thought to originate in the intestine and pancreatic epithelium, and are transported to the liver via the hepatic portal system.
  • a suspension of hepatocytes is injected into a suitable acceptor organ, where the cells are able to engraft.
  • Organs that can be seeded with hepatocytes include the spleen and liver. The liver may be directly seeded by injection into the portal vein. Injection into one organ may result in translocation of cells to the other organs.
  • intrasplenic implantation which allows the injected hepatocytes to seed the splenic parenchyma or home to the liver stroma via the hepatic portal system.
  • pieces of intact hepatic tissue may be implanted directly into the host liver, or isolated hepatocytes injected directly into the host liver.
  • the cells After injection of the donor hepatocytes, the cells engraft in the host liver, pancreas and/or spleen.
  • the engrafted cells may be either randomly distributed in the host liver, or grow as discrete acini.
  • the hepatic cells may be used after at least about 2 weeks and will remain functional for at least about 6 weeks, or more.
  • the engrafted hepatocytes are functional.
  • Assays for function may include responsiveness to insulin and glucagon, the ability to produce liver specific proteins, e.g. human serum albumin, c- reactive protein in response to IL-6, and the like.
  • the chimeric animal provides an environment for the introduction of a number of agents that are suspected of causing or contributing to hepatic disease, as well as the appropriate antagonists and blocking agents.
  • the human hepatocytes may be fresh tissue, obtained within about 48 hours of death, or freshly frozen tissue, tissue frozen within about 12 hours of death and maintained at below about -10°C, usually at about liquid nitrogen temperature (- 120°C) indefinitely. Normally the tissue will not have been subject to culture in vitro for an extended period of time.
  • the donor tissue will be human, although cells from sources other than members of the same family as the host animal may find use.
  • the source of the tissue will usually be fetal.
  • the tissue will be from a child of less than about 3 years, preferably less than about 1 year and at or younger than neonate, more preferably being fetal tissue of from about 7 to 24 weeks. In some cases adult human hepatocytes may be implanted.
  • the tissue will generally be normal, e.g. non-transformed and non-malignant, except in those cases where study of a particular malignancy is desired.
  • Immunocompromised mammalian hosts suitable for implantation and having the desired immune incapacity exist or can be created.
  • the significant factor is that the immunocompromised host is incapable naturally, or in conjunction with the introduced organs, of mounting an immune response against the xenogeneic tissue or cells. Therefore it is not sufficient that a host be immunocompromised, but that the host may not be able to mount an immune response after grafting, as evidenced by the inability to produce functional syngeneic host B-cells, particularly plasma cells, and/or T-cells, particularly CD4 + and/or CD8 + T-cells after implantation.
  • B-cells particularly plasma cells
  • T-cells particularly CD4 + and/or CD8 + T-cells after implantation.
  • small mammals e.g.
  • murines e.g. mouse and rat, which are immunocompromised due to a genetic defect which results in an inability to undergo germline DNA rearrangement at the loci encoding immunoglobulins and T-cell antigen receptors.
  • mice that have been genetically engineered by transgenic disruption to lack the recombinase function associated with RAG-1 and/or RAG-2 (e.g. commercially available TIMTM RAG-2 transgenic), to lack Class I and/or Class II MHC antigens (e.g. the commercially available CID and C2D transgenic strains), or to lack expression of the Bcl-2 proto-oncogene.
  • RAG-1 and/or RAG-2 e.g. commercially available TIMTM RAG-2 transgenic
  • Class I and/or Class II MHC antigens e.g. the commercially available CID and C2D transgenic strains
  • Bcl-2 proto-oncogene e.g. the commercially available mice that have a homozygous mutation at the scid locus, causing a severe combined immunodeficiency which is manifested by a lack of functionally recombined immunoglobulin and T-cell receptor genes.
  • the scid/scid mutation is available or may be bred into a number of different genetic backgrounds, e.g. CB.17, ICR (outbred), C3H, BALB/c, C57B1/6, AKR, BA, BIO, 129, etc.
  • Other mice which are useful as recipients are NOD scid/scid; SGB scid/scid, bh/bh; CB.17 scid/hr; NIH-3 bg/nu/xid and META nu/nu.
  • Transgenic mice, rats and pigs are available which lack functional B cells and T cells due to a homozygous disruption in the CD3e gene.
  • Immunocompromised rats include HsdHan:RNU-r « «; HsdHan:RNU-rm//+; HsdHan:NZNU-rm.; HsdHa NZNU- rnu/+; LEW/HanHsd-r « «; LEW/HanHsd-rn «/+; WAG/HanHsd-r»« and WAG/HanHsd- rnu/+.
  • Additional loss of immune function in the host animal may be achieved by decreasing the number of endogenous macrophages before, during, or after implantation of the xenogeneic tissue.
  • reduction of macrophages by administration of dichloromethylene diphosphonate (Cl-MDP) encapsulated in liposomes, as described in co-pending application no. 08/169,293.
  • the host will usually be of an age less than about 25% of the normal lifetime of an immunocompetent host, usually about 1 to 20% of the normal lifetime. Generally, the host will be at least about four weeks old and large enough to manipulate for introduction of the donor tissue at the desired site. For example, mice are usually used at about 4 to 6 weeks of age. Growth of the tissue within the host will vary with the organ.
  • the mammalian host will be grown in conventional ways. Depending on the degree of immunocompromised status of the mammalian host, it may be protected to varying degrees from infection. An aseptic environment is indicated. Prophylactic antibiosis for protection from Pneumocystis infection may be achieved for scid/scid mice with 25-75 mg trimethoprim and 100-300 mg sulfamethoxazole in 5 ml of suspension, given three days each week, or in impregnated food pellets. Alternatively, it may be satisfactory to isolate the potential hosts from other animals in gnotobiotic environments after cesarean derivation. The feeding and maintenance of the chimeric host will for the most part follow gnotobiotic techniques.
  • tissue may be transplanted into the host in addition to the hepatocytes.
  • hematopoietic components may be included, such as stem cells, lymph nodes, embryonic yolk sac, fetal liver, pancreatic tissue, appendix tissue, tonsil tissue and the like, which may serve to provide human lymphoid and granulocytic cells in the immunocompromised host.
  • Sites for introduction of additional tissue may include under the spleen capsule, abdominal wall muscle, under the renal capsule, in the anterior chamber of the eye, the peritoneum, the peritoneal lining, brain, subcutaneous, vascular system, spinal cord, membranous sacs or capsules of various tissue, the retroperitoneal space, reproductive organs, ear pinnae, etc.
  • tissue or cells of interest will generally be normal, e.g. non-transformed and non-malignant tissue or cells.
  • tissue or cells of interest will generally be normal, e.g. non-transformed and non-malignant tissue or cells.
  • the surrounding tissue may comprise connective tissue, or portions of blood and lymphatic vessels.
  • whole organ grafts may be transplanted by anastomosing donor and host blood vessels, lymphatic vessels, and the like.
  • normal cells, tissue, and/or organs may be stably maintained and functional for at least about 3-6 months and frequently for at least about 10 months.
  • a mixed population of cells in suspension may be enriched for the particular cells of interest.
  • the suspension may be enriched for T cells by Ficoll-hypaque density gradient centrifugation, fluorescence activated cell sorting, panning, magnetic bead separation, elutriation within a centrifugal field, or rosetting.
  • Immunoaffinity columns may be employed which allow for specific separation of either the desired or undesired cells, depending on the antibodies or fragments thereof used for separation, and the nature of the mixture.
  • dispersed cells are employed, where the relevant organs are teased apart to yield viable cells in suspension.
  • Cells of particular interest as a secondary implant are human hematopoietic cells, particular T cells, neutrophils, and other granulocytic and myeloid cells. Such cells may be obtained from an immunocompetent human donor.
  • the hematopoietic cells may be mismatched as to HLA type with the hepatocytes, so as to provide a marker for the source, or may be matched as to HLA type in order to provide T cells that recognize antigen presented by the hepatocytes.
  • the presence of the human hepatocytes in an immunocompromised host may be used to study the effect of various compounds on the growth, viability, differentiation, maturation, transformation, or the like, of the human cells in a live host.
  • the chimeric host may be used to study the effect of a variation of a condition on a symptom or indication of a disease.
  • condition it is intended a physical, chemical or biological property, e.g. temperature, electric potential, ionic strength, drugs, transformation, etc.
  • hepatic pathogens e.g. viruses, protozoans and bacteria
  • drugs and toxins e.g. isoniazid, carbon tetrachloride, and ethanol.
  • the effect of such agents of human hepatocellular tissue may be investigated with the subject animals.
  • Viruses of interest include the human hepatitis viruses A, B, C, D and E, particularly HBV and HDV, which cannot be grown in culture.
  • Other hepatic viruses are Epstein-Barr virus, cytomegalovirus, varicella-zoster virus and yellow fever viruses. Infection of hepatocytes is an essential feature of Plasmodium falciparum, P. vivax, P. ovale and P. malariae infection. The worldwide incidence of malaria provides a strong incentive for the study of its disease etiology.
  • the pathogen may be wild-type, e.g. clinical isolates, conventional strains, etc.; attenuated strains; or may be genetically engineered to enhance or reduce infectivity, pathogenicity, etc.
  • modifications in the genome may include deletion of virulence genes, mutations in coat proteins that alter the host range, change in viral nucleic acid polymerases, alterations in proteins that affect integration into the host genome, etc. Mutations introduced into the pathogen genome are useful to map the functions of various proteins, and to determine which domains are responsible for various aspects of the infection, i.e. in establishing latency, transforming cells, replication, etc.
  • a liver implant is inoculated with the pathogen, usually at an infectious level.
  • the effect of the pathogen is determined, in most cases as a function of time.
  • Data may be obtained as to the response of human cells to the pathogen; products which are secreted by infected or involved cells in response to infection, e.g. cytokines, interferons, etc.; the viability and growth of the human hepatocytes; and pathogen replication, e.g. release of new infectious particles or cells.
  • Infection may be achieved by direct injection of the pathogen.
  • the injection will involve at least about 10 2 infectious units, preferably from about 10 3 to 10 5 infectious units.
  • the pathogen may be a clinical isolate, a cloned clinical isolate, a genetically modified isolate, or the like.
  • administration may be via injection of infected cells, where the injected cells will produce infectious pathogens over time.
  • the cells will deliver a dose of at least about 10 2 infectious units, preferably from about 10 3 to 10 5 infectious units.
  • Non-invasive techniques include NMR, CAT scans, fluoroscopy, roentgenography, radionuclide scanning, ultrasonography, electrocardiography, electroencephalography, evoked potentials, etc.
  • Invasive techniques include biopsy, autopsy, laparotomy, intermittent intravenous blood sampling, or intravenous catheterization, etc. Convenient placement of various devices, e.g. catheters, electrodes, etc. may be performed for continuous monitoring.
  • the host may be used to determine the carcinogenicity of various compounds, the effect on growth and viability of hepatic tissue, the effect of combinations of compounds, e.g. drugs, or the like.
  • the effect of various drugs in protecting the host tissue from the pathogen, as well as being cytotoxic to or suppressive of the pathogen in a cellular environment can be determined.
  • Use of the chimeric animal in studying the effect of drugs on infection may begin with administration of the drug prior to, substantially concomitant with, and/or subsequent to the administration of the infectious dose of pathogen.
  • Administration of the drug will usually begin not earlier than 7 days prior to infection, more usually not more than about 1 day prior to infection. In most cases, administration of the drug will begin not later than about 7 days after infection, more usually not later than about 1 day after infection.
  • drug treatment may be started after as much as one year after infection, usually after six months, more usually after one month. After initial screening, different periods of time may be of interest in establishing the effectiveness of the drug.
  • the manner of administration will vary greatly, depending upon the nature of the drug. It may be provided orally, ad libitum, intraperitoneally, intravascularly, subcutaneously, intrathymically, or the like. Usually, different dosage levels will be employed, based on past experience with the drug, anticipated levels with human treatment, toxicity or side effects, experience with the particular chimeric host, and the like.
  • the effect of the drug may be monitored for any convenient time, usually at least 1 week from the initiation of administration of the drug, more usually at least 2 weeks, and at times for periods as long as 6 weeks or more. Preferably, determinations will be made in the period from about 2-6 weeks.
  • Phenotyping of the xenogeneic cells to verify their origin and stage of developmental progression may be performed by standard histological methods, by immunohistochemistry, antibody staining or in situ hybridization with RNA and/or DNA probes. The exact method is not critical to the invention.
  • HLA markers may be used to distinguish the established xenogeneic organ transplants. The HLA type can be readily determined by staining with an appropriate antibody directed against any of the alleles of the human HLA locus, including Class I and Class II antigens.
  • physiologic products of human hepatocytes distinguishable from their murine analogs by immunologic or quantitative criteria, e.g. expression of human serum albumin, or expression of c- reactive protein in response to IL-6, etc., can be used to determine the presence of human cells without sacrifice of the recipient.
  • Placement of isolated hepatocytes into the mouse hepatic environment will provide growth factors, nutrients and other influences to the liver cells without the addition of other human tissue such as pancreas or intestine.
  • Splenic injection allows injected hepatocytes the option of either proliferating into microtubules in the splenic parenchyma, or homing to the liver through the hepatic portal system.
  • Partial hepatectomy induces expression of hepatic growth factors, because of the autonomous signaling that occurs following reduction in functional liver mass.
  • Partial hepatectomy and splenic injection can be performed via a single surgical approach in minimal time. The transplantation was done into animals that are 4-6 week old C.B-17 scid/scid mice of either sex.
  • Tissue from a 20-23 week old human fetal liver is collected into Hanks' balanced salts. 1.5 to 2 grams of the liver is minced by sharp dissection, then incubated in RPMI with 100 IU/ml type VI collagenase to enzymatically dissociate hepatocytes. All cells are pelleted at 500 x g for 10 minutes at 10°C and the connective tissue remnant discarded. The pellet is resuspended in cold RPMI.
  • the resuspended pellet is centrifuged at 50 x g for 5 minutes.
  • the pellet (small mononuclear cells) and supernatant (large hepatocytes in suspension) are separated, saving both fractions for later testing.
  • the hepatocyte enrichment is repeated by light centrifugation of supernatant, and the second pellet resuspended in RPMI.
  • the cell suspension can then be held on wet ice for up to 2 hours until injected.
  • the hepatocyte yield and concentration is then checked visually by Wright-Giemsa staining, and the cell viability is checked by trypan-blue dye exclusion.
  • the cell suspension for injection is prepared from the fraction with the highest concentration of hepatocytes.
  • the fraction with the highest hepatocyte concentration is resuspended in normal saline for injection.
  • the dose is calculated by the number of mice to be injected x 0.1 ml per injection.
  • An optimal injection dose is 10* to 10 7 viable hepatocytes per injection.
  • a 28 gauge needle attached to a 0.5 ml tuberculin syringe is used for injections.
  • the cell suspension is transported to surgery in ice.
  • Mice are anesthetized with either metafane using an open-drop method, or ketamine/xylazine cocktail using an intramuscular injection.
  • the lateral abdomen is clipped and prepared for surgery.
  • a left lateral paracostal abdominal incision is made over the normal position of the spleen.
  • the left caudate lobe of the liver is located and exteriorized through the incision, making sure that the stomach, pancreas, proximal duodenum and biliary tract are not pulled or handled.
  • the base of the lobe is elevated and ligated with 6-0 Vicryl or other absorbable suture.
  • the ligature is tightened with multiple surgical knots.
  • the lobe is transected by sharp dissection, and the exposed liver surface packed with sterile gel-foam. After holding the stump briefly to be certain of hemostasis, it is returned to the abdomen. The spleen is carefully located and exteriorized through the incision without disrupting any mesenteric or vascular attachments. 0.1 ml of the hepatocyte suspension is injected with a 28 guage needle, which punctures the splenic capsule on its antimesenteric surface.
  • the needle should be parallel to the long axis of the spleen.
  • the spleen is returned to the abdominal cavity, and the body wall closed with one or two simple interrupted sutures using 5-0 or 6-0 Vicryl, followed by a steel clip closure of the skin incision.
  • liver fragments are implanted. Tissue from a 20-23 week old human fetal liver is cut with a sharp blade into approximately l l x l mm pieces. The mice are anesthetized and a 1 cm incision made in the liver at a site separate from the hepatectomy. One or two of these fragments are implanted with an 18 gauge trocar.
  • Animals are held in recovery and observed to rouse from anesthesia, and then checked every 24 hours for 3 days post-surgery. Starting at two weeks post surgery, blood samples may be collected for screening.
  • Serum samples are collected to screen for the success of engraftment by human hepatocytes.
  • the samples are tested for the presence of human albumin (produced solely by hepatocytes) by RIA.
  • Human albumin produced solely by hepatocytes
  • Polyclonal anti-human serum albumin is commercially available (United States Biochemical).
  • the antisera may be cleared of mouse cross-reactive antibodies by affinity purification against mouse serum albumin.
  • spleens and livers are removed and fixed in 10% neutral buffered formalin (10% formalin in phosphate or carbonate buffer, pH 7.4) following collection of heart blood. Following fixation, tissues are processed for histologic and immunohistologic examination. Immunostaining is performed using rabbit a-human HLA (pan class I) antibody on formalin fixed, dewaxed sections, with secondary staining with FITC or peroxidase conjugated goat a-rabbit IgG.
  • HLA pan class I
  • Hepatic cells derived from diseased liver may be introduced into previously implanted liver grafts to study malignancy and the effects of growth factors and/or drugs which might modulate normal liver function or disease states.
  • this model can also serve as a valuable system to test the long-term expression of exogenous genes introduced into human hepatic cells.
  • the model is also a valuable system for observing the effects of human tropic viral infection and therapy in an in vivo system. It provides an alternative to animal hepatitis viruses and in vitro culture for the investigation of hepatocellular function during acute and/or chronic infection.

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Abstract

Immunocompromised hosts are provided, comprising functional human hepatocytes. A suspension of isolated human hepatocytes is injected into an acceptor organ in conjunction with hepatocyte growth factors. The hepatocytes survive and function in the host animal. The chimeric animal is useful in the study of human hepatic diseases. Particularly, liver specific pathogens such as Plasmodium sp. and hepatitis viruses may be studied.

Description

HUMAN HEPATOCELLULAR TISSUE IN CHIMERIC IMMUNOCOMPROMISED ANIMALS
The field of this invention is immunocompromised mammals comprising xenogeneic hepatocellular tissue.
The liver is a critically important organ for monitoring and adjusting plasma constituents. Hepatocytes are active in controlling levels of blood glucose, lipids and cholesterol, and a number of plasma proteins. The plasma proteins include albumin, fibrinogen and prothrombin, and several complement factors. The structure of a liver lobule is that of a hexagon with portal triads at each corner, where each triad contains branches of the hepatic portal vein, hepatic artery and bile duct, so that each hepatocyte is in a close association with the vascular system.
Hepatocytes synthesize triglycerides, cholesterol and phospholipids. Much of the lipid synthesized is then packaged with proteins and released into the circulation as VLDLs, providing a source of fatty acids for all cells. Hepatocytes also synthesize the enzyme essential for formation of cholesteryl esters in HDL, remove chylomicron fragments from the circulation, and are an indirect source of LDLs, which are formed in plasma from VLDLs depleted of fatty acids. Balancing the lipoprotein levels and cholesterol content in the circulation has proven to be a critical factor in vascular disease.
Glucose from the blood is stored by hepatocytes in the form of glycogen. This is a major source of glucose for other cells in the body. During meals with high glucose, insulin increases the ability of hepatocytes to synthesize glycogen. As blood glucose drops, glucagon and epinephrine increase the ability of hepatocytes to degrade glycogen. Enzyme deficiencies associated with glycogen deficiencies can result in storage diseases.
Despite its specialized functions, the liver has a unique regenerative capacity. After partial hepatectomy, the liver mass is restored by division of fully differentiated hepatocytes. Even in adults, these cells have a tremendous replicative ability. The existence of liver stem cells remains controversial, but such cells may be active in liver growth after severe injury.
The response of hepatocytes to tissue damage is mediated by several cytokines. Immediately after an injury, hepatocytes undergo a priming phase in which they become competent to enter the cell cycle. This phase is characterized by expression of the proto-oncogenes c-myc and c-jun. The primed cells are then able to respond to cytokines such as epidermal growth factor (EGF), tumor growth factor (TGFa), and hepatocyte growth factor (HGF). TGFa is synthesized by hepatocytes and acts as an autocrine factor.
Certain viruses show great specificity for infecting hepatocytes. The only animals that can be infected with human hepatitis B virus (HBV) are humans and chimpanzees, and the major tissue that is productively infected is the liver, althou there have been reports of infected stromal cells. At the present time there is no good in vitro culture model for HBV. Primary hepatocyte cultures are susceptible to infection for only a few days, if at all, and do not produce the characteristic infectious particles. Human hepatitis D virus (HDV) requires envelope proteins produced by HBV, and therefore can only infect cells susceptible to HBV. The need for a good experimental system having cells that are susceptible to productive infection by viruses such as the hepatitis viruses, and other hepatic pathogens, remains.
The field of medicine relies heavily on animal models. These models provide a means of analyzing the effect of viruses and other pathogens, cytokines, environmental factors, hormones, diet, and the like. Without animal models, it is extremely difficult to perform controlled experiments. Having viable human tissue in an animal model provides numerous advantages. One can investigate the effect of agents on the tissue at various stages in the development of the disease. The interactions of cells, secreted agents and tissue can also be analyzed. A xenogeneic animal model further provides a means of testing the effect of factors and other agents on cells that are difficult to maintain in culture. Short-lived lymphocyte subsets, neural cells, complex tissues, neutrophils, etc. that cannot easily be grown in culture for extended periods of time may be examined.
In view of the many important functions performed by the liver, it is of substantial interest to develop and provide animal models comprising functional human hepatocytes that remain viable for extended periods of time. Such an animal model would permit investigation of the function and dysfunction of hepatocytes, the etiology of disease and the effect of pathogens and therapeutic drugs.
A description of the SCID-hu mouse may be found in J.M. McCune et al. (1988) Science 241:1632-1639; R. Namikawa et al. (1990) J. Exp. Med. 172:1055-1063 and J.M. McCune et al. (1991) Ann. Rev. Immunol. 9:395-429. Immunocompromised mouse strains are described in S. Nonoyama et al. (1993) J. Immunol 150:3817-3824; I. Gerling et al. (1994) Diabetes 43:433-440; Bosma, et al. (1983) Nature 301:52; and P. Mombaerts et al. (1992) Cell 68:869-877. Sacci et al. (1992) P.N.A.S. 89:3701-3705 describes a mouse model for the exoerythrocytic stages of Plasmodium falciparum.
The hepatocyte stimulatory effect of FK506 is described in Sakata et al. (1994) Int. Hepatol Commun. 2:67-69. Hepatic cell transplantation in the mouse is disclosed in Rhim et al. (1994) Science 263:1149-1152. The in vivo response of hepatocytes to growth factors is discussed in Webber et al. (1994) Hepatol 19:489-497. Engraftment of hepatocytes after intra-splenic injection is described in Gupta et al. (1991) Hepatologv 14:144-149. The growth of liver cells in the pancreas after intra-splenic transplantation is discussed in Jaffe et al. (1991) Int. J. Exp. Path. 72:289-299. Transplantation of isolated hepatocytes into the pancreas is describedby Vroemen et al. (1988) Eur. Surg. Res. 20:1-1 1. The isolation and in vitro culture of mouse hepatocytes is described in Klaunig et al (1981) In Vitro 17:913-925. In vitro hepatocyte culture systems for hepatitis B and hepatitis D virus are described in Sureau (1993) Arch. Virol. 8:3-14.
Immunocompromised non-human mammalian hosts are provided, comprising functional human hepatocytes. Isolated human hepatocytes or fragments of human hepatic tissue are introduced into the xenogeneic host at a site that permits survival of the cells, in conjunction with hepatocyte growth factors. The hepatocytes are able to survive and function in the host animal. The chimeric animal has broad applicability in the study of human hepatic diseases. The chimeric animal has broad applicability in the study of human infectious diseases with hepatocellular tropism, degenerative and metabolic diseases of the human liver, and toxic or carcinogenic agents that target the human liver.
Methods and compositions are provided for the growth of hepatocytes in an immunocompromised heterologous mammalian host, particularly a mouse, for extended periods of time. The method comprises implanting isolated human hepatocytes in an appropriate site in an immunocompromised host, in conjunction with growth factors. The chimeric animal provides an easily manipulated experimental model that is useful for studying human hepatic diseases.
Hepatocytes are isolated from human liver tissue by any convenient method, as known in the art. The liver tissue is dissociated mechanically or enzymatically to provide a suspension of single cells. Alternatively, fragments of intact human hepatic tissue may be used. The suspension may be enriched for hepatic precursors by Ficoll-hypaque density gradient centrifugation, fluorescence activated cell sorting, panning, magnetic bead separation, elutriation within a centrifugal field, or rosetting. Generally a suspension of partially purified hepatocytes, usually at least about 50% hepatocytes by number, more usually at least about 80% hepatocytes by number, is used for implantation.
The ability of hepatocytes to engraft is increased by providing growth factors involved in liver regeneration. The factors may be administered from an exogenous source, or expression may be induced in the animal host. Factors that increase proliferation of hepatocytes include insulin, epidermal growth factor (EGF), tumor growth factor a (TGFa) and hepatocyte growth factor (HGF), as well as a wide variety of factors produced by the gut, pancreas and liver. The factors may be administered singly, particularly HGF, or as a cocktail. Preferably human factors will be used, although cross- reactive factors from other species may be used in place of human. Administration may be through any convenient method, e.g. i.v., i.p., through an osmotic pump or sustained release implant, etc. Preferably, the animal host may be stimulated to produce growth factors by a partial hepatectomy. Removal of one-third to two-thirds of the host liver provides adequate hepatocyte growth factors for engraftment. These factors may be those previously characterized, as well as chalones, a class of molecules believed to be responsible for liver regeneration. The hepatocyte growth factors produced by partial hepatectomy of the animal host are sufficiently active on human hepatocytes to provide for engraftment.
The site of implantation is important for successful engraftment of hepatocytes, such that the liver cells will be placed in contact with the host growth factors, nutrients, and other factors required by the human hepatocytes. For the most part, factors regulating the total functional liver mass are thought to originate in the intestine and pancreatic epithelium, and are transported to the liver via the hepatic portal system. A suspension of hepatocytes is injected into a suitable acceptor organ, where the cells are able to engraft. Organs that can be seeded with hepatocytes include the spleen and liver. The liver may be directly seeded by injection into the portal vein. Injection into one organ may result in translocation of cells to the other organs. Of particular interest is intrasplenic implantation, which allows the injected hepatocytes to seed the splenic parenchyma or home to the liver stroma via the hepatic portal system. Alternatively, pieces of intact hepatic tissue may be implanted directly into the host liver, or isolated hepatocytes injected directly into the host liver.
After injection of the donor hepatocytes, the cells engraft in the host liver, pancreas and/or spleen. The engrafted cells may be either randomly distributed in the host liver, or grow as discrete acini. The hepatic cells may be used after at least about 2 weeks and will remain functional for at least about 6 weeks, or more. The engrafted hepatocytes are functional. Assays for function may include responsiveness to insulin and glucagon, the ability to produce liver specific proteins, e.g. human serum albumin, c- reactive protein in response to IL-6, and the like. The chimeric animal provides an environment for the introduction of a number of agents that are suspected of causing or contributing to hepatic disease, as well as the appropriate antagonists and blocking agents.
The human hepatocytes may be fresh tissue, obtained within about 48 hours of death, or freshly frozen tissue, tissue frozen within about 12 hours of death and maintained at below about -10°C, usually at about liquid nitrogen temperature (- 120°C) indefinitely. Normally the tissue will not have been subject to culture in vitro for an extended period of time.
For the most part the donor tissue will be human, although cells from sources other than members of the same family as the host animal may find use. The source of the tissue will usually be fetal. Preferably the tissue will be from a child of less than about 3 years, preferably less than about 1 year and at or younger than neonate, more preferably being fetal tissue of from about 7 to 24 weeks. In some cases adult human hepatocytes may be implanted. The tissue will generally be normal, e.g. non-transformed and non-malignant, except in those cases where study of a particular malignancy is desired.
Immunocompromised mammalian hosts suitable for implantation and having the desired immune incapacity exist or can be created. The significant factor is that the immunocompromised host is incapable naturally, or in conjunction with the introduced organs, of mounting an immune response against the xenogeneic tissue or cells. Therefore it is not sufficient that a host be immunocompromised, but that the host may not be able to mount an immune response after grafting, as evidenced by the inability to produce functional syngeneic host B-cells, particularly plasma cells, and/or T-cells, particularly CD4+ and/or CD8+ T-cells after implantation. Of particular interest are small mammals, e.g. rabbits, gerbils, hamsters, guinea pigs, etc., particularly murines, e.g. mouse and rat, which are immunocompromised due to a genetic defect which results in an inability to undergo germline DNA rearrangement at the loci encoding immunoglobulins and T-cell antigen receptors.
Presently available hosts include mice that have been genetically engineered by transgenic disruption to lack the recombinase function associated with RAG-1 and/or RAG-2 (e.g. commercially available TIM™ RAG-2 transgenic), to lack Class I and/or Class II MHC antigens (e.g. the commercially available CID and C2D transgenic strains), or to lack expression of the Bcl-2 proto-oncogene. Of particular interest are mice that have a homozygous mutation at the scid locus, causing a severe combined immunodeficiency which is manifested by a lack of functionally recombined immunoglobulin and T-cell receptor genes. The scid/scid mutation is available or may be bred into a number of different genetic backgrounds, e.g. CB.17, ICR (outbred), C3H, BALB/c, C57B1/6, AKR, BA, BIO, 129, etc. Other mice which are useful as recipients are NOD scid/scid; SGB scid/scid, bh/bh; CB.17 scid/hr; NIH-3 bg/nu/xid and META nu/nu. Transgenic mice, rats and pigs are available which lack functional B cells and T cells due to a homozygous disruption in the CD3e gene. Immunocompromised rats include HsdHan:RNU-r««; HsdHan:RNU-rm//+; HsdHan:NZNU-rm.; HsdHa NZNU- rnu/+; LEW/HanHsd-r««; LEW/HanHsd-rn«/+; WAG/HanHsd-r»« and WAG/HanHsd- rnu/+.
Additional loss of immune function in the host animal may be achieved by decreasing the number of endogenous macrophages before, during, or after implantation of the xenogeneic tissue. Of particular interest is the reduction of macrophages by administration of dichloromethylene diphosphonate (Cl-MDP) encapsulated in liposomes, as described in co-pending application no. 08/169,293.
The host will usually be of an age less than about 25% of the normal lifetime of an immunocompetent host, usually about 1 to 20% of the normal lifetime. Generally, the host will be at least about four weeks old and large enough to manipulate for introduction of the donor tissue at the desired site. For example, mice are usually used at about 4 to 6 weeks of age. Growth of the tissue within the host will vary with the organ.
The mammalian host will be grown in conventional ways. Depending on the degree of immunocompromised status of the mammalian host, it may be protected to varying degrees from infection. An aseptic environment is indicated. Prophylactic antibiosis for protection from Pneumocystis infection may be achieved for scid/scid mice with 25-75 mg trimethoprim and 100-300 mg sulfamethoxazole in 5 ml of suspension, given three days each week, or in impregnated food pellets. Alternatively, it may be satisfactory to isolate the potential hosts from other animals in gnotobiotic environments after cesarean derivation. The feeding and maintenance of the chimeric host will for the most part follow gnotobiotic techniques.
Other tissues may be transplanted into the host in addition to the hepatocytes. For example, hematopoietic components may be included, such as stem cells, lymph nodes, embryonic yolk sac, fetal liver, pancreatic tissue, appendix tissue, tonsil tissue and the like, which may serve to provide human lymphoid and granulocytic cells in the immunocompromised host. Sites for introduction of additional tissue may include under the spleen capsule, abdominal wall muscle, under the renal capsule, in the anterior chamber of the eye, the peritoneum, the peritoneal lining, brain, subcutaneous, vascular system, spinal cord, membranous sacs or capsules of various tissue, the retroperitoneal space, reproductive organs, ear pinnae, etc.
Introduction of the secondary tissue may be achieved by injection, implantation, or joining blood vessels (and other vessels if necessary) of the donor and host, using intravenous catheters, trocars, and/or surgical incision, or the like. The tissue or cells of interest will generally be normal, e.g. non-transformed and non-malignant tissue or cells. With various organs one may include native surrounding tissue with the organ tissue itself. The surrounding tissue may comprise connective tissue, or portions of blood and lymphatic vessels. In some cases, whole organ grafts may be transplanted by anastomosing donor and host blood vessels, lymphatic vessels, and the like. For the most part, normal cells, tissue, and/or organs may be stably maintained and functional for at least about 3-6 months and frequently for at least about 10 months.
A mixed population of cells in suspension may be enriched for the particular cells of interest. For example, with bone marrow cells, the suspension may be enriched for T cells by Ficoll-hypaque density gradient centrifugation, fluorescence activated cell sorting, panning, magnetic bead separation, elutriation within a centrifugal field, or rosetting. In some instances it may be desirable to enrich cells by killing or removing other cells. This may be achieved by employing monoclonal antibodies specific for the undesired cells in the presence of complement or linked to a cytotoxic agent, such as a toxin, e.g. ricin, abrin, diphtheria toxin, or a radiolabel, e.g. "'I, or the like. Immunoaffinity columns may be employed which allow for specific separation of either the desired or undesired cells, depending on the antibodies or fragments thereof used for separation, and the nature of the mixture.
As appropriate, dispersed cells are employed, where the relevant organs are teased apart to yield viable cells in suspension. Cells of particular interest as a secondary implant are human hematopoietic cells, particular T cells, neutrophils, and other granulocytic and myeloid cells. Such cells may be obtained from an immunocompetent human donor. The hematopoietic cells may be mismatched as to HLA type with the hepatocytes, so as to provide a marker for the source, or may be matched as to HLA type in order to provide T cells that recognize antigen presented by the hepatocytes.
The presence of the human hepatocytes in an immunocompromised host may be used to study the effect of various compounds on the growth, viability, differentiation, maturation, transformation, or the like, of the human cells in a live host. The chimeric host may be used to study the effect of a variation of a condition on a symptom or indication of a disease. By condition, it is intended a physical, chemical or biological property, e.g. temperature, electric potential, ionic strength, drugs, transformation, etc.
There are a number of agents known to cause hepatitis in humans, including hepatic pathogens, e.g. viruses, protozoans and bacteria, as well as drugs and toxins, e.g. isoniazid, carbon tetrachloride, and ethanol. The effect of such agents of human hepatocellular tissue may be investigated with the subject animals. Viruses of interest include the human hepatitis viruses A, B, C, D and E, particularly HBV and HDV, which cannot be grown in culture. Other hepatic viruses are Epstein-Barr virus, cytomegalovirus, varicella-zoster virus and yellow fever viruses. Infection of hepatocytes is an essential feature of Plasmodium falciparum, P. vivax, P. ovale and P. malariae infection. The worldwide incidence of malaria provides a strong incentive for the study of its disease etiology.
The pathogen may be wild-type, e.g. clinical isolates, conventional strains, etc.; attenuated strains; or may be genetically engineered to enhance or reduce infectivity, pathogenicity, etc. Such modifications in the genome may include deletion of virulence genes, mutations in coat proteins that alter the host range, change in viral nucleic acid polymerases, alterations in proteins that affect integration into the host genome, etc. Mutations introduced into the pathogen genome are useful to map the functions of various proteins, and to determine which domains are responsible for various aspects of the infection, i.e. in establishing latency, transforming cells, replication, etc.
To study the effects of of infection on human cells, a liver implant is inoculated with the pathogen, usually at an infectious level. The effect of the pathogen is determined, in most cases as a function of time. Data may be obtained as to the response of human cells to the pathogen; products which are secreted by infected or involved cells in response to infection, e.g. cytokines, interferons, etc.; the viability and growth of the human hepatocytes; and pathogen replication, e.g. release of new infectious particles or cells.
Infection may be achieved by direct injection of the pathogen. Usually, the injection will involve at least about 102 infectious units, preferably from about 103 to 105 infectious units. The pathogen may be a clinical isolate, a cloned clinical isolate, a genetically modified isolate, or the like. Alternatively, administration may be via injection of infected cells, where the injected cells will produce infectious pathogens over time. The cells will deliver a dose of at least about 102 infectious units, preferably from about 103 to 105 infectious units.
Various drugs may be administered to the host and the effect on hepatocytes determined by invasive or non-invasive techniques. Non-invasive techniques include NMR, CAT scans, fluoroscopy, roentgenography, radionuclide scanning, ultrasonography, electrocardiography, electroencephalography, evoked potentials, etc. Invasive techniques include biopsy, autopsy, laparotomy, intermittent intravenous blood sampling, or intravenous catheterization, etc. Convenient placement of various devices, e.g. catheters, electrodes, etc. may be performed for continuous monitoring. Thus, the host may be used to determine the carcinogenicity of various compounds, the effect on growth and viability of hepatic tissue, the effect of combinations of compounds, e.g. drugs, or the like. In addition, by providing for pathogenic infection of the xenogeneic tissue, the effect of various drugs in protecting the host tissue from the pathogen, as well as being cytotoxic to or suppressive of the pathogen in a cellular environment can be determined.
Use of the chimeric animal in studying the effect of drugs on infection may begin with administration of the drug prior to, substantially concomitant with, and/or subsequent to the administration of the infectious dose of pathogen. Administration of the drug will usually begin not earlier than 7 days prior to infection, more usually not more than about 1 day prior to infection. In most cases, administration of the drug will begin not later than about 7 days after infection, more usually not later than about 1 day after infection. However, for studies of chronic infections, drug treatment may be started after as much as one year after infection, usually after six months, more usually after one month. After initial screening, different periods of time may be of interest in establishing the effectiveness of the drug.
The manner of administration will vary greatly, depending upon the nature of the drug. It may be provided orally, ad libitum, intraperitoneally, intravascularly, subcutaneously, intrathymically, or the like. Usually, different dosage levels will be employed, based on past experience with the drug, anticipated levels with human treatment, toxicity or side effects, experience with the particular chimeric host, and the like. The effect of the drug may be monitored for any convenient time, usually at least 1 week from the initiation of administration of the drug, more usually at least 2 weeks, and at times for periods as long as 6 weeks or more. Preferably, determinations will be made in the period from about 2-6 weeks.
Phenotyping of the xenogeneic cells to verify their origin and stage of developmental progression may be performed by standard histological methods, by immunohistochemistry, antibody staining or in situ hybridization with RNA and/or DNA probes. The exact method is not critical to the invention. HLA markers may be used to distinguish the established xenogeneic organ transplants. The HLA type can be readily determined by staining with an appropriate antibody directed against any of the alleles of the human HLA locus, including Class I and Class II antigens. Additionally, physiologic products of human hepatocytes distinguishable from their murine analogs by immunologic or quantitative criteria, e.g. expression of human serum albumin, or expression of c- reactive protein in response to IL-6, etc., can be used to determine the presence of human cells without sacrifice of the recipient.
Example 1
Construction of a biologically relevant immunocompromised mouse with intact functional human hepatocellular populations.
Placement of isolated hepatocytes into the mouse hepatic environment will provide growth factors, nutrients and other influences to the liver cells without the addition of other human tissue such as pancreas or intestine. Splenic injection allows injected hepatocytes the option of either proliferating into microtubules in the splenic parenchyma, or homing to the liver through the hepatic portal system. Partial hepatectomy induces expression of hepatic growth factors, because of the autonomous signaling that occurs following reduction in functional liver mass. Partial hepatectomy and splenic injection can be performed via a single surgical approach in minimal time. The transplantation was done into animals that are 4-6 week old C.B-17 scid/scid mice of either sex. Tissue from a 20-23 week old human fetal liver is collected into Hanks' balanced salts. 1.5 to 2 grams of the liver is minced by sharp dissection, then incubated in RPMI with 100 IU/ml type VI collagenase to enzymatically dissociate hepatocytes. All cells are pelleted at 500 x g for 10 minutes at 10°C and the connective tissue remnant discarded. The pellet is resuspended in cold RPMI.
To enrich for hepatocytes, the resuspended pellet is centrifuged at 50 x g for 5 minutes. The pellet (small mononuclear cells) and supernatant (large hepatocytes in suspension) are separated, saving both fractions for later testing. The hepatocyte enrichment is repeated by light centrifugation of supernatant, and the second pellet resuspended in RPMI. The cell suspension can then be held on wet ice for up to 2 hours until injected. For both the pellets and supernatants, the hepatocyte yield and concentration is then checked visually by Wright-Giemsa staining, and the cell viability is checked by trypan-blue dye exclusion. The cell suspension for injection is prepared from the fraction with the highest concentration of hepatocytes. The fraction with the highest hepatocyte concentration is resuspended in normal saline for injection. The dose is calculated by the number of mice to be injected x 0.1 ml per injection. An optimal injection dose is 10* to 107 viable hepatocytes per injection. A 28 gauge needle attached to a 0.5 ml tuberculin syringe is used for injections. The cell suspension is transported to surgery in ice.
Mice are anesthetized with either metafane using an open-drop method, or ketamine/xylazine cocktail using an intramuscular injection. The lateral abdomen is clipped and prepared for surgery. A left lateral paracostal abdominal incision is made over the normal position of the spleen. The left caudate lobe of the liver is located and exteriorized through the incision, making sure that the stomach, pancreas, proximal duodenum and biliary tract are not pulled or handled.
While applying gentle traction on the lobe, the base of the lobe is elevated and ligated with 6-0 Vicryl or other absorbable suture. The ligature is tightened with multiple surgical knots. After the capsule has been ligated, the lobe is transected by sharp dissection, and the exposed liver surface packed with sterile gel-foam. After holding the stump briefly to be certain of hemostasis, it is returned to the abdomen. The spleen is carefully located and exteriorized through the incision without disrupting any mesenteric or vascular attachments. 0.1 ml of the hepatocyte suspension is injected with a 28 guage needle, which punctures the splenic capsule on its antimesenteric surface. The needle should be parallel to the long axis of the spleen. The spleen is returned to the abdominal cavity, and the body wall closed with one or two simple interrupted sutures using 5-0 or 6-0 Vicryl, followed by a steel clip closure of the skin incision.
Alternatively, instead of isolating hepatocytes, intact liver fragments are implanted. Tissue from a 20-23 week old human fetal liver is cut with a sharp blade into approximately l l x l mm pieces. The mice are anesthetized and a 1 cm incision made in the liver at a site separate from the hepatectomy. One or two of these fragments are implanted with an 18 gauge trocar.
Animals are held in recovery and observed to rouse from anesthesia, and then checked every 24 hours for 3 days post-surgery. Starting at two weeks post surgery, blood samples may be collected for screening.
Serum samples are collected to screen for the success of engraftment by human hepatocytes. The samples are tested for the presence of human albumin (produced solely by hepatocytes) by RIA. Polyclonal anti-human serum albumin is commercially available (United States Biochemical). The antisera may be cleared of mouse cross-reactive antibodies by affinity purification against mouse serum albumin.
For terminal examination, spleens and livers are removed and fixed in 10% neutral buffered formalin (10% formalin in phosphate or carbonate buffer, pH 7.4) following collection of heart blood. Following fixation, tissues are processed for histologic and immunohistologic examination. Immunostaining is performed using rabbit a-human HLA (pan class I) antibody on formalin fixed, dewaxed sections, with secondary staining with FITC or peroxidase conjugated goat a-rabbit IgG.
Results:
Injection of hepatocyte pools directly into the liver of newborn scid/scid mice failed to produce detectable levels of human serum albumin three weeks after injection.
Following the above protocol of partial hepatectomy and intrasplenic injection of isolated hepatocytes, three 6 week old female C.B-17 scid/scid mice were injected with 21 week old fetal hepatocytes. A single cohort was sham injected with sterile RPMI following partial hepatectomy. Two weeks post surgery, all three animals that received human hepatocytes were positive for the presence of human serum albumin by RIA (0.32 μg/ml; 1.87 μg/ml; and 2.49 μg/ml for the three mice respectively). These results were positive and significant, given a zero assay for the sham injected mouse and other control mouse sera.
Example 2.
Hepatitis B Virus Infection of an Immunocompromised Mouse with Functional Human
Hepatocytes
Primary isolates of human HBV from acutely infected patient serum is used used to infect the human hepatocytes. Chimeric hepatocellular mice are made as described in Example 1. At least 2 weeks after engraftment, the host liver is exposed with an incision, and is inoculated via the portal vein using a 30-gauge needle with clinical isolates of human HBV. The virus is able to grow in the implant. The host animals are bled to assay for the presence of infectious HBV in the blood, assayed by the ability to infect primary human hepatocytes, or by immunological methods, e.g. using an antigen- capture assay that utilizes anti-HBV surface antigen antibodies coated to a plate. At 7 and 15 days post inoculation, animals are sacrificed and the tissue removed, minced, sonicated and titered for the presence of human HBV by ELISA for HBV antigens, and by PCR for the presence of viral genes. This subject chimeric system provides a small animal model for the analysis of human hepatic function, and its disease states. After engraftment, the implanted human tissue can be manipulated in a systematic way. The consequences of such manipulations can be read out by various methods, as described. Hepatic cells derived from diseased liver, e.g., as in hepatomas or genetic disorders, may be introduced into previously implanted liver grafts to study malignancy and the effects of growth factors and/or drugs which might modulate normal liver function or disease states. For human gene therapy trials, this model can also serve as a valuable system to test the long-term expression of exogenous genes introduced into human hepatic cells. The model is also a valuable system for observing the effects of human tropic viral infection and therapy in an in vivo system. It provides an alternative to animal hepatitis viruses and in vitro culture for the investigation of hepatocellular function during acute and/or chronic infection.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A non-human mammalian host lacking functional syngeneic B-cells and T- cells, comprising: engrafted functional human hepatocytes grown for at least two weeks in said host, wherein said human hepatocytes are injected into a host acceptor organ in conjunction with the provision of hepatocyte growth factors.
2. A host according to Claim 1, wherein said host acceptor organ is the spleen.
3. A host according to Claim 1, wherein said host acceptor organ is the liver.
4. A host according to Claim 1 , wherein said injection is via the portal vein.
5. A host according to Claim 1, wherein a suspension of isolated hepatocytes comprises said human fetal hepatocytes.
6. A host according to Claim 1 , wherein a fragment of intact liver tissue comprises said human fetal hepatocytes.
7. A host according to Claim 1, wherein said provision of hepatocyte growth factors comprises the induction of autologous factors by partial hepatectomy of said non- human mammalian host.
8. A host according to Claim 1, wherein said host is a mouse.
9. A host according to Claim 8, wherein said mouse has a homozygous mutation at the scid locus.
10. A host according to Claim 8, wherein said mouse lacks expression at least one of functional RAG-1 or RAG-2.
11. A method for producing a chimeric immunocompromised non-human mammalian host comprising functional human hepatocytes, said method comprising: injecting human fetal hepatocytes into a host acceptor organ in conjunction with the provision of hepatocyte growth factors, wherein said host is an immunocompromised non-human mammalian host lacking functional syngeneic B- and T-cells; and maintaining said host for a period of at least 2 weeks, wherein said human fetal hepatocytes are engrafted.
12. A method according to Claim 11, wherein said host acceptor organ is the spleen.
13. A method according to Claim 11, wherein said host acceptor organ is the liver.
14. A method according to Claim 11, wherein said injection is via the portal vein.
15. A method according to Claim 1 1, wherein a suspension of isolated hepatocytes comprises said human fetal hepatocytes.
16. A method according to Claim 11, wherein a fragment of intact liver tissue comprises said human fetal hepatocytes.
17. A method according to Claim 1 1, wherein said provision of hepatocyte growth factors comprises the induction of autologous factors by partial hepatectomy of said host.
18. A method according to Claim 17, wherein said host is a mouse.
19. A method according to Claim 18, wherein said mouse has a homozygous mutation at the scid locus.
20. A method according to Claim 18, wherein said mouse lacks expression of at least one of functional RAG-1 or RAG-2.
21. A method for determining the effect of a hepatic pathogen on human hepatocytes, the method comprising: administering said hepatic pathogen to a non-human mammalian host lacking functional syngeneic B-cells and T-cells, comprising engrafted functional human fetal hepatocytes grown for at least two weeks in said host, wherein said human fetal hepatocytes injected into a host acceptor organ in conjunction with the provision of hepatocyte growth factors; and determining the effect of said hepatic pathogen on said human hepatocytes.
22. A method according to Claim 21 further comprising administration to the host of a test compound (before or after administration of the hepatic pathogen) to determine the efficacy of the test compound in treating or preventing infection by the hepatic pathogen.
23. A method according to Claim 21, wherein said host acceptor organ is the spleen.
24. A method according to Claim 21, wherein said host acceptor organ is the liver.
25. A method according to Claim 21, wherein said injection is via the portal vein.
26. A method according to Claim 21, wherein a suspension of isolated hepatocytes comprises said human fetal hepatocytes.
27. A method according to Claim 21, wherein a fragment of intact liver tissue comprises said human fetal hepatocytes.
28. A method according to Claim 21, wherein said provision of hepatocyte growth factors comprises the induction of autologous factors by partial hepatectomy of said host.
29. A method according to Claim 21, wherein said host is a mouse.
30. A method according to Claim 29, wherein said mouse has a homozygous mutation at the scid locus.
31. A method according to Claim 29, wherein said mouse lacks expression of at least one of functional RAG-1 or RAG-2.
32. A method according to Claim 21, wherein said hepatic pathogen is a hepatitis virus.
33. A method according to Claim 32, wherein said hepatitis virus is hepatitis B virus.
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WO2000040083A1 (en) * 1999-01-05 2000-07-13 Surrogen, Inc. Chimeric animal with engrafted xenogeneic tissue and method for producing same
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WO2000040083A1 (en) * 1999-01-05 2000-07-13 Surrogen, Inc. Chimeric animal with engrafted xenogeneic tissue and method for producing same
US6660905B1 (en) 1999-07-14 2003-12-09 The Board Of Trustees Of The Leland Stanford Junior University Mice comprising engrafted functional human hepatocytes
WO2001005955A3 (en) * 1999-07-14 2001-08-16 Univ Leland Stanford Junior Animals comprising human hepatocellular tissue
WO2001005955A2 (en) * 1999-07-14 2001-01-25 The Board Of Trustees Of The Leland Stanford Junior University Animals comprising human hepatocellular tissue
WO2001032009A1 (en) * 1999-11-02 2001-05-10 The University Of Connecticut Propagation of human hepatocytes in non-human animals
US6525242B1 (en) 1999-11-02 2003-02-25 The University Of Connecticut Propagation of human hepatocytes in non-human mammals
US6995299B2 (en) 1999-11-02 2006-02-07 University Of Connecticut Propagation of human hepatocytes in non-human animals
US7498479B2 (en) 2000-03-17 2009-03-03 Kmt Hepatech, Inc. Animal model having a chimeric human liver
US7781642B2 (en) 2000-03-17 2010-08-24 Kmt Hepatech, Inc. Animal model having a chimeric human liver and susceptible to human hepatitis C virus infection
US8212106B2 (en) 2000-03-17 2012-07-03 Kmt Hepatech, Inc. Animal model having a chimeric human liver and susceptible to human hepatitis C virus infection
US8445745B2 (en) 2000-03-17 2013-05-21 Kmt Hepatech, Inc. Animal model having a chimeric human liver and susceptible to human hepatitis C virus infection
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EP1784640A4 (en) * 2004-08-20 2010-10-06 Kmt Hepatech Inc Malarial animal model having a chimeric human liver

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