Methods for introducing heterologous cells into fish

Abstract

The invention provides methods of introducing heterologous cells into fish. After introduction cells remain viable, and in some instances proliferate, for sufficient time to conduct a variety of analyses on the heterologous cells or the fish or both. Such methods are useful for screening potential drugs for toxicity toward introduced cells or for capacity to stimulate differentiation and/or proliferation of introduced cells. Such methods are also useful for diagnosing the presence of small quantities of cancerous cells or pathogens in patient tissue samples. Such methods are also useful for culturing cells for subsequent use in cell or tissue engineering.

1 . A method of cellular analysis, the method comprising: introducing a heterologous cell into a fish; and analyzing a property of the cell or the fish. 
     
     
         2 . The method of  claim 1 , wherein the fish is a teleost. 
     
     
         3 . The method of  claim 2 , wherein the teleost is a zebrafish, medaka, Giant rerio, or puffer fish. 
     
     
         4 . The method of  claim 1 , wherein the fish is a larva or an adult. 
     
     
         5 . The method of  claim 1 , wherein the fish is a wild-type strain. 
     
     
         6 . The method of  claim 1 , wherein the fish is a mutant strain. 
     
     
         7 . The method of  claim 1 , wherein the heterologous cell is a stem cell. 
     
     
         8 . The method of  claim 7 , wherein the stem cell is a pluripotent or mesenchymal stem cell. 
     
     
         9 . The method of  claim 1 , wherein the heterologous cell is a differentiated cell. 
     
     
         10 . The method of  claim 1 , wherein the heterologous cell is a myocyte. 
     
     
         11 . The method of  claim 1 , wherein the heterologous cell is a chondrocyte. 
     
     
         12 . The method of  claim 1 , wherein the heterologous cell is a pancreatic, renal, hepatic, glial, endothelial, or epidermal cell. 
     
     
         13 . The method of  claim 1 , wherein the heterologous cell is a human cell. 
     
     
         14 . The method of  claim 1 , wherein the heterologous cell is a bacterial cell. 
     
     
         15 . The method of  claim 1 , further comprising infecting the heterologous cell with a virus. 
     
     
         16 . The method of  claim 15 , wherein the virus infects the heterologous cell without infecting cells of the fish. 
     
     
         17 . A method of cellular analysis, the method comprising: introducing a heterologous cell into a fish; contacting the fish with an agent; and analyzing a property of the cell or the fish, wherein said analyzing comprises determining whether the property is responsive to administration of the agent. 
     
     
         18 . The method of  claim 17 , wherein the fish is a teleost. 
     
     
         19 . The method of  claim 18 , wherein the teleost is a zebrafish, medaka, Giant rerio, or puffer fish. 
     
     
         20 . The method of  claim 17 , wherein the fish is an embryo. 
     
     
         21 . The method of  claim 17 , wherein the fish is a larva or an adult. 
     
     
         22 . The method of  claim 17 , wherein the fish is a wild-type strain. 
     
     
         23 . The method of  claim 17 , wherein the fish is a mutant strain. 
     
     
         24 . The method of  claim 17 , wherein a plurality of cells are introduced into the fish. 
     
     
         25 . The method of  claim 17 , wherein a plurality of cells are introduced at multiple sites of the fish. 
     
     
         26 . The method of  claim 17 , wherein the heterologous cell is undifferentiated. 
     
     
         27 . The method of  claim 17 , wherein the heterologous cell is terminally differentiated. 
     
     
         28 . The method of  claim 17 , wherein the agent is from a combinatorial library of peptides or small molecules. 
     
     
         29 . The method of  claim 17 , wherein the agent is a naturally occurring molecule. 
     
     
         30 . The method of  claim 17 , wherein the agent is a hormone, growth factor, or cytokine. 
     
     
         31 . The method of  claim 17 , wherein agent is a polypeptide, beta-turn mimetic, polysaccharide, phospholipid, prostaglandin, steroid, aromatic compound, heterocyclic compound, benzodiazepine, oligomeric N-substituted glycine, or oligocarbamate. 
     
     
         32 . The method of  claim 17 , wherein the agent is administered by adding the agent to media containing the fish. 
     
     
         33 . The method of  claim 17 , wherein the agent is administered by microinjecting the agent into the fish. 
     
     
         34 . The method of  claim 17 , wherein the heterologous cell is a mammalian cell, bacterial cell, plant cell, or bird cell. 
     
     
         35 . The method of  claim 34 , wherein the cell is a mammalian cell. 
     
     
         36 . The method of  claim 35 , wherein the mammalian cell is from a cat, dog, horse, bovine, mouse, rat, rabbit, guinea pig, or primate. 
     
     
         37 . The method of  claim 36 , wherein the mammalian cell is a human cell. 
     
     
         38 . The method of  claim 17 , wherein the heterologous cell is a cancer cell. 
     
     
         39 . The method of  claim 38 , wherein the property is a differentiation marker, movement of the cancer cell relative to an initial site of introduction, death of the cancer cell, or proliferation of the cancer cell. 
     
     
         40 . The method of  claim 38 , which comprises labeling the cancer cell. 
     
     
         41 . The method of  claim 40 , wherein the labeling comprises immunostaining with an antibody specific to a cancer cell antigen. 
     
     
         42 . The method of  claim 40 , wherein the cancer cell is labeled after introducing the cancer cell into the fish. 
     
     
         43 . The method of  claim 38 , wherein the property is tumor-induced angiogenesis. 
     
     
         44 . The method of  claim 43 , which comprises identifying a profile of differentially expressed genes. 
     
     
         45 . The method of  claim 44 , which further comprises using the profile of differentially expressed genes to determine the presence of angiogenesis in another animal or in a human. 
     
     
         46 . The method of  claim 38 , which comprises monitoring the fish for a deleterious side effect of the agent on fish cells and/or embryogenesis. 
     
     
         47 . The method of  claim 46 , wherein the deleterious side effect comprises cell death activity in the fish. 
     
     
         48 . The method of  claim 47 , wherein the cell activity comprises apoptosis or necrosis. 
     
     
         49 . The method of  claim 47 , wherein the monitoring of cell death activity comprises staining the fish with a membrane-impermeant, nuclear-staining fluorescent dye. 
     
     
         50 . The method of  claim 49 , wherein the membrane-impermeant, nuclear-staining dye is a dye of the quinolium dye family. 
     
     
         51 . The method of  claim 50 , wherein the quinolium dye is a benzothiazolium-4-quinolium dye. 
     
     
         52 . The method of  claim 46 , wherein the monitoring comprises in situ hybridization of RNA or antibody staining of a protein. 
     
     
         53 . The method of  claim 46 , wherein the monitoring comprises detection of carboxilase enzymes using a streptavidin-conjugated reporter enzyme. 
     
     
         54 . The method of  claim 17 , wherein the heterologous cell is a pathogenic cell. 
     
     
         55 . The method of  claim 54 , wherein the pathogenic cell is a bacterial cell or a fungal cell. 
     
     
         56 . The method of  claim 17 , further comprising contacting the heterologous cell with virus before or after introducing the heterologous cell into the fish, wherein the property to be analyzed in viral infection or pathogenesis of the heterologous cell. 
     
     
         57 . The method of  claim 56 , further comprising propagating the virus in the heterologous cell. 
     
     
         58 . The method of  claim 56 , wherein the virus is a HBV, HCV, HPV, HIV, or herpes virus. 
     
     
         59 . The method of  claim 56 , wherein the fish is contacted with the agent before introduction of the virus, and wherein the method comprises testing the agent for prophylactic activity against the virus. 
     
     
         60 . The method of  claim 56 , wherein the fish is contacted with the agent after introduction of the virus, and wherein the method comprises testing the agent for therapeutic activity against the virus. 
     
     
         61 . The method of  claim 17 , wherein the property analyzed is differentiation or proliferation of the heterologous cell. 
     
     
         62 . The method of  claim 61 , wherein the agent is a growth factor. 
     
     
         63 . The method of  claim 62 , wherein the growth factor is GM-CSF, EPO, FGF, PDGF, VEGF, or stem cell factor. 
     
     
         64 . The method of  claim 61 , wherein the property analyzed is differentiation of the heterologous cell. 
     
     
         65 . The method of  claim 64 , wherein analyzing differentiation of the heterologous cell comprises observing a change in a set of one or more differentiation markers. 
     
     
         66 . The method of  claim 61 , which comprises screening a plurality of agents for activity in promoting at least one of cell differentiation and proliferation. 
     
     
         67 . The method of  claim 66 , further comprising identifying an agent that promotes at least one of cell differentiation and proliferation. 
     
     
         68 . The method of  claim 66 , wherein the heterologous cell is a stem cell. 
     
     
         69 . The method of  claim 68 , wherein the stem cell is a totipotent stem cell. 
     
     
         70 . The method of  claim 68 , wherein the stem cell is a pluripotent or mesenchymal stem cell. 
     
     
         71 . The method of  claim 68 , wherein the stem cell is a neural stem cell and the property is differentiation of the neural stem cell to a neuron. 
     
     
         72 . The method of  claim 71 , wherein analyzing the differentiation of the neural stem cell comprises monitoring neural transduction or differentiation markers of mature cells. 
     
     
         73 . The method of  claim 68 , wherein the stem cell is a cardiac stem cell and the property is differentiation of the cardiac stem cell to a myocyte. 
     
     
         74 . The method of  claim 73 , wherein analyzing the differentiation of the cardiac stem cell comprises monitoring excitation level, capacity to beat, or differentiation markers of mature cells. 
     
     
         75 . The method of  claim 61 , which comprises screening a plurality of agents for activity in promoting or inhibiting a function of a differentiated cell. 
     
     
         76 . The method of  claim 75 , wherein the differentiated cell function comprises expression or secretion of a protein. 
     
     
         77 . The method of  claim 76 , wherein the differentiated cell function is secretion of insulin from an islet cell. 
     
     
         78 . The method of  claim 76 , wherein the heterologous cell is a human stem cell and the differentiated cell function is expression of an exogenous enzyme. 
     
     
         79 . A method for propagating cells, the method comprising: introducing one or more heterologous cells into a fish; and culturing the fish under conditions in which the one or more heterologous cells propagate in the fish. 
     
     
         80 . The method of  claim 79 , wherein the fish is a teleost. 
     
     
         81 . The method of  claim 80 , wherein the teleost is a zebrafish, medaka, Giant rerio, or puffer fish. 
     
     
         82 . The method of  claim 79 , wherein the one or more heterologous cells are human cells. 
     
     
         83 . The method of  claim 79 , wherein the one or more heterologous cells are stem cells. 
     
     
         84 . The method of  claim 79 , wherein the one or more heterologous cells are selected from the group consisting of osteoblast, osteoclast, chondrocytes, myocytes, neurons, pancreatic cells, hepatic cells, glial cells, and renal cells. 
     
     
         85 . The method of  claim 79 , wherein the one or more heterologous cells proliferate in the fish. 
     
     
         86 . The method of  claim 85 , wherein the one or more heterologous cells proliferate without reaching a terminal differentiation state. 
     
     
         87 . The method of  claim 86 , further comprising recovering the heterologous cells from the fish. 
     
     
         88 . The method of  claim 87 , further comprising introducing the heterologous cells into a patient at a site at which tissue regeneration is needed. 
     
     
         89 . The method of  claim 85 , wherein the cells differentiate in the course of proliferation. 
     
     
         90 . The method of  claim 89 , further comprising recovering the cells from the fish as a differentiated tissue. 
     
     
         91 . The method of  claim 90 , further comprising introducing the recovered cells into a patient. 
     
     
         92 . The method of  claim 91 , wherein the one or more heterologous cells are obtained from the same patient into which the recovered cells are introduced. 
     
     
         93 . The method of  claim 90 , wherein the differentiated tissue is skin tissue. 
     
     
         94 . The method of  claim 93 , further comprising recovering the skin tissue from the fish and introducing the skin tissue into a patient. 
     
     
         95 . The method of  claim 89 , wherein the one or more heterologous cells are hematopoietic human stem cells. 
     
     
         96 . The method of  claim 95 , wherein the hematopoietic human stem cells differentiate into human platelets and red blood cells. 
     
     
         97 . The method of  claim 96 , further comprising recovering the human platelets and red blood cells from the fish. 
     
     
         98 . The method of  claim 97 , further comprising introducing the human platelets and red blood cells into a patient. 
     
     
         99 . The method of  claim 79 , wherein the one or more heterologous cells are transformed with a nucleic acid sequence suitable for gene therapy before introducing the heterologous cells into the fish. 
     
     
         100 . The method of  claim 99 , further comprising recovering the transformed heterologous cells from the fish and introducing the cells into a patient. 
     
     
         101 . The method of  claim 85 , wherein the one or more heterologous cells are bone marrow cells. 
     
     
         102 . The method of  claim 101 , further comprising recovering the bone marrow cells from the fish following proliferation. 
     
     
         103 . The method of  claim 102 , further comprising introducing the recovered bone marrow cells into a patient. 
     
     
         104 . The method of  claim 85 , wherein the one or more heterologous cells are cancer cells obtained from a patient biopsy. 
     
     
         105 . The method of  claim 104 , further comprising recovering the cancer cells following proliferation. 
     
     
         106 . The method of  claim 105 , further comprising killing the recovered cancer cells and reintroducing the killed cancer cells into the patient. 
     
     
         107 . The method of  claim 105 , further comprising immunizing an animal with the recovered cancer cells to generate antibodies. 
     
     
         108 . The method of  claim 79 , wherein a plurality of heterologous cells are introduced into a plurality of fish, said plurality of heterologous cells comprising different clonal isolates from a mixed population of cancerous and non-cancerous cells obtained from a patient, and wherein different clonal isolates are introduced into different fish. 
     
     
         109 . The method of  claim 108 , further comprising screening the heterologous cells for the presence of a cancer specific antigen. 
     
     
         110 . The method of  claim 109 , further comprising recovering cells that lack the cancer specific antigen from their host fish and reintroducing said cells into the patient. 
     
     
         111 . The method of  claim 110 , wherein the patient is a bone marrow transplant patient. 
     
     
         112 . A method of diagnosing a sample for a cancerous cell or pathogen, the method comprising: obtaining a sample from a patient containing a population of cells; introducing the population of cells into a fish; and detecting a property of the population of cells to indicate whether the population comprises a cancerous cell or pathogen. 
     
     
         113 . The method of  claim 112 , wherein the fish is a teleost. 
     
     
         114 . The method of  claim 113 , wherein the teleost is a zebrafish, medaka, Giant rerio, or puffer fish. 
     
     
         115 . The method of  claim 112 , wherein the property detected is proliferation or metastasis of the cells. 
     
     
         116 . The method of  claim 115 , further comprising determining the rate of proliferation or metastasis. 
     
     
         117 . The method of  claim 112 , wherein the sample comprises residual lymphoma cells, and wherein the method comprises determining the clonogenic potential of the residual lymphoma cells. 
     
     
         118 . The method of  claim 112 , wherein the method comprises detecting a cellular pathogen. 
     
     
         119 . The method of  claim 112 , wherein the method comprises detecting a viral pathogen. 
     
     
         120 . A method of screening cancerous cells from a patient for an antineoplastic treatment appropriate for the patient, the method comprising: obtaining a sample from a patient containing cancerous cells; introducing the cancerous cells into fish; subjecting the cancerous cells to different antineoplastic treatments; and detecting a property of the cancerous cells to determine the most appropriate antineoplastic treatment for the patient. 
     
     
         121 . A method for monitoring differentiation of a cell lineage in isolation of other cell types from the same organism, the method comprising: introducing nonterminally differentiated, heterologous cells into a plurality of fish; and monitoring markers of differentiation in at least one of the heterologous cells and the fish. 
     
     
         122 . The method of  claim 121 , wherein the markers of differentiation are monitored with an array of nucleic acid probes complementary to mRNA of the fish or heterologous cells.