Avian induced pluripotent stem cells and their use

Abstract

The present invention relates to the production of avian induced pluripotent stem cells from non-pluripotent somatic cells, including embryonic fibroblasts and adult somatic cells. In this method, avian (including quail or chicken) somatic cells are reprogrammed into a state closely resembling embryonic stem cells including the expression of key stem cell markers alkaline phosphatase, etc. by transfecting/transducing the non-stem cells with genes (preferably using a non-integrating vector as otherwise described herein or alternatively an integrating vector, such a lentiviral vector, retroviral vector or inducible lentiviral vector, among others) which express at least nanog, Lin28 and cMyc. In preferred aspects of the invention, the transfected/transduced vectors express nanog, Lin28, cMyc, Oct 4 (POU5F1 or PouV), SOX2 and KLF4. The induced stem cells which are produced contribute to all 3 germ layers, the trophectoderm and in certain aspects, the gonad in chimeric offspring.

1 . A population of avian induced pluripotent stem cells (aiPSCs) or avian induced pluripotent germ cells (aiPGCs) reprogrammed from avian somatic cells, said somatic cells being reprogrammed with at least the three reprogramming genes Nanog, LIN28 and c-MYC and optionally, at least one reprogramming gene selected from the group consisting of OCT4, SOX2 and KLF4 to produce said aiPSCs and said somatic cells being reprogrammed with the following four reprogramming genes Oct 4, SOX2, Nanog and LIN28, and optionally c-MYC to produce said aiPGCs. 
     
     
         2 . The population of aiPSCs according to  claim 1  wherein said cells have been reprogrammed with Nanog, LIN28, c-MYC, Oct4 and optionally SOX 2, KLF4 or a mixture of SOX 2 and KLF4. 
     
     
         3 . The population of aiPSCs according to  claim 1  wherein said cells have been reprogrammed with Nanog, LIN28, c-MYC, SOX2 and optionally Oct4 and/or KLF4. 
     
     
         4 . The population of aiPSCs according to  claim 3  wherein said cells have been reprogrammed with Nanog, LIN28, c-MYC and SOX2. 
     
     
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         9 . The population of cells according to  claim 1  wherein said somatic cells are embryonic somatic cells. 
     
     
         10 . The population of cells according to  claim 1  wherein said somatic cells are embryonic fibroblast cells. 
     
     
         11 . The population of cells according to  claim 1  wherein said somatic cells are adult somatic cells. 
     
     
         12 . The population of cells according to  claim 1  wherein said somatic cells are adult fibroblast cells. 
     
     
         13 . The population according to  claim 1  wherein said somatic cells are reprogrammed by transfecting or transducing said cells with at least one vector comprising said reprogramming genes. 
     
     
         14 . The population according to  claim 1  wherein said reprogramming genes are avian or mammalian genes each of which has a sequence homology of at least about 50% of the identical gene of the avian species from which the somatic cells are reprogrammed. 
     
     
         15 . The population according to  claim 1  wherein said reprogramming genes are chicken, mouse, human, pig or cow genes. 
     
     
         16 . The population according to  claim 1  wherein said cells are transfected or transduced with vectors wherein each of said vectors comprises a single reprogramming gene. 
     
     
         17 . The population according to  claim 1  wherein said avian induced pluripotent stem or germ cell is a quail, chicken, turkey, duck or goose pluripotent cell. 
     
     
         18 . The population according to  claim 17  wherein said avian induced pluripotent stem or germ cell is a chicken plurirpotent cell. 
     
     
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         21 . A population of avian induced pluripotent stem cells according to  claim 1  exhibiting the following four characteristics:
 form colonies with cells with a high nuclear to cytoplasmic ratio ranging from about 4:1 to about 1:2; 
 express alkaline phosphatase; 
 are developmentally identical to avian embryonic stem cells (aESCs); and 
 are capable of in vitro differentiation to all three germ layer types (mesoderm, endoderm and ectoderm) as determined by gene expression; 
 incorporate tranfection/transduction vectors which express reprogramming genes. 
 
     
     
         22 . The population of pluripotent stem cells according to  claim 21  wherein said cells exhibit one or more of the following characteristics:
 express the following genes and proteins: Nanog, PouV, SOX2; 
 exhibit a normal karyotype; 
 express high levels of telomerase; and 
 express Periodic Acid Schiff (PAS). 
 
     
     
         23 . The population of pluripotent stem cells according to  claim 21  wherein said cells exhibit one or more of the following characteristics:
 express at least one of the cell surface markers SSEA1, SSEA3, SSEA4, TRA 1-61 and TRA 1-81; 
 exhibit stable/robust pluripotency and can differentiate or contribute to chimeras even after more than 25 passages; 
 fast cell cycle with high population doubling;
 when injected into avian embryos, contribute to all germ cell lineages during development; 
 
 chimera generate protein and morphologically distinct cells in vitro such as neurons and muscle cells, among numerous others; and 
 the cells are clonigenic. 
 
     
     
         24 . A population of avian induced pluripotent germ cells according to  claim 1  exhibiting at four or more of the following characteristics:
 Morphologically possess high nucleus to cytoplasm ratio and large nucleoli; 
 express Alkaline Phosphotase, SSEA1, EMA1, POU5F1, SOX2, NANOG, Chicken Vasa Homolog (CVH), DAZL, CXCR4, C-KIT and express high levels of telomerase; 
 migrate to the gonad; 
 can be maintained in adherent or suspension culture; traditional PGCs are cultured in suspension 
 can be derived from any somatic cell of the body; 
 show robust differentiation in vitro; 
 incorporate tranfection/transduction vectors which express reprogramming genes 
 intermediary PSCs are capable of differentiating into all 3 germ layers in vitro
 can be used to generate gametes (ova and sperm) 
 
 can be used to generate chimeric animals; and 
 can be expanded in adherent cultures. 
 
     
     
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         26 . The population of induced pluripotent stem or germ cells according to  claim 1  which have been instilled with disease resistance from a disease agent. 
     
     
         27 . The population of pluripotent stem cells according to  claim 26  wherein said disease agent is Newcastle disease virus or avian influenza virus. 
     
     
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         30 . A chimeric bird produced from a population of pluripotent stem cells according to  claim 1 . 
     
     
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         34 . The chimeric bird according to  claim 30  which exhibit resistance to a disease agent. 
     
     
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         36 . A method comprising reprogramming an avian embryonic or adult somatic cell to an avian induced pluripotent stem cell (aiPSC) or an avian induced pluripotent germ cell (aiGSC), said method comprising transfecting or transducing said somatic cell with the three reprogramming genes Nanog, LIN28 and c-Myc and optionally, at least one reprogramming gene selected from the group consisting of Oct 4, SOX2 and KLF4, or four reprogramming genes Oct4, SOX2, Nanog and LIN28 and optionally c-MYC, allowing said genes to reprogram said somatic cells into pluripotent stem cells or pluripotent germ cells and optionally, isolating and/or expanding said stem or germ cells, wherein each of said reprogramming genes is an avian or mammalian gene which exhibits at least about 50% sequence homology with the identical gene from the species from which the somatic are reprogrammed. 
     
     
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         61 . A method of genetically manipulating avian species, especially chickens, comprising selecting and introducing genes into somatic cells to be reprogrammed into avian iPSCs or PGCs or directly into avian iPSCs or iPGCs to produce genetically manipulated iPSCs or iPGCs, introducing the genetically manipulated avian iPSCs or iPGCs cells into avian embryos to form chimeric embryos; and generating offspring with said chimeric embryos, wherein said offspring exhibit a phenotype consistent with said introduced genes. 
     
     
         62 . The method according to  claim 61  wherein said genes are introduced directly into said iPSCs or iPGCs. 
     
     
         63 . The method according to  claim 61  wherein said genes are introduced into said somatic cells before being reprogrammed to iPSCs or iPGCs. 
     
     
         64 . The method according to  claim 61  wherein said genetically manipulated iPSCs or iPGCs are introduced into a subgerminal cavity of said embryos and said embryos are allowed to mature. 
     
     
         65 . (canceled) 
     
     
         66 . (canceled) 
     
     
         67 . A method of instilling disease resistance in an avian induced pluripotent stem cell or avian induced pluripotent germ cell, said method comprising producing an avian induced pluripotent stem cell or avian induced pluripotent germ cell from somatic cells according to the method of  claim 36 , exposing said pluripotent stem or germ cells to a disease agent for a period of time sufficient to produce resistance to said disease agent in said cells so that more than 99% of the population of said pluripotent stem or germ cells succumb to said disease agent, isolating the remaining viable cells, optionally testing said cells for resistance to said disease agent and optionally, propagating said resistant cells to provide a population of avian induced pluripotent stem or germ cells exhibiting resistance to said disease agent. 
     
     
         68 . A method of instilling disease resistance into a population of avian induced pluripotent stem cells or avian induced pluripotent germ cells from disease resistant somatic cells, said method comprising producing disease resistant somatic cells by exposing somatic cells to a disease agent for a period of time sufficient to produce resistance to said disease agent in said cells, more than 99% of the population of which succumb to said disease agent, isolating said resistant somatic cells, propagating said cells to provide a population of resistant somatic and producing a population of disease resistant avian induced pluripotent stem cells or avian induced pluripotent germ cells from said disease resistant somatic cells according to the method of  claim 36 . 
     
     
         69 . The method according to  claim 67  wherein said resistant aiPSCs or aiPGCs are injected into an avian embryo to produce a chimeric bird, said bird exhibiting resistance to said disease agent. 
     
     
         70 . (canceled) 
     
     
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         72 . A method of producing a chimeric avian gamete comprising administering into a germline cell an avian induced pluripotent stem or germ cell according to  claim 1  to produce a chimeric germline cell, introducing said chimeric germline cell into the testes or ovaries of an adult bird and isolated said chimeric gamete after a period sufficient to produce said gamete from said germline in said adult bird. 
     
     
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         75 . The population according to  claim 26  wherein said disease agent is an agent against which a vaccine is to be made. 
     
     
         76 . A method of producing immunogenic material for a vaccine comprising providing a population of aiPSCs or aiPGCs resistant to a disease agent according to  claim 75 , growing said disease agent in said population of aiPSCs or aiPGCs, removing said disease agent from said population of aiPSCs or aiGSCs after said growing step, and optionally inactivating said disease agent after said removing step. 
     
     
         77 . The method according to  claim 76  wherein said disease agent is influenza virus. 
     
     
         78 . The method according to  claim 76  wherein said disease state is Newcastle disease virus avian influenza virus. 
     
     
         79 . (canceled) 
     
     
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         81 . A vaccine comprising immunogenic material produced according to the methods of  claim 76 . 
     
     
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         94 . A method of producing an avian gamete comprising introducing an avian induced pluripotent germ cell into the testes or ovaries of an adult bird and isolating said avian gamete after a period sufficient to produce said gamete from said testes or said ovaries in said adult bird. 
     
     
         95 - 98 . (canceled) 
     
     
         99 . The method according to  claim 68  wherein said resistant aiPSCs or aiPGCs are injected into an avian embryo to produce a chimeric bird, said bird exhibiting resistance to said disease agent.