Compositions and methods for organoid generation and disease modeling

Abstract

Provided are pancreatic islet organoid and pancreatic organoid, and the cell culture and method that are useful for the rapid and reliable production of pancreatic islet and pancreatic islet organoid.Also provided are the method for treating pancreatic disease such as type 2 diabetes and pancreatic cancer that uses pancreatic islet organoid and pancreatic organoid of the present invention, and the method for identifying the active substance that is useful for treating said pancreatic disease. [Solution] Provided are methods for producing pancreatic islet organoids, including culturing induced pluripotent stem cell (iPSC)-derived β-like cells in a three-dimensional matrix containing gellan gum, thereby producing pancreatic islet organoids. Also provided are cell cultures containing iPSC-derived β-like cells in a three-dimensional matrix containing gellan gum.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 341,461, filed May 25, 2016, the entire contents of which are incorporated herein by reference.

[0002] Statement of Rights to Inventions Made Under Federally Sponsored Research This invention was made with Government support under Grant Nos. DK057978 and DK0909962 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Although animal disease models can provide insight into disease pathogenesis, drugs screened and selected using animal models often cannot be adopted in human patients. Because evolutionary biology, molecular biology, and genetic studies show that animals and humans can differ greatly, there is an urgent need to recapitulate human diseases using human cells and generate functional human organs. Summary of the Invention

[0004] As described below, the present invention features compositions and methods for generating organoids, including pancreatic islet organoids or pancreatic organoids.

[0005] In one aspect, the present invention provides a method for producing pancreatic islet organoids, comprising culturing induced pluripotent stem cell (iPSC)-derived beta-like cells in a three-dimensional matrix containing gellan gum, thereby producing pancreatic islet organoids.

[0006] In another aspect, the present invention provides a cell culture comprising iPSC-derived β-like cells in a three-dimensional matrix containing gellan gum.

[0007] In another aspect, the present invention provides a cell culture comprising human iPSC-derived β-like cells, human adipose-derived stem cells (hADSCs), and human umbilical vein endothelial cells (HUVECs) in a three-dimensional matrix containing gellan gum.

[0008] In various embodiments of any of the aspects detailed herein, the cell culture comprises adipose-derived stem cells and / or endothelial cells.

[0009] In another aspect, the present invention provides pancreatic islet organoids containing iPSC-derived β-like cells, which are vascularized and exhibit glucose-stimulated insulin secretion (GSIS).

[0010] In another aspect, the present invention provides a pancreatic islet organoid comprising iPSC-derived β-like cells, iPSC-derived α cells, iPSC-derived δ cells, iPSC-derived ductal cells, adipose-derived stem cells (hADSCs), and endothelial cells, the pancreatic islet organoid being vascularized and exhibiting glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, and glucagon secretion.

[0011] In a related aspect, the invention provides a non-human organism implanted with an organoid of any aspect described in detail herein.

[0012] In another aspect, the present invention provides a method for identifying an agent that regulates pancreatic activity and / or treats pancreatic disease, comprising contacting a candidate agent with pancreatic islet organoid or pancreatic organoid; measuring the activity of the organoid that is contacted with the candidate agent, and if the activity of the organoid changes compared to the reference, the candidate agent is identified as an agent that regulates pancreatic activity and / or treats pancreatic disease.

[0013] In another aspect, the present invention provides a method for identifying an agent that regulates pancreatic activity and / or treats pancreatic disease, comprising administering a candidate agent to a non-human subject that is transplanted with pancreatic islet organoid or pancreatic organoid; and measuring the pancreatic activity of the non-human subject, and if the pancreatic activity of the non-human subject is changed compared with a reference, the candidate agent is identified as an agent that regulates pancreatic activity and / or treats pancreatic disease.

[0014] In another aspect, the present invention provides a method for treating a pancreatic disease in a subject, comprising transplanting a pancreatic islet organoid into the subject, wherein the pancreatic islet organoid contains iPSC-derived β-like cells, is vascularized, and exhibits glucose-stimulated insulin secretion (GSIS).

[0015] In another aspect, the present invention provides a method for treating type 1 diabetes in a subject, comprising transplanting a pancreatic islet organoid into the subject, wherein the pancreatic islet organoid contains iPSC-derived β-like cells, is vascularized, and exhibits glucose-stimulated insulin secretion (GSIS).

[0016] In another aspect, the present invention provides pancreatic islet organoids generated by culturing induced pluripotent stem cell (iPSC)-derived β-like cells in a three-dimensional matrix containing gellan gum.

[0017] In another aspect, the present invention provides pancreatic organoids produced by culturing induced pluripotent stem cell (iPSC)-derived beta-like cells and iPSC-derived exocrine component cells in a three-dimensional matrix containing gellan gum.

[0018] In another aspect, the present invention provides a liver organoid produced by culturing hepatocytes derived from induced pluripotent stem cells (iPSCs) in a three-dimensional matrix containing gellan gum.

[0019] In another aspect, the present invention provides a cardiac organoid produced by culturing induced pluripotent stem cell (iPSC)-derived cardiomyocytes in a three-dimensional matrix containing gellan gum.

[0020] In another aspect, the present invention provides an intestinal organoid produced by culturing intestinal cells derived from induced pluripotent stem cells (iPSCs) in a three-dimensional matrix containing gellan gum.

[0021] In various embodiments of any aspect described in detail herein, the method includes culturing the iPSC-derived β-like cells with adipose-derived stem cells and / or endothelial cells. In various embodiments of any aspect described in detail herein, the method includes culturing the iPSC-derived β-like cells with iPSC-derived α-like cells, iPSC-derived δ-like cells, and / or iPSC-derived duct-like cells.

[0022] In various embodiments of any aspect described herein in detail, pancreatic islet organoid contains iPSC-derived alpha-like cells, iPSC-derived delta-like cells, and / or iPSC-derived duct-like cells.In various embodiments of any aspect described herein in detail, pancreatic islet organoid comprises adipose-derived stem cells and / or endothelial cells.In various embodiments of any aspect described herein in detail, pancreatic islet organoid shows KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, and / or glucagon secretion.In various embodiments of any aspect described herein in detail, pancreatic islet organoid expresses one or more of β cell transcription factors Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, and Foxa2. In certain embodiments, pancreatic islet organoid comprises iPSC-derived β-like cells, iPSC-derived α-cells, iPSC-derived δ-cells, iPSC-derived ductal cells, adipose-derived stem cells (hADSCs) and endothelial cells, and in this case, said organoid is vascularized and shows glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion and glucagon secretion.In various embodiments of any aspect described herein in detail, pancreatic islet organoid is surrounded by iPSC-derived exocrine components.In various embodiments, iPSC-derived exocrine components express one or more of markers PDX1, Nkx6-1 and Ptf1.

[0023] In various embodiments of any aspect described in detail herein, the method includes inducing or mimicking pancreatic disease in organoids or non-human subjects.In various embodiments of any aspect described in detail herein, the disease is induced by contacting organoids or administering to non-human subjects one or more of the following agents: free fatty acid (FFA), glucose, and cytokines.In various embodiments of any aspect described in detail herein, the disease is mimicked by culturing organoids with pancreatic cancer cells, stellate cells, and immune cells to create a human pancreatic cancer microenvironment.In various embodiments of any aspect described in detail herein, the pancreatic activity is one or more of insulin secretion, beta cell apoptosis, expression or activity of NDUFA4, ESRRG, G6PC2, MDH1, LDHA, KCNK3, or MAFA polypeptide or polynucleotide, amylase secretion, apoptosis of exocrine components, collagen synthesis, and stellate cell activation.In various embodiments, the non-human subject is also transplanted with liver organoids.

[0024] In various embodiments of any aspect described herein in detail, the candidate agent increases insulin secretion.In various embodiments of any aspect described herein in detail, the candidate agent identified as the agent that regulates pancreatic activity is tested for its ability to treat pancreatic disease.In various embodiments, the pancreatic disease is type 2 diabetes or pancreatic cancer.

[0025] In various embodiments of any aspect described in detail herein, the organoid is an organoid according to any aspect described in detail herein.

[0026] In various embodiments, the non-human organism is a mammal (eg, a mouse).

[0027] In various embodiments of any aspect described in detail herein, the pancreatic islet organoids contain iPSC-derived alpha-like cells, iPSC-derived delta-like cells, and / or iPSC-derived duct-like cells.

[0028] In various embodiments of any of the aspects detailed herein, the pancreatic islet organoids contain adipose-derived stem cells and / or endothelial cells.

[0029] In various embodiments of any aspect described herein in detail, pancreatic disease is induced or simulated in the subject.In certain embodiments, the pancreatic disease is type 1 diabetes or type 2 diabetes.In certain embodiments, the subject is a mammal (e.g., human).In various embodiments of any aspect described herein in detail, the subject is administered an immunosuppressant.

[0030] In various embodiments of any aspect as described herein in detail, liver organoid expresses one or more of markers AFP, ALB and Cyp3a7.In various embodiments of any aspect as described herein in detail, liver organoid shows insulin signal transduction, palmitic acid insulin resistance and lipid accumulation.

[0031] In various embodiments of any aspect described herein in detail, cardiac organoid expresses one or more of markers: hMlc2a, hNkx2-5, αMHC and KCNQ1.In various embodiments of any aspect described herein in detail, cardiac organoid shows heartbeat.

[0032] In various embodiments of any aspect described herein in detail, intestinal organoid expresses one or more of markers CDX2, Muc2 and Lgr5.In various embodiments of any aspect described herein in detail, intestinal organoid shows budding in response to R-spondin.

[0033] In various embodiments of any aspect described in detail herein, the iPSC-derived β-like cells, iPSC-derived α-like cells, iPSC-derived δ-like cells, and / or iPSC-derived duct-like cells are human. In various embodiments of any aspect described in detail herein, the iPSC-derived β-like cells, iPSC-derived exocrine component cells, iPSC-derived hepatocytes, iPSC-derived cardiomyocytes, or iPSC-derived intestinal cells are human. In various embodiments, the adipose-derived stem cells are human adipose-derived stem cells (hADSCs). In various embodiments of any aspect described in detail herein, the endothelial cells are human umbilical vein endothelial cells (HUVECs).

[0034] In various embodiments of any aspect described herein in detail, pancreatic islet organoid, pancreatic organoid, liver organoid, heart organoid or intestinal organoid contains adipose-derived stem cell and / or endothelial cell.In various embodiments of any aspect described herein in detail, pancreatic islet organoid, pancreatic organoid, liver organoid, heart organoid or intestinal organoid is vascularized.

[0035] In another aspect, the present invention provides a method for producing pancreatic islet organoid, comprising culturing induced pluripotent stem cell (iPSC) derived β-like cells in a medium containing Wnt4 or Wnt5a protein.In one embodiment, induced pluripotent stem cell (iPSC) derived β-like cells are cultured in a three-dimensional matrix.In one embodiment of the above aspect, Wnt4 or Wnt5a protein is recombinant human Wnt4 or Wnt5a protein.In a particular embodiment, the medium contains recombinant human Wnt4 protein.In another particular embodiment, the medium contains recombinant human Wnt5a protein.

[0036] In another aspect, the present invention provides a cell culture comprising human iPSC-derived β-like cells and Wnt4 or Wnt5a protein.In one embodiment, the human iPSC-derived β-like cells are present in a three-dimensional matrix comprising gellan gum.In one embodiment, the Wnt4 or Wnt5a protein is recombinant human Wnt4 or Wnt5a protein.In a particular embodiment, the medium comprises recombinant human Wnt4 protein.In another particular embodiment, the medium comprises recombinant human Wnt5a protein.

[0037] In another aspect, the present invention provides pancreatic islet organoid comprising iPSC-derived β-like cells cultured in a medium comprising Wnt4 or Wnt5a protein, the pancreatic islet organoid being vascularized and exhibiting glucose-stimulated insulin secretion (GSIS).In one embodiment, the organoid further exhibits KCl-stimulated insulin secretion or glucose-stimulated insulin secretion.In one embodiment, the pancreatic islet organoid expresses Fltp gene and Esrrg gene.In one embodiment, the Wnt4 or Wnt5a protein is recombinant human Wnt4 or Wnt5a protein.In a particular embodiment, the medium comprises recombinant human Wnt4 protein.In another particular embodiment, the medium comprises recombinant human Wnt5a protein.

[0038] In another aspect, the present invention provides a non-human organism implanted with an organoid as defined in the above-described aspect.

[0039] In another aspect, the present invention provides a method for enhancing the self-organization of adipose-derived stem cells (ADSCs) to generate organoids derived from induced pluripotent stem cells (iPSCs), comprising culturing ADSCs in a three-dimensional (3D) culture matrix medium comprising Wnt5a protein.In one embodiment of the method, ADSCs are cultured in a 3D culture matrix medium comprising gellan gum.In one embodiment, ADSCs are cultured in a 3D culture matrix medium comprising Wnt5 protein and iPSC-derived cells selected from iPSC-derived β-like cells, iPSC-derived exocrine component cells, iPSC-derived hepatocytes, iPSC-derived cardiomyocytes, or iPSC-derived intestinal cells.In one embodiment of the method, iPSC-derived organoids are selected from pancreatic islet organoids, pancreatic organoids, liver organoids, cardiac organoids, or intestinal organoids. In one embodiment of the method, the organoid derived from induced pluripotent stem cells (iPSCs) is an organoid derived from human induced pluripotent stem cells (hiPSCs). In one embodiment of the method, the Wnt5a protein is a recombinant human Wnt5a protein. In one embodiment of the method, the pancreatic islet organoid, pancreatic organoid, liver organoid, cardiac organoid, or intestinal organoid is derived from an iPSC-derived cell selected from an iPSC-derived β-like cell, an iPSC-derived exocrine component cell, an iPSC-derived hepatocyte, an iPSC-derived cardiomyocyte, or an iPSC-derived intestinal cell, respectively. In one embodiment of any of the above, the iPSC-derived cell is human. In another aspect, the present invention provides a method for enhancing the self-organization of adipose-derived stem cells (ADSCs) to generate pancreatic islet organoids or pancreatic organoids, comprising culturing the ADSCs in a medium comprising Wnt5a protein. In one embodiment, the ADSCs are cultured in a three-dimensional matrix comprising gellan gum. In another embodiment, Wnt5a protein is recombinant human Wnt5a protein.In another aspect, the present invention provides the pancreatic islet organoid, pancreatic organoid, liver organoid, heart organoid or intestinal organoid produced by any of the above-described method and its embodiments.

[0040] The compositions and articles defined by the present invention have been isolated or otherwise prepared in connection with the examples provided below. Other features and advantages of the present invention will become apparent from the detailed description, and from the claims.

[0041] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994);The Cambridge Dictionary of Science and Technology (Walker ed., 1988);The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991);and Hale & Marham, The Harper Collins Dictionary of Biology (1991).As used herein, the following terms have the meanings assigned to them below, unless otherwise specified.

[0042] By "AFP polypeptide" or "alpha-fetoprotein" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_001125.1 and having the biological activity of an AFP polypeptide. Exemplary biological activities of an AFP polypeptide include binding to copper, nickel, fatty acids, and bilirubin. The amino acid sequence provided in NCBI Accession No. NP_001125.1 is shown below. TIFF0007608402000001.tif49146

[0043] By "AFP polynucleotide" is meant a polynucleotide that encodes an AFP polypeptide or a fragment thereof. Exemplary AFP polynucleotide sequences are provided in NCBI Ref: NM_001134.2. The sequences provided in NCBI Ref: NM_001134.2 are reproduced below. TIFF0007608402000002.tif159148

[0044] By "ALB polypeptide" or "albumin" is meant a protein or fragment thereof having at least 85% amino acid sequence identity with the sequence provided in NCBI Accession No. NP_000468.1 and having the biological activity of an ALB polypeptide. Exemplary biological activities of an ALB polypeptide include binding to fatty acids, calcium ions, sodium ions, potassium ions, hormones, and bilirubin; stabilizing extracellular fluid volume; and transporting plasma zinc. The amino acid sequence provided in NCBI Accession No. NP_000468.1 is shown below. TIFF0007608402000003.tif49146

[0045] By "ALB polynucleotide" is meant a polynucleotide encoding an ALB polypeptide or a fragment thereof. Exemplary AFP polynucleotide sequences are provided in NCBI Ref: NM_000477.5. The sequences provided in NCBI Ref: NM_000477.5 are reproduced below. TIFF0007608402000004.tif173148

[0046] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0047] By "ameliorate" is meant to reduce, inhibit, attenuate, ameliorate, arrest, or stabilize the onset or progression of a disease.

[0048] By "change" is meant an increase or decrease. An increase is any positive change, for example, at least about 5%, 10%, or 20%; at least about 25%, 50%, 75%, or even 100%, 200%, 300%, or more. A decrease is a negative change, for example, at least about 5%, 10%, or 20%; at least about 25%, 50%, 75% decrease; or even 100%, 200%, 300%, or more increase.

[0049] In this disclosure, "comprises," "comprising," "containing," "having," and the like may have the meaning assigned to them in United States patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meaning assigned to them in United States patent law, and the terms are open-ended and permit the presence of other than what is recited, but exclude prior art aspects, so long as the basic or novel characteristics of the recited items are not altered by the presence of other than what is recited.

[0050] By "CDX2 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_001256.3 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_001256.3 is shown below. TIFF0007608402000005.tif26146

[0051] By "CDX2 polynucleotide" is meant a polynucleotide encoding a CDX2 polypeptide or a fragment thereof. An exemplary CDX2 polynucleotide sequence is provided in NCBI Ref: NM_001265.4. The sequence provided in NCBI Ref: NM_001265.4 is reproduced below. TIFF0007608402000006.tif183148

[0052] By "CYP3A7 polypeptide" or "cytochrome P450" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_000756.3 and having monooxygenase activity. The amino acid sequence provided in NCBI Accession No. NP_000756.3 is shown below. TIFF0007608402000007.tif40146

[0053] "CYP3A7 polynucleotide" refers to a polynucleotide encoding a CYP3A7 polypeptide or a fragment thereof. Exemplary AFP polynucleotide sequences are provided in NCBI Ref: NM_00765.4. The sequences provided in NCBI Ref: NM_00765.4 are reproduced below. TIFF0007608402000008.tif160148

[0054] "Detecting" refers to identifying the presence, absence, or amount of the analyte being detected.

[0055] "Detectable label" refers to a composition that, when linked to a molecule of interest, allows the latter to be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, or haptens.

[0056] "Differentiation" refers to the developmental process of lineage commitment. Differentiation can be assayed by measuring the increase in one or more cell-specific markers compared to their expression in corresponding undifferentiated control cells. "Lineage" refers to a pathway of cell development in which precursor or "progenitor" cells undergo progressive physiological changes to become a specific cell type with a characteristic function. In some embodiments, the cell type is a beta cell. In some embodiments, the cell type is an alpha cell, a delta cell, or a duct cell. In some other embodiments, the cell type is a hepatocyte. In still other embodiments, the cell type is a cardiomyocyte. In some embodiments, the cell type is an intestinal cell. Differentiation occurs in stages, whereby cells become progressively more specialized until they reach full maturity, also referred to as "terminal differentiation." A "terminally differentiated cell" is a cell that is committed to a particular lineage and has reached the final stage of differentiation (i.e., a fully mature cell). In some embodiments, the induced pluripotent stem cells (iPSCs) are differentiated into beta-like cells, alpha-like cells, delta-like cells, or duct-like cells. In some other embodiments, the induced pluripotent stem cells (iPSCs) are differentiated into hepatocytes, cardiomyocytes, or intestinal cells.

[0057] A "dedifferentiated cell" is a cell in which the process of differentiation has been reversed at least to some extent. Dedifferentiation can be assayed, for example, by identifying a decrease in the expression of one or more cell-specific markers compared to expression in corresponding control cells. Alternatively, dedifferentiation can be assayed by measuring an increase in one or more markers typically expressed in embryonic stem cells, pluripotent or multipotent cell types, or expressed at earlier stages of development. In some embodiments, the dedifferentiated cell is an induced pluripotent stem cell (iPSC). In certain embodiments, the dedifferentiated cell is a human induced pluripotent stem cell (iPSC).

[0058] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include type 1 diabetes, type 2 diabetes, and pancreatic cancer.

[0059] "Effective amount" refers to the amount of therapeutic agent or organoid required to improve the symptoms of disease in a subject compared to untreated subjects.The effective amount of therapeutic agent used to carry out the present invention for the purpose of therapeutically treating disease varies depending on the mode of administration, the age, weight and general health of the subject.The attending physician or veterinarian will ultimately determine the appropriate amount and administration schedule.Such amount is referred to as "effective" amount.In some embodiments, the therapeutic organoid is pancreatic islet organoid.In some other embodiments, an effective amount of pancreatic islet organoid is administered to a subject with type 1 or type 2 diabetes.

[0060] "ESRRG polypeptide" refers to a protein or fragment thereof having at least 85% amino acid sequence identity with the sequence provided in NCBI Accession No. NP_001230448.1 and having nuclear hormone receptor activity. The amino acid sequence provided in NCBI Accession No. NP_001230448.1 is shown below. TIFF0007608402000009.tif36146

[0061] "ESRRRG polynucleotide" refers to a polynucleotide that encodes an ESRRG polypeptide or a fragment thereof. An exemplary ESRRG polynucleotide sequence is provided in NCBI Ref: NM_001243519.1. The sequence provided in NCBI Ref: NM_001243519.1 is reproduced below. TIFF0007608402000010.tif141148TIFF0007608402000011.tif233148TIFF0007608402000012.tif49148

[0062] As used herein, "endocrine" refers to the secretion of an agent (e.g., a hormone) into the bloodstream. "exocrine" refers to the secretion of an agent through a duct to an epithelial surface.

[0063] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, the portion containing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0064] By "FOXA2 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_068556.2 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_068556.2 is shown below. TIFF0007608402000013.tif35146

[0065] "FOXA2 polynucleotide" refers to a polynucleotide that encodes a FOXA2 polypeptide or a fragment thereof. Exemplary FOXA2 polynucleotide sequences are provided in NCBI Ref: NM_021784.4. The sequences provided in NCBI Ref: NM_021784.4 are reproduced below. TIFF0007608402000014.tif187148

[0066] "GATA6 polypeptide" refers to a protein or fragment thereof having at least 85% amino acid sequence identity with the sequence provided in NCBI Accession No. NP_005248.2 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_005248.2 is shown below. TIFF0007608402000015.tif45146

[0067] "GATA6 polynucleotide" refers to a polynucleotide encoding a GATA6 polypeptide or a fragment thereof. An exemplary KCNK3 polynucleotide sequence is provided in NCBI Ref: NM_005257.5. The sequence provided in NCBI Ref: NM_005257.5 is reproduced below. TIFF0007608402000016.tif169148TIFF0007608402000017.tif122148

[0068] By "gellan gum" is meant a polysaccharide having linear chains with repeating units having any one of the following molecular structures: Gellan gum - high acyl type TIFF0007608402000018.tif29128 Gellan gum - low acyl type TIFF0007608402000019.tif20128

[0069] In the above structures, "Ac" refers to an acetate group, "Gly" refers to a glycerate group, and "M+" is a monovalent cation. In some embodiments, the gellan gum is KELCOGEL® gellan gum.

[0070] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0071] "Immunosuppressant" or "immunosuppressant drug" refers to an agent that suppresses or prevents an immune response, such as rejection, of a transplanted organ or organoid in a subject. Examples of immunosuppressants include, but are not limited to, basilizimab, antithymocyte globulin, alemtuzumab, prednisone, azathioprine, mycophenolic acid, cyclosporine, sirolimus, methotrexate, interferon, and tacrolimus.

[0072] "Induced pluripotent stem cells" or "iPSCs" refer to differentiated somatic cells that have acquired pluripotency by exogenously expressing one or more transcription factors in the cells. "iPSC-derived cells" are cells derived from induced pluripotent stem cells. "iPSC-derived beta-like cells", "iPSC-derived alpha-like cells", "iPSC-derived delta-like cells" or "iPSC-derived ductal-like cells" are cells that are derived from induced pluripotent stem cells and have characteristics of beta cells, alpha cells, delta cells, or ductal cells, respectively.

[0073] The terms "isolated," "purified," or "biologically pure" refer to a material that has been removed to various degrees from components that normally accompany it as found in its native state. "Isolate" refers to some degree of separation from the original source or surroundings. "Purify" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein has been sufficiently removed from other materials such that no impurity substantially affects the biological properties of the protein or causes other deleterious events. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or if chemically synthesized, if it is substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. With respect to proteins that may be subject to modifications, such as phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which may be purified separately.

[0074] "Isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank it in the native genome of the organism from which the nucleic acid molecule of the present invention is derived. Thus, the term includes recombinant DNA that exists, for example, in a vector; in an autonomously replicating plasmid or virus; or integrated into the genomic DNA of a prokaryotic or eukaryotic organism; or as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene that codes for additional polypeptide sequences.

[0075] By "isolated polypeptide" is meant a polypeptide of the invention that is separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preparations may be at least 75%, at least 90%, and at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity may be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0076] By "KCNK3 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_002237.1 and having potassium channel activity. The amino acid sequence provided in NCBI Accession No. NP_002237.1 is shown below. TIFF0007608402000020.tif31146

[0077] "KCNK3 polynucleotide" refers to a polynucleotide encoding a KCNK3 polypeptide or a fragment thereof. Exemplary KCNK3 polynucleotide sequences are provided in NCBI Ref: NM_002246.2. The sequences provided in NCBI Ref: NM_002246.2 are reproduced below. TIFF0007608402000021.tif238148TIFF0007608402000022.tif68148

[0078] By "KCNQ1 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_000209.2 (isoform 1) or NP_861463.1 (isoform 2) and having potassium channel activity. The amino acid sequence provided in NCBI Accession No. NP_000209.2 is shown below. TIFF0007608402000023.tif54146

[0079] "KCNQ1 polynucleotide" refers to a polynucleotide encoding a KCNQ1 polypeptide or a fragment thereof. Exemplary KCNQ1 polynucleotide sequences are provided in NCBI Ref: NM_00218.2. The sequences provided in NCBI Ref: NM_00218.2 are reproduced below. TIFF0007608402000024.tif22146TIFF0007608402000025.tif229148

[0080] By "LGR5 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_003658.1 (isoform 1), NP_001264155.1 (isoform 2), or NP_001264156.1 (isoform 3) and having transmembrane signaling receptor activity or G protein-coupled receptor activity. The amino acid sequence provided in NCBI Accession No. NP_003658.1 is shown below. TIFF0007608402000026.tif72146

[0081] "LGR5 polynucleotide" refers to a polynucleotide that encodes an LGR5 polypeptide or a fragment thereof. Exemplary LGR5 polynucleotide sequences are provided in NCBI Ref: NM_003667.3. The sequences provided in NCBI Ref: NM_003667.3 are reproduced below. TIFF0007608402000027.tif77146TIFF0007608402000028.tif233148TIFF0007608402000029.tif44148

[0082] By "LDHA polypeptide" or "lactate dehydrogenase A polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_005557.1 (isoform 1), NP_001128711.1 (isoform 2), NP_001158886.1 (isoform 3), NP_001158887.1 (isoform 4), or NP_001158888.1 (isoform 5) and having dehydrogenase activity. The amino acid sequence provided in NCBI Accession No. NP_005557.1 is shown below. TIFF0007608402000030.tif26146

[0083] "LDHA polynucleotide" or "lactate dehydrogenase A polynucleotide" refers to a polynucleotide that encodes a LDHA polypeptide or a fragment thereof. Exemplary LDHA polynucleotide sequences are provided in NCBI Ref: NM_005566.3. The sequences provided in NCBI Ref: NM_005566.3 are reproduced below. TIFF0007608402000031.tif173148

[0084] By "MAFA polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_963883.2 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_963883.2 is shown below. TIFF0007608402000032.tif26146

[0085] "MAFA polynucleotide" refers to a polynucleotide that encodes a MAFA polypeptide or a fragment thereof. Exemplary MAFA polynucleotide sequences are provided in NCBI Ref: NM_201589.3. The sequences provided in NCBI Ref: NM_201589.3 are reproduced below. TIFF0007608402000033.tif183148

[0086] As used herein, "marker" refers to any protein or polynucleotide whose expression level or activity changes in association with a disease or disorder or in association with a particular cell type. In some embodiments, the marker for beta cells is Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, or Foxa2. In some embodiments, the marker for hepatocytes is AFP, ALB, or Cyp3a7. In some other embodiments, the marker for cardiomyocytes is hM1c2a, hNkx2-5, αMHC, or KCNQ1. In still other embodiments, the marker for small intestinal cells is CDX2, Muc2, or Lgr5.

[0087] By "αMHC polypeptide" or "myosin heavy chain (MHC) α polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_002462.2 and having actin-binding activity. The amino acid sequence provided in NCBI Accession No. NP_002462.2 is shown below. TIFF0007608402000034.tif150148

[0088] By "αMHC polynucleotide" is meant a polynucleotide that encodes an αMHC polypeptide or a fragment thereof. Exemplary αMHC polynucleotide sequences are provided in NCBI Ref: NM_002471.3. The sequences provided in NCBI Ref: NM_002471.3 are reproduced below. TIFF0007608402000035.tif22146TIFF0007608402000036.tif233148TIFF0007608402000037.tif200148

[0089] By "MLC2A polypeptide" or "human MLSC2A (hMLC2A) polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_067046.1 and having calcium binding activity. The amino acid sequence provided in NCBI Accession No. NP_067046.1 is shown below. TIFF0007608402000038.tif13146

[0090] "MLC2A polynucleotide" refers to a polynucleotide encoding an MLC2A polypeptide or a fragment thereof. Exemplary MLC2A polynucleotide sequences are provided in NCBI Ref: NM_021223.2. The sequences provided in NCBI Ref: NM_021223.2 are reproduced below. TIFF0007608402000039.tif49146

[0091] By "MUC2 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_002448.3 and having the biological activity of a MUC2 polypeptide. Exemplary biological activities of MUC2 polypeptide include polymerization into a gel and coating the epithelium of the intestine and other mucosa-containing organs. The amino acid sequence provided in NCBI Accession No. NP_002448.3 is shown below. TIFF0007608402000040.tif68146TIFF0007608402000041.tif233148TIFF0007608402000042.tif95148

[0092] By "MUC2 polynucleotide" is meant a polynucleotide that encodes a MUC2 polypeptide or a fragment thereof. Exemplary MUC2 polynucleotide sequences are provided in NCBI Ref: NM_002457.3. The sequences provided in NCBI Ref: NM_002457.3 are reproduced below. TIFF0007608402000043.tif100148TIFF0007608402000044.tif233148TIFF0007608402000045.tif233148 TIFF0007608402000046.tif233150TIFF0007608402000047.tif233150TIFF0007608402000048.tif164150

[0093] By "NKX2-5 polypeptide" or "human NKX2-5 (hNKX2-5) polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_004378.1 (isoform 1), NP_001159647.1 (isoform 2), or NP_001159648.1 (isoform 3) and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_004378.1 is shown below. TIFF0007608402000049.tif26146

[0094] "NKX2-5 polynucleotide" refers to a polynucleotide encoding a NKX2-5 polypeptide or a fragment thereof. An exemplary NKX2-5 polynucleotide sequence is provided in NCBI Ref: NM_004387.3. The sequence provided in NCBI Ref: NM_004387.3 is reproduced below. TIFF0007608402000050.tif127148

[0095] "NEUROD1 polypeptide" refers to a protein or fragment thereof having at least 85% amino acid sequence identity with the sequence provided in NCBI Accession No. NP_002491.2 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_002491.2 is shown below. TIFF0007608402000051.tif26146

[0096] "NEUROD1 polynucleotide" refers to a polynucleotide encoding a NEUROD1 polypeptide or a fragment thereof. Exemplary NEUROD1 polynucleotide sequences are provided in NCBI Ref: NM_002500.4. The sequences provided in NCBI Ref: NM_002500.4 are reproduced below. TIFF0007608402000052.tif232148

[0097] By "NKX6-1 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity with the sequence provided in NCBI Accession No. NP_006159.2 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_006159.2 is shown below. TIFF0007608402000053.tif31146

[0098] "NKX6-1 polynucleotide" refers to a polynucleotide encoding a NKX6-1 polypeptide or a fragment thereof. An exemplary NKX6-1 polynucleotide sequence is provided in NCBI Ref: NM_006168.2. The sequence provided in NCBI Ref: NM_006168.2 is reproduced below. TIFF0007608402000054.tif86148

[0099] By "NDUFA4 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_002480.1 and having NADH dehydrogenase activity and oxidoreductase activity. The amino acid sequence provided in NCBI Accession No. NP_002480.1 is shown below. TIFF0007608402000055.tif26146

[0100] "NDUFA4 polynucleotide" refers to a polynucleotide that encodes an NDUFA4 polypeptide or a fragment thereof. Exemplary NDUFA4 polynucleotide sequences are provided in NCBI Ref: NM_002489.3. The sequences provided in NCBI Ref: NM_002489.3 are reproduced below. TIFF0007608402000056.tif160148

[0101] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0102] "Organ" refers to a collection of cells that perform biological functions.In one embodiment, organs include, but are not limited to, bladder, brain, nerve tissue, glial tissue, esophagus, fallopian tube, heart, pancreas, intestine, gallbladder, kidney, liver, lung, ovary, prostate, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, breast, skeletal muscle, skin, bone and cartilage.The biological functions of organs can be assayed using standard methods known to those skilled in the art.

[0103] "Organoid" refers to an in vitro generated body that mimics the structure and function of an organ. "Organoid" and "mini-organ" are used interchangeably herein. "Pancreatic islet organoid" is an in vitro generated cell mass that mimics the structure and function of a pancreatic islet. Exemplary functions of a pancreatic islet include, but are not limited to, glucose stimulated insulin secretion (GSIS), potassium chloride (KCl) stimulated insulin secretion, GLP-1 stimulated insulin secretion, somatostatin secretion, or glucagon secretion. "Pancreatic islet organoid" and "mini-islet" are used interchangeably herein. "Pancreatic organoid" is an in vitro generated body that mimics the structure and function of a pancreas. Exemplary functions of a pancreas include, but are not limited to, endocrine secretion of hormones such as glucose and glucagon that regulate glucose metabolism and blood glucose concentration, and exocrine secretion of digestive enzymes that aid in the breakdown of carbohydrates, proteins, and lipids. "Pancreatic organoid" and "mini-pancreas" are used interchangeably herein.

[0104] By "PAX4 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_006184.2 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_006184.2 is shown below. TIFF0007608402000057.tif26146

[0105] By "PAX4 polynucleotide" is meant a polynucleotide that encodes a PAX4 polypeptide or a fragment thereof. Exemplary PAX4 polynucleotide sequences are provided in NCBI Ref: NM_006193.2. The sequences provided in NCBI Ref: NM_006193.2 are reproduced below. TIFF0007608402000058.tif155148

[0106] By "PAX6 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_001297090.1 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_001297090.1 is shown below. TIFF0007608402000059.tif22146

[0107] "PAX6 polynucleotide" refers to a polynucleotide encoding a PAX6 polypeptide or a fragment thereof. An exemplary PAX6 polynucleotide sequence is provided in NCBI Ref: NM_001310161.1. The sequence provided in NCBI Ref: NM_001310161.1 is reproduced below. TIFF0007608402000060.tif155148TIFF0007608402000061.tif233148TIFF0007608402000062.tif128148

[0108] By "PDX1 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity with the sequence provided in NCBI Accession No. NP_000200.1 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_000200.1 is shown below. TIFF0007608402000063.tif22146

[0109] "PDX1 polynucleotide" refers to a polynucleotide encoding a PDX1 polypeptide or a fragment thereof. Exemplary PDX1 polynucleotide sequences are provided in NCBI Ref: NM_000209.3. The sequences provided in NCBI Ref: NM_000209.3 are reproduced below. TIFF0007608402000064.tif196148

[0110] By "PTF1 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI Accession No. NP_835455.1 and having transcription factor activity. The amino acid sequence provided in NCBI Accession No. NP_835455.1 is shown below. TIFF0007608402000065.tif26146

[0111] By "PTF1 polynucleotide" is meant a polynucleotide that encodes a PTF1 polypeptide or a fragment thereof. Exemplary PTF1 polynucleotide sequences are provided in NCBI Ref: NM_178161.2. The sequences provided in NCBI Ref: NM_178161.2 are reproduced below. TIFF0007608402000066.tif104148

[0112] "Wnt3a polynucleotide" refers to a polynucleotide that encodes a Wnt3a polypeptide or a fragment thereof, or has at least 85% sequence identity with a human Wnt3a polynucleotide sequence. An exemplary human Wnt3a polynucleotide sequence is provided in NCBI GenBank Accession No. AB060284.1. The polynucleotide sequence provided in NCBI GenBank Accession No. AB060284.1 is reproduced below. TIFF0007608402000067.tif141148

[0113] "Wnt3a polypeptide" refers to a Wnt3a polypeptide or a fragment thereof, or a polypeptide having at least 85% sequence identity with a human Wnt3a polypeptide sequence. An exemplary human Wnt3a polypeptide sequence is provided in NCBI GenBank: AAI03924.1. The sequence provided in GenBank: AAI03924.1 is reproduced below. TIFF0007608402000068.tif36146

[0114] "Wnt4 polynucleotide" refers to a polynucleotide that encodes a Wnt4 polypeptide or a fragment thereof, or has at least 85% sequence identity with human Wnt4 polynucleotide sequence. An exemplary human Wnt4 polynucleotide sequence is provided in NCBI GenBank Accession No. AY009398.1. Accession No. NCBI Ref NG_008974.1 is the reference standard Wnt4a polynucleotide sequence. The polynucleotide sequence provided in NCBI GenBank Accession No. AY009398.1 is reproduced below. TIFF0007608402000069.tif97148

[0115] "Wnt4 polypeptide" refers to a Wnt4 polypeptide or a fragment thereof, or a polypeptide having at least 85% sequence identity with the human Wnt4 polypeptide sequence. An exemplary human Wnt4 polypeptide sequence is provided in NCBI GenBank Accession No. AAG38658.1. The sequence provided in GenBank Accession No. AAG38658.1 is reproduced below. TIFF0007608402000070.tif31146

[0116] By "Wnt5a polynucleotide" is meant a polynucleotide encoding a Wnt5a polypeptide or a fragment thereof, or a polynucleotide having at least 85% sequence identity to a human Wnt5a polynucleotide sequence. An exemplary polynucleotide sequence encoding human Wnt5a is provided in the reference standard sequence NCBI Ref: GenBank NM_003392. Nucleotides 658-1800 of the Wnt5a genomic sequence, which has 6194 nucleotides, encode the human Wnt5a polypeptide. The polynucleotide sequence of the human Wnt5a coding sequence provided at bases 658-1800 of NCBI Ref: GenBank NM_003392 is reproduced below. TIFF0007608402000071.tif117148

[0117] By "Wnt5a polypeptide" is meant a Wnt5a polypeptide or a fragment thereof, or a polypeptide having at least 85% sequence identity with a human Wnt5a polypeptide sequence. An exemplary human Wnt5a (isoform 1) polypeptide sequence is provided in UniProtKB Identifier: P41221-1. The sequence provided in UniProtKB Identifier: P41221-1 is reproduced below. TIFF0007608402000072.tif39140

[0118] By "progenitor cell" is meant a multipotent stem cell that can give rise to an endothelial cell (e.g., by differentiation or division). A progenitor cell capable of giving rise to an endothelial cell can exhibit this capability when grown under appropriate in vitro or in vivo conditions, as described herein.

[0119] By "progeny" is meant cells derived from the pluripotent stem cells of the present invention. Progeny include, but are not limited to, progenitor cells, differentiated cells, and terminally differentiated cells.

[0120] "Derived from" refers to the process by which progeny cells are derived.

[0121] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0122] "Reference" or "control" refers to a standard condition, e.g., an untreated cell, tissue, or organ is used as a reference.

[0123] A "reference sequence" is a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, or at least about 25 amino acids. The length of a reference polypeptide sequence may be about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, or at least about 75 nucleotides. The length of a reference nucleic acid sequence may be about 100 nucleotides, about 300 nucleotides, or any integer before, after, or between them.

[0124] A "somatic" cell refers to a cell obtained from the tissue of a subject. Such a subject is at a postnatal stage of development (e.g., an adult, an infant, or a child). In contrast, an "embryonic cell" or "embryonic stem cell" is derived from an embryo at a prenatal stage of development.

[0125] By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the invention, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the invention.

[0126] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence typically can hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence typically can hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means the pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0127] For example, stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, and high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions usually include temperatures of at least about 30°C, at least about 37°C, and at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are achieved by combining these various conditions as necessary. In one embodiment, hybridization is performed at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization is performed at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In yet another embodiment, hybridization is performed at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to one of skill in the art.

[0128] For most applications, the washing step following hybridization also varies in stringency. Stringency conditions for washing can be defined by salt concentration and by temperature. As mentioned above, washing stringency can be increased by lowering salt concentration or by increasing temperature. For example, stringent salt concentrations for washing steps are less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps usually include temperatures of at least about 25°C, at least about 42°C, and at least about 68°C. In one embodiment, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another embodiment, the washing step is carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0129] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such a sequence is at least 60%, at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% identical at the amino acid or nucleic acid level to the sequence being compared.

[0130] Sequence identity is typically measured using sequence analysis software (e.g., the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, sequence analysis software packages, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach for determining the degree of identity, the BLAST program can be used, e.g., -3 ~e -100 A probability score of 0.05 indicates closely related sequences.

[0131] As used herein, the term "self-renewal" refers to the process by which a stem cell divides to produce one (asymmetric division) or two (symmetric division) daughter cells with developmental potential indistinguishable from that of the parent cell. Self-renewal includes both proliferation and maintenance of an undifferentiated state.

[0132] The term "stem cell" refers to a pluripotent or multipotent stem cell that has the capacity to self-renew and differentiate into multiple cell lineages.

[0133] By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, rodent, or cat.

[0134] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0135] By "tissue" is meant an aggregation of cells having similar morphology and function.

[0136] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms. Although not excluded, it is recognized that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0137] "Vascularized" means having blood vessels. In some embodiments, the pancreatic islet organoids or pancreatic organoids are vascularized.

[0138] Unless specifically stated otherwise or clear from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated otherwise or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0139] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the range of normal acceptable values ​​in the art, for example, within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0140] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0141] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein. [The present invention 1001] 1. A method for producing pancreatic islet organoids, comprising culturing induced pluripotent stem cell (iPSC) derived beta-like cells in a three-dimensional matrix comprising gellan gum, thereby producing pancreatic islet organoids. [The present invention 1002] The method of claim 1001, further comprising culturing the iPSC-derived β-like cells with adipose-derived stem cells and / or endothelial cells. [The present invention 1003] The method of any one of claims 1001 and 1002, further comprising culturing the iPSC-derived β-like cells with iPSC-derived α-like cells, iPSC-derived δ-like cells, or iPSC-derived duct-like cells. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the iPSC-derived β-like cells, iPSC-derived α-like cells, iPSC-derived δ-like cells, or iPSC-derived duct-like cells are human. [The present invention 1005] The method of claim 1002, wherein the adipose-derived stem cells are human adipose-derived stem cells (hADSCs). [The present invention 1006] The method of claim 1002, wherein the endothelial cells are human umbilical vein endothelial cells (HUVEC). [The present invention 1007] Cell culture containing iPSC-derived β-like cells in a 3D matrix containing gellan gum. [The present invention 1008] The cell culture of claim 1007, further comprising adipose-derived stem cells and / or endothelial cells. [The present invention 1009] The cell culture of the present invention 1007 or 1008, wherein the iPSC-derived β-like cells are human iPSC-derived β-like cells. [The present invention 1010] The cell culture of claim 1008 or 1009, wherein the adipose-derived stem cells are human adipose-derived stem cells (hADSC). [The present invention 1011] The cell culture of any of claims 1008 to 1010, wherein the endothelial cells are human umbilical vein endothelial cells (HUVEC). [The present invention 1012] Cell cultures containing human iPSC-derived β-like cells, human adipose-derived stem cells (hADSCs), and human umbilical vein endothelial cells (HUVECs) in a 3D matrix containing gellan gum. [The present invention 1013] A pancreatic islet organoid comprising iPSC-derived β-like cells, which is vascularized and exhibits glucose-stimulated insulin secretion (GSIS). [The present invention 1014] The pancreatic islet organoid of the present invention 1013, further comprising iPSC-derived alpha-like cells, iPSC-derived delta-like cells, or iPSC-derived duct-like cells. [The present invention 1015] The pancreatic islet organoid of the present invention 1013, further comprising adipose-derived stem cells and / or endothelial cells. [The present invention 1016] The pancreatic islet organoid of the present invention 1015, wherein the adipose-derived stem cells are human adipose-derived stem cells (hADSC) and / or the endothelial cells are human umbilical vein endothelial cells (HUVEC). [The present invention 1017] 1014. The pancreatic islet organoid of the present invention, further exhibiting KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, or glucagon secretion. [The present invention 1018] The pancreatic islet organoid of the present invention 1013, which expresses a beta cell transcription factor selected from the group consisting of Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, and Foxa2. [The present invention 1019] A pancreatic islet organoid comprising iPSC-derived beta-like cells, iPSC-derived alpha cells, iPSC-derived delta cells, iPSC-derived ductal cells, adipose-derived stem cells (hADSCs), and endothelial cells, the pancreatic islet organoid being vascularized and exhibiting glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, and glucagon secretion. [The present invention 1020] A pancreatic organoid comprising any one of the islet organoids of the present invention 1013 to 1019 surrounded by iPSC-derived exocrine components. [The present invention 1021] The pancreatic organoid of the present invention 1020, wherein the iPSC-derived exocrine component expresses a marker selected from the group consisting of PDX1, Nkx6-1, and Ptf1. [The present invention 1022] A non-human organism transplanted with any one of the organoids of the present invention 1013 to 1021. [The present invention 1023] The non-human organism of the present invention 1022 which is a mammal. [The present invention 1024] The non-human organism of the present invention 1023 which is a mouse. [The present invention 1025] 1. A method for identifying an agent that modulates pancreatic activity and / or treats a pancreatic disease, comprising: (a) contacting a candidate agent with a pancreatic islet organoid or pancreatic organoid; and (b) measuring the activity of the organoid contacted with the candidate agent. wherein if the activity of the organoid is altered compared to the reference, the candidate agent is identified as an agent that regulates pancreatic activity and / or treats a pancreatic disease. [The present invention 1026] 1. A method for identifying an agent that modulates pancreatic activity and / or treats a pancreatic disease, comprising: (a) administering a candidate agent to a non-human subject into which pancreatic islet organoids or pancreatic organoids have been transplanted; and (b) measuring the pancreatic activity in said non-human subject wherein if the pancreatic activity of the non-human subject is altered compared to the reference, the candidate agent is identified as an agent that modulates pancreatic activity and / or treats a pancreatic disease. [The present invention 1027] The method of claim 1025 or 1026, further comprising inducing or mimicking a pancreatic disease in the organoid or non-human subject. [The present invention 1028] The method of claim 1025 or 1026, wherein the non-human subject is further transplanted with a liver organoid. [The present invention 1029] The method of invention 1027 or invention 1028, wherein the disease is induced by contacting the organoid with or administering to the non-human subject any one or more of the following agents: free fatty acids (FFAs), glucose, and cytokines. [The present invention 1030] The method of claim 1027 or 1028, wherein the disease is mimicked by culturing the organoids with pancreatic cancer cells, astrocytes, and immune cells to create a human pancreatic cancer microenvironment. [The present invention 1031] The method of any one of claims 1025 to 1026, wherein the pancreatic activity is selected from the group consisting of insulin secretion, beta cell apoptosis, expression or activity of NDUFA4, ESRRG, G6PC2, MDH1, LDHA, KCNK3, or MAFA polypeptide or polynucleotide, amylase secretion, apoptosis of exocrine components, collagen synthesis, and stellate cell activation. [The present invention 1032] The method of any of claims 1025 to 1031, wherein the candidate agent increases insulin secretion. [The present invention 1033] The method of any of claims 1025-1031, wherein the candidate agents identified as agents that modulate pancreatic activity are further tested for the ability to treat a pancreatic disease. [The present invention 1034] The method according to any one of claims 1025 to 1033, wherein the pancreatic disease is type 2 diabetes or pancreatic cancer. [The present invention 1035] The method of any one of claims 1025 to 1034, wherein the organoid is any one of claims 1013 to 1020. [The present invention 1036] The method of claim 1026, wherein the non-human subject is a mouse. [The present invention 1037] 1. A method for treating a pancreatic disease in a subject, comprising transplanting a pancreatic islet organoid into the subject, the pancreatic islet organoid comprising iPSC-derived beta-like cells, being vascularized, and exhibiting glucose-stimulated insulin secretion (GSIS). [The present invention 1038] A method for treating type 1 diabetes in a subject, comprising transplanting a pancreatic islet organoid into the subject, the pancreatic islet organoid comprising iPSC-derived beta-like cells, being vascularized, and exhibiting glucose-stimulated insulin secretion (GSIS). [The present invention 1039] The method of any one of claims 1037 to 1038, wherein the pancreatic islet organoids further comprise iPSC-derived alpha-like cells, iPSC-derived delta-like cells, or iPSC-derived duct-like cells. [The present invention 1040] The method of any one of claims 1037 to 1038, wherein the pancreatic islet organoids further comprise adipose-derived stem cells and / or endothelial cells. [The present invention 1041] The method of claim 1040, wherein the adipose derived stem cells are human adipose derived stem cells (hADSC) and / or the endothelial cells are human umbilical vein endothelial cells (HUVEC). [The present invention 1042] The method of any one of claims 1037 to 1038, wherein the pancreatic islet organoids further exhibit KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, or glucagon secretion. [The present invention 1043] The method of any of claims 1037 to 1042, wherein the pancreatic islet organoid expresses a beta cell transcription factor selected from the group consisting of Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, and Foxa2. [The present invention 1044] Any of the methods of the present invention 1037 to 1043, wherein the pancreatic islet organoid comprises iPSC-derived β-like cells, iPSC-derived α cells, iPSC-derived δ cells, iPSC-derived duct cells, adipose-derived stem cells (hADSCs), and endothelial cells, and the organoid is vascularized and exhibits glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, and glucagon secretion. [The present invention 1045] The method of any one of claims 1037 to 1044, wherein the pancreatic islet organoid is surrounded by iPSC-derived exocrine components. [The present invention 1046] The method of claim 1045, wherein the iPSC-derived exocrine components express a marker selected from the group consisting of PDX1, Nkx6-1, and Ptf1. [The present invention 1047] The method of any one of claims 1037 to 1046, wherein the pancreatic islet organoid is any one of the organoids of claims 1013 to 1021. [The present invention 1048] The method of any one of claims 1037 to 1047, wherein the subject is administered an immunosuppressant. [The present invention 1049] The method of any one of claims 1037 to 1048, wherein the subject is a human. [The present invention 1050] The method according to any one of claims 1037 to 1049, wherein the pancreatic disease is type 1 diabetes or type 2 diabetes. [The present invention 1051] Pancreatic islet organoids generated by culturing induced pluripotent stem cell (iPSC)-derived beta-like cells in a 3D matrix containing gellan gum. [The present invention 1052] Pancreatic organoids were generated by culturing induced pluripotent stem cell (iPSC)-derived beta-like cells and iPSC-derived exocrine component cells in a 3D matrix containing gellan gum. [The present invention 1053] Liver organoids generated by culturing induced pluripotent stem cell (iPSC)-derived hepatocytes in a 3D matrix containing gellan gum. [The present invention 1054] Cardiac organoids generated by culturing induced pluripotent stem cell (iPSC)-derived cardiomyocytes in a 3D matrix containing gellan gum. [The present invention 1055] Intestinal organoids were generated by culturing induced pluripotent stem cell (iPSC)-derived intestinal cells in a 3D matrix containing gellan gum. [The present invention 1056] The pancreatic islet organoid, pancreatic organoid, liver organoid, cardiac organoid, or intestinal organoid of any of the present inventions 1051 to 1055, wherein the iPSC-derived β-like cells, iPSC-derived exocrine component cells, iPSC-derived hepatic cells, iPSC-derived cardiomyocytes, or iPSC-derived intestinal cells are human. [The present invention 1057] The pancreatic islet organoid, pancreatic organoid, liver organoid, cardiac organoid, or intestinal organoid of any of 1051 to 1055, further comprising adipose-derived stem cells and / or endothelial cells. [The present invention 1058] The pancreatic islet organoid, pancreatic organoid, liver organoid, cardiac organoid, or intestinal organoid of the present invention 1057, wherein the adipose-derived stem cells are human adipose-derived stem cells (hADSC) and / or the endothelial cells are human umbilical vein endothelial cells (HUVEC). [The present invention 1059] A pancreatic islet organoid, a pancreatic organoid, a liver organoid, a cardiac organoid, or an intestinal organoid according to any one of 1051 to 1055 of the present invention, which undergoes angiogenesis. [The present invention 1060] The liver organoid of the present invention 1053 expresses a marker selected from the group consisting of AFP, ALB, and Cyp3a7. [The present invention 1061] 1053 liver organoids of the present invention showing insulin signaling, insulin resistance by palmitic acid, and lipid accumulation. [The present invention 1062] The cardiac organoid of the present invention 1054 expresses a marker selected from the group consisting of hMlc2a, hNkx2-5, αMHC, and KCNQ1. [The present invention 1063] 1054, a cardiac organoid of the present invention, showing cardiac beating. [The present invention 1064] The intestinal organoid of the present invention 1055, which expresses a marker selected from the group consisting of CDX2, Muc2, and Lgr5. [The present invention 1065] Intestinal organoids of the present invention 1055 exhibiting sprouting in response to R-spondin. [The present invention 1066] A method for producing pancreatic islet organoids, comprising culturing induced pluripotent stem cell (iPSC)-derived beta-like cells in a medium containing Wnt4 or Wnt5a protein. [The present invention 1067] The method of claim 1066, wherein induced pluripotent stem cell (iPSC)-derived beta-like cells are cultured in a three-dimensional matrix. [The present invention 1068] The method of claim 1066 or 1067, wherein the Wnt4 or Wnt5a protein is a recombinant human Wnt4 or Wnt5a protein. [The present invention 1069] The method of any one of claims 10 to 68, wherein the medium comprises recombinant human Wnt4 protein. [The present invention 1070] The method of any one of claims 1068 to 1069, wherein the medium comprises recombinant human Wnt5a protein. [The present invention 1071] A cell culture comprising human iPSC-derived β-like cells and Wnt4 or Wnt5a protein. [The present invention 1072] The cell culture of the present invention 1071, wherein the human iPSC-derived beta-like cells are present in a three-dimensional matrix comprising gellan gum. [The present invention 1073] The cell culture of claim 1071 or 1072, wherein the Wnt4 or Wnt5a protein is a recombinant human Wnt4 or Wnt5a protein. [The present invention 1074] The cell culture of claim 1073, wherein the medium comprises recombinant human Wnt4 protein. [The present invention 1075] The cell culture of claim 1073, wherein the medium comprises recombinant human Wnt5a protein. [The present invention 1076] A pancreatic islet organoid comprising iPSC-derived β-like cells cultured in a medium containing Wnt4 or Wnt5a protein, the pancreatic islet organoid being vascularized and exhibiting glucose-stimulated insulin secretion (GSIS). [The present invention 1077] 1076 of the pancreatic islet organoid of the present invention, further exhibiting KCl-stimulated insulin secretion or glucose-stimulated insulin secretion. [The present invention 1078] The pancreatic islet organoid of the present invention 1076 or 1077, which expresses the Fltp gene and the Esrrg gene. [The present invention 1079] The pancreatic islet organoid of any of claims 1076 to 1078, wherein the Wnt4 or Wnt5a protein is a recombinant human Wnt4 or Wnt5a protein. [The present invention 1080] 1079. The pancreatic islet organoid of the present invention, wherein the culture medium comprises recombinant human Wnt4 protein. [The present invention 1081] 1079. The pancreatic islet organoid of the present invention, wherein the culture medium comprises recombinant human Wnt5a protein. [The present invention 1082] A non-human organism transplanted with any one of the organoids of the present invention 1076 to 1081. [The present invention 1083] A method for enhancing self-organization of adipose-derived stem cells (ADSCs) to generate organoids derived from induced pluripotent stem cells (iPSCs), the method comprising culturing ADSCs in a three-dimensional (3D) culture matrix medium containing Wnt5a protein. [The present invention 1084] The method of claim 1083, wherein the ADSCs are cultured in a 3D culture matrix comprising gellan gum. [The present invention 1085] The method of claim 1083 or 1084, wherein the 3D culture matrix medium for ADSCs comprises iPSC-derived cells selected from iPSC-derived beta-like cells, iPSC-derived exocrine component cells, iPSC-derived hepatic cells, iPSC-derived cardiomyocytes, or iPSC-derived intestinal cells. [The present invention 1086] The method of any of claims 1083 to 1085, wherein the iPSC-derived organoid is selected from a pancreatic islet organoid, a pancreatic organoid, a liver organoid, a cardiac organoid, or an intestinal organoid. [The present invention 1087] The method of claim 1085 or 1086, wherein the organoid derived from induced pluripotent stem cells (iPSCs) is an organoid derived from human induced pluripotent stem cells (hiPSCs). [The present invention 1088] The method of any of claims 1083 to 1087, wherein the Wnt5a protein is a recombinant human Wnt5a protein. [The present invention 1089] Any of the methods of the present invention 1086 to 1088, wherein the pancreatic islet organoid, pancreatic organoid, liver organoid, cardiac organoid, or intestinal organoid is derived from an iPSC-derived cell selected from iPSC-derived β-like cells, iPSC-derived exocrine component cells, iPSC-derived hepatic cells, iPSC-derived cardiomyocytes, or iPSC-derived intestinal cells, respectively. [The present invention 1090] The method of claim 1089, wherein the iPSC-derived cells are human. [The present invention 1091] A pancreatic islet organoid, a pancreatic organoid, a liver organoid, a cardiac organoid, or an intestinal organoid produced by any of the methods of the present invention 1083 to 1090. [Brief description of the drawings]

[0142] [Figure 1] 1 is a schematic showing various symptoms of human disease that can be modeled in a dish. The creation of functional human organs provides new therapeutic strategies in drug screening and disease modeling. A novel technique for creating 3D human mini-organs in a dish is described herein. This technique can be used to model human type 2 diabetes in a dish to find effective drugs in gene-, patient-, or environment-specific diseases such as human type 2 diabetes. [Figure 2-1]FIG. 2A is a series of micrographs showing the utility of human adipose-derived stem cells (hADSCs) in organogenesis. In the studies herein, human adipose-derived stem cells were found to be a novel source for generating self-organized organoids. FIG. 2 compares the appearance of human iPSC-derived pancreatic progenitor cells (PP, days 15-19), human umbilical vein endothelial cells (HUVECs), and hADSCs cultured on plates (2D) with those cultured in 3D Matrigel (Matrigel® system, 24 hours in PP cell differentiation medium, endothelial cell growth medium, and hADSC growth medium, respectively). FIG. 2B is a series of micrographs demonstrating the ability of ADSCs to gradually self-organize when cultured in Matrigel. FIG. 2C is a series of images of hADSCs seeded at the indicated densities demonstrating the minimum cell number required for sphere formation when grown in Matrigel. Figure 2D shows the transcriptional changes occurring in hADSCs during 48 h of culture in Matrigel, shown as a heat map. Statistically distinct changes in gene expression were determined by RNA-Seq analysis of cells after the indicated times in Matrigel. Biological pathways altered during culture in Matrigel were identified using DAVID software. Figure 2E is a schematic diagram describing the generation of human islet-like organoids by culturing in Matrigel. Figure 2F is an image of a human islet-like organoid in a single well of a 24-well plate. Figure 2G is a series of images of human islet-like organoids generated by co-culturing hiPSC-derived pancreatic progenitor cells, HUVECs, and hADSCs in Matrigel for 1–5 days. GFP was used to show human insulin expression (green, first panel) and mCherry was used to label HUVEC cells (red, second panel), as well as brightfield images (third panel), and overlay of GFP and mCherry images (fourth panel). [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 2-4] See description of Figure 2-1. [Figure 2-5] See description of Figure 2-1. [Figure 2-6] See description of Figure 2-1. [Diagram 2-7] See description of Figure 2-1. [Figure 3-1]Figures 3A-3F are plots, images, and schematics showing the characterization of insulin secretion in INS-1 cells, mouse islets, and human islets using the proinsulin luciferase reporter system as a quantitative insulin secretion assay. Figure 3A is a schematic showing the creation of a stably expressed insulin reporter in rat beta cell line INS-1 cells, where luciferase expression is under the control of the proinsulin promoter. Figure 3B is a plot showing induced luciferase activity in INS-1 reporter cells in response to treatment with 3 mM glucose (G3), 20 mM glucose (G20), 20 mM glucose (G20) + 100 nM exendin-4 (Ex-4), or 20 mM potassium chloride (KCl). Figure 3C is an image showing a single mouse islet in one well of a 96-well plate. Mouse islets were infected with a proinsulin-luciferase lentiviral reporter construct two days prior to the assay. FIG. 3D is a series of plots showing luciferase activity in response to 3 mM glucose (G3mM) and 20 mM glucose (G20mM) from individual mouse islets infected with a proinsulin luciferase reporter (left) and the average of individual assays (right). FIG. 3E is an image showing a single human islet in one well of a 96-well plate. Two days prior to the assay, the human islet was infected with a proinsulin-luciferase lentiviral reporter construct. FIG. 3F is a series of plots showing luciferase activity in response to 3 mM glucose (G3mM) and 20 mM glucose (G20mM) from individual human islets infected with a proinsulin luciferase reporter (left) and the average of individual assays (right). FIGs. 3D and 3F show that glucose-stimulated insulin secretion (GSIS) can be measured from single mouse and human islets, respectively, after infection with a lentiviral luciferase reporter. [Figure 3-2] See description of Figure 3-1. [Figure 4-1]Figures 4A-4I are plots, images, graphs, and schematics showing the generation of functional, vascularized human islets in a dish. Figure 4A is a schematic showing a scheme for generating functional, vascularized human islets in gellan gum. Figure 4B shows human islet-like mini-organs. The top panel shows insulin-positive cells (green fluorescent protein expression driven by the insulin promoter (left side)) and phase contrast images (left side) of islet-like organoids (lower panel) grown in 3D gellan gum suspension. Electron microscopy images reveal insulin granules in β-like cells and lipid droplets in hADSCs. Figure 4C shows that human islet-like mini-organs generated by the methods described herein are morphologically identical to human islets. Figures 4D and 4E show the relative expression of genes associated with β-cell determination (Figure 4D) and mitochondrial function (Figure 4E) as measured by qPCR. Islet-like cell clusters (derived from pancreatic progenitor cells cultured in gellan gum, day 35) as well as islet-like organoids (derived from pancreatic progenitor cells co-cultured with HUVECs and hADSCs in gellan gum) were FACS sorted into insulin expressing (GFP positive) and non-expressing (GFP negative) cells before analysis and compared to gene expression in human islets. Figure 4F shows glucose-stimulated insulin secretion, measured by fold change in c-peptide secretion after 30 minutes of exposure to 20 mM glucose, in selected islet-like organoid preparations prepared in 3D gellan gum culture and in human islets. Figure 4G shows that human islet-like mini-organs produced by the methods described herein can develop functional vascularization. Human islet-like organoids were transferred to Matrigel and grown in the presence of endothelial growth medium. Cells expressing insulin are visualized as green fluorescence. The top panel is a fluorescent image of cells showing HUVEC cell outgrowth after 24 and 48 hours of stimulation with endothelial cell growth medium (ECM). The bottom panel is a schematic diagram summarizing the experiment and findings. Figure 4H shows a graph illustrating that human islet-like mini-organs generated by the methods described herein can regulate blood glucose in NODSCID, a known mouse model of type 1 diabetes.In this mouse model, a SCID (severe combined immunodeficiency) mutation has been introduced into a diabetes-prone non-obese diabetic (NOD) background. The multiple immunological deficiencies in this mouse model provide a system for reconstituting the animal with human hematopoietic cells. The graph in Figure 4H shows blood glucose levels in NODSCID mice treated with streptozotocin (STZ) (180 mg / kg) to induce type 1 diabetes after transplantation of hiPSC-derived human islet-like organoids (n=1000), (dotted line with squares); human islets (n=1000), (dashed line) into the kidney capsule; or mock treatment (solid line with open circles). Figure 4I shows a bar graph illustrating that human islet-like mini-organs ("human islet organoids") generated by the methods described herein can secrete insulin after a meal. The graph in Figure 4I shows serum levels of human c-peptide (pmol / L) in NODSCID mice 4 weeks after transplantation described in Figure 4H under random feeding conditions (left bar), 8 hour fasting conditions (middle bar), and re-fed conditions (right bar). Human c-peptide levels provide a measure of insulin secretion from the graft, distinct from endogenous mouse insulin. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 4-5] See description of Figure 4-1. [Figure 4-6] See description of Figure 4-1. [Figure 5-1]Figures 5A-5E are diagrams and plots showing the generation of functional islet-like organoids in 3D gellan gum culture. Figure 5A is a schematic describing the generation of hiPSCs stably incorporating dual reporters for insulin expression (GFP) and insulin secretion (luciferase). Figure 5B is a bar graph showing increased expression of human insulin during differentiation of hiPSCs incorporating the dual reporter. Figure 5C is a bar graph comparing glucose stimulated insulin secretion (GSIS) from human islet-like organoids generated using the methods described herein and human islets. GSIS was measured using secreted luciferase in single islet-like organoids or human islets in response to 3 mM and 20 mM glucose. Arrows indicate functional organoids that can increase insulin secretion in response to a glucose load. Figure 5D is a bar graph comparing insulin secretion from individual batches of islet-like organoids prepared as described herein and mouse islets as a negative control. Insulin secretion was measured in response to 3 mM glucose (G3mM), 20 mM glucose (G20mM), 20 mM glucose (G20mM) and exendin-4 (Ex4), or 20 mM potassium chloride (KCl20mM) after 133 days of culture. Responses were measured as secreted luciferase activity from pooled organoids (100 organoids / sample). Figure 5E shows intracellular luciferase activity as a measure of intracellular insulin (100 organoids / sample). [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 6A]Figures 6A-6H are schematics and images showing the generation of functional human mini-organs in dishes. Figure 6A is a schematic showing the generation of functional human mini-organs including human islets, pancreas, liver, heart, and intestine. Figures 6B-6C show the generation of human cardiac organoids. Figure 6B (top) provides a schematic illustrating the protocol for differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes (when cultured in 2D) or mini-hearts (when co-cultured with hADSCs and HUVECs in 3D). Figure 6B (bottom left) provides plots showing the relative expression of cardiomyocyte-specific genes human MLC2a (hMLC2a), human Nkx2-5 (hNkx2-5), α-myosin heavy chain (αMHC), and KCNQ1 before (day 0) and at day 18 of the differentiation protocol with and without the PPARδ agonist GW501516. The bottom right of Figure 6B is a video micrograph showing beating hiPSC-derived cardiomyocytes. Figure 6C shows images of human mini-heart-like organoids generated by culturing hiPSC-derived cardiomyocytes with hADSCs and HUVECs as described in the schematic above. Figures 6D-6F show the generation of human liver organoids. Figure 6D (top) provides a schematic illustrating the protocol for differentiating human pluripotent stem cells (hPSCs) into hepatocytes. Figure 6D (bottom left) provides a plot showing the relative expression of hepatocyte-specific genes AFP, ALB, and Cyp3a7 during differentiation of six independent preparations. The bottom right of Figure 6D is a micrograph showing hiPSC-derived hepatocytes. Figure 6E is a series of micrographs showing hiPSC-derived hepatocytes. The top panels show the expression of Cyp7a1 (Cyp7a1-GFP reporter, left) and SREBP1c (SREBP1c-GFP reporter, right) in hiPSC-derived hepatocytes, indicating functional maturation. The bottom panels show hiPSC-derived hepatocytes cultured overnight with (right) or without (left) phosphatidic acid (PA). High magnification images shown at the bottom left reveal the accumulation of lipid droplets in hepatocytes treated with phosphatidic acid. Figure 6F shows human mini-liver-like organoids generated by culturing hiPSC-derived hepatocytes with hADSCs and HUVECs in a gellan gum 3D culture system.FIG. 6G (top) provides a schematic illustrating the protocol for differentiating human pluripotent stem cells (hPSCs) into intestinal organoids when co-cultured with hADSCs and HUVECs in a gellan gum 3D culture system. The bottom image shows the budding of human intestinal organoid cultures, consistent with crypt-like structures and showing functional organoids. FIG. 6H (top) provides a schematic illustrating the protocol for differentiating human pluripotent stem cells (hPSCs) into mini-pancreases when co-cultured with hADSCs and HUVECs in a gellan gum 3D culture system. The middle panel shows an image of insulin-positive beta cells, indicated by the expression of green fluorescent protein driven by the insulin promoter, and the corresponding light microscope image of the pancreatic organoids. Exocrine cells are the cells that remain unlabeled. The bottom panel shows an image of a single pancreatic organoid in a 96-well plate. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. [Figure 7]Schematic diagram showing modeling human type 2 diabetes in a dish. Creation of functional human organs such as liver, pancreas, and islets provides a new strategy in drug screening and modeling human type 2 diabetes. For example, lipid-laden liver organoids can be used to study early insulin resistance phenotypes, and human islet-like organoids can be used to study β-cell death seen in late stage type 2 diabetes. The inserted graph shows the response of islet-like organoids to increasing concentrations of human amyloid polypeptide (hIAPP). Increased propidium iodide (PI) staining in cells in the G0 / G1 phase indicates that hIAPP induces apoptosis in islet-like organoids. [Figure 8] Figures 8A and 8B are schematics showing a platform for drug screening and subsequent evaluation of potential candidates for human type 2 diabetes and pancreatic cancer. Figure 8A shows a schematic for screening potential drugs in a dish for use in human type 2 diabetes or human pancreatic cancer tumorigenesis. Prior to screening a drug library for compounds that reverse or reduce disease manifestations, organoid cultures are exposed to appropriate stresses (e.g., high levels of free fatty acids (FFA), high glucose levels, or related cytokines) to induce disease-like phenotypes. Figure 8B shows an approach for evaluating potential drug candidates for type 2 diabetes and pancreatic cancer tumorigenesis / metastasis in mice. Mice transplanted with individual human organs (e.g., pancreas) or combinations of human organs (e.g., pancreas and liver) are exposed to appropriate disease-inducing stressors (e.g., high fat / high cholesterol (HF / HC) diet) prior to treatment with potential disease-altering drug candidates. [Figure 9]9 is a set of schematic diagrams and images showing pancreatic structure and pancreatic tissue, as well as images of islet-like organoids and pancreatic organoids produced herein.The schematic diagram on the left side of FIG. 9 represents the anatomical morphology and structure of pancreas (top) and islet (bottom).The image on the top right corner of FIG. 9 shows iPSC-derived pancreatic organoids with islet (indicated by green fluorescent protein driven by insulin promoter) and exocrine / ductal components as indicated.The image on the right center of FIG. 9 shows optical microscope images corresponding to the fluorescent images above.The image on the bottom right corner of FIG. 9 shows a single islet organoid. [Figure 10A] Figures 10A-10D are a series of schematics, images, heat maps, and bar graphs summarizing the generation of islet-like organoids from PSCs. Figure 10A is a schematic describing the protocol for generating islet-like organoids by culturing in gellan gum in 3D. Figure 10B is a series of images documenting the proliferation of hPSCs and their differentiation into pancreatic lineage in 3D gellan gum culture as described herein. Insulin expression is indicated by green fluorescence seen on day 21. Figure 10C is a heat map representation of the changes in gene expression during the differentiation of hiPSCs into islet-like organoids. Figure 10D is a series of bar graphs reporting the changes in relative expression of the pluripotency marker Nanog, the endocrine hormones insulin, somatostatin, and glucagon, and the beta cell lineage marker Nkx6-1 in hiPSCs and two stem cell lines (HuES8 and H1ES) during differentiation as described in the methods herein. Gene expression was measured by qPCR. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11]Figure 11A and Figure 11B show heatmaps and graphs that illustrate the role of WNT protein in 3D cultured hADSC. Figure 11A shows the heatmap representation of gene expression changes in hADSC during spontaneous self-organization that occurs in 3D culture. Listed are the genes that are induced in the WNT5a pathway. Figure 11B shows the graphs that show the relative levels of several individual WNT proteins in hADSC 3D culture over time, which identifies WNT5a protein as the main protein that is expressed. [Figure 12-1]Figure 12A-H are a series of bar graphs and images illustrating the role of WNT proteins in the metabolic maturation of iPSC-derived islet organoids. Figure 12A is a bar graph comparing the expression of Fltp and Esrrg genes in iPSC-derived islet organoids (day 21, generated in the absence of hADSCs and HUVECs) after treatment with PBS, WNT3a (500 ng / ml), WNT4 (100 ng / ml), or WNT5a (400 ng / ml) for 5 days. Figure 12B is a bar graph showing induction of Esrrg gene expression in hiPSC-derived islet organoids generated in the absence of supporting hADSCs and HUVECs in response to increasing doses of WNT4 (0, 10, 25, 50, 100, 200 ng / ml) and WNT5a (0, 25, 50, 100, 200, 400 ng / ml). Figure 12C is a bar graph showing induction of mitochondrial genes involved in oxidative phosphorylation (Cox7a2, Ndufal, Ndufa7), lactate dehydrogenase (Ldha), and Fltp (effector and reporter gene of Wnt / planar cell polarity (PCP)) in response to increasing doses of WNT4 (0, 10, 25, 50, 100, 200, 400 ng / ml) and WNT5a (0, 25, 50, 100, 200, 400 ng / ml) in hiPSC-derived islet organoids generated in the absence of supporting hADSCs and HUVECs. Figure 12D shows fluorescent images showing levels of mitochondria (Mitotracker; Mito-Red) and insulin (Insulin-GFP) in hiPSC-derived islet organoids (day 27) after 8 days of treatment with PBS or WNT4 (100 ng / ml). Figure 12E shows fluorescent images of FACS analysis of hiPSC-derived islet organoids (day 27) after 8 days of treatment with PBS or WNT4 (100 ng / ml). Figures 12F, 12G, and 12H show a series of bar graphs illustrating the results of FACS analysis of hiPSC-derived islet organoids (day 27) after 8 days of treatment with PBS, WNT4 (100 ng / ml), WNT5a (400 ng / ml), control fibroblast conditioned medium (50%), or conditioned medium of fibroblasts secreting WNT5a (50%).The WNT protein used was recombinant human (rh) protein. [Figure 12-2] See description of Figure 12-1. [Figure 12-3] See description of Figure 12-1. [Figure 13] 1 shows a bar graph demonstrating the role of WNT4 in the functional maturation of hiPSC-derived islet organoids. Human iPSC (hiPSC)-derived islet organoids (day 22) were treated with PBS (vehicle, "Veh") or WNT4 (100 ng / ml) for 12 days, and secretion of human c-peptide was measured in response to low glucose levels (3 mM, "G3mM"), high glucose levels (20 mM, "G20mM"), or high KCl levels (20 mM, "KCL20mM"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0143] Detailed Description of the Invention The present invention features compositions and methods that are useful for generating expandable, functional, vascularized organoids, particularly human pancreatic or islet organoids in vitro.The present invention is based, at least in part, on the discovery that culturing iPSC-derived β-like cells together with human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (Huvecs) in a three-dimensional matrix containing gellan gum generates functional pancreatic and islet organoids.

[0144] The generated organoids were vascularized and showed functional properties such as glucose-stimulated insulin secretion (GSIS). Islet transplantation is known as the best treatment to cure insulin-deficient diabetes, such as type 1 diabetes and late-stage type 2 diabetes. Recent studies have demonstrated the feasibility of generating glucose-responsive insulin-producing β-like cells from human pluripotent stem cells (PSCs), but the generation of functional, vascularized islets from PSCs that can secrete insulin, glucagon, and somatostatin in response to nutrients has not been achieved so far.

[0145] The studies described herein demonstrate that the self-organizing capabilities of human adipose-derived stem cells (hADSCs), HUVECs, and human iPSC-derived β-like cells allow the in vitro generation of glucose-responsive insulin-secreting islet-like organoids with the ability to form functional vasculature. The studies herein further demonstrate the successful expansion of islet-like organoid generation by using a gellan gum-based 3D culture system. The functional heterogeneity in hiPSC-derived islet-like organoids was characterized using Gaussia luciferase reporter to measure insulin secretion. Without intending to be bound by theory, the results herein suggest that novel human islet-like organoids may provide therapeutic treatment for diabetes and may provide a platform for drug screening, genome editing, and modeling of organogenesis and pathogenesis of diabetes.

[0146] pancreas In some aspects, the present invention provides pancreatic organoids or pancreatic islet organoids. The pancreas is an organ located in the abdomen and has endocrine and exocrine functions. Figure 9 provides a schematic diagram showing the structure of the pancreas. The part of the pancreas that has an endocrine role is a mass of cells called "islets" (also known as islets of Langerhans). The endocrine secretions of the pancreas include hormones that regulate glucose metabolism and blood glucose concentration. There are four main cell types in the islets: α cells, which secrete glucagon (a hormone that increases blood glucose concentration); β cells, which secrete insulin (a hormone that decreases blood glucose concentration); δ cells, which secrete somatostatin (a hormone that regulates α cells and β cells); and γ cells, which secrete pancreatic polypeptide.

[0147] The portion of the pancreas that has an exocrine role is called the exocrine component. The exocrine pancreatic secretions reach the small intestine and contain digestive enzymes that aid in the breakdown of carbohydrates, proteins, and lipids. The exocrine component has ducts arranged in clusters called pancreatic acini. The exocrine pancreatic secretions are secreted into the lumen of the acini, which accumulate and drain into the pancreatic duct and duodenum.

[0148] The pancreatic islet organoid and pancreatic organoid of the present invention mimic the structure of pancreatic islet and pancreas, respectively.In some embodiments, the pancreatic islet organoid and pancreatic organoid of the present invention contain any one or more of the following cells: iPSC-derived β-like cells, iPSC-derived α-like cells, iPSC-derived δ-like cells, and iPSC-derived duct-like cells.In some embodiments, the pancreatic organoid of the present invention contains iPSC-derived exocrine components.In some embodiments, the iPSC is human iPSC (hiPSC).Human embryonic stem cells and human induced pluripotent stem cells are commercially available (e.g., from WiCell, which provides iPS(IMR-90)-1, iPS(IMR-90)-4, and iPS(foreskin)-1). Human induced pluripotent stem cells can also be generated from a variety of somatic cell types using methods known in the art (Yu, J., K. Hu, et al. (2009). "Human induced pluripotent stem cells free of vector and transgene sequences." Science 324(5928): 797-801).

[0149] The pancreatic islet organoid and pancreatic organoid of the present invention also exhibit the function of pancreatic islet and pancreas.In certain embodiments, the pancreatic islet organoid or pancreatic organoid exhibits any one or more of the following functions: glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, and glucagon secretion.In some embodiments, the pancreatic islet organoid or pancreatic organoid expresses any one or more of the following transcription factors: Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, and Foxa2.

[0150] Generation of pancreatic or islet organoids In some other aspects, the present invention features a method for producing pancreatic organoids or pancreatic islet organoids. Although recent studies have shown that it is possible to produce glucose-responsive insulin-producing β-like cells, attempts to produce pancreatic islets that can secrete insulin, glucagon, and somatostatin in response to nutrients, as well as attempts to obtain vascularization from stem cells, have not been successful. Results are described herein that demonstrate the successful in vitro production of glucose-responsive insulin-secreting islet-like organoids capable of functional vascularization using the self-organizing function of human adipose-derived stem cells (hADSCs), human umbilical vein endothelial cells (HUVECs), and human iPSC-derived β-like cells. Furthermore, the method for producing islet-like organoids has been successfully scaled up using a gellan gum-based 3D culture system. Functional heterogeneity in hiPSC-derived human islet-like organoids has also been investigated using a Gaussia luciferase reporter to measure insulin secretion.

[0151] The generation of functional human organs provides new therapeutic strategies in drug screening, disease modeling, and arresting or preventing end-point organ failure. Efficient stepwise differentiation methods from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) into insulin-producing β-like cells have been previously demonstrated. D'Amour et al and Kroon E et al reported efficient differentiation of hESCs into insulin-producing cells capable of secreting insulin in response to glucose after 4-5 months of in vivo maturation (D'Amour et al., 2006, Nature biotechnology 24, 1392-1401;Kroon et al., 2008, Nature biotechnology 26, 443-452). Recently, Rezania et al. and Pagliuca et al. reported a differentiation method to induce the formation of mature human β-like cells in vitro, as they expressed the definitive β-cell markers MAFA and Nkx6-1 and showed partial functionality (e.g., insulin secretion) (Rezania et al., 2014, Nature Biotechnology Nov; 32(11):1121-33;Pagliuca et al., 2014, Cell 159, 428-439). However, in contrast to cadaveric human islets, their β-like cells required several months of functional maturation in vivo and lacked functionality provided by other islet cell types, such as glycemic control by α-cells (glucagon secretion) and δ-cells (somatostatin secretion). Moreover, β-like cells lacked both mesenchyme and vascularized endothelial cells that human islets naturally possess. These critical differences between hPSC-derived β-like cells and human islets may impair the ability of hPSC-based therapies to treat insulin-dependent diabetes (such as type 1 diabetes or late-stage type 2 diabetes).

[0152] Previously, we found that a metabolic transition occurs during neonatal to adult maturation of β cells, in which the orphan nuclear receptor estrogen-related receptor gamma (ERRγ) regulates the increase in oxidative metabolism required for fully functional β cells. Consistent with this result, human iPSC-derived β-like cells expressing insulin, MAFA, and Nkx6-1 can be metabolically matured by overexpression of ERRγ to increase their oxidative metabolism and thereby enhance their glucose-stimulated insulin secretion (GSIS) functionality. These results indicate that in addition to the expression of lineage determining factors such as PDX1, MAFA, Nkx6-1, and insulin, additional cellular signaling that matures β cell metabolism is required to generate fully functional β cells.

[0153] During early pancreatic organogenesis, newly specified pancreatic cells arise from a foregut endoderm sheet to form a condensed tissue mass, the pancreatic bud, which soon becomes vascularized. A similar progression has been observed during liver organogenesis. Such large-scale morphogenetic changes depend on an exquisite orchestration of signals between endodermal epithelial progenitors, mesenchymal progenitors, and endothelial progenitors before blood perfusion. Takebe et al. successfully generated liver organ primordia that rapidly vascularized and functionally matured in vivo by culturing hepatic endoderm cells together with endothelial and mesenchymal lineages (Takebe et al., 2013, Nature 499, 481-484).

[0154] Previous studies have not demonstrated the feasibility of generating mature, functional, and vascularized organoids in vitro, such as other organoid tissue types, such as pancreatic organoids. Furthermore, previous studies have demonstrated a lack of scalability because the organoids were generated using MATRIGEL® matrix, which is not efficient for use in scale-up production.

[0155] Described herein are studies that demonstrate the successful large-scale generation of human islet-like organoids that can secrete insulin and are vascularized as seen in human islets.(1) Human adipose-derived stem cells (hADSCs) have the ability to self-organize;(2) Late pancreatic progenitor cells can form islet-like clusters with an efficiency comparable to β-like cells when co-cultured with HUVECs and hADSCs;(3) Human islet-like organoids have improved expression of lineage determining factors and metabolic regulators, including ERRγ;(4) Islet insulin secretion assays measured using the Gaussia luciferase-proinsulin system revealed that human islet-like organoids contain functional cells that can secrete insulin in response to glucose;(5) Human islet-like organoids exhibit vascularization;(6) Human islet-like organoids derived from hiPSCs by the methods described herein recapitulated human islet organogenesis and type 1 and type 2 diabetes pathogenesis in a dish;(7) It is demonstrated herein that the human islet-like organoids derived from hiPSCs by the method described herein provide a new source that can replace human islet transplantation for treating type 1 and type 2 diabetes; and (8) that the human islet-like organoids transplanted into the STZ-induced NODSCID mouse model of type 1 diabetes ameliorated type 1 diabetes in recipient animals (Figure 4H and 4I).

[0156] Also described herein is a study that evaluates the role of certain Wnt (also referred to herein as "WNT") proteins in developing human islet-like organoids that can secrete insulin and vascularize as seen in human islets. The WNT gene family consists of structurally related genes that code for secreted signaling proteins that have been implicated in several developmental processes, including oncogenesis, and the regulation of cell fate and pattern formation during embryonic development. Wnt proteins constitute a major family of signaling molecules that orchestrate and affect various cell biological and developmental processes. Wnt proteins undergo a complex series of post-translational modifications involving several highly specialized processing enzymes. Once released from cells, Wnt proteins interact with several molecules in the extracellular environment, such as glycans, protein binding partners (e.g., WIF, Sfrp), and cell surface receptors. (Willert, K. et al., 2012, Cold Spring Harbor, Perspectives in Biology, 2012). It is demonstrated herein that (1) Wnt5a is the major Wnt protein that induces self-organization of hADSCs (Figures 11A and 11B); (2) Wnt5a and Wnt4 activate the ERRγ mitochondrial metabolic pathway (Figures 12A-12H); and (3) Wnt4 is sufficient to induce functional maturation of hiPSC-derived islet-like organoids in vitro in the absence of additional cell types such as hADSCs and HUVECs (Figure 13).

[0157] Treatment Method Islet transplantation is a therapeutic method for treating insulin-deficient diabetes, such as type 1 diabetes and late type 2 diabetes.Therefore, in another aspect, the present invention provides a method for treating pancreatic disease, such as type 1 or type 2 diabetes, comprising administering the pancreatic organoid or islet organoid of the present invention to a subject (e.g., a mammal, such as a human) by transplantation.One embodiment is a method for treating a subject suffering from or susceptible to pancreatic disease (e.g., type 1 diabetes) or disorder or symptoms thereof.The method comprises transplanting the pancreatic organoid or islet organoid of the present invention, which is sufficient to treat disease or disorder or symptoms thereof, into a mammal under conditions such that disease or disorder is treated.

[0158] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms. Although not excluded, it is recognized that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0159] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of or susceptible to developing it.

[0160] The therapeutic method (including preventive treatment) of the present invention generally comprises administering (particularly transplanting) an effective amount of pancreatic organoid or pancreatic islet organoid to the subject (for example, animal, human) that needs it, for example, mammal, particularly human.The administration of pancreatic organoid or pancreatic islet organoid can be by any suitable means, which, in combination with other components, results in the amount of organoid that is effective for improving, alleviating or stabilizing pancreatic disease, such as type 1 or type 2 diabetes.

[0161] In some aspects, the subject is further administered with an immunosuppressant.The immunosuppressant can be administered to the subject before, during or after the organoid is administered (e.g., transplanted) to the subject.The immunosuppressant can be an agent that suppresses or prevents the rejection (e.g., acute rejection) of transplanted organoid during transplantation, or an agent that maintains immunosuppression after transplantation.The immunosuppressant includes, but is not limited to, basilizimab, antithymocyte globulin, alemtuzumab, prednisone, azathioprine, mycophenolic acid, cyclosporine, sirolimus, and tacrolimus.

[0162] In some embodiments, at least about 100,000, at least about 200,000, at least about 300,000, at least about 400,000, at least about 500,000, at least about 600,000, at least about 700,000, at least about 800,000, at least about 900,000, or at least about 1 million pancreatic islet organoids are transplanted into the subject.In some embodiments, before transplanting the organoids of the present invention, the islets of the subject are removed.In some other embodiments, the pancreatic islet organoids are transplanted into the subject by injecting them into the upper abdomen of the subject.In some embodiments, the pancreatic islet organoids are injected into the liver.The pancreatic islet organoids can be injected into the subject using a catheter.In some other embodiments, the pancreatic organoids or pancreatic islet organoids are administered to the subject by surgery.In another embodiment, the pancreatic islet organoids are transplanted onto the omentum. Omental transplantation can use layering technique, in which islet organoid (or its cells) is mixed with autologous plasma and layered on the omentum under laparoscopy.Then, a solution (20 ml) containing recombinant thrombin (1000 U / ml) is layered on the islet organoid, followed by another layer of autologous plasma to generate a biodegradable biological scaffold that can remain and function in the patient for at least one year (see, for example, Baidal, D. et al., 2017, N. Engl. J. Med., 376:19).In another embodiment, hydrogel biomaterials that mitigate the immune response by the recipient can be used for islet organoid transplantation (see, for example, Vegas, A. et al., 2016, Nature Biotechnology, 34:345-352).

[0163] To further reduce the immune response to transplanted organoids in a subject, the organoids can be encapsulated in hydrogel and then transplanted into a subject. Such transplantation methods are further described in Vegas et al., Nature Medicine 2016, doi:10.1038 / nm.4030;Vegas et al., Nature Biotechnology 2016, doi:10.1038 / nbt.3462. In some embodiments, the hydrogel contains alginic acid or alginic acid derivatives (e.g., triazole-thiomorpholine dioxide). Various modifications of alginic acid hydrogels have also been identified that substantially reduce the inflammatory or fibrotic effects of alginic acid hydrogels (Vegas et al., Nature Biotechnology 2016, doi:10.1038 / nbt.3462). Thus, in some other embodiments, the hydrogel contains chemical modifications that reduce the inflammatory effects of transplanted organoids in a subject.

[0164] Screening assays The pancreatic islet organoid and pancreatic organoid of the present invention can be useful for modeling pancreatic disease in vitro or in vivo.Such pancreatic disease model can be used to identify the drug that is useful for treating pancreatic disease.Thus, in some aspects, the present invention provides a method for identifying the regulator, i.e., candidate or test compound or candidate or test agent (e.g., protein, peptide, peptidomimetic, peptoid, polynucleotide, small molecule, or other drug) that is useful for treating pancreatic disease, particularly type 2 diabetes and / or pancreatic cancer.In one embodiment, the agent regulates the activity of the organoid of the present invention.

[0165] Test agents of the present invention can be obtained alone or using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules with novel non-peptide backbones that have peptide functionality but are resistant to enzymatic digestion and yet retain biological activity; see, e.g., Zuckermann, RN (1994) et al., J. Med. Chem. 37:2678-85); spatially addressable parallel solid-phase or solution-phase libraries; synthetic library methods that require deconvolution; "one bead one compound" library methods; and synthetic library methods that use affinity chromatography selection. While the biological library and peptoid library approaches are limited to peptide libraries, the other four approaches are applicable to peptide, non-peptide oligomer, or small compound libraries (Lam (1997) Anticancer Drug Des. 12:145).

[0166] Examples of methods for synthesizing molecular libraries can be found in the art, for example, in DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0167] Libraries of compounds may be presented in solution (e.g., Houghten (1992), Biotechniques 13:412-421), or on beads (Lam (1991), Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869), or phages (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra).

[0168] Compounds to be used as test agents (i.e. potential inhibitors, antagonists, agonists) can be obtained from commercial sources or can be synthesized from readily available starting materials using standard synthesis techniques and methodologies known to those skilled in the art.Synthetic chemical transformations and protecting group methodologies (protection and deprotection) useful in synthesizing compounds identified by the methods described herein are known in the art, including, for example, those described in R. Larock (1989) Comprehensive Organic Transformations, VCH Publishers;TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991);L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994);and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and successive editions thereof.

[0169] Combinations of substituents and variables in the compounds envisioned by the present invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that have sufficient stability to permit their manufacture and maintain their integrity for a sufficient period of time to be useful for the purposes detailed herein (e.g., transportation, storage, assays, therapeutic administration to a subject).

[0170] The compounds described herein may contain one or more asymmetric centers, and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.All such isomers of these compounds are expressly included in the present invention.The compounds described herein may also be represented in multiple tautomers, all of which are included herein.The compounds may also exist as cis- or trans-, or E- or Z-double bond isomers.All such isomers of such compounds are expressly included in the present invention.

[0171] A test agent of the present invention can also be a peptide (eg, a growth factor, a cytokine, a receptor ligand), or a polynucleotide encoding such a peptide.

[0172] The screening method of the present invention identifies the agent that increases or decreases the biological activity of the islet organoid and pancreatic organoid of the present invention.In some embodiments, pancreatic disease, such as type 2 diabetes or pancreatic cancer, is induced or mimicked in islet organoid or pancreatic organoid.Type 2 diabetes in islet organoid or pancreatic organoid can be induced, for example, by contacting organoid with free fatty acid (FFA), glucose, and cytokines (particularly high glucose and / or high FFA).In one embodiment, pancreatic organoid is co-cultured with pancreatic cancer cells, astrocytes, and immune cells to create human pancreatic cancer microenvironment in vitro.

[0173] In some embodiments, the organoid is contacted with a candidate agent, and the effect of the candidate agent on biological activity, function or event is assayed.In some embodiments, the candidate agent is a drug approved by the Food and Drug Administration (FDA).For example, the biological activity of the pancreatic islet organoid or pancreatic organoid that is assayed in the screening method of the present invention includes insulin secretion (e.g., glucose-stimulated insulin secretion (GSIS)), beta cell apoptosis, LDHA activity, K(ATP) channel activity, mitochondrial function, NDUFA4, ESRRG, KCNK3 or MAFA polypeptide or polynucleotide level or activity, cell death, cell proliferation, and metastasis.In some embodiments, the agent increases GSIS.

[0174] In some other embodiments, the organoid of the present invention (for example, pancreatic islet organoid or pancreatic organoid) is transplanted into host to model pancreatic disease, such as type 2 diabetes or pancreatic cancer in vivo.The method of transplanting organ or organoid is known in the art.Host can be any non-human mammal, such as rat or mouse.

[0175] To reduce the immune response to the transplanted organoid in the host afterwards, the organoid can be encapsulated in hydrogel and then transplanted into the host. Such transplantation methods are further described in Vegas et al., Nature Medicine 2016, doi:10.1038 / nm.4030;Vegas et al., Nature Biotechnology 2016, doi:10.1038 / nbt.3462. In some embodiments, the hydrogel contains alginic acid or alginic acid derivatives (e.g., triazole-thiomorpholine dioxide). Various modifications of alginic acid hydrogels have also been identified that substantially reduce the inflammatory or fibrotic effects of alginic acid hydrogels (Vegas et al., Nature Biotechnology 2016, doi:10.1038 / nbt.3462). Thus, in some other embodiments, the hydrogel contains chemical modifications that reduce the inflammatory effects of transplanted organoid in the host.

[0176] In some embodiments, pancreatic organoid and liver organoid are transplanted together in host.Liver is the main target organ of metastasis of pancreatic cancer.In vivo in mice, the endothelial cells in mini pancreas and mini liver are related to each other, and create pancreatic-liver vasculature network for pancreatic cancer metastasis.Therefore, the host that is transplanted together with pancreatic organoid and liver organoid can be useful for studying human pancreatic cancer metastasis to human liver.

[0177] In some embodiments, the host that the organoid of the present invention is transplanted is subjected to environmental stress (for example, high fat / high glucose diet or administer pancreatic cancer cell) to induce or mimic pancreatic disease in the host.In some other embodiments, the pancreatic islet organoid or pancreatic organoid and / or liver organoid that are induced with disease (for example, type 2 diabetes or pancreatic cancer) are transplanted into the host.

[0178] In some embodiments, a candidate agent is administered to the host. In certain embodiments, the candidate agent is a drug approved by the U.S. Food and Drug Administration (FDA). In some embodiments, the effect of the candidate agent on the host phenotype, such as pancreas-related biological activity or function, or disease-related activity, is assayed. Exemplary biological activities include insulin secretion (e.g., glucose-stimulated insulin secretion (GSIS)), beta cell apoptosis, LDHA activity, K(ATP) channel activity, mitochondrial function, NDUFA4, ESRRG, or MAFA polypeptide or polynucleotide level or activity, cell death, cell proliferation, and metastasis. In some embodiments, the agent increases GSIS.

[0179] In any one of the embodiments herein, the effect of candidate agent (i.e., the ability to regulate pancreatic activity or function) is measured in comparison with reference.Reference can be, for example, untreated pancreatic islet organoid or pancreatic organoid.In some embodiments, reference is the host that the organoid of the present invention is transplanted and is not administered with candidate agent.

[0180] Agents useful in the methods of the present invention can also be detected by identifying an increase in the expression of a desired marker (e.g., MAFA as a β-cell fate marker). The level of expression can be measured in several ways, including, but not limited to, measuring the mRNA encoded by the genetic marker; measuring the amount of the protein encoded by the genetic marker; or measuring the activity of the protein encoded by the genetic marker.

[0181] The level of mRNA corresponding to a marker can be determined by in situ and in vitro formats. The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern analysis, polymerase chain reaction analysis, and probe arrays. In one format, the mRNA (or cDNA) is immobilized on a surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In another format, the probe is immobilized on a surface and the mRNA (or cDNA) is contacted with the probe, for example in a two-dimensional gene chip array described below. Those skilled in the art can adapt known mRNA detection methods for use in detecting the level of the mRNA encoded by the markers described herein.

[0182] Levels of mRNA in a sample can be determined using a variety of techniques, including, for example, rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1989) Bio / Technology 6:1197), and ribosomal amplification (Ribosomal amplification). al., U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as a pair of nucleic acid molecules that can anneal to the 5' or 3' region of a gene (the plus and minus strands, respectively, or vice versa) and contain a short region between them. Generally, amplification primers are about 10-30 nucleotides in length and flank a region about 50-200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers allow for the amplification of a nucleic acid molecule that contains the nucleotide sequence flanked by the primers.

[0183] The practice of the present invention will utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections.

[0184] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening, and therapeutic methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0185] Example 1: Generation and characterization of pancreatic organoids and islet organoids Although animal disease models can provide insight into disease pathogenesis, drugs identified from screening using animal models often cannot be adopted in human patients. The creation of functional human organoids provides new therapeutic strategies in drug screening and disease modeling (Figure 1). A novel technique for creating 3D human "mini-organs" or organoids in a dish is described herein. This technique can be used to model diseases such as human type 2 diabetes in a dish to find drugs that are effective in gene-, patient-, or environment-specific diseases such as human type 2 diabetes.

[0186] Development of a gellan gum-based 3D culture system for the differentiation of β-like cells It is known that three-dimensional (3D) culture systems contribute to promoting cell self-organization and integration. Therefore, MATRIGEL® matrix, which contains extracellular matrix components such as collagen and fibronectin, is often used as the base of 3D culture systems. However, MATRIGEL® matrix-based 3D culture systems are not ideal for large-scale human organoid production due to their cost and difficulty in scale-up. A cost-effective and easily scalable gellan gum-based 3D culture system and method for differentiating β-like cells are described herein. Using a fully chemically defined stepwise differentiation protocol (Figure 10A), human pluripotent cells (hPSCs) differentiate into insulin-producing islet-like spherical cell clusters with high efficiency and reproducibility in a gellan gum-based 3D culture system (Figure 10B). Single dissociated pluripotent stem cells (PSCs) successfully formed spheres within 5 days in STEMCELL™ TeSR™ medium containing gellan gum. After 15-21 days of differentiation in gellan gum-containing custom TeSR™ with defined small molecule stimulation, insulin-positive GFP clusters were observed (Figure 10B). Global transcriptome analysis by RNA-seq revealed stepwise differentiation of hiPSCs into insulin-positive cells expressing β-cell lineage-specific marker genes including Pdx1, Nkx6-1, GATA6, and MAFB (Figure 10C). The differentiation of hiPSCs and human ESC lines HuES8 and H1ES into islet-like cell clusters was further confirmed by the progressive loss of pluripotency marker Nanog, induction of β-cell specific marker Nkx6-1, and progressive induction of endocrine hormones insulin, somatostatin, and glucagon, as determined by qPCR (Figure 10D). These results demonstrate that the gellan gum-based 3D culture system is suitable for generating large-scale islet-like organoids from hPSCs.

[0187] In vitro generation of expandable human islet-like organoids Beta-like cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) have limited functionality and lack the morphological and functional characteristics of human islets. Previous studies have demonstrated that co-culture of hiPSC-derived hepatocytes with human umbilical vein endothelial cells (HUVECs) and human bone marrow-derived mesenchymal stem cells (hMSCs) results in self-organized 3D hepatospheres within Matrigel (Takebe et al., 2013, Nature 499, 481-484). The study found that liver "organoids" had superior expression of lineage-determining factors compared to differentiation of isolated hepatocytes, and that these organoids rapidly vascularized and functionally matured in vivo.

[0188] The study herein found that hiPSC-derived pancreatic progenitor cells (hiPSC-PPs) generated using a 2D differentiation protocol (Yoshihara et al, Cell Metab. 23, 622-634) did not self-organize in 3D MATRIGEL® matrix (Figure 2A). In contrast, HUVEC cells rapidly formed vasculature-like structures and human adipose-derived stem cells (hADSCs) self-organized in 3D MATRIGEL® matrix (Figure 2A). In MATRIGEL® matrix, dispersed hADSC cells protruded processes within 4 hours, formed cloth-like wrappers within 12 hours, and adopted sphere-like structures within 24-48 hours (Figure 2B). Furthermore, a minimum cell density for self-organization was identified (i.e., approximately 2 cm). 2 Approximately 10,000-20,000 cells in 300 μl of MATRIGEL® matrix in a well (Figure 2C). RNA-seq analysis identified dynamic transcriptional changes during hADSC 3D self-organization, suggesting that the ability to self-organize under 3D culture conditions is an inherent feature of naive hADSCs (Figure 2D). These results identify mesenchymal hADSCs as a source for generating self-organizing organoids.

[0189] To investigate pancreatic organogenesis, hiPSC-PPs (cells 1x10 6 ) cells were cultured in a Matrigel matrix with HUVEC (7x10 cells). 5 ) and hADSCs (1–2 x 10 cells 5 The human islet-like organoids were co-cultured with human islet-like cells (Figure 2E). This co-culture gave rise to macroscopic 3D cell masses 48 hours after seeding (Figure 2F). Furthermore, insulin expression based on GFP reporter expression was detected 5 days after seeding and increased with time in culture in the human islet-like organoids. In addition, HUVEC-based endothelial cells were incorporated inside the organoids, as shown by fluorescently labeled (mCherry) HUVECs (Figure 2G).

[0190] Limitations of MATRIGEL® Matrix for organoid generation include high cost, difficulty in recovering organoids, limited expansion, and batch-to-batch variability.

[0191] Described herein is a method to generate morphologically identical human islet-like organoids using gellan gum-based 3D culture (Figures 4A-4G). Figure 4A shows a schematic for generating functional, vascularized human pancreatic islets in a dish. Human induced pluripotent stem cell-derived pancreatic progenitor cells (hiPSC-PPs) (1x10 cells) were cultured in 50 ml of gellan gum-based 3D culture medium. 8 ) and human umbilical vein endothelial cells (HUVECs) (2–7 x 10 cells 6 ) and human adipose-derived stem cells (hADSCs) (2–7x10 6The hiPSC-PPs were cultured together with stromal cell populations such as β-cells (Figure 4B). Figure 4B shows that within 5 days after seeding in gellan gum-based 3D culture medium, hiPSC-PPs rapidly formed islet-like sphere structures together with HUVECs and hADSCs. The human islet-like mini-organs expressed human insulin GFP reporter within 5 days after seeding, and the GFP intensity gradually increased. By co-culturing hiPSC-PPs, hADSCs, and HUVECs according to this method, human islet-like organoids morphologically similar to human islets were generated with high reproducibility (Figure 4C). In addition, the generated human islet-like organoids contained insulin granules in β-like cells (Figure 4B). Gene expression analysis revealed that the islet-like organoids showed increased expression of β-cell fate-determining genes (insulin, Nkx6-1, PCSK1, and UCN3) and mitochondria-related metabolic genes (Esrrg, Ndufa1, Ndufa 12, Cox7a2. Atp5b) in the insulin-expressing cell population (GFP-enriched (GFP+)) compared to those prepared without co-culture with hADSCs and HUVECs (Figure 4D and 4E). Glucose-stimulated human c-peptide secretion assay revealed that the islet-like organoids generated by this method could secrete human c-peptide in response to high (20 mM) glucose (Figure 4F).

[0192] Then, an in vitro functional vascularization test was performed. Figure 4G shows the in vitro functional vascularization test performed. The islet-like mini-organs created in gellan gum were transferred to MATRIGEL® matrix and cultured in endothelial growth medium (EGM). Green fluorescence indicates the expression of insulin gene. Within 24-48 hours of stimulation with EGM, the elongation of HUVEC cells was observed, indicating that the human islet-like organoids created by the method described herein have the ability to form vascular structures.

[0193] Establishment of a single islet insulin secretion assay using the proinsulin-NanoLuc Gaussia luciferase assay system It was previously reported that a reporter construct in which Gaussia luciferase was placed within the c-peptide portion of proinsulin accurately measured insulin secretion without affecting β-cell function (Burns et al., 2015, Cell metabolism 21, 126-137). Using a lentiviral system, we generated INS-1 cells stably expressing this Gaussia luciferase (Figures 3A-3F). Luciferase secretion from INS-1 cells stably expressing proinsulin-NanoLuc increased with high glucose (20 mM), high glucose and exendin-4 (G20 mM+Ex4), and the depolarizing agent potassium chloride (Figures 3A-3B), confirming the usefulness of this reporter system. We next evaluated the usefulness of this reporter for measuring insulin secretion in mouse or human islets transiently infected with the proinsulin-NanoLuc reporter. Luciferase secretion in response to 20 mM high glucose was detected in both transiently infected mouse and human islets (Figures 3C-3F). Importantly, the assay sensitivity was sufficient such that insulin secretion could be qualified at the single islet level. These results indicate that the insulin secretion assay based on the proinsulin-NanoLuc luciferase reporter is applicable not only to the rat beta cell line INS-1 cells but also to primary mouse and human primary beta cells.

[0194] Establishment of hiPSC and hESC cells incorporating dual lineage and functional reporters Human iPSCs and hESCs, respectively, stably expressing a reporter for β-cell lineage (human insulin reporter) and a reporter for β-cell function (proinsulin-NanoLuc reporter) hINS-GFP / Sec-Luc and hESCs hINS-GFP / Sec-Luc(Figure 5A). First, a neomycin-resistant construct of human insulin GFP reporter was generated by inserting the human insulin promoter sequence of pGreenZeo lentireporter (SR10028PA-1, System Bioscience) into pGreenFire lentireporter plasmid (TR019PA-1, System Bioscience) (named hINS-GFP-EF1a-Neo). hINS-GFP-EF1a-Neo lentivirus was infected into hiPSCs and hESCs by spinfection (800g, 1 hour, 37 degrees), and then the medium was replaced with fresh STEMCELL™ TeSR™ medium. Three days after the first infection, the cells were treated with 100μg / ml G418 in STEMCELL™ TeSR™ medium for 7 days. Selected hiPSC and hESC cells stably expressing hINS-GFP-EF1a-Neo were then infected with proinsulin-NanoLuc (Addgene, plasmid #62057) lentivirus by spinfection (800g, 1 hour, 37 degrees) and then replaced with fresh STEMCELL™ TeSR™ medium. Three days after the second infection, cells were treated with 5μg / ml blasticidin and 100μg / ml G418 in STEMCELL™ TeSR™ medium for 7 days. Cells were then maintained in STEMCELL™ TeSR™ medium (Figure 5A). The generated stable cell lines incorporating the dual reporter maintained self-renewal and pluripotency capabilities, as well as the ability to differentiate into insulin-producing β-like cells (Figure 5B).

[0195] Pooled human islet-like organoid cultures show consistent insulin secretion despite variable functionality observed in individual organoids Recent studies have reported the generation of insulin-producing β-like cells from hESCs and hiPSCs that can secrete insulin in response to glucose (Pagliuca et al. 2014, Cell 159, 428-439;Rezania et al., 2014, Nature Biotechnology Nov; 32(11):1121-33;Russ et al., 2015, EMBO Journal 34, 1759-1772). However, fully functional human islet-like clusters that can properly secrete insulin in response to nutrient signals including glucose, amino acids, fatty acids, and incretins such as GLP-1 have not been demonstrated so far. To date, efforts have been focused on generating insulin-producing β-like cells, glucagon-producing α-like cells, and somatostatin-producing δ-like cells individually from hPSCs. However, these approaches lack supporting cells that are important for regulation, such as mesenchymal cells, adipocytes, and vasculature cells. These missing cellular components may impair the functionality of islet-like cell masses, as the 3D structure of islets naturally enhances their function. In addition, pancreatic islet organogenesis involves clonal expansion of β-cells, suggesting that these cells may have multiple functions in islet-like organoids. To test this idea, a single organoid proinsulin secretion assay was performed. Human islet-like organoids generated by the methods described herein are morphologically identical to human islets (Figure 4C). However, there was a marked variability in the glucose-stimulated insulin secretion (GSIS) capacity of individual human islet-like organoids compared to human islets, as measured by a proinsulin luciferase secretion assay (Figure 5C). Pooled organoids demonstrated consistent GSIS functionality (10-100 organoids per assay) (Figure 5D). Furthermore, pooled human islet-like organoids exhibit enhanced GSIS when co-stimulated with GLP-1, and robust KCl-stimulated insulin secretion (Figure 5D).

[0196] The in vitro cultured iPSC-derived human islet-like organoids generated herein maintained the ability to respond to glucose, GLP1, and KCl after long-term (133 days) culture (Figure 5D).

[0197] Example 2: Generation and Characterization of Human Organoids Using the methods described herein, functional human mini-organs can be generated, including human islets, pancreas, liver, heart, and intestine (Figure 6A). Figure 9 shows the structure of a human pancreas and human islets within the pancreas. Using the methods described herein, human islet mini-organs or organoids were generated in approximately 30 days. The generated islets contained human induced pluripotent stem cell (hiPSC)-derived beta cells, alpha cells, delta cells, duct cells, as well as endothelial cells and hADSCs. The generated islet organoids express key beta cell transcription factors, such as insulin, Nkx6-1, PCSK1, and UCN3, as well as key mitochondrial metabolism genes, including Esrrg, Ndufa 1, Ndufa 12, Cox7a2, and Atp5b (Figure 4D-4E). The islet organoids exhibited at least partial GSIS, KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, vascularization, somatostatin secretion, and glucagon secretion (Figures 4G, 5D).

[0198] Human mini-pancreases or human pancreatic organoids were generated in approximately 30 days (Figure 6H). Human pancreatic organoids contained hiPSC-derived islets clustered inside the organoids and hiPSC-derived exocrine components surrounding the islets (Figure 6H). Pancreatic organoids also contained endothelial cells and hADSC. The functionality of human pancreatic organoids was demonstrated using an amylase secretion test, among other tests.

[0199] Human mini-livers were generated in approximately 15 days (Figures 6D-6F). The human mini-livers contained hiPSC-derived hepatocytes, endothelial cells, and hADSCs. Analysis of the functional characteristics of the human mini-livers revealed that they expressed AFP, ALB, and Cyp3a7 (Figure 6D), as well as the mature hepatocyte marker Cyp7a1 and the lipogenic marker SREBP1c (Figure 6E). The human mini-livers also exhibited insulin signaling, insulin resistance with palmitate, and lipid accumulation. The human mini-livers will be further tested for metabolic functions regarding gluconeogenesis, and drug metabolism.

[0200] Human mini-hearts were generated in approximately 15 days (Figures 6B-6C). Human mini-pancreases contained hiPSC-derived cardiomyocytes, endothelial cells, and hADSCs. Human mini-pancreases expressed key cardiomyocyte genes such as hMlc2a, hNkx2-5, αMHC, and KCNQ1 (Figure 6B) and were observed to beat in culture.

[0201] Human mini-intestines were generated in approximately 30 days (Figure 6G). Human mini-intestines contained hiPSC-derived enterocytes, endothelial cells, and hADSCs. Human mini-intestines expressed small intestinal markers CDX2, Muc2, and Lgr5, and showed intestinal organoid budding in response to R-spondin.

[0202] Example 3: Transplantation of functional pancreatic islet organoids rescued type 1 diabetic mice In hiPSC-derived human islet-like organoids, expression of specific functional islet markers, such as MAFA, UCN3, and mitochondrial oxidation genes, such as ERRγ (Esrrg), Ndufa 1, Ndufa 12, Cox7a2, and Atp5b, was observed. Remarkably, these islet-like organoids recapitulate both human islet development and diabetic pathogenesis in a dish. Transplantation of these functional islet-like organoids rescues type 1 diabetic mice with long-term survival, rapid vascularization, and reduced immune rejection.

[0203] Example 4: Drug screening and disease modeling in human islet-like organoids The creation of functional human organs by the methods described herein provides a new strategy for drug screening and disease modeling. Specifically, functional organoids can be used as a model of type 2 diabetes for drug screening (Figure 7). Human islet-like organoids respond to hepatic amyloid polypeptide (hIAPP) toxicity, an inducer of beta cell loss in type 2 diabetes patients and islet dysfunction after transplantation in hyperglycemic patients, and hIAPP induced G0 / G1 arrest in a dose-dependent manner within 24 hours in human islet-like organoids (Figure 7).

[0204] Figure 8A provides a schematic diagram showing the experimental approach to model type 2 diabetes and potentially screen drugs using the islet organoids and / or pancreatic organoids produced by the methods herein.In an exemplary assay, 3D mini-organs are exposed to stress factors that induce type 2 diabetes, such as high levels of free fatty acids (FFA) and / or glucose and selected cytokines.The stressed 3D mini-organs are then treated with various drugs.In some embodiments, the drugs are approved by the Food and Drug Administration (FDA).

[0205] As outputs, the following are assayed in human islet organoids: insulin secretion, beta cell apoptosis (PI staining), lactate dehydrogenase A (LDHA) expression via a luciferase reporter, and changes in expression of marker genes including NDUFA4 (mitochondrial oxidative phosphorylation), ESRRG (mitochondrial function), KCNK3 (Katp channel activity), and MAFA (beta cell fate marker). For human pancreatic organoids, amylase secretion and apoptosis of exocrine cells (PI staining) are assayed. For liver organoids, lipid accumulation is assayed using Oil Red O or histological examination. In the case of cardiac organoids, cardiac beating and cardiac size (hypertrophy) are measured. Intestinal organoids are analyzed by measuring lipid accumulation using Oil Red O or histological examination.

[0206] Figure 8A also shows the possibility of modeling human pancreatic cancer tumorigenesis and metastasis in a dish, and screening drugs that target these diseases. In an exemplary assay, 3D mini human pancreas is co-cultured with pancreatic cancer cells, stellate cells, and immune cells to create a human pancreatic cancer microenvironment in a dish. Then, various drugs (e.g., FDA-approved drugs) are screened to find compounds that effectively suppress pancreatic cancer growth or metastasis in the mini human pancreas microenvironment. As output, the following are measured for pancreatic organoids: apoptosis of exocrine cells (PI staining), collagen synthesis (Trichrome staining), and stellate cell activation (GFAP-reporter). Potential candidate drugs identified in these assays are tested in a mouse model of pancreatic cancer tumorigenesis and metastasis. Gene expression and morphology are examined, as well as the extent of cell death, cell proliferation, and metastasis.

[0207] Figure 8B provides a schematic diagram showing modeling of human type 2 diabetes in mice. In an exemplary assay, human islet organoids and / or human liver organoids are transplanted into mice. The mice are then subjected to various stress factors that induce type 2 diabetes, such as a high-fat diet (HFD) or cytokine injection. Potential candidate drugs identified in this assay are further tested in human type 2 diabetes mouse models. Gene expression and morphology as well as the degree of diabetes are examined.

[0208] Figure 8B also shows the modeling of tumor formation and metastasis of human pancreatic cancer in mice. In an exemplary assay, human pancreatic organoids and / or human liver organoids are transplanted into mice. The mouse with mini-pancreas transplantation is used to study human pancreatic cancer growth in human pancreatic microenvironment. In another exemplary assay, mini-pancreas and mini-liver are co-transplanted into mice. Liver is the main site of metastasis of pancreatic cancer. In vivo, endothelial cells in mini-pancreas and mini-liver create pancreatic-liver vasculature network for pancreatic cancer metastasis. Thus, the mouse with mini-pancreas and mini-liver co-transplantation is used to study metastasis of human pancreatic cancer to human liver.

[0209] The ultimate goal of generating functional organ-like clusters from pluripotent stem cells (PSCs) is to gain insight into the mechanisms underlying human disease. Although great progress has been made in terms of developing disease models in animals, many of these models fail to faithfully recapitulate the human condition. In the case of pancreatic islets, their development, cytoarchitecture, and physiology in rodents and humans differ significantly.

[0210] The results herein were obtained using the following materials and methods.

[0211] 3D Kelcogel® (3DKG) Culture Medium Kelcogel® F low acyl gellan gum (GG-LA) from Modernist Pantry is suspended in purified water at 0.3% (w / v) and dissolved by stirring or in a microwave at 90°C. The aqueous solution is sterilized in an autoclave at 121°C for 20 minutes. This solution is then diluted with TeSR™ (Ludwid et al., Nature methods 3, 637-646) or custom TeSR™ medium (800 ml DMEM / F12, 13.28 g BSA, 10 ml Glutamax, 560 mg NaHCO 3 , 330mg thiamine, 100mg reduced glutathione, 3300mg vitamin C, 14μg selenium, 10ml NEAA, 2ml trace element B, 1ml trace element C, 7μl β-ME, 2ml DLC, 2ml GABA, 2ml LiCl, 129.7μg pipecolic acid, insulin 2mg, adjust to 1000ml) to a final concentration of 0.015%. Add methylcellulose (MC) stock to a final concentration of 0.3% (R&D systems) (e.g., 0.3% Kelcogel® stock: 300mg Kelcogel® F low acyl GG-LA + 100ml MilliQ water; 3DKG Stem TeSR™ Basal Medium: 95ml STEMCELL™ TeSR™ + 5ml 0.3% Kelcogel® stock + 300ul MC stock; 3DKG Custom TeSR™ Basal Medium: 95ml Custom TeSR™ medium + 5ml 0.3% Kelcogel® stock + 300ul MC stock; add penicillin / streptozocin to 3DKG medium to a final concentration of 1%).

[0212] Preparation of human pancreatic endocrine precursor cells and β-like cells in vitro Pancreatic endocrine cells (hiPSC-PE) were prepared from human iPSCs using differentiation methods previously described. Briefly, human induced pluripotent stem cells (hiPSCs) derived from HUVECs were obtained from Stem Cell Core (Salk Institute). Cells were cultured on MATRIGEL® (BD)-coated dishes in complete STEMCELL™ TeSR™ medium at 37 °C in humidified 5% CO2 at 27 °C. 2 They were maintained at 37° in an incubator. For pancreatic differentiation, hiPSCs were infected with human insulin reporter lentivirus (pGreenZero lentireporter human insulin, System Biosciences) by spinfection (800g, 1 h). Method 1: The medium was changed to custom TeSR™ medium (800ml DMEM / F12, 13.28g BSA, 10ml Glutamax, 560mg NaHCO ) containing 100ng / ml human activin (R&D Systems), 25ng / ml recombinant human Wnt3a (R&D Systems). 3, 330 mg thiamine, 100 mg reduced glutathione, 3300 mg vitamin C, 14 μg selenium, 10 ml NEAA, 2 ml trace elements B, 1 ml trace elements C, 7 μl β-ME, 2 ml DLC, 2 ml GABA, 2 ml LiCl, 129.7 μg PA, insulin 2 mg, adjusted to 1000 ml) for 2 days, then differentiation medium containing 100 ng / ml human activin for an additional 2 days (stage 1, pancreatic endoderm). The medium was then replaced with custom TeSR™ medium with 1 uM dorsomorphin (Calbiochem), 2 μM retinoic acid (Sigma), 10 μM SB431542, and 1% B27 supplement for 7 days (stage 2). The medium was then replaced with custom TeSR™ medium with 10uM forskolin (Sigma), 10μM dexamethasone (Stemgent), 10μM TGFβ RI kinase inhibitor II / Alk5 inhibitor II (Calbiochem or Enzo), 10μM nicotinamide (Sigma), 1μM 3,3',5-triiodo-L-thyronine sodium salt (T3), and 1% B27 supplement for 4-5 days (days 15-21, pancreatic endocrine precursor cells). Medium was replaced daily (stage 1) or every other day (stages 2 and 3).

[0213] Method 2: The medium was replaced with differentiation medium (S1) containing 100 ng / ml human activin (R&D Systems), 25 ng / ml recombinant human Wnt3a (R&D Systems) or 3 μM CHIR99021 (Axon or Selleckchem) for 1 day, then with differentiation medium (S1) containing 100 ng / ml human activin for an additional 2 days (stage 1, pancreatic endoderm). The medium was then replaced with differentiation medium (S2) with 50 ng / ml FGF7 (R&D Systems) for 2 days, then with differentiation medium (S3) with 50 ng / ml FGF7, 0.25 μM SANT-1 (Sigma), 1 μM retinoic acid (Sigma), 100 nM LDN193189, and 100 nM α-amyloid precursor protein regulator TPB for 3 days. The medium was then replaced with differentiation medium (S4) with 0.25 μM SANT-1, 50 nM retinoic acid, 10 μM Alk5 inhibitor II, and 1 μM T3 for 3 days. The medium was then replaced with differentiation medium (S5) with 100 nM LDN193189, 100 nM gamma-secretase inhibitor XX GSiXX (Millipore), 10 μM Alk5 inhibitor II, and 1 μM T3 for 7 days. The medium was then replaced with differentiation medium (S5) with 10 μM Trolox (Calbiochem), 2 μM R428 (Selleckchem), 1 mM N-acetylcysteine, 10 μM Alk5 inhibitor II, and 1 μM T3 for an additional 7 to 20 days.

[0214] S1 medium (MCDB 131 medium, 8 mM glucose, 2.46 g / L NaHC03, 2% fatty acid-free BSA, 0.25 mM L-ascorbic acid, 0.002% insulin-transferrin-selenium ITS-X (GIBCO), 2 mM Glutamax, 1% penicillin-streptomycin), S2 medium (MCDB 131 medium, 8 mM glucose, 1.23 g / L NaHC03, 2% fatty acid-free BSA, 0.25 mM L-ascorbic acid, 0.002% insulin-transferrin-selenium ITS-X (GIBCO), 2 mM Glutamax, 1% penicillin-streptomycin), S3 medium (MCDB 131 medium, 8 mM glucose, 1.23 g / L NaHC03, 2% fatty acid-free BSA, 0.25 mM L-ascorbic acid, 0.5% insulin-transferrin-selenium ITS-X (GIBCO), 2mM Glutamax, 1% penicillin-streptomycin), S4 medium (MCDB 131 medium, 8mM glucose, 1.23g / L NaHC03, 2% fatty acid-free BSA, 0.25mM L-ascorbic acid, 0.002% insulin-transferrin-selenium ITS-X (GIBCO), 2mM Glutamax, 1% penicillin-streptomycin, 10μg / ml heparin, 10μM zinc sulfate), S5 medium (MCDB 131 medium or BLAR medium, 20mM glucose, 1.754g / L NaHC03, 2% fatty acid-free BSA, 0.25mM L-ascorbic acid, 0.002% insulin-transferrin-selenium ITS-X (GIBCO), 2 mM Glutamax, 1% penicillin-streptomycin). For 3D culture, hiPSCs or hESCs were cultured in 3DKG Stem TeSR™ basal medium with 10 μM Y-27632 for 5-7 days, and then the medium was replaced with the respective differentiation medium with 0.015% Kelcogel and 0.3% methylcellulose.

[0215] Generation of 3D pancreatic islet buds in vitro: islet-like organoids in Matrigel by co-culture with hADSCs and HUVECs Primary HUVECs and human adipose-derived stem cells (hADSCs) (Invitrogen or PromoCell) were cultured in 15 cm dishes in EBM medium (Ronza, cc-3121) or MesenProRS™ medium (GIBCO, 12747-010, or Adipose Progenitor Cell Growth Medium Kit, C-27417), respectively, at 37 °C in a humidified 5% CO 2 The cells were cultured at 37°C in an incubator. For co-culture experiments, human iPSC-derived pancreatic endocrine precursor cells were treated with Accutase, HUVECs and hADSCs were treated with TrypLE (GIBCO, 12604-013), and the cells were collected in 50 ml tubes, respectively. After counting the cells, 1x10 cells were collected. 6 hiPS-PP, cells 7x10 6 1 x HUVECs, and 1-2 x 10 cells 5 hADSCs were co-cultured in one well of a 24-well plate with 300 ul of MATRIGEL® matrix. For the scalable generation of human islet-like organoids, 1x10 cells were cultured. 6 hiPS-PPs (days 15-21), 7x10 cells 6 1 x HUVECs, and 1-2 x 10 cells 5hADSCs were co-cultured in 3DKG custom TeSR® medium with 10 uM forskolin (Sigma), 10 μM dexamethasone (Stemgent), 10 uM TGFβ RI kinase inhibitor II / Alk5 inhibitor II (Calbiochem or Enzo), 10 uM nicotinamide (Sigma), 1 uM 3,3′,5-triiodo-L-thyronine sodium salt (T3), and 1% B27 supplement, R428 (2 μM), zinc sulfate (10 μM), and N-Cys (1 mM) (Method 1) or in differentiation medium (S5) with 100 nM LDN193189, 100 nM gamma-secretase inhibitor XX GSiXX (Millipore), 10 μM Alk5 inhibitor II, 1 μM T3 for 7 days. The medium was then replaced with differentiation medium (S5) with 10 μM Trolox (Calbiochem), 2 μM R428 (Selleckchem), 1 mM N-acetylcysteine, 10 μM Alk5 inhibitor II, and 1 μM T3 for an additional 7–20 days (Method 2). The mixed cells formed spherical, islet-like clusters within a few days. The medium was changed every other day.

[0216] Generation of 3D (three-dimensional) pancreatic islet buds in vitro: Islet-like organoids in expandable gellan gum by co-culture with hADSCs and HUVECs Cells were prepared as described above. Briefly, 1x10 cells were 8 hiPS-PP, cells 2-7x10 7 10 HUVECs, and 5-7x10 cells 6hADSCs were co-cultured in 60-100 ml of 3DKG custom TeSR™ with 10 μM forskolin (Sigma), 10 μM dexamethasone (Stemgent), 10 μM TGFβ RI kinase inhibitor II / Alk5 inhibitor II (Calbiochem or Enzo), 10 μM nicotinamide (Sigma), 1 μM 3,3',5-triiodo-L-thyronine sodium salt (T3), and 1% B27 supplement, R428 (2 μM), zinc sulfate (10 μM), and N-Cys (1 mM) (Method 1) or in differentiation medium (S5) with 100 nM LDN193189, 100 nM γ-secretase inhibitor XX GSiXX (Millipore), 10 μM Alk5 inhibitor II, 1 μM T3 for 7 days. The medium was then replaced with differentiation medium (S5) with 10 μM Trolox (Calbiochem), 2 μM R428 (Selleckchem), 1 mM N-acetylcysteine, 10 μM Alk5 inhibitor II, and 1 μM T3 for an additional 7–20 days (Method 2). The mixed cells formed spherical, islet-like clusters within a few days. The medium was changed daily or every other day.

[0217] Generation of 3D (three-dimensional) pancreatic islet buds in vitro: Islet-like organoids in an expandable gellan gum 3D culture method without (w / o) hADSCs and HUVECs Human PSCs, including iPSCs or ESCs, were first cultured in Matrigel-coated plates (two-dimensional (2D) culture). Cells were then treated with Accutase (Innovative Cell Technologies, Inc., San Diego, CA) to create a single cell suspension, washed with PBS, and centrifuged at 1000–1300 rpm for 5 min to pellet the cells. Cells were suspended in 3DKG Stem TeSR™ basal medium (Stemcell Technologies, Cambridge, MA) with 10 μM Y-27632 (a RHO / ROCK pathway inhibitor compound) and cultured for an additional 5–7 days until PSC sphere growth reached a diameter of 50–100 μm. The medium was then replaced with differentiation medium supplemented with 0.015% Kelcogel and 0.3% methylcellulose. Culture medium was changed to differentiation medium (S1) containing 100 ng / ml human activin (R&D Systems), 25 ng / ml recombinant human Wnt3a (R&D Systems) or 3 μM CHIR99021, a glycogen synthase kinase GSK-3 inhibitor (Axon Medchem, Reston, VA; or Selleckchem) for 1 day, then changed to differentiation medium (S1) containing 100 ng / ml human activin for an additional 2 days (stage 1, pancreatic endoderm). The medium was then replaced with differentiation medium (S2) containing 50ng / ml FGF7 (R&D Systems) for 2 days, followed by differentiation medium (S3) containing 50ng / ml FGF7, 0.25uM SANT-1 (Sigma), 1uM retinoic acid (Sigma), 100nM LDN193189 (ALK2 and ALK3 inhibitor, Sigma), and 100nM α-amyloid precursor protein regulator TPB for 3 days. This medium was then replaced with differentiation medium (S4) containing 0.25uM SANT-1, 50nM retinoic acid, 10uM Alk5 inhibitor II, and 1uM T3 for 3 days.The medium was then replaced with differentiation medium (S5) containing 100 nM LDN193189, 100 nM γ-secretase inhibitor XX GSiXX (Millipore), 10 μM Alk5 inhibitor II, and 1 μM T3 for 7 days, followed by differentiation medium (S5) containing 10 μM Trolox (Calbiochem), 2 μM R428 (Selleckchem), 1 mM N-acetylcysteine, 10 μM Alk5 inhibitor II, and 1 μM T3 for an additional 7–20 days. After insulin gene expression was confirmed by either reporter expression or qPCR (typically on days 20–30), the medium was replaced with differentiation medium (S5) containing 10 μM Trolox (Calbiochem), 2 μM R428 (Selleckchem), 1 mM N-acetylcysteine, 10 μM Alk5 inhibitor II, 1 μM T3, and 100 ng / ml recombinant human (rh)Wnt4 (R&D Systems), 400 ng / ml rhWnt5a, or 50% Wnt5a-conditioned medium for 1–20 days. Wnt5a-conditioned medium was prepared by culturing the L-Wnt5a cell line (ATCC, CRL-2814) in DMEM with 10% FBS, 1% penicillin-streptomycin for 4 days after the cells reached 70–100% confluence in T175-T225 Frasko cell culture flasks.

[0218] Generation of 3D (three-dimensional) liver buds in vitro: organ primordia Human iPSC-derived hepatocytes (hiPSC-HE) were prepared using a previously described differentiation method. Briefly, hiPSCs were cultured on MATRIGEL® (BD)-coated dishes in complete STEMCELL™ TeSR™ medium at 37 °C in a humidified 5% CO atmosphere. 2They were maintained in an incubator at 37°C. For liver differentiation, hiPSCs (90% confluence in 6 wells) were cultured with 100ng / ml human activin (Sigma), and 25ng / ml recombinant human Wnt3a (R&D Systems) or 3μM CHIR99021 in RPMI1640 medium, and 1% insulin-free B27 supplement for 1 day, then with 100ng / ml human activin and 1% insulin-free B27 supplement in RPMI medium for another 4 days (stage 1 liver-endoderm). The medium was then replaced with differentiation medium with 10ng / ml bFGF, 20ng / ml BMP4, and 1% B27 supplement in RPMI1640 medium for 3 days (stage 2). The medium was then replaced with differentiation medium with 0.1 μM dexamethasone, 20 ng / ml oncostatin M (R&D Systems), and 10–20 ng / ml hepatic growth factor (HGF, R&D Systems) in hepatocyte culture medium (Lonza, MD, CC-3198, minus EGF and gentamicin / amphotericin-B) and 1% B27 supplement for 4–22 days (days 15–19, pancreatic endocrine progenitor cells). Medium was replaced daily (stage 1) or every other day (stages 2 and 3). Primary HUVEC cells and human adipose-derived stem cells (hADSCs) (Invitrogen or PromoCell) were cultured in 15 cm dishes in EBM medium (Ronza, cc-3121) or MesenProRS medium (GIBCO, 12747-010, or Adipose Progenitor Cell Growth Medium Kit, C-27417), respectively, at 37 °C in a humidified 5% CO 2 The cells were cultured at 37°C in an incubator. For co-culture experiments, the 10-day hepatocytes derived from human iPSCs were treated with Accutase, and the HUVECs and hADSCs were treated with TrypLE (GIBCO, 12604-013), and the cells were collected in 50 ml tubes, respectively. After counting the cells, 1x10 cells were collected. 6 hiPS-PP, cells 7x10 6 1 x HUVECs, and 1-2 x 10 cells 5hADSCs were co-cultured in one well of a 24-well plate with 300 ul of Matrigel. Liver-like organoids were formed within 1-2 days. The liver-like organoids were then removed from the MATRIGEL® matrix and cultured in a 3DKG custom TeSR™.

[0219] Creating 3D heart buds in vitro: organ primordia Cardiomyocytes (hiPSC-CDs) were prepared from human iPSCs using a previously described differentiation method. Briefly, hiPSCs were cultured on MATRIGEL® (BD)-coated dishes in complete Stemcell™ TeSR™ medium at 37 °C in a humidified 5% CO atmosphere. 2 They were maintained at 37°C in an incubator. For hepatic differentiation, hiPSCs (90% confluence in 6 wells) were cultured with 100ng / ml human activin (R&D Systems) and 10uM CHIR99021 and 1% insulin-free B27 supplement in RPMI1640 medium for 1 day, then with 1% insulin-free B27 supplement in RPMI medium for another 2 days (stage 1 heart-mesoderm). The medium was then replaced with RPMI1640 medium with 5uM IWP-2 and 1% insulin-free B27 supplement in RPMI medium for 1 day (stage 2). The medium was then replaced with RPMI medium with 1% insulin-free B27 supplement for 6 or more days (stage 3). Cardiac contractions began around day 13. The medium was replaced daily (stage 1) or every other day (stages 2 and 3). Primary HUVEC cells and human adipose-derived stem cells (hADSCs) (Invitrogen or PromoCell) were cultured in 15 cm dishes in EBM medium (Ronza, cc-3121) or MesenProRS™ medium (GIBCO, 12747-010, or Adipose Progenitor Cell Growth Medium Kit, C-27417), respectively, at 37 °C in a humidified 5% CO 2The cells were cultured at 37°C in an incubator. For co-culture experiments, human iPSC-derived cardiomyocytes from days 13 to 15 were treated with dispase, HUVECs and hADSCs were treated with TrypLE (GIBCO, 12604-013), and the cells were collected in 50 ml tubes, respectively. After counting the cells, 1x10 cells were collected. 6 hiPS-PP, cells 7x10 6 1 x HUVECs, and 1-2 x 10 cells 5 hADSCs were co-cultured in 3DKG custom TeSR™ medium. Contractile heart-like mini-organs were formed within a few days.

[0220] Generation of 3D intestine buds in vitro: organ primordia Intestinal cells (hiPSC-IT) were prepared from human iPSCs using a previously described differentiation method. Briefly, hiPSCs were cultured on Matrigel® (BD)-coated dishes in complete Stemcell™ TeSR™ medium at 37 °C in a humidified 5% CO atmosphere. 2They were maintained in an incubator at 37°C. For hepatic differentiation, hiPSCs (90% confluence in 6 wells) were cultured with 100ng / ml human activin (R&D Systems), 3uM CHIR99021, 2mM Glutamax, and 1% insulin-free B27 supplement in RPMI1640 medium for 1 day, then cultured with 100ng / ml human activin (R&D Systems), 2mM Glutamax, and 1% insulin-free B27 supplement in RPMI1640 medium for an additional 3 days (stage 1 foregut-endoderm). The medium was then replaced with RPMI 1640 medium containing 500ng / ml Wnt3a, 500ng / ml FGF4, and 1% B27 supplement for 4 days (stage 2). The cells were transferred to Matrigel® matrix, and then 3D-spheroid Matrigel® dorms were created at the bottom of 24-well plates. The medium was then replaced with DMEM / F12 medium containing 1% B27 supplement, 1% N2 supplement, 500ng / ml R-spondin, 100ng / ml noggin, 50ng / ml EGF, 2mM Glutamazx™ supplement, 10uM HEPES for 7 days or more (stage 3). Intestinal-like organoid spheroids were observed within one week. The medium was replaced every day (stage 1) and every other day (stages 2 and 3). Primary HUVEC cells and human adipose-derived stem cells (hADSCs) (Invitrogen or PromoCell) were cultured in 15 cm dishes in EBM medium (Ronza, cc-3121) or MesenProRS™ medium (GIBCO®, 12747-010, or Adipose Progenitor Cell Growth Medium Kit, C-27417), respectively, at 37 °C in a humidified 5% CO 2 The cells were cultured at 37°C in an incubator. For co-culture experiments, human iPSC-derived intestinal progenitor cells (day 7) were treated with Accutase, HUVECs and hADSCs were treated with TrypLE (GIBCO®, 12604-013), and the cells were collected in 50 ml tubes, respectively. After counting the cells, 1x10 cells were collected. 6 hiPS-PP, 7x10 6HUVEC cells, and 1-2x10 5 hADSC cells were co-cultured in 3DKG custom TeSR™ medium.

[0221] Insulin secretion assay (mouse and human primary islets and human iPSC-derived cells) Insulin release from intact islets was monitored using a batch incubation method (Yoshihara et al., 2010, Nat. Commun. 1:127). Briefly, isolated islets cultured overnight (RPMI-1640 supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) antibacterial and antimycotic (Gibco)) were preincubated for 30 min at 37°C in 129.4 mM NaCl, 3.7 mM KCl, 2.7 mM CaCl 2 , 1.3 mM KH 2 PO 4 , 1.3 mM MgSO 4 , 24.8 mM NaHCO 3 (5% CO 2 , 95% O 2 Krebs-Ringer bicarbonate buffer (KRBB) (KRBH) containing 0.2% (v / v) BSA (V fraction, Sigma) equilibrated with 0.5% CO, pH 7.4), 10 mM HEPES, and 0.2% (v / v) BSA (V fraction, Sigma) and 3 mM glucose. To determine insulin secretion levels, islets were then incubated in KRBH buffer with 3 mM or 20 mM glucose (500 μl / 10 islets). After 30 min, islets were pelleted by centrifugation and insulin levels were determined by ELISA (Rat / Mouse Insulin ELISA Kit (Millpore) and Human Insulin ELISA Kit (Millpore) for mouse and human islets, respectively). For human iPSC-derived cells, cells (1x10 cells per well of a 24-well plate) were incubated in KRBH buffer with 3 mM or 20 mM glucose (500 μl / 10 islets). 6) were precultured in 3 mM glucose KRBH buffer (500 μl / well). Then, to determine c-peptide secretion levels as an index of insulin secretion levels, cells were incubated in KRBH with 3 mM or 20 mM glucose (200 μl / well). After 30 min, cells were pelleted by centrifugation and c-peptide levels were determined by human c-peptide ELISA kit (Millpore).

[0222] Quantitative RT-PCR analysis Total RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy kit (Qiagen). Reverse transcription was performed using SuperScript III First Strand Synthesis System kit (Invitrogen) or PrimeScript RT Reagent kit (TAKARA). Real-time quantitative RT-PCR (qPCR) was performed using SYBR Green (Bio-Rad).

[0223] Lentivirus generation for Proinsulin-NanoLuc Proinsulin-NanoLuc in pLX304 (Addgene, #62057) was obtained from Addgene. Proinsulin-NanoLuc lentivirus was generated using a second generation viral packaging system. Briefly, 14 μg of proinsulin-NanoLuc, 6.6 μg of PsPAX2 packaging plasmid (Addgene 12260), 5.4 μg of pMD2.G envelope plasmid (Addgene 12259), and 54 μl Lipofectamin2000 (Invitrogen) were used to transfect HEK293LTV packaging cells in a T75 flask. 24 hours after transfection, the medium was replaced with fresh DMEM with 10% FBS and 1% penicillin / streptozocin. Viruses were harvested 48 and 96 hours after transfection, on days 1 and 3, respectively, and passed through a 0.2 μm cellulose acetate filter (VWR). Viruses were aliquoted and stored frozen at −80°C until use.

[0224] Gaussia luciferase assay for measuring insulin secretion Mouse islets, human islets, and human islet-like organoids were plated in their respective growth medium with 10 μg / ml Polybrene® polymer (Santacruz). Virus was then added. After overnight culture, cells were placed in fresh growth medium. 48-72 hours after infection, mouse islets, human islets, and organoids for human islets were manually picked and then placed into 96-wells as single islets or organoids. Insulin secretion assays were then performed. Briefly, single islets or organoids were pre-incubated with 3 mM glucose KRBB at 37°C for 30 min to 1 h. Cells were then incubated in KRBB with 3 mM (100 μl / well) for 30 min, then sequentially incubated with 20 mM glucose with or without 100 nM exendin-4, or 3 mM glucose with 20 mM KCl (100 μl / well). To determine Gaussia luciferase activity as an indicator of insulin secretion levels, 10 μl of the sample was used for luciferase assay using the Pierce Gaussia Luciferase Flash Assay Kit (Prod#16159, Thermo Scientific).

[0225] INS-1 cells were infected with the virus by spinfection (800g, 1 h, 37°C) and then replaced with fresh INS-1 growth medium. 72 h after transfection, INS-1 cells were treated with 5 μg / ml blasticidin (Invitrogen) for 7 days to select for proinsulin-NanoLuc expressing cells. For insulin secretion assay, cells (5x10 cells per well of a 96-well plate) were cultured in 100% PBS. 4 ~1x10 5) were pre-cultured in 3 mM glucose KRBB (100 μl / well). The cells were then incubated in KRBB with 3 mM (100 μl / well) and then successively incubated with 20 mM glucose with or without 100 nM exendin-4, or 3 mM glucose with 20 mM KCl (100 μl / well). To determine Gaussia luciferase activity as an indicator of insulin secretion levels, 10 μl of the sample was used for luciferase assay using Pierce Gaussia luciferase Flash Assay Kit (Prod#16159, Thermo Scientific).

[0226] In vitro angiogenesis assay Human islet-like organoids were embedded in one well of a 24-well plate using 300 μl of Matrigel® matrix with EBM medium (Ronza, cc-3121). Vascularization was observed within 24-72 hours.

[0227] 3D culture of hADSCs and WNT protein expression During spontaneous self-organization in 3D culture, hADSCs undergo changes in the expression of Wnt genes, particularly genes in the Wnt5a pathway (Figure 11A). Wnt5a was found to be the predominant Wnt protein expressed in hADSC 3D cultures over time (Figure 11B).

[0228] Example 5: Wnt proteins in the metabolic maturation of iPSC-derived islet organoids Fltp and Esrrg genes were found to be expressed in iPSC-derived islet organoids (day 21, generated in the absence of co-culture with hADSCs and HUVECs) after treatment with PBS, WNT3a (500 ng / ml), recombinant human (rh) WNT4 (100 ng / ml), or rhWNT5a (400ng / ml) for 5 days (Figure 12A). As shown in Figure 12B, Esrrg gene expression was induced in hiPSC-derived islet organoids generated in the absence of supporting hADSCs and HUVECs in response to increasing doses of rhWNT4 (0, 10, 25, 50, 100, 200 ng / ml) and rhWNT5a (0, 25, 50, 100, 200, 400 ng / ml). In addition, mitochondrial genes involved in oxidative phosphorylation (Cox7a2, Ndufal, Ndufa7), lactate dehydrogenase (Ldha), and Fltp (an effector and reporter gene for Wnt / planar cell polarity (PCP)) were also induced in response to increasing doses of rhWNT4 (0, 10, 25, 50, 100, 200 ng / ml) and rhWNT5a (0, 25, 50, 100, 200, 400 ng / ml) in hiPSC-derived islet organoids generated in the absence of supporting hADSCs and HUVECs (Figure 12C). The levels of mitochondria (Mitotracker; Mito-Red) and insulin (Insulin-GFP) were increased in hiPSC-derived islet organoids (day 27) after treatment with PBS or WNT4 (100 ng / ml) for 8 days (Figure 12D). Human iPSC-derived islet organoids (day 27) were generated after 8 days of treatment with PBS or WNT4 (100 ng / ml) (Figure 12E). Insulin production was found in hiPSC-derived islet organoids (day 27) after 8 days of treatment with rhWNT4 (100 ng / ml), rhWNT5a (400 ng / ml), or conditioned medium from fibroblasts secreting WNT5a (50%) compared to PBS and control fibroblast conditioned medium (50%) (Figures 12F-12H).Human iPSC (hiPSC)-derived islet organoids treated with rhWnt4 (100 ng / ml) for 12 days (day 22) exhibited functional maturation based on secretion of human c-peptide measured in response to low glucose levels (3 mM, "G3mM"), high glucose levels (20 mM, "G20mM"), or high KCl levels (20 mM, "KCL20mM") (Figure 13).

[0229] Other Aspects From the foregoing description it will be apparent that the invention described herein can be adapted for use in a variety of applications and conditions, and such embodiments are also within the scope of the following claims.

[0230] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0231] All patents and publications cited in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0232] Sequence information SEQUENCE LISTING <110> SALK INSTITUTE FOR BIOLOGICAL STUDIES <120> COMPOSITIONS AND METHODS FOR ORGANOID GENERATION AND DISEASE MODELING <150> US 62 / 341,461 <151> 2016-05-25 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 609 <212> PRT <213> Homo sapiens <400> 1 Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr 1 5 10 15 Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30 Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45 Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60 Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp 65 70 75 80 Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu 85 90 95 Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp 100 105 110 Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His 115 120 125 Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro 130 135 140 Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn 145 150 155 160 Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175 Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190 Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200 205 Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys 210 215 220 Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val 225 230 235 240 Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln 245 250 255 Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly 260 265 270 Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285 Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300 Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp 305 310 315 320 Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp 325 330 335 Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala 340 345 350 Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser 355 360 365 Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys 370 375 380 Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu 385 390 395 400 Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415 Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430 Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440 445 Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu 450 455 460 Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile 465 470 475 480 Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly 485 490 495 Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525 Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540 Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys 545 550 555 560 Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser 565 570 575 Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe 580 585 590 Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly 595 600 605 Val <210> 2 <211> 2057 <212> DNA <213> Homo sapiens <400> 2 atattgtgct tccaccactg ccaataacaa aataactagc aaccatgaag tgggtggaat 60 caattttttt aatttccta ctaaatttta ctgaatccag aacactgcat agaaatgaat 120 atggaatagc ttccatattg gattcttacc aatgtactgc agagataagt ttagctgacc 180 tggctaccat atttttgcc cagtttgttc aagaagccac ttacaaggaa gtaagcaaaa 240 tggtgaaaga tgcattgact gcaattgaga aacccactgg agatgaacag tcttcagggt 300 gttagaaaa ccagctacct gcctttctgg aagaactttg ccatgagaaa gaaatttttgg 360 agaagtacgg acattcagac tgctgcagcc aaagtgaaga gggaagacat aactgttttc 420 ttgcacacaa aaagcccact ccagcatcga tcccacttttt ccaagttcca gaacctgtca 480 caagctgtga agcatatgaa gaagacaggg agacattcat gaacaaattc attatgaga 540 tagcaagaag gcatcccttc ctgtatgcac ctacaattct tctttgggct gctcgctatg 600 aaaataat tccatcttgc tgcaagctg aaaatgcagt tgaatgctc caacaagg 660 cagcacagt tashaaagaa ttaagaaa gcagcttgtt aaatcaacat gcatgtgcag 720 taatgaaaaa ttttgggacc cgaactttcc aagccataac tgttactaaa ctgagtcaga 780 agtttaccaa agttaatttt actgaaatcc agaaactagt cctggatgtg gcccatgtac 840 atgagcactg ttgcagagga gatgtgctgg attgtctgca ggatggggaa aaatcatgt 900 cctacatatg ttctcaacaa 960 ccacgctgga acgtggtcaa tgtataattc atgcagaaaa tgatgaaaaa cctgaaggtc 1020 tatctccaaa tctaaacagg tattttaggag atagagattt taaccaattt tctcagggg aaaaaaatat cttcttgca agttttgttc atgaatattc agagacat cctcagcttg 1140 ctgtctcagt aattctaaga gttgctaag gataccagga gttattggag aagtgtttcc 1200 agactgaaaa ccccttgaa tgccaagata aaggagaga agattacag aaatacatcc 1260 aggagagcca agcattggca aagcgaagct gcggccctt ccagaaacta ggagaatatt 1320 acttacaaaa tgcgtttctc gttgcttaca caaagaaagc cccccagctg acctcgtcgg 1380 agctgatggc catcaccaga aaaatggcag ccacagcagc cacttgttgc caactcagtg 1440 aggacaaact attggcctgt ggcgagggag cggctgacat tattatcgga cacttatgta 1500 tcagacatga aatgactcca gtaaaccctg gtgttggcca gtgctgcact tcttcatatg 1560 ccaacaggag gccatgcttc agcagcttgg tggtggatga aacatatgtc cctcctgcat 1620 tctctgatga caagttcatt ttccataagg atctgtgcca agctcagggt gtagcgctgc 1680 aaacgatgaa gcaagagttt ctcattaacc ttgtgaagca aaagccacaa ataacagagg 1740 aacaacttga ggctgtcatt gcagatttct caggcctgtt ggagaaatgc tgccaaggcc 1800 aggaacagga agtctgcttt gctgaagagg gacaaaaact gatttcaaaa actcgtgctg 1860 ctttgggagt ttaaattact tcaggggaag agaagacaaa acgagtcttt cattcggtgt 1920 gaacttttct cttaatttt aactgattta acactttttg tgaattaatg aaatgataaa 1980 gacttttatg tgagatttcc ttatcacaga aataaaatat ctccaaatgt ttccttttca 2040 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA It It It takes 2057 <210> 3 <211> 609 <212> PRT <213> Homo sapiens <400> 3 Met Lys Trp Val Thr Phe Ile Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Thr Thr Thr Thr Thr Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 595 600 605<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 2264<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agtatattag tgctaatttc cctccgtttg tcctagcttt tctcttctgt caaccccaca 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgcctttggc acaatgaagt gggtaacctt tatttccctt ctttttctct ttagctcggc 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttattccagg ggtgtgtttc gtcgagatgc acacaagagt gaggttgctc atcggtttaa 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agatttggga gaagaaaatt tcaaagcctt ggtgttgatt gcctttgctc agtatcttca 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcagtgtcca tttgaagatc atgtaaaatt agtgaatgaa gtaactgaat ttgcaaaaac 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atgtgttgct gatgagtcag ctgaaaattg tgacaaatca cttcataccc tttttggaga 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> caaattatgc acagttgcaa ctcttcgtga aacctatggt gaaatggctg actgctgtgc 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaaacaagaa cctgagagaa atgaatgctt cttgcaacac aaagatgaca acccaaacct 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cccccgattg gtgagaccag aggttgatgt gatgtgcact gcttttcatg acaatgaaga 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gacatttttg aaaaaatact tatatgaaat tgccagaaga catccttact tttatgcccc 600<h2 style=";text-align:left;direction:ltr"> ggaactcctt ttctttgcta aaaggtataa agctgctttt acagaatgtt gccaagctgc 660 tgataaagct gcctgcctgt tgccaaagct cgatgaactt cgggaatgaag ggaaggcttc 720 gtctgccaaa cagagactca agtgtgccag tctccaaaaa tttggagaaa gagctttcaa 780 agcatgggca gtagctcgcc tgagccagag attcccaaa gctgagtttg cagaagtttc 840 900 atgtgctgat gacagggcgg accttgccaa gtatatctgt gaaaatcaag attcgatctc 960 footaactg aaaggaatgct gtgaaaaacc tctgttggaa aaatcccact gcattgccga 1020 agtggaaaat gatgagatgc ctgctgactt gccttcatta gctgctgatt ttgttgaaag 1080 taggatgtt tgcaaaaact atgctgaggc aaaggatgtc ttcctgggca tgttttgta 1140 tgaatatgca agaaggcatc ctgattactc tgtcgtgctg ctgctgagac ttgccaagac 1200 atatgaaacc actctagaga agtgctgtgc cgctgcagat cctcatgaat gctatgccaa 1260 1320 tgagctttt gagcagcttg gagagtaca attccagaat gcgctattag ttcgttacac 1380 cagaaagta cccaagtgt caacccaac tcttgtagag gtctcagaa acctaggaaa 1440 agtgggcagc aaatgttgta aacatcctga agcaaaaga atgccctgtg cagaagacta 1500 tctatccgtg gtcctgaacc agttatgtgt gttgcatgag aaacgccag taagtgacag 1560 agtcaccaaa tgctgcacag aatccttggt gaacaggcga ccatgctttt cagctctgga 1620 agtcgatgaa acatacgttc ccaagagtt taatgctgaa acatcacct tccatgcaga 1680 tatatgcaca ctttctgaga aggagaca aatcaagaa CAACGCAC ttgttgagct 1740 cgtgaaacac aagcccaagg ccaaaaa gcaactgaa gctgttatgg atgatttcgc 1800 agcttttgta gagaagtgct gcaggctga cgataggag acctgctttg ccgaggaggg 1860 taaaaaactt gttgctgcaa gtcaagctgc cttaggctta taacacaca ttaaagca 1920 tctcagccta ccatgagaat agagaaaga aaatgaagat caagctta ttcatctgtt 1980 tttcttttc gttggtgtaa agccaacc ctgtctaaaaaacataatt tctttaatca 2040 <h2 style=";text-align:left;direction:ltr">ttttgcctct tttctctgtg cttcaattaa taaaaaatgg aaagaatcta atagagtggt 2100<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acagcactgt tatttttcaa agatgtgttg ctatcctgaa aattctgtag gttctgtgga 2160<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agttccagtg ttctctctta ttccacttcg gtagaggatt tctagtttct tgtgggctaa 2220<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttaaataaat cattaatact cttctaaaaaaaaaaaaaaaaaaa 2264<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 313<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Tyr Val Ser Tyr Leu Leu Asp Lys Asp Val Ser Met Tyr Pro Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Val Arg His Ser Gly Gly Leu Asn Leu Ala Pro Gln Asn Phe Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Pro Pro Gln Tyr Pro Asp Tyr Gly Gly Tyr His Val Ala Ala Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Ala Ala Ala Ala Asn Leu Asp Ser Ala Gln Ser Pro Gly Pro Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Trp Pro Ala Ala Tyr Gly Ala Pro Leu Arg Glu Asp Trp Asn Gly Tyr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 65 70 75 80<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Pro Gly Gly Ala Ala Ala Ala Ala Asn Ala Val Ala His Gly Leu 85 90 95 Asn Gly Gly Ser Pro Ala Ala Ala Met Gly Tyr Ser Ser Pro Ala Asp 100 105 110 Tyr His Pro His His His Pro His His His Pro His His Pro Ala Ala 115 120 125 Ala Pro Ser Cys Ala Ser Gly Leu Leu Gln Thr Leu Asn Pro Gly Pro 130 135 140 Pro Gly Pro Ala Ala Thr Ala Ala Ala Glu Gln Leu Ser Pro Gly Gly 145 150 155 160 Gln Arg Arg Asn Leu Cys Glu Trp Met Arg Lys Pro Ala Gln Gln Ser 165 170 175 Leu Gly Ser Gln Val Lys Thr Arg Thr Lys Asp Lys Tyr Arg Val Val 180 185 190 Tyr Thr Asp His Gln Arg Leu Glu Leu Glu Lys Glu Phe His Tyr Ser 195 200 205 Arg Tyr Ile Thr Ile Arg Arg Lys Ala Glu Leu Ala Ala Thr Leu Gly 210 215 220 Leu Ser Glu Arg Gln Val Lys Ile Trp Phe Gln Asn Arg Arg Ala Lys 225 230 235 240 <h2 style=";text-align:left;direction:ltr">Glu Arg Lys Ile Asn Lys Lys Lys Leu Gln Gln Gln Gln Gln Gln Gln<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 245 250 255<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Pro Pro Gln Pro Pro Pro Pro Pro Pro Pro Gln Pro Pro Gln Pro Gln Pro<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 260 265 270<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Pro Leu Arg Ser Val Pro Glu Pro Leu Ser Pro Val Ser Ser Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 275 280 285<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gln Ala Ser Val Ser Gly Ser Val Pro Gly Val Leu Gly Pro Thr Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 290 295 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Val Leu Asn Pro Thr Val Thr Gln<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 305 310<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 2370<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctccaaccat tggtgtctgt gtcattacta atagagtctt gtaaacactc gttaatcacg 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaaggccgcc ggcctggggc tccgcacgcc agcctgtggc gggtcttccc cgcctctgca 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcctagtggg aaggaggtgg gaggaaagaa ggaagaaagg gagggaggga ggaggcaggc 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagagggagg gaccgcctcg gaggcagaag agccgcgagg agccagcgga gcaccgcggg 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctggggcgca gccacccgcc gctcctcgag tcccctcgcc cctttccctt cgtgcccccc 300 ggcagcctcc agcgtcggtc cccaggcagc atggtgaggt ctgctcccgg accctcgcca 360 ccatgtacgt gagctacctc ctggacaagg acgtgagcat gtaccctagc tccgtgcgcc 420 actctggcgg cctcaacctg gcgccgcaga acttcgtcag ccccccgcag tacccggact 480 acggcggtta ccacgtggcg gccgcagctg cagcggcagc gaacttggac agcgcgcagt 540 ccccggggcc atcctggccg gcagcgtatg gcgccccact ccgggaggac tggaatggct 600 acgcgcccgg aggcgccgcg gccgccgcca acgccgtggc tcacggcctc aacggtggct 660 ccccggccgc agccatgggc tacagcagcc ccgcagacta ccatccgcac caccacccgc 720 atcaccaccc gcaccacccg gccgccgcgc cttcctgcgc ttctgggctg ctgcaaacgc 780 tcaaccccgg ccctcctggg cccgccgcca ccgctgccgc cgagcagctg tctcccggcg 840 gccagcggcg gaacctgtgc gagtggatgc ggaagccggc gcagcagtcc ctcggcagcc 900 aagtgaaaac caggacgaaa gacaaatatc gagtggtgta cacggaccac cagcggctgg 960 agctggagaa ggagtttcac tacagtcgct acatcaccat ccggaggaaa gccgagctag 1020 ccgccacgct ggggctctct gagaggcagg ttaaatctg gtttcagac cgcagagcaa 1080 agghaggaaaatcaagg aagaagttgc aggshaaca gagghaggaaaaaccaaaaaaaaaaaaactice cgcctccgcc gccaccacag cctccccagc ctcagccagg tcctctgaga agtgtcccag 1200 agcccttgag tccggtgtct tccctgcaag cctcagtgtc tggctctc cctggggttc 1260 tggggccaac tgggggggtg ctaaacccca ccgtcaccca gtgaccacc gggttctgca 1320 gcggcagagc aattccaggc tgagccatga ggagcgtgga ctctgctaga ctcctcagga 1380 gagaccccctc ccccccacc cacagccata gacctacaga cctggctc agaggaaaaa 1440 tgggagccag gagtaagaca agtgggattt ggggcctcaa gaaatact ctcccagatt 1500 tttactttt cccatctggc ttttctgcc actgaggaga cagaagcct ccgctggggct 1560 tcattccgga ctggcagaag cattgctgg actgaccaca ccaaccaggc cttcatccctc 1620 ctccccagct cttctctcc tagatctgca ggctgcacct ctggctagag ccgagggg 1680 agagggactc aagggaagg caagctgag gccaagatgg ctgctgcctg ctcatggccc 1740 tcggaggtcc agctgggcct cctgcctccg ggcaggcaag gttacactg cggaagccaa 1800 aggcagctaa gatagaaagc tggactgacc aaagactgca gaacccccag gtggcctgcg 1860 tcttttttct cttcccttcc cagaccagga aaggcttggc tggtgtatgc acagggtgtg 1920 gtatgagggg gtggttattg gactccaggc ctgaccaggg ggcccgaaca gggacttgtt 1980 tagagagcct gtcaccagag cttctctggg ctgaatgtat gtcagtgcta taaatgccag 2040 agccaacctg gacttcctgt cattttcaca atcttggggc tgatgaagaa gggggtgggg 2100 ggagtttgtg ttgttgttgc tgctgtttgg gttgttggtc tgtgtaacat ccaagccaga 2160 gttttaaag ccttctggat ccatgggggg agaagtgata tggtgaaggg aagtggggag 2220 tatttgaaca cagttgaatt ttttctaaaa agaaaaagag ataaatgagc tttccagatt 2280 tcagattctg tattatctt cagattttgt ctgcaactat ttttatttt ttaaagaaat 2340 gaaatatctt caaaaaaaaaaaaaaaaaaa 2370 <210> 7 <211> 503 <212> PRT <213> Homo sapiens <400> 7 Met Asp Leu Ile Pro Asn Leu Ala Val Glu Thr Trp Leu Leu Leu Ala 1 5 10 15 Val Ser Leu Ile Leu Leu Tyr Leu Tyr Gly Thr Arg Thr His Gly Leu 20 25 30 Phe Lys Lys Leu Gly Ile Pro Gly Pro Thr Pro Leu Pro Phe Leu Gly 35 40 45 Asn Ala Leu Ser Phe Arg Lys Gly Tyr Trp Thr Phe Asp Met Glu Cys 50 55 60 Tyr Lys Lys Tyr Arg Lys Val Trp Gly Ile Tyr Asp Cys Gln Gln Pro 65 70 75 80 Met Leu Ala Ile Thr Asp Pro Asp Met Ile Lys Thr Val Leu Val Lys 85 90 95 Glu Cys Tyr Ser Val Phe Thr Asn Arg Arg Pro Phe Gly Pro Val Gly 100 105 110 Phe Met Lys Asn Ala Ile Ser Ile Ala Glu Asp Glu Glu Trp Lys Arg 115 120 125 Ile Arg Ser Leu Leu Ser Pro Thr Phe Thr Ser Gly Lys Leu Lys Glu 130 135 140 Met Val Pro Ile Ile Ala Gln Tyr Gly Asp Val Leu Val Arg Asn Leu 145 150 155 160 Arg Arg Glu Ala Glu Thr Gly Lys Pro Val Thr Leu Lys His Val Phe 165 170 175 Gly Ala Tyr Ser Met Asp Val Ile Thr Ser Thr Ser Phe Gly Val Ser 180 185 190 Ile Asp Ser Leu Asn Asn Pro Gln Asp Pro Phe Val Glu Asn Thr Lys 195 200 205 Lys Leu Leu Arg Phe Asn Pro Leu Asp Pro Phe Val Leu Ser Ile Lys 210 215 220 Val Phe Pro Phe Leu Thr Pro Ile Leu Glu Ala Leu Asn Ile Thr Val 225 230 235 240 Phe Pro Arg Lys Val Ile Ser Phe Leu Thr Lys Ser Val Lys Gln Ile 245 250 255 Lys Glu Gly Arg Leu Lys Glu Thr Gln Lys His Arg Val Asp Phe Leu 260 265 270 Gln Leu Met Ile Asp Ser Gln Asn Ser Lys Asp Ser Glu Thr His Lys 275 280 285 Ala Leu Ser Asp Leu Glu Leu Met Ala Gln Ser Ile Ile Phe Ile Phe 290 295 300 Ala Gly Tyr Glu Thr Thr Ser Ser Val Leu Ser Phe Ile Ile Tyr Glu 305 310 315 320 Leu Ala Thr His Pro Asp Val Gln Gln Lys Val Gln Lys Glu Ile Asp 325 330 335 Thr Val Leu Pro Asn Lys Ala Pro Pro Thr Tyr Asp Thr Val Leu Gln 340 345 350 Leu Glu Tyr Leu Asp Met Val Val Asn Glu Thr Leu Arg Leu Phe Pro 355 360 365 Val Ala Met Arg Leu Glu Arg Val Cys Lys Lys Asp Val Glu Ile Asn 370 375 380 Gly Met Phe Ile Pro Lys Gly Val Val Val Met Ile Pro Ser Tyr Val 385 390 395 400 Leu His His Asp Pro Lys Tyr Trp Thr Glu Pro Glu Lys Phe Leu Pro 405 410 415 Glu Arg Phe Ser Lys Lys Asn Lys Asp Asn Ile Asp Pro Tyr Ile Tyr 420 425 430 Thr Pro Phe Gly Ser Gly Pro Arg Asn Cys Ile Gly Met Arg Phe Ala 435 440 445 Leu Val Asn Met Lys Leu Ala Leu Val Arg Val Leu Gln Asn Phe Ser 450 455 460 Phe Lys Pro Cys Lys Glu Thr Gln Ile Pro Leu Lys Leu Arg Phe Gly 465,470,475,480 Gly Leu Leu Leu Thr Glu Lys Pro Ile Val Leu Lys Ala Glu Ser Arg 485 490 495 Asp Glu Thr Val Ser Gly Ala 500 <210> 8 <211> 2099 <212> DNA <213> Homo sapiens <400> 8 aatcactgct gtgcagggca ggaaagctcc acacacacag cccagcaaac agcagcacgc 60 tgctgaaaaa aagactcaga ggagagagat aaggaaggaa agtagtgatg gatctcatcc 120 caaacttggc cgtggaaacc tggcttctc tgctgtcag cctgatactc ctctatctat 180 atggaacccg tacacatgga cttttaaga agcttggaat tccagggccc acacctctgc 240 cttttttggg aaatgctttg tccttccgta agggctattg gacgtttgac atggaatgtt 300 ataaaaagta tagaaaagtc tggggtattt atgactgtca acagcctatg ctggctatca 360 cagatcccga catgatcaaa acagtgctag tgaaagaatg ttattctgtc ttcacaaacc 420 ggaggccttt cgggccagtg ggatttatga aaaatgccat cctatagct gaggatgaag 480 aatggaagag aatacgatca ttgctgtctc caacattcac cagcggaaaa ctcaaggaga 540 tggtccctat cattgcccag tatggagatg tgttggtgag aaatctgagg cgggaagcag 600 agacaggcaa gcctgtcacc ttgaaacacg tctttggggc ctacagcatg gatgtgatca 660 ctagcacatc atttggagtg agcatcgact ctctcaacaa tccacaagac ccctttgtgg 720 aaaacaccaa gaagctttta agatttaatc cattagatcc attcgttctc tcaataaaag 780 tctttccatt ccttacccca attcttgaag cattaaatat cactgtgttt ccaagaaaag 840 ttataagttt tctaacaaaa tctgtaaaac agataaaaga aggtcgcctc aaagagacac 900 aaaagcaccg agtggatttc cttcagctga tgattgactc tcagaattca aaagactctg 960 agacccacaa agctctgtct gatctggagc tcatggccca atcaattatc tttatttttg 1020 ctggctatga aaccacgagc agtgttctct ccttcattat atatgaactg gccactcacc 1080 ctgatgtcca gcagaaagtg cagaaggaaa ttgatacagt tttacccaat aaggcaccac 1140 ccacctatga tactgtgcta cagttggagt atcttgacat ggtggtgaat gaaacactca 1200 gattattccc agttgctatg agacttgaga gggtctgcaa aaaagatgtt gaaatcaatg 1260 ggatgttat tcccaaaggg gtggtggtga tgattccaag ctatgttctt catcatgacc 1320 caaagtactg gacagagcct gagaagttcc tcctgaaag gttcagtaaa aagaacaagg 1380 acaacataga tccttacata tacacaccct ttggaagtgg acccagaaac tgcattggca 1440 tgaggttgc tctcgtgaac atgaaacttg ctctagtcag agtccttcag aacttctcct 1500 tcaaaccttg taaagaaaca cagatccccc tgaaattacg cttggagga cttcttctaa 1560 cagaaaaacc cattgttcta aaggctgagt caagggatga gaccgtaagt ggagcctgat 1620 ttccctaagg acttctggtt tgctctttaa gaaagctgtg ccccagaaca ccagagacct 1680 caaattactt tacaataga accctgaaat gaagacgggc ttcatccaat gtgctgcata 1740 aataatcagg gattctgtac gtgcattgtg ctctctcatg gtctgtatag agtgttatac 1800 ttggtaatat agaggagatg accaaatcag tgctggggaa gtagatttgg cttctctgct 1860 tctcatagga ctatctccac cacccccagt tagcaccatt aactcctcct gagctctgat 1920 aacatatta acatttctca ataatttca accaatcat taaaaaat aggaattatt 1980 ttgatggctc taacagtgac atttattaca tgtgttatat ctgtagtatt ctatagtaag 2040 ctttatatta agcaatca taaaaacctc tttacaaag taaaaaaaaaaaaaaaaaaaaaaaaaaa 2099 <210> 9 <211> 435 <212> PRT <213> Homo sapiens <400> 9 Met Ser Asn Lys Asp Arg His Ile Asp Ser Ser Cys Ser Phe Ile 1 5 10 15 Lys Thr Glu Pro Ser Ser Pro Ala Ser Leu Thr Asp Ser Val Asn His 20 25 30 His Ser Pro Gly Gly Ser Ser Asp Ala Ser Gly Ser Tyr Ser Ser Thr 35 40 45 Met Asn Gly His Gln Asn Gly Leu Asp Ser Pro Pro Leu Tyr Pro Ser 50 55 60 Ala Pro Ile Leu Gly Gly Ser Gly Pro Val Arg Lys Leu Tyr Asp Asp 65 70 75 80 Cys Ser Thr Ile Val Glu Asp Pro Gln Thr Lys Cys Glu Tyr Met 85 90 95 Leu Asn Ser Met Pro Lys Arg Leu Cys Leu Val Cys Gly Asp Ile Ala 100 105 110 Ser Gly Tyr His Tyr Gly Val Ala Ser Cys Glu Ala Cys Lys Ala Phe 115 120 125 Phe Lys Arg Thr with Gln Gly Asn and Glu Tyr Ser Cys Pro Ala Thr 130 135 140 Asn Glu Cys Glu Ile Thr Lys Arg Arg Lys Ser Cys Gln Ala Cys 145 150 155 160 Arg Phe Met Lys Cys Leu Lys Val Gly Met Leu Lys Glu Gly Val Arg 165 170 175 Leu Asp Arg Val Arg Gly Gly Arg Gln Lys Tyr Lys Arg Arg Ile Asp 180 185 190 Glu Asn Pro Tyr Leu Asn Pro Gln Leu Val Gln Pro Ala Lys 195 200 205 Lys Pro Tyr Asn Lys Ile Will Be His Leo Leo Will Have Glu Pro Glu 210 215 220 Lys Ile Tyr Ala Met Pro Asp Pro Thr Val Pro Asp Ser Asp Ile Lys 225 230 235 240 Only Leu Thr Thr Leu Cys Asp Leu Asp Ala Leu Arg Glu Leu Val Val Ile 245 250 255 Ile Gly Trp Ala Lys His Ile Pro Gly Phe Ser Thr Leu Ser Leu Ala 260 265 270 Asp Gln Met Ser Leu Leu Gln Ser Ala Trp Met Glu Ile Leu Ile Leu 275 280 285 Gly Val Val Tyr Arg Ser Leu Ser Phe Glu Asp Glu Leu Val Tyr Ala 290 295 300 Asp Asp Tyr Ile Met Asp Glu Asp Gln Ser Lys Leu Ala Gly Leu Leu 305 310 315 320 Asp Leu Asn Asn Ala Ile Leu Gln Leu Val Lys Lys Tyr Lys Ser Met 325 330 335 Lys Leu Glu Lys Glu Glu Phe Val Thr Leu Lys Ala Ile Ala Leu Ala 340 345 350 Asn Ser Asp Ser Met His Ile Glu Asp Val Glu Ala Val Gln Lys Leu 355 360 365 Gln Asp Val Leu His Glu Ala Leu Gln Asp Tyr Glu Ala Gly Gln His 370 375 380 Met Glu Asp Pro Arg Arg Ala Gly Lys Met Leu Met Thr Leu Pro Leu 385 390 395 400 Leu Arg Gln Thr Ser Thr Lys Ala Val Gln His Phe Tyr Asn Ile Lys 405 410 415 Leu Glu Gly Lys Val Pro Met His Lys Leu Phe Leu Glu Met Leu Glu 420 425 430 Here Lys Val 435 <210> 10 <211> 5559 <212> DNA <213> Homo sapiens <400> 10 aagctccaat cggggcttta agtccttgat taggagagtg tgagagcttt ggtcccaact 60 ggctgtgcct ataggcttgt cactaggaga acatttgtgt taattgcact gtgctctgtc 120 aaggaaactt tgatttatag ctggggtgca caaataatgg ttgccggtcg cacatggatt 180 cggtagaact ttgccttcct gaatcttttt ccctgcacta cgaggaagag tagacttgaa 240 tgagacctgc ctcatcagtc atgggatcat agtgtcacag atggaaaagc aactatcagc 300 tgaattgtac tgaactacac acttggctaa ttcatcttat tgctctacac atctaaagga 360 aggctcattc tgttcttgga gtctagacag catcaggagt tgggctcagt gaacaaaact 420 ttaatgtcta gagcatttat gagggtttta atgattggaa aatctatcct gagaatgtgg 480 tcaccatatg tgacagcctt gctttctatc ttgtcttcag tttctggggc ttctctgcag 540 aatgtcaaac aaagatcgac acattgattc cagctgttcg tccttcatca agacggaacc 600 ttccagccca gcctccctga cggacagcgt caaccaccac agccctggtg gctcttcaga 660 cgccagtggg agctacagtt caaccatgaa tggccatcag aacggacttg actcgccacc 720 tctctaccct tctgctccta tcctgggagg tagtgggcct gtcaggaaac tgtatgatga 780 ctgctccagc accattgttg aagatcccca gaccaagtgt gaatacatgc tcaactcgat 840 gcccaagaga ctgtgtttag tgtgtggtga catcgcttct gggtaccact atggggtagc 900 atcatgtgaa gcctgcaagg cattcttcaa gaggacaatt caaggcaata tagaatacag 960 ctgccctgcc acgaatgaat gtgaaatcac aaagcgcaga cgtaaatcct gccaggcttg 1020 ccgcttcatg aagtgtttaa aagtgggcat gctgaaagaa ggggtgcgtc ttgacagagt 1080 acgtggaggt cggcagaagt acaagcgcag gatagatgcg gagaacagcc catacctgaa 1140 ccctcagctg gttcagccag ccaaaaagcc atataacaag attgtctcac atttgttggt 1200 ggctgaaccg gagagaatct atgccatgcc tgaccctact gtccccgaca gtgacatcaa 1260 agccctcact acactgtgtg acttggccga ccgagagttg gtggttatca ttggatgggc 1320 gaagcatatt ccaggcttct ccacgctgtc cctggcggac cagatgagcc ttctgcagag 1380 tgcttggatg gaaattttga tccttggtgt cgtataccgg tctctttcgt ttgaggatga 1440 acttgtctat gcagcgatt atataatgga cgaagaccag tccaaattag caggccttct 1500 tgatctaaat aatgctatcc tgcagctggt aaagaaatac aagagcatga agctggaaaa 1560 agaagaattt gtcaccctca aagctatagc tcttgctaat tcagactcca tgcacataga 1620 agatgttgaa gccgttcaga agcttcagga tgtcttacat gaagcgctgc aggattatga 1680 agctggccag cacatggaag accctcgtcg agctggcaag atgctgatga cactgccact 1740 cctgaggcag acctctacca aggccgtgca gcatttctac aacatcaaac tagaaggcaa 1800 agtcccaatg cacaaacttt ttttggaaat gttggaggcc aaggtctgac taaaagctcc 1860 ctgggccttc ccatccttca tgttgaaaaa gggaaaataa acccaagagt gatgtcgaag 1920 aaacttagag tttagttaac aacatcaaaa atcaacagac tgcactgata atttagcagc 1980 aagactatga agcagctttc agattcctcc ataggttcct gatgagtttc tttctacttt 2040 ctccatcatc ttctttcctc tttcttccca catttctctt tctctttatt ttttctcctt 2100 ttctctttc acctccctta tttctttgct tctttcattc ctagttccca ttctccttta 2160 tttcttccc gtctgcctgc cttctttcttt ttctttacct actctcattc ctctctttc 2220 tcatccttcc ccttttttct aaatttgaaa tagctttagt ttaaaaaaaa atcctccctt 2280 ccccctttcc tttccctttc tttcctttt cctttcctt ttccctttcc tttccttttcc 2340 tcttgacctt ctttccatct ttctttttct tccttctgct gctgaacttt taaaagaggt 2400 ctctaactga agagagatgg aagccagccc tgccaaagga tggagatcca tatatggat 2460 gccagtgaac ttattgtgaa catactgtc cccaatgact aaggaatcaa agagagagaa 2520 ccaacgttcc taaaagtaca gtgcaacata tacaaattga ctgagtgcag tattagattt 2580 catgggagca gcctctaatt agacaactta agcaacgttg catcggctgc ttcttatcat 2640 tgcttttcca tctagatcag ttacagccat ttgattcctt aattgtttt tcaagtcttc 2700 caggtatttg ttagtttagc tactatgtaa cttttcagg gaatagttta agctttattc 2760 attcatgcaa tactaaagag aaataagaat actgcaattt tgtgctggct ttgaacaatt 2820 acgaacaata atgaaggaca aatgaatcct gaaggaagat ttttaaaaat gttttgtttc 2880 ttcttacaaa tggagattt tttgtaccag cttaccact tttcagccat ttattaatat 2940 gggaatttaa cttactcaag caatagttga agggaaggtg catattatca cggatgcaat 3000 ttatgttgtg tgccagtctg gtcccaaaca tcaatttctt aacatgagct ccagtttacc 3060 taaatgttca ctgacacaaa ggatgagatt acacctacag tgactctgag tagtcacata 3120 tataagcact gcacatgaga tatagatccg tagaattgtc aggagtgcac ctctctactt 3180 gggaggtaca attgccatat gatttctagc tgccatggtg gttaggaatg tgatactgcc 3240 tgtttgcaaa gtcacagacc ttgcctcaga aggagctgtg agccagtatt catttaagag 3300 gcaataaggc aaatgccaga attaaaaaaa aaaatcatca aagacagaaa atgcctgacc 3360 aaattctaaa acctaatcca tataagttta ttcatttagg aatgttcgtt taaattaatc 3420 tgcagtttt accaagagct aagccaatat atgtgctttt caaccagtat tgtcacagca 3480 tgaaagtcaa gtcaggttcc agactgttaa gaggtgtaat ctaatgaaga aatcaattag 3540 atgccccgaa atctacagtc gctgaataac caataaacag taacctccat caaatgctat 3600 accaatggac cagtgttagt agctgctccc tgtattatgt gaacagtctt attctatgta 3660 cacagatgta attaaaattg taatcctaac aaacaaaaga aatgtagttc agcttttcaa 3720 tgttcatgt ttgctgtgct tttctgaatt tttgttgca ttcaaagact gttgtctttgt 3780 tcttgtggtg tttggattct tgtggtgtgt gctttagac acagggtaga attagagaca 3840 atattggatg tacaattcct caggagacta cagtagtata ttctattcct taccagtaat 3900 aaggttcttc ctaataataa ttaagagatt gaaactccaa acaagtattc attatgaaca 3960 gatacacatc aaaatcataa tatattttc aaaacaagga ataatttctc taatggttta 4020 ttatagaata ccaatgtata gcttagaaat aaaactttga atatttcaag aatagata 4080 agtctaattt ttaaatgctg tatatatggc ttcactca tcatctca gatgttgtta 4140 ttaactcgct ctgtgttgtt gcaaaacttt ttggtgcaga tcgtttcca aaactattgc 4200 tactttgtgt gctttaaaca aaataccttg ggttgatgaa acatcaaccc agtgctagga 4260 atactgtgta tctatcatta gctatatggg actatattgt agatgtggt ttctcagtag 4320 agaagtgact gtagtgtgat tctagactaa tcatcattag cattcattc agatggtca 4380 taacttgaaa ttatagctg tgataggagt tcagaaattg gcacatccct ttaaaaataa 4440 siacagaaaa tacaacct gggaaaaag gtgctgattc tataagatta tttatatatg 4500 taagtgttta aaagattat ttccagaaa gtttgtgcag ggtttaagtt gctactattc 4560 aactacacta tatatata aaatatatac atatataca ttgttttcac tgtatcacat 4620 taaagtactt gggcttcaga agtagagcc aaccaactga aaacctgaga tggatatg 4680 ttcaagaat gagatacaat ttttagtt tcagtttaag taactcag cattacaaa 4740 gaggtaagtat ctcacaata ggaaaaaa ctaaaacgtg gatttaaaaa gaactgcacg 4800 ggctttaggg taaatgctca tcttaaacct cactagaggg aagtcttctc aagttttcaag 4860 caagaccatt tacttaatgt gaagttttgg aaagttataa aggtgtatgt tttagccata 4920 tgatttaat tttaatttg cttcttttag gttcgttctt atttaaagca atatgattgt 4980 gtgactcctt gtagttacac ttgtgtttca atcagatcag attgttgtat ttattccact 5040 atttgcatt taaatgataa cataaaagat ataaaaaatt taaaactgct atttttctta 5100 tagaagagaa aatgggtgtt ggtgattgta ttttaattat ttaagcgtct ctgtttacct 5160 gcctaggaaa acattttatg gcagtcttat gtgcaaagat cgtaaaagga caaaaaattt 5220 aaactgctta tataatcca ggagttgcat tatagccagt agtaaaaata ataataataa 5280 tataaaacc atgtctatag ctgtagatgg gcttcacatc tgtaaagcaa tcaattgtat 5340 atttttgtga tgtgtaccat actgtgtgct ccagcaaatg tccatttgtg taaatgtatt 5400 tattttatat tgtatatatt gttaaatgca aaaaggagat atgattctgt aactccaatc 5460 agttcagatg tgtaactcaa attattgc ctttcaggat gatggtagag caatattaaa 5520 caagcttcca cttttgactg ctaaaaaaaaa aaaaaaaaa 5559 <210> 11 <211> 463 <212> PRT <213> Homo sapiens <400> 11 With His Ser Ala Ser Ser With Leu Gly Ala Val Lys With Glu Gly His 1 5 10 15 Glu Pro Ser Asp Trp Ser Ser Tyr Tyr Ala Glu Pro Glu Gly Tyr Ser 20 25 30 Ser Val Ser Asn Met Asn Ala Gly Leu Gly Met Asn Gly Met Asn Thr 35 40 45 Tyr Met Ser Met Ser Ala Ala Ala Met Gly Ser Gly Ser Gly Asn Met 50 55 60 Ser Ala Gly Ser Met Asn Met Ser Ser Tyr Val Gly Ala Gly Met Ser 65 70 75 80 Pro Ser Leu Ala Gly Met Ser Pro Gly Ala Gly Ala Met Ala Gly Met 85 90 95 Gly Gly Ser Ala Gly Ala Ala Gly Val Ala Gly Met Gly Pro His Leu 100 105 110 Ser Pro Ser Leu Ser Pro Leu Gly Gly Gln Ala Ala Gly Ala With Gly 115 120 125 Gly Leu Ala Pro Tyr Ala Asn Met Asn Ser Met Ser Pro Met Tyr Gly 130 135 140 Gln Ala Gly Leu Ser Arg Ala Arg Asp Pro Lys Thr Tyr Arg Arg Ser 145 150 155 160 Tyr Thr His Ala Lys Pro Pro Tyr Ser Tyr Ile Ser Leu Ile Thr Met 165 170 175 Ala Ile Gln Gln Ser Pro Asn Lys Met Leu Thr Leu Ser Glu Ile Tyr 180 185 190 Gln Trp Ile Met Asp Leu Phe Pro Phe Tyr Arg Gln Asn Gln Gln Arg 195 200 205 Trp Gln Asn Ser Ile Arg His Ser Leu Ser Phe Asn Asp Cys Phe Leu 210 215 220 Lys Val Pro Arg Ser Pro Asp Lys Pro Gly Lys Gly Ser Phe Trp Thr 225 230 235 240 Leu His Pro Asp Ser Gly Asn Met Phe Glu Asn Gly Cys Tyr Leu Arg 245 250 255 Arg Gln Lys Arg Phe Lys Cys Glu Lys Gln Leu Ala Leu Lys Glu Ala 260 265 270 Ala Gly Ala Ala Gly Ser Gly Lys Lys Ala Ala Ala Gly Ala Gln Ala 275 280 285 Ser Gln Ala Gln Leu Gly Glu Ala Ala Gly Pro Ala Ser Glu Thr Pro 290 295 300 Ala Gly Thr Glu Ser Pro His Ser Ser Ala Ser Pro Cys Gln Glu His 305 310 315 320 Lys Arg Gly Gly Leu Gly Glu Leu Lys Gly Thr Pro Ala Ala Ala Leu 325 330 335 Ser Pro Pro Glu Pro Ala Pro Ser Pro Gly Gln Gln Gln Gln Ala Ala 340 345 350 Ala His Leu Leu Gly Pro Pro His His Pro Gly Leu Pro Pro Glu Ala 355 360 365 His Leu Lys Pro Glu His His Tyr Ala Phe Asn His Pro Phe Ser Ile 370 375 380 Asn Asn Leu Met Ser Ser Glu Gln Gln His His His Ser His His His 385 390 395 400 His Gln Pro His Lys Met Asp Leu Lys Ala Tyr Glu Gln Val Met His 405 410 415 Tyr Pro Gly Tyr Gly Ser Pro Met Pro Gly Ser Leu Ala Met Gly Pro 420 425 430 Val Thr Asn Lys Thr Gly Leu Asp Ala Ser Pro Leu Ala Ala Asp Thr 435 440 445 Ser Tyr Tyr Gln Gly Val Tyr Ser Arg Pro Ile Met Asn Ser Ser 450 455 460 <210> 12 <211> 2428 <212> DNA <213> Homo sapiens <400> 12 cccgcccact tccaactacc gcctccggcc tgcccaggga gagagagggga gtggagccca 60 gggagaggga gcgcgagaga gggaggggagg aggggacggt gctttggctg actttttttt 120 aaaagagggt gggggtgggg ggtgattgct ggtcgtttgt tgtggctgtt aaatttaaa 180 ctgccatgca ctcggcttc agtatgctgg gagcggtgaa gatggaaggg cacgagccgt 240 ccgactggag cagctactat gcagagccg agggctactc ctccgtgagc aacatgaacg 300 ccggcctggg gatgaacggc atgaacacgt acatgagcat gtcggcggcc gccatgggca 360 gcggctcggg caacatgagc gcgggctcca tgaacatgtc gtcgtacgtg ggcgctggca 420 tgagcccgtc cctggcgggg atgtccccg gcgcgggcgc catggcgggc atgggcggct 480 cggccggggc ggccggcgtg gcgggcatgg ggccgcactt gagtcccagc ctgagcccgc 540 tcggggggca ggcggccggg gccatggggcg gcctggcccc ctacgccaac atgaactcca 600. tgagccccat gtacgggcag gcggggcctga gccgcgcccg cgaccccag acctacaggc 660 gcagctacac gcacgcaaag ccgccctact cgtacatctc gctcatcacc atggccatcc 720 agcagagccc caacaagatg ctgacgctga gcgagatcta ccagtggatc atggacctct tccccttcta ccggcagaac cagcagcgct ggcagaactc catccgccac tcgctctcct 840 tcaacgactg tttcctgag gtgccccgct cgcccgacaa gcccggcaag ggctccttct ggaccctgca ccctgactcg ggcaacatgt tcgagaacgg ctgctacctg cgccgccaga 960 agcgcttcaa gtgcgagaag cagctggcgc tgaaggaggc cgcaggcgcc gccggcagcg gcaagaaggc ggccgccgga gcccaggcct cacaggctca actcggggag gccgccggggc 1080 cggcctccga gactccggcg ggcaccgagt cgcctcactc gagcgcctcc ccgtgccagg 1140 agcacaagcg agggggcctg ggagagctga aggggacgcc ggctgcggcg ctgagccccc cagagccggc gccctctccc gggcagcagc agcaggccgc ggcccacctg ctgggcccgc 1260 cccaccaccc gggcctgccg cctgaggccc acctgaagcc ggaacaccac tacgccttca 1320 accaccgtt ctccatcaac aacctcatgt cctcggagca gcagcaccac cacagccacc 1380 accaccacca accccacaaa atggacctca aggcctacga acaggtgatg cactaccccg 1440 gctacggttc ccccatgcct ggcagcttg ccatgggccc ggtcacgaac aaaacgggcc 1500 tggacgcctc gcccctggcc gcagatacct cctactacca gggggtgtac tcccggccca 1560 tttgaactc ctcttaagaa gacgacggct tcaggcccgg ctaactctgg caccccggat 1620 cgaggacaag tgagagagca agtgggggtc gagactttgg ggagacggtg ttgcagagac 1680 gcaagggaga agaaatccat aacaccccca ccccaacacc cccaagacag cagtcttctt 1740 cacccgctgc agccgttccg tcccaaacag agggccacac agatacccca cgttctatat 1800 aaggaggaaa acgggaaaga atataaagtt aaaaaaaagc ctccggtttc cactactgtg 1860 tagactcctg cttcttcaag cacctgcaga ttctgattttt tttgttgttg ttgttctcct 1920 ccattgctgt tgttgcaggg aagtcttact taaaaaaaa aaaaaatttt gtgagtgact 1980 cggtgtaaaa ccatgtagtt ttaacagaac cagagggttg tactattgtt taaaacagg 2040 aaaaaaata atgtaagggt ctgttgtaaa tgaccaagaa aaagaaaaaa aaagcattcc 2100 caatcttgac acggtgaaat ccaggtctcg ggtccgatta attatggtt tctgcgtgct 2160 ttatttatgg cttataaatg tgtattctgg ctgcaagggc cagagttcca caaatctata 2220 ttaaagtgtt atacccggtt ttatcccttg aatcttttct tccagatttt tctttttcttt 2280 acttggctta caaaatac aggcttggaa attatttcaa gaaggaggga gggataccct 2340 gtctggttgc aggttgtatt ttattttggc ccagggagtg ttgctgtttt cccaacattt 2400 tattaataaa atttcagac ataaaaaa 2428 <210> 13 <211> 595 <212> PRT <213> Homo sapiens <400> 13 Met Ala Leu Thr Asp Gly Gly Trp Cys Leu Pro Lys Arg Phe Gly Ala 1 5 10 15 Ala Gly Ala Asp Ala Ser Asp Ser Arg Ala Phe Pro Ala Arg Glu Pro 20 25 30 Ser Thr Pro Pro Ser Pro Ile Ser Ser Ser Ser Ser Ser Cys Ser Arg 35 40 45 Gly Gly Glu Arg Gly Pro Gly Gly Ala Ser Asn Cys Gly Thr Pro Gln 50 55 60 Leu Asp Thr Glu Ala Ala Ala Gly Pro Pro Ala Arg Ser Leu Leu Leu 65 70 75 80 Ser Ser Tyr Ala Ser His Pro Phe Gly Ala Pro His Gly Pro Ser Ala 85 90 95 Pro Gly Val Ala Gly Pro Gly Gly Asn Leu Ser Ser Trp Glu Asp Leu 100 105 110 Leu Leu Phe Thr Asp Leu Asp Gln Ala Ala Thr Ala Ser Lys Leu Leu 115 120 125 Trp Ser Ser Arg Gly Ala Lys Leu Ser Pro Phe Ala Pro Glu Gln Pro 130 135 140 Glu Glu Met Tyr Gln Thr Leu Ala Ala Leu Ser Ser Gln Gly Pro Ala 145 150 155 160 Ala Tyr Asp Gly Ala Pro Gly Gly Phe Val His Ser Ala Ala Ala Ala 165 170 175 Ala Ala Ala Ala Ala Ala Ala Ser Ser Pro Val Tyr Val Pro Thr Thr 180 185 190 Arg Val Gly Ser Met Leu Pro Gly Leu Pro Tyr His Leu Gln Gly Ser 195 200 205 Gly Ser Gly Pro Ala Asn His Ala Gly Gly Ala Gly Ala His Pro Gly 210 215 220 Trp Pro Gln Ala Ser Ala Asp Ser Pro Pro Tyr Gly Ser Gly Gly Gly 225 230 235 240 Ala Ala Gly Gly Gly Ala Ala Gly Pro Gly Gly Ala Gly Ser Ala Ala 245 250 255 Ala His Val Ser Ala Arg Phe Pro Tyr Ser Pro Ser Pro Pro Met Ala 260 265 270 Asn Gly Ala Ala Arg Glu Pro Gly Gly Tyr Ala Ala Ala Gly Ser Gly 275 280 285 Gly Ala Gly Gly Val Ser Gly Gly Gly Ser Ser Leu Ala Ala Met Gly 290 295 300 Gly Arg Glu Pro Gln Tyr Ser Ser Leu Ser Ala Ala Arg Pro Leu Asn 305 310 315 320 Gly Thr Tyr His His His His His His His His His His Pro Ser Pro 325 330 335 Tyr Ser Pro Tyr Val Gly Ala Pro Leu Thr Pro Ala Trp Pro Ala Gly 340 345 350 Pro Phe Glu Thr Pro Val Leu His Ser Leu Gln Ser Arg Ala Gly Ala 355 360 365 Pro Leu Pro Val Pro Arg Gly Pro Ser Ala Asp Leu Leu Glu Asp Leu 370 375 380 Ser Glu Ser Arg Glu Cys Val Asn Cys Gly Ser Ile Gln Thr Pro Leu 385 390 395 400 Trp Arg Arg Asp Gly Thr Gly His Tyr Leu Cys Asn Ala Cys Gly Leu 405 410 415 Tyr Ser Lys Met Asn Gly Leu Ser Arg Pro Leu Ile Lys Pro Gln Lys 420 425 430 Arg Val Pro Ser Ser Arg Arg Leu Gly Leu Ser Cys Ala Asn Cys His 435 440 445 Thr Thr Thr Thr Thr Leu Trp Arg Arg Asn Ala Glu Gly Glu Pro Val 450 455 460 Cys Asn Ala Cys Gly Leu Tyr Met Lys Leu His Gly Val Pro Arg Pro 465 470 475 480 Leu Ala Met Lys Lys Glu Gly Ile Gln Thr Arg Lys Arg Lys Pro Lys 485 490 495 Asn Ile Asn Lys Ser Lys Thr Cys Ser Gly Asn Ser Asn Asn Ser Ile 500 505 510 Pro Met Thr Pro Thr Ser Thr Ser Ser Asn Ser Asp Asp Cys Ser Lys 515 520 525 Asn Thr Ser Pro Thr Thr Gln Pro Thr Ala Ser Gly Ala Gly Ala Pro 530 535 540 Val Met Thr Gly Ala Gly Glu Ser Thr Asn Pro Glu Asn Ser Glu Leu 545 550 555 560 Lys Tyr Ser Gly Gln Asp Gly Leu Tyr Ile Gly Val Ser Leu Ala Ser 565 570 575 Pro Ala Glu Val Thr Ser Ser Val Arg Pro Asp Ser Trp Cys Ala Leu 580 585 590 Ala Leu Ala 595 <210> 14 <211> 3785 <212> DNA <213> Homo sapiens <400> 14 agttccgacc cacagcctgg cacccttcgg cgagcgctgt ttgtttaggg ctcggtgagt 60 ccaatcagga gcccaggctg cagttttccg gcagagcagt aagaggcgcc tcctctctcc 120 tttttattca ccagcagcgc ggcgcagacc ccggactcgc gctcgcccgc tggcgccctc 180 ggcttctctc cgcgcctggg agcaccctcc gccgcggccg ttctccatgc gcagcgcccg 240 cccgaggagc tagacgtcag cttggagcgg cgccggaccg tggatggcct tgactgacgg 300 cggctggtgc ttgccgaagc gcttcggggc cgcgggtgcg gacgccagcg actccagagc 360 ctttccagcg cgggagccct ccacgccgcc ttcccccatc tcttcctcgt cctcctcctg 420 ctcccggggc ggagagcggg gccccggcgg cgccagcaac tgcgggacgc ctcagctcga 480 cacggaggcg gcggccggac ccccggcccg ctcgctgctg ctcagttcct acgcttcgca 540 tcccttcggg gctccccacg gaccttcggc gcctggggtc gcgggccccg ggggcaacct 600 gtcgagctgg gaggacttgc tgctgttcac tgacctcgac caagccgcga ccgccagcaa 660 gctgctgtgg tccagccgcg gcgccaagct gagccccttc gcacccgagc agccggagga 720 gatgtaccag accctcgccg ctctctccag ccagggtccg gccgcctacg acggcgcgcc 780 cggcggcttc gtgcactctg cggccgcggc ggcagcagcc gcggcggcgg ccagctcccc 840 ggtctacgtg cccaccaccc gcgtgggttc catgctgccc ggcctaccgt accacctgca 900 ggggtcgggc agtgggccag ccaaccacgc gggcggcgcg ggcgcgcacc ccggctggcc 960 tcaggcctcg gccgacagcc ctccatacgg cagcggaggc ggcgcggctg gcggcggggc 1020 cgcggggcct ggcggcgctg gctcagccgc ggcgcacgtc tcggcgcgct tcccctactc 1080 tcccagcccg cccatggcca acggcgccgc gcgggagccg ggaggctacg cggcggcggg 1140 cagtgggggc gcgggaggcg tgagcggcgg cggcagtagc ctggcggcca tgggcggccg 1200 cgagccccag tacagctcgc tgtcggccgc gcggccgctg aacgggacgt accaccacca 1260 ccaccaccac caccaccacc atccgagccc ctactcgccc tacgtggggg cgccactgac 1320 gcctgcctgg cccgccggac ccttcgagac cccggtgctg cacagcctgc agagccgcgc 1380 cggagccccg ctcccggtgc cccggggtcc cagtgcagac ctgctggagg acctgtccga 1440 gagccgcgag tgcgtgaact gcggctccat ccagacgccg ctgtggcggc gggacggcac 1500 cggccactac ctgtgcaacg cctgcgggct ctacagcaag atgaacggcc tcagccggcc 1560 cctcatcaag ccgcagaagc gcgtgccttc atcacggcgg cttggattgt cctgtgccaa 1620 ctgtcacacc acaactacca ccttatggcg cagaaacgcc gagggtgaac ccgtgtgcaa 1680 tgcttgtgga ctctacatga aactccatgg ggtgcccaga ccacttgcta tgaaaaaaga 1740 gggaattcaa accaggaaac gaaaacctaa gaacataaat aaatcaaaga cttgctctgg 1800 taatagcaat aattccattc ccatgactcc aacttccacc tcttctaact cagatgattg 1860 cagcaaaaat acttccccca caacacaacc tacagcctca ggggcgggtg ccccggtgat 1920 gactggtgcg ggagagagca ccaatcccga gaacagcgag ctcaagtatt cgggtcaaga 1980 tgggctctac ataggcgtca gtctcgcctc gccggccgaa gtcacgtcct ccgtgcgacc 2040 ggattcctgg tgcgccctgg ccctggcctg agcccacgcc gccaggaggc agggagggct 2100 ccgccgcggg cctcactcca ctcgtgtctg cttttgtgca gcggtccaga cagtggcgac 2160 tgcgctgaca gaacgtgatt ctcgtgcctt tattttgaaa gagatgtttt tcccaagagg 2220 cttgctgaaa gagtgagaga agatggaagg gaagggccag tgcaactggg cgcttgggcc 2280 actccagcca gcccgcctcc ggggcggacc ctgctccact tccagaagcc aggactagga 2340 cctgggcctt gcctgctatg gaatattgag agagattttt taaaaaagat tttgcatttt 2400 gtccaaaatc atgtgcttct tctgatcaat tttggttgtt ccagaatttc ttcatacctt 2460 ttccacatcc agatttcatg tgcgttcatg gagaagatca cttgaggcca tttggtacac 2520 atctctggag gctgagtcgg ttcatgaggt ctcttatcaa aaatattact cagtttgcaa 2580 gactgcattg taactttaac atacactgtg actgacgttt ctcaaagttc atattgtgtg 2640 gctgatctga agtcagtcgg aatttgtaaa cagggtagca aacaagatat ttttcttcca 2700 tgtatacaat aattttttta aaaagtgcaa tttgcgttgc agcaatcagt gttaaatcat 2760 ttgcataaga tttaacagca ttttttataa tgaatgtaaa cattttaact taatggtact 2820 taaaataatt taaaagaaaa atgttaactt agacattctt atgcttcttt tacaactaca 2880 tcccatttta tatttccaat tgttaaagaa aaatatttca agaacaaatc ttctctcagg 2940 aaaattgcct ttctctattt gttaagaatt tttatacaag aacaccaata tacccccttt 3000 attttactgt ggaatatgtg ctggaaaaat tgcaacaaca ctttactacc taacggatag 3060 catttgtaaa tactctaggt atctgtaaac actctgatga agtctgtata gtgtgactaa 3120 cccacaggca ggttggttta cattaatttt ttttttgaa tgggatgtcc tatggaaacc 3180 tatttcacca gagttttaaa aataaaaagg gtattgtttt gtcttctgta cagtgagttc 3240 cttcccttt caaagctttc tttttatgct gtatgtgact atagatattc atataaaaca 3300 agtgcacgtg aagtttgcaa aatgctttaa ggccttcctt tcaaagcata gtccttttgg 3360 agccgttttg taccttttat accttggctt atttgaagtt gacacatggg gttagttact 3420 actctccatg tgcattgggg acagttttta taagtgggaa ggactcagta ttattatatt 3480 tgagatgata agcatttgtgt ttgggaacaa tgcttaaaaa tattccagaa agttcagatt 3540 ttttttcttt gtgaatgaaa tatattctgg cccacgaaca gggcgatttc ctttcagttt 3600 tttccttttg caacgtgcct tgaagtctca aagctcacct gaggttgcag acgttacccc 3660 caacagaaga taggtagaaa tgattccagt ggcctctttg tattttcttc attgttgagt 3720 agatttcagg aaatcaggag gtgtttcaca atacagaatg atggccttta actgtgaaaa 3780 aaaaa 3785 <210> 15 <211> 394 <212> PRT <213> Homo sapiens <400> 15 Met Lys Arg Gln Asn Val Arg Thr Leu Ala Leu Ile Val Cys Thr Phe 1 5 10 15 Thr Tyr Leu Leu Val Gly Ala Ala Val Phe Asp Ala Leu Glu Ser Glu 20 25 30 Pro Glu Leu Ile Glu Arg Gln Arg Leu Glu Leu Arg Gln Gln Glu Leu 35 40 45 Arg Ala Arg Tyr Asn Leu Ser Gln Gly Gly Tyr Glu Glu Leu Glu Arg 50 55 60 Val Val Leu Arg Leu Lys Pro His Lys Ala Gly Val Gln Trp Arg Phe 65 70 75 80 Ala Gly Ser Phe Tyr Phe Ala Ile Thr Val Ile Thr Thr Ile Gly Tyr 85 90 95 Gly His Ala Ala Pro Ser Thr Asp Gly Gly Lys Val Phe Cys Met Phe 100 105 110 Tyr Ala Leu Leu Gly Ile Pro Leu Thr Leu Val Met Phe Gln Ser Leu 115 120 125 Gly Glu Arg Ile Asn Thr Leu Val Arg Tyr Leu Leu His Arg Ala Lys 130 135 140 Lys Gly Leu Gly Met Arg Arg Ala Asp Val Ser Met Ala Asn Met Val 145 150 155 160 Leu Ile Gly Phe Phe Ser Cys Ile Ser Thr Leu Cys Ile Gly Ala Ala 165 170 175 Ala Phe Ser His Tyr Glu His Trp Thr Phe Phe Gln Ala Tyr Tyr Tyr 180 185 190 Cys Phe Ile Thr Leu Thr Thr Ile Gly Phe Gly Asp Tyr Val Ala Leu 195 200 205 Gln Lys Asp Gln Ala Leu Gln Thr Gln Pro Gln Tyr Val Ala Phe Ser 210 215 220 Phe Val Tyr Ile Leu Thr Gly Leu Thr Val Ile Gly Ala Phe Leu Asn 225 230 235 240 Leu Val Val Leu Arg Phe Met Thr Met Asn Ala Glu Asp Glu Lys Arg 245 250 255 Asp Ala Glu His Arg Ala Leu Leu Thr Arg Asn Gly Gln Ala Gly Gly 260 265 270 Gly Gly Gly Gly Gly Ser Ala His Thr Thr Asp Thr Ala Ser Ser Thr 275 280 285 Ala Ala Ala Gly Gly Gly Gly Phe Arg Asn Val Tyr Ala Glu Val Leu 290,295,300 His Phe Gln Ser Met Cys Ser Cys Leu Trp Tyr Lys Ser Arg Glu Lys 305 310 315 320 Leu Gln Tyr Ser Ile Pro Met Ile Ile Pro Arg Asp Leu Ser Thr Ser 325 330 335 Asp Thr Cys Val Glu Gln Ser His Ser Ser Pro Gly Gly Gly Gly Arg 340 345 350 Tyr Ser Asp Thr Pro Ser Arg Arg Cys Leu Cys Ser Gly Ala Pro Arg 355 360 365 To be Ala Ile To be Val To be Thr Gly Leu His To be Leu To be Thr Phe 370 375 380 Arg Gly Leu Met Lys Arg Arg Ser Ser Val 385,390 <210> 16 <211> 3978 <212> DNA <213> Homo sapiens <400> 16 ggcggcggcg gcggcggcgg ccccgggcgc tgagcgggtg cccggcgcgg agagcggcga 60 gcgcagccat gccccaggcc gcctcgggg cagcagcagc ggcggccggg gccgaggcggc 120 gggccggggg cgccgggggg ccggcggcgg cccgggcggg acgatgaagc ggcagaacgt 180 gcgcacgctg gcgctcatcg tgtgcacctt cacctacctg ctggtgggcg ccgcggtctt 240 cgacgcgctg gagtcggagc ccgagctgat cgagcggcag cggctggagc tgcggcagca 300 ggagctgcgg gcgcgctaca acctcagcca gggcggctac gaggagctgg agcgcgtcgt 360 gctgcgcctc aagccgcaca aggccggcgt gcagtggcgc ttcgccggct ccttctactt 420 cgccatcacc gtcatcacca ccatcggcta cgggcacgcg gcacccagca cggatggcgg 480 caaggtgttc tgcatgttct acgcgctgct gggcatcccg ctcacgctcg tcatgttcca 540 gagcctgggc gagcgcatca acaccttggt gaggtacctg ctgcaccgcg ccaagaaggg 600 gctgggcatg cggcgcgccg acgtgtccat ggccaacatg gtgctcatcg gcttcttctc 660 gtgcatcagc acgctgtgca tcggcgccgc cgccttctcc cactacgagc actggacctt 720 cttccaggcc tactactact gcttcatcac cctcaccacc atcggcttcg gcgactacgt 780 ggcgctgcag aaggaccagg ccctgcagac gcagccgcag tacgtggcct tcagcttcgt 840 ctacatcctt acgggcctca cggtcatcgg cgccttcctc aacctcgtgg tgctgcgctt 900 catgaccatg aacgccgagg acgagaagcg cgacgccgag caccgcgcgc tgctcacgcg 960 caacgggcag gcgggcggcg gcggaggggg tggcagcgcg cacactacgg acaccgcctc 1020 atccacggcg gcagcgggcg gcggcggctt ccgcaacgtc tacgcggagg tgctgcactt 1080 ccagtccatg tgctcgtgcc tgtggtacaa gagccgcgag aagctgcagt actccatccc 1140 catgatcatc ccgcgggacc tctccacgtc cgacacgtgc gtggagcaga gccactcgtc 1200 gccgggaggg ggcggccgct acagcgacac gccctcgcga cgctgcctgt gcagcggggc 1260 gccacgctcc gccatcagct cggtgtccac gggtctgcac agcctgtcca ccttccgcgg 1320 cctcatgaag cgcaggagct ccgtgtgact gccccgaggg gcctggagca cctgggggcg 1380 cgggcggggg acccctgctg ggaggccagg agactgcccc tgctgccttc tgcccagtgg 1440 gaccccgcac aacatccctc accactctcc cccagcaccc ccatctccga ctgtgcctgc 1500 ttgcaccagc cggcaggagg ccgggctctg aggacccctg gggcccccat cggagccctg 1560 caaattccga gaaatgtgaa acttggtggg gtcagggagg aaaggcagaa gctgggagcc 1620 tccctccct ttgaaaatct aagaagctcc cagtcctcag agaccctgct ggtacccaga 1680 cccccacctt cggaggggac ttcatgttcc gtgtacgttt gcatctctat ttatacctct 1740 gtcctgctag gtctcccacc ttcccttggt tccaaaagcc agggtgcta tgtccaagtc 1800 acccctactc agccccactc cccttcctca tccccagctg tgctcccaa cctcccttg 1860 tgttgttttg catggctttg cagttatgga gaaagtggaa acccagcagt ccctaaagct 1920 ggtccccaga aagcaggaca gaaagaagga gggacaggca ggcagcagga ggggcgagct 1980 gggaggcagg aggcagcggc ctgtcagtct gcagaatggt cgcactggag gttcaagcta 2040 actggcctcc agccacattc tcatagcagg taggacttca gccttccaga cactgccctt 2100 agaatctgga acagaagact tcagactcac cataattgct gataattacc cactcttaaa 2160 tttgtcgagt gatttttagc ctctgaaaac tctatgctgg ccactgattc ctttgagtct 2220 cacaaaaccc tacttaggtc atcagggcag gagttctcac tcccatttta cagatgagaa 2280 tactgaggcc tggacaggtg aagtgaccag agagcaaaag gcaaaggggt gggggctggg 2340 tgcagtggct cacacctgta ttcccaacac ttttggaggc tgaggttgga ggattgcttg 2400 agcccaggaa tttgagacca gcctaggtga catagtgaga ccccatctct acaaaaaata 2460 aaaaattaac caggtgtggt ggcacgtgcc tgggagtccc agcgacttgg gaggctgagg 2520 tgggaggatt gtttgagcct gggaggtcga ggctgtagtg agccctgatt gcaccactgt 2580 actccagcct gggtgacagg gcaagaccct gtctcaaaaa aaaaaaaaaa aatggcaaag 2640 ggagacaaga gcccagcctg cttgttgcta gccaaagtgt tctttccttc cagcttggcc 2700 tgctcttaaa agcaaagctc ctgcagtgta catcctggca ttgtgtggct acctgggttt 2760 taaaccagaa tcagaagtcc cggatcagag ggcactgctg aggttcagcc tcttctcttc 2820 ttggccagga ggcagcagct ctgaatgggc ccctgaggct gcacaggggc ctttgtcact 2880 ggggcgcatg cttacaaaca gtgcagttct tgggaccgag gtaagcaggg ctgggtctca 2940 tggcagaaag gccaggatct ggggctctag gaatttggga attgggcaga gtggccaaga 3000 aagctggcag gcatatccta tgggacatca cacctggcac cattgtcatt gttggtgcct 3060 gtgtcccaag tagctagtga taagctgagg ctgcagcaag aaacaccctt cccaggtggg 3120 ggagtttgga ccagaggtgc cctctgccca ccacacctgc aacccagaag cccagatgga 3180 acgcagctga cgaaggtgat gcttgaggct cacttttggg gccccacagc tggagccggt 3240 ataatgactg ggacaacatc aaggggtgga tgaggggcct ctcctcccgc aacactgcct 3300 tcccatgctg ttcccctgcc agctccttaa cactgccgac caaggccagc cctggcattc 3360 agggaaattg gagggcagca cccgtagggt ggccagcctc aggccccacc ccagctgtgt 3420 cctctagtct ctggggaccc ctggggggaa gaagtctacc ctgcttgtga gtcccgtctc 3480 agtgtggagg aactggctgc acgtgggacc tgaaggtgcc ctctgtgttt atgttggggg 3540 tgggggggca gtgctggctg cctctgtcct gtgtgtgacc ctgccctcga agggtcctgt 3600 cctgtcagtc ccgagggagc cacaaccaaa gctgcggaga gaaggtgggg aagggtgcag 3660 aatggccgtg gggcacagcg tggcagactg ttcagtctct gctgggtctt tcctagggac 3720 ctggaaggcc agtgttgctt cccctcact ccctttcact gcaggcagcc tctctgcttc 3780 cccaatgcct tatgcctggg cacactgcca cagaatatgc aatatgtgtg ggtgaccatg 3840 ccctcacgac cacaccccca ccccgggcag cccccggact ccaaaggtcg tggctgccac 3900 agcctccctc agctcttcct gcctatctgt cttcacactg agaatggcgc ccaataaatg 3960 ctatccacgg agaccagg 3978 <210> 17 <211> 676 <212> PRT <213> Homo sapiens <400> 17 Put Ala Ala Ala Sir Sir Pro Pro Arg Ala Glu Arg Smooth Arg Trp Gly 1 5 10 15 Trp Gly Arg Leu Pro Gly Ala Arg Arg Gly Ser Ala Gly Leu Ala Lys 20 25 30 Lys Cys Pro Phe Ser Leu Glu Leu Ala Glu Gly Gly Pro Ala Gly Gly 35 40 45 Ala Leu Tyr Ala Pro Ile Ala Pro Gly Ala Pro Gly Pro Ala Pro Pro 50 55 60 Here Dear Pro Here Here Pro Here Here Pro Pro Val Here Dear Asp Leu Gly 65 70 75 80 Pro Arg Pro Pro Val Ser Leu Asp Pro Arg Val Ser Ile Tyr Ser Thr 85 90 95 Arg Arg Pro Val Leu Ala Arg Thr His Val Gln Gly Arg Val Tyr Asn 100 105 110 Phe Leu Glu Arg Pro Thr Gly Trp Lys Cys Phe Val Tyr His Phe Ala 115 120 125 Val Phe Leu Ile Val Leu Val Cys Leu Ile Phe Ser Val Leu Ser Thr 130 135 140 Ile Glu Gln Tyr Ala Ala Leu Ala Thr Gly Thr Leu Phe Trp Met Glu 145 150 155 160 Ile Val Leu Val Val Phe Phe Gly Thr Glu Tyr Val Val Arg Leu Trp 165 170 175 Ser Ala Gly Cys Arg Ser Lys Tyr Val Gly Leu Trp Gly Arg Leu Arg 180 185 190 Phe Ala Arg Lys Pro Ile Ser Ile Ile Asp Leu Ile Val Val Val Ala 195 200 205 Ser Met Val Val Leu Cys Val Gly Ser Lys Gly Gln Val Phe Ala Thr 210 215 220 Ser Ala Ile Arg Gly Ile Arg Phe Leu Gln Ile Leu Arg Met Leu His 225 230 235 240 Val Asp Arg Gln Gly Gly Thr Trp Arg Leu Leu Gly Ser Val Val Phe 245 250 255 Ile His Arg Gln Glu Leu Ile Thr Thr Leu Tyr Ile Gly Phe Leu Gly 260 265 270 Leu Ile Phe Ser Ser Tyr Phe Val Tyr Leu Ala Glu Lys Asp Ala Val 275 280 285 Asn Glu Ser Gly Arg Val Glu Phe Gly Ser Tyr Ala Asp Ala Leu Trp 290 295 300 Trp Gly Val Val Thr Val Thr Thr Ile Gly Tyr Gly Asp Lys Val Pro 305 310 315 320 Gln Thr Trp Val Gly Lys Thr Ile Ala Ser Cys Phe Ser Val Phe Ala 325 330 335 Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser Gly Phe Ala 340 345 350 Leu Lys Val Gln Gln Lys Gln Arg Gln Lys His Phe Asn Arg Gln Ile 355 360 365 Pro Ala Ala Ala Ser Leu Ile Gln Thr Ala Trp Arg Cys Tyr Ala Ala 370 375 380 Glu Asn Pro Asp Ser Ser Thr Trp Lys Ile Tyr Ile Arg Lys Ala Pro 385 390 395 400 Arg Ser His Thr Leu Leu Ser Pro Ser Pro Lys Pro Lys Lys Ser Val 405 410 415 Val Val Lys Lys Lys Lys Phe Lys Leu Asp Lys Asp Asn Gly Val Thr 420 425 430 Pro Gly Glu Lys Met Leu Thr Val Pro His Ile Thr Cys Asp Pro Pro 435 440 445 Glu Glu Arg Arg Leu Asp His Phe Ser Val Asp Gly Tyr Asp Ser Ser 450 455 460 Val Arg Lys Ser Pro Thr Leu Leu Glu Val Ser Met Pro His Phe Met 465 470 475 480 Arg Thr Asn Ser Phe Ala Glu Asp Leu Asp Leu Glu Gly Glu Thr Leu 485 490 495 Leu Thr Pro Ile Thr His Ile Ser Gln Leu Arg Glu His His Arg Ala 500 505 510 Thr Ile Lys Val Ile Arg Arg Met Gln Tyr Phe Val Ala Lys Lys Lys 515 520 525 Phe Gln Gln Ala Arg Lys Pro Tyr Asp Val Arg Asp Val Ile Glu Gln 530 535 540 Tyr Ser Gln Gly His Leu Asn Leu Met Val Arg Ile Lys Glu Leu Gln 545 550 555 560 Arg Arg Leu Asp Gln Ser Ile Gly Lys Pro Ser Leu Phe Ile Ser Val 565 570 575 Ser Glu Lys Ser Lys Asp Arg Gly Ser Asn Thr Ile Gly Ala Arg Leu 580 585 590 Asn Arg Val Glu Asp Lys Val Thr Gln Leu Asp Gln Arg Leu Ala Leu 595 600 605 Ile Thr Asp Met Leu His Gln Leu Leu Ser Leu His Gly Gly Ser Thr 610 615 620 Pro Gly Ser Gly Gly Pro Pro Arg Glu Gly Gly Ala His Ile Thr Gln 625 630 635 640 Pro Cys Gly Ser Gly Gly Ser Val Asp Pro Glu Leu Phe Leu Pro Ser 645 650 655 Asn Thr Leu Pro Thr Tyr Glu Gln Leu Thr Val Pro Arg Arg Gly Pro 660 665 670 Asp Glu Gly Ser 675 <210> 18 <211> 3262 <212> DNA <213> Homo sapiens <400> 18 gcggcggggc tggcagcagt ggctgcccgc actgcgcccg ggcgctcgcc ttcgctgcag 60 ctcccggtgc cgccgctcgg gccggccccc cggcaggccc tcctcgttat ggccgcggcc 120 tcctccccgc ccagggccga gaggaagcgc tggggttggg gccgcctgcc aggcgcccgg 180 cggggcagcg cgggcctggc caagaagtgc cccttctcgc tggagctggc ggagggcggc 240 ccggcgggcg gcgcgctcta cgcgcccatc gcgcccggcg ccccaggtcc cgcgccccct 300 gcgtccccgg ccgcgcccgc cgcgccccca gttgcctccg accttggccc gcggccgccg 360 gtgagcctag acccgcgcgt ctccatctac agcacgcgcc gcccggtgtt ggcgcgcacc 420 cacgtccagg gccgcgtcta caacttcctc gagcgtccca ccggctggaa atgcttcgtt 480 taccacttcg ccgtcttcct catcgtcctg gtctgcctca tcttcagcgt gctgtccacc 540 atcgagcagt atgccgccct ggccacgggg actctcttct ggatggagat cgtgctggtg 600 gtgttcttcg ggacggagta cgtggtccgc ctctggtccg ccggctgccg cagcaagtac 660 gtgggcctct gggggcggct gcgctttgcc cggaagccca tttccatcat cgacctcatc 720 gtggtcgtgg cctccatggt ggtcctctgc gtgggctcca aggggcaggt gtttgccacg 780 tcggccatca ggggcatccg cttcctgcag atcctgagga tgctacacgt cgaccgccag 840 ggaggcacct ggaggctcct gggctccgtg gtcttcatcc accgccagga gctgataacc 900 accctgtaca tcggcttcct gggcctcatc ttctcctcgt actttgtgta cctggctgag 960 aaggacgcgg tgaacgagtc aggccgcgtg gagttcggca gctacgcaga tgcgctgtgg 1020 tggggggtgg tcacagtcac caccatcggc tatggggaca aggtgcccca gacgtgggtc 1080 gggaagacca tcgcctcctg cttctctgtc tttgccatct ccttctttgc gctcccagcg 1140 gggattcttg gctcggggtt tgccctgaag gtgcagcaga agcagaggca gaagcacttc 1200 aaccggcaga tcccggcggc agcctcactc attcagaccg catggaggtg ctatgctgcc 1260 gagaaccccg actcctccac ctggaagatc tacatccgga aggccccccg gagccacact 1320 ctgctgtcac ccagccccaa acccaagaag tctgtggtgg taaagaaaaa aaagttcaag 1380 ctggacaaag acaatggggt gactcctgga gagaagatgc tcacagtccc ccatatcacg 1440 tgcgaccccc cagaagagcg gcggctggac cacttctctg tcgacggcta tgacagttct 1500 gtaaggaaga gcccaacact gctggaagtg agcatgcccc atttcatgag aaccaacagc 1560 ttcgccgagg acctggacct ggaaggggag actctgctga cacccatcac ccacatctca 1620 cagctgcggg aacaccatcg ggccaccatt aaggtcattc gacgcatgca gtactttgtg 1680 gccaagaaga aattccagca agcgcggaag ccttacgatg tgcgggacgt cattgagcag 1740 tactcgcagg gccacctcaa cctcatggtg cgcatcaagg agctgcagag gaggctggac 1800 cagtccattg ggaagccctc actgttcatc tccgtctcag aaaagagcaa ggatcgcggc 1860 agcaacacga tcggcgcccg cctgaaccga gtagagaca aggtgacgca gctggaccag 1920 aggctggcac tcatcaccga catgcttcac cagctgctct ccttgcacgg tggcagcacc 1980 cccggcagcg gcggcccccc cagagaggc ggggcccaca tcacccagcc ctgcggcagt 2040 ggcggctccg tcgaccctga gctcttcctg cccagcaaca ccctgcccac ctacgagcag 2100 ctgaccgtgc ccaggagggg ccccgatgag gggtcctgag gaggggatgg ggctggggga 2160 tgggcctgag tgagaggga ggccaagagt ggccccacct ggccctctct gaaggaggcc 2220 acctcctaaa aggcccagag agaagagccc cactctcaga ggccccaata ccccatggac 2280 catgctgtct ggcacagcct gcacttgggg gctcagcaag gccacctctt cctggccggt 2340 gtgggggccc cgtctcaggt ctgagttgtt accccaagcg ccctggcccc cacatggtga 2400 tgttgacatc actggcatgg tggttgggac ccagtggcag ggcacagggc ctggcccatg 2460 tatggccagg aagtagcaca ggctgagtgc aggcccaccc tgcttggccc agggggcttc 2520 ctgaggggag acagagcaac ccctggaccc cagcctcaaa tccaggaccc tgccaggcac 2580 aggcagggca ggaccagccc acgctgacta cagggccgcc ggcaataaaa gcccaggagc 2640 ccatttggag ggcctgggcc tggctccctc actctcagga aatgctgacc catgggcagg 2700 agactgtgga gactgctcct gagcccccag cttccagcag gagggacagt ctcaccattt 2760 ccccagggca cgtggttgag tggggggaac gcccacttcc ctgggttaga ctgccagctc 2820 ttcctagctg gagaggagcc ctgcctctcc gcccctgagc ccactgtgcg tggggctccc 2880 gcctccaacc cctcgcccag tcccagcagc cagccaaaca cacagaaggg gactgccacc 2940 <h2 style=";text-align:left;direction:ltr">tccccttgcc agctgctgag ccgcagagaa gtgacggttc ctacacagga caggggttcc 3000<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttctgggcat tacatcgcat agaaatcaat aatttgtggt gatttggatc tgtgttttaa 3060<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgagtttcac agtgtgattt tgattattaa ttgtgcaagc ttttcctaat aaacgtggag 3120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aatcacaggc tggggctggggc actgctctca ccttggttcc tggggcatcc atggggtctc 3180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcacagacag gacccctgca gttcccctgg aagcagtgcc caggtggctg tggaatagga 3240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acgctaaaaa aaaaaaa aa 3262<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 19<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 907<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 19<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Asp Thr Ser Arg Leu Gly Val Leu Leu Ser Leu Pro Val Leu Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gln Leu Ala Thr Gly Gly Ser Ser Pro Arg Ser Gly Val Leu Leu Arg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Cys Pro Thr His Cys His Cys Glu Pro Asp Gly Arg Met Leu Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Arg Val Asp Cys Ser Asp Leu Gly Leu Ser Glu Leu Pro Ser Asn Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> Ser Val Phe Thr Ser Tyr Leu Asp Leu Ser Met Asn Asn Ile Ser Gln 65 70 75 80 Leu Leu Pro Asn Pro Leu Pro Ser Leu Arg Phe Leu Glu Glu Leu Arg 85 90 95 Leu Ala Gly Asn Ala Leu Thr Tyr Ile Pro Lys Gly Ala Phe Thr Gly 100 105 110 Leu Tyr Ser Leu Lys Val Leu Met Leu Gln Asn Asn Gln Leu Arg His 115 120 125 Val Pro Thr Glu Ala Leu Gln Asn Leu Arg Ser Leu Gln Ser Leu Arg 130 135 140 Leu Asp Ala Asn His Ile Ser Tyr Val Pro Pro Ser Cys Phe Ser Gly 145 150 155 160 Leu His Ser Leu Arg His Leu Trp Leu Asp Asp Asn Ala Leu Thr Glu 165 170 175 Ile Pro Val Gln Ala Phe Arg Ser Leu Ser Ala Leu Gln Ala Met Thr 180 185 190 Leu Ala Leu Asn Lys Ile His His Ile Pro Asp Tyr Ala Phe Gly Asn 195 200 205 Leu Ser Ser Leu Val Val Leu His Leu His Asn Asn Arg Ile His Ser 210 215 220 Leu Gly Lys Lys Cys Phe Asp Gly Leu His Ser Leu Glu Thr Leu Asp 225 230 235 240 Leu Asn Tyr Asn Asn Leu Asp Glu Phe Pro Thr Ala Ile Arg Thr Leu 245 250 255 Ser Asn Leu Lys Glu Leu Gly Phe His Ser Asn Asn Ile Arg Ser Ile 260 265 270 Pro Glu Lys Ala Phe Val Gly Asn Pro Ser Leu Ile Thr Ile His Phe 275 280 285 Tyr Asp Asn Pro Ile Gln Phe Val Gly Arg Ser Ala Phe Gln His Leu 290 295 300 Pro Glu Leu Arg Thr Leu Thr Leu Asn Gly Ala Ser Gln Ile Thr Glu 305 310 315 320 Phe Pro Asp Leu Thr Gly Thr Ala Asn Leu Glu Ser Leu Thr Leu Thr 325 330 335 Gly Ala Gln Ile Ser Ser Leu Pro Gln Thr Val Cys Asn Gln Leu Pro 340 345 350 Asn Leu Gln Val Leu Asp Leu Ser Tyr Asn Leu Leu Glu Asp Leu Pro 355 360 365 Ser Phe Ser Val Cys Gln Lys Leu Gln Lys Ile Asp Leu Arg His Asn 370 375 380 Glu Ile Tyr Glu Ile Lys Val Asp Thr Phe Gln Gln Leu Leu Ser Leu 385 390 395 400 Arg Ser Leu Asn Leu Ala Trp Asn Lys Ile Ala Ile Ile His Pro Asn 405 410 415 Ala Phe Ser Thr Leu Pro Ser Leu Ile Lys Leu Asp Leu Ser Ser Asn 420 425 430 Leu Leu Ser Ser Phe Pro Ile Thr Gly Leu His Gly Leu Thr His Leu 435 440 445 Lys Leu Thr Gly Asn His Ala Leu Gln Ser Leu Ile Ser Ser Glu Asn 450 455 460 Phe Pro Glu Leu Lys Val Ile Glu Met Pro Tyr Ala Tyr Gln Cys Cys 465 470 475 480 Ala Phe Gly Val Cys Glu Asn Ala Tyr Lys Ile Ser Asn Gln Trp Asn 485 490 495 Lys Gly Asp Asn Ser Ser Met Asp Asp Leu His Lys Lys Asp Ala Gly 500 505 510 Met Phe Gln Ala Gln Asp Glu Arg Asp Leu Glu Asp Phe Leu Leu Asp 515 520 525 Phe Glu Glu Asp Leu Lys Ala Leu His Ser Val Gln Cys Ser Pro Ser 530 535 540 Pro Gly Pro Phe Lys Pro Cys Glu His Leu Leu Asp Gly Trp Leu Ile 545 550 555 560 Arg Ile Gly Val Trp Thr Ile Ala Val Leu Ala Leu Thr Cys Asn Ala 565 570 575 Leu Val Thr Ser Thr Val Phe Arg Ser Pro Leu Tyr Ile Ser Pro Ile 580 585 590 Lys Leu Leu Ile Gly Val Ile Ala Ala Val Asn Met Leu Thr Gly Val 595 600 605 Ser Ser Ala Val Leu Ala Gly Val Asp Ala Phe Thr Phe Gly Ser Phe 610 615 620 Ala Arg His Gly Ala Trp Trp Glu Asn Gly Val Gly Cys His Val Ile 625 630 635 640 Gly Phe Leu Ser Ile Phe Ala Ser Glu Ser Ser Val Phe Leu Leu Thr 645 650 655 Leu Ala Ala Leu Glu Arg Gly Phe Ser Val Lys Tyr Ser Ala Lys Phe 660 665 670 Glu Thr Lys Ala Pro Phe Ser Ser Leu Lys Val Ile Ile Leu Leu Cys 675 680 685 Ala Leu Leu Ala Leu Thr Met Ala Ala Val Pro Leu Leu Gly Gly Ser 690 695 700 Lys Tyr Gly Ala Ser Pro Leu Cys Leu Pro Leu Pro Phe Gly Glu Pro 705 710 715 720 Ser Thr Met Gly Tyr Met Val Ala Leu Ile Leu Leu Asn Ser Leu Cys 725 730 735 Phe Leu Met Met Thr Ile Ala Tyr Thr Lys Leu Tyr Cys Asn Leu Asp 740 745 750 Lys Gly Asp Leu Glu Asn Ile Trp Asp Cys Ser Met Val Lys His Ile 755 760 765 Ala Leu Leu Leu Phe Thr Asn Cys Ile Leu Asn Cys Pro Val Ala Phe 770 775 780 Leu Ser Phe Ser Ser Leu Ile Asn Leu Thr Phe Ile Ser Pro Glu Val 785 790 795 800 Ile Lys Phe Ile Leu Leu Val Val Val Pro Leu Pro Ala Cys Leu Asn 805 810 815 Pro Leu Leu Tyr Ile Leu Phe Asn Pro His Phe Lys Glu Asp Leu Val 820 825 830 Ser Leu Arg Lys Gln Thr Tyr Val Trp Thr Arg Ser Lys His Pro Ser 835 840 845 Leu Met Ser Ile Asn Ser Asp Asp Val Glu Lys Gln Ser Cys Asp Ser 850 855 860 Thr Gln Ala Leu Val Thr Phe Thr Ser Ser Ser Ile Thr Tyr Asp Leu 865,870,875,880 Pro Pro Ser Ser Val Pro Ser Pro Ala Tyr Pro Val Thr Glu Ser Cys 885 890 895 His Leu Ser Ser Val Ala Phe Val Pro Cys Leu 900 905 <210> 20 <211> 4625 <212> DNA <213> Homo sapiens <400> 20 aaaaaacgag cgtgcaagca gagatgctgc tccacaccgc tcaggccgcg agcagcagca 60 aggcgcaccg ccactgtcgc cgctgcagcc agggctgctc cgaaggccgg cgtggcggca 120 accggcacct ctgtccccgc cgcgctctc ctcgccgccc acgccgtggg gtcaggaacg 180 cggcgtctgg cgctgcagac gccgctgag ttgcagaagc ccacggagcg gcgcccggcg 240 cgccacggcc cgtagcagtc cggtgctgct ctccgcccgc gtccggctcg tggccccta 300 cttcgggcac catggacacc tcccggctcg gtgtgctcct gtccttgcct gtgctgctgc 360 agctggcgac cgggggcagc tctcccaggt ctggtgtgtt gctgaggggc tgccccacac 420 actgtcattg cgagcccgac ggcaggatgt tgctcagggt ggactgctcc gacctggggc 480 tctcggagct gccttccaac ctcagcgtct tcacctccta cctagacctc agtatgaaca 540 acatcagtca gctgctcccg aatcccctgc ccagtctccg cttcctggag gagttacgtc 600 ttgcgggaaa cgctctgaca tacattccca agggagcatt cactggcctt tacagtctta 660 aagttcttat gctgcagaat aatcagctaa gacacgtacc cacagaagct ctgcagaatt 720 tgcgaagcct tcaatccctg cgtctggatg ctaaccacat cagctatgtg cccccaagct 780 gtttcagtgg cctgcattcc ctgaggcacc tgtggctgga tgacaatgcg ttaacagaaa 840 tccccgtcca ggcttttaga agtttatcgg cattgcaagc catgaccttg gccctgaaca 900 aaatacacca cataccagac tatgcctttg gaaacctctc cagcttggta gttctacatc 960 tccataacaa tagaatccac tccctgggaa agaaatgctt tgatgggctc cacagcctag 1020 agactttaga tttaaattac aataaccttg atgaattccc cactgcaatt aggacactct 1080 ccaaccttaa agaactagga tttcatagca acaatatcag gtcgatacct gagaaagcat 1140 ttgtaggcaa cccttctctt attacaatac atttctatga caatcccatc cagtttgttg 1200 ggagatctgc ttttcaacat ttacctgaac taagaacact gactctgaat ggtgcctcac 1260 aaataactga atttcctgat ttaactggaa ctgcaaacct ggagagtctg actttaactg 1320 gagcacagat ctcatctctt cctcaaaccg tctgcaatca gttacctaat ctccaagtgc 1380 tagatctgtc ttacaaccta ttagaagatt tacccagttt ttcagtctgc caaaagcttc 1440 agaaaattga cctaagacat aatgaaatct acgaaattaa agttgacact ttccagcagt 1500 tgcttagcct ccgatcgctg aatttggctt ggaacaaaat tgctattatt caccccaatg 1560 cattttccac tttgccatcc ctaataaagc tggacctatc gtccaacctc ctgtcgtctt 1620 ttcctataac tgggttacat ggtttaactc acttaaaatt aacaggaaat catgccttac 1680 agagcttgat atcatctgaa aactttccag aactcaaggt tatagaaatg ccttatgctt 1740 accagtgctg tgcatttgga gtgtgtgaga atgcctataa gatttctaat caatggaata 1800 aaggtgacaa cagcagtatg gacgaccttc ataagaaaga tgctggaatg tttcaggctc 1860 aagatgaacg tgaccttgaa gatttcctgc ttgactttga ggaagacctg aaagcccttc 1920 attcagtgca gtgttcacct tccccaggcc ccttcaaacc ctgtgaacac ctgcttgatg 1980 gctggctgat cagaattgga gtgtggacca tagcagttct ggcacttact tgtaatgctt 2040 tggtgacttc aacagttttc agatcccctc tgtacatttc ccccattaaa ctgttaattg 2100 gggtcatcgc agcagtgaac atgctcacgg gagtctccag tgccgtgctg gctggtgtgg 2160 atgcgttcac ttttggcagc tttgcacgac atggtgcctg gtgggagaat ggggttggtt 2220 gccatgtcat tggttttttg tccatttttg cttcagaatc atctgttttc ctgcttactc 2280 tggcagccct ggagcgtggg ttctctgtga aatattctgc aaaatttgaa acgaaagctc 2340 cattttctag cctgaaagta atcattttgc tctgtgccct gctggccttg accatggccg 2400 cagttcccct gctgggtggc agcaagtatg gcgcctcccc tctctgcctg cctttgcctt 2460 ttggggagcc cagcaccatg ggctacatgg tcgctctcat cttgctcaat tccctttgct 2520 tcctcatgat gaccattgcc tacaccaagc tctactgcaa tttggacaag ggagacctgg 2580 agaatatttg ggactgctct atggtaaaac acattgccct gttgctcttc accaactgca 2640 tcctaaactg ccctgtggct ttcttgtcct tctcctcttt aataaacctt acatttatca 2700 gtcctgaagt aattaagttt atccttctgg tggtagtccc acttcctgca tgtctcaatc 2760 cccttctcta catcttgttc aatcctcact ttaaggagga tctggtgagc ctgagaaagc 2820 aaacctacgt ctggacaaga tcaaaacacc caagcttgat gtcaattaac tctgatgatg 2880 tcgaaaaaca gtcctgtgac tcaactcaag ccttggtaac cttaccagc tccagcatca 2940 cttatgacct gcctcccagt tccgtgccat caccagctta tccagtgact gagagctgcc 3000 atctttcctc tgtggcattt gtcccatgtc tctaattaat atgtgaagga aaatgttttc 3060 aaaggttgag aacctgaaaa tgtgagattg agtatatcag agcagtaatt aataagaaga 3120 gctgaggtga aactcggttt aaaaaccaaa aaagaatctc tcagttagta agaaaaggct 3180 gaaaacctct tgatacttga gagtgaatat aagtctaaat gctgctttgt ataatttgtt 3240 cagctaaggg atagatcgat cacactattt aagtgagccc agatcaaaaa agcagattga 3300 aattttcttt agaaaagatt ctccatgatt tgaattgcat tctctttaaa ctcaccaatg 3360 taatcatttt gggaggaggg agaacccact tgctttccaa atgggtttat ttaaacccac 3420 aaactcaaga ggttgttggg ggaattagga aaataagggt tttcaatgac ctacattgct 3480 aggtagaggc tgtgatccat gggatttcat tctaatgacc atgtgaagat gtttgagtcc 3540 tcctttgcct ttcctcagaa agaatccttc taaggcacaa atcccttaga tggataatgt 3600 aaggtattgt taactcactc atattgagat catttttaga gataccaggt tttatgtatc 3660 agcactagat ggttccaccc tcatgggata aaactgctta caagtatttt gaaagaaaaa 3720 ctgaccaaaa ttcttaaatt gttactaagg caatcatgca caggtgacgt atgtcttatc 3780 tgatttgttt ttaactcctt ggtgcccaaa gctcagaagg gaattccact gccagcaatg 3840 aacatacctg gaaaagaaag taagcaatct gggatttttt ttctgggtta gtaaagaatt 3900 tttgcaataa gttttatcag ttgattcaaa ctgatgtgca tcttaatgat caaatgtgca 3960 cattacataa attaagtcca ctgatacaac ttcttacaca tgtatctcta gtagctctgg 4020 caaacccaat atctgacacc actttggact caagagactc agtaacgtat tatcctgttt 4080 atttagcttg gttttagctg tgttctctct ggataaccca cttgatgtta ggaacattac 4140 ttctctgctt attccatatt aatactgtgt taggtatttt aagaagcaag ttattaaata 4200 agaaaagtca aagtattaat tcttacctt tattatccta tattagcttc aatacatcca 4260 aaccaaatgg ctgttaggta gatttttt tatataagca tgtttttt gatcagatgt 4320 tttaacttgg atttgaaaaa atacatttat gagatgttt ataagatgtg taaatataga 4380 actgtattta ttactatagt aaaggttcag taacattaag gaccatgata atgataataa 4440 accttgtaca gtggcatatt ctttgattta tattgtgttt ctctgcccat tttctttaaa 4500 ttcattaact gtatatatgt aaatatatag tacttgtaaa tagattccaa atttgctttt 4560 ctattgggta aaaaataaat ttgtaataaa atgtgtgact atgaaacaaa aaaaaaaaaa 4620 aaaaa 4625 <210> 21 <211> 332 <212> PRT <213> Homo sapiens <400> 21 Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu Glu 1 5 10 15 Gln Thr Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly 20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu 35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met Asp 50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly 65 70 75 80 Lys Asp Tyr Asn Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr Ala 85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg 100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr Ser 115 120 125 Pro Asn Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Val Leu Gly Glu 180 185 190 His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Met Asn Val Ala Gly 195 200 205 Val Ser Leu Lys Thr Leu His Pro Asp Leu Gly Thr Asp Lys Asp Lys 210 215 220 Glu Gln Trp Lys Glu Val His Lys Gln Val Val Glu Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro 260 265 270 Val Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Asp Asp Val Phe 275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Leu Val 290 295 300 Lys Val Thr Leu Thr Ser Glu Glu Glu Ala Arg Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe 325 330 <210> 22 <211> 2226 <212> DNA <213> Homo sapiens <400> 22 gtctgccggt cggttgtctg gctgcgcgg ccacccgggc ctctccagtg cccgcctgg 60 ctcggcatcc acccccagcc cgactcacac gtgggttccc gcacgtccgc cggccccccc 120 cgctgacgtc agcatagctg ttccacttaa ggcccctccc gcgcccagct cagagtgctg 180 cagccgctgc cgccgattc ggatctcatt gccacgcgcc cccgacgacc gccgacgtg 240 cattcccgat tccttttggt tccaagtcca atatggcaac tctaaaggat cagctgattt 300 ataatcttct aaaggaagaa cagacccccc agaataagat tacagttgtt ggggttggtg 360 ctgttggcat ggcctgtgcc atcagtatct taatgaagga cttggcagat gaacttgctc 420 ttgttgatgt catcgaagac aaattgaagg gagagatgat ggatctccaa catggcagcc 480 ttttccttag aacaccaaag attgtctctg gcaaagacta taatgtaact gcaaactcca 540 agctggtcat tatcacggct ggggcacgtc agcaagaggg agaaagccgt cttaatttgg 600 tccagcgtaa cgtgaacatc tttaaattca tcattcctaa tgttgtaaaa tacagcccga 660 actgcaagtt gcttattgtt tcaaatccag tggatatctt gacctacgtg gcttggaaga 720 taagtggttt tcccaaaac cgtgttattg gaagcggttg caatctggat tcagcccgat 780 tccgttacct aatggggaa aggctgggag ttcacccatt aagctgtcat gggtgggtcc 840 ttgggggaaca tggagattcc agtgtgcctg tatggagtgg aatgaatgtt gctggtgtct 900 ctctgaagac tctgcaccca gatttaggga ctgataaaga taggaacag tggaaagagg 960 ttcacaagca ggtggttgag agtgcttatg aggtgatcaa actcaaaggc tacacatcct 1020 gggctattgg actctctgta gcagatttgg cagagagtat aatgaagaat cttaggcggg 1080 tgcacccagt ttccaccatg attaagggtc tttacggaat aaaggatgat gtttcctta 1140 gtgttccttg cattttggga agaatggaa tctcagacct tgtgaaggtg actctgactt 1200 ctgaggaaga ggcccgtttg aagaagagtg cagatacact ttgggggatc caaaaggagc 1260 tgcaatttta aagtcttctg atgtcatatc atttcactgt ctaggctaca acaggattct 1320 aggtggaggt tgtgcatgtt gtccttttta tctgatctgt gattaaagca gtaatatttt 1380 aagatggact gggaaaaaca tcaactcctg aagttagaaa taagaatggt ttgtaaaatc 1440 cacagctata tcctgatgct ggatggtatt aatcttgtgt agtcttcaac tggttagtgt 1500 gaaatagttc tgccacctct gacgcaccac tgccaatgct gtacgtactg catttgcccc 1560 ttgagccagg tggatgttta ccgtgtgtta tataacttcc tggctccttc actgaacatg 1620 cctagtccaa cattttttcc cagtgagtca catcctggga tccagtgtat aaatccaata 1680 tcatgtcttg tgcataattc ttccaaagga tcttattttg tgaactatat cagtagtgta 1740 cattaccata taatgtaaaa agatctacat acaaacaatg caaccaacta tccaagtgtt 1800 ataccaacta aaacccccaa taaaccttga acagtgacta ctttggttaa ttcattatat 1860 taagatataa agtcataaag ctgctagtta ttatattaat ttggaaatat taggctattc 1920 ttgggcaacc ctgcaacgat tttctac agggatatta tgactaata gcagaggatg 1980 taatagtcaa ctgagttgta ttggtaccac ttccattgta agtcccaag tattatatat 2040 ttgataataa tgctaatcat attggaag taacattcta tatgtaatg taaaatttat 2100 ttgccaactg atataggca atgatagtgt gtcactatag ggaacacaga tttttgagat 2160 cttgtcctct ggaagctggt aaaaaaaaaaaaaaaaaaaaaaaaa 2220 AAAAA 2226 <210> 23 <211> 353 <212> PRT <213> Homo sapiens <400> 23 Met Ala Ala Glu Leu Ala Met Gly Ala Glu Leu Pro Ser Ser Pro Leu 1 5 10 15 Ala Ile Glu Tyr Val Asn Asp Phe Asp Leu Met Lys Phe Glu Val Lys 20 25 30 Lys Glu Pro Pro Glu Ala Glu Arg Phe Cys His Arg Leu Pro Pro Gly 35 40 45 Ser Leu Ser Ser Thr Pro Leu Ser Thr Pro Cys Ser Ser Val Pro Ser 50 55 60 Ser Pro Ser Phe Cys Ala Pro Ser Pro Gly Thr Gly Gly Gly Gly Gly 65 70 75 80 Ala Gly Gly Gly Gly Gly Ser Ser Gln Ala Gly Gly Ala Pro Gly Pro 85 90 95 Pro Ser Gly Gly Pro Gly Ala Val Gly Gly Thr Ser Gly Lys Pro Ala 100 105 110 Leu Glu Asp Leu Tyr Trp Met Ser Gly Tyr Gln His His Leu Asn Pro 115 120 125 Glu Ala Leu Asn Leu Thr Pro Glu Asp Ala Val Glu Ala Leu Ile Gly 130 135 140 Ser Gly His His Gly Ala His His Gly Ala His His Pro Ala Ala Ala 145 150 155 160 Ala Ala Tyr Glu Ala Phe Arg Gly Pro Gly Phe Ala Gly Gly Gly Gly 165 170 175 Ala Asp Asp Met Gly Ala Gly His His His Gly Ala His His Ala Ala 180 185 190 His His His His Ala Ala His His His His His His His His His His 195 200 205 Gly Gly Ala Gly His Gly Gly Gly Ala Gly His His Val Arg Leu Glu 210 215 220 Glu Arg Phe Ser Asp Asp Gln Leu Val Ser Met Ser Val Arg Glu Leu 225 230 235 240 Asn Arg Gln Leu Arg Gly Phe Ser Lys Glu Glu Val Ile Arg Leu Lys 245 250 255 Gln Lys Arg Arg Thr Leu Lys Asn Arg Gly Tyr Ala Gln Ser Cys Arg 260 265 270 Phe Lys Arg Val Gln Gln Arg His Ile Leu Glu Ser Glu Lys Cys Gln 275 280 285 Leu Gln Ser Gln Val Glu Gln Leu Lys Leu Glu Val Gly Arg Leu Ala 290 295 300 Lys Glu Arg Asp Leu Tyr Lys Glu Lys Tyr Glu Lys Leu Ala Gly Arg 305 310 315 320 Gly Gly Pro Gly Ser Ala Gly Gly Ala Gly Phe Pro Arg Glu Pro Ser 325 330 335 Pro Pro Gln Ala Gly Pro Gly Gly Ala Lys Gly Thr Ala Asp Phe Phe 340 345 350 Leu <210> 24 <211> 2373 <212> DNA <213> Homo sapiens <400> 24 gcgcggccgg gcgcgggccc cgggcgatgg ccgcggagct ggcgatgggc gccgagctgc 60 ccagcagccc gctggccatc gagtacgtca acgacttcga cctgatgaag ttcgaggtga 120 agaaggagcc tcccgaggcc gagcgcttct gccaccgcct gccgccaggc tcgctgtcct 180 cgacgccgct cagcacgccc tgctcctccg tgccctcctc gcccagcttc tgcgcgccca 240 gcccgggcac cggcggcggc ggcggcgcgg ggggcggcgg cggctcgtct caggccgggg 300 gcgcccccgg gccgccgagc gggggccccg gcgccgtcgg gggcacctcg gggaagccgg 360 cgctggagga tctgtactgg atgagcggct accagcatca cctcaacccc gaggcgctca 420 acctgacgcc cgaggacgcg gtggaggcgc tcatcggcag cggccaccac ggcgcgcacc 480 acggcgcgca ccacccggcg gccgccgcag cctacgaggc tttccgcggc ccgggcttcg 540 cgggcggcgg cggagcggac gacatgggcg ccggccacca ccacggcgcg caccacgccg 600 cccaccatca ccacgccgcc caccaccacc accaccacca ccaccaccat ggcggcgcgg 660 gacacggcgg tggcgcgggc caccacgtgc gcctggagga gcgcttctcc gacgaccagc 720 tggtgtccat gtcggtgcgc gagctgaacc ggcagctccg cggcttcagc aaggaggag 780 tcatccggct caagcaag cggcgcacgc tcaagaaccg cggctacgcg cagtcctgcc 840 gcttcaagcg ggtgcagcag cggcacattc tggagagcga gaagtgccaa ctccagagcc 900 aggtggagca gctgaagctg gaggtggggc gcctggccaa agagcgggac ctgtacaagg 960 agaatacga gaagctggcg ggccggggcg gccccgggag cgcgggcggg gccggtttcc 1020 cgcgggagcc ttcgccgccg caggccggtc ccggcggggc caagggcacg gccgacttct 1080 tcctgtaggc gccggacccc gagcccgcgc cgccgtcgcc ggggacaagt tcgcgcaggc 1140 ctctcggggc ctcggctcgg actccgcggt agaggacgtg gacaccaggc ccggcccggc 1200 cgtgctggcc ccggtgccaa gtctgcgggc gcggggctgg aggccccttc gctcccggtc 1260 cccgttcgcg cgcgtcggcc cgggtcgccg tcctgaggtt gagcggagaa cggtgatttc 1320 taggaaact tgagccaggt ctaacttctt tccaagcgtc cgcttgtaca tacgttgaac 1380 gtggttctcc gttcccacct tcgccctgcc agcctagagg gaccgcgctg ccgtcccttc 1440 ccggggtggcc cctgcctgcc cccgccctcc ttcgttctct tctcagcctc cctttccttg ccttttttaa cttcccctcc ccgttttaa atcggtctta ttttcgaagt atttataatt attatgcttg gtgattagaa aagaaaacct tggaggaagc cccttctttc cccagccggg gtccgccctc agtcgcgagt cacagcatga gtcgctcgcc aggaggggcc cggcccctgc 1680 ctgccccctc cccgcttgcc cccgaccctg ctaccggcgt tccttggagg tcgaagccag 1740. ggacgtcacc cgtgctgtgt ccaggcctgc tgtcctacta tgctcaaccg ggggtggggg 1800. gaggggggtg agtcctgtgc tcagtcgggt gggggctggc ccggatcccg agctgctgtc 1860 tctctatgca ccagaacata tctgtaactc ctggggaaat acatcttgtt ttaaccttca aagaagaagtga aagaaaaaag taatgcacag tatttctagc agaaaatttt tttttttaag aggaggcttg ggccagagcc ttctggcatg gggcgggtgg agaaagtgtt tttattttaa tttaaattgt gtttcgtttt gtttgtgga tctttcttta atgcttcgtc gctctttgga ctagccgggga gagagggcgga ggaggcggggt gctccaggcc ctgtaggctg ggccaggcgc 2160 ctgggggatc tgcccgtttt cggaggccct caggggccat caggggatt cggaccgctc 2220 cacacccctc ccctgagcac tcggagtgga aggcgccg actcgttgaa agttttgttg 2280 tgtagttggt ttcgttgag ttctttttc atttgctacg aaactgagaa aaagaaaaaa 2340 attackaaaaaatctgtt international tca 2373 <210> 25 <211> 1939 <212> PRT <213> Homo sapiens <400> 25 Met Thr Asp Ala Gln Met Ala Asp Phe Gly Ala Ala Gln Tyr Leu 1 5 10 15 Arg Lys Ser Glu Lys Glu Arg Leu Glu Ala Gln Thr Arg Pro Phe Asp 20 25 30 Ile Arg Thr Glu Cys Phe Val Pro Asp Asp Lys Glu Glu Phe Val Lys 35 40 45 Only Lys Ile Leu Serves Arg Glu Gly Gly Lys Val Ile Only Glu Thr Glu 50 55 60 Asn Gly Lys Thr Val Thr Val Lys Glu Asp Gln Val Leu Gln Gln Asn 65 70 75 80 Pro Pro Lys Phe Asp Lys Ile Glu Asp Met Ala Met Leu Thr Phe Leu 85 90 95 His Glu Pro Ala Val Leu Phe Asn Leu Lys Glu Arg Tyr Ala Ala Trp 100 105 110 Met Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Thr Val Asn Pro Tyr 115 120 125 Lys Trp Leu Pro Val Tyr Asn Ala Glu Val Val Ala Ala Tyr Arg Gly 130 135 140 Lys Lys Arg Ser Glu Ala Pro Pro His Ile Phe Ser Ile Ser Asp Asn 145 150 155 160 Ala Tyr Gln Tyr Met Leu Thr Asp Arg Glu Asn Gln Ser Ile Leu Ile 165 170 175 Thr Gly Glu Ser Gly Ala Gly Lys Thr Val Asn Thr Lys Arg Val Ile 180 185 190 Gln Tyr Phe Ala Ser Ile Ala Ala Ile Gly Asp Arg Gly Lys Lys Asp 195 200 205 Asn Ala Asn Ala Asn Lys Gly Thr Leu Glu Asp Gln Ile Ile Gln Ala 210 215 220 Asn Pro Ala Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Arg Asn Asp 225 230 235 240 Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile His Phe Gly Ala Thr 245 250 255 Gly Lys Leu Ala Ser Ala Asp Ile Glu Thr Tyr Leu Leu Glu Lys Ser 260 265 270 Arg Val Ile Phe Gln Leu Lys Ala Glu Arg Asn Tyr His Ile Phe Tyr 275 280 285 Gln Ile Leu Ser Asn Lys Lys Pro Glu Leu Leu Asp Met Leu Leu Val 290 295 300 Thr Asn Asn Pro Tyr Asp Tyr Ala Phe Val Ser Gln Gly Glu Val Ser 305 310 315 320 Val Ala Ser Ile Asp Asp Ser Glu Glu Leu Met Ala Thr Asp Ser Ala 325 330 335 Phe Asp Val Leu Gly Phe Thr Ser Glu Glu Lys Ala Gly Val Tyr Lys 340 345 350 Leu Thr Gly Ala Ile Met His Tyr Gly Asn Met Lys Phe Lys Gln Lys 355 360 365 Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Asp Ala Asp Lys 370 375 380 Ser Ala Tyr Leu Met Gly Leu Asn Ser Ala Asp Leu Leu Lys Gly Leu 385 390 395 400 Cys His Pro Arg Val Lys Val Gly Asn Glu Tyr Val Thr Lys Gly Gln 405 410 415 Ser Val Gln Gln Val Tyr Tyr Ser Ile Gly Ala Leu Ala Lys Ala Val 420 425 430 Tyr Glu Lys Met Phe Asn Trp Met Val Thr Arg Ile Asn Ala Thr Leu 435 440 445 Glu Thr Lys Gln Pro Arg Gln Tyr Phe Ile Gly Val Leu Asp Ile Ala 450 455 460 Gly Phe Glu Ile Phe Asp Phe Asn Ser Phe Glu Gln Leu Cys Ile Asn 465 470 475 480 Phe Thr Asn Glu Lys Leu Gln Gln Phe Phe Asn His His Met Phe Val 485 490 495 Leu Glu Gln Glu Glu Tyr Lys Lys Glu Gly Ile Glu Trp Thr Phe Ile 500 505 510 Asp Phe Gly Met Asp Leu Gln Ala Cys Ile Asp Leu Ile Glu Lys Pro 515 520 525 Met Gly Ile Met Ser Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ala 530 535 540 Thr Asp Met Thr Phe Lys Ala Lys Leu Tyr Asp Asn His Leu Gly Lys 545 550 555 560 Ser Asn Asn Phe Gln Lys Pro Arg Asn Ile Lys Gly Lys Gln Glu Ala 565 570 575 His Phe Ser Leu Ile His Tyr Ala Gly Thr Val Asp Tyr Asn Ile Leu 580 585 590 Gly Trp Leu Glu Lys Asn Lys Asp Pro Leu Asn Glu Thr Val Val Ala 595 600 605 Leu Tyr Gln Lys Ser Ser Leu Lys Leu Met Ala Thr Leu Phe Ser Ser 610 615 620 Tyr Ala Thr Ala Asp Thr Gly Asp Ser Gly Lys Ser Lys Gly Gly Lys 625 630 635 640 Lys Lys Gly Ser Ser Phe Gln Thr Val Ser Ala Leu His Arg Glu Asn 645 650 655 Leu Asn Lys Leu Met Thr Asn Leu Arg Thr Thr His Pro His Phe Val 660 665 670 Arg Cys Ile Ile Pro Asn Glu Arg Lys Ala Pro Gly Val Met Asp Asn 675 680 685 Pro Leu Val Met His Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile 690 695 700 Arg Ile Cys Arg Lys Gly Phe Pro Asn Arg Ile Leu Tyr Gly Asp Phe 705 710 715 720 Arg Gln Arg Tyr Arg Ile Leu Asn Pro Val Ala Ile Pro Glu Gly Gln 725 730 735 Phe Ile Asp Ser Arg Lys Gly Thr Glu Lys Leu Leu Ser Ser Leu Asp 740 745 750 Ile Asp His Asn Gln Tyr Lys Phe Gly His Thr Lys Val Phe Phe Lys 755 760 765 Ala Gly Leu Leu Gly Leu Leu Glu Glu Met Arg Asp Glu Arg Leu Ser 770 775 780 Arg Ile Ile Thr Arg Met Gln Ala Gln Ala Arg Gly Gln Leu Met Arg 785 790 795 800 Ile Glu Phe Lys Lys Ile Val Glu Arg Arg Asp Ala Leu Leu Val Ile 805 810 815 Gln Trp Asn Ile Arg Ala Phe Met Gly Val Lys Asn Trp Pro Trp Met 820 825 830 Lys Leu Tyr Phe Lys Ile Lys Pro Leu Leu Lys Ser Ala Glu Thr Glu 835 840 845 Lys Glu Met Ala Thr Met Lys Glu Glu Phe Gly Arg Ile Lys Glu Thr 850 855 860 Leu Glu Lys Ser Glu Ala Arg Arg Lys Glu Leu Glu Glu Lys Met Val 865 870 875 880 Ser Leu Leu Gln Glu Lys Asn Asp Leu Gln Leu Gln Val Gln Ala Glu 885 890 895 Gln Asp Asn Leu Asn Asp Ala Glu Glu Arg Cys Asp Gln Leu Ile Lys 900 905 910 Asn Lys Ile Gln Leu Glu Ala Lys Val Lys Glu Met Asn Glu Arg Leu 915 920 925 Glu Asp Glu Glu Glu Met Asn Ala Glu Leu Thr Ala Lys Lys Arg Lys 930 935 940 Leu Glu Asp Glu Cys Ser Glu Leu Lys Lys Asp Ile Asp Asp Leu Glu 945 950 955 960 Leu Thr Leu Ala Lys Val Glu Lys Glu Lys His Ala Thr Glu Asn Lys 965 970 975 Val Lys Asn Leu Thr Glu Glu Met Ala Gly Leu Asp Glu Ile Ile Ala 980 985 990 Lys Leu Thr Lys Glu Lys Lys Ala Leu Gln Glu Ala His Gln Gln Ala 995 1000 1005 Leu Asp Asp Leu Gln Val Glu Glu Asp Lys Val Asn Ser Leu Ser 1010 1015 1020 Lys Ser Lys Val Lys Leu Glu Gln Gln Val Asp Asp Leu Glu Gly 1025 1030 1035 Ser Leu Glu Gln Glu Lys Lys Val Arg Met Asp Leu Glu Arg Ala 1040 1045 1050 Lys Arg Lys Leu Glu Gly Asp Leu Lys Leu Thr Gln Glu Ser Ile 1055 1060 1065 Met Asp Leu Glu Asn Asp Lys Leu Gln Leu Glu Glu Lys Leu Lys 1070 1075 1080 Light Light Glu Phe Asp Ile Asn Gln Gln Asn Ser Light Ile Glu Asp 1085 1090 1095 Glu Gln Val Leu Ala Leu Gln Leu Gln Lys Lys Leu Lys Glu Asn 1100 1105 1110 Gln Ala Arg Ile Glu Glu Leu Glu Glu Glu Leu Glu Ala Glu Arg 1115 1120 1125 Thr Ala Arg Ala Lys Val Glu Lys Leu Arg Ser Asp Leu Ser Arg 1130 1135 1140 Glu Leu Glu Glu Ile Ser Glu Arg Leu Glu Glu Ala Gly Gly Ala 1145 1150 1155 Thr Ser Val Gln Ile Glu Met Asn Lys Lys Arg Glu Ala Glu Phe 1160 1165 1170 Gln Lys Met Arg Arg Asp Leu Glu Glu Ala Thr Leu Gln His Glu 1175 1180 1185 Ala Thr Ala Ala Ala Leu Arg Lys Lys His Ala Asp Ser Val Ala 1190 1195 1200 Glu Leu Gly Glu Gln Ile Asp Asn Leu Gln Arg Val Lys Gln Lys 1205 1210 1215 Leu Glu Lys Glu Lys Ser Glu Phe Lys Leu Glu Leu Asp Asp Val 1220 1225 1230 Thr Ser Asn Met Glu Gln Ile Ile Lys Ala Lys Ala Asn Leu Glu 1235 1240 1245 Lys Val Ser Arg Thr Leu Glu Asp Gln Ala Asn Glu Tyr Arg Val 1250 1255 1260 Lys Leu Glu Glu Ala Gln Arg Ser Leu Asn Asp Phe Thr Thr Gln 1265 1270 1275 Arg Ala Lys Leu Gln Thr Glu Asn Gly Glu Leu Ala Arg Gln Leu 1280 1285 1290 Glu Glu Lys Glu Ala Leu Ile Ser Gln Leu Thr Arg Gly Lys Leu 1295 1300 1305 Ser Tyr Thr Gln Gln Met Glu Asp Leu Lys Arg Gln Leu Glu Glu 1310 1315 1320 Glu Gly Lys Ala Lys Asn Ala Leu Ala His Ala Leu Gln Ser Ala 1325 1330 1335 Arg His Asp Cys Asp Leu Leu Arg Glu Gln Tyr Glu Glu Glu Thr 1340 1345 1350 Glu Ala Lys Ala Glu Leu Gln Arg Val Leu Ser Lys Ala Asn Ser 1355 1360 1365 Glu Val Ala Gln Trp Arg Thr Lys Tyr Glu Thr Asp Ala Ile Gln 1370 1375 1380 Arg Thr Glu Glu Leu Glu Glu Ala Lys Lys Lys Leu Ala Gln Arg 1385 1390 1395 Leu Gln Asp Ala Glu Glu Ala Val Glu Ala Val Asn Ala Lys Cys 1400 1405 1410 Ser Ser Leu Glu Lys Thr Lys His Arg Leu Gln Asn Glu Ile Glu 1415 1420 1425 Asp Leu Met Val Asp Val Glu Arg Ser Asn Ala Ala Ala Ala Ala 1430 1435 1440 Leu Asp Lys Lys Gln Arg Asn Phe Asp Lys Ile Leu Ala Glu Trp 1445 1450 1455 Lys Gln Lys Tyr Glu Glu Ser Gln Ser Glu Leu Glu Ser Ser Gln 1460 1465 1470 Lys Glu Ala Arg Ser Leu Ser Thr Glu Leu Phe Lys Leu Lys Asn 1475 1480 1485 Ala Tyr Glu Glu Ser Leu Glu His Leu Glu Thr Phe Lys Arg Glu 1490 1495 1500 Asn Lys Asn Leu Gln Glu Glu Ile Ser Asp Leu Thr Glu Gln Leu 1505 1510 1515 Gly Glu Gly Gly Lys Asn Val His Glu Leu Glu Lys Val Arg Lys 1520 1525 1530 Gln Leu Glu Val Glu Lys Leu Glu Leu Gln Ser Ala Leu Glu Glu 1535 1540 1545 Ala Glu Ala Ser Leu Glu His Glu Glu Gly Lys Ile Leu Arg Ala 1550 1555 1560 Gln Leu Glu Phe Asn Gln Ile Lys Ala Glu Ile Glu Arg Lys Leu 1565 1570 1575 Ala Glu Lys Asp Glu Glu Met Glu Gln Ala Lys Arg Asn His Gln 1580 1585 1590 Arg Val Val Asp Ser Leu Gln Thr Ser Leu Asp Ala Glu Thr Arg 1595 1600 1605 Ser Arg Asn Glu Val Leu Arg Val Lys Lys Lys Met Glu Gly Asp 1610 1615 1620 Leu Asn Glu Met Glu Ile Gln Leu Ser His Ala Asn Arg Met Ala 1625 1630 1635 Ala Glu Ala Gln Lys Gln Val Lys Ser Leu Gln Ser Leu Leu Lys 1640 1645 1650 Asp Thr Gln Ile Gln Leu Asp Asp Ala Val Arg Ala Asn Asp Asp 1655 1660 1665 Leu Lys Glu Asn Ile Ala Ile Val Glu Arg Arg Asn Asn Leu Leu 1670 1675 1680 Gln Ala Glu Leu Glu Glu Leu Arg Ala Val Val Glu Gln Thr Glu 1685 1690 1695 Arg Ser Arg Lys Leu Ala Glu Gln Glu Leu Ile Glu Thr Ser Glu 1700 1705 1710 Arg Val Gln Leu Leu His Ser Gln Asn Thr Ser Leu Ile Asn Gln 1715 1720 1725 Lys Lys Lys Met Glu Ser Asp Leu Thr Gln Leu Gln Ser Glu Val 1730 1735 1740 Glu Glu Ala Val Gln Glu Cys Arg Asn Ala Glu Glu Lys Ala Lys 1745 1750 1755 Lys Ala Ile Thr Asp Ala Ala Met Met Ala Glu Glu Leu Lys Lys 1760 1765 1770 Glu Gln Asp Thr Ser Ala His Leu Glu Arg Met Lys Lys Asn Met 1775 1780 1785 Glu Gln Thr Ile Lys Asp Leu Gln His Arg Leu Asp Glu Ala Glu 1790 1795 1800 Gln Ile Ala Leu Lys Gly Gly Lys Lys Gln Leu Gln Lys Leu Glu 1805 1810 1815 Ala Arg Val Arg Glu Leu Glu Gly Glu Leu Glu Ala Glu Gln Lys 1820 1825 1830 Arg Asn Ala Glu Ser Val Lys Gly Met Arg Lys Ser Glu Arg Arg 1835 1840 1845 Ile Lys Glu Leu Thr Tyr Gln Thr Glu Glu Asp Lys Lys Asn Leu 1850 1855 1860 Leu Arg Leu Gln Asp Leu Val Asp Lys Leu Gln Leu Lys Val Lys 1865 1870 1875 Ala Tyr Lys Arg Gln Ala Glu Glu Ala Glu Glu Gln Ala Asn Thr 1880 1885 1890 Asn Leu Ser Lys Phe Arg Lys Val Gln His Glu Leu Asp Glu Ala 1895 1900 1905 Glu Glu Arg Ala Asp Ile Ala Glu Ser Gln Val Asn Lys Leu Arg 1910 1915 1920 Ala Lys Ser Arg Asp Ile Gly Ala Lys Gln Lys Met His Asp Glu 1925 1930 1935 Glu <210> 26 <211> 5941 <212> DNA <213> Homo sapiens <400> 26 agatagagag actcctgcgg cccagattct tcaggattct ccgtgaaggg ataaccaggg 60 gaagcaccaa gatgaccgat gcccagatgg ctgactttgg ggcagcggcc cagtacctcc 120 gcaagtcaga gaaggagcgt ctagaggccc agacccggcc ctttgacatt cgcactgagt 180 gcttcgtgcc cgatgacaag gaagagtttg tcaaagccaa gattttgtcc cgggagggag 240 gcaaggtcat tgctgaaacc gagaatggga agacggtgac tgtgaaggag gaccaggtgt 300 tgcagcagaa cccacccaag ttcgacaaga ttgaggacat ggccatgctg accttcctgc 360 acgagcccgc ggtgcttttc aacctcaagg agcgctacgc ggcctggatg atatatacct 420 actcgggcct cttctgtgtc actgtcaacc cctacaagtg gctgccggtg tacaatgccg 480 aggtggtggc cgcctaccgg ggcaagaaga ggagtgaggc cccgccccac atcttctcca 540 tctccgacaa cgcctatcag tacatgctga cagatcggga gaaccagtcc atcctcatca 600 cgggagaatc cggggcgggg aagactgtga acaccaagcg tgtcatccag tactttgcca 660 gcattgcagc cataggtgac cgtggcaaga aggacaatgc caatgcgaac aagggcaccc 720 tggaggacca gatcatccag gccaaccccg ctctggaggc cttcggcaat gccaagactg 780 tccggaacga caactcctcc cgctttggga aattcattag gatccacttt ggggccactg 840 gaaagctggc ttctgcagac atagagacct acctgctgga gaagtcccgg gtgatcttcc 900 agctgaaagc tgagagaaac taccacatct tctaccagat tctgtccaac aagaagccgg 960 agttgctgga catgctgctg gtcaccaaca atccctacga ctacgccttc gtgtctcagg 1020 gagaggtgtc cgtggcctcc attgatgact ccgaggagct catggccacc gatagtgcct 1080 ttgacgtgct gggcttcact tcagaggaga aagctggcgt ctacaagctg acgggagcca 1140 tcatgcacta cgggaacatg aagttcaagc agaagcagcg ggaggagcag gcggagccag 1200 acggcaccga agatgctgac aagtcggcct acctcatggg gctgaactca gctgacctgc 1260 tcaaggggct gtgccaccct cgggtgaaag tgggcaacga gtatgtcacc aagggcaga 1320 gcgtgcagca ggtgtactac tccatcgggg ctctggccaa ggcagtgtat gagaagatgt 1380 tcaactggat ggtgacgcgc atcaacgcca ccctggagac caagcagcca cgccagtact 1440 tcataggagt cctggacatc gctggcttcg agatcttcga cttcaacagc tttgagcagc 1500 tctgcatcaa cttcaccaac gagaagctgc agcagttctt caaccaccac atgttcgtgc 1560 tggagcagga ggagtacaag aagggca ttgagtggac attcattgac tttggcatgg 1620 acctgcaggc ctgcattgac ctcatcgaga agcccatggg catcatgtcc atcctggagg 1680 aggagtgcat gttccccaag gccactgaca tgaccttcaa ggccaagctg tacgacaacc 1740 acctgggcaa gtccaacaat ttccagaagc cacgcaacat caaggggaag caggaagccc 1800 acttctccct gatccactac gccggcactg tggactacaa catcctgggc tggctgggaaa 1860 aaaacaagga tcctctcaac gagactgttg tggccctgta ccagaagtcc tccctcaagc 1920 tcatggccac tctcttctcc tcctacgcaa ctgccgatac tggggacagt ggtaaaagca 1980 aaagggcaa gaaaaagggc tcatccttcc agacggtgtc ggctctccac cgggaaaatc 2040 tcaacaagct aatgaccaac ctgaggacca cccatcctca ctttgtgcgt tgcatcatcc 2100 ccaatgagcg gaaggctcca ggggtgatgg acaaccccct ggtcatgcac cagctgcgct 2160 gcaatggcgt gctggagggc atccgcatct gcaggaaggg cttccccaac cgcatcctct 2220 acggggactt ccggcagagg tatcgcatcc tgaacccagt ggccatccct gagggacagt 2280 tcattgatag caggaaggg agagaagc tgctcagctc tctggacatt gatcacaacc 2340 agtacaagtt tggccacacc aaggtgttct tcaaggcagg gctgcttggg ctgctgggagg 2400 agatgcggga tgagaggctg agccgcatca tcacgcgcat gcaggcccaa gcccggggcc 2460 agctcatgcg cattgagttc aagaagatag tggaacgcag ggatgccctg ctggtaatcc 2520 agtggaacat tcgggccttc atggggtca agaattggcc ctggatgaag ctctacttca 2580 agatcaagcc gctgctgaag agcgcagaga cggagaaagga gatggccacc atgaaggaag 2640 agttcgggcg catcaaagag acgctggagaga agtccgaggc tcgccgcaag gagctgggagg 2700 agaagagatgt gtccctgctg caggagaga atgacctgca gctccaagtg caggcggaac 2760 aagacaacct caatgatgct gaggagcgct gcgaccagct gatxaaaac aagattcagc 2820 tggaggccaa aggaagagag atgaatgaga ggctggagga tgaggag atgaacgcgg 2880 agctcactgc caaagcgc aagctggaag acgagtgctc agagctcaag aaggacattg 2940 atgacctgga gctgacactg gccaaggtgg agagagaa gcatgcaca gagaacaagg 3000 tgagaacct aacagaggag atggctgggc tggatgaat catcgctaag ctgaccagg 3060 agaagaaagc tctacaagag gcccatcagc agccctgga tgaccttcag gttgaggaag 3120 acaaggtcaa cagcctgtcc aagtctagg tcaagctgga gcagcaggtg gatgatctgg 3180 agggatccct aaggaagagag aggaggtgc gcatggacct ggagcgagca agcggaaac 3240 tggaggcga cctgaagctg acccaggaga gcatcatgga cctggaaaat gataaactgc 3300 agctggaaga aaagcttaag aagaaggt ttgacattaa tcagcagaac agtaagattg 3360 aggatgagca gtgctggcc cttcactac agagaact gagagaaac caggcacgca 3420 tcgaggagct ggaggaggg ctggaggccg agcgcaccgc caggggctag gtggagaagc 3480 tgcgctcaga cctgtctcgg gagctggagg agatcagcga gcggctggaa gaggccggcg 3540 gggccacgtc cgtgcagatc gagatgaca agaagcgcga ggccgagttc cagaagagatgc 3600 ggcgggacct ggaggaggcc acgctgcagc acgaggccac tgccgcggcc ctgcgcaaga 3660 agcacgccga cagcgtggcc gagctggggcg agcagatcga caacctgcag cgggtgaagc 3720 agaagctgga gaaggagaag agcgagttca agctggagct ggatgacgtc acctccaaca 3780 tggagcagat catcaggcc aaggcaacc tggagaagt gtctcggacg ctggaggacc 3840 aggccaatga gtaccgcgtg aagctagaag agccccaacg ctccctcaat gatttcacca 3900 cccagcgagc caagctgcag accgagaatg gagagttggc ccggcagcta gaggaaaagg 3960 aggcgctaat ctcgcagctg acccggggga agctctctta tacccagcaa atggaggacc 4020 tcaaaggca gctggaggag gaggggcagg cgagaacgc cctggcccat gcactgcagt 4080 cggcccggca tgactgcgac ctgctgcggg agcagtacga ggaggagaca gaggccagg 4140 ccgagctgca gcgcgtcctg tccaggcca actcggaggt ggcccagtgg aggaccagt 4200 atgagacgga cgccattcag cggactgagg agctcgaaga ggccaaaag aagctggccc 4260 agcggctgca ggatgccgag gaggccgtgg aggctgttaa tgccaagtgc tcctcactgg 4320 agaagaccaa gcaccggcta cagaatgaga tagaggactt gatggtggac gtagagcgct 4380 ccaatgctgc tgctgcagcc ctggacaaga agcagagaaa ctttgacaag atcctggccg 4440 agtggaagca gaagtatgag gagtcgcagt ctgagctgga gtcctcacag aaggaggctc 4500 gctccctcag cacagagctc ttcaagctca agaacgccta cgaggagtcc ctggagcacc 4560 tagagacctt caagcgggag aacaagaacc ttcaggagga aatctcggac cttactgagc 4620 agctaggaga aggaggaag aatgtgcatg agctggagaa ggtccgcaaa cagctggagg 4680 tggagaagct ggagctgcag tcagccctgg aggaggcag ggcctccctg gagcacgagg 4740 agggcagat cctccgggcc cagcagagt tcaccagat cctcgggc atcgagcga 4800 agctggcaga gaggacgag gagatgaac agccaagcg caccaccag cgggtggtgg 4860 actcgctgca gacctccctg gatgcagaga cacgcagccg caacgaggtc ctgagggtga 4920 agaagaagat ggaaggagac ctcaatgaga tggagatcca gctcagccac gccaaccgca 4980 tggctgccga ggcccagaag caagtcaaga gcctccagag cttgctgaag gacacccaga 5040 tccagctgga cgatgcggtc cgtgccaacg acgacctgaa ggagaacatc gccatcgtgg 5100 agcggcgcaa caacctgctg caggctgagc tggaggagct gcgtgccgtg gtggagcaga 5160 cagagcggtc ccggaagctg gcggagcagg agctgattga gaccagcgag cgggtgcagc 5220 tgctgcattc ccagaacacc agcctcatca accagaagaa gaagatggag tcggatctga 5280 cccagctcca gtcggaagtg gaggaggcag tgcaggagtg cagaaacgcc gaggagaagg 5340 ccaagaaggc catcacggat gccgccatga tggcagagga gctgaagaag gagcaggaca 5400 ccagcgccca cctggagcgc atgaagaaga acatggagca gaccattaag gacctgcagc 5460 accggctgga cgaggccgag cagatcgccc tcaagggagg caagaagcag ctgcagaagc 5520 tggaagcgcg ggtgcgggag ctggagggtg agctggaggc cgagcagaag cgcaacgcag 5580 agtcggtgaa gggcatgagg aagagcgagc ggcgcatcaa ggagctcacc taccagacag 5640 aggaagacaa aaagaacctg ctgcggctac aggacctggt ggacaagctg caactgaagg 5700 tcaaggccta caagcgccag gccgaggagg cggaggagca agccaacacc aacctgtcca 5760 agttccgcaa ggtgcagcat gagctggatg aggcagagga gcgggcggac atcgctgagt 5820 cccaggtcaa caagcttcga gccaagagcc gtgacattg tgccaagcaa aaaatgcacg 5880 atgaggagtg acactgcctc gggaacctca cctttgccaa cctgtaataa atatgagtgc 5940 c 5941 <210> 27 <211> 175 <212> PRT <213> Homo sapiens <400> 27 Met Ala Ser Arg Lys Ala Gly Thr Arg Gly Lys Val Ala Ala Thr Lys 1 5 10 15 Gln Ala Gln Arg Gly Ser Ser Asn Val Phe Ser Met Phe Glu Gln Ala 20 25 30 Gln Ile Gln Glu Phe Lys Glu Ala Phe Ser Cys Ile Asp Gln Asn Arg 35 40 45 Asp Gly Ile Ile Cys Lys Ala Asp Leu Arg Glu Thr Tyr Ser Gln Leu 50 55 60 Gly Lys Val Ser Val Pro Glu Glu Glu Leu Asp Ala Met Leu Gln Glu 65 70 75 80 Gly Lys Gly Pro Ile Asn Phe Thr Val Phe Leu Thr Leu Phe Gly Glu 85 90 95 Lys Leu Asn Gly Thr Asp Pro Glu Glu Ala Ile Leu Ser Ala Phe Arg 100 105 110 Met Phe Asp Pro Ser Gly Lys Gly Val Val Asn Lys Asp Glu Phe Lys 115 120 125 Gln Leu Leu Leu Thr Gln Ala Asp Lys Phe Ser Pro Ala Glu Val Glu 130 135 140 Gln Met Phe Ala Leu Thr Pro Met Asp Leu Ala Gly Asn Ile Asp Tyr 145 150 155 160 Lys Ser Leu Cys Tyr Ile Ile Thr His Gly Asp Glu Lys Glu Glu 165 170 175 <210> 28 <211> 619 <212> DNA <213> Homo sapiens <400> 28 tctgcagaga gaatggccag caggaaggcg gggacccggg gcaaggtggc agccaccaag 60 caggcccaac gtggttcttc caacgcttt tccatgtttg aacaagccca gatacaggag 120 ttcaagaag ccttcagctg tatcgaccag aatcgtgat gcatcatctg caagcagac 180 ctgagggaga cctactccca gctggggaag gtgagtgtcc cagaggagga gctggacgcc 240 atgctgcaag agggcaaggg cccatcaac ttcaccgtct tcctcacgct ctttgggag 300 aagctcaatg ggacagaccc cgaggaagcc atcctgagtg ccttccgcat gttgacccc 360 agcggcaaag gggtggtgaa caagtgag ttcaagcagc ttctcctgac ccaggcagac 420 aagttctctc cagctgaggt ggagcagatg ttcgccctga cacccatgga cctggcgggg 480 aacatcgact acagtcact gtgctacatc atcaccatg gagacgagaa agaggaatga 540 ggggcagggc caggcccacg ggggggcacc tcaataact ctgttgcaaa attggaaaaa 600 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 619 <210> 29 <211> 5179 <212> PRT <213> Homo sapiens <400> 29 Met Gly Leu Pro Leu Wing Arg Leu Wing Val Cys Leu Wing Leu Ser 1 5 10 15 Leu Ala Gly Gly Ser Glu Leu Gln Thr Glu Gly Arg Thr Arg Asn His 20 25 30 Gly His Asn Val Cys Ser Thr Trp Gly Asn Phe His Tyr Lys Thr Phe 35 40 45 Asp Gly Asp Val Phe Arg Phe Pro Gly Pro Cys Asp Tyr Asn Phe Ala 50 55 60 Ser Asp Cys Arg Gly Ser Tyr Lys Glu Phe Ala Val His Leu Lys Arg 65 70 75 80 Gly Pro Gly Gln Ala Glu Ala Pro Ala Gly Val Glu Ser Ile Leu Leu 85 90 95 Thr Ile Lys Asp Asp Thr Ile Tyr Leu Thr Arg His Leu Ala Val Leu 100 105 110 Asn Gly Ala Val Val Ser Thr Pro His Tyr Ser Pro Gly Leu Leu Ile 115 120 125 Glu Lys Ser Asp Ala Tyr Thr Lys Val Tyr Ser Arg Ala Gly Leu Thr 130 135 140 Leu Met Trp Asn Arg Glu Asp Ala Leu Met Leu Glu Leu Asp Thr Lys 145 150 155 160 Phe Arg Asn His Thr Cys Gly Leu Cys Gly Asp Tyr Asn Gly Leu Gln 165 170 175 Ser Tyr Ser Glu Phe Leu Ser Asp Gly Val Leu Phe Ser Pro Leu Glu 180 185 190 Phe Gly Asn Met Gln Lys Ile Asn Gln Pro Asp Val Val Cys Glu Asp 195 200 205 Pro Glu Glu Glu Val Ala Pro Ala Ser Cys Ser Glu His Arg Ala Glu 210 215 220 Cys Glu Arg Leu Leu Thr Ala Glu Ala Phe Ala Asp Cys Gln Asp Leu 225 230 235 240 Val Pro Leu Glu Pro Tyr Leu Arg Ala Cys Gln Gln Asp Arg Cys Arg 245 250 255 Cys Pro Gly Gly Asp Thr Cys Val Cys Ser Thr Val Ala Glu Phe Ser 260 265 270 Arg Gln Cys Ser His Ala Gly Gly Arg Pro Gly Asn Trp Arg Thr Ala 275 280 285 Thr Leu Cys Pro Lys Thr Cys Pro Gly Asn Leu Val Tyr Leu Glu Ser 290 295 300 Gly Ser Pro Cys Met Asp Thr Cys Ser His Leu Glu Val Ser Ser Leu 305 310 315 320 Cys Glu Glu His Arg Met Asp Gly Cys Phe Cys Pro Glu Gly Thr Val 325 330 335 Tyr Asp Asp Ile Gly Asp Ser Gly Cys Val Pro Val Ser Gln Cys His 340 345 350 Cys Arg Leu His Gly His Leu Tyr Thr Pro Gly Gln Glu Ile Thr Asn 355 360 365 Asp Cys Glu Gln Cys Val Cys Asn Ala Gly Arg Trp Val Cys Lys Asp 370 375 380 Leu Pro Cys Pro Gly Thr Cys Ala Leu Glu Gly Gly Ser His Ile Thr 385 390 395 400 Thr Phe Asp Gly Lys Thr Tyr Thr Phe His Gly Asp Cys Tyr Tyr Val 405 410 415 Leu Ala Lys Gly Asp His Asn Asp Ser Tyr Ala Leu Leu Gly Glu Leu 420 425 430 Ala Pro Cys Gly Ser Thr Asp Lys Gln Thr Cys Leu Lys Thr Val Val 435 440 445 Leu Leu Ala Asp Lys Lys Lys Asn Val Val Val Phe Lys Ser Asp Gly 450 455 460 Ser Val Leu Leu Asn Glu Leu Gln Val Asn Leu Pro His Val Thr Ala 465 470 475 480 Ser Phe Ser Val Phe Arg Pro Ser Ser Tyr His Ile Met Val Ser Met 485 490 495 Ala Ile Gly Val Arg Leu Gln Val Gln Leu Ala Pro Val Met Gln Leu 500 505 510 Phe Val Thr Leu Asp Gln Ala Ser Gln Gly Gln Val Gln Gly Leu Cys 515 520 525 Gly Asn Phe Asn Gly Leu Glu Gly Asp Asp Phe Lys Thr Ala Ser Gly 530 535 540 Leu Val Glu Ala Thr Gly Ala Gly Phe Ala Asn Thr Trp Lys Ala Gln 545 550 555 560 Ser Thr Cys His Asp Lys Leu Asp Trp Leu Asp Asp Pro Cys Ser Leu 565 570 575 Asn Ile Glu Ser Ala Asn Tyr Ala Glu His Trp Cys Ser Leu Leu Lys 580 585 590 Lys Thr Glu Thr Pro Phe Gly Arg Cys His Ser Ala Val Asp Pro Ala 595 600 605 Glu Tyr Tyr Lys Arg Cys Lys Tyr Asp Thr Cys Asn Cys Gln Asn Asn 610 615 620 Glu Asp Cys Leu Cys Ala Ala Leu Ser Ser Tyr Ala Arg Ala Cys Thr 625 630 635 640 Ala Lys Gly Val Met Leu Trp Gly Trp Arg Glu His Val Cys Asn Lys 645 650 655 Asp Val Gly Ser Cys Pro Asn Ser Gln Val Phe Leu Tyr Asn Leu Thr 660 665 670 Thr Cys Gln Gln Thr Cys Arg Ser Leu Ser Glu Ala Asp Ser His Cys 675 680 685 Leu Glu Gly Phe Ala Pro Val Asp Gly Cys Gly Cys Pro Asp His Thr 690 695 700 Phe Leu Asp Glu Lys Gly Arg Cys Val Pro Leu Ala Lys Cys Ser Cys 705 710 715 720 Tyr His Arg Gly Leu Tyr Leu Glu Ala Gly Asp Val Val Val Arg Gln 725 730 735 Glu Glu Arg Cys Val Cys Arg Asp Gly Arg Leu His Cys Arg Gln Ile 740 745 750 Arg Leu Ile Gly Gln Ser Cys Thr Ala Pro Lys Ile His Met Asp Cys 755 760 765 Ser Asn Leu Thr Ala Leu Ala Thr Ser Lys Pro Arg Ala Leu Ser Cys 770 775 780 Gln Thr Leu Ala Ala Gly Tyr Tyr His Thr Glu Cys Val Ser Gly Cys 785 790 795 800 Val Cys Pro Asp Gly Leu Met Asp Asp Gly Arg Gly Gly Cys Val Val 805 810 815 Glu Lys Glu Cys Pro Cys Val His Asn Asn Asp Leu Tyr Ser Ser Gly 820 825 830 Ala Lys Ile Lys Val Asp Cys Asn Thr Cys Thr Cys Lys Arg Gly Arg 835 840 845 Trp Val Cys Thr Gln Ala Val Cys His Gly Thr Cys Ser Ile Tyr Gly 850 855 860 Ser Gly His Tyr Ile Thr Phe Asp Gly Lys Tyr Tyr Asp Phe Asp Gly 865 870 875 880 His Cys Ser Tyr Val Ala Val Gln Asp Tyr Cys Gly Gln Asn Ser Ser 885 890 895 Leu Gly Ser Phe Ser Ile Ile Thr Glu Asn Val Pro Cys Gly Thr Thr 900 905 910 Gly Val Thr Cys Ser Lys Ala Ile Lys Ile Phe Met Gly Arg Thr Glu 915 920 925 Leu Lys Leu Glu Asp Lys His Arg Val Val Ile Gln Arg Asp Glu Gly 930 935 940 His His Val Ala Tyr Thr Thr Arg Glu Val Gly Gln Tyr Leu Val Val 945 950 955 960 Glu Ser Ser Thr Gly Ile Ile Val Ile Trp Asp Lys Arg Thr Thr Val 965 970 975 Phe Ile Lys Leu Ala Pro Ser Tyr Lys Gly Thr Val Cys Gly Leu Cys 980 985 990 Gly Asn Phe Asp His Arg Ser Asn Asn Asp Phe Thr Thr Arg Asp His 995 1000 1005 Met Val Val Ser Ser Glu Leu Asp Phe Gly Asn Ser Trp Lys Glu 1010 1015 1020 Ala Pro Thr Cys Pro Asp Val Ser Thr Asn Pro Glu Pro Cys Ser 1025 1030 1035 Leu Asn Pro His Arg Arg Ser Trp Ala Glu Lys Gln Cys Ser Ile 1040 1045 1050 Leu Lys Ser Ser Val Phe Ser Ile Cys His Ser Lys Val Asp Pro 1055 1060 1065 Lys Pro Phe Tyr Glu Ala Cys Val His Asp Ser Cys Ser Cys Asp 1070 1075 1080 Thr Gly Gly Asp Cys Glu Cys Phe Cys Ser Ala Val Ala Ser Tyr 1085 1090 1095 Ala Gln Glu Cys Thr Lys Glu Gly Ala Cys Val Phe Trp Arg Thr 1100 1105 1110 Pro Asp Leu Cys Pro Ile Phe Cys Asp Tyr Tyr Asn Pro Pro His 1115 1120 1125 Glu Cys Glu Trp His Tyr Glu Pro Cys Gly Asn Arg Ser Phe Glu 1130 1135 1140 Thr Cys Arg Thr Ile Asn Gly Ile His Ser Asn Ile Ser Val Ser 1145 1150 1155 Tyr Leu Glu Gly Cys Tyr Pro Arg Cys Pro Lys Asp Arg Pro Ile 1160 1165 1170 Tyr Glu Glu Asp Leu Lys Lys Cys Val Thr Ala Asp Lys Cys Gly 1175 1180 1185 Cys Tyr Val Glu Asp Thr His Tyr Pro Pro Gly Ala Ser Val Pro 1190 1195 1200 Thr Glu Glu Thr Cys Lys Ser Cys Val Cys Thr Asn Ser Ser Gln 1205 1210 1215 Val Val Cys Arg Pro Glu Glu Gly Lys Ile Leu Asn Gln Thr Gln 1220 1225 1230 Asp Gly Ala Phe Cys Tyr Trp Glu Ile Cys Gly Pro Asn Gly Thr 1235 1240 1245 Val Glu Lys His Phe Asn Ile Cys Ser Ile Thr Thr Arg Pro Ser 1250 1255 1260 Thr Leu Thr Thr Phe Thr Thr Ile Thr Leu Pro Thr Thr Pro Thr 1265 1270 1275 Thr Phe Thr Thr Thr Thr Thr Thr Thr Thr Pro Thr Ser Ser Thr 1280 1285 1290 Val Leu Ser Thr Thr Pro Lys Leu Cys Cys Leu Trp Ser Asp Trp 1295 1300 1305 Ile Asn Glu Asp His Pro Ser Ser Gly Ser Asp Asp Gly Asp Arg 1310 1315 1320 Glu Thr Phe Asp Gly Val Cys Gly Ala Pro Glu Asp Ile Glu Cys 1325 1330 1335 Arg Ser Val Lys Asp Pro His Leu Ser Leu Glu Gln Leu Gly Gln 1340 1345 1350 Lys Val Gln Cys Asp Val Ser Val Gly Phe Ile Cys Lys Asn Glu 1355 1360 1365 Asp Gln Phe Gly Asn Gly Pro Phe Gly Leu Cys Tyr Asp Tyr Lys 1370 1375 1380 Ile Arg Val Asn Cys Cys Trp Pro Met Asp Lys Cys Ile Thr Thr 1385 1390 1395 Pro Ser Pro Pro Thr Thr Thr Pro Ser Pro Pro Pro Thr Ser Thr 1400 1405 1410 Thr Thr Leu Pro Pro Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr 1415 1420 1425 Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Pro Pro Ile Thr 1430 1435 1440 Thr Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Pro Pro Ile 1445 1450 1455 Ser Thr Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Pro Pro 1460 1465 1470 Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr Pro Ser Pro Pro Thr 1475 1480 1485 Thr Thr Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Pro Pro 1490 1495 1500 Thr Thr Thr Pro Ile Thr Pro Pro Ala Ser Thr Thr Thr Leu Pro 1505 1510 1515 Pro Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr Thr Thr Thr Pro 1520 1525 1530 Pro Pro Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr Pro Ile Thr 1535 1540 1545 Pro Pro Thr Ser Thr Thr Thr Leu Pro Pro Thr Thr Thr Pro Ser 1550 1555 1560 Pro Pro Pro Thr Thr Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro 1565 1570 1575 Ser Pro Pro Thr Thr Thr Thr Pro Ser Pro Pro Thr Ile Thr Thr 1580 1585 1590 Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr 1595 1600 1605 Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Pro Pro Thr Thr 1610 1615 1620 Thr Pro Ile Thr Pro Pro Thr Ser Thr Thr Thr Leu Pro Pro Thr 1625 1630 1635 Thr Thr Pro Ser Pro Pro Pro Thr Thr Thr Thr Thr Pro Pro Pro 1640 1645 1650 Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr Thr Pro Ser Pro Pro 1655 1660 1665 Ile Thr Thr Thr Thr Thr Pro Pro Pro Thr Thr Thr Pro Ser Ser 1670 1675 1680 Pro Ile Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr Met Thr Thr 1685 1690 1695 Pro Ser Pro Thr Thr Thr Pro Ser Ser Pro Ile Thr Thr Thr Thr 1700 1705 1710 Thr Pro Ser Ser Thr Thr Thr Pro Ser Pro Pro Pro Thr Thr Met 1715 1720 1725 Thr Thr Pro Ser Pro Thr Thr Thr Pro Ser Pro Pro Thr Thr Thr 1730 1735 1740 Met Thr Thr Leu Pro Pro Thr Thr Thr Ser Ser Pro Leu Thr Thr 1745 1750 1755 Thr Pro Leu Pro Pro Ser Ile Thr Pro Pro Thr Phe Ser Pro Phe 1760 1765 1770 Ser Thr Thr Thr Pro Thr Thr Pro Cys Val Pro Leu Cys Asn Trp 1775 1780 1785 Thr Gly Trp Leu Asp Ser Gly Lys Pro Asn Phe His Lys Pro Gly 1790 1795 1800 Gly Asp Thr Glu Leu Ile Gly Asp Val Cys Gly Pro Gly Trp Ala 1805 1810 1815 Ala Asn Ile Ser Cys Arg Ala Thr Met Tyr Pro Asp Val Pro Ile 1820 1825 1830 Gly Gln Leu Gly Gln Thr Val Val Cys Asp Val Ser Val Gly Leu 1835 1840 1845 Ile Cys Lys Asn Glu Asp Gln Lys Pro Gly Gly Val Ile Pro Met 1850 1855 1860 Ala Phe Cys Leu Asn Tyr Glu Ile Asn Val Gln Cys Cys Glu Cys 1865 1870 1875 Val Thr Gln Pro Thr Thr Met Thr Thr Thr Thr Thr Glu Asn Pro 1880 1885 1890 Thr Pro Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr 1895 1900 1905 Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro 1910 1915 1920 Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly 1925 1930 1935 Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val 1940 1945 1950 Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr 1955 1960 1965 Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr 1970 1975 1980 Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr 1985 1990 1995 Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr 2000 2005 2010 Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro 2015 2020 2025 Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr 2030 2035 2040 Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr 2045 2050 2055 Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr 2060 2065 2070 Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr 2075 2080 2085 Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro 2090 2095 2100 Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro 2105 2110 2115 Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr 2120 2125 2130 Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr 2135 2140 2145 Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr 2150 2155 2160 Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr 2165 2170 2175 Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln 2180 2185 2190 Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro 2195 2200 2205 Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile 2210 2215 2220 Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr 2225 2230 2235 Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr 2240 2245 2250 Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro 2255 2260 2265 Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly 2270 2275 2280 Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val 2285 2290 2295 Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr 2300 2305 2310 Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr 2315 2320 2325 Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr 2330 2335 2340 Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr 2345 2350 2355 Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro 2360 2365 2370 Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr 2375 2380 2385 Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr 2390 2395 2400 Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr 2405 2410 2415 Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr 2420 2425 2430 Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro 2435 2440 2445 Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro 2450 2455 2460 Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr 2465 2470 2475 Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr 2480 2485 2490 Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr 2495 2500 2505 Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr 2510 2515 2520 Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln 2525 2530 2535 Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro 2540 2545 2550 Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile 2555 2560 2565 Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr 2570 2575 2580 Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr 2585 2590 2595 Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro 2600 2605 2610 Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly 2615 2620 2625 Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val 2630 2635 2640 Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr 2645 2650 2655 Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr 2660 2665 2670 Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr 2675 2680 2685 Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr 2690 2695 2700 Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr Pro 2705 2710 2715 Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr 2720 2725 2730 Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro Thr 2735 2740 2745 Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro Thr 2750 2755 2760 Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr Thr 2765 2770 2775 Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr Pro 2780 2785 2790 Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr Pro 2795 2800 2805 Thr Pro Thr Gly Thr Gln Thr Pro Thr Thr Thr Pro Ile Thr Thr 2810 2815 2820 Thr Thr Thr Val Thr Pro Thr Pro Thr Pro Thr Gly Thr Gln Thr 2825 2830 2835 Pro Thr Thr Thr Pro Ile Thr Thr Thr Thr Thr Val Thr Pro Thr ...

Claims

1. Culturing induced pluripotent stem cell (iPSC)-derived pancreatic islet organoids, including iPSC-derived beta-like cells, in a medium containing a Wnt4 polypeptide or a Wnt5a polypeptide to generate functional and metabolically mature pancreatic islet organoids.

1. A method for producing functionally and metabolically mature pancreatic islet organoids, comprising: The functionally and metabolically mature pancreatic islet organoids are iPSC-derived β-like cells expressing estrogen-related receptor γ (Esrrg or ERRγ), express one or more mitochondrial genes involved in oxidative phosphorylation selected from the group consisting of Cox7a2, Ndufal, Ndufa7 and lactate dehydrogenase (Ldha); and / or exhibiting one or more of glucose stimulated insulin secretion (GSIS), c-peptide secretion, KCl stimulated insulin secretion, GLP-1 stimulated insulin secretion, somatostatin secretion, and glucagon secretion; The iPSC-derived pancreatic islet organoids are generated in the absence of adipose-derived stem cells or human umbilical vein endothelial cells (HUVECs) as feeder cells; The method.

2. The method of claim 1, wherein induced pluripotent stem cell (iPSC)-derived beta-like cells are cultured in a three-dimensional matrix to generate pancreatic islet organoids.

3. 3. The method of claim 1 or 2, wherein the iPSC-derived pancreatic islet organoid is a human iPSC (hiPSC)-derived pancreatic islet organoid.

4. The method of claim 1, wherein the Wnt4 polypeptide or Wnt5a polypeptide is a recombinant human Wnt4 polypeptide or a recombinant human Wnt5a polypeptide.

5. The method of claim 4, wherein the medium comprises a recombinant human Wnt4 polypeptide.

6. The method of claim 1, wherein the Wnt4 polypeptide or the Wnt5a polypeptide is present in the culture medium of a cell line that produces Wnt4 or a cell line that produces Wnt5a.

7. The method of any one of claims 1 to 6, wherein the culture medium comprises a Wnt4 polypeptide or a Wnt5a polypeptide at a concentration of 10 to 400 ng / ml.

8. The method of any one of claims 1 to 7, wherein the pancreatic islet organoid is cultured in culture medium for at least 4 days.

9. The method of claim 1, wherein said pancreatic islet organoid further comprises one or more cell types selected from the group consisting of iPSC-derived alpha cells, iPSC-derived delta cells, and iPSC-derived duct cells.

10. The method of claim 1, wherein the pancreatic islet organoid is vascularized.

11. The method of claim 1, wherein the pancreatic islet organoid is surrounded by iPSC-derived exocrine components.

12. The method of claim 1, wherein said pancreatic islet organoid expresses a beta cell transcription factor selected from the group consisting of PDX1, MAFA, PAX4, PAX6, NEUROD1, NKX6-1, GATA6 and FOXA2.

13. The method of claim 1, wherein the iPSC-derived beta-like cells are human iPSC-derived beta-like cells.

14. 10. The method of claim 9, wherein one or more of the cell types selected from the group consisting of iPSC-derived beta-like cells, iPSC-derived alpha cells, iPSC-derived delta cells, and iPSC-derived ductal cells are human.

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