Stem cell culture system for columnar epithelial stem cells and related methods of use

Abstract

Methods of isolating normal stem cells in culture derived from embryonic epithelial tissue and cancer stem cells derived from epithelial cancers and culture medium systems useful for epigenetically stable growth SOLUTION: A method for isolating stem cells from epithelial tissue, preferably columnar epithelial tissue, comprising: (I) culturing dissociated epithelial cells from a columnar epithelial tissue sample to form a stem cell colony; (ii) isolating single stem cells from the cell colony; and (iii) individually culturing the isolated single stem cells from step (ii) to form a culture of purified stem cell clones, wherein: Wherein each of the stem cell clones represents a clonal expansion of epithelial stem cells present in the columnar epithelial tissue sample, thereby isolating epithelial stem cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 611,176, filed December 28, 2017, and U.S. Provisional Patent Application No. 62 / 724,937, filed August 30, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention The isolation and long-term expansion of primary cells, specifically stem / progenitor cell populations, is a fundamental and important foundational technique in various areas of biology, including developmental and stem cell biology, as well as in medical science. Cells within epithelial tissues are highly regenerative and are disproportionately involved in many human cancers and inflammatory / autoimmune diseases. Epithelial cells come in three main forms: squamous, columnar, and cuboidal. They can be arranged in layers of a single cell, such as simple squamous epithelium, simple columnar epithelium, simple cuboidal epithelium, or pseudostratified epithelium, or in layers two or more cells thick, such as stratified (multilayered) squamous epithelium, stratified (multilayered) columnar epithelium, or stratified (multilayered) cuboidal epithelium. All glands are composed of epithelial cells. Their functions include secretion, selective absorption, protection, transcytosis, and sensing. For example, the intestinal epithelium is a layer of cells that forms the luminal surface or lining of both the small and large intestines (colons) of the gastrointestinal tract. It is composed of simple columnar epithelium. It has two important functions: absorbing beneficial substances and providing a barrier against harmful substances. Some diseases and conditions are caused by dysfunction of the intestinal epithelium, and some diseases and conditions cause problems with these cells, which in turn lead to further complications.

[0003] Stem cells from the gastrointestinal tract, pancreas, liver, and other columnar epithelia collectively resist cloning in their elemental state. The isolation and long-term expansion of primary cells, specifically stem / progenitor cell populations, is a fundamental and important fundamental technique in various areas of biology, including developmental biology and stem cell biology, as well as in medical science. Cells from stratified and columnar epithelial tissues are highly regenerative and disproportionately implicated in many human cancers, yet cloning of adult stem cells has been limited by the difficulty of maintaining these cells in their immature state.

[0004] Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising long-term strategies for regenerative medicine, but face difficult challenges, including the risk of teratomas, complex guide protocols for lineage specificity, and limited regenerative capacity of the resulting lineages. Muller et al. Development (1993) 118:1343-51 (Non-Patent Document 1); Helgason et al. Blood (1996) 87:2740-9 (Non-Patent Document 2); Bonde et al. Transplantation (2008) 86:1803-9 (Non-Patent Document 3); Iuchi et al. PNAS (2006) 103:1792-7 (Non-Patent Document 4); Amabile et al. Blood (2013) 121:1255-64 (Non-Patent Document 5); and Suzuki et al. Mol Ther (2013) 21:1424-31 (Non-Patent Document 6). The success and promise of iPSCs has cast a shadow over efforts to harness stem cells native to regenerative tissues. Green et al. developed a method for cloning epidermal stem cells that form stratified epithelia upon engraftment, and these methods have been successfully applied to corneal, thymic, and respiratory epithelia. Rama et al. NEJM (2010) 363:147-155 (Non-Patent Document 7); Senoo et al. Cell (2007) 129:523-536 (Non-Patent Document 8); and Kumar et al. Cell (2011) 147:525-538 (Non-Patent Document 9). However, stem cells from columnar epithelial tissues resist cloning as they maintain their immaturity during proliferative expansion, and instead must progress as regenerative, differentiated "organoids."Matsuura et al.Stem Cells(2006)24:624-630(Non-patent document 10);Sato et al.Nature(2009)459:262-5(Non-patent document 11);Ootani et al.Nat Med(2009)15:701-706(Non-patent document 12);Sato et al. al.Nature (2011) 469:415-418 (Non-Patent Document 13); Fordham et al. Cell Stem Cell (2013) 13:734-744 (Non-Patent Document 14); and Middendorp et al. Stem Cells (2014) 32:1083-1091 (Non-Patent Document 15). Despite their clear potential and constant improvements in regenerative medicine (Yin et al. Nat Methods (2014) 11:106-112), the extremely low percentage of clonogenic cells in organoids limits their growth kinetics and their potential for exploring fundamental stem cells.

[0005] Equally problematic is the limited ability to isolate stem cells from diseased epithelial tissues (i.e., cancer or inflammatory diseases such as IBD, asthma, and COPD). The majority of human cancers originate from epithelial tissues. Since the concept of cancer stem cells ("CSCs") was introduced in the late 1990s, it has become accepted as the underlying mechanism for tumor initiation, growth, and ultimately, drug resistance. These stem cells have influenced all approaches to cancer research and treatment because they help explain the mechanistic progression of more benign forms of cancer to more aggressive forms. While the majority of anticancer drugs kill a large proportion of tumor cells, they ultimately fail to induce a sustained clinical response because they are unable to eliminate critical CSCs that are often resistant to existing cancer treatments, including targeted drugs, chemotherapy, and radiation therapy. Surviving CSCs then generate new tumors and metastases, leading to disease recurrence. Recurrent tumors become more malignant, spread rapidly, and become resistant to radiation therapy and previously used drugs, thereby resulting in a poor prognosis for cancer patients.

[0006] A complicating factor is that many tumors are thought to contain heterogeneous populations of CSCs, which exhibit a range of tumor-promoting activities and drug sensitivities. Therefore, the special survival of CSCs or subsets of CSCs from heterogeneous CSC populations could explain many treatment failures and highlight new directions for enhancing cancer therapy. To develop truly effective treatments that can result in durable clinical responses, it is crucial to develop drugs that can target and kill CSCs. Due to technological advances that facilitate the identification, isolation, and characterization of distinct tumor cell subpopulations that differ in their ability to form and perpetuate tumors, CSCs have only recently begun to be accurately identified. Therefore, methods and reagents for the isolation and stable passage and expansion of columnar CSCs are needed for their usefulness in drug screening.

[0007] It is an object of the present invention to provide systems and reagents for the rapid isolation / cloning of columnar epithelial stem cells, particularly from small biopsies, under conditions that preserve the epigenetic memory of the stem cells as they existed in the tissue biopsy and faithfully preserve their in vivo characteristics during repeated expansion and passaging in culture, so that they are sufficiently scalable, efficient, and ultimately affordable to be performed on a patient-by-patient basis for patient-specific diagnostic and treatment strategies (e.g., inflammatory diseases and metaplasia / tumor) or for regenerative medicine. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Muller et al. Development(1993)118:1343-51 [Non-patent document 2] Helgason et al.Blood(1996)87:2740-9 [Non-patent document 3] Bonde et al.Transplantation(2008)86:1803-9

Non-Patent Document 4

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[0010] In certain preferred embodiments, the medium comprises (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; and (h) a Notch agonist.

[0011] In certain preferred embodiments, the culture medium comprises (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; and (h) a Notch agonist, and the cells derived from the tissue sample are in fluid or direct contact with division-inactive feeder cells.

[0012] In certain preferred embodiments, the medium comprises (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; (h) a Notch agonist; (i) a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); and (j) a bone morphogenetic protein (BMP) antagonist.

[0013] In certain preferred embodiments, the culture medium comprises (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; (h) a Notch agonist; (i) a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); and (j) a bone morphogenetic protein (BMP) antagonist, and the cells derived from the tissue sample are in fluid or direct contact with division-inactive feeder cells.

[0014] In certain preferred embodiments, the medium comprises: (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; (h) a Notch agonist; (i) a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); (j) a bone morphogenetic protein (BMP) antagonist; (k) an Oct4 activator; (l) a PDGFRα / β inhibitor, preferably a selective PDGFRα / β inhibitor; and (m) a JNK inhibitor.

[0015] In certain preferred embodiments, the culture medium comprises (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; (h) a Notch agonist; (i) a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); (j) a bone morphogenetic protein (BMP) antagonist; (k) an Oct4 activator; (l) a PDGFRα / β inhibitor, preferably a selective PDGFRα / β inhibitor; and (m) a JNK inhibitor, and the cells derived from the tissue sample are in fluid or direct contact with division-inactive feeder cells.

[0016] In certain preferred embodiments, the culture medium comprises (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) insulin or IGF; (e) a BRAF inhibitor; (f) a VEGF inhibitor; (g) nicotinamide; (h) a Notch agonist; (i) a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); (j) a bone morphogenetic protein (BMP) antagonist; (k) an Oct4 activator; (l) a PDGFRα / β inhibitor, preferably a selective PDGFRα / β inhibitor; and (m) a JNK inhibitor, and the culture system does not contain feeder cells (i.e., contains only cells derived from the tissue sample and their progeny).

[0017] The phrase "feeder cell-free," as used herein, refers to a culture medium and / or cell culture that is devoid of feeder cells, and / or the conditioned medium produced thereby.

[0018] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. (1) culturing dissociated epithelial cells derived from a columnar epithelial tissue sample to form stem cell colonies, wherein the dissociated cells and cell colonies (a) ROCK (Rho kinase) inhibitors; (b) Wnt agonists; (c) mitogenic growth factors; (d) insulin or IGF; (e) BRAF inhibitors; and (f) VEGF inhibitors. Including, optionally, further comprising nicotinamide; optionally, further comprising a Notch agonist; optionally, further comprising an Oct4 activator; optionally, further comprising a PDGFRα / β inhibitor, preferably a selective PDGFRα / β inhibitor; optionally, further comprising a JNK inhibitor; optionally, further comprising a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); Optionally, cultured in a medium further comprising a bone morphogenetic protein (BMP) antagonist; the culture system does not contain feeder cells (i.e., contains only cells derived from the tissue sample and their progeny); cells from the tissue sample are optionally in contact with an extracellular matrix (such as a basement membrane matrix) or other biomatrix or synthetic matrix; (2) isolating a single stem cell from the cell colony; and (3) individually culturing the isolated single stem cells from step (2) to form a culture of purified stem cell clones in a medium and (optionally) in contact with feeder cells and / or a basement membrane matrix; each of the stem cell clones represents a clonal expansion of epithelial stem cells present in a columnar epithelial tissue sample; thereby isolating columnar epithelial stem cells. Feeder-free method for isolating stem cells from epithelial tissue, preferably columnar epithelial tissue, such as normal or diseased tissue to provide.

[0019] In certain embodiments, the Notch agonist is Jagged-1 and is provided in the culture medium at a concentration of 0.1 μM to 50 μM, preferably 0.1 μM to 10 μM, and more preferably 0.5 μM to 5 μM. In other embodiments, the Notch agonist is other than Jagged-1 and is provided in the culture medium at a concentration equivalent to the EC50 of 0.1 μM to 50 μM Jagged-1, preferably 0.1 μM to 10 μM Jagged-1, and more preferably 0.5 μM to 5 μM Jagged-1.

[0020] In certain embodiments, the ROCK inhibitor is Y-27632 and is provided in the culture medium at a concentration of 0.25 μM to 125 μM, preferably 0.25 μM to 25 μM, and more preferably 1.25 μM to 10 μM. In other embodiments, the ROCK inhibitor is other than Y-27632 and is provided in the culture medium at a concentration equivalent to the EC50 of 0.25 μM to 125 μM Y-27632, preferably 0.25 μM to 25 μM Y-27632, and more preferably 1.25 μM to 10 μM Y-27632.

[0021] In certain embodiments, the ROCK inhibitor is GSK429286A and is provided in the culture medium at a concentration of 25 nM to 12.5 μM, preferably 25 nM to 2.5 μM, and more preferably 125 nM to 1.25 μM. In other embodiments, the ROCK inhibitor is other than GSK429286A and is provided in the culture medium at a concentration equivalent to the EC50 of 25 nM to 12.5 μM GSK429286A, preferably 25 nM to 2.5 μM GSK429286A, and more preferably 125 nM to 1.25 μM GSK429286A.

[0022] In certain embodiments, the ROCK inhibitor is a combination of Y-27632 and GSK429286A at the concentrations described above, or one or more other ROCK inhibitors at EC50 equivalent concentrations.

[0023] In certain embodiments, the BMP antagonist is Noggin and is provided in the culture medium at a concentration of 10 ng / mL to 5 μg / mL, preferably 10 ng / mL to 1 μg / mL, and more preferably 50 ng / mL to 500 ng / mL. In other embodiments, the BMP antagonist is other than Noggin and is provided in the culture medium at a concentration equivalent to the EC50 of 10 ng / mL to 5 μg / mL Noggin, preferably 10 ng / mL to 1 μg / mL Noggin, and more preferably 50 ng / mL to 500 ng / mL Noggin.

[0024] In certain embodiments, the WNT agonist is R-spondin1 and is provided in the culture medium at a concentration of 12.5 ng / mL to 6.25 μg / mL, preferably 12.5 ng / mL to 1.25 μg / mL, more preferably 62.5 ng / mL to 625 ng / mL. In other embodiments, the WNT agonist is other than R-spondin1 and is provided in the culture medium at a concentration equivalent to the EC50 of 12.5 ng / mL to 6.25 μg / mL R-spondin1, preferably 12.5 ng / mL to 1.25 μg / mL R-spondin1, more preferably 62.5 ng / mL to 625 ng / mL R-spondin1.

[0025] In certain embodiments, the mitogenic growth factor is EGF and is provided in the culture medium at a concentration of 1 ng / mL to 500 ng / mL, preferably 1 ng / mL to 100 ng / mL, and more preferably 5 ng / mL to 50 ng / mL. In other embodiments, the mitogenic growth factor is other than EGF and is provided in the culture medium at a concentration equivalent to the EC50 of 1 ng / mL to 500 ng / mL EGF, preferably 1 ng / mL to 100 ng / mL EGF, and more preferably 5 ng / mL to 50 ng / mL EGF.

[0026] In certain embodiments, the TGFβ signaling pathway inhibitor is SB431542 and is provided in the culture medium at a concentration of 0.2 μM to 100 μM, preferably 0.2 μM to 20 μM, and more preferably 1.0 μM to 10 μM. In other embodiments, the TGFβ signaling pathway inhibitor is other than SB431542 and is provided in the culture medium at a concentration equivalent to the EC50 of 0.2 μM to 100 μM SB431542, preferably 0.2 μM to 20 μM SB431542, and more preferably 1.0 μM to 10 μM SB431542.

[0027] In certain embodiments, the culture contains insulin at a concentration of 0.5 μg / mL to 250 μg / mL, preferably 0.5 μg / mL to 50 μg / mL, and more preferably 2.5 μg / mL to 25 μg / mL. In other embodiments, instead of insulin, the culture medium contains an IGF or insulin mimetic at an EC50 equivalent concentration of 0.5 μg / mL to 250 μg / mL insulin, preferably 0.5 μg / mL to 50 μg / mL insulin, and more preferably 2.5 μg / mL to 25 μg / mL insulin.

[0028] In certain embodiments, the VEGF inhibitor is tivozanib and is provided in the culture medium at a concentration of 50 nM to 25 μM, preferably 50 nM to 5 μM, and more preferably 250 nM to 2500 μM. In other embodiments, the VEGF inhibitor is other than tivozanib and is provided in the culture medium at a concentration equivalent to the EC50 of 50 nM to 25 μM tivozanib, preferably 50 nM to 5 μM tivozanib, and more preferably 250 nM to 2500 μM tivozanib.

[0029] In certain embodiments, the B-raf inhibitor is GDC-0879 and is provided in the culture medium at a concentration of 50 nM to 25 μM, preferably 50 nM to 5 μM, and more preferably 250 nM to 2500 μM. In other embodiments, the B-raf inhibitor is other than GDC-0879 and is provided in the culture medium at a concentration equivalent to the EC50 of 50 nM to 25 μM GDC-0879, preferably 50 nM to 5 μM GDC-0879, and more preferably 250 nM to 2500 μM GDC-0879.

[0030] In certain embodiments, nicotinamide is provided in the culture medium at a concentration of 1 nM to 500 nM, preferably 1 nM to 100 nM, and more preferably 5 nM to 50 nM.

[0031] In certain embodiments, the PDGFRα / β inhibitor is CP673451 and is provided in the culture medium at a concentration of 0.1 μM to 50 μM, preferably 0.1 μM to 10 μM, and preferably 0.5 μM to 5 μM. In other embodiments, the PDGFRα / β inhibitor is other than CP673451 and is provided in the culture medium at a concentration equivalent to the EC50 of 0.1 μM to 50 μM CP673451, preferably 0.1 μM to 10 μM CP673451, and more preferably 0.5 μM to 5 μM CP673451.

[0032] In certain embodiments, the OCT4 activator is OAC1 and is provided in the culture medium at a concentration of 0.1 μM to 50 μM, preferably 0.1 μM to 10 μM, and more preferably 0.5 μM to 5 μM. In other embodiments, the OCT4 activator is other than OAC1 and is provided in the culture medium at a concentration equivalent to the EC50 of 0.1 μM to 50 μM OAC1, preferably 0.1 μM to 10 μM OAC1, and more preferably 0.5 μM to 5 μM OAC1.

[0033] In certain embodiments, the JNK inhibitor is JNK-IN-8 and is provided in the culture medium at a concentration of 0.1 μM to 50 μM, preferably 0.1 μM to 10 μM, and more preferably 0.5 μM to 5 μM. In other embodiments, the JNK inhibitor is other than JNK-IN-8 and is provided in the culture medium at a concentration equivalent to the EC50 of 0.1 μM to 50 μM JNK-IN-8, preferably 0.1 μM to 10 μM JNK-IN-8, and more preferably 0.5 μM to 5 μM JNK-IN-8.

[0034] As used herein, "EC50 equivalent concentration" refers to the concentration of a drug relative to a reference drug that provides the same biological effect on cultured cells after adjusting for differences in the EC50s of the two drugs on cultured cells. Thus, for example, a ROCK inhibitor with an EC50 on cultured cells that is 5-fold higher (i.e., less effective) than Y-27632 may require a concentration of 6.25 μM to 50 μM to provide the same range of biological effect on cell culture as Y-27632, which is 1.25 μM to 10 μM. In the case of drugs that are inhibitors of specific receptors, enzymes, pathways, etc., IC50 can be used instead of EC50.

[0035] In certain embodiments, the epithelial tissue is derived from a patient with a disease, disorder, or abnormal condition, the patient being affected by the disease, disorder, or abnormal condition. In certain embodiments, the columnar epithelial stem cells are adult columnar epithelial stem cells. In certain embodiments, the columnar epithelial stem cells are fetal columnar epithelial stem cells.

[0036] In certain embodiments, the medium does not contain a Notch agonist.

[0037] In certain embodiments, in step (1), the (epithelial) cells are dissociated from the tissue through enzymatic digestion with enzymes, such as collagenase, protease, dispase, pronase, elastase, hyaluronidase, accutase, or trypsin.

[0038] In certain embodiments, in step (1), the (epithelial) cells are dissociated from the tissue through dissolution of the extracellular matrix surrounding the (epithelial) cells.

[0039] In certain embodiments, the mitotically inactivated cells are mitotically inactivated fibroblasts, preferably human or mouse fibroblasts, such as 3T3-J2 cells. Mitotic inactivation can be achieved by administration of mitomycin C or other chemical mitotic inhibitors, gamma irradiation, X-ray irradiation, and / or UV light irradiation.

[0040] In certain embodiments, the extracellular matrix is ​​a basement membrane matrix, such as a laminin-containing basement membrane matrix (e.g., MATRIGEL™ basement membrane matrix (BD Biosciences)), preferably growth factor-reduced. In other embodiments, the biopolymer is selected from the group consisting of collagen, chitosan; fibronectin, fibrin, and mixtures thereof.

[0041] In certain embodiments, the basement membrane matrix does not support three-dimensional growth or does not form the three-dimensional matrix necessary to support three-dimensional growth.

[0042] In certain embodiments, the medium further comprises serum, preferably FBS (more preferably non-heat-inactivated FBS), for example, at a concentration of 5% to 15%, e.g., 10% FBS.

[0043] In certain embodiments, ROCK inhibitors include Rho kinase inhibitor VI (Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide)), Fasudil, or HA1077 (5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), or HI 152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride).

[0044] In certain embodiments, the BMP antagonist includes noggin, DAN, DAN-like proteins containing a DAN cystine-knot domain (e.g., Cerberus and Gremlin), chordin, chordin-like proteins containing a chordin domain, follistatin, follistatin-related proteins containing a follistatin domain, sclerostin / SOST, decorin, or a-2 macroglobulin. In certain preferred embodiments, the BMP antagonist is noggin.

[0045] In certain embodiments, the Wnt agonist includes R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, an R-spondin mimetic, a Wnt family protein (e.g., Wnt-3a, Wnt-5, Wnt-6a), Norrin, or a GSK inhibitor (e.g., CHIR99021).

[0046] In certain embodiments, the mitogenic growth factors include EGF, keratinocyte growth factor (KGF), TGFa, BDNF, HGF, and / or FGF (eg, FGF7 or FGF10).

[0047] In certain embodiments, TGFβ receptor inhibitors include SB431542 (4-(4-(5-benzo[1,3]dioxol-5-yl)-4-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide), A83-01, SB-505124, SB-525334, LY 364947, SD-208, or SJN 2511.

[0048] In certain embodiments, the TGFβ (signaling) inhibitor binds to and reduces the activity of one or more serine / threonine protein kinases selected from the group consisting of ALK5, ALK4, TGFβ receptor kinase 1, and ALK7.

[0049] In certain embodiments, the TGFβ (signaling) inhibitor is added at a concentration of 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 10 μM, or approximately 1 μM.

[0050] In certain embodiments, the BRAF inhibitor is AMG542, ARQ197, ARQ736, AZ628, CEP-32496, GDC-0879, GSK1120212, GSK2118436 (dabrafenib, Tafinlar), LGX818 (encorafenib), NMS-P186, NMS-P349, NMS-P383, NMS-P396, NMS-P730, PLX360 3 (RO5212054), PLX4032 (vemurafenib, Zelboraf), PLX4720 (difluorophenyl-sulfonamine), PF-04880594, PLX4734, RAF265 (CHIR-265), RO4987655, SB590885, sorafenib, sorafenib tosylate, and XL281 (BMS-908662). Exemplary BRAF inhibitors are also available from Selleckchem (http: / / www.selleckchem.com / BRAF.html) and include vemurafenib (PLX4032, RG7204); sorafenib tosylate; PLX-4720; dabrafenib (GSK2118436); GDC-0879; lifirafenib (BGB-283); CCT196969; RAF265 (CHIR-265); AZ 628; NVP-BHG712; SB590885; ZM 336372; sorafenib; GW5074; TAK-632; Raf265 derivatives; CEP-32496; encorafenib (LGX818); PLX7904; LY3009120; RO5126766 (CH5126766), and MLN2480.

[0051] In certain embodiments, the VEGF inhibitor is aflibercept, pegaptanib, tivozanib, 3-(4-bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride, axitinib, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl-)methoxy]quinazolin-4-amine, an inhibitor of VEGF-R2 and VEGF-R1, axitinib, N,2-dimethyl-6-(2-(1-methyl-1H-imidazol-2-yl)thieno[ [3,2-b]pyridin-7-yloxy)benzo[b]thiophene-3-carboxamide, a tyrosine kinase inhibitor of RET / PTC oncogenic kinase, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine, a pan-VEGF-R kinase inhibitor; a protein kinase inhibitor, a multi-target human epithelial growth factor receptor (HER)1 / 2 and vascular endothelial growth factor receptor (VEGFR)1 / 2 receptor family tyrosine kinase inhibitor, cediranib, sorafenib, vatalanib, glufanide disodium, a VEGFR2-selective monoclonal antibody, angiozyme, an siRNA-based VEGFR1 inhibitor, 5-((7-benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol hydrochloride, derivatives thereof, and combinations thereof.

[0052] In certain preferred embodiments, the VEGF inhibitor is a VEGF receptor inhibitor, more preferably a VEGF receptor kinase inhibitor such as tivozanib (AV-951), AZD2932, midostaurin (pkc412), BAW2881 (NVP-BAW2881), nintedanib (BIBF 1120), SU5402, SU1498, BFH772, sorafenib, sunitinib, dovitinib (TKI258), semaxanib (SU5416), hypericin, vatalanib, ZM306416, AAL993, SU4312, DMXAA, or foretinib.

[0053] In certain embodiments, the BRAF inhibitor and the VEGF receptor kinase inhibitor are the same compound, such as sorafenib, which is a dual inhibitor of VEGFR kinase and RAF kinase.

[0054] An exemplary selective inhibitor of PDGFRα / β is CP-673451 The file is TIFF2026034588000001.tif37128.

[0055] Exemplary JNK inhibitors include, but are not limited to, SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one), JNK-IN-8 (3-[[4-(dimethylamino)-1-oxo-2-buten-1-yl]amino]-N-[3-methyl-4-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide); and JNK inhibitor IX (N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thien-2-yl)-1-naphthalenecarboxamide).

[0056] In certain embodiments, the Oct4 activator is TIFF2026034588000002.tif110128.

[0057] In another aspect, the present invention provides single cell clones of epithelial stem cells, or in vitro cultures thereof, such as those comprising the medium of the present invention, wherein the epithelial stem cells substantially lack expression of markers associated with differentiated cell types of the epithelial tissue from which they were derived.

[0058] In another aspect, the present invention provides single cell clones of non-embryonic epithelial stem cells or in vitro cultures thereof, such as those comprising the medium of the present invention, wherein the non-embryonic epithelial stem cells have an immature, undifferentiated morphology characterized by small, round cell shape with a high nucleus / cytoplasm ratio.

[0059] In a related aspect, the present invention also provides libraries or collections of single-cell clones of the present invention, or in vitro cultures thereof (e.g., comprising the media of the present invention). In certain embodiments, the library or collection may contain single-cell clones derived from the same tissue / organ type. In certain embodiments, the library or collection may contain single-cell clones isolated from the same type of tissue / organ type but derived from different members of the population. In certain embodiments, one or more (preferably each) members of the population are homozygous at at least one tissue-typing locus (e.g., HLA-A, HLA-B, and HLA-D). In certain embodiments, at least one tissue-typing locus (e.g., an HLA locus described above) is modified in cloned stem cells, e.g., via TALEN technology or CRISPR technology (see below), to generate a universal donor cell line (e.g., hepatocytes) lacking tissue antigens encoded by the tissue-typing loci (e.g., HLA-A, HLA-B, and HLA-D, etc.). See Torikai et al. (Blood, 122(8):1341-1349, 2013, incorporated herein by reference). In certain embodiments, populations can be defined by ethnicity, age, sex, disease status, or common characteristics of the population. A library or collection can have at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, or more members.

[0060] In another aspect, the present invention provides a method of treating a subject having a disease, disorder, or abnormal condition in need of treatment, comprising the steps of: (1) isolating epithelial stem cells from tissue corresponding to tissue affected by the disease, disorder, or abnormal condition in the subject using any of the methods of the present invention; (2) optionally altering the expression of at least one gene in the epithelial stem cells to generate altered epithelial stem cells; and (3) reintroducing the isolated or altered epithelial stem cells or clonal expansion thereof into the subject, wherein at least one adverse effect or symptom of the disease, disorder, or abnormal condition in the subject is alleviated.

[0061] In certain embodiments, the expression of at least one gene in an epithelial stem cell is genetically, recombinantly, and / or epigenetically altered to generate an altered epithelial stem cell.

[0062] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from a healthy adult or fetal (ie, non-embryonic) subject.

[0063] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from a subject, hi certain embodiments, the tissue from which the epithelial stem cells are isolated is diseased tissue affected by a disease, disorder, or abnormal condition.

[0064] In certain embodiments, the tissue from which the epithelial stem cells are isolated is adjacent to diseased tissue affected by a disease, disorder, or abnormal condition.

[0065] In certain embodiments, at least one gene is underexpressed in tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is enhanced in the altered epithelial stem cells.

[0066] In certain embodiments, at least one gene is overexpressed in a tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is reduced in the altered epithelial stem cells.

[0067] In certain embodiments, step (2) is accomplished by introducing exogenous DNA or RNA into the epithelial stem cells.

[0068] In yet another aspect, the present invention provides a method for screening compounds, comprising the steps of: (1) isolating epithelial stem cells from a subject using any of the methods of the present invention; (2) generating a cell line of epithelial stem cells through single-cell clonal expansion; (3) contacting test cells derived from the cell line with a plurality of candidate compounds; and (4) identifying one or more compounds that produce a predetermined phenotypic change in the test cells.

[0069] Another aspect of the present invention provides the use of epithelial stem cells or their progeny isolated from diseased epithelial tissue using the culture medium system of the present invention to identify agents that selectively inhibit the growth or proliferation of stem cells or their progeny compared to normal regenerative epithelial stem cells, or that restore epithelial stem cells to a normal epigenetic state so that they differentiate into normal epithelial tissue. The diseased epithelial tissue can be derived, for example, from a patient with an inflammatory disease or tumor. In certain embodiments, methods are further provided in which the identified agents are formulated for administration to a mammalian subject, such as a human patient, for example, by formulation with a pharmaceutically acceptable excipient.

[0070] Another aspect of the present invention provides the use of epithelial stem cells or their progeny isolated from normal epithelial tissue using the culture medium system of the present invention for identifying agents that promote the growth, proliferation, and / or regenerative capacity of stem cells. In certain embodiments, a method is further provided in which the identified agent is formulated for administration to a mammalian subject, such as a human patient, for example, by formulation with a pharmaceutically acceptable excipient.

[0071] [The present invention 1001] 1. A method for isolating stem cells from epithelial tissue, preferably columnar epithelial tissue, comprising: (i) culturing dissociated epithelial cells derived from a columnar epithelial tissue sample to form stem cell colonies, wherein the dissociated cells and cell colonies are cultured in a medium comprising a ROCK (Rho kinase) inhibitor, a Wnt agonist, a mitogenic growth factor, insulin (or an insulin mimetic) or IGF, a BRAF inhibitor, a VEGF inhibitor, nicotinamide, a Notch agonist, a TGFβ signaling pathway inhibitor, and a bone morphogenetic protein (BMP) antagonist; (ii) isolating a single stem cell from the cell colony; and (iii) individually culturing the isolated single stem cells from step (ii) to form a culture of purified stem cell clones, each of the stem cell clones represents a clonal expansion of epithelial stem cells present in the columnar epithelial tissue sample. thereby isolating epithelial stem cells. [The present invention 1002] 1001. The method of claim 1001, wherein the cells derived from the tissue sample are in fluid or direct contact with division-inactive feeder cells. [The present invention 1003] 10. The method of claim 1001 or 1002, wherein the cells derived from the tissue sample are in contact with an extracellular matrix or a synthetic matrix. [The present invention 1004] 1001. The method of claim 1001, wherein the culture medium further comprises an Oct4 activator, a PDGFRα / β inhibitor (preferably a selective PDGFRα / β inhibitor), and a JNK inhibitor. [The present invention 1005] The method of claim 1004, wherein the medium does not contain feeder cells. [The present invention 1006] The method of any one of claims 1004 to 1005, wherein the cells derived from the tissue sample are in contact with a biomatrix (such as an extracellular matrix) or a synthetic matrix. [The present invention 1007] The method of any of claims 1001 to 1006, wherein the stem cells are isolated from a tissue sample taken from normal epithelial tissue. [The present invention 1008] The method of any of claims 1001 to 1006, wherein the stem cells are isolated from a tissue sample taken from diseased epithelial tissue, such as from an inflammatory or autoimmune patient. [The present invention 1009] The method of any of claims 1001 to 1006, wherein the stem cells are isolated from a tissue sample taken from the tumor. [The present invention 1010] An epithelial stem cell isolated by any of the methods of the present inventions 1001 to 1009. [The present invention 1011] At least 10% of epithelial stem cells isolated from a tissue sample by any of the methods of the present inventions 1001 to 1009. 6 1. A stem cell culture comprising progeny cells of the A stem cell culture, wherein the progeny cells are epithelial stem cells and maintain the epigenetic and genetic traits of the epithelial stem cells isolated from the tissue sample. [The present invention 1012] A stem cell culture comprising progeny cells of at least passage 10 (P10) of epithelial stem cells isolated from a tissue sample by any one of the methods of the present inventions 1001 to 1009, A stem cell culture, wherein the progeny cells are epithelial stem cells and maintain the epigenetic and genetic traits of the epithelial stem cells isolated from the tissue sample. [The present invention 1013] A differentiated tissue isolated in culture, differentiated from epithelial stem cells isolated by any of the methods of the present inventions 1001 to 1009. [The present invention 1014] 1. A defined culture medium for isolating columnar epithelial stem cells and stably maintaining their epigenetics over multiple passages in culture, comprising: A defined culture medium comprising a ROCK (Rho kinase) inhibitor; a Wnt agonist; a mitogenic growth factor; insulin (or insulin mimetic) or IGF; a BRAF inhibitor; a VEGF inhibitor; nicotinamide; a Notch agonist, a TGFβ signaling pathway inhibitor; and a bone morphogenetic protein (BMP) antagonist, which supports epigenetically stable growth and proliferation of stem cells of columnar epithelial tissue origin in the presence of co-cultured feeder cells. [The present invention 1015] 1. A defined culture medium for isolating columnar epithelial stem cells and stably maintaining their epigenetics over multiple passages in culture, comprising: A defined culture medium comprising a basal medium; and each of a ROCK (Rho kinase) inhibitor, a Wnt agonist, a mitogenic growth factor, insulin or IGF, a BRAF inhibitor, a VEGF inhibitor, an Oct4 activator, a PDGFRα / β inhibitor, a JNK inhibitor, and (optionally) a TGFβ signaling pathway inhibitor, wherein the defined culture medium supports epigenetically stable growth and proliferation of stem cells of columnar epithelial tissue origin in the absence of co-cultured feeder cells. [The present invention 1016] A clonal columnar epithelial stem cell isolated using the culture medium of the present invention 1014 or 1015 or by any of the methods of the present invention 1001 to 1009. [The present invention 1017] Use of epithelial stem cells or their progeny isolated from diseased epithelial tissue using the culture medium of invention 1014 or 1015 or by any of the methods of inventions 1001 to 1009, for identifying agents that selectively inhibit the growth or proliferation of the stem cells or their progeny compared to normal regenerative epithelial stem cells, or that restore the epithelial stem cells to a normal epigenetic state that will differentiate into normal epithelial tissue. [The present invention 1018] The use of the present invention 1017, wherein the diseased epithelial tissue is derived from a patient with an inflammatory disease or a tumor. [The present invention 1019] Use of epithelial stem cells or their progeny isolated from normal epithelial tissue using the culture medium of the present invention 1014 or 1015 or by any of the methods of the present invention 1001 to 1009, for identifying agents that promote the growth, proliferation, and / or regenerative capacity of stem cells. [The present invention 1020] The use of any of inventions 1017, 1018, or 1019, wherein the identified agent is formulated for administration to a mammalian subject. It is contemplated that any embodiment described herein, including those described in the examples and figures / drawings, as well as those described under different aspects of the invention, may be combined with one or more other embodiments, where applicable. [Brief explanation of the drawings]

[0072] [Figure 1] Representative images of stem cells derived from various human columnar epithelia, including liver, intestine, pancreas, and stomach, as well as diseased epithelia such as Barrett's esophagus and esophageal cancer. [Figure 2] Epithelial stem cells are highly clonogenic: when single cells are sorted into individual wells of tissue culture plates, approximately 70% of the single cells can give rise to colonies that can then be expanded into lineages. [Figure 3A] Single-cell-derived human colonic stem cell lineages differentiate into all intestinal cell types. This figure shows colonic stem cells cultured in MGM medium were plated onto a Transwell membrane and allowed to reach confluence. An air-liquid interface was then created by removing the medium from the well. [Figure 3B] Single-cell-derived human colonic stem cell lineages differentiate into all intestinal cell types. This figure illustrates that after cell polarization, a single stem cell-derived lineage differentiates into goblet cells (MUC2-positive), endocrine cells (CHGA-positive), Paneth cells (DEFA6-positive), and enterocytes (villin-positive). [Figure 4]Figure 4A: Starting from a single ISCGS colony, one billion ISCGS cells could be generated in approximately 6 days from all 30 patients, regardless of age. Figure 4B: ISCGS from all ages showed indistinguishable morphology and identical pluripotency. ISCGS lineages from 16-, 56-, and 77-year-old patients were differentiated in air-liquid interface (ALI) culture for 10 days. [Figure 5A] In a copy number polymorphism study, we demonstrate polyclonality in the intestinal epithelium by sampling ISCGS clones from elderly patients (aged 40-70 years). We first demonstrated that ISCGS clones from all 30 patients were highly clonogenic. Clonogenicity was observed at 50-70% in different patients. Single-cell-derived colonies could be expanded into single-cell-derived lineages containing several thousand cells, providing sufficient DNA for routine genomic analysis. We used high-density SNP arrays to sample 1-23 clones from 11 adult patients with and without UC. We found that the majority of clones displayed few chromosomal alterations compared with blood samples from the same patients. However, one of 23 clones from a 44-year-old non-IBD patient displayed amplification of two putative oncogenes, SOS1 and XPO1, while the remaining clones were all wild-type. Furthermore, one of seven clones from a 56-year-old UC patient displayed significantly more significant chromosomal alterations. As a result, 16 genes were amplified, including putative oncogenes such as ERBB4, ALK, and MYCN. [Figure 5B]In a copy number polymorphism study, we demonstrate polyclonality in the intestinal epithelium by sampling ISCGS clones from elderly patients (aged 40-70 years). We first demonstrated that ISCGS clones from all 30 patients were highly clonogenic. Clonogenicity was observed at 50-70% in different patients. Single-cell-derived colonies could be expanded into single-cell-derived lineages containing several thousand cells, providing sufficient DNA for routine genomic analysis. We used high-density SNP arrays to sample 1-23 clones from 11 adult patients with and without UC. We found that the majority of clones displayed few chromosomal alterations compared with blood samples from the same patients. However, one of 23 clones from a 44-year-old non-IBD patient displayed amplification of two putative oncogenes, SOS1 and XPO1, while the remaining clones were all wild-type. Furthermore, one of seven clones from a 56-year-old UC patient displayed significantly more significant chromosomal alterations. As a result, 16 genes are amplified, including putative oncogenes such as ERBB4, ALK, and MYCN. [Figure 5C] Polyclonality in the intestinal epithelium is illustrated by sampling ISCGS clones from elderly patients (ages 40-70) in a copy number polymorphism study. The figure shows that several other clones from the same patient from Figures 5A and 5B displayed wild-type genomes. [Figure 6]Figure 6A: To investigate genomic alterations in wild-type and mutant clones derived from UC patients, we performed exome sequencing on ISCGS pools and lineages. Our genomic analysis of these cells consisted of assessment of copy number variation (CNV) and point mutations using exome sequencing. We determined CNV and point mutations using DNA samples from the same patient derived from mutant and wild-type lineages, pooled cells, and venous blood. 28 Significantly, pooled ISCGSs exhibited very low CNV in the form of interstitial deletions and amplifications. This degree of CNV in pooled stem cells was within the range of that observed in wild-type stem cell lineages from the same patients. Figure 6B: To investigate genomic alterations in wild-type and mutant clones derived from UC patients, we performed exome sequencing on ISCGS pools and lineages. Our genomic analysis of these cells consisted of assessment of copy number variation (CNV) and point mutations using exome sequencing. We determined CNVs and point mutations using mutant and wild-type lineages, as well as DNA samples from the same patient derived from pooled cells and venous blood. 28 Significantly, the pooled ISCGSs showed very low CNVs in the form of interstitial deletions and amplifications. This degree of CNV in the pooled stem cells was within the range of that observed in the wild-type stem cell lineages of the same patient. Figure 6C: To investigate genomic alterations in wild-type and mutant clones derived from UC patients, we performed exome sequencing on ISCGS pools and lineages. Our genomic analysis of these cells consisted of assessment of copy number variation (CNV) and point mutations using exome sequencing. We determined CNVs and point mutations using mutant and wild-type lineages, as well as DNA samples from the same patient derived from pooled cells and venous blood. 28 Significantly, the pooled ISCGSs showed very low CNVs in the form of interstitial deletions and amplifications. This degree of CNV in pooled stem cells was within the range of that observed in wild-type stem cell lineages of the same patients. [Figure 7] FIG. 1 is a schematic diagram showing the process of screening cultured intestinal stem cells prior to transplantation for safety concerns. [Figure 8] Figure 8A: Clonal analysis of colonic stem cells derived from endoscopic biopsies. Workflow for generating a "library" of single-cell-derived colonies from a 1 mm endoscopic biopsy and subsequently generating a three-dimensional intestinal epithelium. White-light imaging of a typical endoscopic biopsy, representative images of 100-300 colonies derived from a typical biopsy, and a plan view of the in vitro intestinal epithelium generated from these stem cells differentiated in an air-liquid interface environment. Scale bar, 1000 μm. Figure 8B: Clonal analysis of colonic stem cells derived from endoscopic biopsies. Individual colonies are sampled from the pool and grown separately as separate lines. Figure 8C: Histological analysis of in vitro differentiated colonic epithelium via hematoxylin-eosin staining and immunofluorescence with antibodies against the secretory cell markers mucin 2, chromogranin A, and defensin α6. Scale bar, 50 μm. [Figure 9] Figure 9A: Immortality and rapid expansion of colonic stem cells in vitro. Clonogenicity of single GFP-labeled colonic stem cells sorted into individual wells. Figure 9B: Immortality and rapid expansion of colonic stem cells in vitro. Clonogenic assay reveals a nearly constant number of rhodamine red-stained colonies grown 10 days after plating 2000 passaged colonic stem cells. Figure 9C: Immortality and rapid expansion of colonic stem cells in vitro. Rapid expansion of a single cell to 1 billion cells in approximately 60 days. DETAILED DESCRIPTION OF THE INVENTION

[0073] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 1. Overview The invention described herein relates to a method for isolating and / or maintaining in culture non-embryonic (e.g., adult or fetal) epithelial stem cells derived from the columnar epithelium of an organ. Epithelial stem cells so isolated from various tissues or organs can self-renew or proliferate indefinitely in vitro, are pluripotent, and can differentiate into the various differentiated cell types normally found in the tissue or organ from which they were isolated. Cultures (including in vitro cultures) containing such isolated epithelial stem cells are also within the scope of the invention.

[0074] Furthermore, isolated epithelial stem cells can be propagated through clonal expansion of a single isolated stem cell to generate clones (e.g., in vitro cultures) in which at least about 40%, 70%, or 90% or more of the cells can be further passaged as clones of single-cell origin. Thus, stem cells isolated using the methods of the present invention can be uniquely manipulated in vitro through standard molecular biology techniques, such as the introduction of exogenous genetic material via infection or transfection.

[0075] As used herein, "epithelial stem cells" include adult stem cells isolated from adult tissues or organs, and fetal stem cells isolated from prenatal tissues or organs.

[0076] In related embodiments, the methods of the invention described herein isolate fetal stem cells from fetal or prenatal tissues or organs. In certain embodiments, when fetal tissues or organs are the source of the stem cells, particularly when the fetus is a human fetus, the methods of the invention do not destroy the fetus or impair its normal development. In other embodiments, the source of fetal tissue is obtained from aborted fetuses, dead fetuses, macerated fetal material, or cells, tissues, or organs excised therefrom.

[0077] The methods of the present invention are applicable to any animal columnar epithelial tissue that contains epithelial stem cells, including tissue from humans, non-human mammals, non-human primates, rodents (including, but not limited to, mice, rats, polecats, hamsters, guinea pigs, and rabbits), livestock (including, but not limited to, pigs, cows, sheep, goats, horses, and camels), birds, reptiles, fish, pet or other companion animals (e.g., cats, dogs, and birds), or other vertebrates.

[0078] "Columnar epithelial cells" are elongated, cylindrical cells with a height at least four times greater than their width. The nucleus is elongated and generally located near the base of the cell. Columnar epithelium forms the lining of the stomach and intestine. Cells here may possess microvilli to maximize surface area for absorption, and these microvilli may form a brush border. Some cells are ciliated to move mucus in the function of mucociliary clearance. Other ciliated cells are found in the fallopian tubes, uterus, and central canal of the spinal cord. Some columnar cells, such as those in the nose, ear, and taste buds, are specialized for sensory reception. Hair cells of the inner ear have stereocilia, which are similar to microvilli. Goblet cells, a modified type of columnar cell, are found between columnar epithelial cells in the duodenum. They secrete mucus, which acts as a lubricant. Simple, non-ciliated columnar epithelium tends to perform absorptive functions.

[0079] Simple columnar epithelium is columnar epithelium that is a single layer. In humans, simple columnar epithelium lines most organs of the digestive tract, including the stomach, small intestine, and large intestine. Simple columnar epithelium lines the uterus. Simple columnar epithelium is further divided into two categories: ciliated and non-ciliated. Ciliated columnar epithelium transports mucus and other substances via cilia and is found in the upper respiratory tract, fallopian tubes, uterus, and central spinal cord.

[0080] Ciliated columnar epithelium lines the lumen of the fallopian tube, and current generated by the cilia propels the egg toward the uterus.

[0081] Non-ciliated epithelium is found lining sections of the gastrointestinal tract and may have a brush border.

[0082] "Pseudostratified columnar epithelium" is columnar epithelium that contains only a single layer of cells but with the cell nuclei arranged in a manner similar to stratified epithelium. Pseudostratified columnar epithelium is found, for example, lining the trachea, bronchi, male urethra, and a few other locations.

[0083] In certain embodiments, the epithelial tissue is isolated from a healthy or normal individual.

[0084] In certain embodiments, the epithelial tissue is isolated from diseased tissue (eg, tissue affected by a disease), disordered tissue (eg, tissue affected by a disorder), or tissue having any other abnormal condition.

[0085] As used herein, the term "disease" includes an abnormal or medical condition that affects the body of an organism and is generally associated with specific symptoms and signs. Diseases can be caused by external factors (such as infectious diseases, including papillomavirus infections or sexually transmitted diseases) or by internal dysfunction (such as autoimmune diseases or cancer). In a broad sense, "disease" can also include conditions that cause pain, disability, suffering, social problems, or death in the affected individual, or similar problems in those who come into contact with that individual. In this broader sense, it can include damage, disability, disorders, syndromes, infections, isolated symptoms, abnormal behavior, and atypical variations in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. In certain preferred embodiments, stem cells are isolated from tumor biopsies.

[0086] In some embodiments, epithelial tissue is isolated from an individual with disease, disorder or other abnormal condition, but the epithelial tissue itself may not be affected by disease, disorder or abnormal condition.For example, epithelial tissue may be isolated from a patient with inflammatory bowel disease or gastric cancer from the healthy part of the intestine (in the case of IBD) or stomach (in the case of tumor) that has not yet been affected by inflammatory condition or cancer.In some embodiments, epithelial tissue may be proximal or distal to disease, disorder or abnormal tissue.

[0087] In certain embodiments, the epithelial tissue is isolated from an individual who is predisposed to or at high risk for developing a disease, disorder, or other abnormal condition, e.g., based on the individual's genetic makeup, family history, lifestyle choices (e.g., smoking, diet, exercise habits), previous viral infections, etc., but who has not yet developed the disease, disorder, or other abnormal condition or exhibits detectable symptoms of the disease, disorder, or other abnormal condition.

[0088] Another aspect of the invention provides an epithelial stem cell isolated by any of the methods of the invention, or an in vitro culture thereof.

[0089] In yet another aspect, the present invention further provides a single cell clone of an isolated epithelial stem cell, or an in vitro culture thereof, wherein at least about 40%, 50%, 60%, 70%, or about 80% of the cells in the single cell clone are capable of expanding to give rise to the single cell clone when isolated as a single cell.

[0090] Each single cell clone may be cultured for at least about 10, 100, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 It may contain one or more cells.

[0091] In a related aspect, the present invention provides single cell clones of isolated epithelial stem cells or in vitro cultures thereof, wherein the epithelial stem cells, when isolated as single cells, are capable of self-renewal for about 50, 70, 100, 150, 200, 250, 300, 350, or about 400 generations or more.

[0092] In certain embodiments, the in vitro culture comprises a medium of the present invention (e.g., a modified medium of the present invention as described below). See the sections below describing the media of the present invention. Each of the media described therein is incorporated herein by reference. In certain embodiments, epithelial stem cells can differentiate into differentiated cell types of the epithelial tissue from which they were originally biopsied, or, in the case of cancer stem cells, into the tumor of that tissue origin. For example, isolated epithelial stem cells of the present invention can differentiate into one or more cell types normally found in the epithelial tissue from which they were derived.

[0093] In certain embodiments, epithelial stem cells can differentiate into organized structures that resemble structures or substructures found in the tissue from which they were derived, for example, isolated hepatic stem cells of the present invention can differentiate into liver tissue-like structures that resemble liver epithelium, and isolated gastrointestinal stem cells of the present invention can differentiate into GI tissue-like structures that resemble gastrointestinal epithelium.

[0094] In certain embodiments, epithelial stem cells have an immature, undifferentiated morphology characterized by a small, round cell shape with a high nuclear / cytoplasmic ratio.

[0095] A further aspect of the present invention provides methods of treating a subject having a disease, disorder, or abnormal condition and in need of treatment, comprising: (1) using any of the methods of the present invention to isolate non-embryonic (e.g., adult) stem cells from regenerative tissue corresponding to the tissue affected by the disease, disorder, or abnormal condition in the subject; (2) altering the expression of at least one gene in the epithelial stem cells to generate altered epithelial stem cells; and (3) reintroducing the altered epithelial stem cells or clonal expansion, or a culture-derived tissue graft thereof, into the subject, whereby at least one adverse effect or symptom of the disease, disorder, or abnormal condition in the subject is alleviated, or as a means of regenerating / replacing damaged regenerative tissue. In other cases, the transplanted cells / tissue may be genetically modified to be resistant to viral infection, such as papillomavirus infection.

[0096] For example, step (2) of the method can be achieved by introducing exogenous DNA or RNA into isolated epithelial stem cells, which increases or decreases the expression of a target gene in the epithelial stem cells. Any art-recognized molecular biology technique can be used to alter gene expression in cells, for example, in vitro or ex vivo. Such methods can include, but are not limited to, transfection or infection with a viral or non-viral vector that may encode a coding sequence for a protein or functional fragment thereof that is dysfunctional or missing in the target cell, or may encode RNA (antisense RNA, siRNA, miRNA, shRNA, ribozyme, etc.) that disrupts the function of the target gene.

[0097] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from a healthy subject, preferably one that is HLA-type matched to the subject in need of treatment.

[0098] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from the subject, and the isolated epithelial stem cells are autologous to the subject.

[0099] In certain embodiments, the tissue from which the epithelial stem cells are isolated is a diseased tissue affected by a disease, disorder, or abnormal condition.

[0100] In certain embodiments, the tissue from which the epithelial stem cells are isolated is adjacent to diseased tissue affected by a disease, disorder, or abnormal condition.

[0101] In certain embodiments, at least one gene is underexpressed in tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is enhanced in the altered epithelial stem cells.

[0102] In certain embodiments, at least one gene is overexpressed in a tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is reduced in the altered epithelial stem cells.

[0103] In another aspect, the present invention also provides a method for screening for agents or conditions that alter the "phenotype" of cells, such as the differentiation, epigenetics, survival, etc., of stem cells in regenerative tissues, whether normal or derived from a cancer / disease state. In an exemplary embodiment, the method includes the steps of: (1) using any of the methods of the present invention to isolate epithelial stem cells (including cancer stem cells) from a subject's regenerative tissue; (2) generating one or more stem cell lines from the epithelial stem cells through single-cell clonal expansion; (3) contacting test cells derived from the cell line with one or more candidate compounds; and (4) identifying compounds that produce a predetermined phenotypic change in the test cells. This screening method of the present invention can be used for target identification and validation. For example, potential target genes in epithelial stem cells isolated from a patient in need of treatment may function abnormally (either overexpressed or underexpressed) to cause a phenotype associated with a disease, disorder, or abnormal condition. Clonal expansion of epithelial stem cells isolated using the methods of the invention can be subjected to the screening methods of the invention to test an array of potential compounds (such as small molecule compounds) to identify one or more compounds that can correct, alleviate, or reverse the phenotype.

[0104] In another embodiment, epithelial stem cells can be isolated from the regenerative tissue of a patient in need of treatment, such as regenerative tissue affected by a disease, disorder, or abnormal condition. Clonal expansion of epithelial stem cells isolated using the methods of the present invention can be subjected to the screening methods of the present invention to test an array of potential compounds (such as small molecule compounds or RNA-based antagonists, such as siRNA libraries) to identify one or more compounds that can correct, alleviate, or reverse the phenotype. Target genes affected by effective compounds can be further identified, for example, by microarray, RNA-Seq, or PCR-based expression profile analysis.

[0105] Epithelial stem cells isolated using the methods of the present invention and their clonal expansion may further be useful for toxicological screening or research, such that toxicological analysis and testing can be personalized for individual patients who will receive certain medications or medical interventions.

[0106] Epithelial stem cells isolated using the methods of the present invention and their clonal expansion may also be useful for regenerative medicine, where either autologous stem cells or stem cells isolated from HLA-matched healthy donors can be induced to differentiate in vitro, ex vivo, or in vivo into regenerative tissues or organs to treat existing conditions or prevent / delay the onset of such conditions. Such stem cells may also be genetically manipulated prior to induced differentiation.

[0107] The epithelial stem cells isolated using the method of the present invention and their clonal expansion can be used in in vitro or in vivo disease models. For example, isolated intestinal stem cells can be induced to differentiate at the air-liquid interface (ALI) to produce intestinal epithelial-like structures, and these structures can be used in any of the screening methods described herein. To establish a suitable humanized disease model for performing in vivo methods such as the screening method of the present invention, isolated epithelial stem cells (e.g., derived from humans) can also be introduced into SCID or nude mice or rats.

[0108] 2. Methods for Obtaining and / or Cultivating Stem Cells One aspect of the present invention relates to a method for isolating epithelial stem cells from epithelial tissue, as generally described above.

[0109] To illustrate, one step of the method involves culturing dissociated epithelial cells derived from epithelial tissue in contact with (optionally) a first population of division-inactive feeder cells and / or an extracellular matrix, e.g., a basement membrane matrix, to form epithelial cell clones.

[0110] In certain embodiments, (epithelial) cells are dissociated from the tissue through enzymatic digestion with enzymes including, but not limited to, one or more of collagenase, protease, dispase, pronase, elastase, hyaluronidase, accutase, and / or trypsin.

[0111] These enzymes or functional equivalents are well known in the art and, in almost all cases, are commercially available.

[0112] In other embodiments, (epithelial) cells can be dissociated from tissue samples through dissolution of the extracellular matrix surrounding the (epithelial) cells. One suitable reagent for this embodiment of the invention includes a non-enzymatic proprietary solution sold by BD Biosciences (San Jose, CA) as BD™ Cell Recovery Solution (BD Catalog No. 354253), which allows for the recovery of cells cultured in BD MATRIGEL™ Basement Membrane Matrix for subsequent biochemical analysis.

[0113] In some embodiments, the culture system includes feeder cells, which may include certain lethally irradiated fibroblasts, such as mouse 3T3-J2 cells. Other feeder cells include human dermal fibroblasts, (human) adipose tissue-derived mesenchymal stem cells, (human) bone marrow-derived mesenchymal stem cells, (human) amniotic epithelial cells, (mouse or human) embryonic feeder cells, (human) bone marrow stromal cells, HELA cells, and (human) amniotic cells. The feeder cells can form a feeder cell layer on the basement membrane matrix.

[0114] In other embodiments, in those embodiments in which feeder cells may be used, feeder cell conditioned medium can be used instead.

[0115] Suitable 3T3-J2 cell clones are well known in the art (see, e.g., Todaro and Green, "Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines," Cell Biol. 17:299-313, 1963) and are readily publicly available. For example, Waisman Biomanufacturing (Madison, Wisconsin) sells irradiated 3T3-J2 feeder cells that are produced and tested according to cGMP guidelines. These cells are initially obtained by the vendor from Dr. Howard Green's laboratory under a Material Transfer Agreement and are of sufficient quality to support, for example, skin gene therapy and wound healing clinical trials. The vendor also states that each vial of 3T3 cells contains a minimum of 3 x 10 cells manufactured in a fully compliant clean room. 6 The bank contains 1000 cells and is certified mycoplasma-free and low-endotoxin. Additionally, the cell bank has been fully tested for adventitious agents, including mouse viruses. These cells have been screened for keratinocyte culture support and are mitomycin C-free.

[0116] The method of the present invention provides for the use of feeder cells, such as the mouse 3T3-J2 clone of fibroblasts. Generally, without being limited to a particular phenotype, feeder cell layers are often used to support the culture of stem cells and / or inhibit differentiation. A feeder cell layer is generally a monolayer of cells that is co-cultured with the cells of interest and provides a suitable surface for their growth. The feeder cell layer provides an environment in which the cells of interest can grow. Feeder cells are often mitotically inactivated (e.g., by (lethal) irradiation or treatment with mitomycin C) to prevent their proliferation.

[0117] In some embodiments, the feeder cells are appropriately screened and are GMP-grade human feeder cells, e.g., sufficient to support the clinical-grade stem cells of the present invention. For GMP-grade human feeder cells grown in a medium containing GMP-quality FBS, see Crook et al. (Cell Stem Cell 1(5):490-494, 2007, incorporated by reference).

[0118] In some embodiments, feeder cells can be labeled with the marker that stem cells lack, so that stem cells can be easily distinguished and isolated from feeder cells.For example, feeder cells can be modified to express fluorescent markers, such as GFP or other similar fluorescent markers.Fluorescently labeled feeder cells can be separated from stem cells, for example, by FACS sorting.

[0119] Any of a number of physical separation methods known in the art can be used to separate the stem cells of the present invention from feeder cells.Other than FACS, such physical methods can include various immunoaffinity methods based on specific expressed markers.For example, the stem cells of the present invention can be isolated based on the specific stem cell markers they express and by using antibodies specific to these markers.

[0120] In one embodiment, the stem cells of the present invention can be isolated by FACS, for example, using an antibody against one of these markers. Fluorescence-activated cell sorting (FACS) can be used to detect markers characteristic of a particular cell type or lineage. As will be apparent to those skilled in the art, this can be achieved through a fluorescently labeled antibody or a fluorescently labeled secondary antibody that has binding specificity for the primary antibody. Examples of suitable fluorescent labels include, but are not limited to, FITC, Alexa Fluor® 488, GFP, CFSE, CFDA-SE, DyLight 488, PE, PerCP, PE-Alexa Fluor® 700, PE-Cy5 (TRI-COLOIT), PE-Cy5.5, PI, PE-Alexa Fluor* 750, and PE-Cy7. This list of fluorescent markers is provided for illustrative purposes only and is not intended to be limiting.

[0121] For example, it will be clear to those skilled in the art that FACS analysis using stem cell-specific antibodies will provide a purified stem cell population. However, in some embodiments, it may be desirable to further purify the cell population by performing additional FACS analysis using one or more other identifiable markers, such as those that select for non-feeders.

[0122] For some competitive methods, the presence of feeder cells can complicate the cell passage in these competitive methods, so the use of feeder cells is considered undesirable.For example, cells must be separated from feeder cells at each passage, and new feeder cells are required at each passage.In addition, the use of feeder cells can cause feeder cells to be mixed with desired cells.

[0123] However, the use of a feeder layer is not necessarily a disadvantage of the present invention, since the isolated stem cells of the present invention can be, and indeed preferably are, passaged as single cells, and thus the potential risk of feeder contamination during passaging is minimized, if not eliminated.

[0124] In certain embodiments, the basement membrane matrix is ​​a laminin-containing basement membrane matrix (eg, MATRIGEL™ basement membrane matrix (BD Biosciences)), preferably growth factor reduced.

[0125] In certain embodiments, basement membrane matrix does not support three-dimensional growth or does not form the three-dimensional matrix necessary to support three-dimensional growth. Thus, when seeding basement membrane matrix, it is generally not required that the basement membrane matrix be deposited on the support in a particular shape or form, e.g., forming a dome shape or form and maintaining such shape or form after solidification, to support three-dimensional growth. In certain embodiments, the basement membrane matrix is ​​distributed or spread evenly over a flat surface or support structure (such as a flat-bottom tissue culture dish or well).

[0126] In certain embodiments, the basement membrane matrix is ​​first thawed, diluted to an appropriate concentration (e.g., 10%) with cold (e.g., about 0-4°C) feeder cell growth medium, plated, and allowed to solidify on a flat surface by warming to 37°C in a tissue culture incubator with an appropriate CO2 content (e.g., about 5%). Lethally irradiated feeder cells are then plated onto the solidified basement membrane matrix at an appropriate density so that the plated feeder cells form a subconfluent or confluent feeder cell layer on the basement membrane matrix overnight. The feeder cells are preferably cultured in a feeder cell medium, such as a medium (e.g., 3T3-J2 growth medium) containing basal tissue culture medium with high glucose (e.g., about 4.5 g / L), no L-glutamine, and no sodium pyruvate (e.g., DMEM (Invitrogen catalog no. 11960; high glucose (4.5 g / L), no L-glutamine, no sodium pyruvate), 10% calf serum (not heat-inactivated), one or more antibiotics (e.g., 1% penicillin-streptomycin), and L-glutamine (e.g., about 1.5 mM, or 1-2 mM, or 0.5-5 mM, or 0.2-10 mM, or 0.1-20 mM).

[0127] According to the method of the present invention, epithelial cell colonies become detectable after dissociated cells derived from the tissue of origin are cultured in the stem cell medium of the present invention for several days (e.g., 3 to 4 days or about 10 days).

[0128] In certain embodiments, single cells can be isolated from these epithelial cell colonies, for example, by enzymatic digestion. Suitable enzymes for this purpose include trypsin, such as warmed 0.25% trypsin (Invitrogen catalog number 25200056). In certain embodiments, the enzymatic digestion is substantially complete, such that essentially all cells in the epithelial cell clone are dissociated from other cells and become single cells. In certain embodiments, the method includes culturing the isolated single cells in modified growth medium (preferably after washing and resuspending the single cells) in contact with a second population of lethally irradiated feeder cells and a second basement membrane matrix in modified growth medium. Optionally, the isolated single cells may be passed through a cell strainer of appropriate size (e.g., 40 microns) before plating the single cells on the feeder cells and basement membrane matrix.

[0129] In certain embodiments, the modified growth medium is changed periodically (e.g., every day, every 2 days, every 3 days, or every 4 days, etc.) until single cell clones or clonal expansions of the isolated single stem cells are formed.

[0130] In certain embodiments, single colonies of stem cells can be isolated, for example, using cloning rings. The isolated stem cell clones can be expanded to develop lineage cell lines, i.e., cell lines derived from a single stem cell.

[0131] In certain embodiments, a single stem cell can be isolated from the clonal expansion of a single stem cell and passaged again as a single stem cell.

[0132] 3. Culture Medium The present invention provides various cell culture media for isolating, culturing, and / or differentiating stem cells of the present invention, including basal media supplemented with numerous factors to create stem cell culture media for regenerative tissue stem cells. Factors that can be added to basal media or modified media are first described below. Then, to illustrate specific, non-limiting aspects of the present invention, several exemplary basal and modified media of the present invention are described in further detail.

[0133] ROCK (Rho kinase) inhibitors Without wishing to be bound by any particular theory, the addition of a ROCK inhibitor may prevent anoikis, particularly when culturing single stem cells. ROCK inhibitors include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, Sigma-Aldrich), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077, Cayman Chemical), and (1S,)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H1 152, Tocris). Bioscience), and N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide (GSK429286A, Stemgent).

[0134] In certain embodiments, the final concentration for Y27632 is about 1-5 μM or 2.5 μM.

[0135] A Rho kinase inhibitor, for example, 'Y-21632, may be added to the culture medium every 1, 2, 3, 4, 5, 6, or 7 days for the first 7 days of stem cell culture.

[0136] Wnt agonists The Wnt signaling pathway is defined by a series of events that occur when a Wnt protein ligand binds to a cell surface receptor that is a member of the Frizzled receptor family. This results in the activation of the Dishevelled (Dsh) family of proteins, which inhibit a complex of proteins containing axin, GSK-3, and the protein APC, to degrade intracellular β-catenin. The resulting concentrated nuclear β-catenin enhances transcription by the TCF / LEF family of transcription factors. As used herein, "Wnt agonist" includes agents that directly or indirectly activate TCF / LEF-mediated transcription in cells, such as by modulating the activity of any of the proteins / genes in the Wnt signaling cascade (e.g., by enhancing the activity of a positive regulator of the Wnt signaling pathway or inhibiting the activity of a negative regulator of the Wnt signaling pathway).

[0137] The Wnt agonist is selected from true Wnt agonists that bind to and activate members of the Frizzled receptor family, including all Wnt family proteins, inhibitors of intracellular beta-catenin degradation, and activators of TCF / LEF. The Wnt agonist can stimulate Wnt activity in cells by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least about 100%, at least about 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold or more, compared to the level of Wnt activity in the absence of the Wnt agonist. As known to those skilled in the art, Wnt activity can be determined by measuring Wnt transcriptional activity, for example, using Tcf luciferase reporter constructs in pTOPFLASH and pFOPFLASH (see Korinek et al., Science 275:1784-1787, 1997, incorporated herein by reference).

[0138] Representative Wnt agonists may include secreted glycoproteins, including Wnt-1 / Int-1, Wnt-2 / Irp (Int-1 related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi et al, Biochem. Biophys. Res. Com., 283:798-805, 2001), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, Wnt-11, and Wnt-16. A summary of human Wnt proteins is provided in "The Wnt Family of Secreted Proteins," R&D Systems Catalog, 2004 (incorporated herein by reference).

[0139] Additionally, Wnt agonists include secreted proteins of the R-spondin family, which are involved in activating and regulating the Wnt signaling pathway and include at least four members: R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 (R&D systems), R-spondin 3, and R-spondin 4. Wnt agonists also include Norrin (R&D systems), a secreted regulatory protein (also known as Norrie Disease Protein or NDP) that functions similarly to Wnt proteins by binding with high affinity to the Frizzled 4 receptor and inducing activation of the Wnt signaling pathway (Kestutis Planutis et al., BMC Cell Biol. 8:12, 2007).

[0140] Wnt agonists include those having the following structure, as described in Liu et al. (Angew Chem. Int. Ed. Engl. 44 13):1987-1990, 2005), which is incorporated herein by reference: Further included is a small molecule agonist of the Wnt signaling pathway, an aminopyrimidine derivative (N4-[(2H-1,3-benzodioxol-5-yl)methyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine) of TIFF2026034588000003.tif30128.

[0141] GSK inhibitors include small interfering RNA (siRNA, Cell Signaling), lithium (Sigma), Kenpaullone (Biomol International, Leost et al., Eur. J. Biochem. 267: 5983-5994, 2000), 6-bromoindirubin-30-acetoxime (Meyer et al., Chem. Biol. 10: 1255-1266, 2003), SB 216763, and SB 415286 (Sigma-Aldrich), as well as FRAT family members and FRAT-derived peptides that prevent the interaction of GSK-3 with axin. A summary is provided by Meijer et al. (Trends in Pharmacological Sciences 25: 471-480, 2004, incorporated herein by reference). Methods and assays for determining the level of GSK-3 inhibition are known in the art and may include, for example, methods and assays such as those described in Liao et al. (Endocrinology 145(6):2941-2949, 2004, incorporated herein by reference).

[0142] In certain embodiments, the Wnt agonist is selected from one or more of a Wnt family member, R-spondins 1-4 (such as R-spondin 1), Norrin, Wnt3a, Wnt-6, and a GSK inhibitor.

[0143] In certain embodiments, the Wnt agonist comprises or consists of R-spondin 1. R-spondin 1 can be added to the culture medium of the present invention at a concentration of at least about 50 ng / mL, at least about 75 ng / mL, at least about 100 ng / mL, at least about 125 ng / mL, at least about 150 ng / mL, at least about 175 ng / mL, at least about 200 ng / mL, at least about 300 ng / mL, or at least about 500 ng / mL. In certain embodiments, R-spondin 1 is about 125 ng / mL.

[0144] In certain embodiments, any of the specific protein-based Wnt agonists mentioned herein, such as R-spondin1 through R-spondin4, any Wnt family member, etc., may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, or 99% of the respective Wnt agonist activity, and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, or 99% amino acid sequence identity, as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm). The sequences of representative Wnt agonists mentioned herein are represented in SEQ ID NOs. 10-17.

[0145] During the culture of the stem cells of the present invention, a Wnt family member can be added to the culture medium every day, every two days, every three days, every four days, every five days, or more, while changing the culture medium every day, every two days, every three days, every four days, every five days, or more.

[0146] In certain embodiments, the Wnt agonist is selected from the group consisting of R-spondin, Wnt-3a, and Wnt-6, or a combination thereof. In certain embodiments, R-spondin and Wnt-3a are used together as Wnt agonists. In certain embodiments, the R-spondin concentration is about 125 ng / mL, and the Wnt3a concentration is about 100 ng / mL.

[0147] mitogenic growth factors Mitogenic growth factors suitable for the present invention may include the family of growth factors including epidermal growth factor (EGF) (Peprotech), transforming growth factor alpha (TGFa, Peprotech), basic fibroblast growth factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), and keratinocyte growth factor (KGF, Peprotech).

[0148] EGF is a potent mitogen for a variety of cultured ectodermal and mesodermal cells and exerts profound effects on the differentiation of certain cells in vivo and in vitro, as well as some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53-amino acid peptide hormone that stimulates cells. EGF can be added to the culture medium of the present invention at a concentration of 1 to 500 ng / mL. In certain embodiments, the final EGF concentration in the medium is at least about 1, 2, 5, 10, 20, 25, 30, 40, 45, or 50 ng / mL, but not more than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 30, or 20 ng / mL. In certain embodiments, the final EGF concentration is about 1-50 ng / mL, or about 2-50 ng / mL, or about 5-30 ng / mL, or about 5-20 ng / mL, or about 10 ng / mL.

[0149] The same concentration can be used for FGFs such as FGF10 or FGF7. When multiple types of FGFs, such as FGF7 and FGF10, are used, the above-mentioned FGF concentrations can refer to the total concentration of all FGFs used in the medium.

[0150] In certain embodiments, any of the specific mitogenic growth factors mentioned herein, such as, for example, EGF, TGFα, bFGF, BDNF, KGF, etc., may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective mitogenic growth factor activity and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0151] The sequences of representative mitogenic growth factors referred to herein are represented in SEQ ID NOs. 18-27.

[0152] During the culture of the stem cells of the present invention, mitogenic growth factors can be added to the medium, for example, every day, every two days, with the medium being changed daily.

[0153] Any member of the bFGF family can be used. In certain embodiments, FGF7 and / or FGF10 are used. FGF7 is also known as KGF (keratinocyte growth factor). In certain embodiments, a combination of mitogenic growth factors such as EGF and KGF or EGF and BDNF is added to the culture medium of the present invention. In certain embodiments, a combination of mitogenic growth factors such as EGF and KGF or EGF and FGF10 is added to the culture medium of the present invention.

[0154] BMP inhibitors Bone morphogenetic proteins (BMPs) bind as dimeric ligands to a receptor complex consisting of two distinct receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, leading to activation of this receptor kinase. The type I receptor then phosphorylates specific receptor substrates (such as SMADs), resulting in a signaling pathway that leads to transcriptional activation.

[0155] As used herein, BMP inhibitors include agents that inhibit BMP signaling through its receptor.In one embodiment, BMP inhibitors bind to BMP molecules to form complexes, for example, by preventing or inhibiting the binding of BMP molecules to BMP receptors, thereby neutralizing BMP activity.Examples of such BMP inhibitors can include antibodies specific to BMP ligands or their antigen-binding portions.Other examples of such BMP inhibitors include dominant-negative mutants of BMP receptors, such as soluble BMP receptors, that bind to BMP ligands and prevent the ligands from binding to natural BMP receptors on cell surfaces.

[0156] Alternatively, BMP inhibitors can include drugs that function as antagonists or inverse agonists.This type of inhibitor binds to BMP receptor and prevents BMP from binding to the receptor.An example of such a drug is an antibody that specifically binds to BMP receptor and prevents BMP from binding to antibody-bound BMP receptor.

[0157] In certain embodiments, BMP inhibitors inhibit the BMP-dependent activity in cells by at most 90%, at most 80%, at most 70%, at most 50%, at most 30%, at most 10%, or about 0% (near complete inhibition), compared to the level of BMP activity in the absence of inhibitor.As known to those skilled in the art, BMP activity can be determined by measuring the transcriptional activity of BMP, for example, as exemplified in Zilberberg et al. ("A rapid and sensitive bioassay to measure bone morphogenetic protein activity," BMC Cell Biology 8:41,2007, incorporated herein by reference).

[0158] Several classes of natural BMP binding proteins are known, including Noggin (Peprotech), Chordin and Chordin-like proteins containing Chordin domains (R&D systems), Follistatin and follistatin-related proteins containing Follistatin domains (R&D systems), DAN and DAN-cystine knot domain-containing DAN-like proteins (e.g., Cerberus and Gremlin) (R&D systems), Sclerostin / SOST (R&D systems), Decorin (R&D systems), and α2 macroglobulin (R&D systems), or as described in US 8,383,349. Exemplary BMP inhibitors for use in the methods of the present invention are selected from Noggin, DAN, and DAN-like proteins containing Cerberus and Gremlin (R&D systems). These diffusible proteins can bind to BMP ligands with varying degrees of affinity and inhibit BMP from accessing signaling receptors.

[0159] Any of the above BMP inhibitors may be added, alone or in combination, to the culture medium of the present invention when desired.

[0160] In certain embodiments, the BMP inhibitor is Noggin, which may be added to the respective culture medium at a concentration of at least about 10 ng / mL, or at least about 20 ng / mL, or at least about 50 ng / mL, or at least about 100 ng / mL (e.g., 100 ng / mL).

[0161] In certain embodiments, any of the specific BMP inhibitors mentioned herein, such as noggin, chordin, follistatin, DAN, cerberus, gremlin, sclerostin / SOST, decorin, and α2 macroglobulin, may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective BMP inhibitory activity and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0162] The sequences of representative BMP inhibitors referred to herein are represented in SEQ ID NOs. 1-9.

[0163] During the culture of the stem cells of the present invention, the BMP inhibitor can be added to the culture medium every day, every two days, every three days, or every four days, while changing the culture medium every day, every two days, every three days, or every four days, as appropriate.

[0164] BRAF inhibitors BRAF inhibitors that can be used in accordance with embodiments described herein include those that inhibit wild-type BRAF or mutant BRAF (e.g., BRAF V600E , BRAF V600K , BRAF V600D , BRAF V600L , BRAF V600R) may include agents that selectively inhibit at least a portion of the biological activity (e.g., signal transduction activity) of BRAF. In some aspects, the BRAF inhibitor may be selective for BRAF alone or may have inhibitory activity against one or more additional targets in the RAF / MEK / ERK pathway. For example, in one aspect, the BRAF inhibitor may be a RAF kinase inhibitor, i.e., the inhibitor may have inhibitory activity against RAF kinases, such as ARAF, CRAF, or both, in addition to BRAF. In certain embodiments, the BRAF inhibitor is selected to have increased paradoxical MAPK activation activity. Thus, the BRAF inhibitor used in accordance with the embodiments described herein may function as a MAPK paradoxical activator, i.e., the BRAF inhibitor causes increased MAPK signaling. In some aspects, the MAPK paradoxical activator is a BRAF inhibitor that exhibits increased MAPK signaling when the target BRAF kinase is wild-type BRAF kinase.

[0165] Several BRAF kinase inhibitors have been described in the art, any of which may be suitable for use in the methods, dressings, and compositions described herein. Suitable BRAF inhibitors include 1,2-dicyclyl-substituted alkyne compounds or derivatives; 1-methyl-5-(2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine; 2,6-disubstituted quinazoline, quinoxaline, quinoline, and isoquinoline compounds or derivatives; 4-amino-5-oxo-8-phenyl-5H-pyrido[2,3-D]-pyrimidine compounds. Compounds or derivatives; 4-amino-thieno[3,2-c]pyridine-7-carboxylic acid compounds or derivatives; 5-(4-aminophenyl)-isoquinoline compounds or derivatives; benzenesulfonamide thiazole compounds or derivatives; benzimidazole compounds or derivatives; bicyclic compounds or derivatives; bridged, bicyclic heterocyclic, or spiro-bicyclic heterocyclic derivatives of pyrazolo[1,5-a]pyrimidine compounds or derivatives; cinnamide and hydro-cinnamide compounds or derivatives; biposition Substituted imidazole compounds or derivatives; Fused tricyclic pyrazolo[1,5-a]pyrimidine compounds or derivatives; Heteroaryl compounds or derivatives; Heterocyclic compounds or derivatives; 1H-Benzo[D]imidazole compounds or derivatives; Imidazo[4,5-B]pyridine compounds or derivatives; N-(6-aminopytidin-3-yl)-3-(sulfonamido)benzamide compounds or derivatives; N-[3-(1-amino-5,6,7,8-tetrahydro-2,4,4B-triazaflurane]

[00100] (9-phenyl-2-olen-9-yl)-benzamide compounds or derivatives; Nitrogen-containing bicyclic heteroaryl compounds or derivatives; N-oxides of heterocyclic substituted bisarylurea compounds or derivatives; ω-carboxyaryl-substituted diphenylurea compounds or derivatives; Oxazole compounds or derivatives; Phenethylamide compounds or derivatives; Phenylsulfonamide-substituted pyrazolo[1,5-a]pyrimidine compounds or derivatives; Phenyltriazole compounds or derivatives; Heterocyclic compounds or derivatives;1h-Pyrazolo[3,4-b]pyridine compounds or derivatives;Purine compounds or derivatives;Pyrazole[3,4-B]pyridine compounds or derivatives;Pyrazole compounds or derivatives;Pyrazolin compounds or derivatives;Pyrazolo[3,4-b]pyridine, pyrrolo[2,3-b]pyridine compounds or derivatives;Pyrazolo[3,4-d]pyrimidine compounds or derivatives;Pyrazolo[5,1-c][1,2,4]triazine compounds or derivatives;Pyrazolyl compounds or derivatives;Pyrimidine compounds or derivatives;Pyrrole compounds or derivatives;Pyrrolo[2,3-B ]pyridine compounds or derivatives; substituted 6-phenyl-pyrido[2,3-D]pyrimidin-7-one compounds or derivatives; substituted benzazole compounds or derivatives; substituted benzimidazole compounds or derivatives; substituted bisarylurea compounds or derivatives; thienopyridine compounds or derivatives; thienopyrimidine, thienopyridine, or pyrrolopyrimidine compounds or derivatives; thiopheneamide compounds or derivatives, and other suitable aryl and / or heteroaryl compounds or derivatives. In some aspects, suitable BRAF inhibitors described herein may include the compounds or derivatives themselves, or may be pharmaceutically acceptable salts or solvates thereof.

[0166] International Patent Application Publication Numbers: WO2011117381, WO2011119894, WO2011117381, WO2011097594, WO2011097526, WO2011085269, WO2011090738, WO2011025968, WO2011025927, WO2011023773, WO2011028540, WO2010111527, WO2010104973, WO2010100127, WO2010078408, WO2011 0065893, WO2010032986, WO2009115572, WO2009108838, WO2009111277, WO2009111278, WO2009111279, WO2009111280, WO2009 108827, WO2009111260, WO2009100536, WO2009059272, WO2009039387, WO2009021869, WO2009006404, WO2009006389, WO200814 0850, WO2008079277, WO2008055842, WO2008034008, WO2008115263, WO2008030448, WO2008028141, WO2007123892, WO2007115 670, WO2007090141, WO2007076092, WO2007067444, WO2007056625, WO2007031428, WO2007027855, WO2007002433, WO20070023 25, WO2006125101, WO2006124874, WO2006124780, WO2006102079, WO2006108482, WO2006105844, WO2006084015, WO200607670 6, WO2006050800, WO2006040569, WO2005112932, WO2005075425, WO2005049603, WO2005037285, WO2005037273, WO2005032548;and several patents and patent applications, including, but not limited to, U.S. Patent Nos. 8,642,759, 8,557,830, 8,504,758, 7,863,288, 7,491,829, 7,482,367, and 7,235,576, disclose exemplary BRAF inhibitors that may be used in accordance with the embodiments described herein; the specifications of all of which are incorporated by reference as if fully set forth herein.

[0167] In certain embodiments, the BRAF inhibitor is selected from the group consisting of AMG542, ARQ197, ARQ736, AZ628, CEP-32496, GDC-0879, GSK1120212, GSK2118436 (dabrafenib, Tafinlar), LGX818 (encorafenib), NMS-P186, NMS-P349, NMS-P383, NMS-P396, NMS-P730, PLX3603 (R O5212054), PLX4032 (vemurafenib, Zelboraf), PLX4720 (difluorophenyl-sulfonamine), PF-04880594, PLX4734, RAF265 (CHIR-265), 804987655, SB590885, sorafenib, sorafenib tosylate, or XL281 (BMS-908662).

[0168] In some embodiments, the BRAF inhibitor is represented by Formula (I) or Formula (II): TIFF2026034588000004.tif35128 or a pharmaceutically acceptable salt thereof, wherein: R 1 is H, C3-C6 cycloalkyl optionally substituted with cyano, C1-C3 alkyl optionally substituted with cyano, -C(O)NH2, hydroxy, -X 1 NHC(O)OR 1 a, -X 1 NHC(O)NHR 1 a and X 1is C1-C4 alkylene optionally substituted with 1 to 3 groups independently selected from halo, C1-C4 alkyl, or halo-substituted C1-C4 alkyl; R 1 a is H, C1-C4 alkyl, or halo-substituted C1-C4 alkyl; R 1 b is H or methyl; R 2 is H or a halogen; R 3 is H, halogen, C1-C4 alkoxy, C1-C4 alkyl, halo-substituted C1-C4 alkoxy, or halo-substituted C1-C4 alkyl; R 4 is halogen, H, or C1-C4 alkyl; R 5 is C1-C6 alkyl, C3-C6 cycloalkyl, C3-C8 branched alkyl, halo-substituted C1-C6 alkyl, halo-substituted C3-C8 branched alkyl, C3-C6 cycloalkyl-(C1-C3)-alkylene, or phenyl, wherein the phenyl is optionally substituted with 1 to 3 substituents each independently selected from halo, CH3, or CF3; R 6 is H, C1-C4 alkyl, or halogen; R 7 is H, C1-C6 alkyl, C3-C6 cycloalkyl, 1-methyl-(C3-C6)-cycloalkyl, 1-(halo-substituted methyl)-(C3-C6)-cycloalkyl, C3-C8 branched alkyl, halo-substituted C1-C6 alkyl, halo-substituted C3-C8 branched alkyl, or phenyl, wherein the phenyl is optionally substituted with 1 to 3 substituents selected from halogen, C1-C4 alkyl, or halo-substituted C1-C4 alkyl, and preferably R 7 is H, C1-C6 alkyl, C3-C6 cycloalkyl, 1-methyl-(C3-C6)-cycloalkyl, C3-C8 branched alkyl, or phenyl, wherein the phenyl is optionally substituted with 1 to 3 substituents selected from halogen, C1-C4 alkyl, or halo-substituted C1-C4 alkyl.

[0169] In one particular embodiment of the compounds of formula (I), R 1 is C1-C3 alkyl optionally substituted with cyano, -C(O)NH2, hydroxy, -X 1 NHC(O)OR 1 a and X 1 is C1-C4 alkylene optionally substituted with 1 to 3 groups independently selected from halo, C1-C4 alkyl, or halo-substituted C1-C4 alkyl; R 1 a is H, C1-C4 alkyl, or halo-substituted C1-C4 alkyl; R 2 is H or a halogen; R 3 is H, halogen, C1-C4 alkoxy, C1-C4 alkyl, halo-substituted C1-C4 alkoxy, or halo-substituted C1-C4 alkyl; R 4 is halogen, H, or C1-C4 alkyl; R 5 is C1-C6 alkyl, C3-C6 cycloalkyl, C3-C8 branched alkyl, halo-substituted C1-C6 alkyl, or halo-substituted C3-C8 branched alkyl; R 6 is H, C1-C4 alkyl, or halogen; R 7 is H, C1-C6 alkyl, C3-C6 cycloalkyl, 1-methyl-(C3-C6)-cycloalkyl, 1-(halo-substituted methyl)-(C3-C6)-cycloalkyl, C3-C8 branched alkyl, halo-substituted C1-C6 alkyl, or halo-substituted C3-C8 branched alkyl or phenyl, wherein the phenyl is optionally substituted with 1 to 3 substituents selected from halogen, C1-C4 alkyl, or halo-substituted C1-C4 alkyl, and preferably R 7 is H, C1-C6 alkyl, C3-C6 cycloalkyl, 1-methyl-(C3-C6 cycloalkyl), or phenyl, wherein the phenyl is optionally substituted with 1 to 3 substituents selected from halogen, C1-C4 alkyl, or halo-substituted C1-C4 alkyl; or a pharmaceutically acceptable salt thereof.

[0170] In a preferred embodiment, R 1 is -CH2-(S)-CH(CH3)NHC(O)OCH3; R 1 b is H; R 2 is H; R 3 is Cl; R 4 is H; R 5 is CH3; R 6 is F; R 7 is isopropyl; Provided is a compound of formula (II) or a pharmaceutically acceptable salt thereof (also referred to herein as "LGX818" or "encorafenib"):

[0171] In another embodiment, R 2 is H or F; R 3 is H, halogen, C1-C2 alkoxyl, C1-C2 alkyl, halo-substituted C1-C2 alkoxyl, or halo-substituted C1-C2 alkyl; R 4 is H or methyl; R 5 is C1-C4 alkyl, C3-C6 cycloalkyl, C3-05 branched alkyl, halo-substituted C1-C4 alkyl, halo-substituted C3-C6 branched alkyl, or C3-C6 cycloalkyl-(C1-C3)-alkylene; R 6 is H, C1-C2 alkyl, or halogen; R 7 is C3-C6 cycloalkyl, 1-methyl-(C3-C6)-cycloalkyl, or C3-C6 branched alkyl; There is provided a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0172] In another embodiment, R2 is H; R 3 is H, Cl, F, methoxy, methyl, or difluoromethoxy; R 4 is H; R 5 is methyl, cyclopropyl, ethyl, propyl, isopropyl, sec-butyl, isobutyl, trifluoromethyl, or 3,3,3-trifluoropropyl; R 6 is H, methyl, F, or Cl; R 7 is t-butyl, cyclopropyl, or 1-methylcyclopropyl; There is provided a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0173] In some embodiments, the BRAF inhibitor has formula (III): TIFF2026034588000005.tif47128, and pharmaceutically acceptable salts thereof, wherein a is 0, 1, 2, or 3; R 1 are the same or different and are halo, alkyl, haloalkyl, -OR 6 , -CO2R 6 , -NR 6 R 7 , and -CN; Ring A is selected from C3-C6 cycloalkyl, phenyl, a 5- to 6-membered heterocycle, and a 5- to 6-membered heteroaryl, wherein the heterocycle and the heteroaryl each have 1 or 2 heteroatoms selected from N, O, and S; Q 1 , Q 2 , Q 3 , and Q 4 are CH and CR respectively 2 , or N and Q 1 , Q 2 , Q 3 , and Q 4 at most one of is N; R 2are each the same or different and include halo, alkyl, haloalkyl, and -OR 6 more independently selected; W is selected from -O- and -S-; R 3 is H, alkyl, haloalkyl-, -alkylene-OH, -NR 6 R 7 , -C3-C6 cycloalkyl, -alkylene-C(O)-OH, -alkylene-NH2, and Het; R 3 is C3-C6 cycloalkyl, the C3-C6 cycloalkyl may be substituted with 1 or 2 substituents, which may be the same or different and independently selected from halo, C1-C3 alkyl, halo-(C1-C3)-alkyl, OH, O-(C1-C3)-alkyl, oxo, S-(C1-C3)-alkyl, SO2, NH2, N(H)(C1-C3)-alkyl, and N(C1-C3 alkyl)2; Het is a 5-6-membered heterocycle having 1 or 2 heteroatoms selected from N, O, and S, which may be the same or different and optionally substituted by 1 or 2 substituents each independently selected from halo, C-C alkyl, halo-(C-C)-alkyl, O—(C-C)-alkyl, C-C alkylene-O—(C-C)-alkyl, OH, C-C alkylene-OH, oxo, SO((C-C)-alkyl), C-C alkylene-SO((C-C)-alkyl), NH, N(H)((C-C)-alkyl), N(C-C alkyl), CN, and —CHCN; R 4 is H, alkyl, haloalkyl, alkenyl, -OR 6 , -R 5 -OR 6 , -R 5 -CO2R 6 , -R 5 -SO2R 6 , -R 5 -Het, -R 5 -C(O)-Het, -N(H)R 8 , -N(CH3)R 8 , and -R5 -NR 6 R 7 Selected from;R 5 are each the same or different and independently C1-C4 alkylene; R 6 and each R 7 are the same or different and are independently selected from H, alkyl, haloalkyl, —C(O)-alkyl, and —C(O)-cycloalkyl; R 8 is H, alkyl (optionally substituted with -OH), haloalkyl, C3-C6 cycloalkyl, -R 5 -(C3-C6)-cycloalkyl, Het 2 , -R 5 -Het 2 , -R 5 -OR 6 , -R 5 -OR 5 -OR 6 , -R 5 -C(O)2R 6 , -R 5 -C(O)NR 6 R 7 , -R 5 -N(H)C(O)-R 6 , -R 5 -N(H)C(O)-R 5 -OR 6 , -R 5 -N(H)C(O)2-R 5 -R 5 -NR 5 R 7 , -R 5 -S(O)2R 6 , -R 5 -CN, and -R 5 -N(H)S(O)2R 6 More selected; R 8is C3-C6 cycloalkyl, the C3-C6 cycloalkyl may be substituted by one or two substituents which are the same or different and independently selected from halo, C1-C3 alkyl, halo-(C1-C3)-alkyl, OH, O-(C1-C3)-alkyl, oxo, S-(C1-C3)-alkyl, SO2(C1-C3)-alkyl, NH2, N(H)-(C1-C3)-alkyl, and N(C1-C3 alkyl)2, and N(H)-SO2-(C1-C3)-alkyl; Het 2 has 1 or 2 heteroatoms selected from N, O, and S, and is 1, 2, 3, 4, or 5 C-C alkyl, or is the same or different and is halo, C-C alkyl, halo-(C-C)-alkyl, O—(C-C)-alkyl, C-C alkylene-O—(C-C alkyl), OH, C-C alkylene-OH, oxo, SO(C- a 4- to 6-membered heterocycle optionally substituted by one or two substituents independently selected from N(H)—(C1-C3 alkyl), C1-C3 alkylene-SO2(C1-C3 alkyl), NH2, N(H)—(C1-C3 alkyl), N(C1-C3 alkyl)2, N(H)SO2—(C1-C3 alkyl), C(O)(C1-C3 alkyl), CO2(C1-C4 alkyl), CN, and —CH2CN; R 9 and R 1 9 is independently selected from H and alkyl.

[0174] In a preferred embodiment, a is 2; R 1 is F; R 2 are each F; R 3 is t-butyl; R 4 N(H)R 8 and; R 8 is H; W is S Provided is a compound of formula (III) or a pharmaceutically acceptable salt thereof (referred to herein as "GSK2118436," "dabrafenib," or "Tafinlar"):

[0175] In some embodiments, the BRAF inhibitor has formula (IV): TIFF2026034588000006.tif39128, wherein R 2 , R 4 , R 5 , and R 6 is hydrogen, halogen, optionally substituted lower alkyl, optionally substituted lower alkenyl, optionally substituted lower alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -CN, -NO2, -CR a R b R 26 , and -LR 26 independently selected from the group consisting of: R 3 is hydrogen, halogen, optionally substituted lower alkyl, optionally substituted lower alkenyl, optionally substituted lower alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -CN, -NO2, -CR a R b R 26 , -LR 26 , and -A-Ar-L1-R 24 selected from the group consisting of: A is -O-, -S-, -CR a R b -, -NR 1 selected from the group consisting of -, -C(O)-, -C(S)-, -S(O)-, and -S(O)-; R 1 is hydrogen, lower alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C(O)R 7 , -C(S)R 7, -S(O)2R 7 , -C(O)NHR 7 , -C(S)NHR 7 , and -S(O)2NHR 7 and lower alkyl is selected from the group consisting of fluoro, -OH, -NH2, lower alkoxy, lower alkylthio, monoalkylamino, dialkylamino, and -NR 8 R 9 and the alkyl chain of lower alkoxyl, lower alkylthio, monoalkylamino, or dialkylamino may be substituted with one or more substituents selected from the group consisting of fluoro, —OH, —NH2, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino, provided that the substituent on the alkyl chain carbon bonded to the O of alkoxy, the S of thioalkyl, or the N of monoalkylamino or dialkylamino is fluoro; and further, R 1 When is lower alkyl, -NR 1 The substituent of the lower alkyl carbon bonded to the N of - is fluoro, and the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl may be substituted with one or more substituents selected from the group consisting of halogen, -OH, -NH2, lower alkyl, fluoro-substituted lower alkyl, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino; R 7 is selected from the group consisting of lower alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein lower alkyl is selected from fluoro, —OH, —NH2, lower alkoxy, lower alkylthio, monoalkylamino, dialkylamino, and —KR 8 R 9 and optionally substituted with one or more substituents selected from the group consisting of: 7 , -C(S)NHR 7 , or -S(O)2NHR 7the substituent of the alkyl carbon bonded to the N of the lower alkoxy, lower alkylthio, monoalkylamino, or dialkylamino is optionally substituted with one or more substituents selected from the group consisting of fluoro, -OH, -NH2, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino, provided that the substituent of the O of the alkoxy, the S of the thioalkyl, or the alkyl chain carbon bonded to the N of the monoalkylamino or dialkylamino is fluoro, and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of halogen, -OH, -NH2, lower alkyl, fluoro-substituted lower alkyl, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino; Ar is selected from the group consisting of optionally substituted arylene and optionally substituted heteroarylene; L at each location is -(alk) a -S-(alk) b -, -(alk) a -O-(alk) b -, -(alk) a -NR 25 -(alk) b -, -(alk) a -C(O)-(alk) b -, -(alk) a -C(S)-(alk) b -, -(aUc) a -S(O)-(alk) b -, -(alk) a -S(O)2-(alk) b -, -(alk) a -OC(O)-(alk) b -, -(alk) a -C(O)O-(alk) b -, -(alk) a -OC(S)-(alk) b -, -(alk)a -C(S)O-(alk) b -, -(alk) a -C(O)NR 25 -(alk) b -, -(alk) a -C(S)NR 25 -(alk) b -, -(alk) a -S(O)2NR 25 -(alk) b -, -(alk) a -NR 25 C(O)-(alk) b -, -(alk) a -NR 25 C(S)-(alk) b -, -(alk) a -NR 25 S(O)2-(alk) b -, -(alk) a -NR 25 C(O)O-(alk) b -, -(alk) a -NR 25 C(S)O-(alk) b -, -(alk) a -OC(O)NR 25 -(alk) b -, -(alk) a -OC(S)NR 25 -(alk) b -, -(alk) a -NR 25 C(O)NR 25 -(alk) b -, -(alk) a -NR 25 C(S)NR 25 -(alk) b - and -(alk) a -NR 25 S(O)NR 25 -(alk) b a and b are independently 0 or 1; alk is C1-C3 alkylene, or fluoro, -OH, -NH2, lower alkyl, lower alkoxy, lower alkylthio, monoalkylamino, dialkylamino, and -NR 8 R9 C1-C3 alkylene substituted with one or more substituents selected from the group consisting of lower alkyl, or the alkyl chain of lower alkoxy, lower alkylthio, monoalkylamino, or dialkylamino may be substituted with one or more substituents selected from the group consisting of fluoro, -OH, -NH2, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino, with the proviso that the substituent on the alkyl chain carbon bonded to O in alkoxy, S in thioalkyl, or N in monoalkylamino or dialkylamino is fluoro; L1 is -(CR a R b )v- or L, where v is 1, 2, or 3; R at each occurrence a and R b is hydrogen, fluoro, -OH, -NH2, lower alkyl, lower alkoxy, lower alkylthio, monoalkylamino, dialkylamino, and -NR 8 R 9 and the alkyl chain of the lower alkyl, lower alkoxy, lower alkylthio, monoalkylamino, or dialkylamino may be optionally substituted with one or more substituents selected from the group consisting of fluoro, -OH, -NH2, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino, with the proviso that the substituent on the alkyl chain carbon bonded to the O of the alkoxy, the S of the thioalkyl, or the N of the monoalkylamino or dialkylamino is fluoro; or R on the same or different carbons a and R b two of these together form a 3- to 7-membered monocyclic cycloalkyl or a 5- to 7-membered monocyclic heterocycloalkyl, and R a and R b The others are hydrogen, fluoro, -OH, -NH2, lower alkyl, lower alkoxy, lower alkylthio, monoalkylamino, dialkylamino, and -NR 8 R9 and the alkyl chain of lower alkyl, lower alkoxy, lower alkylthio, monoalkylamino, or dialkylamino is optionally substituted with one or more substituents selected from the group consisting of fluoro, -OH, -NH2, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino, provided that the substituent on the alkyl chain carbon bonded to O of alkoxy, S of thioalkyl, or N of monoalkylamino or dialkylamino is fluoro, and the 3- to 7-membered monocyclic cycloalkyl or 5- to 7-membered monocyclic heterocycloalkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, -OH, -NH2, lower alkyl, fluoro-substituted lower alkyl, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, fluoro-substituted lower alkylthio, monoalkylamino, dialkylamino, and cycloalkylamino; R 8 and R 9 together with the nitrogen to which they are attached form a 5- to 7-membered heterocycloalkyl optionally substituted with one or more substituents selected from the group consisting of fluoro, -OH, -NH2, lower alkyl, fluoro-substituted lower alkyl, lower alkoxy, fluoro-substituted lower alkoxy, lower alkylthio, and fluoro-substituted lower alkylthio; R at each location 25 are independently selected from the group consisting of hydrogen, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; R at each location 24 and R 26 is hydrogen (provided that the hydrogen is not bonded to any of S(O), S(O)2, C(O), or C(S) of L or Li), optionally substituted lower alkyl, optionally substituted lower alkenyl (provided that R 24 or R 26is optionally substituted lower alkenyl, the alkene carbons are not bonded to N, S, O, S(O), S(O)2, C(O), or C(S) of L or L1), optionally substituted lower alkynyl (provided that R 24 or R 26 When is an optionally substituted lower alkynyl, the alkyne carbons are independently selected from the group consisting of N, S, O, S(O), S(O), C(O), or C(S) of L or L), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl.

[0176] In a preferred embodiment, R 2 is H; R 3 Ga-A-Ar-L1-R 24 and; A is -C(O)-; Ar is 2,4-difluorophenyl; L1 is -SO2-; R 4 is H; R 5 is 4-chlorophenyl; R 6 is H; R 24 is n-propyl Provided is a compound of formula (III) or a pharmaceutically acceptable salt thereof (referred to herein as "PLX4032," "vemurafenib," or "Zelboraf"):

[0177] In other embodiments, those skilled in the art can generate or identify new BRAF inhibitors using in vitro, in vivo, in silico, or other screening methods known in the art.For example, BRAF inhibitors of wild-type BRAF can be identified from a training set of small molecules, peptides, or nucleic acids using an assay to detect the phosphorylation of molecules downstream of BRAF in the MAPK signaling cascade (for example, MEK and / or ERK).BRAF inhibitors can suppress or inhibit BRAF expression and / or signaling function, thereby reducing the phosphorylation of MEK and ERK.Several phosphorylation assays are available that can be used in such embodiments, including but not limited to kinase activity assays (for example, those sold by R&D Systems, Promega, Life Technologies); phosphorylation-specific antibodies for use in immunoassays such as Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunocytochemistry, immunohistochemistry, etc.; mass spectrometry, proteomics, and phosphorylated protein multiplex assays. In certain embodiments, BRAF inhibitors for use in the embodiments described herein may be identified using screening methods that measure the ability of candidate inhibitors to activate the MAPK pathway.

[0178] VEGF inhibitors In certain embodiments, the VEGF inhibitor is aflibercept, pegaptanib, tivozanib, 3-(4-bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride, axitinib, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl-)methoxy]quinazolin-4-amine, an inhibitor of VEGF-R2 and VEGF-R1, axitinib, N,2-dimethyl-6-(2-(1-methyl-1H-imidazol-2-yl)thieno[3,2-b]pyridin-7-yloxy)benzo[b]thiophene-3-carboxamide, a tyrosine kinase inhibitor of the RET / PTC oncogenic kinase. and a combination thereof.

[0179] In certain preferred embodiments, the VEGF inhibitor is a VEGF receptor inhibitor, more preferably a VEGF receptor kinase inhibitor such as tivozanib (AV-951), AZD2932, midostaurin (pkc412), BAW2881 (NVP-BAW2881), nintedanib (BIBF 1120), SU5402, SU1498, BFH772, sorafenib, sunitinib, dovitinib (TKI258), semaxanib (SU5416), hypericin, vatalanib, ZM306416, AAL993, SU4312, DMXAA, or foretinib.

[0180] In certain embodiments, the VEGF receptor inhibitor is afatinib, imatinib, dacomitinib, dasatinib, ponatinib, KD-019, bosutinib, lapatinib ditosylate, AZD9291, neratinib, poziotinib, S-222611, suramin hexasodium, AL-6802, BGB-102, PB357, pyrotinib, nib), sunitinib, sorafenib tosylate, pazopanib, regorafenib, apatinib, axitinib, carbozantinib, lenvatinib, nintedanib, vandetanib, tivozanib, anlotinib, midostaurin, muparfostat, BMS-690514, ENMD-2076 , golvatinib, lucitanib, motesanib, necuparinib, RAF265, famitinib, telatinib, X82, ALNVSP, altiratinib, ABT348, MGCD516, OB318, ODM203, HHGV678, LY-3012207, CS2164, ilorasertib, radotinib, bafetinib, NRCAN-019, ABL001, metatinib tromethamine, rebastinib tosylate, or a multi-tyrosine kinase inhibitor such as VX-15.

[0181] TGFβ or TGFβ receptor inhibitors TGF-β signaling is involved in many cellular functions, including cell growth, cell fate, and apoptosis. Signaling typically begins with the binding of a TGF-β superfamily ligand to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates SMADs, which function as nuclear transcription factors and regulate target gene expression. Alternatively, TGF-β signaling can activate the MAP kinase signaling pathway, for example, via p38 MAP kinase.

[0182] TGFβ superfamily ligands include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, and TGFβ.

[0183] TGFβ inhibitors, as used herein, include agents that reduce the activity of the TGFβ signaling pathway. There are many different ways of disrupting the TGFβ signaling pathway known in the art, any of which can be used in the present invention. For example, TGFβ signaling can be disrupted by inhibiting TGFβ expression using small interfering RNA strategies; inhibiting furin (a TGFβ-activating protease); inhibiting the pathway with physiological inhibitors, such as inhibiting BMPs with noggin, DAN, or DAN-like proteins; neutralizing TGFβ with monoclonal antibodies; inhibiting TGFβ receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7, or other TGFβ-related receptor kinases with small molecule inhibitors; inhibiting Smad2 and Smad3 signaling by overexpressing the physiological inhibitor Smad7 or by using thioredoxin as a Smad anchor, rendering the Smads inactivatable (Fuchs, Inhibition of TGFβ Signaling for the Treatment of Tumor Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy 6(1):29-43(15), 2011).

[0184] For example, a TGFβ inhibitor may target a serine / threonine protein kinase selected from TGFβ receptor kinase 1, ALK4, ALK5, ALK7, or p38. ALK4, ALK5, and ALK7 are all closely related receptors of the TGFβ superfamily. ALK4 has a GI number of 91; ALK5 (also known as TGFβ receptor kinase 1) has a GI number of 7046; and ALK7 has a GI number of 658. An inhibitor of any of these kinases reduces the enzymatic activity of one or more of these kinases. Inhibition of ALK and p38 kinase has previously been shown to be associated with B-cell lymphoma (Bakkebo et al., "TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad 1 / 5 signaling and constitutively active p38MAPK," BMC Immunol. 11:57, 2010).

[0185] In certain embodiments, a TGFβ inhibitor can bind to and inhibit the activity of a Smad protein, such as R-SMAD or SMAD1-5 (ie, SMAD1, SMAD2, SMAD3, SMAD4, or SMAD5).

[0186] In certain embodiments, the TGFβ inhibitor may bind to and reduce the activity of a Ser / Thr protein kinase selected from TGFβ receptor kinase 1, ALK4, ALK5, ALK7, or p38.

[0187] In certain embodiments, the media of the present invention comprises an inhibitor of ALK5.

[0188] In certain embodiments, the TGFβ inhibitor or TGFβ receptor inhibitor does not include a BMP antagonist (ie, is an agent other than a BMP antagonist).

[0189] Various methods are known for determining whether a substance is a TGFβ inhibitor.For example, a cell assay can be used in which cells are stably transfected with a reporter construct containing the human PAI-1 promoter or Smad binding site that drives a luciferase reporter gene.The inhibition of luciferase activity compared to a control group can be used as a measure of the activity of a compound (De Gouville et al., Br.J.Pharmacol.145(2):166-177,2005, incorporated herein by reference).Another example is the ALPHASCREEN® phosphosensor assay for measuring kinase activity (Drew et al., J.Biomol.Screen.16(2):164-173,2011, incorporated herein by reference).

[0190] The TGFβ inhibitor useful for the present invention can be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer, antibody or antigen-binding portion thereof. The inhibitor can be naturally occurring or synthetic. Examples of small molecule TGFβ inhibitors that can be used in the context of the present invention include, but are not limited to, the small molecule inhibitors listed in Table 1 below.

[0191] Table 1: Small molecule TGF inhibitors targeting receptor kinases TIFF2026034588000007.tif105144

[0192] One or more of the inhibitors listed in Table 1 above, or a combination thereof, can be used as a TGFβ inhibitor in the present invention. In certain embodiments, the combination can include SB-525334 and SD-208 and A83-01; SD-208 and A83-01; or SD-208 and A83-01.

[0193] Those skilled in the art will recognize that there are numerous other small molecule inhibitors that are primarily designed to target other kinases but also inhibit TGFβ receptor kinase at high concentrations. For example, SB-203580 is a p38 MAP kinase inhibitor that can inhibit ALK5 at high concentrations (e.g., approximately 10 μM or higher). Such inhibitors that inhibit the TGFβ signaling pathway can also be used in the present invention. In certain embodiments, A83-01 can be added to the culture medium at a concentration of 10 nM to 10 μM, or 20 nM to 5 μM, or 50 nM to 1 μM. In certain embodiments, A83-01 can be added to the culture medium at approximately 500 nM. In certain embodiments, A83-01 can be added to the culture medium at a concentration of 350 to 650 nM, 450 to 550 nM, or approximately 500 nM. In certain embodiments, A83-01 can be added to the culture medium at a concentration of 25-75 nM, 40-60 nM, or about 50 nM.

[0194] SB-431542 can be added to the culture medium at a concentration of 80 nM to 80 μM, 100 nM to 40 μM, 500 nM to 10 μM, or 1 to 5 μM. For example, SB-431542 can be added to the culture medium at about 2 μM.

[0195] SB-505124 can be added to the culture medium at a concentration of 40 nM to 40 μM, 80 nM to 20 μM, or 200 nM to 1 μM. For example, SB-505124 can be added to the culture medium at about 500 nM.

[0196] SB-525334 can be added to the culture medium at a concentration of 10 nM to 10 μM, or 20 nM to 5 μM, or 50 nM to 1 μM. For example, SB-525334 can be added to the culture medium at about 100 nM.

[0197] LY 364947 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, LY 364947 can be added to the culture medium at about 500 nM.

[0198] SD-208 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, SD-208 can be added to the culture medium at about 500 nM.

[0199] S JN 2511 can be added to the culture medium at a concentration of 20 nM to 20 μM, or 40 nM to 10 μM, or 100 nM to 1 μM. For example, A83-01 can be added to the culture medium at approximately 200 nM.

[0200] p38 inhibitors "p38 inhibitors" may include inhibitors that directly or indirectly negatively regulate p38 signaling, such as agents that bind to and reduce the activity of at least one p38 isoform. p38 protein kinase (see GI No. 1432) is part of the mitogen-activated protein kinase (MAPK) family. MAPKs are serine / threonine-specific protein kinases that respond to extracellular stimuli, such as environmental stress and inflammatory cytokines, and regulate various cellular activities, such as gene expression, differentiation, division, proliferation, and cell survival / apoptosis. p38 MAPK exists as α, β, β2, γ, and δ isoforms.

[0201] Various methods are known to determine whether a substance is a p38 inhibitor, such as the detection of phosphorylation at Thrl80 / Tyrl82 with a phospho-specific antibody, which provides a well-established measure of cellular p38 activation or inhibition; biochemical recombinant kinase assays; tumor necrosis factor alpha (TNFα) secretion assays; and the DiscoverRx high-throughput screening platform for p38 inhibitors. Several p38 activity assay kits also exist (e.g., Millipore, Sigma-Aldrich).

[0202] In certain embodiments, high concentrations (e.g., greater than 100 nM, greater than 1 μM, greater than 10 μM, or greater than 100 μM) of p38 inhibitors can have the effect of inhibiting TGFβ. In other embodiments, p38 inhibitors do not inhibit TGFβ signaling.

[0203] Various p38 inhibitors are known in the art (see, e.g., Table 1). In some embodiments, the inhibitor that directly or indirectly negatively regulates p38 signaling is selected from the group consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257, and BIRB-796.

[0204] In certain embodiments, the medium contains both (a) an inhibitor that binds to and reduces the activity of one or more kinases from the group consisting of ALK4, ALK5, and ALK7; and (b) an inhibitor that binds to and reduces the activity of p38.

[0205] In certain embodiments, the medium comprises an inhibitor that binds to and reduces the activity of ALK5 and an inhibitor that binds to and reduces the activity of p38.

[0206] In one embodiment, the inhibitor binds to its target (e.g., TGFβ and / or p38) and reduces its activity by more than 10%, more than 30%, more than 60%, more than 80%, more than 90%, more than 95%, or more than 99% compared to a control, as assessed by a cellular assay. Examples of cellular assays for measuring target inhibition are well known in the art, as described above.

[0207] TGFβ and / or p38 inhibitors may have IC50 values ​​of 2000 nM or less; less than 1000 nM; less than 100 nM; less than 50 nM; less than 30 nM; less than 20 nM, or less than 10 mM. The IC50 value refers to the effectiveness of an inhibitor in inhibiting the biological or biochemical function of its target. IC50 indicates the amount of a specific inhibitor required to inhibit a kinase by 50%. The IC50 value can be calculated according to the assay method described above. TGFβ and / or p38 inhibitors can exist in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules, such as small natural or synthetic organic molecules up to 2000 Da, preferably less than 800 Da, peptidomimetics, inorganic molecules, peptides, polypeptides, antisense oligonucleotide aptamers, and structural or functional mimetics thereof, including small molecules.

[0208] In certain embodiments, the inhibitor of TGFβ and / or p38 may be an aptamer. As used herein, the term "aptamer" refers to a strand of oligonucleotide (DNA or RNA) that can adopt a highly specific three-dimensional conformation. Aptamers are designed to have high binding affinity and specificity for certain target molecules, including extracellular and intracellular proteins. Aptamers can be generated, for example, using the SELEX process (see, for example, Tuerk and Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA Polymerase. Science 249:505-510, 1990, incorporated herein by reference).

[0209] In certain embodiments, the inhibitor of TGFβ and / or p38 can be a small synthetic molecule having a molecular weight of 50-800 Da, 80-700 Da, 100-600 Da, or 150-500 Da.

[0210] In certain embodiments, the inhibitor of TGFβ and / or p38 includes a pyridinylimidazole or a 2,4-disubstituted teridine or quinazoline, e.g., Includes TIFF2026034588000008.tif20128.

[0211] Specific examples of inhibitors of TGFβ and / or p38 that may be used in accordance with the present invention include, but are not limited to, SB-202190, SB-203580, SB-206718, SB-227931, VX-702, VX-745, PD-169316, RO-4402257, BIRB-796, A83-01 SB-431542, SB-505124, SB-525334, LY 364947, SD-208, SJ 2511 (see Table 2).

[0212] For example, SB-202190 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-202190 can be added to the culture medium at approximately 10 μM.

[0213] SB-203580 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-203580 can be added to the culture medium at approximately 10 μM.

[0214] VX-702 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 25 μM. For example, VX-702 can be added to the culture medium at approximately 5 μM.

[0215] VX-745 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 250 nM to 10 μM. For example, VX-745 can be added to the culture medium at approximately 1 μM.

[0216] PD-169316 can be added to the culture medium at a concentration of 100 nM to 200 μM, or 200 nM to 100 μM, or 1 μM to 50 μM. For example, PD-169316 can be added to the culture medium at approximately 20 μM.

[0217] RO-4402257 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 500 nM to 10 μM. For example, RO-4402257 can be added to the culture medium at approximately 1 μM.

[0218] BIRB-796 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 500 nM to 10 μM. For example, BIRB-796 can be added to the culture medium at approximately 1 μM.

[0219] For applicable concentrations for other factors in Table 2, see Table 1 and associated text above.

[0220] Table 2: Exemplary TGFβ and / or p38 inhibitors TIFF2026034588000009.tif211148TIFF2026034588000010.tif63148

[0221] Thus, in some embodiments, inhibitors that directly or indirectly negatively regulate TGFβ and / or p38 signaling are added to the culture medium at a concentration of 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM. For example, the total concentration of one or more inhibitors is 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM.

[0222] Oct4 activator The Oct4 activator is a drug that can activate a reporter gene driven by the Oct4 promoter, such as a luciferase gene under the transcriptional control of the Oct4 promoter, and more preferably, can activate both a reporter gene driven by the Oct4 promoter and a reporter gene driven by the Nanog promoter. Furthermore, when added to the reprogramming mixture together with the reprogramming factor tetrad (Oct4, Sox2, c-Myc, and Klf4), the Oct4 activator enhances iPSC reprogramming efficiency and accelerates the reprogramming process. Exemplary Oct4 activators are taught, for example, in U.S. Patent Application No. 20150191701 and Li et al. (2012) "Identification of Oct4-activating compounds that enhance reprogramming efficiency", PNAS 109(51):20853-8.

[0223] In certain embodiments, the Oct4 activator has the following formula: TIFF2026034588000011.tif35128, wherein: X 1 is C(R 12 ) or N; X 2 is C(R 4 ) or N; X 3 is C(R 5 ) or N; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12are independently selected from hydrogen, halogen, —CN, —NO2, —NH2, —CF3, —CCl3, —OH, —SH, —SO3H, —C(O)OH, —C(O)NH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 2 and R 3 may together form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl.

[0224] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO, —NH, —CF, —CCl, —OH, —SH, —SOH, —C(O)OH, —C(O)NH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted heterocycloalkyl.

[0225] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO 2 , —NH 2 , —CF 3 , —CCl 3 , —OH, —SH, —SO 3 H, —C(O)OH, —C(O)NH 2 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

[0226] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, substituted or unsubstituted C1 to C 10 and independently selected from alkyl, substituted or unsubstituted 2- to 10-membered heteroalkyl, or substituted or unsubstituted 3- to 8-membered heterocycloalkyl.

[0227] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, substituted or unsubstituted C1 to C 10 alkyl, or substituted or unsubstituted 2- to 10-membered heteroalkyl.

[0228] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12are independently selected from hydrogen, halogen, —CN, —NO 2 , —NH 2 , —CF 3 , —CCl 3 , —OH, —SH, —SO 3 H, —C(O)OH, —C(O)NH 2 , unsubstituted alkyl, unsubstituted heteroalkyl, or substituted heterocycloalkyl.

[0229] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO 2 , —NH 2 , —CF 3 , —CCl 3 , —OH, —SH, —SO 3 H, —C(O)OH, —C(O)NH 2 , unsubstituted alkyl, or unsubstituted heteroalkyl.

[0230] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 represents hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, unsubstituted C1 to C 10 and independently selected from alkyl, unsubstituted 2- to 10-membered heteroalkyl, or unsubstituted 3- to 8-membered heterocycloalkyl.

[0231] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R9 , R 10 , R 11 , and R 12 represents hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, unsubstituted C1 to C 10 alkyl, or unsubstituted 2-10 membered heteroalkyl.

[0232] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, unsubstituted C1-C 10 alkyl, or unsubstituted 2-10 membered heteroalkyl.

[0233] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —N(CH 3 ) 2 , unsubstituted C 1 -C 5 alkyl, or unsubstituted C 1 -C 5 alkoxy.

[0234] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R12 are independently selected from hydrogen, halogen, —N(CH 3 ) 2 , unsubstituted C 1 -C 5 alkyl, methoxy, ethoxy, or propoxy.

[0235] In certain embodiments, the Oct4 activator is: TIFF2026034588000012.tif110128.

[0236] In certain embodiments, the Oct4 activator has the structure: OAC1 with TIFF2026034588000013.tif19128.

[0237] PDGFRα / β inhibitors In certain embodiments, the medium comprises a PDGFR inhibitor, preferably a PDGFRα / β inhibitor.

[0238] An exemplary PDGFRα / β inhibitor is GZD856 (Zhang et al. Cancer Lett. 2016 May 28;375(1):172-178): The file is TIFF2026034588000014.tif23128.

[0239] In certain embodiments, the PDGFRα / β inhibitor is a potent inhibitor with an IC50 of 250 nM or less (in a cell-free assay), more preferably 100 nM or less, and may be selected from sunitinib malate, ponatinib (AP24534), telatinib, amuvatinib (MP-470), Ki8751, regorafenib, crenolanib (CP-868596), CP-673451, axitinib, and nintedanib (BIBF 1120).

[0240] In certain embodiments, the PDGFRα / β inhibitor is a potent, selective inhibitor of PDGFRα / β with an IC50 (in a cell-free assay) of 250 nM or less, more preferably 100 nM or less, and exhibits greater than 100-fold selectivity over other angiogenic receptors, more preferably greater than 200-fold, 300-fold, or even 400-fold selectivity over other angiogenic receptors. An exemplary selective inhibitor of PDGFRα / β is CP-673451: The file is TIFF2026034588000015.tif37128.

[0241] JNK inhibitors In certain embodiments, the culture medium contains a JNK inhibitor. Mitogen-activated kinases JNK1 / 2 / 3 are key enzymes in a signaling module that transduces and integrates extracellular stimuli into coordinated cellular responses. In certain embodiments, the JNK inhibitor inhibits JNK kinase, i.e., inhibits the phosphorylation of c-Jun, a direct substrate of JNK kinase, in cells exposed to the inhibitor with an IC50 of 250 nM or less, more preferably 100 nM.

[0242] In certain embodiments, at least one apoptosis inhibitor is a JNK inhibitor.Any JNK inhibitor is intended for use in the formulation, composition, method of the present invention.JNK inhibitors are generally known to those skilled in the art (see, for example, U.S. Patent No. 6,949,544; No. 7,129,242; No. 7,326,418, No. 8,143,271 and No. 8,530,480).

[0243] In certain embodiments, the JNK inhibitor is preferably a selective JNK inhibitor that inhibits phosphorylation of c-Jun in a manner that is dependent on the covalent modification of a conserved cysteine ​​residue within the JNK kinase.

[0244] In certain embodiments, the JNK inhibitor is JNK-IN-5, JNK-IN-6, JNK-IN-7, JNK-IN-8, JNK-IN-9, JNK-IN-10, JNK-IN-11, JNK-IN-12, SP-600125, or AS601245.

[0245] Exemplary JNK inhibitors include, but are not limited to, SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one), JNK-IN-8 (3-[[4-(dimethylamino)-1-oxo-2-buten-1-yl]amino]-N-[3-methyl-4-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide); and JNK inhibitor IX (N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thien-2-yl)-1-naphthalenecarboxamide).

[0246] Notch agonists The culture medium of the present invention may additionally contain a Notch agonist. Notch signaling has been shown to play an important role in both cell fate determination and cell survival and proliferation. Notch receptor proteins can interact with a number of surface-bound or secreted ligands, including, but not limited to, Jagged-1, Jagged-2, Delta-1, or Delta-like-1, Delta-like-3, and Delta-like-4. Upon ligand binding, Notch receptors are activated by sequential cleavage events involving members of the ADAM protease family, as well as by intramembrane cleavage regulated by the gamma-secretase presinilin. This results in the translocation of the intracellular domain of Notch to the nucleus, where it transcriptionally activates downstream genes.

[0247] "Notch agonist," as used herein, includes molecules that stimulate Notch activity in cells by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least about 100%, at least about 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more, compared to the level of Notch activity in the absence of the Notch agonist. As is known in the art, Notch activity can be determined by measuring the transcriptional activity of Notch, for example, by the 4xwtCBF1-luciferase reporter construct described by Hsieh et al. (Mol. Cell. Biol. 16:952-959, 1996, incorporated herein by reference).

[0248] In certain embodiments, the Notch agonist is selected from Jagged-1, Delta-1, and Delta-like-4, or active fragments or derivatives thereof. In certain embodiments, the Notch agonist is the DSL peptide (Dontu et al., Breast Cancer Res., 6:R605-R615, 2004) having the amino acid sequence CDDYYYGFGCNKFCRPR (SEQ ID NO:36). The DSL peptide (ANA spec) may be used at a concentration of 10 μM to 100 nM, or at least 10 μM, but not more than 100 nM. In certain embodiments, the final concentration of Jagged-1 is about 0.1 to 10 μM; or about 0.2 to 5 μM; or about 0.5 to 2 μM; or about 1 μM.

[0249] In certain embodiments, any of the specific Notch agonists mentioned herein, such as Jagged-1, Jagged-2, Delta-1, and Delta-like-4, may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective Wnt agonist activity, and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0250] Representative Notch agonist sequences referred to herein are represented in SEQ ID NOs. 28-35.

[0251] Notch agonists can be added to the culture medium every 1, 2, 3, or 4 days for the first 1-2 weeks of stem cell culture.

[0252] Nicotinamide The culture medium of the present invention may be supplemented with nicotinamide or its analogs, precursors, or mimetics, such as methylnicotinamide, benzamide, pyrazinamide, thymine, or niacin. Nicotinamide may be added to the culture medium at a final concentration of 1 to 100 mM, 5 to 50 mM, or preferably 5 to 20 mM. For example, nicotinamide may be added to the culture medium at a final concentration of approximately 10 mM. Similar concentrations of nicotinamide analogs, precursors, or mimetics may also be used, alone or in combination.

[0253] Extracellular matrix (ECM) Extracellular matrix (ECM), used interchangeably herein with "basement membrane matrix," is secreted by connective tissue cells and comprises a variety of polysaccharides, water, elastin, and proteins, which may include proteoglycans, collagen, entactin (nidogen), fibronectin, fibrinogen, fibrillin, laminin, and hyaluronic acid. The ECM can provide a suitable substrate and microenvironment useful for the selection and culture of stem cells of the present invention.

[0254] In certain embodiments, the stem cells of the present invention are attached to or in contact with ECM.Various types of ECM are known in the art and can contain different compositions, including different types of proteoglycans and / or different combinations of proteoglycans.ECM can be provided by culturing ECM-producing cells, such as certain fibroblasts.Examples of extracellular matrix-producing cells include chondrocytes, which mainly produce collagen and proteoglycans; fibroblasts, which mainly produce type IV collagen, laminin, interstitial procollagen, and fibronectin; and colonic myofibroblasts, which mainly produce collagen (type I, type III, and type V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C.

[0255] In certain embodiments, at least some of the ECM is produced by a murine 3T3-J2 clone that can be grown on MATRIGEL™ basement membrane matrix (BD Biosciences) as a feeder cell layer.

[0256] Alternatively, ECM can be commercially available. Examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen) and MATRIGEL™ basement membrane matrix (BD Biosciences). The use of ECM to culture stem cells can enhance the long-term survival of stem cells and / or the continued existence of undifferentiated stem cells. Alternatives can be scaffolds such as fibrin matrix or fibrin gel, or glycerol-treated allografts that have been initially depleted of cells.

[0257] In certain embodiments, the ECM for use in the methods of the present invention comprises at least two distinct glycoproteins, such as two different types of collagen, or collagen and laminin. The ECM may be a synthetic hydrogel extracellular matrix or a naturally occurring ECM. In certain embodiments, the ECM is provided by MATRIGEL™ basement membrane matrix (BD Biosciences), which comprises laminin, entactin, and collagen IV.

[0258] Culture medium The cell culture medium used in the methods of the present invention can include any cell culture medium, such as a culture medium buffered to about pH 7.4 (e.g., about pH 7.2 to 7.6) with a carbonate-based buffer. Many commercially available tissue culture media may be suitable for the methods of the present invention, including, but not limited to, Dulbecco's Modified Eagle's Medium (DMEM, e.g., DMEM without L-glutamine and containing high glucose), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Eagle's Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Medium, and Minimal Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI 1640 medium.

[0259] Cells may be cultured in an atmosphere containing 5-10% CO2 (e.g., at least about 5%, 10% or less CO2, or about 5% CO2). In certain embodiments, the cell culture medium is DMEM / F12 (e.g., a 3:1 mixture) or RPMI 1640 supplemented with L-glutamine, insulin, penicillin / streptomycin, and / or transferrin. In certain embodiments, Advanced DMEM / F12 or Advanced RPMI, which are optimized for serum-free culture and already contain insulin, are used. Advanced DMEM / F12 or Advanced RPMI medium may be further supplemented with L-glutamine and penicillin / streptomycin. In certain embodiments, the cell culture medium is supplemented with one or more purified, natural, semi-synthetic, and / or synthetic factors described herein. In certain embodiments, the cell culture medium is supplemented with about 10% fetal bovine serum (FBS) that is not heat-inactivated prior to use. For example, additional supplements such as B-27® serum-free supplement (Invitrogen), N-acetylcysteine ​​(Sigma), and / or N2 serum-free supplement (Invitrogen), or Neurobasal (Gibco), TeSR (StemGent), etc. may be added to the culture medium.

[0260] In certain embodiments, the medium may contain one or more antibiotics (such as penicillin / streptomycin) to prevent contamination. In certain embodiments, the medium may have an endotoxin content of less than 0.1 endotoxin units / mL, or may have an endotoxin content of less than 0.05 endotoxin units / mL. Methods for determining the endotoxin content of culture medium are known in the art.

[0261] The cell culture medium according to the present invention allows the survival and / or proliferation and / or differentiation of epithelial stem cells on an extracellular matrix. As used herein, the term "cell culture medium" is synonymous with "culture medium," "culture medium," or "cell culture medium."

[0262] The modified (growth) medium of the present invention comprises, in a basal medium, (a) a ROCK (Rho kinase) inhibitor; (b) a Wnt agonist; (c) a mitogenic growth factor; (d) a TGFβ signaling pathway inhibitor, such as a TGFβ inhibitor or a TGFβ receptor inhibitor; and (e) insulin or IGF; the medium optionally further comprises a bone morphogenetic protein (BMP) antagonist.

[0263] Thus, in one aspect, the present invention provides a basal medium (basal medium) comprising insulin or an insulin-like growth factor; T3 (3,3',5-triiodo-L-tyrosine); hydrocortisone; adenine; EGF; and 10% fetal bovine serum (not heat-inactivated) in a DMEM:F12 3:1 medium supplemented with L-glutamine.

[0264] In certain embodiments, the basal medium comprises about 5 μg / mL insulin; about 2×10 M T3 (3,3′,5-triiodo-L-tyrosine); about 400 ng / mL hydrocortisone; about 24.3 μg / mL adenine; about 10 ng / mL EGF; and 10% fetal bovine serum (not heat inactivated) in DMEM:F12 3:1 medium supplemented with about 1.35 mM L-glutamine.

[0265] In certain embodiments, the concentration for each of the media components mentioned in the immediately preceding paragraph is independently 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% higher or lower than the respective stated value, or 2-fold, 3-fold, 5-fold, 10-fold, 20-fold higher than the respective stated value. For example, in an exemplary media, the insulin concentration may be 6 μg / mL (20% higher than the stated 5 μg / mL), the EGF concentration may be 5 ng / mL (50% lower than the stated 10 ng / mL), and each remaining component has the same concentration as the stated concentration.

[0266] In a related aspect, the invention provides a basal medium containing cholera enterotoxin. In another embodiment, the basal medium does not contain cholera enterotoxin.

[0267] The basal medium may further contain one or more antibiotics such as penicillin / streptomycin and / or gentamicin.

[0268] Basal medium can be used to create modified growth medium (or simply, modified medium) by adding one or more of the factors listed above.

[0269] 4. Protein sequences of representative culture medium factors Some representative (non-limiting) protein factors used in the media and methods of the present invention are provided below. For each listed factor, numerous homologs or functional equivalents are known in the art and can be easily retrieved from public databases, such as GenBank, EMBL, and / or NCBI RefSeq, to ​​name a few. Additional proteins or their peptide fragments or the polynucleotides encoding them, for example, functional homologs from human or non-human mammals, can be easily retrieved from public sources, for example, through sequence-based searches such as NCBI BLASTp or BLASTn, or both.

[0270] TIFF2026034588000016.tif124144TIFF2026034588000017.tif212144TIFF2026034588000018.tif223144TIFF20260345880 00019.tif222144TIFF2026034588000020.tif218144TIFF2026034588000021.tif222144TIFF2026034588000022.tif139144

[0271] 5. How to differentiate stem cells Isolated stem cells (e.g., epithelial stem cells) can be induced to differentiate into differentiated cells normally present in the tissue or organ from which they were derived or isolated. Other tissues include the fallopian tubes, endometrium (uterus), male ductus efferentus, male epididymis, male vas deferens, male ejaculatory duct, male bulbourethral gland, and seminal vesicles. Differentiated cells may express markers characteristic of differentiated cells and can be easily distinguished from stem cells that do not express such differentiated cell markers.

[0272] 6. Markers Generally, for all of the markers described below, gene expression can be measured at the RNA level. Additionally, expression of certain markers can also be detected by protein expression, for example, using antibodies specific for the protein encoded by the marker gene.

[0273] 7. How to use In a further aspect, the present invention provides for the use of stem cells of the present invention isolated from various cultures in drug discovery screens, toxicity assays, animal-based disease models, or medicine, such as regenerative medicine.

[0274] Genetic manipulation of cloned stem cells For example, stem cells isolated by the methods of the present invention are suitable for many types of genetic manipulation, including the introduction of exogenous genetic material capable of modulating the expression of one or more target genes of interest. Such types of gene therapy can be used, for example, in methods for repairing damaged or diseased tissues. Briefly, appropriate vectors, including adenoviral, lentiviral, or retroviral gene delivery vehicles (see below), can be used to deliver genetic information such as DNA and / or RNA to any of the stem cells of the present invention. Those skilled in the art can replace or repair specific genes targeted by gene therapy. For example, to replace a non-functional gene, a normal gene can be inserted into a non-specific location in the genome of the affected cell. In another example, an abnormal gene sequence can be replaced with a normal gene sequence through homologous recombination. Alternatively, selective reversion may restore a gene to its normal function. Another example is the alteration of the regulation (the degree to which a gene is turned on or off) of a specific gene. Preferably, stem cells are treated ex vivo using a gene therapy approach and then transferred into a mammal, preferably a human in need of treatment.

[0275] Art-recognized methods for genetic manipulation, including transfection and infection with various types of nucleic acid constructs (e.g., with viral vectors), can be applied to stem cells so isolated.

[0276] For example, heterologous nucleic acids (e.g., DNA) can be introduced into the stem cells of the present invention using chemical materials or biological vectors (viruses), by physical processes (e.g., electroporation, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, nanoparticles, magnetofection). Chemical-based transfection can be based on calcium phosphate, cyclodextrins, polymers (e.g., cationic polymers such as DEAE-dextran or polyethyleneimine), highly branched organic compounds such as dendrimers, liposomes (e.g., cationic liposomes, lipofection such as lipofection using lipofectamine), or nanoparticles (with or without chemical or viral functionality).

[0277] The nucleic acid construct comprises a nucleic acid molecule of interest and is generally capable of expressing the nucleic acid molecule of interest in a cell into which it is introduced.

[0278] In certain embodiments, the nucleic acid construct is an expression vector in which a nucleic acid molecule encoding a gene product such as a polypeptide, or a nucleic acid that antagonizes expression of a polypeptide (e.g., an siRNA, miRNA, shRNA, antisense sequence, aptamer, ribozyme, etc.), is operably linked to a promoter capable of expressing the nucleic acid molecule in a target cell (e.g., an isolated stem cell).

[0279] The term "expression vector" generally refers to a nucleic acid molecule capable of achieving expression of a gene / nucleic acid molecule it contains in a cell compatible with such sequence. These expression vectors typically contain at least a suitable promoter sequence and, optionally, a transcription termination signal. The nucleic acid or DNA or nucleotide sequence encoding the polypeptide is incorporated into a DNA / nucleic acid construct capable of introduction into and expression in an in vitro cell culture as identified in the methods of the present invention.

[0280] DNA constructs prepared for introduction into specific cells typically contain a replication system recognized by the cell, a DNA segment intended to encode the desired polypeptide, and transcriptional and translational initiation and termination control sequences operably linked to the polypeptide-encoding segment. A DNA segment is "operably linked" when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of that sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide. Generally, operably linked DNA sequences are contiguous, and in reading phase, in the case of a signal sequence. However, enhancers need not be contiguous with the coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or by adapters or linkers inserted instead.

[0281] The selection of an appropriate promoter sequence generally depends on the host cell selected for expression of the DNA segment. Examples of suitable promoter sequences include eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, 2001). Transcriptional control sequences typically include a heterologous enhancer or promoter recognized by the cell. Suitable promoters include the CMV promoter. Expression vectors may contain a replication system and utilize transcriptional and translational control sequences along with an insertion site for a polypeptide-encoding segment. Examples of viable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334:31-36.

[0282] Some aspects of the present invention relate to the use of a nucleic acid construct or expression vector comprising a nucleotide sequence as defined above, wherein the vector is a vector suitable for gene therapy. Anderson(Nature 392:25-30,1998);Walther and Stein(Drugs 60:249-71,2000);Kay et al.(Nat.Med.7:33-40,2001);Russell(J.Gen.Virol.81:2573-604,2000);Amado and Chen(Science 285:674-6,1999);Federico(Curr.Opin.Biotechnol.10:448-53,1999);Vigna and Naldini(J.Gene Med.2:308-16,2000);Marin et al.(Mol.Med.Today 3:396-403,1997);Peng and Suitable vectors for gene therapy are known in the art, such as those described in Russell (Curr. Opin. Biotechnol. 10:454-7, 1999); Sommerfeld (J. Gen. Virol. 80:3049-64, 1999); Reiser (Gene Ther. 7:910-3, 2000); and the references cited therein (all incorporated by reference). Examples include integrating and non-integrating vectors, such as those based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, poxviruses, alphaviruses, and herpesviruses.

[0283] Particularly suitable gene therapy vectors include adenoviral (Ad) and adeno-associated viral (AAV) vectors. These vectors infect a wide variety of dividing and non-dividing cell types. Furthermore, adenoviral vectors are capable of high-level transgene expression. However, due to the episomal nature of adenoviral and AAV vectors after cell entry, these viral vectors are best suited for therapeutic applications requiring only transient expression of the transgene, as described above (Russell, J. Gen. Virol. 81:2573-2604, 2000; Goncalves, Virol J. 2(1):43, 2005). Preferred adenoviral vectors have been modified to reduce host responses, as outlined by Russell (2000, supra). The safety and efficacy of AAV gene transfer has been extensively studied in humans, with promising results in the liver, muscle, CNS, and retina (Manno et al., Nat. Medicine 2006; Stroes et al., ATYB 2008; Kaplitt, Feigin, Lancet 2009; Maguire, Simonelli et al. NEJM 2008; Bainbridge et al., NEJM 2008).

[0284] AAV2 is the most well-characterized serotype for gene transfer research in both humans and experimental models.AAV2 exhibits natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes.Other examples of adeno-associated virus-based non-integrating vectors include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and pseudotyped AAV.The use of non-human serotypes such as AAV8 and AAV9 may be useful to overcome these immunological responses in subjects, and clinical trials have just begun (ClinicalTrials.gov identification number: NCT00979238). For gene transfer into hepatocytes, adenovirus serotype 5, or AAV serotypes 2, 7, or 8 have been shown to be effective vectors and are therefore preferred Ad or AAV serotypes (Gao, Molecular Therapy 13:77-87, 2006).

[0285] An exemplary retroviral vector for application in the present invention is a lentivirus-based expression construct. Lentivirus vectors have the unique ability to infect non-dividing cells (Amado and Chen, Science 285:674-676, 1999). The construction and use of lentivirus-based expression constructs are described in U.S. Patent Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as Federico (Curr. Opin. Biotechnol. 10:448-53, 1999) and Vigna et al. (J. Gene Med. 2:308-16, 2000). Generally, gene therapy vectors are expression vectors in the sense that they contain a nucleotide sequence encoding the gene product (e.g., polypeptide) of the present invention to be expressed, and the nucleotide sequence is operably linked to an appropriate control sequence as described above. Such control sequences will at least include a promoter sequence. Suitable promoters for expression of a nucleotide sequence encoding a polypeptide from a gene therapy vector include, for example, the cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs) such as those derived from murine Moloney leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, the simian virus 40 (SV40) early promoter, and the herpes simplex virus thymidine kinase promoter. Additional suitable promoters are described below.

[0286] Several inducible promoter systems have been described that can be induced by administration of organic or inorganic small molecule compounds. Such inducible promoters include those regulated by heavy metals, such as the metallothionein promoter (Brinster et al., Nature 296:39-42, 1982; Mayo et al., Cell 29:99-108, 1982), those regulated by RU-486 (a progesterone antagonist) (Wang et al., Proc. Natl. Acad. Sci. USA 91:8180-8184, 1994), those regulated by steroids (Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607, 1993), and those regulated by tetracycline (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; U.S. Patent No. 5,464,758; Furth et al., U.S. Patent No. 5,464,758). al, Proc. Natl. Acad. Sci. USA 91:9302-9306, 1994; Howe et al, J. Biol. Chem. 270:14168-14174, 1995; Resnitzky et al, Mol. Cell. Biol. 14:1669-1679, 1994; Shockett et al, Proc. Natl. Acad. Sci. USA 92:6522-6526, 1995), and the tTAER system, which is based on a multi-chimeric transactivator composed of the tetR polypeptide as the activation domain of VP16 and the ligand-binding domain of the estrogen receptor (Yee et al, 2002, US 6,432,705).

[0287] Suitable promoters for nucleotide sequences encoding small RNAs for knockdown of specific genes by RNA interference (see below) include polymerase III promoters in addition to the polymerase II promoters mentioned above. RNA polymerase III (pol III) is responsible for the synthesis of a variety of small nuclear and cytoplasmic non-coding RNAs, including 5S, U6, adenovirus VA1, vault, telomerase RNA, and tRNA. The promoter structures of many genes encoding these RNAs have been determined, and RNA pol III promoters have been found to fall into three types of structures (for reviews, see Geiduschek and Tocchini-Valentini, Annu. Rev. Biochem. 57:873-914, 1988; Willis, Eur. J. Biochem. 212:1-11, 1993; Hernandez, J. Biol. Chem. 276:26733-36, 2001). Particularly suitable for siRNA expression are type 3 RNA pol III promoters, in which transcription is driven by cis-acting elements found only in the 5'-flanking region, i.e., upstream of the transcription start site. Upstream sequence elements include the traditional TATA box (Mattaj et al., Cell 55:435-442, 1988), proximal sequence element, and distal sequence element (DSE; Gupta and Reddy, Nucleic Acids Res. 19:2073-2075, 1991).

[0288] Examples of genes under the control of type 3 pol III promoters are the U6 small nuclear RNA (U6 snRNA) gene, the 7SK gene, the Y gene, the MRP gene, the HI gene, and the telomerase RNA gene (see, e.g., Myslinski et al., Nucl. Acids Res. 21:2502-09, 2001).

[0289] The gene therapy vector may optionally contain one or more additional nucleotide sequences encoding a second or additional polypeptide. The second or additional polypeptide may be a (selectable) marker polypeptide that allows for identification, selection, and / or screening of cells containing the expression construct. Suitable marker proteins for this purpose include, for example, the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection with HAT medium), bacterial hygromycin B phosphotransferase (for selection with hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection with G418), and dihydrofolate reductase (DHFR) (for selection with methotrexate), CD20, and the low-affinity nerve growth factor gene. The sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, 2001.

[0290] Alternatively, the second or additional nucleotide sequence may encode a polypeptide derived from the transgenic cells that provides a safety mechanism that allows the subject to be cured if deemed necessary. Such a nucleotide sequence, often referred to as a suicide gene, encodes a polypeptide capable of converting a prodrug into a toxic substance capable of killing the transgenic cells in which the polypeptide is expressed. Suitable examples of such suicide genes include, for example, the Escherichia coli (E. coli) cytosine deaminase gene or one of the thymidine kinase genes derived from herpes simplex virus, cytomegalovirus, and varicella-zoster virus; in this case, ganciclovir may be used as a prodrug to kill IL-10 transgenic cells in the subject (see, e.g., Clair et al., Antimicrob. Agents Chemother. 31:844-849, 1987).

[0291] To knock down the expression of a specific polypeptide, gene therapy vectors or other expression constructs are used to express the desired nucleotide sequence, preferably encoding an RNAi agent, i.e., an RNA molecule capable of RNA interference or a part of an RNA molecule capable of RNA interference. Such RNA molecules are called siRNA (for example, small interfering RNA, including short hairpin RNA). The desired nucleotide sequence comprises antisense-coded DNA encoding antisense RNA for a region of target gene mRNA, and / or sense-coded DNA encoding sense RNA for the same region of target gene mRNA. In the DNA construct of the present invention, antisense-coded DNA and sense-coded DNA are operably linked to one or more promoters as defined herein above, which can express antisense RNA and sense RNA, respectively. "siRNA" includes small interfering RNA, which is a short double-stranded RNA that is not toxic in mammalian cells (Elbashir et al., Nature 411:494-98, 2001; Caplen et al., Proc. Natl. Acad. Sci. USA 98:9742-47, 2001). The length of siRNA is not necessarily limited to 21-23 nucleotides. As long as it does not exhibit toxicity, the length of siRNA is not particularly limited. "siRNA" can be, for example, at least about 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides in length. Alternatively, the double-stranded RNA portion of the final transcription product of the expressed siRNA can be, for example, at least about 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides in length.

[0292] "Antisense RNA" preferably refers to an RNA strand that has a sequence complementary to a target gene mRNA and is thought to induce RNAi by binding to the target gene mRNA.

[0293] A "sense RNA" is complementary to an antisense RNA and has a sequence that anneals to its complementary antisense RNA to form an siRNA.

[0294] In this context, the term "target gene" includes the gene whose expression is to be silenced by the siRNA expressed by the system of the present invention, and can be selected arbitrarily.For example, as the target gene, a gene whose sequence is known but whose function is not yet elucidated, and a gene whose expression is thought to cause a disease, are preferably selected.As long as the partial sequence of the mRNA of the gene, which has at least 15 nucleotides or more in length that can be bound to one strand of siRNA (antisense RNA strand), has been determined, the genome sequence of the target gene may not be completely elucidated.Therefore, even if the full-length sequence has not been determined, some sequences (preferably at least 15 nucleotides) of the gene, expressed sequence tags (ESTs), and parts of mRNA can be selected as "target genes".

[0295] The double-stranded RNA portion of an siRNA in which two RNA strands are paired is not limited to being perfectly paired, and may contain unpaired portions due to mismatches (corresponding nucleotides are not complementary) or bulges (corresponding complementary nucleotides are missing in one strand). Unpaired portions may be present to the extent that they do not interfere with siRNA formation. As used herein, a "bulge" includes one to two unpaired nucleotides, and the double-stranded RNA region of an siRNA in which two RNA strands are paired preferably contains one to seven, and more preferably one to five, bulges.

[0296] As used herein, the term "mismatch" refers to a mismatch that may be present in the double-stranded RNA region of an siRNA in which two RNA strands are paired. In certain mismatches, one nucleotide is guanine and the other is uracil. Such mismatches may be due to, but are not limited to, a C to T mutation, a G to A mutation, or a mixture thereof in the DNA encoding the sense RNA. Furthermore, in the present invention, the double-stranded RNA region of an siRNA in which two RNA strands are paired may contain both bulges and mismatches, preferably 1 to 7, more preferably 1 to 5, in total. Such mismatches (e.g., mismatches or bulges) suppress the recombination between the antisense-encoding DNA and the sense-encoding DNA described below, stabilizing the siRNA expression system described below. Furthermore, although it is difficult to sequence stem-loop DNA that does not contain a mismatch in the double-stranded RNA region of an siRNA in which two RNA strands are paired, introducing mismatches or bulges as described above makes sequencing possible. Furthermore, siRNAs containing mismatches or bulges in the paired double-stranded RNA region have the advantage of being stable in E. coli or animal cells.

[0297] The terminal structure of siRNA may be blunt or sticky (overhanging) as long as the siRNA is capable of silencing target gene expression through the RNAi effect. The sticky (overhanging) end structure is not limited to 3' overhangs but can also include 5' overhangs, as long as they are capable of inducing the RNAi effect. Furthermore, the number of overhanging nucleotides is not limited to the previously reported 2 or 3, but can be any number as long as the overhang is capable of inducing the RNAi effect. For example, the overhang may consist of 1 to 8 nucleotides, preferably 2 to 4 nucleotides. Herein, the total length of an siRNA having a sticky end structure is expressed as the sum of the length of the paired double-stranded portion and the length of the pair, including the overhanging single strands at both ends. For example, in the case of a 19-bp double-stranded RNA portion with 4-nucleotide overhangs at both ends, the total length is expressed as 23 bp. Furthermore, because this overhanging sequence has low specificity for the target gene, it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence. Furthermore, as long as the siRNA can maintain its gene silencing effect on the target gene, the siRNA may contain, for example, a low-molecular-weight RNA (which may be a natural RNA molecule such as tRNA, rRNA, or viral RNA, or an artificial RNA molecule) in the protruding portion at one end.

[0298] Furthermore, the terminal structure of "siRNA" is necessarily a cutoff structure at both ends as described above, and may have a stem-loop structure in which one end of the double-stranded RNA is connected by a linker RNA ("shRNA"). The length of the double-stranded RNA region (stem-loop portion) may be, for example, at least 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides. Alternatively, the length of the double-stranded RNA region that is the final transcription product of the expressed siRNA may be, for example, at least 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides.

[0299] Furthermore, the length of the linker is not particularly limited, as long as it has a length that does not interfere with the pairing of the stem portion.For example, the linker portion can have a cloverleaf tRNA structure to ensure stable pairing of the stem portion and to prevent recombination between the DNA encoding that portion.Even if the linker has a length that interferes with the pairing of the stem portion, it is possible to construct the linker portion with an intron, for example, so that the intron is excised during the processing of precursor RNA into mature RNA, thereby allowing the pairing of the stem portion.In the case of stem-loop siRNA, either end (head or tail) of the RNA without loop structure can have a low molecular weight RNA.As mentioned above, this low molecular weight RNA can be a natural RNA molecule such as tRNA, rRNA, snRNA, or viral RNA, or an artificial RNA molecule.

[0300] The DNA construct of the present invention contains a promoter as defined above to express antisense and sense RNAs from the antisense and sense coding DNAs, respectively. In principle, the number and location of promoters within the construct can be freely selected, as long as they are capable of expressing the antisense and sense coding DNAs. As a simple example of the DNA construct of the present invention, a tandem expression system can be formed in which a promoter is located upstream of the antisense and sense coding DNAs. This tandem expression system can produce siRNAs with the above-mentioned cutoff structures at both ends. In a stem-loop siRNA expression system (stem expression system), the antisense and sense coding DNAs are arranged in opposite directions and connected via a linker DNA to construct a single unit. A promoter is linked to one side of this unit to construct the stem-loop siRNA expression system. The length and sequence of the linker DNA are not particularly limited herein, and the linker DNA can have any length and sequence, as long as it is not a termination sequence and does not interfere with the pairing of the stem portion during the production of mature RNA as described above. As an example, DNA encoding the aforementioned tRNA or the like can be used as linker DNA.

[0301] In both the tandem expression system and the stem-loop expression system, the 5' end has a sequence that can promote transcription from the promoter. More specifically, in the case of tandem siRNA, the efficiency of siRNA production can be improved by adding a sequence that can promote transcription from the promoter to the 5' end of the antisense and sense coding DNA. In the case of stem-loop siRNA, such a sequence can be added to the 5' end of the above-mentioned unit. The transcript from such a sequence can be used in a state attached to siRNA as long as it does not interfere with target gene silencing by siRNA. If this condition interferes with gene silencing, it is preferable to trim the transcript using a trimming means (e.g., a ribozyme known in the art). It will be clear to those skilled in the art that antisense and sense RNAs can be expressed in the same vector or in different vectors. To avoid adding excessive sequences downstream of the sense and antisense RNAs, it is preferable to place a transcription terminator at the 3' end of each strand (the strand encoding the antisense and sense RNAs). A terminator can be a sequence of four or more consecutive adenine (A) nucleotides.

[0302] Genome editing Genome editing can be used to change the genomic sequence of the cloned stem cells of the present invention, for example, cloned cancer (or other disease) stem cells, by introducing heterologous transgenes or inhibiting the expression of target endogenous genes.Such genetically modified stem cells can be used for regenerative medicine (see below) or wound healing.Therefore, in certain embodiments, the regenerative medicine (see below) method of the present invention comprises the use of the stem cells of the present invention whose genomic sequence is modified by genome editing.

[0303] Genome editing can be carried out using art-recognized technologies such as ZFN / TALEN technology or CRISPR technology (see review by Gaj et al., Trends in Biotech. 31(7):397-405, 2013, the entire text and all references cited therein are incorporated herein by reference). Such technology allows virtually any gene to be manipulated in a wide range of cell types and organisms by inducing DNA double-strand (DSB) breaks that stimulate error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) at specific genomic locations, thereby enabling a wide range of gene modifications.

[0304] Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are chimeric nucleases composed of a programmable sequence-specific DNA binding module linked to a nonspecific DNA cleavage domain. They are artificial restriction enzymes (REs) created by fusing the zinc finger or TAL effector DNA binding domain with a DNA cleavage domain. Zinc finger (ZFs) or transcription activator-like effectors (TALEs) can be engineered to bind to any desired target DNA sequence and fused to the DNA cleavage domain of the RE, thereby creating engineered restriction enzymes (ZFNs or TALENs) specific to the desired target DNA sequence. When ZFNs / TALENs are introduced into cells, they can be used for in situ genome editing. Indeed, the versatility of ZFNs and TALENs can be extended to effector domains other than nucleases, such as transcriptional activators and repressors, recombinases, transposases, DNA and histone methyltransferases, and histone acetyltransferases, to affect genome structure and function.

[0305] The Cys2-His2 zinc finger domain is one of the most common types of DNA-binding motifs found in eukaryotes and represents the second most frequently encoded protein domain in the human genome. Individual zinc fingers contain approximately 30 amino acids in a conserved ββα configuration. Key to the application of zinc finger proteins for specific DNA recognition was the development of non-natural arrays containing more than three zinc finger domains. This advance was facilitated by the structure-based discovery of highly conserved linker sequences, which enabled the construction of synthetic zinc finger proteins that recognize DNA sequences 9–18 bp long. This design proved to be the optimal strategy for constructing zinc finger proteins that recognize specific continuous DNA sequences in complex genomes. Suitable zinc fingers can be obtained by a modular assembly approach (e.g., by selection of large combinatorial libraries or using preselected libraries of zinc finger modules generated by rational design). Zinc finger domains that recognize nearly all 64 possible nucleotide triplets have been developed, and preselected zinc finger modules can be linked in tandem to target DNA sequences containing a set of these DNA triplets. Alternatively, selection-based approaches such as oligomerized pool engineering (OPEN), which take into account environment-dependent interactions between neighboring fingers, can be used to select new zinc finger arrays from randomized libraries. A combination of the two approaches can also be used.

[0306] Engineered zinc fingers are commercially available. Sangamo Biosciences (Richmond, CA, USA) has developed a proprietary platform for zinc finger construction (CompoZr) in collaboration with Sigma-Aldrich (St. Louis, MO, USA) that allows researchers to bypass zinc finger construction and validation altogether, and thousands of proteins are already available. Broadly, zinc finger protein technology allows for the targeting of virtually any sequence.

[0307] TAL effectors are proteins secreted by the plant pathogenic bacterium Xanthomonas that contain a DNA-binding domain containing a repeat of a highly conserved 33–34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable (Repeat Variable Diresidues, or RVDs) and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has enabled the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs. Similar to zinc fingers, modular TALE repeats are linked together to recognize contiguous DNA sequences. Numerous effector domains, including nucleases, transcriptional activators, and site-specific recombinases, have been made available for fusion with TALE repeats for targeted gene modification. Rapid assembly of custom TALE arrays can be achieved by using strategies including "Golden Gate" molecular cloning, high-throughput solid-phase assembly, and ligation-independent cloning techniques, all of which can be used in the present invention for genome editing of cloned stem cells.

[0308] TALE repeats can be easily assembled using numerous tools available in the art, such as a library of TALENs targeting 18,740 human protein-coding genes (Kim et al., Nat. Biotechnol. 31, 251-258, 2013). Custom-designed TALE arrays are also commercially available, for example, through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).

[0309] Nonspecific DNA cleavage domains derived from the ends of REs, such as FokI endonuclease (or FokI cleavage domain variants, such as Sharkey, with mutations designed to improve cleavage specificity and / or activity), can be used to construct hybrid nucleases that are active in yeast assays (and also active in plant and animal cells). To improve ZFN activity, transient low-temperature culture conditions can be used to increase nuclease expression levels; co-delivery of site-specific nucleases with DNA end-processing enzymes, as well as the use of fluorescent surrogate reporter vectors that allow enrichment of cells modified by ZFNs and TALENs, can also be used. The specificity of ZFN-mediated genome editing can also be improved by the use of zinc finger nickases (ZF nickases), which exploit the observation that induction of nicked DNA stimulates HDR without activating the error-prone NHEJ repair pathway.

[0310] The simple relationship between the amino acid sequence of a TALE binding domain and DNA recognition allows for designable proteins. A publicly available software program (DNAWorks) can be used to calculate appropriate oligonucleotides for assembly in two-step PCR. Numerous modular assembly schemes for generating modified TALE constructs have also been reported and are known in the art. Both methods offer a systematic approach to modifying DNA binding domains that is conceptually similar to the modular assembly method for generating zinc finger DNA recognition domains.

[0311] After the TALEN gene is assembled, it is introduced into target cells by a vector using any art-recognized method (such as electroporation or transfection using cationic lipid-based reagents, or various viral vectors such as plasmid vectors, adenovirus vectors, AAV vectors, and integrase-deficient lentivirus vectors (IDLV)). Alternatively, TALENs can be delivered to cells as mRNA, which eliminates the possibility of genomic integration of TALEN-expressed proteins. This can also dramatically increase the level of homology-directed repair (HDR) during gene editing and the success of gene transfection. Finally, direct delivery of purified ZFN / TALEN proteins into cells can also be used. This approach does not involve the risk of insertional mutagenesis and results in fewer off-target effects than delivery systems that rely on expression from nucleic acids, making it ideal for use in studies requiring precise genome modification in cells such as the stem cells of the present invention.

[0312] TALENs can be used to edit genomes by inducing double-strand breaks (DSBs), to which cells respond through repair mechanisms. Non-homologous end joining (NHEJ) reconnects DNA from either side of a double-strand break where there is little or no sequence overlap for annealing. A simple heteroduplex break assay can be performed to detect differences between two PCR-amplified alleles. Cleavage products can be visualized in simple agarose gel or slab gel systems. Alternatively, DNA can be introduced into the genome through NHEJ in the presence of an exogenous double-stranded DNA fragment.

[0313] Homologous recombination repair can also introduce foreign DNA at DSBs because the transfected double-stranded sequence is used as a template for repair enzymes. TALENs have been used to generate knockout C. elegans, rats, and zebrafish, and to generate stably modified human embryonic stem cells and induced pluripotent stem cell (iPSC) clones.

[0314] For stem cell-based therapy, ZFNs and TALENs can correct the underlying cause of disease through precise genome modification, thus permanently eliminating symptoms. For example, ZFN-induced HDR can be used to directly correct disease-causing mutations associated with X-linked severe combined immunodeficiency (SCJD), hemophilia B, sickle cell disease, α1-antitrypsin deficiency, and many other genetic diseases by either repairing defective target genes or knocking out target genes. Furthermore, these site-specific nucleases can also be used to safely insert therapeutic transgenes into the stem cells of the present invention at specific "safe harbor" locations within the human genome. Such technology can be used in combination with the stem cells of the present invention in gene therapy, including autologous stem cell transplantation-based treatments, in which one or more genes of cloned (diseased or normal) stem cells are manipulated to increase or reduce / eliminate target gene expression.

[0315] Alternatively, the CRISPR / Cas system may be used to efficiently induce targeted genetic modifications in stem cells of the present invention. The CRISPR / Cas (CRISPR-related) system, or "Clustered Regulatory Interspaced Short Palindromic Repeats," is a genetic locus that contains multiple short direct repeats and provides adaptive immunity to bacteria and archaea. The CRISPR system relies on crRNA and tracrRNA for sequence-specific silencing of invading foreign DNA. The term "tracrRNA" refers to a non-coding RNA that promotes crRNA processing and is a trans-activating chimeric RNA required to activate RNA-guided Cas9 cleavage. CRISPR RNA or crRNA base-pairs with tracrRNA to form a two-RNA structure that guides Cas9 endonuclease to the complementary DNA site for cleavage.

[0316] There are three types of CRISPR / Cas systems: in Type II systems, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition. In bacteria, CRISPR systems provide adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage. By redesigning the crRNA, CRISPR / Cas systems can be retargeted to cleave virtually any DNA sequence. In fact, it has been shown that CRISPR / Cas systems can be directly transferred into human cells by co-delivering a plasmid expressing the Cas9 endonuclease and the necessary crRNA components. These programmable RNA-guided DNA endonucleases have demonstrated multiple gene disruption capabilities and target-specific integration in iPS cells and therefore can be used in the stem cells of the present invention as well.

[0317] Cancer stem cells The methods and reagents of the present invention also enable the culture and isolation of cancer-derived cancer stem cells (CSCs) derived from epithelial tissue samples / biopsies or from other columnar regenerative tissues, which CSCs can be used in a number of applications that were previously impossible or impractical, in part due to the inability to obtain such CSCs as single-cell clones in large quantities.

[0318] For example, a library of CSCs established from a patient using the method of the present invention allows comparison between sensitive and resistant clones from the same patient for directed drug discovery efforts. Certain genes may be up-regulated or down-regulated in resistant clones compared to sensitive clones. Inhibitors of up-regulated genes can be further validated as drug target genes, for example, by testing the ability to down-regulate target genes in resistant clones and determining the effect on drug resistance. Conversely, restoring or overexpressing down-regulated genes in resistant clones can also overcome drug resistance.

[0319] Thus, in one aspect, the present invention provides a drug discovery method using CSCs isolated using the method and medium of the present invention to identify genes up- or down-regulated in drug-resistant CSC clones, the method comprising: (1) obtaining a large number of cell clones from cancer tissue (such as those derived from a cancer patient) using the method of the present invention; (2) contacting the large number of cell clones with one or more chemical compounds (e.g., anticancer drugs) under conditions in which a small percentage (e.g., at most 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.01%, or less) of drug-resistant clones survive; and (3) comparing the gene expression profile of the drug-resistant clones with that of sensitive clones (e.g., one or more cell clones randomly selected before step (2) that are suspected to be sensitive to drug treatment), thereby identifying genes that are up- or down-regulated in the surviving drug-resistant clones.

[0320] In certain embodiments, the method further comprises inhibiting the expression of up-regulated genes in surviving drug-resistant clones.For example, the up-regulated genes can be commonly up-regulated in two or more surviving drug-resistant clones, which are derived from the same or different tumor types, and from the same or different patients.In certain embodiments, the up-regulated genes can be specific to the patient from which CSCs are isolated.This can be useful in designing personalized medicine or treatment plans for patients.

[0321] In certain embodiments, the method further comprises restoring or increasing the expression of down-regulated genes in surviving drug-resistant clones.For example, the down-regulated genes can be commonly down-regulated in two or more remaining drug-resistant clones from the same type of tumor or different types of tumor, from the same patient, or from different patients.In certain embodiments, the down-regulated genes can be specific for the patient from which CSCs are isolated.This can also be useful in designing personalized medicine or treatment plans for patients.

[0322] In a related aspect, the present invention provides a drug discovery method using CSCs isolated using the method and medium of the present invention to identify candidate compounds that inhibit the growth of drug-resistant CSCs or promote their death, the method comprising: (1) obtaining a large number of cell clones from cancer tissue (such as those derived from a cancer patient) using the method of the present invention; (2) contacting the large number of cell clones with one or more compounds (e.g., anticancer drugs) under conditions in which a small percentage of drug-resistant clones remains (e.g., at most 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.01%, or less); (3) contacting the remaining drug-resistant clones with a large number of candidate compounds; and (4) identifying one or more candidate compounds that inhibit the growth of drug-resistant CSCs or promote their death. In certain embodiments, the method is carried out using a high-throughput screening format for candidate drugs that target resistant cells.

[0323] In certain embodiments, the method further comprises testing the general toxicity of the identified candidate compound in matched sensitive clones (e.g., one or more cell clones randomly selected before step (2) that are suspected to be sensitive to drug treatment) and / or matched normal cells derived from the same patient from which the CSCs were isolated. Preferably, the identified candidate compound specifically or preferentially inhibits the growth of or promotes the death of drug-resistant CSCs compared to matched sensitive clones and / or matched normal cells.

[0324] In certain embodiments, the healthy cells are patient-matched normal stem cells similarly isolated using the methods and reagents of the present invention.

[0325] The above-described embodiment is based in part on the discovery that, in many cases, drug-resistant CSCs grow slower than drug-sensitive clones. Without wishing to be bound by a particular theory, the applicant believes that the slow growth is likely the result of altered gene expression in drug-resistant CSCs to avoid chemotherapy. Therefore, certain drugs are expected to preferentially inhibit the growth or kill drug-resistant cells, but be less toxic than standard chemotherapy drugs (such as cisplatin or paclitaxel) that are primarily used to treat cancer.

[0326] In another aspect, the present invention provides a method for identifying an appropriate or effective treatment for a patient in need of treating a disease, the method comprising the steps of: (1) obtaining multiple stem cell clones from diseased tissue (such as cancer tissue) from the patient using the method of the present invention; (2) subjecting the multiple cell clones to one or more candidate treatments; and (3) determining the effectiveness of each of the one or more candidate treatments, thereby identifying an appropriate or effective treatment for the patient in need of treating the disease. This can be useful, for example, when a patient has several possible treatment options, each of which may or may not be appropriate or effective for the patient.

[0327] In a related aspect, the present invention provides a method for screening for the most appropriate or effective treatment among multiple candidate treatments for treating a patient in need of disease, the method comprising: (1) obtaining multiple stem cell clones from diseased tissue (such as tissue) from the patient using the method of the present invention; (2) subjecting the multiple cell clones to the candidate treatments; and (3) comparing the relative effectiveness of one or more candidate treatments, thereby identifying the most appropriate or effective treatment for the patient. For example, this can be useful when a patient has several alternative treatment options, each of which is effective for a specific patient population but not necessarily for other populations.

[0328] In certain embodiments, the disease is cancer, eg, any of the cancers from which cancer stem cells can be isolated.

[0329] In certain embodiments, the treatment is a chemotherapy regimen, such as one utilizing one or more chemotherapeutic agents. In certain embodiments, the treatment is radiation therapy. In certain embodiments, the treatment is immunotherapy, such as one using a cell-binding agent (e.g., an antibody) that specifically binds to a surface ligand (e.g., a surface antigen) of a cancer cell. In certain embodiments, the treatment is a combination of surgery, chemotherapy, radiation therapy, and / or immunotherapy.

[0330] In certain embodiments, the disease is an inflammatory disease, a disease from which disease-associated stem cells can be isolated, or any of the diseases mentioned herein.

[0331] In certain embodiments, the methods further comprise treating the patient with an appropriate or effective treatment for one or more of the identified diseases.

[0332] In certain embodiments, the method further comprises generating a report providing the effectiveness of each of the candidate treatments, such as the effectiveness of each of the candidate chemotherapeutic agents tested individually or in combination (e.g., serially or simultaneously).

[0333] In certain embodiments, the method further comprises providing a recommendation for the most effective treatment.

[0334] In a related aspect, the invention provides kits and reagents for carrying out the methods of the invention.

[0335] In certain embodiments, the general screening method of the present invention (not necessarily limited to cancer stem cells) is performed in a high-throughput / automated manner. For high-throughput, the expanded stem cell population can be cultured in multi-well plates, e.g., 96-well or 384-well plates. A library of molecules is used to identify molecules that affect the plated stem cells. Preferred libraries include, but are not limited to, antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g., LOPAP™, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g., LOP AC™, Sigma Aldrich), or natural compound libraries (Specs, TimTec). In addition, gene libraries can be used to induce or suppress the expression of one or more genes in stem cell progeny. These gene libraries include cDNA libraries, antisense libraries, and siRNA or other non-coding RNA libraries.

[0336] The stem cells are preferably exposed to multiple concentrations of the test / candidate agent for a period of time, and at the end of the exposure period, the cultures are evaluated for a predetermined effect, such as changes in the cells, including, but not limited to, reduction or loss of proliferation, morphological changes, and cell death.

[0337] The expanded stem cell population may also be used to identify drugs that specifically target epithelial cancer cells or stem cells isolated therefrom, rather than the expanded stem cell population itself.

[0338] Rapid cloning of cancer stem cells also enables immunological approaches to tumor destruction. The technology described herein allows for highly efficient cloning of CSCs and may therefore provide information to aid approaches to eradicate these cells via immune activation.

[0339] For example, when CSCs (either drug-sensitive or drug-resistant) are isolated, one or more epitopes of such CSCs, preferably epitopes specific to CSCs compared to healthy controls (e.g., epitopes on the cell surface or secretome of CSCs), can be used to vaccinate antigen-presenting cells (APCs) to instruct lymphocytes to target these CSCs. Immunological approaches can include identifying and targeting molecules on the cell surface or secretome of CSCs that suppress immune surveillance, as has been done for melanoma.

[0340] Regenerative Medicine The stem cells of the present invention may be useful in regenerative medicine, for example, in the post-traumatic, post-radiation, and / or post-surgical repair of various damaged regenerative tissues or organs.

[0341] In yet another embodiment, small biopsies or tissue samples can be taken from adult donors, and stem cells therein can be isolated, expanded, and optionally differentiated to generate transplantable epithelium for regeneration. The fact that the stem cells of the present invention can be frozen, thawed, and returned to culture without loss of stem cell properties and without significant cell death further increases the applicability of the stem cells of the present invention for transplantation purposes.

[0342] Thus, the present invention provides stem cells or their expanded clones or their differentiation products (or collectively "stem cells" for use in regenerative medicine) for use in transplantation into a mammal, preferably a human. Also provided is a method of treating a patient in need of transplantation comprising transplanting into the patient a population of stem cells of the present invention, wherein the patient is a mammal, preferably a human.

[0343] Accordingly, another aspect of the present invention provides a method for treating a human or non-human animal patient through cell therapy. Such cell therapy involves the application or administration of stem cells of the present invention (such as tissue-matched stem cells of the present invention) to the patient through appropriate means. Specifically, such methods of treatment include the regeneration of injured tissue or wound healing. According to the present invention, a patient can be treated with allogeneic or autologous stem cells or their clonal expansion. "Autologous" cells are cells derived from the same organism as those being reintroduced for cell therapy, e.g., to enable tissue regeneration. However, the cells are not necessarily isolated from the same tissue as the tissue into which they are introduced. Autologous cells do not require a patient match to overcome rejection issues. "Allogeneic" cells are cells derived from the same species but from a different individual than the individual into which the cells are being introduced for cell therapy, e.g., to enable tissue regeneration. Nevertheless, some degree of patient match may be required to prevent rejection issues.

[0344] Generally, the stem cells of the present invention are introduced into a patient's body by injection or transplantation. Generally, the cells will be injected directly into the tissue they are intended to affect. Alternatively, the cells will be injected through the portal vein. A syringe containing the cells of the present invention and a pharmaceutically acceptable carrier is included within the scope of the present invention. A catheter attached to a syringe containing the cells of the present invention and a pharmaceutically acceptable carrier is also included within the scope of the present invention.

[0345] The stem cells of the present invention can also be used in tissue regeneration. To accomplish this function, the cells can be injected or implanted directly into damaged tissue, where they can proliferate and ultimately differentiate into the required cell type, depending on their location in the body and / or after homing to the tissue of origin.

[0346] Alternatively, the stem cells of the present invention may be directly injected or implanted into damaged tissues.Tissues that can be treated include all damaged tissues, particularly those damaged by disease, injury, trauma, autoimmune reaction, or viral or bacterial infection.In some embodiments of the present invention, the stem cells of the present invention are used to regenerate the lung, esophagus, stomach, small intestine, colon, intestinal metaplasia, fallopian tube, kidney, pancreas, bladder, liver, or gastric system, or parts / sections thereof.

[0347] In certain embodiments, the patient is a human, but may alternatively be a non-human mammal such as a cat, dog, horse, cow, pig, sheep, rabbit, or mouse.

[0348] In certain embodiments, the stem cells of the present invention are injected into a patient using a syringe, such as a Hamilton syringe. One of skill in the art would know what the appropriate dosage of the stem cells of the present invention is for the particular condition being treated.

[0349] In certain embodiments, the stem cells of the present invention are administered in solution, in microspheres, or in microparticles of various compositions into an artery that supplies blood to a tissue or portion of an injured organ in need of regeneration.

[0350] Generally, such administration will be performed using a catheter, which may be one of a variety of balloon catheters used for plastic surgery and / or cell delivery, or a catheter designed for the specific purpose of delivering cells to a particular location in the body.

[0351] For certain uses, stem cells may be encapsulated in microspheres approximately 15 μm in diameter made from a number of different biodegradable compounds. This method may allow intravascularly administered stem cells to remain at the site of injury and not enter the systemic circulation on their first pass through the capillary network. Retention on the arterial side of the capillary network may also facilitate migration into the extravascular space.

[0352] In certain embodiments, stem cells may be injected retrogradely into the vascular tree through veins for delivery systemically or locally, specifically to specific veins that drain the tissue or body part to which the stem cells are directed.

[0353] In another embodiment, the stem cells of the present invention may be attached to a biocompatible implant and implanted into injured tissue. In this embodiment, the cells may be attached to the biocompatible implant in vitro before implantation into a patient. As will be apparent to one skilled in the art, any of a number of adhesives may be used to attach the cells to the implant before implantation. By way of example only, such adhesives may include fibrin, one or more members of the integrin family, one or more members of the cadherin family, one or more members of the selectin family, one or more cell adhesion molecules (CAMs), one or more members of the immunoglobulin family, and one or more artificial adhesives. This list is provided for illustrative purposes only and is not intended to be limiting. It will be apparent to one skilled in the art that combinations of one or more adhesives may also be used.

[0354] In another embodiment, the stem cells of the present invention may be embedded in a matrix before implantation of the matrix into a patient. Generally, the matrix will be implanted into the patient's injured tissue. Examples of matrices include collagen-based matrices, fibrin-based matrices, laminin-based matrices, fibronectin-based matrices, and artificial matrices. This list is provided for illustrative purposes only and is not intended to be limiting. In a further embodiment, the stem cells of the present invention may be implanted or injected into a patient along with matrix-forming components. This allows the cells to form a matrix after injection or transplantation, ensuring that the stem cells remain in the appropriate location in the patient. Examples of matrix-forming components include fibrin glue, liquid alkyl acrylates, cyanoacrylate monomers, plasticizers, polysaccharides such as dextran, ethylene oxide-containing oligomers, block copolymers such as poloxamers and Pluronics, non-ionic surfactants such as Tween and Triton 8, and artificial matrix-forming components. This list is provided for illustrative purposes only and is not intended to be limiting. It will be apparent to those skilled in the art that combinations of one or more matrix-forming components may be used.

[0355] In a further embodiment, the stem cells of the present invention may be contained within microspheres. In this embodiment, the cells may be encapsulated in the center of the microsphere. In this embodiment, the cells may be embedded in the matrix material of the microsphere. The matrix material may include any suitable biodegradable polymer, including, but not limited to, alginate, polyethylene glycol (PLGA), and polyurethane. This list is provided for illustrative purposes only and is not intended to be limiting.

[0356] In a further embodiment, the stem cells of the present invention may be attached to a medical device intended for implantation. Examples of such medical devices include stents, pins, sutures, splits, pacemakers, artificial joints, artificial skin, and rods. This list is provided for illustrative purposes only and is not intended to be limiting. It will be apparent to those skilled in the art that cells may be attached to a medical device in a variety of ways. For example, stem cells may be attached using fibrin, one or more members of the integrin family, one or more members of the cadherin family, one or more members of the selectin family, one or more cell adhesion molecules (CAMs), one or more members of the immunoglobulin family, and one or more artificial adhesives. This list is provided for illustrative purposes only and is not intended to be limiting. It will be apparent to those skilled in the art that a combination of one or more adhesives may be used.

[0357] Thus, methods of treating human or animal patients by cell therapy are included within the scope of the present invention. The term "animal" herein refers to all mammalian, preferably human, patients. It also includes individual animals at all stages of development, including embryonic and fetal stages. For example, the patient may be an adult, or the treatment may be for pediatric use (e.g., neonate, child, or adolescent). Such cell therapy involves administering stem cells generated by the present invention to the patient via appropriate means. Specifically, such methods of treatment include regeneration of injured tissue or wound healing. The term "administration," as used herein, refers to well-recognized forms of administration, such as intravenous or injection, and also refers to administration by transplantation, e.g., surgery, transplantation, or grafting, of tissue-engineered livers obtained from stem cells according to the present invention. In the case of cells, systemic administration to an individual may be possible, for example, by injection via the thoracic duct into the superior mesenteric artery, celiac artery, subclavian vein, injection into the heart via the superior vena cava, or injection into the peritoneal cavity with subsequent movement of the cells via the subdiaphragmatic lymphatics, or directly to the liver site via injection into the hepatic arterial blood supply or portal vein.

[0358] 10 per 100 kg of body weight 4 ~10 13 Cells may be administered in one injection, preferably about 1-5 x 10 cells per 100 kg body weight. 4 and 1 to 5 × 10 7 Preferably, about 1 x 10 cells per 100 kg of body weight can be injected intravenously. 4 and 1 × 10 6 Cells may be injected intravenously. In some embodiments, a single administration of the stem cells of the present invention is provided. In other embodiments, multiple administrations are used. Multiple administrations may be provided, for example, in an initial treatment regimen of 3-7 consecutive days, and then repeated at other times.

[0359] It will be clear to those skilled in the art that gene therapy can also be used in methods directed to repairing damaged or diseased tissues. For example, adenovirus or retrovirus gene delivery vehicles can be used to deliver genetic information such as DNA and / or RNA to stem cells. Those skilled in the art can replace or repair specific genes targeted in gene therapy. For example, normal genes can be inserted into non-specific locations in the genome to replace non-functional genes. In another example, abnormal gene sequences can be replaced with normal gene sequences through homologous recombination. Alternatively, selective reversion can restore genes to normal function. Another example is the alteration of the regulation (degree to which a gene is turned on or off) of specific genes. Preferably, stem cells are treated ex vivo by gene therapy approaches, and then transferred into mammals, preferably humans, who require treatment. For example, cells derived from stem cells can be genetically modified in culture before transplantation into patients.

[0360] Toxicity assay Expanded stem cell populations can further replace the use of cell lines such as Caco-2 cells in toxicity assays of potential new drugs or known or new nutritional supplements. Such toxicity assays can be performed using patient-matched or tissue / organ-matched stem cells, which can be useful in personalized medicine.

[0361] Cell-based toxicity tests are used to determine organ-specific cytotoxicity.

[0362] Compounds that can be tested include cancer chemopreventive agents, environmental chemicals, dietary supplements, and potential toxicants. Cells are exposed to multiple concentrations of the test agent for a set period of time. The concentration range of the test agent in the assay is determined in a preliminary assay using a 5-day exposure and logarithmic dilutions from the highest soluble concentration. At the end of the exposure period, cultures are assessed for growth inhibition. Data are analyzed to determine the concentration that inhibited the endpoint by 50 percent (TC50).

[0363] For high-throughput purposes, epithelial stem cells are cultured in multiwell plates, such as 96-well or 384-well plates. Molecular libraries are used to identify molecules that affect stem cells. Preferred libraries include antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g., LOPAP™, Sigma-Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g., LOP AC™, Sigma-Aldrich), or natural compound libraries (Specs, TimTec). Additionally, gene libraries that induce or suppress the expression of one or more genes in the progeny of adenoma cells can be used. These gene libraries include cDNA libraries, antisense libraries, and libraries of siRNA or other non-coding RNA. Cells are preferably exposed to multiple concentrations of the test agent for a set period of time. At the end of the exposure period, the cultures are evaluated. The term "affect" is used to cover any change in cells, including, but not limited to, a reduction or loss of proliferation, morphological changes, and cell death.

[0364] Animal models Another aspect of the present invention provides animal models comprising the stem cells of the present invention, such as the cancer stem cells of the present invention.

[0365] In some embodiments, the animal is an immunodeficient non-human animal (such as a rodent, for example, a mouse or a rat), because such an animal is less likely to cause rejection.As the immunodeficient animal, it is preferable to use a non-human animal that lacks functional T cells, such as nude mice and nude rats, and a non-human animal that lacks functional T cells and B cells, such as SCID mice and NOD-SCID mice.In particular, mice that lack T cells, B cells, and NK cells (for example, severe immunodeficient mice obtained by crossbreeding SCID mice, RAG2KO mice, or RAG1KO mice with IL-2Rgnu11 mice, including NOD / SCID / gammacnu11 mice, NOD-scid, IL-2Rgnu11 mice, and BALB / c-Ragnu11, IL-2Rgnu11 mice) that exhibit excellent transplantability.

[0366] Regarding the age of the non-human animal, when athymic nude mice, SCID mice, NOD / SCID mice, or NOG mice are used, those aged 4 to 100 weeks are preferably used.

[0367] NOG mice may be generated, for example, by the methods described in WO 2002 / 043477 (incorporated by reference) or may be obtained from the Central Institute for Experimental Animals or the Jackson Laboratory (NSG mice).

[0368] The transplanted cells can be any type of cell, including stem cell clumps / clones, tissue sections differentiated from the stem cells of the present invention, monodispersed stem cells, stem cells cultured after isolation or freeze / thawing, and stem cells transplanted into another animal and re-isolated from the animal. The number of cells transplanted can be 10 6The number of cells may be less than 100, or more than 100. In some embodiments, subcutaneous transplantation is preferred because of its simple transplantation technique. However, the site of transplantation is not particularly limited, and preferably is appropriately selected according to the animal used. The method for transplanting established cancer cell lines into NOG is not particularly limited, and any conventional transplantation method can be used.

[0369] Such animal model can be used to, for example, explore drug target molecules and evaluate drugs.Drug evaluation methods include drug screening and anti-cancer drug screening.The method of exploring target molecules includes, but is not limited to, using gene chip analysis to identify genes (such as cancer stem cell markers) that are highly expressed in cancer stem cells, such as DNA and RNA, and using proteomics to identify proteins, peptides or metabolites that are highly expressed in cancer stem cells.

[0370] Screening methods for searching for target molecules include those that use cell proliferation inhibition assays to screen for substances that inhibit the growth of cancer stem cells from small molecule libraries, antibody libraries, microRNA libraries, RNAi libraries, etc. After an inhibitor is obtained, its target can be identified.

[0371] Therefore, the present invention also provides a method for identifying target molecules of drugs, which includes the steps of: (1) generating a non-human animal model by transplanting the cancer stem cells of the present invention into a non-human animal (e.g., an immunodeficient mouse or rat); (2) collecting tissue sections that exhibit tissue structures characteristic of the cancer development process of cancer stem cell populations or exhibit their biological properties before and after drug administration; (3) investigating / comparing the tissue sections (before and after) collected in (2) for expression of DNA, RNA, proteins, peptides, or metabolites; and (4) identifying DNA, RNA, proteins, peptides, or metabolites in the tissue sections that vary depending on the structures formed from cancer stem cells, the cancer development process caused by cancer stem cells, or the biological properties of cancer stem cells.

[0372] The present invention also provides a method for evaluating a drug, which includes the steps of: (1) generating a non-human animal model by transplanting the cancer stem cells of the present invention into a non-human animal (e.g., an immunodeficient mouse or rat); (2) administering a test substance to the non-human animal model of (1); (3) collecting tissue sections that exhibit tissue structures characteristic of the cancer development process of cancer stem cell populations or that exhibit their biological properties; (4) observing changes in the cancer stem cells, the cancer development process, or their biological properties over time in the tissue sections; and (5) identifying structures formed from cancer stem cells, cancer development processes caused by cancer stem cells, or biological properties of cancer stem cells that are inhibited by the test substance.

[0373] The present invention also provides a method for screening drugs, which includes the steps of: (1) generating a non-human animal model by transplanting the cancer stem cells of the present invention into a non-human animal (e.g., an immunodeficient mouse or rat); (2) administering a test substance to the non-human animal model of (1); (3) collecting tissue sections that exhibit tissue structures characteristic of the cancer development process of cancer stem cell populations or that exhibit their biological properties; (4) observing changes in the cancer stem cells over time, the cancer development process, or their biological properties in the tissue sections; and (5) identifying a test substance that inhibits the structure formed from cancer stem cells, the cancer development process caused by cancer stem cells, or the biological properties of cancer stem cells. [Example]

[0374] 8. Illustrative Examples The media described herein have been tested and demonstrated to support robust growth of epithelial stem cells derived from columnar epithelial tissues from humans and other mammals, for example, colonic stem cells have been cloned (see Figures 1, 2, 3A, and 3B).

[0375] A. MGM medium An exemplary system is a medium called MGM. MGM medium has been tested and proven to support robust growth of epithelial stem cells derived from human tissue or other mammals. For example, columnar lung stem cells, esophageal stem cells, gastrointestinal stem cells, cancer stem cells, hepatic stem cells, and pancreatic stem cells can all grow robustly in this culture system containing MGM medium, along with irradiated 3T3-J2 feeders, in the illustrated example.

[0376] MGM medium starts with a base medium as follows: Per liter of medium: DMEM 645ml F12 215ml FBS 100ml L-Glutamine 10ml Adenine 10ml Penicillin / streptomycin 10ml Insulin 1ml T3 1ml 2ml of hydrocortisone Cholera enterotoxin 1ml EGF 1ml Gentamicin 5ml Fungizone 1ml Additional ingredients: 1: R-spondin 1 (Cat. 4645-RS, R&D; final concentration: 125ng / ml, stock: 25ug / vial) 2:AV-951 (Cat. S1207, Selleckchem Inc.; final concentration: 500 nM, stock: 10 mM) 3:GDC-0879 (Cat. S1104, Selleckchem Inc.; final concentration: 500 nM, stock: 10 mM) 3: Human Noggin (Cat. 120-10c, Peprotech; final concentration: 100ng / ml, stock: 100ug / ml) (For stock, dissolve 500µg in 5ml H2O.) 4: ROCK inhibitors (Cat. 688000, Calbiochem; final concentration: 2.5 μM, stock: 2.5 mM) (For stock, dissolve 5 mg in 5.912 ml H2O.) 5:SB431542 (Cat. 13031, Cayman Chemical Company; final concentration: 2 μM, stock: 2 mM) (For stock, dissolve 5 mg in 6.5 ml DMSO.) 6: Nicotinamide (Sigma, Cat. N0636-100G; final concentration: 10 mM, stock: 5 M) (For stock, dissolve 6g in 10ml H2O.) 7:GSK429286A (Cat. S1474, Selleckchem Inc.; final concentration: 500 nM, stock: 10 mM)

[0377] Filter and store at 4°C.

[0378] Epithelial stem cells derived from a variety of different tissues, including lung and cervix, have been passaged in SAM medium for more than 25 passages and maintain self-renewal and multipotency both in vitro and in xenograft models using NSG mice.

[0379] Filter and store at 4°C.

[0380] B.SGM-88 + Feeder-free system An exemplary feeder-free system is a medium called SGM-88+. SGM-88+ medium has been tested and demonstrated to support robust growth of epithelial stem cells derived from human tissue or other mammals without the need for co-culture with feeder cells. It is produced as described above with the addition of the following ingredients 8-11:

[0381] SGM-88+ medium (1 liter): DMEM 645ml F12 215ml FBS 100ml L-Glutamine 10ml Adenine 10ml Penicillin / streptomycin 10ml Insulin 1ml T3 1ml 2ml of hydrocortisone Cholera enterotoxin 1ml EGF 1ml Gentamicin 5ml Fungizone 1ml Additional ingredients: 1: R-spondin 1 (Cat. 4645-RS, R&D; final concentration: 125ng / ml, stock: 25ug / vial) 2:AV-951 (Cat. S1207, Selleckchem Inc.; final concentration: 500 nM, stock: 10 mM) 3:GDC-0879 (Cat. S1104, Selleckchem Inc.; final concentration: 500 nM, stock: 10 mM) 3: Human Noggin (Cat. 120-10c, Peprotech; final concentration: 100ng / ml, stock: 100ug / ml) (For stock, dissolve 500µg in 5ml H2O.) 4:Y-27632 (Cat. 688000, Calbiochem; final concentration: 2.5 μM, stock: 2.5 mM) (For stock, dissolve 5 mg in 5.912 ml H2O.) 5:SB431542 (Cat. 13031, Cayman Chemical Company; final concentration: 2 μM, stock: 2 mM) (For stock, dissolve 5 mg in 6.5 ml DMSO.) 6: Nicotinamide (Sigma, Cat. N0636-100G; final concentration: 10 mM, stock: 5 M) (For stock, dissolve 6g in 10ml H2O.) 7:GSK429286A (Cat. S1474, Selleckchem Inc.; final concentration: 250 nM, stock: 10 mM) 8: CP673451 (Cat. S1536, Selleckchem Inc; final concentration: 1 μM, stock: 10 mM) 9:OAC1 (Cat.S7217, Selleckchem Inc; final concentration: 1μM, stock: 10mM) 10: JNK-IN-8 (Selleckchem Inc; final concentration: 1 μM, stock: 10 mM) 11: Jagged-1 (Cat. 61298, AnaSpec Inc; final concentration: 1 μM, stock: 1 mg / vial)

[0382] Filter and store at 4°C.

[0383] Ingredient preparation DMEM (Invitrogen 11960) High glucose (4.5 g / l), no L-glutamine, no sodium pyruvate.

[0384] F-12 Nutrient Mixture (HAM) (Invitrogen 11765) Contains L-glutamine.

[0385] Adenine (Calbiochem 1152 10g) Add 243 mg of adenine to 100 ml of 0.05 M HCl (dilute 0.4 ml of concentrated HCl with 100 ml of distilled HO). Stir at RT for about 1 h to dissolve. Filter sterilize. Divide into 10.0 ml aliquots. Final concentration: 1.8×10 -4 M. Store at -20°C.

[0386] FBS (Hyclone SH30910.03 500mL) Do not heat inactivate serum. Serum is thawed, aliquoted into 50 ml / tube and stored at -20°C.

[0387] L-Glutamine (GIBCO 25030-081 100ml) Thaw and divide into 10.0 ml aliquots. Store at -20°C.

[0388] Penicillin / Streptomycin (GIBCO 15140-122 100mL)

[0389] Fungizone (Gibco, 15290-018)

[0390] Gentamicin (Gibco, 15710-064)

[0391] Insulin (Sigma I-5500 50mg) Dissolve 50 mg in 10 ml of 0.005 N HCl (stock 5 mg / ml). Dispense 1 ml aliquots and store at -20°C. Final concentration 5ug / ml.

[0392] T3 (3,3',5-Triiodo-L-tyrosine) (Sigma T-2752 100mg) Dissolve 13.6 mg in 15 ml of 0.02 N NaOH. Bring the volume to 100 ml with PBS (concentrated stock 2 x 10 -4 M). Dispense 10 ml aliquots and store at -20°C. Take 0.1 ml of concentrated stock and bring the volume to 10 ml with PBS. Distribute into 1 ml aliquots and store at -20 °C (stock 2 x 10 -6 M). Final concentration 2 x 10 -9 M.

[0393] Hydrocortisone (Sigma H-0888 1g or Calbiochem / EMD386698) Dissolve 25 mg in 5 ml 95% ETOH (concentrated stock 5 mg / ml). Store at -20°C. Take 0.4 ml of the concentrated stock and make up to 10 ml with serum-free SBM medium. Distribute into 1 ml aliquots and store at -20°C (stock 200 μg / ml). Final concentration 0.4ug / ml.

[0394] Cholera enterotoxin (MP Biomedicals 190329 1mg or Calbiochem / EMD227036) Dissolve 1 mg (1 vial) in 1.18 ml distilled HO (concentrated stock 10 -5 M). Store at 4°C (do not freeze). Add 0.1 ml of concentrated stock to 10 ml SBM medium containing 10% FBS. Distribute into 1 ml aliquots and store at -20 °C (Stock 10 -7 M). Final concentration 10 -10 M.

[0395] EGF(Upstate Biotechnology 01-107) Preparation of 0.1% BSA: 100 mg BSA (SigmaA-2058; IgG-free, 5 g of cell culture tested). Dissolve in 100 ml distilled H2O. Sterile filter through a 0.22μ Nalgene. Store at either 4°C or -20°C depending on frequency of use. Preparation of EGF: 1 mg EGF is dissolved in 1 ml 0.1% BSA. Distribute into 100 μl aliquots and store at −80° C. (concentrated stock 100 μg / 100 μl). Make up the 100 μg concentrated stock to 10 ml with 0.1% BSA. Sterile filter using a 0.22μ Millipore Millex-GV. Distribute into 1 ml aliquots and store at -20°C (stock 10 μg / ml). Final concentration 10ng / ml.

[0396] C. Stemness and genomic stability of ground-state intestinal stem cells are age-independent Adult stem cells of the intestinal epithelium proliferate frequently. Mutations may accumulate in normal stem cells with age. Recent technological advances in cloning and culturing ground-state intestinal stem cells provide an opportunity to accurately assess age-related effects on the function and genome of these highly proliferative intestinal stem cells. Our ability to robustly expand lineages derived from single stem cells in vitro indefinitely provides sufficient DNA to perform reliable analyses and complete genome coverage of intestinal stem cells at the clonal level. Using exome sequencing analysis, we found that chromosomal deletions, amplifications, and gene mutations occur in intestinal stem cell clones derived from older individuals and diseased individuals. Interestingly, intestinal stem cell clones with wild-type genomes were identified in all donors, regardless of age. These wild-type stem cells can be cloned in vitro and expanded to one billion cells in approximately six weeks, maintaining a stable genome and without changes in stemness as evidenced by clonogenicity and pluripotency. Our studies suggest that wild-type stem cell clones exist in elderly and diseased patients and can be cloned and expanded in vitro with the same efficiency and stability as those derived from much younger individuals. Our results highlight the importance of screening for wild-type stem cell clones in elderly or diseased patients for autologous transplantation and support the promise of adult stem cell-based personalized medicine.

[0397] Autologous transplantation using wild-type or transgenic epidermal stem cells has proven highly successful in patients with severe burns, chronic wounds, and junctional epidermolysis bullosa. Conceivably, adult stem cells derived from other regenerative tissues, such as the intestine, can be used to restore intestinal epithelial function after autologous transplantation in patients with severe short bowel syndrome (SBS), those with congenital disorders, or those with inflammatory bowel disease (IBD).

[0398] However, caution is needed when using these patient-derived adult stem cells for autologous transplantation. Although there is a compelling amount of evidence suggesting that stem cells present in the intestinal tissue of elderly individuals are still highly competent, it is unclear whether their stem cell behavior is similar to that of those harvested from younger individuals. Whether aged stem cells are intrinsically dysfunctional is a question of considerable relevance for the practical development of autologous transplantation-based stem cell therapy for people of all ages. Furthermore, accumulated cytotoxicity in intestinal stem cells from elderly patients can lead to genomic alterations that may render these stem cells precancerous or transform them (Hsieh et al., 2013, Aging Cell (2013) 12, pp. 269-279). Furthermore, some intestinal disorders, such as ulcerative colitis (UC), are associated with the development of colorectal cancer (O'Conor Pm et al., Bowel Dis. 16, 1411-1420). Therefore, it is relevant to utilize intestinal stem cells derived from elderly or diseased patients for autologous transplantation without prior screening and selection of wild-type intestinal stem cells.

[0399] Cloning, screening, and expansion of wild-type intestinal stem cells is challenging due to significant barriers in adult stem cell research: the inability to clone stem cells from columnar epithelial tissue and maintain their immaturity during in vitro expansion. Therefore, intestinal stem cells must be developed as regenerative, differentiating "organoids" with an extremely low percentage of clonogenic cells, limiting their proliferation dynamics and availability for exploring basal stem cells. Recently, new techniques have been developed to support the cloning of ground-state intestinal stem cells (ISCGs) in their highly immature, clonogenic state. These cultured ISCGS demonstrated remarkable stability of genomic and epigenetic commitment programs, maintained clonogenicity, and unlimited replicative expansion, suggesting great potential for the selective cultivation of wild-type ISCGS for personalized regenerative medicine.

[0400] In this study, we used ground-state stem cell cloning technology to study intestinal stem cells derived from a wide range of patients. We found that although the probability of obtaining ISCGS with genetic mutations increases in elderly patients and those with UC, it is still possible to clone wild-type ISCGS from them. Furthermore, after being removed from the aged cellular environment, their behavior is identical to that of those obtained from younger individuals. Thus, our study suggests that wild-type ISCGS are present in patients of all ages, even in the setting of UC, and that they can be robustly and stably passaged in vitro, suggesting that the inherent immortality of intestinal stem cells is age-independent.

[0401] result ISCGS from patients with a wide range of ages To understand whether ground state intestinal stem cells (ISCGS) could be successfully cloned and cultured from patients of all ages, we selected 10 patients aged 10–20 years, 10 patients aged 30–50 years, and 10 patients aged 50–80 years. One-mm biopsies from these patients' intestinal epithelia were enzymatically digested and plated on a system containing 3T3J2 feeders and specialized media. We detected that approximately 50 colonies could be derived from each of the 30 patients. Starting from a single ISCGS colony, 1 billion ISCGS cells could be generated from all 30 patients, regardless of age, in approximately 6 days (Figure 4A). ISCGS derived from all ages exhibited indistinguishable morphology and identical pluripotency. ISCGS lineages from 16-, 56-, and 77-year-old patients were differentiated in air-liquid interface (ALI) culture for 10 days (Figure 4B). All ISCGS formed a highly uniform 3D serpentine pattern. Histological sections of these differentiated ISCGS revealed columnar epithelium with villous structures characterized by goblet cells (mucin 2+), endocrine cells (chromogranin A+), and Paneth cells (defensin α6+), demonstrating that the progeny of a single ISCGS from a wide range of patients (10-80 years old) can give rise to all epithelial lineages typically found in the intestine.

[0402] Genomic diversity of cloned ISCGS We next addressed polyclonality in the intestinal epithelium by sampling ISCGS clones from elderly patients (aged 40–70 years) for copy number polymorphism studies. We first demonstrated that ISCGS clones from all 30 patients were highly clonogenic. 50–70% clonogenicity was observed between patients (Figures 5A and 5B). Thus, single-cell-derived colonies can be expanded into single-cell-derived lineages containing thousands of cells, which provide sufficient DNA for routine genomic analysis. We used high-density SNP arrays to sample 1–23 clones from 11 adult patients with and without UC. We found that the majority of clones displayed few chromosomal alterations compared with patient-matched blood. However, one of 23 clones from a 44-year-old non-IBD patient displayed amplification of two putative oncogenes, SOS1 and XPO1, while the remainder of the clones were all wild-type. Furthermore, one of seven clones from a 56-year-old UC patient showed significantly more significant chromosomal alterations, resulting in the amplification of 16 genes, including putative oncogenes such as ERBB4, ALK, and MYCN. Interestingly, several other clones from the same patient displayed wild-type genomes (see Figure 5C). Our data suggest that both wild-type and mutant ISCGS can be cloned and expanded in vitro, and thus prior elimination of mutant ISCGS is an essential step before use for autologous transplantation.

[0403] Long-term culture of wild-type ISCGS To further investigate the genomic alterations in the wild-type and mutant clones derived from this UC patient, we performed exome sequencing on the ISCGS pool and lineages. Genomic analysis of these cells consisted of assessment of copy number variations (CNVs) and point mutations using exome sequencing. We determined CNVs and point mutations using mutant and wild-type lineages, as well as patient-matched DNA samples derived from pooled cells and venous blood. 28 Significantly, the pooled ISCGS exhibited very low CNVs in the form of interstitial deletions and amplifications. This degree of CNV in the pooled stem cells was within the range of that observed in the wild-type stem cell lineage of the same patient (Figures 6A, 6B, and 6C). In striking contrast, CNVs in the mutant stem cell lineages exhibited many more interstitial deletions and amplifications affecting a series of cancer-related genes, such as FHIT, PTPRD, p15 / p16, and ERBB4. The exomes of these stem cell lineages had point mutation allele frequencies clustered at approximately 0.4–0.5, as expected for a clonal population, while point mutation allele frequencies were undetectable or approximately 0.05 in pooled stem cells. These allele frequencies highlight the robustness of genomic analysis in stem cell lineages. Consistent with the CNV data, wild-type lineages showed no nonsynonymous mutations compared to blood. The mutant lineages showed significantly more nonsynonymous mutations at an allele frequency of 0.5, suggesting the absence of loss of heterozygosity. These SNVs include Notch and Ras mutations, which have been implicated as drivers of carcinogenesis. Taken together, the significantly higher number of CNV events and nonsynonymous mutations in this ulcerative colitis patient's mutant clone suggests that stem cells from this mutant clone may not be suitable for an autologous transplant approach for this patient. Therefore, cloning wild-type stem cell clones in polyclonal intestinal epithelia and expanding them for transplantation is important. We next investigated the genomic and functional stability of these wild-type stem cells in culture. We compared stem cells of normal stem cell clones at early passage (p1) and late passage (p10).Each passage involved 10 days of in vitro culture, encompassing approximately 17 cell divisions. Regardless of the number of passages, stem cells were able to properly differentiate into embryonic (Muc2+), endocrine (chromogranin A+), and Paneth (DEFA6) cells and maintained high clonogenic potential (>60%). To assess the genomic stability of normal ISCGS clones in vitro, we investigated copy number variations (CNVs) and single nucleotide variations (SNVs) in ISCGS clones after 100 days of continuous expansion using whole-exome sequencing (average 150x) (Figure 6A). At P10, when a single ISCGS lineage can expand to an estimated 1 billion cells, no copy number abnormalities were detected. Thus, this low level of structural variation was maintained up to the 10th passage. Compared with blood, ISCGS lineages exhibited a small number of point mutations (three) by the 10th passage, including two common SNPs and one synonymous SNP (Figure 6B). No new deletions or loss-of-heart events were observed during passage. These results suggest that these lineages sustain few genomic changes within the first 100 days of expansion. This result is consistent with what we observed in in vitro expansion of human fetal ISCGS (Wang and Yamamoto et al., 2015). Thus, stable and robust wild-type cultures are age-independent, providing a safe and reliable stem cell source for personalized regenerative medicine.

[0404] Consideration Stem cell-based autologous transplantation may improve outcomes for patients with a wide range of gastrointestinal disorders characterized by impaired mucosal barrier function, including IBD, necrotizing enterocolitis, fistulas, NSAID-induced injury, or gastroduodenal bleeding (Hong et al., "Concise review: The potential use of intestinal stem cells to treat patients with intestinal failure", Stem Cells Translational Medicine, 2017; Fredrik EO Holmberg et al., 2017; "Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease", EMBO, March 10, 2017). Important unanswered questions regarding elderly or diseased patients include whether ISCGS derived from these individuals can expand to sufficient numbers to functionally regenerate the intestinal epithelium and whether age- or disease-related genomic alterations pose safety concerns when ISCGS are used for therapeutic purposes.

[0405] The current therapeutic direction is to use a patient's own stem cells for autologous transplantation. If aged stem cells were inherently dysfunctional, this would greatly limit the ability to use this type of therapy for elderly people. However, if aged stem cells still maintain full stemness—in other words, if the inherent immortality of ISCGS is age-independent—this approach to regenerative medicine for age-related diseases may be extremely promising. Here, we demonstrate that ISCGS can be cloned from a wide range of patients, from 10 to 80 years of age. We did not detect any age-related loss of self-renewal or differentiation capacity. In approximately 60 days, single ISCGS could expand to approximately 1 billion cells for all 30 patients included in this study, each with a remarkably stable wild-type genome, suggesting that they serve as an ideal stem cell source for autologous transplantation targeted at patients with intestinal disorders.

[0406] In the 1980s, Howard Green and colleagues demonstrated the first example of cell therapy using cultured stem cells. They showed that human epidermis could be grown in the laboratory and transplanted into burn patients to reconstitute a functional epidermis. Since then, this technique has repeatedly been shown to be lifesaving for patients with severe burns. Furthermore, the long-term efficacy and safety of genetically modified epidermal stem cells for healing the severe skin blistering disease, epidermolysis bullosa, has been clinically demonstrated. The successful clinical use of epidermal stem cells has demonstrated a close correlation between the number of long-lived stem cells used in the technique and their ability to extensively self-renew both in vitro and in vivo.

[0407] Whether autologous transplantation of cultured intestinal cells can achieve the same success in a clinical setting remains unclear. Although successful transplantation of organoids containing a small proportion of intestinal stem cells has been claimed to be possible in a mouse model of experimental colitis, showing that these organoids attach and become an integrated part of the epithelium, it is likely that a very limited number of stem cells within the organoid structure can support long-term intestinal epithelial regeneration in humans.

[0408] Compared to the approximately 1% intestinal stem cells present within organoid structures, ground-state ISC cultures contain over 70% ISCGS. Based on previous lessons learned through the clinical use of cultured epidermal stem cells, we believe the use of ISCGS will significantly improve the efficacy and success of transplantation. Another key advantage of ISCGS technology is our ability to establish lineages derived from a single cell and rapidly expand them to 1 billion cells in approximately 60 days. We anticipate that aging or intestinal disorders may result in genomic changes in some intestinal stem cell clones, rendering these mutant cells unsuitable for transplantation. In our study, using the example of a 56-year-old patient with UC, we demonstrated the polyclonal complexity of cultured ISCGS, demonstrating the coexistence of wild-type and mutant clones in one patient. By screening single-cell-derived lineages, we established a lineage with a wild-type genome and showed that this lineage could self-renew, differentiate, and expand in vivo over long periods without alarming genomic changes.

[0409] Taken together, our data support the importance of screening cultured intestinal stem cells prior to transplantation due to safety concerns and provide a solution for efficient and reliable stem cell sources for personalized regenerative medicine (see Figure 7).

[0410] D. An efficient method for cloning gastrointestinal stem cells derived from patients via endoscopic biopsies Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are generally considered and treated as disorders of the immune system. However, recent studies suggest that the intestinal epithelium may be a key, perhaps even primary, player in the pathogenesis of Crohn's disease and ulcerative colitis. To assess the precise role of the intestinal epithelium in inflammatory bowel disease, a system for isolating, cloning, and studying mucosal stem cells without the complex influence of immune, stromal, and microbial cells is needed. Research on intestinal stem cells is progressing at a rapid pace, driven by the discovery of stem cell markers, such as Lgr5, Bmi1, and others, used as stable lineage tracers in mouse models. Furthermore, methods for isolating and analyzing gastrointestinal epithelial cells have been refined by coordinating induced pluripotent stem cells toward the intestinal lineage or by developing so-called organoids or mini-guts. Despite the remarkable properties of intestinal stem cells revealed by these tracing and in vitro organoid studies, the field as a whole suffers from an inability to maintain patient-specific human intestinal stem cells in an immature state that would allow for the large-scale amplification of such clones for a range of studies addressing functional defects, drug discovery, and regenerative medicine, as well as the analysis of possible pathogenic heterogeneity.

[0411] As described herein, we have developed a robust method for generating libraries of 100–300 unrelated stem cell clones derived from clinically standard 1 mm biopsies of human intestinal mucosa (Figure 8). Briefly, biopsies were enzymatically digested and plated on irradiated 3T3-J2 feeder cells (or feeder-free when using SGM-88 medium) in the presence of MGM medium as described above. To induce differentiation, stem cells were plated onto Transwell inserts (Corning, Corning, NY). At confluency, the luminal medium was removed, and culture was continued for an additional 6–12 days (MGM medium). Our method allows these clones to be expanded as immature cells in vitro to virtually unlimited numbers, reaching approximately 1 billion cells in less than 60 days (Figure 9). Importantly, we are able to maintain each of these stem cell clones in a highly immature, clonogenic state, which offers numerous advantages over either the induced pluripotent stem cell approach or the mini-gut approach, both of which yield relatively few stem cells among many differentiated cells. Furthermore, these stem cells can be induced to differentiate into intestinal-like structures containing all cell lineages, such as enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, regardless of the number of stem cell passages sustained. In summary, the advantages of this system include: (1) a highly uniform, homogenous population of immature cells; (2) rapid, uniform proliferation; (3) the ability to generate topologically precise, region-specific, and region-committed stem cells using endoscopy-assisted biopsy retrieval; (4) the ability to easily generate single-cell "lineages" for analysis of homogenous somatic genotypes and cross-study studies; and (5) the ability to assess both stem cell lineages and corresponding tissues for disease signatures. We anticipate that our research will ultimately provide disease-related stem cell lineages for extensive analysis in multiple laboratories to resolve the basis of intestinal diseases and ultimately identify means to treat them.

[0412] summary Single intestinal stem cells derived from 1 mm biopsies can be sampled and form colonies capable of independently expanding as pure "lineages." These single-cell-derived lineages fulfill all of the key stem cell criteria, including long-term self-renewal (intrinsic immortality) and pluripotency. These cells exhibit a remarkable clonogenic rate of >70% upon subsequent passage and thus represent a nearly homogeneous population of so-called ground-state stem cells, in contrast to "organoids," which have a clonogenic rate of <1%. The high clonogenicity of ground-state stem cells goes beyond academic merit, as it confers a significantly higher "expansion potential" than organoids, a significant 250-fold increase. Thus, a single ground-state stem cell can expand to 1 billion cells in less than 60 days, enough to establish 10,000 three-dimensional intestinal cultures in an air-liquid interface system. Another significant property of these highly immature stem cells is that they possess all the information necessary to autonomously form the complex three-dimensional epithelium of the native mucosa from which they originate. Taken together, this is a remarkably simple process for generating unlimited patient-derived, genetically stable, and regionally committed stem cells for analysis via multiple technologies and by multiple laboratories.

[0413] E. Maintenance of immaturity of human gastrointestinal stem cells in a feeder-free system Gastrointestinal stem cells drive the extremely rapid process of tissue regeneration and are central to the concept of adult stem cells based on engineered mouse models. The ability to clone and maintain human intestinal stem cells in a ground state in a feeder-dependent manner complements in vitro studies. Here, we present our efforts to establish a feeder-free system for cloning human gastrointestinal stem cells, establish lineages derived from single cells, and demonstrate long-term self-renewal of these lineages while maintaining their deeply related multipotency to reconstitute in vitro intestinal villi, including the formation of enterocytes, goblet cells, neuroendocrine cells, and Paneth cells or gastric pits, including mucosal, parietal, chief, and neuroendocrine cells. Despite the stable and deep commitment of these gastrointestinal stem cells to the intestinal or gastric lineages, respectively, complete genome expression analysis reveals striking similarities to each other, consistent with similar strategies for stem cell maintenance. Feeder-independence to maintain the immaturity of adult stem cells in vitro for long periods without genomic abnormalities offers certain advantages for use in regenerative medicine and disease modeling.

[0414] Tissue-specific epithelial stem cells are promising tools for regenerative medicine. Cultured epidermal stem cells, corneal epithelial stem cells, and lung stem cells have been successfully used for engraftment in clinical and mouse models. Stem cells from columnar epithelial tissues, such as the human intestine and colon, have recently been cloned in highly immature forms using feeder-based methods. Compared to technologies that channel induced pluripotent stem cells (iPSCs) into the intestinal lineage or the development of regenerative differentiated organoids (e.g., "mini-guts"), "ground state" stem cells cloned in feeder systems have been shown to be inherently immortal, demonstrating self-renewal, pluripotency, and genomic stability despite long-term culture. Furthermore, the ability to derive intestinal epithelial stem cells from standard 1 mm endoscopic biopsies makes this technology compatible with standard patient monitoring protocols. However, the preparation of mouse-derived feeder cells in this system requires significant time and effort. Furthermore, the involvement of feeders may be incompatible with the use of these cells for high-throughput drug screening, genomic analysis, and regenerative medicine. Therefore, transitioning to a feeder-free culture system for adult stem cells would represent a necessary and important improvement. The present study reports the development and validation of a feeder-free system for expanding "ground state" human gastrointestinal stem cells (GSCGS and ISCGS). This technology provides an easily usable, robust, and replicable system for using adult stem cells derived from columnar epithelium in research and clinical applications.

[0415] result Human gastrointestinal stem cells self-renew in a feeder-free system A specialized medium (designated SGM-88, as described above) was developed to support the maintenance of a ground state and highly clonogenic form of human gastrointestinal stem cells in the absence of mouse fibroblast feeder cells. It contains a novel combination of growth factors, including regulators of FLT (vascular endothelial growth factor receptor), TGF-β / BMP (transforming growth factor-β / bone morphogenetic protein), EGF (epidermal growth factor), IGF (insulin-like growth factor), Wnt / β-catenin, and the Notch pathway. Therefore, ISCGS and GSCGS previously established on feeder cells can be maintained in this medium as highly immature cells without expressing differentiation markers.

[0416] The clonogenicity of the cells, as determined by single-cell transfer, is greater than 50%. The lineages could be propagated for several months without any change in clonogenicity. This high clonogenicity allows us to rapidly generate single-cell "lineage" lines for amplification.

[0417] Pluripotent differentiation of intestinal and gastric stem cells ISCGS and GSCGS lineages were differentiated in air-liquid interface (ALI) culture for 10–30 days. ISCGS formed a highly uniform 3D snake-like pattern. Histological analysis of differentiated ISCGS sections revealed a columnar epithelium with villus-like structures composed of germ cells (Muc2+), endocrine cells (chromogranin A+), Paneth cells (DEFA6+), and polarized villin expression. In contrast, GSCGS gave rise to a 3D glandular pattern composed of pepsinogen-producing zymogen (chief) cells, hydrochloric acid-secreting parietal cells, gastric gland neck mucous cells, gastrin-producing cells (G cells), and glucagon-expressing cells (A cells). These results indicated that the progeny of a single ISCGS or GSCGS can give rise to all epithelial lineages typically found in the small intestine or stomach. Significantly, ground state stem cells differentiated by polarization after exposure to ALI instead of relying on the removal of factors such as Wnt or the addition of factors such as γ-secretase inhibitors (see reference).

[0418] Transcriptome analysis of ground-state stem cells and tissues differentiated by ALI demonstrated gene expression differences, as expected for intestinal and gastric epithelia, although the gene expression profiles of undifferentiated ISCGS and GSCGS differed by less than 1% (>2.0-fold, P<0.5). ISCGS showed high expression of intestinal stem cell markers, such as CD133, Lgr5, and Lrig1, whereas those derived from the stomach possessed typical stem cell markers of the gastric epithelium.

[0419] Feeder-independent genome and lineage stability To assess the genomic stability of ISCGS and GSCGS in this feeder-free system, we investigated copy number variations (CNVs) and single nucleotide variations (SNVs) by whole-exome sequencing (average 150x) in ISCGS and GSCGS lineages after 20 days (second passage: P2), 40 days (P3), 60 days (P6), 80 days (P8), and 100 days (P11) of continuous expansion. At P10, when a single ISCGS or GSCGS lineage can expand to an estimated 1–10 billion cells, no copy number abnormalities were detected. Thus, this low level of structural variation was maintained through the 10th passage. By comparison with P2, ISCGS and GSCGS lineages exhibited several point mutations (0–3), of which two SNPs were common variants and one SNP was a synonymous mutation, up to the 10th passage. No new deletions or LOH events were found during passage, suggesting that these lineages sustain few genomic changes within the first 100 days of expansion.

[0420] We next compared early and late passages of ISCGS and GSCGS lineages during ALI differentiation. Based on histological criteria, including staining for gastric and intestinal markers, we were unable to distinguish ALI-differentiated epithelia derived from P2 and P10. Furthermore, we found that ISCGS and GSCGS lineages did not lose (or gain) clonogenicity and stably persisted at greater than 50% when tested at P2 and P10. Finally, we found no evidence of tumorigenicity of these ground-state intestinal and gastric stem cells after subcutaneous implantation in immunodeficient (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice. In contrast, ISCGS and GSCGS lineages generated well-differentiated epithelia resembling the respective epithelia (intestinal and gastric) from which they were derived.

[0421] Sequence information SEQUENCE LISTING <110> TRACT PHARMACEUTICALS, INC. UNIVERSITY OF HOUSTON SYSTEM <120> STEM CELL CULTURE SYSTEMS FOR COLUMNAR EPITHELIAL STEM CELLS, AND USES RELATED THERETO <150> US 62 / 611,176 <151> 2017-12-28 <150> US 62 / 724,937 <151> 2018-08-30 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 232 <212> PRT <213> Homo sapiens <400> 1 Met Glu Arg Cys Pro Ser Leu Gly Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 Val Leu Gly Leu Arg Ala Thr Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30 Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35 40 45 His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50 55 60 Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp Pro Gly Phe Met Ala 65 70 75 80 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly Gly Ala Ala Gly 85 90 95 Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln Leu Leu Arg Gln Arg Pro 100 105 110 Ser Gly Ala Met Pro Ser Glu Ile Lys Gly Leu Glu Phe Ser Glu Gly 115 120 125 Leu Ala Gln Gly Lys Lys Gln Arg Leu Ser Lys Lys Leu Arg Arg Lys 130 135 140 Leu Gln Met Trp Leu Trp Ser Gln Thr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 Trp Asn Asp Leu Gly Ser Arg Phe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175 Ser Cys Phe Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val Cys 180 185 190 Lys Pro Ser Lys Ser Val His Leu Thr Val Leu Arg Trp Arg Cys Gln 195 200 205 Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro Ile Gln Tyr Pro Ile 210 215 220 Ile Ser Glu Cys Lys Cys Ser Cys 225 230 <210> 2 <211> 955 <212> PRT <213> Homo sapiens <400> 2 Met Pro Ser Leu Pro Ala Pro Pro Ala Pro Leu Leu Leu Leu Gly Leu 1 5 10 15 Leu Leu Leu Gly Ser Arg Pro Ala Arg Gly Ala Gly Pro Glu Pro Pro 20 25 30 Val Leu Pro Ile Arg Ser Glu Lys Glu Pro Leu Pro Val Arg Gly Ala 35 40 45 Ala Gly Cys Thr Phe Gly Gly Lys Val Tyr Ala Leu Asp Glu Thr Trp 50 55 60 His Pro Asp Leu Gly Glu Pro Phe Gly Val Met Arg Cys Val Leu Cys 65 70 75 80 Ala Cys Glu Ala Pro Gln Trp Gly Arg Arg Thr Arg Gly Pro Gly Arg 85 90 95 Val Ser Cys Lys Asn Ile Lys Pro Glu Cys Pro Thr Pro Ala Cys Gly 100 105 110 Gln Pro Arg Gln Leu Pro Gly His Cys Cys Gln Thr Cys Pro Gln Glu 115 120 125 Arg Ser Ser Ser Glu Arg Gln Pro Ser Gly Leu Ser Phe Glu Tyr Pro 130 135 140 Arg Asp Pro Glu His Arg Ser Tyr Ser Asp Arg Gly Glu Pro Gly Ala 145 150 155 160 Glu Glu Arg Ala Arg Gly Asp Gly His Thr Asp Phe Val Ala Leu Leu 165 170 175 Thr Gly Pro Arg Ser Gln Ala Val Ala Arg Ala Arg Val Ser Leu Leu 180 185 190 Arg Ser Ser Leu Arg Phe Ser Ile Ser Tyr Arg Arg Leu Asp Arg Pro 195 200 205 Thr Arg Ile Arg Phe Ser Asp Ser Asn Gly Ser Val Leu Phe Glu His 210 215 220 Pro Ala Ala Pro Thr Gln Asp Gly Leu Val Cys Gly Val Trp Arg Ala 225 230 235 240 Val Pro Arg Leu Ser Leu Arg Leu Leu Arg Ala Glu Gln Leu His Val 245 250 255 Ala Leu Val Thr Leu Thr His Pro Ser Gly Glu Val Trp Gly Pro Leu 260 265 270 Ile Arg His Arg Ala Leu Ala Ala Glu Thr Phe Ser Ala Ile Leu Thr 275 280 285 Leu Glu Gly Pro Pro Gln Gln Gly Val Gly Gly Ile Thr Leu Leu Thr 290 295 300 Leu Ser Asp Thr Glu Asp Ser Leu His Phe Leu Leu Leu Phe Arg Gly 305 310 315 320 Leu Leu Glu Pro Arg Ser Gly Gly Leu Thr Gln Val Pro Leu Arg Leu 325 330 335 Gln Ile Leu His Gln Gly Gln Leu Leu Arg Glu Leu Gln Ala Asn Val 340 345 350 Ser Ala Gln Glu Pro Gly Phe Ala Glu Val Leu Pro Asn Leu Thr Val 355 360 365 Gln Glu Met Asp Trp Leu Val Leu Gly Glu Leu Gln Met Ala Leu Glu 370 375 380 Trp Ala Gly Arg Pro Gly Leu Arg Ile Ser Gly His Ile Ala Ala Arg 385 390 395 400 Lys Ser Cys Asp Val Leu Gln Ser Val Leu Cys Gly Ala Asp Ala Leu 405 410 415 Ile Pro Val Gln Thr Gly Ala Ala Gly Ser Ala Ser Leu Thr Leu Leu 420 425 430 Gly Asn Gly Ser Leu Ile Tyr Gln Val Gln Val Val Gly Thr Ser Ser 435 440 445 Glu Val Val Ala Met Thr Leu Glu Thr Lys Pro Gln Arg Arg Asp Gln 450 455 460 Arg Thr Val Leu Cys His Met Ala Gly Leu Gln Pro Gly Gly His Thr 465 470 475 480 Ala Val Gly Ile Cys Pro Gly Leu Gly Ala Arg Gly Ala His Met Leu 485 490 495 Leu Gln Asn Glu Leu Phe Leu Asn Val Gly Thr Lys Asp Phe Pro Asp 500 505 510 Gly Glu Leu Arg Gly His Val Ala Ala Leu Pro Tyr Cys Gly His Ser 515 520 525 Ala Arg His Asp Thr Leu Pro Val Pro Leu Ala Gly Ala Leu Val Leu 530 535 540 Pro Pro Val Lys Ser Gln Ala Ala Gly His Ala Trp Leu Ser Leu Asp 545 550 555 560 Thr His Cys His Leu His Tyr Glu Val Leu Leu Ala Gly Leu Gly Gly 565 570 575 Ser Glu Gln Gly Thr Val Thr Ala His Leu Leu Gly Pro Pro Gly Thr 580 585 590 Pro Gly Pro Arg Arg Leu Leu Lys Gly Phe Tyr Gly Ser Glu Ala Gln 595 600 605 Gly Val Val Lys Asp Leu Glu Pro Glu Leu Leu Arg His Leu Ala Lys 610 615 620 Gly Met Ala Ser Leu Leu Ile Thr Thr Lys Gly Ser Pro Arg Gly Glu 625 630 635 640 Leu Arg Gly Gln Val His Ile Ala Asn Gln Cys Glu Val Gly Gly Leu 645 650 655 Arg Leu Glu Ala Ala Gly Ala Glu Gly Val Arg Ala Leu Gly Ala Pro 660 665 670 Asp Thr Ala Ser Ala Ala Pro Pro Val Val Pro Gly Leu Pro Ala Leu 675 680 685 Ala Pro Ala Lys Pro Gly Gly Pro Gly Arg Pro Arg Asp Pro Asn Thr 690 695 700 Cys Phe Phe Glu Gly Gln Gln Arg Pro His Gly Ala Arg Trp Ala Pro 705 710 715 720 Asn Tyr Asp Pro Leu Cys Ser Leu Cys Thr Cys Gln Arg Arg Thr Val 725 730 735 Ile Cys Asp Pro Val Val Cys Pro Pro Pro Ser Cys Pro His Pro Val 740 745 750 Gln Ala Pro Asp Gln Cys Cys Pro Val Cys Pro Glu Lys Gln Asp Val 755 760 765 Arg Asp Leu Pro Gly Leu Pro Arg Ser Arg Asp Pro Gly Glu Gly Cys 770 775 780 Tyr Phe Asp Gly Asp Arg Ser Trp Arg Ala Ala Gly Thr Arg Trp His 785 790 795 800 Pro Val Val Pro Pro Phe Gly Leu Ile Lys Cys Ala Val Cys Thr Cys 805 810 815 Lys Gly Gly Thr Gly Glu Val His Cys Glu Lys Val Gln Cys Pro Arg 820 825 830 Leu Ala Cys Ala Gln Pro Val Arg Val Asn Pro Thr Asp Cys Cys Lys 835 840 845 Gln Cys Pro Val Gly Ser Gly Ala His Pro Gln Leu Gly Asp Pro Met 850 855 860 Gln Ala Asp Gly Pro Arg Gly Cys Arg Phe Ala Gly Gln Trp Phe Pro 865 870 875 880 Glu Ser Gln Ser Trp His Pro Ser Val Pro Pro Phe Gly Glu Met Ser 885 890 895 Cys Ile Thr Cys Arg Cys Gly Ala Gly Val Pro His Cys Glu Arg Asp 900 905 910 Asp Cys Ser Leu Pro Leu Ser Cys Gly Ser Gly Lys Glu Ser Arg Cys 915 920 925 Cys Ser Arg Cys Thr Ala His Arg Arg Pro Ala Pro Glu Thr Arg Thr 930 935 940 Asp Pro Glu Leu Glu Lys Glu Ala Glu Gly Ser 945 950 955 <210> 3 <211> 344 <212> PRT <213> Homo sapiens <400> 3 Met Val Arg Ala Arg His Gln Pro Gly Gly Leu Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Cys Gln Phe Met Glu Asp Arg Ser Ala Gln Ala Gly Asn Cys 20 25 30 Trp Leu Arg Gln Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr 35 40 45 Glu Leu Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser Thr Ser 50 55 60 Trp Thr Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile 65 70 75 80 Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Thr Cys Glu 85 90 95 Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Arg Met Asn Lys Lys Asn 100 105 110 Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys 115 120 125 Gly Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala 130 135 140 Leu Leu Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr 145 150 155 160 Gln Gly Arg Cys Lys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser 165 170 175 Ser Thr Cys Val Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys 180 185 190 Asn Arg Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly 195 200 205 Asn Asp Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr 210 215 220 Cys Leu Leu Gly Arg Ser Ile Gly Leu Ala Tyr Glu Gly Lys Cys Ile 225 230 235 240 Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Thr Gly Gly Lys Lys Cys 245 250 255 Leu Trp Asp Phe Lys Val Gly Arg Gly Arg Cys Ser Leu Cys Asp Glu 260 265 270 Leu Cys Pro Asp Ser Lys Ser Asp Glu Pro Val Cys Ala Ser Asp Asn 275 280 285 Ala Thr Tyr Ala Ser Glu Cys Ala Met Lys Glu Ala Ala Cys Ser Ser 290 295 300 Gly Val Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn Ser Ile Ser 305 310 315 320 Glu Asp Thr Glu Glu Glu Glu Glu Asp Glu Asp Gln Asp Tyr Ser Phe 325 330 335 Pro Ile Ser Ser Ile Leu Glu Trp 340 <210> 4 <211> 180 <212> PRT <213> Homo sapiens <400> 4 Met Leu Arg Val Leu Val Gly Ala Val Leu Pro Ala Met Leu Leu Ala 1 5 10 15 Ala Pro Pro Pro Ile Asn Lys Leu Ala Leu Phe Pro Asp Lys Ser Ala 20 25 30 Trp Cys Glu Ala Lys Asn Ile Thr Gln Ile Val Gly His Ser Gly Cys 35 40 45 Glu Ala Lys Ser Ile Gln Asn Arg Ala Cys Leu Gly Gln Cys Phe Ser 50 55 60 Tyr Ser Val Pro Asn Thr Phe Pro Gln Ser Thr Glu Ser Leu Val His 65 70 75 80 Cys Asp Ser Cys Met Pro Ala Gln Ser Met Trp Glu Ile Val Thr Leu 85 90 95 Glu Cys Pro Gly His Glu Glu Val Pro Arg Val Asp Lys Leu Val Glu 100 105 110 Lys Ile Leu His Cys Ser Cys Gln Ala Cys Gly Lys Glu Pro Ser His 115 120 125 Glu Gly Leu Ser Val Tyr Val Gln Gly Glu Asp Gly Pro Gly Ser Gln 130 135 140 Pro Gly Thr His Pro His Pro His Pro His Pro His Pro Gly Gly Gln 145 150 155 160 Thr Pro Glu Pro Glu Asp Pro Pro Gly Ala Pro His Thr Glu Glu Glu 165 170 175 Gly Ala Glu Asp 180 <210> 5 <211> 267 <212> PRT <213> Homo sapiens <400> 5 Met His Leu Leu Leu Phe Gln Leu Leu Val Leu Leu Pro Leu Gly Lys 1 5 10 15 Thr Thr Arg His Gln Asp Gly Arg Gln Asn Gln Ser Ser Leu Ser Pro 20 25 30 Val Leu Leu Pro Arg Asn Gln Arg Glu Leu Pro Thr Gly Asn His Glu 35 40 45 Glu Ala Glu Glu Lys Pro Asp Leu Phe Val Ala Val Pro His Leu Val 50 55 60 Ala Thr Ser Pro Ala Gly Glu Gly Gln Arg Gln Arg Glu Lys Met Leu 65 70 75 80 Ser Arg Phe Gly Arg Phe Trp Lys Lys Pro Glu Arg Glu Met His Pro 85 90 95 Ser Arg Asp Ser Asp Ser Glu Pro Phe Pro Pro Gly Thr Gln Ser Leu 100 105 110 Ile Gln Pro Ile Asp Gly Met Lys Met Glu Lys Ser Pro Leu Arg Glu 115 120 125 Glu Ala Lys Lys Phe Trp His His Phe Met Phe Arg Lys Thr Pro Ala 130 135 140 Ser Gln Gly Val Ile Leu Pro Ile Lys Ser His Glu Val His Trp Glu 145 150 155 160 Thr Cys Arg Thr Val Pro Phe Ser Gln Thr Ile Thr His Glu Gly Cys 165 170 175 Glu Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser 180 185 190 Val His Phe Pro Gly Ala Ala Gln His Ser His Thr Ser Cys Ser His 195 200 205 Cys Leu Pro Ala Lys Phe Thr Thr Met His Leu Pro Leu Asn Cys Thr 210 215 220 Glu Leu Ser Ser Val Ile Lys Val Val Met Leu Val Glu Glu Cys Gln 225 230 235 240 Cys Lys Val Lys Thr Glu His Glu Asp Gly His Ile Leu His Ala Gly 245 250 255 Ser Gln Asp Ser Phe Ile Pro Gly Val Ser Ala 260 265 <210> 6 <211> 184 <212> PRT <213> Homo sapiens <400> 6 Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu Leu Leu Gly 1 5 10 15 Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys Lys Gly Ser Gln Gly Ala 20 25 30 Ile Pro Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu Gln Thr Gln 35 40 45 Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg Gly Arg Gly Gln Gly Arg 50 55 60 Gly Thr Ala Met Pro Gly Glu Glu Val Leu Glu Ser Ser Gln Glu Ala 65 70 75 80 Leu His Val Thr Glu Arg Lys Tyr Leu Lys Arg Asp Trp Cys Lys Thr 85 90 95 Gln Pro Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn Ser Arg Thr 100 105 110 Ile Ile Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro 115 120 125 Arg His Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys Ser Phe Cys 130 135 140 Lys Pro Lys Lys Phe Thr Thr Met Met Val Thr Leu Asn Cys Pro Glu 145 150 155 160 Leu Gln Pro Pro Thr Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys 165 170 175 Arg Cys Ile Ser Ile Asp Leu Asp 180 <210> 7 <211> 213 <212> PRT <213> Homo sapiens <400> 7 Met Gln Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr 1 5 10 15 Ala Phe Arg Val Val Glu Gly Gln Gly Trp Gln Ala Phe Lys Asn Asp 20 25 30 Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 80 Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val 130 135 140 Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 150 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln 165 170 175 Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gln Lys Gly 180 185 190 Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gln Ala Glu 195 200 205 Leu Glu Asn Ala Tyr 210 <210> 8 <211> 70 <212> PRT <213> Homo sapiens <400> 8 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Serum Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu 65 70 <210> 9 <211> 1474 <212> PRT <213> Homo sapiens <400> 9 Met Gly Lys Asn Lys Leu Leu His Pro Ser Leu Val Leu Leu Leu Leu 1 5 10 15 Val Leu Leu Pro Thr Asp Ala Ser Val Ser Gly Lys Pro Gln Tyr Met 20 25 30 Val Leu Val Pro Ser Leu Leu His Thr Glu Thr Thr Glu Lys Gly Cys 35 40 45 Val Leu Leu Ser Tyr Leu Asn Glu Thr Val Thr Val Ser Ala Ser Leu 50 55 60 Glu Ser Val Arg Gly Asn Arg Ser Leu Phe Thr Asp Leu Glu Ala Glu 65 70 75 80 Asn Asp Val Leu His Cys Val Ala Phe Ala Val Pro Lys Ser Ser Ser 85 90 95 Asn Glu Glu Val Met Phe Leu Thr Val Gln Val Lys Gly Pro Thr Gln 100 105 110 Glu Phe Lys Lys Arg Thr Thr Val Met Val Lys Asn Glu Asp Ser Leu 115 120 125 Val Phe Val Gln Thr Asp Lys Ser Ile Tyr Lys Pro Gly Gln Thr Val 130 135 140 Lys Phe Arg Val Val Ser Met Asp Glu Asn Phe His Pro Leu Asn Glu 145 150 155 160 Leu Ile Pro Leu Val Tyr Ile Gln Asp Pro Lys Gly Asn Arg Ile Ala 165 170 175 Gln Trp Gln Ser Phe Gln Leu Glu Gly Gly Leu Lys Gln Phe Ser Phe 180 185 190 Pro Leu Ser Ser Glu Pro Phe Gln Gly Ser Tyr Lys Val Val Val Gln 195 200 205 Lys Lys Ser Gly Gly Arg Thr Glu His Pro Phe Thr Val Glu Glu Phe 210 215 220 Val Leu Pro Lys Phe Glu Val Gln Val Thr Val Pro Lys Ile Ile Thr 225 230 235 240 Ile Leu Glu Glu Glu Met Asn Val Ser Val Cys Gly Leu Tyr Thr Tyr 245 250 255 Gly Lys Pro Val Pro Gly His Val Thr Val Ser Ile Cys Arg Lys Tyr 260 265 270 Ser Asp Ala Ser Asp Cys His Gly Glu Asp Ser Gln Ala Phe Cys Glu 275 280 285 Lys Phe Ser Gly Gln Leu Asn Ser His Gly Cys Phe Tyr Gln Gln Val 290 295 300 Lys Thr Lys Val Phe Gln Leu Lys Arg Lys Glu Tyr Glu Met Lys Leu 305 310 315 320 His Thr Glu Ala Gln Ile Gln Glu Glu Gly Thr Val Val Glu Leu Thr 325 330 335 Gly Arg Gln Ser Ser Glu Ile Thr Arg Thr Ile Thr Lys Leu Ser Phe 340 345 350 Val Lys Val Asp Ser His Phe Arg Gln Gly Ile Pro Phe Phe Gly Gln 355 360 365 Val Arg Leu Val Asp Gly Lys Gly Val Pro Ile Pro Asn Lys Val Ile 370 375 380 Phe Ile Arg Gly Asn Glu Ala Asn Tyr Tyr Ser Asn Ala Thr Thr Asp 385 390 395 400 Glu His Gly Leu Val Gln Phe Ser Ile Asn Thr Thr Asn Val Met Gly 405 410 415 Thr Ser Leu Thr Val Arg Val Asn Tyr Lys Asp Arg Ser Pro Cys Tyr 420 425 430 Gly Tyr Gln Trp Val Ser Glu Glu His Glu Glu Ala His His Thr Ala 435 440 445 Tyr Leu Val Phe Ser Pro Ser Lys Ser Phe Val His Leu Glu Pro Met 450 455 460 Ser His Glu Leu Pro Cys Gly His Thr Gln Thr Val Gln Ala His Tyr 465 470 475 480 Ile Leu Asn Gly Gly Thr Leu Leu Gly Leu Lys Lys Leu Ser Phe Tyr 485 490 495 Tyr Leu Ile Met Ala Lys Gly Gly Ile Val Arg Thr Gly Thr His Gly 500 505 510 Leu Leu Val Lys Gln Glu Asp Met Lys Gly His Phe Ser Ile Ser Ile 515 520 525 Pro Val Lys Ser Asp Ile Ala Pro Val Ala Arg Leu Leu Ile Tyr Ala 530 535 540 Val Leu Pro Thr Gly Asp Val Ile Gly Asp Ser Ala Lys Tyr Asp Val 545 550 555 560 Glu Asn Cys Leu Ala Asn Lys Val Asp Leu Ser Phe Ser Pro Ser Gln 565 570 575 Ser Leu Pro Ala Ser His Ala His Leu Arg Val Thr Ala Ala Pro Gln 580 585 590 Ser Val Cys Ala Leu Arg Ala Val Asp Gln Ser Val Leu Leu Met Lys 595 600 605 Pro Asp Ala Glu Leu Ser Ala Ser Ser Val Tyr Asn Leu Leu Pro Glu 610 615 620 Lys Asp Leu Thr Gly Phe Pro Gly Pro Leu Asn Asp Gln Asp Asp Glu 625 630 635 640 Asp Cys Ile Asn Arg His Asn Val Tyr Ile Asn Gly Ile Thr Tyr Thr 645 650 655 Pro Val Ser Ser Thr Asn Glu Lys Asp Met Tyr Ser Phe Leu Glu Asp 660 665 670 Met Gly Leu Lys Ala Phe Thr Asn Ser Lys Ile Arg Lys Pro Lys Met 675 680 685 Cys Pro Gln Leu Gln Gln Tyr Glu Met His Gly Pro Glu Gly Leu Arg 690 695 700 Val Gly Phe Tyr Glu Ser Asp Val Met Gly Arg Gly His Ala Arg Leu 705 710 715 720 Val His Val Glu Glu Pro His Thr Glu Thr Val Arg Lys Tyr Phe Pro 725 730 735 Glu Thr Trp Ile Trp Asp Leu Val Val Val Asn Ser Ala Gly Val Ala 740 745 750 Glu Val Gly Val Thr Val Pro Asp Thr Ile Thr Glu Trp Lys Ala Gly 755 760 765 Ala Phe Cys Leu Ser Glu Asp Ala Gly Leu Gly Ile Ser Ser Thr Ala 770 775 780 Ser Leu Arg Ala Phe Gln Pro Phe Phe Val Glu Leu Thr Met Pro Tyr 785 790 795 800 Ser Val Ile Arg Gly Glu Ala Phe Thr Leu Lys Ala Thr Val Leu Asn 805 810 815 Tyr Leu Pro Lys Cys Ile Arg Val Ser Val Gln Leu Glu Ala Ser Pro 820 825 830 Ala Phe Leu Ala Val Pro Val Glu Lys Glu Gln Ala Pro His Cys Ile 835 840 845 Cys Ala Asn Gly Arg Gln Thr Val Ser Trp Ala Val Thr Pro Lys Ser 850 855 860 Leu Gly Asn Val Asn Phe Thr Val Ser Ala Glu Ala Leu Glu Ser Gln 865 870 875 880 Glu Leu Cys Gly Thr Glu Val Pro Ser Val Pro Glu His Gly Arg Lys 885 890 895 Asp Thr Val Ile Lys Pro Leu Leu Val Glu Pro Glu Gly Leu Glu Lys 900 905 910 Glu Thr Thr Phe Asn Ser Leu Leu Cys Pro Ser Gly Gly Glu Val Ser 915 920 925 Glu Glu Leu Ser Leu Lys Leu Pro Pro Asn Val Val Glu Glu Ser Ala 930 935 940 Arg Ala Ser Val Ser Val Leu Gly Asp Ile Leu Gly Ser Ala Met Gln 945 950 955 960 Asn Thr Gln Asn Leu Leu Gln Met Pro Tyr Gly Cys Gly Glu Gln Asn 965 970 975 Met Val Leu Phe Ala Pro Asn Ile Tyr Val Leu Asp Tyr Leu Asn Glu 980 985 990 Thr Gln Gln Leu Thr Pro Glu Ile Lys Ser Lys Ala Ile Gly Tyr Leu 995 1000 1005 Asn Thr Gly Tyr Gln Arg Gln Leu Asn Tyr Lys His Tyr Asp Gly 1010 1015 1020 Ser Tyr Ser Thr Phe Gly Glu Arg Tyr Gly Arg Asn Gln Gly Asn 1025 1030 1035 Thr Trp Leu Thr Ala Phe Val Leu Lys Thr Phe Ala Gln Ala Arg 1040 1045 1050 Ala Tyr Ile Phe Ile Asp Glu Ala His Ile Thr Gln Ala Leu Ile 1055 1060 1065 Trp Leu Ser Gln Arg Gln Lys Asp Asn Gly Cys Phe Arg Ser Ser 1070 1075 1080 Gly Ser Leu Leu Asn Asn Ala Ile Lys Gly Gly Val Glu Asp Glu 1085 1090 1095 Val Thr Leu Ser Ala Tyr Ile Thr Ile Ala Leu Leu Glu Ile Pro 1100 1105 1110 Leu Thr Val Thr His Pro Val Val Arg Asn Ala Leu Phe Cys Leu 1115 1120 1125 Glu Ser Ala Trp Lys Thr Ala Gln Glu Gly Asp His Gly Ser His 1130 1135 1140 Val Tyr Thr Lys Ala Leu Leu Ala Tyr Ala Phe Ala Leu Ala Gly 1145 1150 1155 Asn Gln Asp Lys Arg Lys Glu Val Leu Lys Ser Leu Asn Glu Glu 1160 1165 1170 Ala Val Lys Lys Asp Asn Ser Val His Trp Glu Arg Pro Gln Lys 1175 1180 1185 Pro Lys Ala Pro Val Gly His Phe Tyr Glu Pro Gln Ala Pro Ser 1190 1195 1200 Ala Glu Val Glu Met Thr Ser Tyr Val Leu Leu Ala Tyr Leu Thr 1205 1210 1215 Ala Gln Pro Ala Pro Thr Ser Glu Asp Leu Thr Ser Ala Thr Asn 1220 1225 1230 Ile Val Lys Trp Ile Thr Lys Gln Gln Asn Ala Gln Gly Gly Phe 1235 1240 1245 Ser Ser Thr Gln Asp Thr Val Val Ala Leu His Ala Leu Ser Lys 1250 1255 1260 Tyr Gly Ala Ala Thr Phe Thr Arg Thr Gly Lys Ala Ala Gln Val 1265 1270 1275 Thr Ile Gln Ser Ser Gly Thr Phe Ser Ser Lys Phe Gln Val Asp 1280 1285 1290 Asn Asn Asn Arg Leu Leu Leu Gln Gln Val Ser Leu Pro Glu Leu 1295 1300 1305 Pro Gly Glu Tyr Ser Met Lys Val Thr Gly Glu Gly Cys Val Tyr 1310 1315 1320 Leu Gln Thr Ser Leu Lys Tyr Asn Ile Leu Pro Glu Lys Glu Glu 1325 1330 1335 Phe Pro Phe Ala Leu Gly Val Gln Thr Leu Pro Gln Thr Cys Asp 1340 1345 1350 Glu Pro Lys Ala His Thr Ser Phe Gln Ile Ser Leu Ser Val Ser 1355 1360 1365 Tyr Thr Gly Ser Arg Ser Ala Ser Asn Met Ala Ile Val Asp Val 1370 1375 1380 Lys Met Val Ser Gly Phe Ile Pro Leu Lys Pro Thr Val Lys Met 1385 1390 1395 Leu Glu Arg Ser Asn His Val Ser Arg Thr Glu Val Ser Ser Asn 1400 1405 1410 His Val Leu Ile Tyr Leu Asp Lys Val Ser Asn Gln Thr Leu Ser 1415 1420 1425 Leu Phe Phe Thr Val Leu Gln Asp Val Pro Val Arg Asp Leu Lys 1430 1435 1440 Pro Ala Ile Val Lys Val Tyr Asp Tyr Tyr Glu Thr Asp Glu Phe 1445 1450 1455 Ala Ile Ala Glu Tyr Asn Ala Pro Cys Ser Lys Asp Leu Gly Asn 1460 1465 1470 Ala <210> 10 <211> 263 <212> PRT <213> Homo sapiens <400> 10 Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His 1 5 10 15 Leu Thr Ile Ser Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile 20 25 30 Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser 35 40 45 Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu 50 55 60 Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro 65 70 75 80 Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys 85 90 95 Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr 100 105 110 Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala 115 120 125 Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser 130 135 140 Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser 145 150 155 160 Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr 165 170 175 Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp 180 185 190 Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu 195 200 205 Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn 210 215 220 Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg 225 230 235 240 Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro 245 250 255 Leu Thr Ser Ala Gly Pro Ala 260 <210> 11 <211> 243 <212> PRT <213> Homo sapiens <400> 11 Met Gln Phe Arg Leu Phe Ser Phe Ala Leu Ile Ile Leu Asn Cys Met 1 5 10 15 Asp Tyr Ser His Cys Gln Gly Asn Arg Trp Arg Arg Ser Lys Arg Ala 20 25 30 Ser Tyr Val Ser Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys 35 40 45 Asp Asn Gly Cys Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg 50 55 60 Arg Glu Gly Met Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser 65 70 75 80 Gly Tyr Tyr Gly His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys 85 90 95 Arg Ile Glu Asn Cys Asp Ser Cys Phe Ser Lys Asp Phe Cys Thr Lys 100 105 110 Cys Lys Val Gly Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys 115 120 125 Pro Asp Gly Phe Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly 130 135 140 Cys Glu Val Gly His Trp Ser Glu Trp Gly Thr Cys Ser Arg Asn Asn 145 150 155 160 Arg Thr Cys Gly Phe Lys Trp Gly Leu Glu Thr Arg Thr Arg Gln Ile 165 170 175 Val Lys Lys Pro Val Lys Asp Thr Ile Leu Cys Pro Thr Ile Ala Glu 180 185 190 Ser Arg Arg Cys Lys Met Thr Met Arg His Cys Pro Gly Gly Lys Arg 195 200 205 Thr Pro Lys Ala Lys Glu Lys Arg Asn Lys Lys Lys Lys Arg Lys Leu 210 215 220 Ile Glu Arg Ala Gln Glu Gln His Ser Val Phe Leu Ala Thr Asp Arg 225 230 235 240 Ala Asn Gln <210> 12 <211> 272 <212> PRT <213> Homo sapiens <400> 12 Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met 1 5 10 15 Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30 Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45 Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60 Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys 65 70 75 80 Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95 Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105 110 Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125 Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135 140 Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr 145 150 155 160 Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val 165 170 175 Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190 Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205 Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215 220 Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser 225 230 235 240 Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys 245 250 255 Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His 260 265 270 <210> 13 <211> 234 <212> PRT <213> Homo sapiens <400> 13 Met Arg Ala Pro Leu Cys Leu Leu Leu Leu Val Ala His Ala Val Asp 1 5 10 15 Met Leu Ala Leu Asn Arg Arg Lys Lys Gln Val Gly Thr Gly Leu Gly 20 25 30 Gly Asn Cys Thr Gly Cys Ile Ile Cys Ser Glu Glu Asn Gly Cys Ser 35 40 45 Thr Cys Gln Gln Arg Leu Phe Leu Phe Ile Arg Arg Glu Gly Ile Arg 50 55 60 Gln Tyr Gly Lys Cys Leu His Asp Cys Pro Pro Gly Tyr Phe Gly Ile 65 70 75 80 Arg Gly Gln Glu Val Asn Arg Cys Lys Lys Cys Gly Ala Thr Cys Glu 85 90 95 Ser Cys Phe Ser Gln Asp Phe Cys Ile Arg Cys Lys Arg Gln Phe Tyr 100 105 110 Leu Tyr Lys Gly Lys Cys Leu Pro Thr Cys Pro Pro Gly Thr Leu Ala 115 120 125 His Gln Asn Thr Arg Glu Cys Gln Gly Glu Cys Glu Leu Gly Pro Trp 130 135 140 Gly Gly Trp Ser Pro Cys Thr His Asn Gly Lys Thr Cys Gly Ser Ala 145 150 155 160 Trp Gly Leu Glu Ser Arg Val Arg Glu Ala Gly Arg Ala Gly His Glu 165 170 175 Glu Ala Ala Thr Cys Gln Val Leu Ser Glu Ser Arg Lys Cys Pro Ile 180 185 190 Gln Arg Pro Cys Pro Gly Glu Arg Ser Pro Gly Gln Lys Lys Gly Arg 195 200 205 Lys Asp Arg Arg Pro Arg Lys Asp Arg Lys Leu Asp Arg Arg Leu Asp 210 215 220 Val Arg Pro Arg Gln Pro Gly Leu Gln Pro 225 230 <210> 14 <211> 172 <212> PRT <213> Homo sapiens <400> 14 Met Arg Ala Pro Leu Cys Leu Leu Leu Leu Val Ala His Ala Val Asp 1 5 10 15 Met Leu Ala Leu Asn Arg Arg Lys Lys Gln Val Gly Thr Gly Leu Gly 20 25 30 Gly Asn Cys Thr Gly Cys Ile Ile Cys Ser Glu Glu Asn Gly Cys Ser 35 40 45 Thr Cys Gln Gln Arg Leu Phe Leu Phe Ile Arg Arg Glu Gly Ile Arg 50 55 60 Gln Tyr Gly Lys Cys Leu His Asp Cys Pro Pro Gly Tyr Phe Gly Ile 65 70 75 80 Arg Gly Gln Glu Val Asn Arg Cys Lys Lys Cys Gly Ala Thr Cys Glu 85 90 95 Ser Cys Phe Ser Gln Asp Phe Cys Ile Arg Cys Lys Arg Gln Phe Tyr 100 105 110 Leu Tyr Lys Gly Lys Cys Leu Pro Thr Cys Pro Pro Gly Thr Leu Ala 115 120 125 His Gln Asn Thr Arg Glu Cys Gln Glu Arg Ser Pro Gly Gln Lys Lys 130 135 140 Gly Arg Lys Asp Arg Arg Pro Arg Lys Asp Arg Lys Leu Asp Arg Arg 145 150 155 160 Leu Asp Val Arg Pro Arg Gln Pro Gly Leu Gln Pro 165 170 <210> 15 <211> 133 <212> PRT <213> Homo sapiens <400> 15 Met Arg Lys His Val Leu Ala Ala Ser Phe Ser Met Leu Ser Leu Leu 1 5 10 15 Val Ile Met Gly Asp Thr Asp Ser Lys Thr Asp Ser Ser Phe Ile Met 20 25 30 Asp Ser Asp Pro Arg Arg Cys Met Arg His His Tyr Val Asp Ser Ile 35 40 45 Ser His Pro Leu Tyr Lys Cys Ser Ser Lys Met Val Leu Leu Ala Arg 50 55 60 Cys Glu Gly His Cys Ser Gln Ala Ser Arg Ser Glu Pro Leu Val Ser 65 70 75 80 Phe Ser Thr Val Leu Lys Gln Pro Phe Arg Ser Ser Cys His Cys Cys 85 90 95 Arg Pro Gln Thr Ser Lys Leu Lys Ala Leu Arg Leu Arg Cys Ser Gly 100 105 110 Gly Met Arg Leu Thr Ala Thr Tyr Arg Tyr Ile Leu Ser Cys His Cys 115 120 125 Glu Glu Cys Asn Ser 130 <210> 16 <211> 352 <212> PRT <213> Homo sapiens <400> 16 Met Ala Pro Leu Gly Tyr Phe Leu Leu Leu Cys Ser Leu Lys Gln Ala 1 5 10 15 Leu Gly Ser Tyr Pro Ile Trp Trp Ser Leu Ala Val Gly Pro Gln Tyr 20 25 30 Ser Ser Leu Gly Ser Gln Pro Ile Leu Cys Ala Ser Ile Pro Gly Leu 35 40 45 Val Pro Lys Gln Leu Arg Phe Cys Arg Asn Tyr Val Glu Ile Met Pro 50 55 60 Ser Val Ala Glu Gly Ile Lys Ile Gly Ile Gln Glu Cys Gln His Gln 65 70 75 80 Phe Arg Gly Arg Arg Trp Asn Cys Thr Thr Val His Asp Ser Leu Ala 85 90 95 Ile Phe Gly Pro Val Leu Asp Lys Ala Thr Arg Glu Ser Ala Phe Val 100 105 110 His Ala Ile Ala Ser Ala Gly Val Ala Phe Ala Val Thr Arg Ser Cys 115 120 125 Ala Glu Gly Thr Ala Ala Ile Cys Gly Cys Ser Ser Arg His Gln Gly 130 135 140 Ser Pro Gly Lys Gly Trp Lys Trp Gly Gly Cys Ser Glu Asp Ile Glu 145 150 155 160 Phe Gly Gly Met Val Ser Arg Glu Phe Ala Asp Ala Arg Glu Asn Arg 165 170 175 Pro Asp Ala Arg Ser Ala Met Asn Arg His Asn Asn Glu Ala Gly Arg 180 185 190 Gln Ala Ile Ala Ser His Met His Leu Lys Cys Lys Cys His Gly Leu 195 200 205 Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Trp Ser Gln Pro Asp Phe 210 215 220 Arg Ala Ile Gly Asp Phe Leu Lys Asp Lys Tyr Asp Ser Ala Ser Glu 225 230 235 240 Met Val Val Glu Lys His Arg Glu Ser Arg Gly Trp Val Glu Thr Leu 245 250 255 Arg Pro Arg Tyr Thr Tyr Phe Lys Val Pro Thr Glu Arg Asp Leu Val 260 265 270 Tyr Tyr Glu Ala Ser Pro Asn Phe Cys Glu Pro Asn Pro Glu Thr Gly 275 280 285 Ser Phe Gly Thr Arg Asp Arg Thr Cys Asn Val Ser Ser His Gly Ile 290 295 300 Asp Gly Cys Asp Leu Leu Cys Cys Gly Arg Gly His Asn Ala Arg Ala 305 310 315 320 Glu Arg Arg Arg Glu Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr 325 330 335 Val Ser Cys Gln Glu Cys Thr Arg Val Tyr Asp Val His Thr Cys Lys 340 345 350 <210> 17 <211> 338 <212> PRT <213> Homo sapiens <400> 17 Ala Val Gly Ser Pro Leu Val Met Asp Pro Thr Ser Ile Cys Arg Lys 1 5 10 15 Ala Arg Arg Leu Ala Gly Arg Gln Ala Glu Leu Cys Gln Ala Glu Pro 20 25 30 Glu Val Val Ala Glu Leu Ala Arg Gly Ala Arg Leu Gly Val Arg Glu 35 40 45 Cys Gln Phe Gln Phe Arg Phe Arg Arg Trp Asn Cys Ser Ser His Ser 50 55 60 Lys Ala Phe Gly Arg Ile Leu Gln Gln Asp Ile Arg Glu Thr Ala Phe 65 70 75 80 Val Phe Ala Ile Thr Ala Ala Gly Ala Ser His Ala Val Thr Gln Ala 85 90 95 Cys Ser Met Gly Glu Leu Leu Gln Cys Gly Cys Gln Ala Pro Arg Gly 100 105 110 Arg Ala Pro Pro Arg Pro Ser Gly Leu Pro Gly Thr Pro Gly Pro Pro 115 120 125 Gly Pro Ala Gly Ser Pro Glu Gly Ser Ala Ala Trp Glu Trp Gly Gly 130 135 140 Cys Gly Asp Asp Val Asp Phe Gly Asp Glu Lys Ser Arg Leu Phe Met 145 150 155 160 Asp Ala Arg His Lys Arg Gly Arg Gly Asp Ile Arg Ala Leu Val Gln 165 170 175 Leu His Asn Asn Glu Ala Gly Arg Leu Ala Val Arg Ser His Thr Arg 180 185 190 Thr Glu Cys Lys Cys His Gly Leu Ser Gly Ser Cys Ala Leu Arg Thr 195 200 205 Cys Trp Gln Lys Leu Pro Pro Phe Arg Glu Val Gly Ala Arg Leu Leu 210 215 220 Glu Arg Phe His Gly Ala Ser Arg Val Met Gly Thr Asn Asp Gly Lys 225 230 235 240 Ala Leu Leu Pro Ala Val Arg Thr Leu Lys Pro Pro Gly Arg Ala Asp 245 250 255 Leu Leu Tyr Ala Ala Asp Ser Pro Asp Phe Cys Ala Pro Asn Arg Arg 260 265 270 Thr Gly Ser Pro Gly Thr Arg Gly Arg Ala Cys Asn Ser Ser Ala Pro 275 280 285 Asp Leu Ser Gly Cys Asp Leu Leu Cys Cys Gly Arg Gly His Arg Gln 290 295 300 Glu Ser Val Gln Leu Glu Glu Asn Cys Leu Cys Arg Phe His Trp Cys 305 310 315 320 Cys Val Val Gln Cys His Arg Cys Arg Val Arg Lys Glu Leu Ser Leu 325 330 335 Cys Leu <210> 18 <211> 288 <212> PRT <213> Homo sapiens <400> 18 Met Val Gly Val Gly Gly Gly Asp Val Glu Asp Val Thr Pro Arg Pro 1 5 10 15 Gly Gly Cys Gln Ile Ser Gly Arg Gly Ala Arg Gly Cys Asn Gly Ile 20 25 30 Pro Gly Ala Ala Ala Trp Glu Ala Ala Leu Pro Arg Arg Arg Pro Arg 35 40 45 Arg His Pro Ser Val Asn Pro Arg Ser Arg Ala Ala Gly Ser Pro Arg 50 55 60 Thr Arg Gly Arg Arg Thr Glu Glu Arg Pro Ser Gly Ser Arg Leu Gly 65 70 75 80 Asp Arg Gly Arg Gly Arg Ala Leu Pro Gly Gly Arg Leu Gly Gly Arg 85 90 95 Gly Arg Gly Arg Ala Pro Glu Arg Val Gly Gly Arg Gly Arg Gly Arg 100 105 110 Gly Thr Ala Ala Pro Arg Ala Ala Pro Ala Ala Arg Gly Ser Arg Pro 115 120 125 Gly Pro Ala Gly Thr Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala 130 135 140 Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys 145 150 155 160 Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile 165 170 175 His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His 180 185 190 Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys 195 200 205 Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu 210 215 220 Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu 225 230 235 240 Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp 245 250 255 Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr 260 265 270 Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 275 280 285 <210> 19 <211> 194 <212> PRT <213> Homo sapiens <400> 19 Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr <210> 20 <211> 208 <212> PRT <213> Homo sapiens <400> 20 Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Val Met Val Ser Pro Glu 35 40 45 Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100 105 110 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 <210> 21 <211> 1207 <212> PRT <213> Homo sapiens <400> 21 Met Leu Leu Thr Leu Ile Ile Leu Leu Pro Val Val Ser Lys Phe Ser 1 5 10 15 Phe Val Ser Leu Ser Ala Pro Gln His Trp Ser Cys Pro Glu Gly Thr 20 25 30 Leu Ala Gly Asn Gly Asn Ser Thr Cys Val Gly Pro Ala Pro Phe Leu 35 40 45 Ile Phe Ser His Gly Asn Ser Ile Phe Arg Ile Asp Thr Glu Gly Thr 50 55 60 Asn Tyr Glu Gln Leu Val Val Asp Ala Gly Val Ser Val Ile Met Asp 65 70 75 80 Phe His Tyr Asn Glu Lys Arg Ile Tyr Trp Val Asp Leu Glu Arg Gln 85 90 95 Leu Leu Gln Arg Val Phe Leu Asn Gly Ser Arg Gln Glu Arg Val Cys 100 105 110 Asn Ile Glu Lys Asn Val Ser Gly Met Ala Ile Asn Trp Ile Asn Glu 115 120 125 Glu Val Ile Trp Ser Asn Gln Gln Glu Gly Ile Ile Thr Val Thr Asp 130 135 140 Met Lys Gly Asn Asn Ser His Ile Leu Leu Ser Ala Leu Lys Tyr Pro 145 150 155 160 Ala Asn Val Ala Val Asp Pro Val Glu Arg Phe Ile Phe Trp Ser Ser 165 170 175 Glu Val Ala Gly Ser Leu Tyr Arg Ala Asp Leu Asp Gly Val Gly Val 180 185 190 Lys Ala Leu Leu Glu Thr Ser Glu Lys Ile Thr Ala Val Ser Leu Asp 195 200 205 Val Leu Asp Lys Arg Leu Phe Trp Ile Gln Tyr Asn Arg Glu Gly Ser 210 215 220 Asn Ser Leu Ile Cys Ser Cys Asp Tyr Asp Gly Gly Ser Val His Ile 225 230 235 240 Ser Lys His Pro Thr Gln His Asn Leu Phe Ala Met Ser Leu Phe Gly 245 250 255 Asp Arg Ile Phe Tyr Ser Thr Trp Lys Met Lys Thr Ile Trp Ile Ala 260 265 270 Asn Lys His Thr Gly Lys Asp Met Val Arg Ile Asn Leu His Ser Ser 275 280 285 Phe Val Pro Leu Gly Glu Leu Lys Val Val His Pro Leu Ala Gln Pro 290 295 300 Lys Ala Glu Asp Asp Thr Trp Glu Pro Glu Gln Lys Leu Cys Lys Leu 305 310 315 320 Arg Lys Gly Asn Cys Ser Ser Thr Val Cys Gly Gln Asp Leu Gln Ser 325 330 335 His Leu Cys Met Cys Ala Glu Gly Tyr Ala Leu Ser Arg Asp Arg Lys 340 345 350 Tyr Cys Glu Asp Val Asn Glu Cys Ala Phe Trp Asn His Gly Cys Thr 355 360 365 Leu Gly Cys Lys Asn Thr Pro Gly Ser Tyr Tyr Cys Thr Cys Pro Val 370 375 380 Gly Phe Val Leu Leu Pro Asp Gly Lys Arg Cys His Gln Leu Val Ser 385 390 395 400 Cys Pro Arg Asn Val Ser Glu Cys Ser His Asp Cys Val Leu Thr Ser 405 410 415 Glu Gly Pro Leu Cys Phe Cys Pro Glu Gly Ser Val Leu Glu Arg Asp 420 425 430 Gly Lys Thr Cys Ser Gly Cys Ser Ser Pro Asp Asn Gly Gly Cys Ser 435 440 445 Gln Leu Cys Val Pro Leu Ser Pro Val Ser Trp Glu Cys Asp Cys Phe 450 455 460 Pro Gly Tyr Asp Leu Gln Leu Asp Glu Lys Ser Cys Ala Ala Ser Gly 465 470 475 480 Pro Gln Pro Phe Leu Leu Phe Ala Asn Ser Gln Asp Ile Arg His Met 485 490 495 His Phe Asp Gly Thr Asp Tyr Gly Thr Leu Leu Ser Gln Gln Met Gly 500 505 510 Met Val Tyr Ala Leu Asp His Asp Pro Val Glu Asn Lys Ile Tyr Phe 515 520 525 Ala His Thr Ala Leu Lys Trp Ile Glu Arg Ala Asn Met Asp Gly Ser 530 535 540 Gln Arg Glu Arg Leu Ile Glu Glu Gly Val Asp Val Pro Glu Gly Leu 545 550 555 560 Ala Val Asp Trp Ile Gly Arg Arg Phe Tyr Trp Thr Asp Arg Gly Lys 565 570 575 Ser Leu Ile Gly Arg Ser Asp Leu Asn Gly Lys Arg Ser Lys Ile Ile 580 585 590 Thr Lys Glu Asn Ile Ser Gln Pro Arg Gly Ile Ala Val His Pro Met 595 600 605 Ala Lys Arg Leu Phe Trp Thr Asp Thr Gly Ile Asn Pro Arg Ile Glu 610 615 620 Ser Ser Ser Leu Gln Gly Leu Gly Arg Leu Val Ile Ala Ser Ser Asp 625 630 635 640 Leu Ile Trp Pro Ser Gly Ile Thr Ile Asp Phe Leu Thr Asp Lys Leu 645 650 655 Tyr Trp Cys Asp Ala Lys Gln Ser Val Ile Glu Met Ala Asn Leu Asp 660 665 670 Gly Ser Lys Arg Arg Arg Leu Thr Gln Asn Asp Val Gly His Pro Phe 675 680 685 Ala Val Ala Val Phe Glu Asp Tyr Val Trp Phe Ser Asp Trp Ala Met 690 695 700 Pro Ser Val Met Arg Val Asn Lys Arg Thr Gly Lys Asp Arg Val Arg 705 710 715 720 Leu Gln Gly Ser Met Leu Lys Pro Ser Ser Leu Val Val Val His Pro 725 730 735 Leu Ala Lys Pro Gly Ala Asp Pro Cys Leu Tyr Gln Asn Gly Gly Cys 740 745 750 Glu His Ile Cys Lys Lys Arg Leu Gly Thr Ala Trp Cys Ser Cys Arg 755 760 765 Glu Gly Phe Met Lys Ala Ser Asp Gly Lys Thr Cys Leu Ala Leu Asp 770 775 780 Gly His Gln Leu Leu Ala Gly Gly Glu Val Asp Leu Lys Asn Gln Val 785 790 795 800 Thr Pro Leu Asp Ile Leu Ser Lys Thr Arg Val Ser Glu Asp Asn Ile 805 810 815 Thr Glu Ser Gln His Met Leu Val Ala Glu Ile Met Val Ser Asp Gln 820 825 830 Asp Asp Cys Ala Pro Val Gly Cys Ser Met Tyr Ala Arg Cys Ile Ser 835 840 845 Glu Gly Glu Asp Ala Thr Cys Gln Cys Leu Lys Gly Phe Ala Gly Asp 850 855 860 Gly Lys Leu Cys Ser Asp Ile Asp Glu Cys Glu Met Gly Val Pro Val 865 870 875 880 Cys Pro Pro Ala Ser Ser Lys Cys Ile Asn Thr Glu Gly Gly Tyr Val 885 890 895 Cys Arg Cys Ser Glu Gly Tyr Gln Gly Asp Gly Ile His Cys Leu Asp 900 905 910 Ile Asp Glu Cys Gln Leu Gly Glu His Ser Cys Gly Glu Asn Ala Ser 915 920 925 Cys Thr Asn Thr Glu Gly Gl...

Claims

1. 1. A defined culture medium for isolating columnar epithelial stem cells and stably maintaining their epigenetics over multiple passages in culture, comprising:

1. A defined culture medium that supports epigenetically stable growth and proliferation of epithelial stem cells of columnar tissue origin in the presence of co-cultured feeder cells, the defined culture medium comprising a ROCK (Rho kinase) inhibitor; a Wnt agonist R-spondin1; an epidermal growth factor (EGF); insulin (or an insulin mimetic) or IGF; an agent that selectively inhibits the biological activity of wild-type BRAF or mutant BRAF; a VEGF receptor inhibitor; nicotinamide; a Notch agonist Jagged-1, a TGFβ receptor inhibitor; and a bone morphogenetic protein (BMP) antagonist Noggin. or （１） 1. A method for isolating epithelial stem cells from columnar epithelial tissue, comprising: (i) culturing dissociated epithelial cells derived from a columnar epithelial tissue sample to form epithelial stem cell colonies, wherein the dissociated cells and cell colonies are cultured in a medium comprising a ROCK (Rho kinase) inhibitor, a Wnt agonist R-spondin 1, epidermal growth factor (EGF), insulin (or an insulin mimetic) or IGF, an agent that selectively inhibits the biological activity of wild-type or mutant BRAF, a VEGF receptor inhibitor, nicotinamide, a Notch agonist Jagged-1, a TGFβ receptor inhibitor, and a bone morphogenetic protein (BMP) antagonist Noggin; (ii) isolating a single epithelial stem cell from the cell colony; and (iii) individually culturing the isolated single epithelial stem cells from step (ii) to form a culture of purified epithelial stem cell clones, each of the epithelial stem cell clones represents a clonal expansion of epithelial stem cells present in the columnar epithelial tissue sample; thereby isolating epithelial stem cells; （２） The method of (1), wherein cells derived from a columnar epithelial tissue sample are in fluid or direct contact with division-inactive feeder cells; （３） The method of (1) or (2), wherein the cells derived from the columnar epithelial tissue sample are in contact with an extracellular matrix or a synthetic matrix; （４） The method of (1), wherein the medium further comprises an agent capable of activating transcription of a gene driven by an Oct4 promoter, a selective PDGFRα / β inhibitor, and a selective JNK inhibitor that inhibits phosphorylation of c-Jun. （５） The method of (4), wherein the medium does not contain feeder cells; （６） The method of (4) or (5), wherein the cells derived from the columnar epithelial tissue sample are in contact with a biomatrix (such as an extracellular matrix) or a synthetic matrix; （７） Any one of the methods (1) to (6), wherein the epithelial stem cells are isolated from a tissue sample taken from normal columnar epithelial tissue; （８） Any one of the methods of (1) to (6), wherein the epithelial stem cells are isolated from a tissue sample taken from a diseased columnar epithelial tissue, such as from an inflammatory or autoimmune patient; and （９） Any one of the methods (1) to (6), wherein the epithelial stem cells are isolated from a columnar epithelial tissue sample taken from a tumor. by any one method selected from the group consisting of The in vitro use of epithelial stem cells or their progeny isolated from diseased columnar epithelial tissue to identify agents that selectively inhibit the growth or proliferation of epithelial stem cells or their progeny compared to normal regenerative epithelial stem cells, or that restore epithelial stem cells to a normal epigenetic state that will differentiate into normal epithelial tissue.

2. 2. The use of claim 1, wherein the diseased columnar epithelial tissue is derived from a patient with an inflammatory disease or a tumor.

3. 1. A defined culture medium for isolating columnar epithelial stem cells and stably maintaining their epigenetics over multiple passages in culture, comprising:

1. A defined culture medium that supports epigenetically stable growth and proliferation of epithelial stem cells of columnar tissue origin in the presence of co-cultured feeder cells, the defined culture medium comprising a ROCK (Rho kinase) inhibitor; a Wnt agonist R-spondin1; an epidermal growth factor (EGF); insulin (or an insulin mimetic) or IGF; an agent that selectively inhibits the biological activity of wild-type BRAF or mutant BRAF; a VEGF receptor inhibitor; nicotinamide; a Notch agonist Jagged-1, a TGFβ receptor inhibitor; and a bone morphogenetic protein (BMP) antagonist Noggin. or （１） 1. A method for isolating epithelial stem cells from columnar epithelial tissue, comprising: (i) culturing dissociated epithelial cells derived from a columnar epithelial tissue sample to form epithelial stem cell colonies, wherein the dissociated cells and cell colonies are cultured in a medium comprising a ROCK (Rho kinase) inhibitor, a Wnt agonist R-spondin 1, epidermal growth factor (EGF), insulin (or an insulin mimetic) or IGF, an agent that selectively inhibits the biological activity of wild-type or mutant BRAF, a VEGF receptor inhibitor, nicotinamide, a Notch agonist Jagged-1, a TGFβ receptor inhibitor, and a bone morphogenetic protein (BMP) antagonist Noggin; (ii) isolating a single epithelial stem cell from the cell colony; and (iii) individually culturing the isolated single epithelial stem cells from step (ii) to form a culture of purified epithelial stem cell clones, each of the epithelial stem cell clones represents a clonal expansion of epithelial stem cells present in the columnar epithelial tissue sample; thereby isolating epithelial stem cells; （２） The method of (1), wherein cells derived from a columnar epithelial tissue sample are in fluid or direct contact with division-inactive feeder cells; （３） The method of (1) or (2), wherein the cells derived from the columnar epithelial tissue sample are in contact with an extracellular matrix or a synthetic matrix; （４） The method of (1), wherein the medium further comprises an agent capable of activating transcription of a gene driven by an Oct4 promoter, a selective PDGFRα / β inhibitor, and a selective JNK inhibitor that inhibits phosphorylation of c-Jun. （５） The method of (4), wherein the medium does not contain feeder cells; （６） The method of (4) or (5), wherein the cells derived from the columnar epithelial tissue sample are in contact with a biomatrix (such as an extracellular matrix) or a synthetic matrix; （７） Any one of the methods (1) to (6), wherein the epithelial stem cells are isolated from a tissue sample taken from normal columnar epithelial tissue; （８） Any one of the methods of (1) to (6), wherein the epithelial stem cells are isolated from a tissue sample taken from a diseased columnar epithelial tissue, such as from an inflammatory or autoimmune patient; and （９） Any one of the methods (1) to (6), wherein the epithelial stem cells are isolated from a columnar epithelial tissue sample taken from a tumor. by any one method selected from the group consisting of 1. The in vitro use of epithelial stem cells or their progeny isolated from normal columnar epithelial tissue for the identification of agents that promote the growth, proliferation, and / or regenerative capacity of epithelial stem cells.

4. The use of any one of claims 1 to 3, wherein the identified agent is formulated for administration to a mammalian subject.

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