Culture medium for stem cells

Abstract

To provide a culture medium for expanding and differentiating a stem cell population and for obtaining an organoid.SOLUTION: The culture medium for expanding and / or differentiating an adult stem cell population comprises i. any one of R-spondin and / or an R-spondin mimetic and ii. one or more inhibitors that directly or indirectly negatively regulate TGF-β signal transduction. The inhibitor binds to one or more serine / threonine protein kinases selected from ALK 5, ALK 4, TGF-β receptor kinase 1 and ALK 7, the activity of which is reduced by the culture medium. The culture medium further comprises one or more additional components selected from BMP inhibitors, Wnt agonists, receptor tyrosine kinase ligands, Bock inhibitors, nicotinamide and gastrin.SELECTED DRAWING: None

Description

Technical Field

[0001] All documents cited in this specification are incorporated herein by reference in their entirety.

[0002] Technical Field The present invention is in the field of culture media and methods for culturing stem cells, and more particularly, culture media and methods for expanding populations of stem cells, such as human epithelial stem cells.

Background Art

[0003] Background There is a high interest in culture media and methods for expanding stem cell populations. Stem cell populations have many uses. For example, stem cells and their differentiated progeny can be used in cell assays, drug screening, and toxicity assays. Stem cells also show the potential for cell therapy, such as in regenerative medicine for treating damaged tissues. Stem cells can also serve as a source of differentiated cells for transplantation purposes, for example, for pancreatic β-cell transplantation for the treatment of diabetes. Furthermore, efficient cell culture media are important for providing and maintaining cell populations for research purposes.

[0004] There is also a high interest in culture media and methods for culturing stem cells for the formation, maintenance, and expansion of organoids such as intestinal crypt-villus organoids, gastric organoids, or pancreatic organoids. Organoids contain stem cells that retain an undifferentiated phenotype and self-renewal ability, such as epithelial stem cells, but also have differentiating progeny that grow into tissue-like structures. Similar to a population consisting of related or identical cells, intestinal crypt-villus organoids, gastric organoids, or pancreatic organoids more closely mimic the basic physiological functions of the tissue from which they are derived and can be used in toxicity assays or assays of drugs or nutraceuticals. Such organoids may also be useful for culturing pathogens for which appropriate tissue culture or animal models are currently unavailable. Furthermore, such organoids may be useful in regenerative medicine, for example, in intestinal epithelial repair after radiation and / or surgery, or in intestinal epithelial repair in patients suffering from inflammatory bowel disease.

[0005] It is clear that stem cells and their differentiated progeny have many clinical and research applications. For all of these applications, a reproducible stem cell culture method is of utmost importance to provide an adequate number of cells of appropriate quality. For example, in the case of effective drug screening, precise culture methods are required to control cell differentiation and proliferation so that a pure population of cells with the same phenotype and karyotype can be generated, and the conditions must be carefully controlled. Similarly, in the case of cell therapy where cultured cells can be directly provided to a patient, the cells must be genetically and phenotypically correct to avoid unwanted immune responses or cell fates when provided to the patient.

[0006] Various culture systems for culturing primary epithelial stem cells, including intestinal epithelial stem cells, have been described (Bjerknes and Cheng, 2006. Methods Enzymol. 419: 337-83 (Non-Patent Document 1)), but a long-term culture system that maintains the differentiation ability and phenotypic and genomic integrity of human epithelial stem cells has not been established to date.

[0007] International Patent Application WO2010 / 090513 (Patent Document 1) discloses a method for culturing epithelial stem cells or isolated tissue fragments. This method is optimized for culturing human colon and intestinal crypts by adding Wnt-3a to the culture medium. This was the first event where human intestinal stem cell cultures were cultured for a long period (up to 3 months), providing the first reproducible human intestinal stem cell culture system. However, there is still a need for improved culture media and methods for culturing stem cells, particularly human stem cells, that improve the growth rate, survival time, and phenotypic and genomic integrity of stem cells grown in culture.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

[0010] The present invention provides improved culture media and methods for stem cells, particularly human epithelial stem cells, and organoids containing such stem cells, which have extremely advantageous points over known culture media and methods. The present invention also provides related culture medium supplements, compositions, and uses.

[0011] Accordingly, the present invention provides a culture medium for expanding a stem cell population, comprising at least one or more inhibitors that bind to one or more serine / threonine protein kinase targets and reduce their activity. This has the effect of enabling continuous growth for at least 3 months at an expansion rate of approximately 5-fold per week. The serine / threonine protein kinase is preferably selected from the group comprising TGFβ receptor kinase 1, ALK4, ALK5, ALK7, p38. Surprisingly, the inventors have discovered that by including an inhibitor of a specific serine / threonine kinase in the culture medium, the performance of the culture medium in expanding the stem cell population has been significantly improved. The stem cell population may be normal (healthy) cells or diseased cells (e.g., cancer stem cells). Specifically, it has been shown that inhibitors of p38 and ALK among all the compounds tested provide the greatest improvement. Since there is no known mechanism for predicting how these specific inhibitors can act, this is unexpected. In fact, some of the small molecule inhibitors selected for testing and functioning in similar pathways did not affect the said method. Therefore, even those skilled in the art could not have predicted that inhibitors of these specific kinases would improve the culture medium so significantly. Even further improvement was observed when two inhibitors, for example, a p38 inhibitor such as SB202190 and an ALK inhibitor such as A83-01, were added together to the culture medium.

[0012] To arrive at this recognition, the inventors examined signal transduction pathways known to be disrupted in a particular cancer, for example, colorectal cancer. The inventors hypothesized that these pathways, which affect cell fate in cancer, may also play a role in determining cell fate under culture conditions. However, it must be emphasized that this hypothesis was entirely new. Considering the state of the art, there was no method for predicting the effect of these additional compounds on the culture medium, nor any prediction that these compounds might actually have a beneficial effect.

[0013] In the first screening experiment, a series of vitamins, hormones, and growth factors were tested in combination with a standard stem cell culture medium. First, gastrin and nicotinamide were identified as those that brought about significantly improved culture conditions. These factors were incorporated into the standard culture conditions to conduct a second screening experiment. In the second screening experiment, small molecule inhibitors related to relevant signaling pathways such as ERK, p38, JNK, PTEN, ROCK, and Hedgehog were tested. These pathways were selected because they are known to be disrupted in certain cancers.

[0014] Previous attempts to culture human intestinal stem cells using a previously described stem cell medium (containing epidermal growth factor (EGF or ("E"), Noggin ("N"), and R-spondin ("R")), optimized with Wnt-3A ("W") (referred to herein as "WENR" medium) resulted in most cells being degraded within 7 days and few cells surviving beyond 1 month. Such attempts were also prone to slow growth times, chromosomal abnormalities, and morphological changes from budding to cystic structures. "Cyst" means that the organoid is mainly spherical. "Budding" means that the organoid has multiple regions growing from the basic structure. Having a budding structure is not always an advantage, but budding structures typically have a large surface area and typically closely resemble the corresponding in vivo tissue.

[0015] The inventors have shown that the improved method enables continuous growth of stem cells for at least 7 months.

[0016] This new method also increased the growth rate of cells in the expanded population. This is clearly very useful when growing cells for commercial and therapeutic purposes.

[0017] This new method also enhanced the quality of the cells in the expanded population. This is a significant advantage because a reproducible stem cell culture method that provides a high-quality cell population is required for applying stem cells and their differentiated progeny to clinical and research settings. Generally, in vitro expansion of stem cells aims to provide a cell population that closely resembles its in vivo counterpart as much as possible. This property is referred to herein as "genomic and phenotypic integrity" of the cells.

[0018] For the first time, the inventors discovered that human epithelial stem cells can be expanded in culture for at least 7 months without losing genomic and phenotypic integrity (see Example 1). Under the improved culture conditions of the present invention, human intestinal organoids showed a budding organoid structure instead of the cystic structure seen under previous culture conditions. Metaphase spreads of organoids over 3 months consistently revealed 46 chromosomes in each of 20 cells collected from three different donors. Furthermore, microarray analysis revealed that the stem cells in culture have a molecular signature similar to that of intestinal crypt cells, including intestinal stem cell genes.

[0019] The inventors also demonstrated that human intestinal organoids produced by the media and methods of the present invention mimicked in vivo cell fate determination in response to external factors. For example, it has previously been shown that Notch inhibition in intestinal stem cells terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. The inventors were also able to show that the intestinal organoids of the present invention terminated proliferation when treated with a Notch inhibitor, and most cells changed into goblet cells within 3 days.

[0020] Similar advantages were observed when TGF-β inhibitors and / or p38 inhibitors were included in the culture medium for expanding stem cells or organoids derived from other epithelial tissues such as the stomach, pancreas, liver, and prostate (see Examples). The tissue may be normal (healthy) tissue or diseased tissue, such as cancer tissue or tissue showing a cystic fibrosis phenotype.

[0021] From these results, it can be seen that the genomic integrity and phenotypic integrity of the stem cells and organoids generated by the method and medium of the present invention are dramatically improved compared to previous methods and media.

[0022] Accordingly, the present invention provides i. any one of R-spondin 1 to 4 and / or an R-spondin mimetic; and ii. one or more inhibitors that directly or indirectly negatively regulate TGF-β signaling and provides a culture medium for expanding and / or differentiating adult stem cell populations.

[0023] The present invention also provides a composition comprising a culture medium according to the present invention and a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as the extracellular matrix or integrin, for example, a laminin-containing extracellular matrix such as Matrigel™ (BD Biosciences).

[0024] The present invention also provides a hermetically sealed container containing a culture medium or composition according to the present invention.

[0025] The present invention also provides the use of a culture medium according to the present invention for expanding and / or differentiating stem cells, stem cell populations, tissue fragments, or organoids.

[0026] The present invention also provides a method for expanding a single stem cell, stem cell population, or tissue fragment, preferably for expanding them to produce organoids, the method comprising culturing a single stem cell or stem cell population in a culture medium according to the present invention.

[0027] The present invention also provides organoids or cell populations obtainable by the method of the present invention.

[0028] The present invention also provides a three-dimensional organoid comprising epithelial cells surrounding a central lumen, optionally wherein the epithelial cells are present in distinct proliferation and differentiation regions, preferably an organoid obtainable by the method of the present invention.

[0029] The present invention also provides a three-dimensional organoid, preferably a three-dimensional organoid comprising epithelial cells surrounding a central lumen, optionally wherein the epithelial cells are present in distinct proliferation and differentiation regions, preferably an organoid obtainable by the method of the present invention, which comprises epithelial cells arranged in a monolayer, optionally regions of folded monolayer and regions of multi-layered cells.

[0030] The present invention also provides i) one or more organoids or cell populations of the present invention; and ii) a culture medium and / or extracellular matrix of the present invention in a composition.

[0031] The present invention also provides an organoid, cell population, or composition according to the present invention for use in drug screening, target validation, target discovery, toxicology, toxicity screening, personalized medicine, regenerative medicine, or ex vivo cell / organ models, for example for use as a disease model.

[0032] The present invention also provides an organoid, cell population, or composition according to the present invention for use in transplantation of an organoid, cell population, or composition into a mammal, preferably a human.

[0033] The present invention also provides a stem cell population or an organoid containing the stem cells obtained using the culture medium of the present invention or that can be obtained using the culture medium of the present invention. The stem cells or the organoid containing the stem cells may be used, for example, for transplantation purposes or other therapeutic applications. For example, the stem cells or the organoid containing the stem cells may be used for drug screening, target validation, target discovery, toxicology and toxicity screening, personalized medicine, regenerative medicine, and ex vivo cell / organ models, such as disease models.

[0034] The present invention also provides a composition containing the culture medium of the present invention.

[0035] The present invention also provides a culture medium supplement containing an inhibitor according to the present invention.

[0036] The present invention also provides a hermetically sealed container containing the culture medium and / or culture medium supplement according to the present invention.

[0037] Depending on specific needs and applications, specific components of the culture medium, supplement, and composition of the present invention can be modified. Similarly, the exact steps of the method of the present invention may vary depending on specific needs and applications.

[0038] The culture medium, supplement, method, composition, and use according to the present invention may also be optimized by routine experiments. For example, if the culture medium, supplement, or composition does not result in the desired level of stem cell expansion, variables such as the amount of each component in the culture medium or supplement, seeding density, culture conditions, culture period, etc. can be changed in further experiments. The amount of each component described herein may be optimized independently of other components by routine optimization, and one or more components may be added or removed. The ability of the medium to support the expansion of stem cells can be tested by conducting tests in parallel with known culture media or methods or instead of known culture media or methods.

[0039] The culture media, supplements, methods, compositions, and uses of the present invention are described in more detail below. The practice of the present invention, unless otherwise specified, uses conventional techniques of cell culture, molecular biology, and microbiology within the skill of the art.

[0040] A number of textbooks can be used as guides for culturing mammalian cells, including textbooks specializing in culture media and methods for culturing stem cells. Such textbooks include "Basic Cell Culture Protocols" by J. Pollard and J. M. Walker (1997), "Mammalian Cell Culture: Essential Techniques" by A. Doyle and J. B. Griffiths (1997), "Culture of Animal Cells: A Manual of Basic Technique" by R. I. Freshney (2005), "Basic Cell Culture Protocols" by C. Helgason and C. L. Miller (2005), "Stem Cells: From Bench to Bedside" by A. Bongso (2005), and "Human Stem Cell Manual: A Laboratory Guide" by J. F. Loring, R. L. Wesselschmidt, and P. H. Schwartz (2007).

[0041] Reagents and instruments for stem cells and cell culture for use in the present invention are commercially available from, for example, Cellartis AB (Goteborg, Sweden), VitroLife AB (Kungsbacka, Sweden), GIBCO® (Invitrogen), Millipore Corporation (Billerica, Massachusetts), Sigma® (St. Louis, Missouri), and Biomol International L.P. (Exeter, UK). [Invention 1001] A culture medium for expanding and / or differentiating an adult stem cell population, comprising: i. Any one of R-spondin 1 to 4 and / or an R-spondin mimetic; and ii. One or more inhibitors that directly or indirectly negatively regulate TGF-β signaling. [Invention 1002] The culture medium of Invention 1001, wherein one or more inhibitors bind to one or more serine / threonine protein kinases selected from the group consisting of ALK5, ALK4, TGF-β receptor kinase 1, and ALK7 and reduce their activity. [Invention 1003] The culture medium of Invention 1001 or Invention 1002, wherein one or more inhibitors that directly or indirectly negatively regulate TGF-β signaling are selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511. [Invention 1004] Any of the above-mentioned culture media of the present invention, further comprising an inhibitor that directly or indirectly negatively regulates p38 signaling. [Invention 1005] The culture medium of Invention 1004, wherein one or more inhibitors that directly or indirectly negatively regulate p38 signaling are selected from the group consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257, and BIRB-796. [Invention 1006] Any of the above-mentioned culture media of the present invention, comprising A83-01 and SB-202190 or A83-01 and SB-203580. [Invention 1007] The inhibitor is added 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. Any of the culture media of the present invention. [Invention 1008] Any of the culture media of the present invention, comprising one or more additional components selected from a BMP inhibitor, a Wnt agonist, a receptor tyrosine kinase ligand, a Rock inhibitor, nicotinamide, and gastrin. [Invention 1009] Any of the culture media of the present invention, comprising any one of R-spondin 1 to 4 and / or an R-spondin mimetic, a BMP inhibitor (e.g., Noggin), a TGF-β inhibitor, a receptor tyrosine kinase ligand (e.g., EGF), nicotinamide, a Wnt agonist (e.g., Wnt(3a)), and optionally, one or more additional components selected from a p38 inhibitor, gastrin, FGF10, HGF, and a Rock inhibitor. [Invention 1010] The BMP inhibitor is selected from the group consisting of Noggin, cordin, a cordin-like protein containing a cordin domain, follistatin, a follistatin-related protein containing a follistatin domain, DAN, a DAN-like protein containing a DAN cystine knot domain, sclerostin / SOST, and α-2 macroglobulin. The culture medium of Invention 1008 or Invention 1009. [Invention 1011] The Wnt agonist is selected from the group consisting of Wnt-3a, a GSK inhibitor (e.g., CHIR99021), Wnt5, Wnt-6a, Norrin, and any other Wnt family protein. The culture medium of Invention 1008 or Invention 1009. [Invention 1012] The culture medium of the present invention 1008 or the present invention 1009, wherein the receptor tyrosine kinase ligand is a mitogenic growth factor selected from the group consisting of mitogenic growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). [The present invention 1013] The culture medium of the present invention 1008 or the present invention 1009, wherein the Rock inhibitor is selected from the group consisting of R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1071), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152). [The present invention 1014] Any of the culture media of the present invention as described above, additionally containing a prostaglandin signaling pathway activator such as PGE2 and / or AA. [The present invention 1015] Any of the culture media of the present invention as described above, additionally containing testosterone such as (dihydro)testosterone. [The present invention 1016] Any of the culture media of the present invention 1001 to 1015 for culturing intestinal cells, containing or consisting of a basal medium, Wnt-3a, EGF, any one of noggin, R-spondin 1 to 4, a TGF-β inhibitor, nicotinamide, and preferably a p38 inhibitor. [The present invention 1017] Any of the culture media of the present invention 1001 to 1015 for culturing gastric cells, containing or consisting of a basal medium, Wnt-3a, EGF, any one of noggin, R-spondin 1 to 4, a TGF-β inhibitor, gastrin, nicotinamide, FGF-10, and preferably a p38 inhibitor. [The present invention 1018] A culture medium according to any one of the present inventions 1001 to 1015 for expanding liver cells, comprising a basal medium, any one of R-spondin 1 to 4, noggin, nicotinamide, EGF, FGF10, HGF, gastrin, a TGF-β inhibitor, and PGE2, and preferably Wnt-3a, or consisting of these. [The present invention 1019] A culture medium according to any one of the present inventions 1001 to 1015 for expanding pancreatic cells, comprising a basal medium, any one of R-spondin 1 to 4, noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, and preferably exendin 4 and Wnt-3a, or consisting of these. [The present invention 1020] A culture medium according to any one of the present inventions 1001 to 1015 for culturing prostate cells, comprising a basal medium, EGF, any one of R-spondin 1 to 4, noggin, nicotinamide, a TGF-β inhibitor, and preferably Wnt-3a and FGF-10, or consisting of these. [The present invention 1021] A culture medium for culturing prostate cells of the present invention 1020, further comprising testosterone, such as (dihydro)testosterone. [The present invention 1022] Comprising or consisting of the components of a culture medium used for culturing cells derived from the corresponding non-cancerous tissue type of interest Optionally, one or more of Wnt-3a, EGF, noggin, R-spondin, a TGF-β inhibitor, a p38 inhibitor, nicotinamide, gastrin, FGF10, and HGF are excluded from the medium used for culturing non-cancerous cells of the tissue type of interest Any of the above-described culture media of the present invention for culturing cancer cells, such as adenocarcinoma cells or carcinoma cells, for example cancer stem cells, derived from the tissue type of interest [The present invention 1023] A culture medium for differentiating stem cells derived from a tissue of interest, which contains or consists of components of a culture medium used for expanding stem cells derived from an organization type of interest, but one or more of Wnt, R-spondin, BMP inhibitor, TGF-β inhibitor, receptor tyrosine kinase ligand, p38 inhibitor, and nicotinamide are excluded. [Inventive Technique 1024] The culture medium of Inventive Technique 1023 for differentiating intestinal cells, which contains or consists of a basal medium, EGF, noggin, a TGF-β inhibitor, and a p38 inhibitor. [Inventive Technique 1025] The culture medium of Inventive Technique 1023 for differentiating liver cells, which contains or consists of a basal medium, noggin, EGF, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor such as DAPT or DBZ, and preferably Wnt-3a. [Inventive Technique 1026] The culture medium of Inventive Technique 1023 for differentiating pancreatic cells, which contains or consists of a basal medium, noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor, and preferably exendin-4. [Inventive Technique 1027] Any of the above-described culture media of the present invention, which is in contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix by interacting with a cell membrane protein such as integrin. [Inventive Technique 1028] The culture medium of Inventive Technique 1027, wherein the extracellular matrix is a laminin-containing extracellular matrix such as Matrigel (trademark) (BD Biosciences). [Inventive Technique 1029] A composition containing any of the culture media of Inventive Techniques 1001 to 1026, and an extracellular matrix or a 3D matrix that mimics the extracellular matrix by interacting with a cell membrane protein such as integrin, for example, a laminin-containing extracellular matrix such as Matrigel (trademark) (BD Biosciences). [The present invention 1030] A hermetically sealed container containing any culture medium or composition of the present invention. [The present invention 1031] Use of any culture medium of the present invention 1001 to 1028 for expanding and / or differentiating stem cells, stem cell populations, tissue fragments, or organoids. [The present invention 1032] Use of the present invention 1031, wherein the stem cells, stem cell population, tissue fragment, or organoid is selected from the group consisting of one or more intestinal stem cells, small intestinal crypts, colonic crypts, gastric stem cells, liver stem cells, pancreatic stem cells, and prostate stem cells. [The present invention 1033] Use of the present invention 1031 or the present invention 1032, wherein the stem cells, stem cell population, tissue fragment, or organoid can be obtained from normal tissue. [The present invention 1034] Use of the present invention 1031 or 1032, wherein the stem cells, stem cell population, tissue fragment, or organoid can be obtained from diseased tissue, such as adenoma, carcinoma, adenocarcinoma, intestine of a patient with cystic fibrosis, or intestine of a patient with inflammatory bowel disease. [The present invention 1035] A method for expanding a single stem cell, stem cell population, or tissue fragment, preferably for obtaining organoids by expanding them, comprising the step of culturing a single stem cell or stem cell population in any culture medium of the present invention 1001 to 1028. [The present invention 1036] The method of the present invention 1035, comprising the following steps: Preparing stem cells, a stem cell population, or an isolated tissue fragment; Preparing any culture medium of the present invention 1001 to 1028; Contacting the stem cells with the culture medium; Culturing the cells under appropriate conditions. [The present invention 1037] A method of the present invention 1035 comprising contacting a stem cell, a stem cell population, or an isolated tissue fragment, and a culture medium with an extracellular matrix, or a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as integrins, for example, a laminin-containing extracellular matrix such as Matrigel™ (BD Biosciences). [The present invention 1038] A method of the present invention 1037, wherein the culture medium diffuses into the extracellular matrix. [The present invention 1039] Any method of the present invention 1035 - 1038 comprising the following steps: Culturing a stem cell, a stem cell population, or a tissue fragment in a first expansion medium; Continuing to culture the stem cell, the stem cell population, or the tissue fragment, and supplementing the medium with a differentiation medium that does not contain one or more, preferably all, of the factors selected from TGF-β inhibitors, p38 inhibitors, nicotinamide, and Wnt. [The present invention 1040] Any method of the present invention 1035 - 1038 for obtaining a small intestine organoid or a colon organoid comprising the following steps: Expanding a small intestine or colon stem cell or tissue fragment in the culture medium of the present invention 1017; and optionally, Differentiating the expanded small intestine or colon stem cell or tissue fragment in the culture medium of the present invention 1024. [The present invention 1041] Any method of the present invention 1035 - 1039 for obtaining a stomach organoid comprising culturing a stomach stem cell or tissue fragment in the culture medium of the present invention 1016. [The present invention 1042] Any method of the present invention 1035 - 1039 for obtaining a liver organoid comprising the following steps: Expanding a liver cell or tissue fragment in the culture medium of the present invention 1019; and optionally, Differentiating the expanded liver cell or tissue fragment in the culture medium of the present invention 1025. [The present invention 1043] Any of the methods of the present invention 1035-1039 for obtaining pancreatic organoids, comprising the following steps: Expanding pancreatic cells or tissue fragments in the culture medium of the present invention 1020; and optionally, Differentiating the expanded pancreatic cells or tissue fragments in the culture medium of the present invention 1026. [The present invention 1044] Any of the methods of the present invention 1035-1039 for obtaining prostate organoids, comprising culturing prostate stem cells or tissue fragments in the culture medium of the present invention 1019. [The present invention 1045] Any of the methods of the present invention 1035-1039 for obtaining adenocarcinoma organoids or carcinoma organoids, comprising culturing adenocarcinoma or carcinoma stem cells or tissue fragments in the culture medium of the present invention 1022. [The present invention 1046] Any of the methods of the present invention 1035-1039, wherein a Rock inhibitor is added to the culture medium for the first 1, 2, 3, 4, 5, 6, or 7 days, optionally every other day. [The present invention 1047] Any of the methods of the present invention 1035-1039, comprising culturing stem cells for 3 months or longer, such as 4, 5, 6, 7, 8, 9 months, or longer, using any of the culture media of the present invention 1001-1028. [The present invention 1048] Organoids or cell populations obtainable by any of the methods of the present invention 1035-1047. [The present invention 1049] The organoids or cell populations of the present invention 1048, which can survive in a cultured state for at least 3 months, such as at least 4, 5, 6, 7, 9 months, or at least 12 months, or longer, when cultured in any of the culture media of the present invention 1001-1028. [The present invention 1050] The organoids or cell populations of the present invention 1048 that have been cultured for at least 3 months, for example, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer. [The present invention 1051] The organoids or cell populations of any one of the present inventions 1048 - 1050 that expand at a rate of at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times per week. [The present invention 1052] The organoids or cell populations of any one of the present inventions 1048 - 1051 that are human organoids or human cell populations. [The present invention 1053] The organoids or cell populations of any one of the present inventions 1048 - 1052 that are normal organoids or normal cell populations, or disease organoids or disease cell populations, for example, disease organoids or disease cell populations obtained by culturing stem cells collected from a human or animal having a disease. [The present invention 1054] The organoids or cell populations of any one of the present inventions 1048 - 1053 that are frozen and stored at a temperature lower than -5°C, lower than -10°C, lower than -20°C, lower than -40°C, lower than -60°C, lower than -80°C, lower than -100°C, or lower than -150°C, for example, at about -180°C. [The present invention 1055] An organoid that is a three - dimensional organoid containing epithelial cells surrounding a central lumen, optionally where the epithelial cells are present in distinct proliferative and differentiative regions, preferably an organoid of any one of the present inventions 1048 - 1054. [The present invention 1056] The organoid of the present invention 1055 that is a three - dimensional organoid containing epithelial cells arranged in a monolayer, optionally in regions of folded monolayers and regions of stratified cells. [The present invention 1057] An organoid according to any one of the present inventions 1048 to 1056, in which non-epithelial cells are absent. [Present Invention 1058] An organoid according to any one of the present inventions 1048 to 1057, in which all differentiated cell types of normal in vivo tissue are present. [Present Invention 1059] An organoid according to any one of the present inventions 1048 to 1058, which is a small intestine organoid, a colon organoid, a stomach organoid, a pancreas organoid, a liver organoid, or a prostate organoid. [Present Invention 1060] A composition comprising: i) one or more organoids or cell populations according to any one of the present inventions 1048 to 1059; and ii) a culture medium and / or an extracellular matrix according to any one of the present inventions 1001 to 1028. [Present Invention 1061] For use in drug screening, target validation, target discovery, toxicology, toxicity screening, personalized medicine, regenerative medicine, or an ex vivo cell / organ model, for example, for use as a disease model, an organoid according to any one of the present inventions 1048 to 1059, or a cell population according to any one of the present inventions 1048 to 1054, or a composition of the present invention 1029 or the present invention 1060. [Present Invention 1062] An organoid, a cell population, or a composition for use in the use of the present invention 1061, wherein regenerative medicine or personalized medicine comprises transplantation of an organoid, a cell population, or a composition into a mammal, preferably a human.

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Mode for Carrying Out the Invention

[0043] Detailed Description According to the present invention, there is provided a culture medium for expanding a stem cell population, which contains one or more inhibitors that bind to one or more serine / threonine protein kinase targets and reduce their activity, and has an effect of enabling continuous proliferation of the stem cell population for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer.

[0044] inhibitor The culture medium used according to the first aspect of the present invention contains any inhibitor that directly or indirectly negatively regulates TGF-β signaling or p38 signaling. In a preferred embodiment, the culture medium of the present invention contains an inhibitor that directly or indirectly negatively regulates TGF-β signaling. In some embodiments, the culture medium of the present invention contains an inhibitor that directly or indirectly negatively regulates TGF-β and an inhibitor that directly or indirectly negatively regulates p38 signaling. In a further embodiment, the culture medium of the present invention additionally contains R-spondin or an R-spondin mimetic.

[0045] One or more inhibitors preferably target serine / threonine protein kinases selected from the group consisting of TGF-β receptor kinase 1, ALK4, ALK5, ALK7, and p38. An inhibitor of any one of these kinases is an inhibitor that reduces the enzymatic activity of any one (or more) of these molecules. Inhibition of ALK and p38 kinases has previously been shown to be associated with B-cell lymphoma (Bakkebo M, Huse K, Hilden VI, Smeland EB, Oksvold MP, "TGF-beta-induced growth inhibition in B-cell lymphoma correlates with Smad1 / 5 signalling and constitutively active p38 MAPK", BMC Immunol. 11:57, 2010). In this publication, it was found that TGF-β-sensitive cell lines expressed high cell surface levels of ALK-5 and that constitutive phosphorylation of p38 was limited to TGF-β-sensitive cell lines. Inhibition of p38 MAPK reduced sensitivity to TGF-β. This suggests that Smad1 / 5 phosphorylation is important for the antiproliferative action of TGF-β in B-cell lymphoma. This result indicates the role of p38 MAPK in the regulation of TGF-β-induced antiproliferative action.

[0046] Without wishing to be bound by theory, the inventors propose that ALK and p38 belong to pathways that negatively regulate the long-term maintenance of stem cells, particularly human epithelial stem cells. The inventors hypothesize that inhibitors acting at any level in this pathway, including those that act, for example, by inhibiting Smad1 / 5 signaling, are also beneficial for stem cell culture. Smad plays an important role in TGF-β signaling.

[0047] In some embodiments, the inhibitors of the invention bind to and reduce the activity of serine / threonine protein kinases selected from the group consisting of TGF-β receptor kinase 1, ALK4, ALK5, ALK7, and p38.

[0048] In some embodiments of the present invention, the culture medium comprises a TGF-β inhibitor. A TGF-β inhibitor means any inhibitor that directly or indirectly negatively regulates TGF-β signaling. In some embodiments, the culture medium of the present invention comprises one or more TGF-β inhibitors that bind to and reduce the activity of one or more serine / threonine protein kinases selected from the group consisting of ALK5, ALK4, TGF-β receptor kinase 1, and ALK7.

[0049] ALK4, ALK5, and ALK7 are all closely related receptors of the TGF-β superfamily. The GI number of ALK4 is 91. The GI number of ALK5 (also known as TGF-β receptor kinase 1) is 7046. The GI number of ALK7 is 658. In one embodiment, the inhibitor according to the present invention binds to and reduces the activity of ALK4, ALK5 (TGF-β receptor kinase 1), and / or ALK7. In another embodiment, the TGF-β receptor binds to and reduces the activity of a Smad protein, such as an R-SMAD or SMAD1-5 (i.e., SMAD1, SMAD2, SMAD3, SMAD4, or SMAD5). In a preferred embodiment, the culture medium of the present invention comprises an ALK5 inhibitor.

[0050] Various methods are known for determining whether a substance is a TGF-β inhibitor. For example, a cell assay may be used in which a reporter construct containing a human PAI-1 promoter or Smad binding site that drives a luciferase reporter gene is stably transfected into cells. Inhibition of luciferase activity compared to a control group can be used as a measure of compound activity (De Gouville et al., Br J Pharmacol. 2005 May; 145(2): 166-177). Another example is the AlphaScreen® phosphosensor assay for measuring kinase activity (Drew A E et al., Comparison of 2 Cell-Based Phosphoprotein Assays to Support Screening and Development of an ALK Inhibitor J Biomol Screen. 16(2) 164-173, 2011).

[0051] Various TGF-β inhibitors are known in the art (see, for example, Table 1). In some embodiments, inhibitors that directly or indirectly negatively regulate TGF-β signaling are selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511.

[0052] In some embodiments of the present invention, the culture medium contains a p38 inhibitor. A p38 inhibitor means any inhibitor that directly or indirectly negatively regulates p38 signaling. In some embodiments, the inhibitor according to the present invention binds to p38 (GI number 1432) and reduces its activity. The p38 protein kinase 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, mitosis, differentiation, proliferation, and cell survival / apoptosis. p38 MAPK exists as α, β, β2, γ, and δ isoforms. A p38 inhibitor is an agent that binds to at least one p38 isoform and reduces its activity. Various methods are known for determining whether a substance is a p38 inhibitor and can be used with the present invention. Examples include phosphorylation site-specific antibody detection of Thr180 / Tyr182 phosphorylation, which provides a well-established method for measuring the activation or inhibition of cellular p38; biochemical recombinant kinase assays; tumor necrosis factor α (TNFα) secretion assays; and the DiscoverRx high-throughput screening platform for p38 inhibitors (see http: / / www.discoverx.com / kinases / literature / biochemical / collaterals / DRx_poster_p38%20KBA.pdf). There are also several p38 activity assay kits available (e.g., Millipore, Sigma-Aldrich).

[0053] The inventors have hypothesized that, in some embodiments, high concentrations (e.g., greater than 100 nM, or greater than 1 μM, greater than 10 μM, or greater than 100 μM) of p38 inhibitors may have a TGF-β inhibitory effect. However, the inventors do not wish to be bound by this hypothesis, and in other embodiments, the p38 inhibitor does not inhibit TGF-β signaling.

[0054] A variety of p38 inhibitors are known in the art (see, for example, Table 1). In some embodiments, inhibitors that directly or indirectly negatively regulate p38 signaling are selected from the group consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257, and BIRB-796. In a further embodiment of the invention, the culture medium comprises both a) an inhibitor that binds to and reduces the activity of any one or more of the kinases derived from the group consisting of ALK4, ALK5, and ALK7; and b) an inhibitor that binds to p38 and reduces its activity. In a preferred embodiment, the culture medium comprises an inhibitor that binds to ALK5 and reduces its activity, and an inhibitor that binds to p38 and reduces its activity.

[0055] In one embodiment, an inhibitor according to the invention, when evaluated by a cell assay, binds to the target of the inhibitor (e.g., TGF-β or p38) and reduces the activity of the target 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. Examples of cell assays for measuring target inhibition are well known in the art as described above.

[0056] The IC50 value of an inhibitor according to the invention may be 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 nM. The IC50 value refers to the effectiveness of the inhibitor in inhibiting the biological or biochemical function of the target by the inhibitor. IC50 indicates how much of a particular inhibitor is required to inhibit the kinase by 50%. The IC50 value can be calculated according to the assay method described above.

[0057] An inhibitor according to the invention may act competitively, non-competitively, uncompetitively, or by mixed inhibition. For example, in certain embodiments, the inhibitor may be a competitive inhibitor of the ATP binding pocket of the target kinase.

[0058] The inhibitors according to the present invention may exist in various forms including natural or modified substrates, enzymes, receptors, small organic molecules, e.g., natural or synthetic small organic molecules up to 2000 Da, preferably 800 Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antisense oligonucleotide aptamers, and structural or functional mimetics of these small molecules. The inhibitors according to the present invention may also be aptamers. 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.

[0059] For example, the inhibitor may be a synthetic small molecule having a molecular weight of 50 to 800 Da, 80 to 700 Da, 100 to 600 Da or 150 to 500 Da.

[0060] In some embodiments, the small molecule inhibitor includes pyridinylimidazole or 2,4-disubstituted pteridine or quinazoline, e.g., including the following. TIFF2025090784000001.tif22128

[0061] Specific examples of inhibitors that can 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, LY364947, SD-208, SJN2511 (see Table 1). The culture medium of the present invention may contain any one or more of the inhibitors listed in Table 1. The culture medium of the present invention may contain any combination of one inhibitor listed and another inhibitor. For example, the culture medium of the present invention may contain SB-202190 or SB-203580 or A83-01. Or, the culture medium of the present invention may contain SB-202190 and A83-01. Or, the culture medium of the present invention may contain SB-203580 and A83-01. Those skilled in the art will recognize that other inhibitors and combinations of inhibitors that bind to the target according to the present invention and reduce its activity may be included in the culture medium or culture medium supplement according to the present invention.

[0062] Taking into account the IC50 value of the inhibitor, the inhibitor according to the present invention may be added to the culture medium to an appropriate final concentration.

[0063] For example, SB-202190 at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM may be added to the culture medium. For example, about 10 μM of SB-202190 may be added to the culture medium.

[0064] SB-203580 at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM may be added to the culture medium. For example, about 10 μM of SB-203580 may be added to the culture medium.

[0065] VX-702 may 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, about 5 μM of VX-702 may be added to the culture medium.

[0066] VX-745 may 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, about 1 μM of VX-745 may be added to the culture medium.

[0067] PD-169316 may 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, about 20 μM of PD-169316 may be added to the culture medium.

[0068] RO-4402257 may 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, about 1 μM of RO-4402257 may be added to the culture medium.

[0069] BIRB-796 may 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, about 1 μM of BIRB-796 may be added to the culture medium.

[0070] A83-01 may 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, about 500 nM of A83-01 may be added to the culture medium.

[0071] SB-431542 may be added to the culture medium at a concentration of 80 nM to 80 μM, or 100 nM to 40 μM, or 500 nM to 10 μM. For example, about 1 μM of SB-431542 may be added to the culture medium.

[0072] SB-505124 may 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, about 500 nM of SB-505124 may be added to the culture medium.

[0073] SB-525334 may 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, about 100 nM of SB-525334 may be added to the culture medium.

[0074] LY36494 may 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, about 500 nM of LY36494 may be added to the culture medium.

[0075] (Table 1) Exemplary inhibitors according to the present invention TIFF2025090784000002.tif209146TIFF2025090784000003.tif132146

[0076] SD-208 may 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, about 500 nM of SD-208 may be added to the culture medium.

[0077] LY364947 may 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, about 500 nM of LY364947 may be added to the culture medium.

[0078] SJN2511 may 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, about 200 nM of SJN2511 may be added to the culture medium.

[0079] Thus, in some embodiments, in the culture medium, an inhibitor that directly or indirectly negatively regulates TGF-β signaling or p38 signaling is added 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.

[0080] In addition to the inhibitor, the cell culture medium generally contains a number of components necessary to support the maintenance and / or expansion of the cultured cells. Thus, the cell culture medium of the present invention usually contains many other components in addition to the inhibitor according to the present invention. In view of the following disclosure, one of ordinary skill in the art can readily formulate an appropriate combination of components. The culture medium according to the present invention is generally a nutrient solution containing standard cell culture components, such as amino acids, vitamins, inorganic salts, carbon energy sources, and buffers, as further detailed below. Other standard cell culture components that may be included in the culture include hormones, such as progesterone, proteins, such as albumin, catalase, insulin, and transferrin. These other standard cell culture components constitute the "basal" medium.

[0081] The culture medium according to the present invention may be prepared by improving an existing cell medium. A person skilled in the art will understand, from general knowledge, the types of culture media that can be used for stem cell culture. Potentially suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco Medium and Minimum Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI1640 medium. Thus, in some embodiments, one of these existing cell culture media is used as a basal medium to which an inhibitor that directly or indirectly negatively regulates TGF-β signaling or p38 signaling is added, and optionally, one or more other components described herein are added.

[0082] In some embodiments, the culture medium of the present invention comprises one or more additional components selected from BMP inhibitors, Wnt agonists, receptor tyrosine kinase ligands, Rock inhibitors, nicotinamide, and gastrin. In some embodiments, the culture medium of the present invention comprises any one of R-spondin 1 to 4 and / or an R-spondin mimetic, a TGF-β inhibitor, a BMP inhibitor (e.g., noggin), and a Wnt agonist (e.g., Wnt(3a)).

[0083] In some embodiments, the culture medium of the present invention comprises any one of R-spondin 1 to 4 and / or an R-spondin mimetic, a BMP inhibitor (e.g., noggin), a TGF-β inhibitor, a receptor tyrosine kinase ligand (e.g., EGF), nicotinamide, a Wnt agonist (e.g., Wnt(3a)), and optionally, one or more additional components selected from a p38 inhibitor, gastrin, FGF10, HGF, and a Rock inhibitor. As will be described in more detail below, any additional components may be added to optimize the culture medium for culturing cells derived from a particular tissue.

[0084] The culture medium of the present invention may contain one or more bone morphogenetic protein (BMP) inhibitors. BMP ligands signal as dimers by constructing a quadripartite transmembrane serine / threonine kinase receptor complex consisting of two type I receptors and two type II receptors. Once the complex is constructed, a phosphorylation cascade is initiated, thereby activating BMP-responsive Smads1 / 5 / 8 and changing transcriptional activity. Conveniently, the inventors have shown that BMP inhibitors promote Lgr5 expression, and thus the presence of BMP inhibitors in the culture medium of the present invention is likely to result in more proliferative organoids than in the absence of BMP inhibitors (see, for example, Example 3). Therefore, BMP inhibitors are advantageous components of the expansion medium of the present invention. Thus, the use of BMP inhibitors is advantageous in the use of an expansion medium when it is desirable to culture cells for at least three months (e.g., at least four months, five months, six months, seven months, eight months, or nine months) without differentiating the cells.

[0085] Several classes of natural BMP-binding proteins are known, including Noggin (Peprotech), Cordonin and Cordonin-like proteins containing Cordonin domains (R&D systems), follistatin and follistatin-related proteins containing follistatin domains (R&D systems), DAN and DAN-like proteins containing DAN cysteine knot domains (R&D systems), sclerostin / SOST (R&D systems), and α-2 macroglobulin (R&D systems). A BMP inhibitor is an agent that binds to a BMP molecule to form a complex with reduced BMP activity, for example, an agent that forms a complex with reduced BMP activity by preventing or inhibiting the binding of a BMP molecule to a BMP receptor. Alternatively, the inhibitor may be an agent that binds to a BMP receptor and prevents the binding of a BMP ligand to the receptor, for example, an antibody that binds to the receptor. The BMP inhibitor may be a protein or a small molecule, natural, modified, and / or partially or fully synthetic. The BMP inhibitor in the culture medium of the present invention may be Noggin, DAN, or a DAN-like protein including Cerberus and Gremlin (R&D systems). These diffusible proteins bind to BMP ligands with varying degrees of affinity and can inhibit the approach of BMP ligands to signaling receptors. A preferred BMP inhibitor for use in the culture medium of the present invention is Noggin. Noggin can be used at any suitable concentration. In some embodiments, the basal medium of the culture medium of the present invention may contain about 10 ng / ml to about 100 ng / ml of Noggin. For example, the culture medium may contain at least 10 ng / ml of Noggin, at least 20 ng / ml of Noggin, at least 50 ng / ml of Noggin, at least 100 ng / ml of Noggin, about 100 ng / ml of Noggin, or 100 ng / ml of Noggin. In some embodiments, the culture medium may contain less than 200 ng / ml of Noggin, less than 150 ng / ml of Noggin, less than 100 ng / ml of Noggin, less than 75 ng / ml of Noggin, less than 50 ng / ml of Noggin, or less than 30 ng / ml of Noggin.A BMP inhibitor may be added to the culture medium every other day during culture, or daily during culture, or every two days, every three days, every four days, or as needed. The BMP inhibitor is a particularly advantageous expansion medium component, for example, a particularly advantageous expansion medium component for expanding pancreatic stem cells, intestinal stem cells, colonic stem cells, liver stem cells, and prostate stem cells. However, noggin has been shown to block some differentiation (see, for example, Example 3). Thus, in some embodiments, the BMP inhibitor is excluded from the differentiation medium of the present invention.

[0086] In some embodiments, cells cultured with a BMP inhibitor have upregulated Lgr5 expression compared to cells cultured without a BMP inhibitor. Thus, addition of a BMP inhibitor typically results in more proliferative organoids. This is surprising since the literature states that BMP activity is useful for differentiating pancreatic cells into ductal cells (see keratin 7 and 19 expression) as well as endocrine cells. Thus, one of ordinary skill in the art would expect to include a BMP inhibitor such as noggin to decrease proliferation and increase differentiation. However, surprisingly, the inventors have discovered that the use of a BMP inhibitor is advantageous as it results in many proliferative organoids and high Lgr5 expression. The culture medium of the present invention may contain one or more Wnt agonists. The Wnt signaling pathway is defined by a series of events that occur when a Wnt protein binds to a cell surface receptor of the Frizzled receptor family. This results in activation of Dishevelled family proteins that inhibit a protein complex containing axin, GSK-3, and the protein APC, which degrades intracellular β-catenin. The resulting high concentration of nuclear β-catenin enhances transcription by TCF / LEF family transcription factors. A Wnt agonist is defined as an agent that activates transcription via TCF / LEF intracellularly. Thus, a Wnt agonist is selected from any and all of a Wnt family protein, an intracellular β-catenin degradation inhibitor, and a TCF / LEF activator and binds to and activates a member of the Frizzled receptor family. The Wnt agonist stimulates intracellular Wnt activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably at least 100% compared to the level of Wnt activity in the absence of the molecule.As is known to those skilled in the art, Wnt activity can be determined by measuring Wnt transcriptional activity, for example, by using pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al, 1997 Science 275 1784-1787).

[0087] In some embodiments, the Wnt agonist includes secreted glycoproteins including Wnt-1 / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D sytems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnM1, and Wnt-16. An overview of human Wnt proteins is shown in "THE WNT FAMILY OF SECRETED PROTEINS", R&D Systems Catalog, 2004. Further, the Wnt agonist includes the R-spondin family of secreted proteins consisting of four members (R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 ((R&D systems), R-spondin 3, and R-spondin-4) that are linked to the activation and regulation of the Wnt signaling pathway, and Norrin, a secreted regulatory protein that functions like a Wnt protein in that it binds to the Frizzled-4 receptor with high affinity and induces activation of the Wnt signaling pathway (also called Nome Disease Protein or NDP) (R&D systems) (Kestutis Planutis et al (2007) BMC Cell Biol 812). In some embodiments, one or more Wnt agonists for use in the present invention are R-spondin mimetics, such as Lgr5 agonists, such as anti-Lgr5 antibodies. Recently, aminopyrimidine derivatives, which are small molecule agonists of the Wnt signaling pathway, have been identified and are also clearly included as Wnt agonists (Lm et al (2005) Angew Chem Int Ed Engl 44, 1987-90).

[0088] In some embodiments, the Wnt agonist is a GSK inhibitor. Known GSK inhibitors include small interfering RNAs (siRNAs, Cell Signaling), lithium (Sigma), kenpaullone (Biomol International, Leost, M et al (2000) Eur J Biochem 267, 5983-5994), 6-bromoindirubin-3'-acetoxime (Meyer, L et al (2003) Chem Biol 10, 1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), and FRAT family members and FRAT-derived peptides that block the interaction between GSK-3 and axin. A review is provided in Meijer et al, (2004) Trends in Pharmacological Sciences 25, 471-480, which is incorporated herein by reference. Methods and assays for determining the level of GSK-3 inhibition are known to those of skill in the art and include, for example, the methods and assays described in Liao et al 2004, Endocrinology, 145(6) 2941-2949.

[0089] In some embodiments, the Wnt agonist is an inhibitor of RNF43 or ZNRF3. The inventors have discovered that RNF43 and ZNRF3 are present in the cell membrane and likely negatively regulate the level of Wnt receptor complexes in the membrane by ubiquitin ligating Frizzled. Thus, the inventors have hypothesized that inhibition of RNF43 or ZNRF3 by antagonist antibodies, RNAi, or small molecule inhibitors indirectly stimulates the Wnt pathway. RNF43 and ZNRF3 have catalytic loop domains (with ubiquitin ligase activity). This catalytic loop domain can be targeted in the design of small molecule inhibitors. Some anti-RNF43 antibodies and some anti-ZNRF3 antibodies are commercially available. In some embodiments, such antibodies are suitable Wnt agonists in the context of the present invention.

[0090] In some embodiments, the Wnt agonist is selected from the group consisting of Wnt-3a, a GSK inhibitor (e.g., CHIR99021), Wnt5, Wnt-6a, Norrin, and any other Wnt family protein.

[0091] In some embodiments, the Wnt agonist comprises any one of R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4, or consists of any one of R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4. In a preferred embodiment, the Wnt agonist is selected from one or more of a Wnt family member, R-spondin 1-4, Norrin, and a GSK inhibitor. In some embodiments, the Wnt agonist is a GSK-3 inhibitor, e.g., CHIR99021 (Stemgent 04-0004). In some embodiments, CHIR99021 is added to the culture medium to a final concentration of 50 nM to 100 μM, e.g., 100 nM to 50 μM, 1 μM to 10 μM, 1 μM to 5 μM, or 3 μM. In some embodiments where a GSK-3 inhibitor is used, the GSK-3 inhibitor is not BIO (6-bromoindirubin-3'-oxime, Stemgent 04-0003). The inventors have discovered that the addition of at least one Wnt agonist to the basal medium is essential for the proliferation of epithelial stem cells or isolated crypts.

[0092] In a further preferred embodiment, the Wnt agonist comprises or consists of R-spondin 1 or R-spondin-4. R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4 is preferably added to the basal medium at a concentration of at least 50 ng / ml, more preferably at least 100 ng / ml, more preferably at least 200 ng / ml, more preferably at least 300 ng / ml, more preferably at least 500 ng / ml. The most preferred concentration of R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4 is about 500 ng / ml or 500 ng / ml. In some embodiments, R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4 is added to the culture medium at a concentration of at least 500 ng / ml, at least 600 ng / ml, at least 700 ng / ml, at least 800 ng / ml, at least 900 ng / ml, at least 1 μg / ml, at least 1.5 μg / ml, or at least 2 μg / ml. In another preferred embodiment, R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4 is added to the culture medium at a concentration of about 1 μg / ml or 1 μg / ml. In some embodiments, R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4 is added to the basal medium at a concentration of less than 1000 ng / ml, for example, less than 800 ng / ml, less than 600 ng / ml, less than 550 ng / ml, less than 500 ng / ml, less than 400 ng / ml, less than 300 ng / ml, or less than 200 ng / ml, or less than 100 ng / ml. In some embodiments, two or more (e.g., 2, 3, or 4) of R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4 ("R-spondin 1-4") are added to the medium. Preferably, when two or more of R-spondin 1-4 are added, the total concentration of R-spondin is the above concentration. When the culture medium described herein is said to "contain R-spondin 1-4", the medium is meant to contain any one or more of R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4.When the culture medium described in this specification is said to contain "R-spondin", the culture medium is meant to contain any one or more of R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, and R-spondin mimics.

[0093] During the culture of stem cells, the Wnt family member is preferably added to the culture medium every other day, while the culture medium is preferably replenished every three days.

[0094] In a preferred embodiment, the Wnt agonist is selected from the group consisting of R-spondin, Wnt-3a, and Wnt-6. More preferably, both R-spondin and Wnt-3a are used as Wnt agonists. Surprisingly, this combination is particularly preferred because it has a synergistic effect on organoid formation. The preferred concentration is about 500 ng / ml or 500 ng / ml for R-spondin and about 100 ng / ml or 100 ng / ml for Wnt3a.

[0095] The culture medium of the present invention may contain one or more receptor tyrosine kinase ligands. An example of a receptor tyrosine kinase ligand for use in the present invention is EGF, which is a ligand for the receptor tyrosine kinase EGFR. Many receptor tyrosine kinase ligands are also mitogenic growth factors.

[0096] The culture medium of the present invention may contain one or more mitogenic growth factors. The one or more mitogenic growth factors may be selected from the growth factor family including epidermal growth factor (EGF, Peprotech), transforming growth factor-α (TGF-α, Peprotech), basic fibroblast growth factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), and keratinocyte growth factor (KGF, Peprotech). EGF is a potent mitogenic factor for various ectodermal and mesodermal cultured cells and has a very large impact on the differentiation of specific cells in vivo and in vitro and the differentiation of some fibroblasts in the cell culture state. The EGF precursor exists as a membrane-bound molecule that is cleaved by proteolysis to yield a 53-amino acid peptide hormone that stimulates cells. The preferred mitogenic growth factor is EGF. EGF is preferably added to the basal medium at a concentration of 5 to 500 ng / ml or at least 5 ng / ml and 500 ng / ml or less. Preferred concentrations are at least 10 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 40 ng / ml, 45 ng / ml, or 50 ng / ml and 500 ng / ml or less, 450 ng / ml or less, 400 ng / ml or less, 350 ng / ml or less, 300 ng / ml or less, 250 ng / ml or less, 200 ng / ml or less, 150 ng / ml or less, or 100 ng / ml or less. A more preferred concentration is at least 50 ng / ml and 100 ng / ml or less. An even more preferred concentration is about 50 ng / ml or 50 ng / ml. In the case of FGF, preferably, in the case of FGF10 or FGF7, the same concentration can be used. If multiple types of FGF, for example, FGF7 and FGF10 are used, the FGF concentration is as defined above and refers to the total concentration of FGF used. During the culture of stem cells, the mitogenic growth factor is preferably added to the culture medium every other day, while the culture medium is preferably replenished every three days. Any member of the FGF family can be used. Preferably, FGF7 and / or FGF10 are used, and FGF7 is also known as KGF (keratinocyte growth factor).In a more preferred embodiment, for example, a combination of mitogenic growth factors such as EGF and KGF, or EGF and BDNF is added to the basal medium. In a more preferred embodiment, for example, a combination of mitogenic growth factors such as EGF and KGF, or EGF and FGF10 is added to the basal medium. The mitogenic growth factor may be added to the culture medium at a concentration of 5 to 500 nanograms / ml or at least 5 nanograms / ml and 500 nanograms / ml or less, for example, at least 10 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 40 ng / ml, 45 ng / ml, or 50 ng / ml, and 500 ng / ml or less, 450 ng / ml or less, 400 ng / ml or less, 350 ng / ml or less, 300 ng / ml or less, 250 ng / ml or less, 200 ng / ml or less, 150 ng / ml or less, or 100 ng / ml or less. The mitogenic growth factor may be selected from the group consisting of EGF, TGF-α, KGF, FGF7, and FGF. Preferably, the mitogenic factor is selected from the group consisting of EGF, TGF-α, and KGF, or from the group consisting of EGF, TGF-α, and FGF7, or from the group consisting of EGF, TGF-α and FGF, or from the group consisting of EGF and KGF, or from the group consisting of EGF and FGF7, or from the group consisting of EGF and FGF, or from the group consisting of TGF-α and KGF, or from the group consisting of TGF-α and FGF7, or from the group consisting of TGF-α and FGF. TGF-α may be used instead of EGF. In some embodiments, the mitogenic growth factor is hepatocyte growth factor (HGF). In some embodiments, HGF is added to the culture medium.

[0097] In some embodiments, the receptor tyrosine kinase ligand is a mitogenic growth factor selected from the family of growth factors consisting of mitogenic growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF).

[0098] Any of the described culture media can, in particular, include a ROCK inhibitor such as Y-27632 (10 μM; Sigma) during the first few days of culturing, especially before performing cell sorting experiments. This is because ROCK inhibitors are known to avoid anoikis, a type of programmed cell death induced by anchorage-dependent cells detached from the surrounding extracellular matrix. Thus, any of the media defined herein may additionally contain a ROCK inhibitor for the first few days. In some embodiments, the culture medium of the present invention additionally contains a ROCK inhibitor such as Y-27632 during the first few days of culturing, for example, before performing cell sorting experiments.

[0099] A further aspect of the method according to the invention comprises a culture medium containing a Rock (Rho-kinase) inhibitor. The addition of the Rock inhibitor has been found to prevent anoikis, especially when culturing a single stem cell. The Rock inhibitor is preferably selected from R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, Sigma-Aldrich), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077, Cayman Chemical), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152, Tocris Bioschience). The Rho-kinase inhibitor, for example, Y-27632, is preferably added to the culture medium every other day for the first 7 days of culturing the stem cells. The Rock inhibitor is preferably included in the medium for the first few days, for example, during the first 1, 2, 3, 4, 5, 6, or 7 days of culture after a single cell seeding or split. Any suitable Rock inhibitor concentration, for example, 1 - 200 uM, 1 - 100 uM, 5 - 50 uM, or about 10 uM can be used. The preferred concentration of Y27632 is 10 uM. Thus, in some aspects, the invention provides a method for culturing stem cells and / or a method for obtaining organoids, wherein the Rock inhibitor is added to the culture medium on the first 1, 2, 3, 4, 5, 6, or 7 days, optionally every other day. In some aspects, the Rock inhibitor is not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 days have passed.

[0100] The addition of a Rock inhibitor is particularly important when culturing a single stem cell (as described above), i.e., when the starting material for the organoid is a single stem cell. Thus, in some embodiments, the present invention includes a step of culturing a stem cell, optionally a single stem cell, wherein the Rock inhibitor is added to the culture medium for the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, optionally every other day, and optionally, the Rock inhibitor is not added to the culture medium after the first 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, to provide a method for obtaining an organoid.

[0101] The Rock inhibitor is less important and sometimes not necessary when culturing multiple types of cells, e.g., when the starting material for the organoid is a tissue fragment. Thus, in some embodiments, the present invention includes a step of culturing a stem cell, optionally a tissue fragment, wherein the Rock inhibitor is not added to the culture medium at all, or is not added to the culture medium after the first 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, to provide a method for obtaining an organoid.

[0102] When the cells are split into multiple cultures, the Rock inhibitor may be added to the culture medium in the same way. This means that after splitting, especially when the split involves taking a single stem cell from the first culture and placing these into the second culture, the Rock inhibitor is added on the first day, second day, third day, fourth day, fifth day, sixth day, or seventh day, optionally every other day. If the split involves taking multiple stem cells from the first culture and placing these into the second culture, the addition of the Rock inhibitor is less important and sometimes not necessary. Thus, in some embodiments, when a method for obtaining an organoid or a method for culturing stem cells involves splitting, optionally when a single cell is involved in the split, the Rock inhibitor is added to the fresh culture medium on the first day, second day, third day, fourth day, fifth day, sixth day, or seventh day, optionally every other day, after splitting. In some embodiments, when a method for obtaining an organoid or a method for culturing stem cells involves splitting, optionally when multiple cells are involved in the split, no Rock inhibitor is added to the culture medium, or it is not added to the culture medium after the first two days, three days, four days, five days, six days, seven days, eight days, nine days, or ten days have passed.

[0103] In a further aspect, the method according to the invention comprises a culture medium further comprising a Notch agonist. Notch signaling has been shown to play an important role in cell fate determination and cell survival and proliferation. The Notch receptor protein can interact with a number of surface-bound or secreted ligands including, but not limited to, Delta1, Jagged1 and 2, and Delta-like1, Delta-like3, Delta-like4. When the ligand binds, the Notch receptor is activated by a series of cleavage events involving members of the ADAM protease family, as well as intramembrane cleavage regulated by the γ-secretase presenilin. The result is the translocation of the Notch intracellular domain to the nucleus, where the Notch intracellular domain activates the transcription of downstream genes. Preferred Notch agonists are selected from Jagged1 and Delta1 or active fragments or derivatives thereof. The most preferred Notch agonist is the DSL peptide having the sequence TIFF2025090784000004.tif3128 (Dontu et al., 2004. Breast Cancer Res 6. R605-R615). The DSL peptide is preferably used at a concentration of 10 μM to 100 nM or at least 10 μM and 100 nM or less. When a Notch agonist is added, particularly when added in the first week of culture, the culture efficiency increases 2- to 3-fold. The Notch agonist is preferably added to the culture medium every other day for the first 7 days of culturing the stem cells. Thus, in some aspects, the invention provides a method for culturing stem cells and / or a method for obtaining an organoid, wherein the Notch agonist is added to the culture medium every other day for 1, 2, 3, 4, 5, 6, or 7 days, optionally. In some aspects, the Notch agonist is not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0104] A Notch agonist is defined as a molecule that stimulates intracellular Notch activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably at least 100% compared to the level of Notch activity in the absence of the molecule. As is known to those skilled in the art, Notch activity can be confirmed by measuring Notch transcriptional activity, for example, by a 4xwtCBF1-luciferase reporter construct as described in (Hsieh et al, 1996 Mol Cell. Biol. 16, 952-959).

[0105] In a further aspect, a γ-secretase inhibitor such as DAPT or DBZ is added to the cell culture medium. γ-secretase inhibitors can affect cell fate determination during differentiation. For example, in some aspects, γ-secretase inhibitors can affect the cell fate towards secretory cells such as goblet cells. Any suitable γ-secretase inhibitor concentration can be used, for example, 1 nM to 10 μM, 1 nM to 1 μM, 1 to 100 nM, or preferably 1 to 20 nM. For example, the γ-secretase inhibitor may be added to the culture medium to a final concentration of about 1 nM.

[0106] In a further aspect, gastrin (or a suitable alternative such as Leu15-gastrin) is added to the cell culture medium. Gastrin (or a suitable alternative) may be added to the culture medium to a final concentration of 1 nM to 10 μM, 1 nM to 1 μM, 5 to 100 nM, or preferably 10 to 50 nM. For example, Leu15-gastrin may be added to the culture medium to a final concentration of about 10 nM. Gastrin is not required in some culture media of the present invention. Thus, in some aspects, the culture media of the present invention do not contain gastrin. In particular, gastrin is not required for culturing intestinal stem cells or for obtaining intestinal (crypt-villus or colonic crypt) organoids. However, even when gastrin is not required, it may still be added to the medium without negative effects.

[0107] In a further aspect, nicotinamide is added to the culture medium of the present invention. The addition of nicotinamide has been found to improve the culture efficiency and lifespan of human colon organoids. Nicotinamide may be added to the culture medium to 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 to a final concentration of about 10 mM.

[0108] In a preferred embodiment of the present invention, nicotinamide and gastrin (or a suitable alternative such as Leu15-gastrin) are added to the culture medium. Nicotinamide and gastrin are added to the culture medium at any of the above concentrations.

[0109] In some embodiments, a prostaglandin signaling pathway activator is added to the culture medium (see Figure 24, Antagonism of the prostaglandin D2 receptors DP1 and CRTH2 as an approach to treat allergic diseases. Roy Pettipher, Trevor T. Hansel & Richard Armer Nature Reviews Drug Discovery 6, 313-325 (April 2007)). For example, any one or more of the compounds selected from the list including the following are added to the culture medium: phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, PGE2 and / or AA are added to the culture medium. In some embodiments, PGE2 is added to the medium to a final concentration of at least 10 nM, such as at least 20 nM, at least 30 nM, at least 40 nM, at least 45 nM, 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 100 nM, 20 nM to 50 nM. In a preferred embodiment, PGE2 is added to the medium to a final concentration of 50 nM. In some embodiments, AA is added to the medium to a final concentration of at least 1 μg / ml, at least 5 μg / ml, at least 8 μg / ml, at least 9 μg / ml, at least 10 μg / ml, such as 1 μg / ml to 1000 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 100 μg / ml, 1 μg / ml to 50 μg / ml, or 5 μg / ml to 20 μg / ml. In a preferred embodiment, AA is added to the medium to a final concentration of 10 μg / ml. AA and PGE2 are interchangeable in the context of the culture medium of the present invention. Thus, if the culture medium described herein is said to contain PGE2, it may instead contain (at an appropriate concentration) AA in place of PGE2.Conversely, when the culture medium described herein is said to contain AA, it may instead contain PGE2 (at an appropriate concentration). Furthermore, those skilled in the art will understand that when PGE2 and / or AA are included in the culture medium of the present invention, instead, the culture medium may contain, in exchange for or in addition to PGE2 and / or AA, any one or more of the compounds selected from the following list: phospholipids, prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), and prostaglandin D2 (PGD2).

[0110] In a further aspect, RANK ligand (also referred to herein as RANKL) is added to the culture medium of the present invention. RANK ligand may be useful for directing differentiation towards a specific cell fate. For example, when RANK ligand is included in a culture medium for intestinal cells, preferably a culture medium for differentiating intestinal cells, a large proportion of the cells differentiate into M cells. Thus, in some aspects, the present invention provides a culture medium containing RANKL. In particular, the present invention provides a medium for culturing intestinal cells, preferably a culture medium for differentiating intestinal cells, containing RANKL. Any suitable RANKL concentration can be used, for example, 10 ng / ml to 1000 ng / ml, 10 to 500 ng / ml, or 50 to 100 ng / ml. For example, RANKL may be added to the culture medium up to a final concentration of about 100 ng / ml.

[0111] A culture medium containing EGF, noggin, and R-spondin is referred to herein as an "ENR medium". A culture medium containing an ENR medium and a Wnt agonist such as Wnt-3a is referred to herein as a "WENR medium". In a preferred aspect of the present invention, the culture medium contains a WENR medium. In the most preferred aspect of the present invention, the culture medium contains a WENR medium to which gastrin and / or nicotinamide has been added (i.e., WENRg or WENR + nicotinamide or WENRg + nicotinamide).

[0112] The pH of the medium may be in the range of about 7.0 to 7.8, in the range of about 7.2 to 7.6, or about 7.4. The pH may be maintained using a buffer solution. Suitable buffer solutions can be easily selected by those skilled in the art. Buffer solutions that can be used include carbonate buffer solutions (e.g., NaHCO3) and phosphate buffer solutions (e.g., NaH2PO4). These buffer solutions are generally used at about 50 to about 500 mg / l. Other buffer solutions such as N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES) and 3-[N-morpholino]-propanesulfonic acid (MOPS) can also usually be used at about 1000 to about 10,000 mg / l. The medium may contain a pH indicator such as phenol red (e.g., about 5 to about 50 mg / litre) so that the pH state of the medium can be easily monitored.

[0113] The culture medium for use in the present invention may contain one or more amino acids. Those skilled in the art understand the appropriate types and amounts of amino acids for use in a stem cell culture medium. Amino acids that may be present include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof. Some culture media contain all of these amino acids. Generally, each amino acid is present at about 0.001 to about 1 g / L of the medium (usually about 0.01 to about 0.15 g / L) when present, except for L-glutamine which is generally present at about 0.05 to about 1 g / L (usually about 0.1 to about 0.75 g / L). The amino acids may be of synthetic origin.

[0114] The culture medium for use in the present invention may contain one or more vitamins. Those skilled in the art will understand the appropriate types and amounts of vitamins for use in a stem cell culture medium. Possible vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0115] The culture medium for use in the present invention may contain one or more inorganic salts. Those skilled in the art will understand the appropriate types and amounts of inorganic salts for use in a stem cell culture medium. Inorganic salts are typically included in the culture medium to help maintain the osmotic balance of the cells and to help regulate the membrane potential. Possible inorganic salts include salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. The salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. Specific salts that may be used include CaCl2, CuSO4-5H2O, Fe(NO3)-9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4-H2O, and ZnSO4-7H2O.

[0116] The weight osmolarity of the medium may be in the range of about 200 to about 400 mOsm / kg, in the range of about 290 to about 350 mOsm / kg, or in the range of about 280 to about 310 mOsm / kg. The weight osmolarity of the medium may be less than about 300 mOsm / kg (for example, about 280 mOsm / kg).

[0117] The culture medium for use in the present invention may contain one or more carbon energy sources in the form of sugars. Those skilled in the art understand the appropriate types and amounts of sugars for use in a stem cell culture medium. Sugars that may be present include glucose, galactose, maltose, and fructose. The sugar is preferably glucose, particularly D-glucose (dextrose). The carbon energy source is usually present at about 1 to about 10 g / L.

[0118] The culture medium of the present invention may contain serum. Serum obtained from any suitable source, including fetal bovine serum (FBS), goat serum, or human serum, can be used. Preferably, human serum is used. Serum may be used at about 1% to about 30% of the medium volume according to conventional techniques.

[0119] In other embodiments, the culture medium of the present invention may contain a serum substitute. Various different serum substitute formulations are commercially available and are known to those skilled in the art. When a serum substitute is used, it may be used at about 1% to about 30% of the medium volume according to conventional techniques.

[0120] In other embodiments, the culture medium of the present invention may be serum-free and / or may not contain a serum substitute. A serum-free medium is a medium that does not contain any type of animal serum. A serum-free medium may be preferred to avoid possible xeno-contamination of the stem cells. A medium that does not contain a serum substitute is a medium to which no commercially available serum substitute formulation has been added.

[0121] In a preferred embodiment, the cell culture medium is supplemented with purified growth factors, natural growth factors, semi-synthetic growth factors, and / or synthetic growth factors, and does not contain undefined components such as fetal bovine serum or bovine fetal serum. For example, supplements such as B27 (Invitrogen), N-acetylcysteine (Sigma), and N2 (Invitrogen) stimulate the growth of some cells. In some embodiments, one or more of these supplements, for example, one of these supplements, any two of these supplements, or all three of these supplements, are added to the cell culture medium.

[0122] In other embodiments, exendin-4 is added to the cell culture medium. Exendin-4 is a 39-amino acid peptide that activates the GLP-1 (glucagon-like peptide-1) receptor, raises intracellular cAMP in pancreatic acinar cells, and has no effect on the VIP (vasoactive intestinal peptide) receptor.

[0123] The culture medium for use in the present invention may contain ions of one or more trace elements, for example, barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, and / or aluminum.

[0124] The medium may contain a reducing agent, for example, β-mercaptoethanol at a concentration of about 0.1 mM.

[0125] The culture medium of the present invention may contain one or more additional agents, for example, nutrients or growth factors reported to improve stem cell culture, such as cholesterol / transferrin / albumin / insulin / progesterone, putrescine, selenite / other factors.

[0126] The culture medium of the present invention may be diffused in the extracellular matrix (ECM). In a preferred method of the present invention, isolated tissue fragments or isolated epithelial stem cells are attached to the ECM. The ECM consists of various polysaccharides, water, elastin, and glycoproteins. The glycoproteins include collagen, entactin (nidogen), fibronectin, and laminin. The ECM is secreted by connective tissue cells. Different types of ECM are known, including different compositions containing different types of glycoproteins and / or different combinations of glycoproteins. The ECM can be provided by culturing ECM-producing cells, such as fibroblasts, in a container, then removing these cells and adding isolated tissue fragments or isolated epithelial stem cells. Examples of extracellular matrix-producing cells are 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 (types I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. Alternatively, the ECM is commercially available. Examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen) and basement membrane preparations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Matrigel™ (BD Biosciences)). Synthetic extracellular matrix materials such as ProNectin (Sigma Z378666) may be used. If desired, a mixture of extracellular matrix materials may be used. Using ECM for culturing stem cells enhanced the long-term survival of stem cells and the continuous presence of undifferentiated stem cells. In the absence of ECM, stem cell cultures could not be maintained for a long period, and the continuous presence of undifferentiated stem cells was not observed. Furthermore, in the presence of ECM, three-dimensional tissue organoids that could not be cultured in the absence of ECM could be cultured. The extracellular matrix material usually sinks to the bottom of the dish in which the cells are suspended.Typically, when the matrix solidifies at 37°C, the medium is added and diffuses into the ECM. Cells in the medium adhere to the ECM by interacting with the surface structure of the ECM, for example, by interacting with integrins. To coat the cell culture vessel, about 1 μg / cm². 2 about 1 mg / ml (stock solution) used in, or about 1 μg / cm² 2 ~ about 250 μg / cm² 2 or about 1 μg / cm² 2 ~ about 150 μg / cm² 2 A fibronectin solution of may be used. In some embodiments, the cell culture vessel is coated with 8 μg / cm² 2 ~ 125 μg / cm² 2 of fibronectin.

[0127] An example of an ECM for use in the method of the present invention contains at least one glycoprotein such as laminin.

[0128] A preferred ECM for use in the method of the present invention contains at least two distinct glycoproteins, for example, two different types of collagen or collagen and laminin. The ECM may be a synthetic hydrogel extracellular matrix or a natural ECM. A more preferred ECM is provided by Matrigel™ (BD Biosciences). Matrigel™ (BD Biosciences) contains laminin, entactin, and collagen IV. In some embodiments, the extracellular matrix is a laminin-containing extracellular matrix such as Matrigel™ (BD Biosciences).

[0129] In some embodiments, a single stem cell, cell population, or tissue fragment is embedded in matrigel. Matrigel is optionally low in growth factors and / or phenol red-free.

[0130] In some embodiments, the culture medium is placed on top of the ECM. Then, the culture medium can be removed and replenished as needed and when necessary. In some embodiments, the culture medium is replenished daily, every two days, every three days, every four days, every five days, every six days, or every seven days. If a component is "added" or "removed" from the medium, this means, in some embodiments, that the medium itself is removed from the ECM and then a new medium containing the "added" component or a new medium from which the "removed" component has been excluded is placed on top of the ECM.

[0131] In some embodiments, the culture medium of the present invention is in contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins, such as integrins.

[0132] In some embodiments, the basal culture medium comprises, or consists of, Advanced DMEM / F12 supplemented with penicillin / streptomycin, 10 mM HEPES, Glutamax, 1xN2, 1xB27 (all obtained from Invitrogen), and 1 mM N-acetylcysteine (Sigma).

[0133] Examples of the culture medium of the present invention In one aspect, the cell culture medium comprises a TGFβ inhibitor that binds to ALK5 and reduces its activity and a p38 inhibitor that binds to p38 and reduces its activity. For example, in one aspect, the cell culture medium comprises A83-01 and / or SB202190, preferably A83-01 + SB202190. Surprisingly, it has been found that using A83-01 + SB202190 together in the culture medium of the present invention multiplicatively increases the number of passages of human colon organoids. In one aspect, the cell culture medium comprises WENR + A83-01 + SB202190. In one aspect, the cell culture medium comprises WENR + A83-01 + SB202190 + nicotinamide. In one aspect, the cell culture medium comprises WENRg + nicotinamide + A83-01 + SB202190 (where "g" is gastrin). In one aspect, the cell culture medium comprises WENR + A83-01 + nicotinamide + FGF10. In one aspect, the cell culture medium comprises WENRg + A83-01 + nicotinamide + FGF10. In one aspect, the cell culture medium comprises WENRg + A83-01 + nicotinamide + FGF10 + SB202190. In one aspect, the cell culture medium is used to obtain colon organoids. Also provided are colon organoids that can be obtained by culturing epithelial cells using the cell culture medium as described in this aspect.

[0134] For example, in one aspect, the cell culture medium comprises WENRg + A83-01 + FGF10. The Wnt agonist is R-spondin, no other Wnt agonists are present, and no nicotinamide is present. For example, in some aspects, the cell culture medium comprises EGF (e.g., 50 ng / ml), R-spondin (e.g., 10% or 1 μg / ml), noggin (e.g., 100 ng / ml), FGF10 (e.g., 100 ng / ml), A8301 (e.g., 500 nM), and gastrin (e.g., 10 nM), and optionally SB202190. These components may be added to a basal medium, e.g., DMEM / F12 medium. In some aspects, one or more (e.g., 1, 2, 3, 4, or 5) or all of the components selected from the list comprising P / S, Glutamax, 10 nM Hepes, B27, N2, and N-acetylcysteine are further added to the basal medium. The use of such a cell culture medium has been found to be useful for obtaining pancreatic organoids. Pancreatic organoids obtained by culturing epithelial cells using the cell culture medium as described in this aspect are also provided. In some aspects, gastrin or nicotinamide or both gastrin and nicotinamide are excluded from the culture medium.

[0135] The tissue-specific culture medium of the present invention Particularly preferred culture media are described in the examples herein. The culture media of the present invention can be adapted for use with different tissues, for example, as follows.

[0136] Intestinal culture medium In some embodiments, the culture medium for small intestinal crypts, e.g., mouse small intestinal crypts, is a basal medium that additionally contains EGF, e.g., mouse EGF; a BMP inhibitor, e.g., mouse noggin; and an R-spondin, e.g., human R-spondin-1 or 4, and includes, or consists of, the basal medium as described above. In some embodiments, this culture medium further contains a TGF-β inhibitor (e.g., A83-01) and / or a p38 inhibitor (e.g., SB202190). In some embodiments, the culture medium for colonic crypts, e.g., mouse colonic crypts, is a basal medium that additionally contains a Wnt agonist, e.g., recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF, e.g., mouse EGF; a BMP inhibitor, e.g., mouse noggin; and an R-spondin, e.g., human R-spondin-1 or 4, and includes, or consists of, the basal medium as described above. In some embodiments, this culture medium further contains a TGF-β inhibitor (e.g., A83-01) and / or a p38 inhibitor (e.g., SB202190).

[0137] In some embodiments, the culture medium for human intestinal stem cells, human small intestinal crypts or human colonic crypts (also known as HISC culture medium) is a basal medium that additionally contains a Wnt agonist, e.g., recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, e.g., noggin; an R-spondin, e.g., human R-spondin-1; a TGF-β inhibitor, e.g., A83-01; a p38 inhibitor, e.g., SB202190; gastrin; and nicotinamide, and includes, or consists of, the basal medium as described above. In some embodiments, the p38 inhibitor and / or gastrin may be excluded from the HISC medium.

[0138] In some embodiments, the present invention provides a culture medium for culturing intestinal cells, which contains, or consists of, a basal medium, Wnt-3a, EGF, noggin, any one of R-spondin 1-4, a TGF-β inhibitor, nicotinamide, and preferably a p38 inhibitor.

[0139] In some embodiments, a culture medium for expanding small intestine stem cells or colon stem cells, such as human small intestine cells or colon cells, comprises a basal medium (e.g., containing Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), and glutamine / glutamax), Wnt3A (optionally, conditioned medium), any one of R-spondin 1-4 (preferably, 1 ug / ml), noggin (preferably, 50-100 ng / ml), nicotinamide (preferably, 10 mM), EGF (preferably, 10-50 ng / ml), gastrin (preferably, 10 nM), a TGF-β inhibitor, such as A83-01 (preferably, 500 nM), or consists of these. In a further embodiment, this culture medium additionally comprises a p38 inhibitor, such as SB202190 (preferably, 100 nM). In a further embodiment, this culture medium additionally comprises a Rock inhibitor, such as LY2157299.

[0140] In some embodiments, the present invention provides a culture medium for differentiating intestinal cells, comprising or consisting of a basal medium, EGF, noggin, a TGF-β inhibitor, and a p38 inhibitor.

[0141] In some embodiments, a culture medium for differentiating small intestinal stem cells or colonic stem cells, e.g., human small intestinal cells or colonic cells, comprises, or consists of, a basal medium (e.g., containing Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), and glutamine / glutamax), noggin (preferably at 50-100 ng / ml), EGF (preferably at 10-50 ng / ml), gastrin (preferably at 10 nM), a TGF-β inhibitor, e.g., A83-01 (preferably at 500 nM), and a p38 inhibitor, e.g., SB202190 (preferably at 100 nM). In some embodiments, gastrin may be excluded from this differentiation medium. In some embodiments, a γ-secretase inhibitor may be added (preferably at a concentration of 1 μM) to the differentiation medium. The γ-secretase inhibitor can affect cell fate determination during differentiation, e.g., cell fate determination towards secretory cells such as goblet cells. In some embodiments, RANKL may be added (e.g., at a concentration of 100 ng / ml) to the differentiation medium. RANKL can affect cell fate determination during differentiation, e.g., cell fate determination towards M cells.

[0142] Cancer culture medium In some embodiments, a culture medium for colon cancer cells comprises, or consists of, a basal medium, e.g., the aforementioned basal medium, additionally containing a Wnt agonist, e.g., recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, e.g., noggin; an R-spondin, e.g., human R-spondin-1; a TGF-β inhibitor, e.g., A83-01; a p38 inhibitor, e.g., SB202190; gastrin; and nicotinamide.

[0143] In one embodiment, a culture medium for colon cancer tumors, e.g., human colon cancer tumors, comprises a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), primocin and / or P / S (antibiotics) (500x), and hepes), R-spondin (optionally, conditioned medium) (preferably, 1 ug / ml), noggin (preferably, 100 ng / ml), nicotinamide (preferably, 10 mM), EGF (preferably, 50 ng / ml), gastrin (preferably, 50 nM), a TGF-β inhibitor, e.g., A83-01 (preferably, 500 nM), a p38 inhibitor, e.g., SB202190 (preferably, 10 uM), optionally, PGE2 (preferably, 10 nM) and / or a Rock inhibitor (preferably, 10 uM).

[0144] In some embodiments, colon cancer cells can also proliferate in HISC culture medium. In some embodiments, colon cancer cells can be cultured in HISC medium from which one or more or all of the following have been excluded: EGF, noggin, R-spondin, TGF-β inhibitor, and p38 inhibitor. Cancer cells may have mutations that constitutively activate or inactivate certain growth pathways. For example, many colon cancers result in constitutive activation of the Wnt pathway. In such cases, the medium does not require a Wnt agonist. With other mutations, other factors can be excluded from the medium as described above. Other epithelial cancers (carcinomas) can also proliferate in the culture medium of the present invention. In a preferred embodiment, cancer organoids obtained from cancer stem cells are optionally grown in a culture medium suitable for the growth of the corresponding normal tissue organoids obtained from normal stem cells, from which certain factors have been excluded. For example, gastric cancer organoids obtained by culturing gastric cancer stem cells may optionally be grown under the same culture conditions as normal gastric organoids obtained by culturing gastric stem cells, from which certain factors have been excluded. In another example, pancreatic cancer organoids obtained by culturing pancreatic cancer stem cells may optionally be grown under the same culture conditions as normal pancreatic organoids obtained by culturing pancreatic stem cells, from which certain factors have been excluded. In another example, prostate cancer organoids obtained by culturing prostate cancer stem cells may optionally be grown under the same culture conditions as normal prostate organoids obtained by culturing prostate stem cells, from which certain factors have been excluded. In another example, liver cancer organoids obtained by culturing liver cancer stem cells may optionally be grown under the same culture conditions as normal liver organoids obtained by culturing liver stem cells, from which certain factors have been excluded. In many cases, it may be preferable (or at least more convenient) to grow cancer organoids in normal tissue medium (in which no factors have been excluded). Without the exclusion of specific cancer mutations, normal tissue medium should be able to grow cancers with all genetic backgrounds.

[0145] Accordingly, in some aspects, the present invention provides a culture medium for culturing cancer cells, such as cancer stem cells, such as adenocarcinoma cells or carcinoma cells derived from a tissue type of interest. The culture medium comprises or consists of the components of a culture medium used to culture cells derived from the corresponding non-cancerous tissue type of interest, and optionally, one or more of the following are excluded from the medium used to culture non-cancerous cells of the tissue type of interest: Wnt-3a, EGF, noggin, R-spondin, TGF-β inhibitor, p38 inhibitor, nicotinamide, gastrin, FGF10, and HGF.

[0146] Adenoma culture medium In some aspects, a culture medium for an intestinal adenoma, such as a mouse intestinal adenoma, comprises a basal medium, such as the basal medium described above, additionally containing EGF, such as mouse EGF.

[0147] Stomach culture medium In some aspects, the present invention provides a medium for culturing gastric cells, comprising or consisting of a basal medium, Wnt-3a, EGF, noggin, any one of R-spondin 1-4, TGF-β inhibitor, gastrin, nicotinamide, FGF-10, and preferably a p38 inhibitor.

[0148] In some embodiments, a culture medium for gastric stem cells, e.g., human gastric stem cells, comprises a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), Primocin and / or P / S (antibiotics) (500x), and glutamine / glutamax), any one of R-spondin 1-4 (optionally, conditioned medium) (preferably, 1 μg / ml), Noggin (optionally, conditioned medium) (preferably, 100 ng / ml), Wnt3A (optionally, conditioned medium), nicotinamide (preferably, 5 mM), EGF (preferably, 50 ng / ml), FGF10 (preferably, 200 ng / ml), gastrin (preferably, 1 nM), a TGF-β inhibitor, e.g., A83-01 (preferably, 2 μM), or consists of these. The culture medium for gastric stem cells optionally further comprises a p38 inhibitor, e.g., SB202190 (preferably, 10 nM). The culture medium for gastric stem cells optionally further comprises PGE2 (preferably, 500 nM). The culture medium for gastric stem cells optionally further comprises a Rock inhibitor (preferably, 10 μM).

[0149] In some embodiments, a culture medium for gastric stem cells, e.g., mouse gastric cells, comprises a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), Primocin and / or P / S (antibiotics) (500x), and glutamine / glutamax), any one of R-spondin 1-4 (optionally, conditioned medium) (preferably, 1 μg / ml), Noggin (optionally, conditioned medium) (preferably, 100 ng / ml), Wnt3A (optionally, conditioned medium), EGF (preferably, 50 ng / ml), FGF10 (preferably, 200 ng / ml), gastrin (preferably, 1 nM), and a Rock inhibitor (preferably, 10 μM), or consists of these. In some embodiments, this culture medium further comprises a TGF-β inhibitor (e.g., A83-01) and / or a p38 inhibitor (e.g., SB202190).

[0150] Prostate culture medium In some embodiments, the culture medium for expanding prostate stem cells contains testosterone, optionally dihydrotestosterone (also referred to herein as DHT). Testosterone is a steroid hormone from the androgen group. In humans, a significant portion of testosterone undergoes 5α reduction to form dihydrotestosterone, a more potent androgen. Testosterone, dihydrotestosterone, or a testosterone mimetic (e.g., a molecule that mimics the activity of testosterone binding to the androgen receptor) can be added to the culture medium of the present invention. Thus, when the term testosterone is used, it can always be replaced with dihydrotestosterone or a testosterone mimetic. The inventors have shown that adding testosterone to the culture medium for prostate stem cells increases not only the differentiation but also the continuous expansion of the stem cell population (see, for example, FIGS. 41 - 45). This is extremely surprising since the literature discloses that testosterone plays an important role in cell differentiation by acting to suppress proliferation and maintain terminal differentiation (Mirochnik et al. PLoS One, 7(3), e31052, 2012; Niu et al. Oncogene 29, 3593 - 3604, 2010). One of ordinary skill in the art would expect that adding testosterone to the culture medium for the prostate would result in fully differentiated organoids with no further expansion capacity. This is similar to what is observed when colon organoids, pancreatic organoids, and liver organoids differentiate in a differentiation medium. However, in contrast, the inventors have discovered that testosterone increases differentiation but also continues to expand the stem cells. Thus, surprisingly, the organoids grown in a culture medium containing testosterone contain stem cells as well as differentiated cells, namely, luminal cells and basal cells.

[0151] In some embodiments, the culture medium for obtaining prostate organoids comprises a basal medium, and testosterone, optionally dihydrotestosterone, and any one of R-spondin 1-4 or an R-spondin mimetic. In some embodiments, the culture medium further comprises a BMP inhibitor, such as noggin. In some embodiments, the culture medium further comprises a tyrosine receptor kinase ligand. Optionally, the tyrosine receptor kinase ligand is a mitogenic growth factor such as EGF, FGF, KGF, or HGF. In some embodiments, the culture medium for obtaining prostate organoids comprises EGF, noggin, any one of R-spondin 1-4, and testosterone.

[0152] In a preferred embodiment, the culture medium for prostate cells comprises a TGF-β inhibitor. In some embodiments, the culture medium for prostate cells comprises EGF, noggin, any one of R-spondin 1-4, a TGF-β inhibitor, and testosterone. In some embodiments, the culture medium for prostate cells further comprises a p38 inhibitor. In some embodiments, the culture medium for obtaining prostate organoids does not comprise an inhibitor of the present invention, such as a TGF-β inhibitor and / or a p38 inhibitor. In some embodiments, the culture medium for prostate stem cells does not comprise testosterone. In some embodiments, the culture medium comprises a basal medium, EGF, noggin, and any one of R-spondin 1-4, optionally a TGF-β inhibitor, and does not comprise testosterone.

[0153] In some embodiments, the present invention provides a culture medium for culturing prostate cells, comprising a basal medium, EGF, any one of R-spondin 1-4, noggin, nicotinamide, a TGF-β inhibitor, preferably including Wnt-3a and FGF-10, or consisting of these. In some embodiments, the culture medium for culturing prostate cells further comprises testosterone, such as (dihydro)testosterone. In some embodiments, the culture medium further comprises a p38 inhibitor. In some embodiments, the culture medium for prostate cells, such as mouse, human, normal or cancerous prostate cells, comprises a basal medium (e.g., containing Advanced DMEM / F12, B27(50x), n-acetylcysteine (1 mM), and glutamine / glutamax), any one of R-spondin 1-4 (optionally, conditioned medium) (preferably, 1 μg / ml), noggin (optionally, conditioned medium) (preferably, 100 ng / ml), nicotinamide (preferably, 10 mM), EGF (preferably, 50 ng / ml), FGF10 (preferably, 100 ng / ml), a TGF-β inhibitor, such as A83-01 (preferably, 500 nM), (dihydro)testosterone (preferably, 1 nM - 10 nM), and optionally Wnt-3a. In some embodiments, this culture medium further comprises a Rock inhibitor (preferably, 10 μM). In some embodiments, the culture medium for prostate cells further comprises a p38 inhibitor, such as SB202190. In some embodiments, when culturing mouse prostate cells, the TGF-β inhibitor may be excluded from the culture medium. In other embodiments, nicotinamide, FGF10, and / or the Rock inhibitor may be excluded from the culture medium.

[0154] Pancreatic culture medium In some embodiments, the present invention provides a culture medium for expanding pancreatic cells, comprising a basal medium, any one of R-spondin 1-4, noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, preferably including exendin-4 and Wnt-3a, or consisting of these.

[0155] In one aspect, a culture medium for expanding pancreatic stem cells, such as human pancreatic stem cells, comprises a basal medium (e.g., containing Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), and glutamine / glutamax), any one of R-spondin 1 to 4 (optionally, conditioned medium) (preferably, 1 μg / ml), noggin (optionally, conditioned medium) (preferably, 100 ng / ml), nicotinamide (preferably, 10 mM), EGF (preferably, 50 ng / ml), FGF10 (preferably, 100 ng / ml), gastrin (preferably, 100 nM), and a TGF-β inhibitor, such as A83-01 (preferably, 2 μM), or consists of these. In a further aspect, this culture medium additionally contains Wnt-3a. In a further aspect, this culture medium additionally contains a p38 inhibitor, such as SB202190 (preferably, 100 nM). In a further aspect, this culture medium additionally contains a Rock inhibitor, such as LY2157299 (preferably, 10 μM). In a further aspect, this culture medium additionally contains exendin-4 (preferably, 50 ng / ml).

[0156] In some embodiments, a culture medium for expanding pancreatic stem cells, such as mouse pancreatic stem cells, comprises a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), Primocin and / or P / S (antibiotics), Hepes, and glutamine / glutamax), any one of R-spondin 1-4 (optionally, conditioned medium) (preferably, 1 μg / ml), Noggin (optionally, conditioned medium) (preferably, 100 ng / ml), nicotinamide (preferably, 10 mM), EGF (preferably, 50 ng / ml), FGF10 (preferably, 100 ng / ml), gastrin (preferably, 100 nM), and a TGF-β inhibitor, such as A83-01 (preferably, 2 μM), or consists of these. In a further embodiment, this culture medium additionally comprises a Rock inhibitor, such as LY2157299 (preferably, 10 μM). In some embodiments, the culture medium for pancreatic cells further comprises a p38 inhibitor, such as SB202190.

[0157] In some embodiments, the present invention provides a culture medium for differentiating pancreatic cells, comprising a basal medium, Noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor, preferably exendin-4, or consisting of these.

[0158] In some embodiments, a culture medium for differentiating pancreatic stem cells, such as human pancreatic stem cells, comprises, or consists of, a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), and glutamine / glutamax), noggin (preferably, 100 ng / ml), EGF (preferably, 50 ng / ml), FGF10 (preferably, 10 nM), gastrin (preferably, 100 nM), a TGF-β inhibitor, such as A83-01 (preferably, 50 nM), and a γ-secretase inhibitor (DAPT / DBZ) (preferably, 10 μM). In a further embodiment, this culture medium additionally comprises exendin-4 (preferably, 50 ng / ml). In some embodiments, the culture medium for pancreatic cells further comprises a p38 inhibitor, such as SB202190.

[0159] In some embodiments, a culture medium for differentiating pancreatic stem cells, such as mouse pancreatic stem cells, comprises, or consists of, a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), and glutamine / glutamax), EGF (preferably, 50 ng / ml), and a γ-secretase inhibitor (e.g., DAPT / DBZ) (preferably, 10 μM).

[0160] Barrett's esophagus culture medium In some embodiments, a culture medium for Barrett's esophagus comprises, or consists of, a basal medium, such as the aforementioned basal medium, additionally containing a Wnt agonist, such as recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as noggin; an R-spondin, such as human R-spondin-1; a TGF-β inhibitor, such as A83-01; a p38 inhibitor, such as SB202190; gastrin; nicotinamide; and an FGF, such as human FGF10 (i.e., HISC+FGF). In some embodiments, gastrin is excluded from this culture medium.

[0161] In some embodiments, the present invention provides a method for obtaining Barrett's esophageal organoids, comprising culturing epithelium isolated from Barrett's esophagus for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer in HISC culture medium, optionally in HISC culture medium additionally containing FGF10; and removing nicotinamide and SB202190 after the first 1 day, 2 days, 3 days, 4 days, or longer. In some embodiments, the culture medium additionally contains a Notch inhibitor such as DBZ. In some embodiments, the Barrett's esophageal organoids cultured in the presence of a Notch inhibitor contain few or no proliferating cells and contain more goblet cells compared to organoids cultured in the absence of a Notch inhibitor (see Figure 5).

[0162] Liver culture medium In some embodiments, liver cells can be grown in a first "expansion" culture medium (also referred to herein as EM) and preferably then cultured in a second "differentiation" culture medium (also referred to herein as DM). However, in some embodiments, the step of differentiating in DM medium is not performed in some methods, for example, where the cells are transplanted and differentiated in vivo. Similarly, there are expansion and differentiation culture media for other tissues such as the pancreas, small intestine, and colon (see above).

[0163] In one embodiment, the liver expansion medium contains EGF, a Wnt agonist, FGF, and nicotinamide. Preferably, the Wnt agonist is R-spondin 1-4 (e.g., any one or more of R-spondin 1, 2, 3, and 4), and thus the expansion medium is referred to as "ERFNic". A particularly preferred expansion medium additionally contains HGF and is referred to as "ERFHNic".

[0164] In some embodiments, a TGFβ inhibitor is added to the liver expansion medium. In some embodiments, TGFβ is present at at least 5 nM, such as at least 50 nM, at least 100 nM, at least 300 nM, at least 450 nM, at least 475 nM, such as 5 nM to 500 mM, 10 nM to 100 mM, 50 nM to 700 μM, 50 nM to 10 μM, 100 nM to 1000 nM, 350 to 650 nM, or more preferably 500 nM. The presence of a TGFβ inhibitor in the expansion medium is particularly preferred for human cell types.

[0165] In some embodiments, the present invention provides a culture medium for expanding liver cells, comprising a basal medium, any one of R-spondin 1-4, noggin, nicotinamide, EGF, FGF10, HGF, gastrin, a TGF-β inhibitor, and PGE2, preferably comprising or consisting of Wnt-3a.

[0166] In some embodiments, the liver expansion medium further comprises a p38 inhibitor.

[0167] In some embodiments, a prostaglandin signaling pathway activator (also referred to as a prostaglandin pathway activator) is added to the liver expansion medium (see FIG. 24). For example, any one or more of the compounds selected from the list including the following may be added to the liver expansion medium: phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, PGE2 and / or AA are added to the liver expansion medium. In some embodiments, PGE2 is added to the liver expansion medium to a final concentration of at least 10 nM, at least 30 nM, at least 40 nM, at least 45 nM, at least 50 nM, for example, 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 100 nM, 20 nM to 50 nM. In a preferred embodiment, PGE2 is added to the liver expansion medium to a final concentration of 50 nM. In some embodiments, AA is added to the liver expansion medium to a final concentration of at least 1 μg / ml, for example, at least 3 μg / ml, at least 5 μg / ml, at least 8 μg / ml, at least 9 μg / ml, at least 10 μg / ml, 1 μg / ml to 1000 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 100 μg / ml, 1 μg / ml to 50 μg / ml, or 5 μg / ml to 10 μg / ml. In a preferred embodiment, AA is added to the medium to a final concentration of 10 μg / ml.

[0168] In a preferred embodiment, a TGF-β inhibitor as well as a prostaglandin signaling pathway activator (e.g., PGE2 and / or AA), optionally a p38 inhibitor, are added to the liver expansion medium.

[0169] In a preferred embodiment, the liver expansion medium additionally contains gastrin.

[0170] In one embodiment, the liver differentiation medium contains EGF, a TGF-β inhibitor, FGF (e.g., FGF10, FGF2, or any other suitable FGF family member), and a Notch inhibitor. In one embodiment, the TGF-β inhibitor is A83-01, and / or the Notch inhibitor is DAPT. This differentiation medium is referred to herein as "EAFD" and is the preferred differentiation medium of the present invention. Optionally, HGF may be used instead of FGF, and both FGF and HGF may or may not be present in the differentiation medium. In some embodiments, instead of EGF, HGF or another receptor tyrosine kinase ligand may be used. Dexamethasone may also be added, for example, at a concentration of 10 nM to 10 μM. The liver differentiation medium may optionally contain a prostaglandin pathway activator such as PGE2 or AA. However, this component may also be excluded from the differentiation medium. In some embodiments, oncostatin M may also be added, for example, at a concentration of 1 ng / ml to 1 mg / ml, to assist in the differentiation towards a hepatocyte fate.

[0171] In some embodiments, the present invention provides a culture medium for differentiating liver cells, comprising or consisting of a basal medium, noggin, EGF, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor such as DAPT or DBZ, preferably Wnt-3a.

[0172] In some embodiments, liver cells may first be cultured in an expansion medium additionally containing Wnt and noggin, for example, in an "ENRW" medium containing EGF, noggin, R-spondin, and Wnt (e.g., Wnt-3A), optionally a prostaglandin pathway activator such as PGE2 or AA, and optionally a TGFβ inhibitor, optionally FGF, HGF, nicotinamide. In a preferred embodiment, to the liver expansion medium, one or more preferably both a TGF-β inhibitor and a prostaglandin signaling pathway activator (e.g., PGE2 and / or AA) are added.

[0173] In some embodiments, the expansion medium for the liver comprises EGF, noggin, gastrin, FGF, nicotinamide, a TGF-β inhibitor such as A83-01, HGF, R-spondin 1-4 (e.g., any one or more of R-spondin 1, 2, 3, and 4), and PGE2.

[0174] In a preferred embodiment, the liver cells may first be cultured in an expansion medium containing any one of EGF, noggin, gastrin, FGF10, nicotinamide, A8301, HGF, and R-spondin 1-4 to which PGE2 and / or AA has been added. R-spondin 1-4 may be provided in the form of Rspo conditioned medium. For example, the expansion medium may contain EGF (100 ng / ml, Invitrogen); noggin (25 ng / ml, peprotech); gastrin (10 nM, Sigma); FGF10 (e.g., 100 ng / ml, peprotech); nicotinamide (10 mM, sigma); A8301 (500 nM, Tocris); HGF (50 ng / ml, peprotech); Rspo conditioned medium (10%, e.g., 1 ug / ml) to which PGE2 (50 nM) and / or AA (10 ug / ml) has been added. The expansion medium may also contain a Rock inhibitor.

[0175] When expanding mouse liver cells, one or more of the following components may be excluded from the culture medium: the TGF-β inhibitor (e.g., A83-01) and PGE2.

[0176] The inventors have found that this medium is optimal for stimulating the initial expansion of cells for the first few days. Accordingly, this first expansion medium is sometimes referred to herein as EM1. In some embodiments, Wnt and noggin are removed after about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or later, for example, after 2 weeks, 1 month, 5 weeks, 8 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or later, or after 14 months, or later. Then, in some embodiments, the cells may be expanded in the expansion medium of the present invention that contains neither Wnt nor noggin. This second expansion medium is sometimes referred to herein as EM2. In some embodiments, the cells are cultured in EM2 for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer, for example, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, or longer. Then, the culture medium may be changed to the optimized differentiation medium containing a TGF-β inhibitor and a Notch inhibitor. Typically, the differentiation medium does not contain a Wnt agonist, an R-spondin, or nicotinamide. In some embodiments, the differentiation medium does not contain a prostaglandin pathway activator such as PGE2 or AA. Thereby, the cells are promoted to differentiate into mature hepatocytes and cholangiocytes. These cells are suitable for transplantation into humans or animals.

[0177] Expansion Medium for Liver (EM2): In one aspect of the present invention, there is provided a cell culture medium comprising, or consisting of, a basal medium for animal cells or human cells to which epidermal growth factor, an FGF capable of binding to FGFR2 or FGFR4 as a mitogenic growth factor, and preferably FGF10, nicotinamide, preferably a Wnt agonist, preferably R-spondin 1-4 are added. This medium is called EM2. This "EM2" medium is preferred for expanding liver cells.

[0178] In some embodiments, EM2 comprises prostaglandin pathway activators such as PGE2 and / or AA.

[0179] In some embodiments, EM2 comprises TGF-β inhibitors such as A83-01.

[0180] Preferably, the Wnt agonist is R-spondin 1-4. A medium containing EGF, R-spondin 1-4, FGF, and nicotinamide is referred to herein as ERFNic.

[0181] In some embodiments, the EM2 medium does not contain noggin, and more preferably does not contain a BMP inhibitor. In some embodiments, the EM2 medium does not contain Wnt, such as Wnt-3a.

[0182] In some embodiments, HGF is present in addition to FGF. A preferred medium containing HGF in addition to FGF is ERFHNic (EGF + R-spondin (preferably, R-spondin 1-4) + FGF (preferably, FGF10) + HGF + nicotinamide + prostaglandin pathway inhibitor, e.g., PGE2 and / or AA, and TGF-β inhibitor. The inventors have found that the ERFHNic medium containing TGF-β inhibitor and prostaglandin pathway activator is an optimal medium for long-term cell expansion. In the absence of HGF, the cells did not survive in culture for more than 3 months. Furthermore, after 10 passages in the absence of HGF, the cells showed a growth disadvantage compared to cells cultured in the presence of HGF, as demonstrated by a low growth ratio. In particular, after 15 passages, the cells did not grow organoids at the same rate ratio as in the presence of HGF. Therefore, HGF has been found to be extremely important for maintaining a good growth rate during long-term culture. Accordingly, the present invention provides the use of the ERFHNic medium of the present invention for culturing cells for at least 2 weeks, at least 1 month, at least 2 months, more preferably at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 24 months, at least 25 months, at least 30 months, or longer, e.g., for more than 3 years. In fact, some embodiments of the present invention include the use of EM2 for subculturing cells about 20-50 times. For example, the cells may be split 4-8 times a week for 7-10 consecutive weeks. Preferably, the cells expand at a rate of about 4-5 times per week or more than 2 population doublings per week.Furthermore, the present invention provides the use of the ERFHNic medium of the present invention for culturing cells for at least 8 passages, for example, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 15 passages, at least 20 passages, at least 25 passages, at least 30 passages, at least 40 passages, at least 50 passages, at least 60 passages, or 15 to 35 passages, for example, about 20 to 30 passages. In some embodiments, a TGF-β inhibitor, such as A83-01, is additionally present in the EM2 medium. This is particularly useful when culturing human cells or organoids. In some embodiments, A83-01 is present at a concentration of 400 to 600 nM, for example, 450 to 550 nM, 470 to 530 nM, or about 500 nM. In embodiments where a TGF-β inhibitor is present in EM2, preferably, no Notch inhibitor is present. In some embodiments, EM2 additionally contains a p38 inhibitor.

[0183] Expansion Medium for Liver (EM1): In one aspect, the present invention provides a cell culture medium comprising, or consisting of, a basal medium for animal or human cells to which EGF, a BMP inhibitor, R-spondin, and Wnt are added. Preferably, the BMP inhibitor is noggin, and the EM1 medium is referred to as "ENRW" (EGF, noggin, R-spondin, and Wnt (e.g., Wnt3A)). This medium is called EM1. In some embodiments, EM1 additionally contains a prostaglandin pathway activator such as PGE2 and / or AA. In some embodiments, EM1 contains a TGF-β inhibitor such as A83-01. More preferably, EM1 additionally contains a prostaglandin pathway activator and a TGF-β inhibitor. The inventors have found that a medium containing Wnt and noggin is ideal for stimulating the initial expansion of cells. Thus, in some embodiments, the EM1 medium is used for only 1 passage or 1 week, but it is also expected to be usable for about 1 year as it is not harmful to the cells. Thus, in some embodiments, the EM1 medium is used to culture cells from day 0 to day 10, e.g., from 0 to 7 days, 0 to 6 days, 0 to 5 days, 0 to 4 days, 0 to 3 days, 0 to 2 days, 0 to 1 day, or for more than 1 week, e.g., 2 weeks, 3 weeks, 4 weeks or more, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. In this case, day 0 is the day when the cells are isolated from the origin tissue of the cells, and day 1 is the next day. In some embodiments, the EM1 medium is used only on the first day of culture or only for the first 2 days. In some embodiments, the EM1 medium is used for 1 passage or more, e.g., 1 passage, 2 passages, 3 passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 15 passages, 20 passages, 25 passages, 30 passages or more, e.g., 20 to 30 passages, 30 to 35 passages, 32 to 40 passages, or longer. In some embodiments, the EM1 medium is used after the freezing step or after any other optional transport step involving a medium or temperature change that is not compatible with optimal growth. This "EM1" medium is preferred for expanding liver cells.

[0184] One or more compounds selected from the group consisting of FGF, HGF, nicotinamide, gastrin, B27, N-acetylcysteine, and N2 are added to the EM1 medium. When starting the culture from a frozen stock or single cells, a ROCK inhibitor is preferably added to the EM1 medium. Y27632 is a preferred ROCK inhibitor for use in the present invention.

[0185] Thus, in one aspect, As mitogenic growth factors, epidermal growth factor, FGF (e.g., FGF that can bind to FGFR2 or FGFR4), preferably FGF10, and HGF, Prostaglandin pathway activators, such as PGE2 and / or AA; TGF-β inhibitor; Gastrin, nicotinamide, B27, N2, and N-acetylcysteine, and preferably; BMP inhibitor, preferably noggin; and Wnt agonist, preferably R-spondin 1 and / or Wnt-3a A cell culture medium is provided that comprises or consists of a basal medium for animal cells or human cells to which is added.

[0186] B27 (Invitrogen), N-acetylcysteine (Sigma) and N2 (Invitrogen), gastrin (Sigma) and nicotinamide (Sigma) are also added to the medium defined above and are believed to control cell growth and assist in DNA stability. In the context of the present invention, nicotinamide is also referred to herein as "Nic".

[0187] The "N2 supplement" can be obtained from Invitrogen, Carlsbad, CA; www.invitrogen.com; catalog number 17502-048; and PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F005-004; Bottenstein & Sato, PNAS, 76(1):514-17, 1979. The N2 supplement is supplied by PAA Laboratories GmbH as a 100x liquid concentrate containing 500 μg / ml human transferrin, 500 μg / ml bovine insulin, 0.63 μg / ml progesterone, 1611 μg / ml putrescine, and 0.52 μg / ml sodium selenite. The N2 supplement may be added to the culture medium as a concentrate or diluted before addition to the culture medium. The N2 supplement may be used at a 1x final concentration or at other final concentrations. The use of the N2 supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine, and sodium selenite into the culture medium of the present invention. In some embodiments where the medium contains B27, the medium may also not contain N2. Thus, where B27 is present, the embodiments of the present invention can be adjusted to exclude N2 if desired.

[0188] To formulate a culture medium containing biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin, a "B27 supplement" (available from Invitrogen, Carlsbad, CA; www.invitrogen.com; currently catalog number 17504-044; and PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F01-002; from Brewer et al., J Neurosci Res., 35(5):567-76, 1993) may be used. The B27 supplement is supplied by PAA Laboratories GmbH as a 50x liquid concentrate containing, among other components, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin. Of these components, at least linolenic acid, retinol, retinyl acetate, and triiodothyronine (T3) are nuclear hormone receptor agonists. The B27 supplement may be added to the culture medium as a concentrate or may be diluted before addition to the culture medium. The B27 supplement may be used at a 1x final concentration or at other final concentrations. The use of the B27 supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin into the culture medium of the present invention.

[0189] For example, the cell culture medium may contain, or consist of, EGF and R-spondin 1 to which FGF10, HGF, and nicotinamide are added; for example, EGF (50 ng / ml) and R-spondin 1 (1 μg / ml) to which FGF10 (100 ng / ml), HGF (25 - 50 ng / ml), nicotinamide (1 - 10 mM), prostaglandin pathway activators, such as PGE2 (50 nM) and / or AA (10 μg / ml), and TGF-β inhibitors, such as A83-01 (500 nM) are added. In some embodiments, the medium additionally contains a p38 inhibitor. The inventors have discovered that this medium can be used for long-term expansion of cells. Thus, this cell culture medium is preferred for use as the EM2 of the present invention. Preferably, B27, N2, and 200 ng / ml N-acetylcysteine are added to the basal medium. In some embodiments, the basal medium is Advanced-DMEM / F12. However, any other suitable basal medium may be used.

[0190] Another example of a cell culture medium and a method of using this medium is Advanced-DMEM / F12, preferably containing, or consisting of, Advanced-DMEM / F12 to which B27, N2, 200 ng / ml N-acetylcysteine, 50 ng / ml EGF, 1 μg / ml R-spondin 1, 10 nM gastrin, 100 ng / ml FGF10, 10 mM nicotinamide, 50 ng / ml HGF, 50% Wnt conditioned medium, prostaglandin pathway activators, such as PGE2 (50 nM) and / or AA (10 μg / ml), and TGF-β inhibitors, such as A83-01 (500 nM), preferably 10 - 100 ng / ml noggin are added. The Wnt conditioned medium also contains Advanced DMEM, P / S, B27, N2, and FCS. 293T cells transfected with a Wnt3A expression plasmid produce Wnt. After several days, all the medium is collected (i.e., together with the secreted Wnt) and used as a Wnt source.

[0191] Therefore, the present invention provides a cell culture medium containing, or consisting of, a basal medium for animal or human cells, to which a mitogenic growth factor capable of binding to epidermal growth factor, FGFR2 or FGFR4, preferably FGF10, and HGF, a prostaglandin pathway activator, such as PGE2 and / or AA, a TGF-β inhibitor; gastrin, nicotinamide, B27, N2, and N-acetylcysteine, and preferably a BMP inhibitor, more preferably noggin, and a Wnt agonist, more preferably R-spondin 1 and / or Wnt-3a are added.

[0192] Therefore, the present invention provides a first preferred culture medium containing, or consisting of, a basal medium for animal or human cells, to which a mitogenic growth factor, such as epidermal growth factor, FGF10, and HGF, a prostaglandin pathway activator, such as PGE2 and / or AA, a TGF-β inhibitor; gastrin, nicotinamide, B27, N2, and N-acetylcysteine, a BMP inhibitor, more preferably noggin, and a Wnt agonist, more preferably R-spondin 1 and Wnt-3a are added.

[0193] In some embodiments, a p38 inhibitor is added to the expansion medium.

[0194] This medium may be used as the EM1 cell culture medium of the present invention to stimulate the initial expansion of cells. In some embodiments, the medium used as the EM1 cell culture medium contains all the components of the EM2 medium of the present invention and additionally contains Wnt-3a and noggin.

[0195] In an embodiment where N-acetylcysteine, B27, and N2 are added to the basal medium, the culture medium preferably has the following added thereto: EGF, R-spondin 1, gastrin, FGF10, nicotinamide, as well as HGF, as well as Wnt conditioned medium, and a prostaglandin pathway activator, for example, PGE2 and / or AA. Preferably, N-acetylcysteine, EGF, R-spondin 1, gastrin, FGF10, nicotinamide, as well as HGF, as well as Wnt conditioned medium, and a prostaglandin pathway activator, for example, PGE2 and / or AA are added to the basal medium according to the amounts described above. Preferably, a TGF-β inhibitor is also present in the amounts described herein.

[0196] For example, in some embodiments, N-acetylcysteine at 150 ng / ml to 250 ng / ml may be added to the basal medium. Preferably, N-acetylcysteine at about 200 ng / ml or exactly 200 ng / ml is added to the basal medium. For example, in some embodiments, EGF at 40 ng / ml to 60 ng / ml may be added to the basal medium. Preferably, EGF at about 50 ng / ml or exactly 50 ng / ml is added to the basal medium. For example, in some embodiments, R-spondin 1 at 0.5 μg / ml to 1.5 μg / ml may be added to the basal medium. Preferably, R-spondin 1 at about 1 μg / ml or exactly 1 μg / ml is added to the basal medium. For example, in some embodiments, gastrin at 5 nM to 15 nM may be added to the basal medium. Preferably, gastrin at about 10 nM or exactly 10 nM is added to the basal medium. For example, in some embodiments, FGF10 at 25 to 200 ng / ml, for example, FGF10 at 70 ng / ml to 130 ng / ml may be added to the basal medium. Preferably, FGF10 at about 100 ng / ml or exactly 100 ng / ml is added to the basal medium. For example, in some embodiments, nicotinamide at 5 mM to 15 mM may be added to the basal medium. Preferably, nicotinamide at about 10 mM or exactly 10 mM is added to the basal medium. For example, in some embodiments, HGF at 25 ng / ml to 100 ng / ml, for example, HGF at 35 ng / ml to 65 ng / ml may be added to the basal medium. Preferably, HGF at about 50 ng / ml or exactly 50 ng / ml is added to the basal medium. For example, in some embodiments, 35% to 65% of Wnt conditioned medium may be added to the basal medium. Preferably, 50% or exactly 50% of Wnt conditioned medium is added to the basal medium.

[0197] For example, in some embodiments, a prostaglandin signaling pathway activator is added to the liver expansion medium (see Figure 24). For example, any one or more of the compounds selected from the list including the following may be added to the liver expansion medium: phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, PGE2 and / or AA are added to the liver expansion medium. In some embodiments, PGE2 is added to the liver expansion medium to a final concentration of at least 10 nM, such as 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 100 nM, 20 nM to 50 nM. In a preferred embodiment, PGE2 is added to the liver expansion medium to a final concentration of 50 nM. In some embodiments, AA is added to the liver expansion medium to a final concentration of at least 1 μg / ml, such as 1 μg / ml to 1000 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 100 μg / ml, 1 μg / ml to 50 μg / ml, or 5 μg / ml to 10 μg / ml. In a preferred embodiment, AA is added to the medium to a final concentration of 10 μg / ml.

[0198] In some embodiments, neither gastrin nor N2 is present in the cell culture medium.

[0199] Preferably, the basal medium is advanced-DMEM / F12.

[0200] This first culture medium (e.g., EM1, EM2, or both EM1 and EM2) is preferably used during the first two weeks of the culturing method of the present invention. However, this first culture medium may be used for a shorter period, for example, 1 day, 2 days, 3 days, 5 days, 7 days, or 10 days, or a longer period, for example, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, or longer, for 5 months or more, for example, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more.

[0201] Differentiation medium for the liver (DM): In another aspect, there is provided a second cell culture medium comprising, or consisting of, a basal medium for animal or human cells to which the following are added: EGF, a TGF-β inhibitor, a Notch inhibitor, and a prostaglandin pathway activator, such as PGE2 and / or AA. The inventors have found that this medium is useful for differentiating cells. The medium used for differentiating cells may sometimes be referred to herein as DM.

[0202] Preferably, the second cell culture medium also contains FGF and / or HGF.

[0203] In one embodiment, the second culture medium contains, as mitogenic growth factors, epidermal growth factor, FGF10, and HGF, a Notch inhibitor; a TGF-β inhibitor; and a prostaglandin pathway activator, such as PGE2 and / or AA and comprises, or consists of, a basal medium for animal or human cells.

[0204] In one aspect, the TGF-β inhibitor is A83-01, and / or the Notch inhibitor is DAPT. In another aspect, the DM cell culture medium additionally contains dexamethasone. In another aspect, the DM cell culture medium additionally contains oncostatin M. In another aspect, the DM cell culture medium additionally contains gastrin.

[0205] Preferred second cell culture media and methods of using the same are described in the Examples and include, or consist of, a basal medium to which 50 ng / ml EGF, 100 ng / ml FGF10, 50 nM A8301, and 10 μM DAPT are added. Advanced-DMEM / F12 may be used as the basal medium, and similarly, any other suitable basal medium may be used.

[0206] In some aspects, a differentiation medium for liver cells, such as human liver cells, comprises, or consists of, a basal medium (e.g., Advanced DMEM / F12, B27 (50x), n-acetylcysteine (1 mM), containing glutamine / glutamax), noggin (preferably 100 ng / ml), EGF (preferably 50 ng / ml), gastrin (preferably 10 nM), a TGF-β inhibitor, such as A83-01 (preferably 50 nM), and a γ-secretase inhibitor (e.g., DAPT / DBZ) (preferably 10 μM).

[0207] In some aspects, a differentiation medium for liver cells, such as mouse liver cells, comprises, or consists of, a basal medium (e.g., Advanced DMEM / F12, B27 (50x), preferably containing n-acetylcysteine (1 mM) glutamine / glutamax), EGF (preferably 50 ng / ml), FGF10 (preferably 100 ng / ml), gastrin (preferably 10 nM), a TGF-β inhibitor, such as A83-01 (preferably 50 nM), and a γ-secretase inhibitor (e.g., DAPT / DBZ) (preferably 10 μM).

[0208] In some embodiments, the second cell culture medium contains neither R-spondin nor Wnt. In some embodiments, the second cell culture medium does not contain a Wnt agonist. In some embodiments, the second cell culture medium does not contain nicotinamide. In some embodiments, the second cell culture medium does not contain a BMP inhibitor. In some embodiments, the second cell culture medium does not contain a prostaglandin pathway activator such as PGE2 and / or AA.

[0209] The inventors discovered that both R-spondin 1 and nicotinamide inhibit the expression of the mature hepatocyte marker CYP3A11, but promote the expression of the hepatoblast marker albumin. Thus, to increase the differentiation of cells towards a mature liver fate, the inventors removed R-spondin and nicotinamide from the cell culture. The inventors also discovered that the expression of certain bile duct transcription factors is highly upregulated in expanded cultures containing R-spondin 1. This indicates that the cultured gene expression was skewed towards a bile duct cell fate. The Notch signaling pathway and the TGF-β signaling pathway have been linked to a bile duct cell fate in vivo. Indeed, abnormal duct formation occurs when Rbpj (which is essential for realizing active Notch signaling) is deleted (Zong Y. Development 2009), and the addition of TGF-β to liver explants promotes bile duct differentiation in vitro (Clotman F. Genes and Development 2005). Since both the Notch signaling pathway and the TGF-β signaling pathway were highly upregulated in liver cultures (Figure 22), the inventors thought that cell differentiation could be induced towards the hepatocyte phenotype by inhibiting the bile duct cell fate. It has been discovered that the addition of a TGF-β inhibitor (e.g., A8301) and a Notch inhibitor (e.g., DAPT) to a differentiation medium, preferably a differentiation medium that contains neither R-spondin nor Wnt, increases the expression of mature hepatocyte markers and the number of hepatocyte-like cells (see, for example, Example 5).

[0210] General culture medium The cell culture medium according to the present invention enables the survival and / or proliferation and / or differentiation of epithelial stem cells or isolated crypts on the extracellular matrix. In some embodiments, the cell culture medium according to the present invention enables the survival and / or proliferation and / or differentiation of the organoids of the present invention on the extracellular matrix, such as crypt-villus organoids, colon organoids, pancreatic organoids, gastric organoids, Barrett's esophagus organoids, adenocarcinoma organoids, or colorectal carcinoma organoids. In some embodiments, the cell culture medium according to the present invention enables the survival and / or proliferation and / or differentiation of the organoids of the present invention on the extracellular matrix, such as small intestine (crypt-villus) organoids, colon organoids, pancreatic organoids, gastric organoids, Barrett's esophagus organoids, adenocarcinoma organoids, carcinoma organoids, colorectal carcinoma organoids, prostate organoids, or prostate carcinoma organoids. Preferably, in embodiments where a TGF-β inhibitor and / or a p38 inhibitor are present, the cell culture medium enables survival and / or proliferation, preferably the survival and proliferation of the cell population or organoids of the present invention. Preferably, initially, a TGF-β inhibitor and / or a p38 inhibitor are present in the cell culture medium, but then are removed from the medium (e.g., by not adding during medium replenishment), such embodiments enable survival and / or differentiation, preferably the survival and differentiation of the cell population or organoids of the present invention.

[0211] In some embodiments, a p38 inhibitor is added to any of the media described herein.

[0212] The term cell culture medium is synonymous with medium, culture medium, or cell medium.

[0213] Use of the culture medium of the present invention The present invention also provides the use of the culture medium of the present invention for expanding and / or differentiating stem cells, stem cell populations, tissue fragments, or organoids.

[0214] In some embodiments, the stem cells, stem cell population, tissue fragment, or organoid is selected from the group consisting of one or more intestinal stem cells, small intestinal crypts, colonic crypts, gastric stem cells, liver stem cells, pancreatic stem cells, and prostate stem cells.

[0215] In some embodiments, the stem cells, stem cell population, tissue fragment, or organoid can be obtained from normal tissue.

[0216] In some embodiments, the stem cells, stem cell population, tissue fragment, or organoid can be obtained from diseased tissue, such as diseased tissue from a patient with an adenoma, carcinoma, adenocarcinoma, intestine of a patient with cystic fibrosis, or intestine of a patient with inflammatory bowel disease.

[0217] Stem cells cultured according to the present invention Stem cells are found in many organs of adult humans and mice. Although there may be significant variation in the exact characteristics of adult stem cells in individual tissues, adult stem cells share at least the following characteristics: maintaining an undifferentiated phenotype; being able to differentiate into all lineages present in the relevant tissue; retaining self-renewal ability throughout life; and being able to regenerate the relevant tissue after injury. Stem cells are present in a special location called the stem cell niche. The stem cell niche provides appropriate cell-cell adhesion and signals for maintaining the stem cell population. The stem cells according to the present invention preferably express Lgr5.

[0218] In one embodiment, the present invention provides a cell population or one or more organoids containing the stem cells produced or obtained by the step of culturing the stem cells or tissue fragments according to the present invention. The stem cells or tissue fragments have been cultured for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer.

[0219] A "population" of cells is any number of cells greater than one, but preferably at least 1x10 3cells, at least 1x10 4 cells, at least 1x10 5 cells, at least 1x10 6 cells, at least 1x10 7 cells, at least 1x10 8 cells, or at least 1x10 9 cells.

[0220] The stem cells of the present invention cultured according to the present invention may be human stem cells. The stem cells of the present invention cultured according to the present invention may be epithelial stem cells.

[0221] In some embodiments, the stem cells of the present invention and / or the stem cells cultured according to the present invention are not embryonic stem cells. In some embodiments, the stem cells of the present invention and / or the stem cells cultured according to the present invention are not human embryonic stem cells. Preferably, the stem cells of the present invention are adult stem cells.

[0222] In a preferred embodiment, the stem cells may be human epithelial stem cells. Human epithelial stem cells include stem cells derived from human epithelial tissues. These include, but are not limited to, tissues selected from the group consisting of pancreatic tissue, small intestine tissue, large intestine tissue, corneal tissue, olfactory tissue, respiratory tissue, stomach tissue, liver tissue and skin tissue, breast tissue and / or prostate tissue, for example, pancreatic tissue, small intestine tissue, large intestine tissue, corneal tissue, olfactory tissue, and respiratory tissue. Epithelial stem cells can form distinct cell types that make up the epithelium. Some epithelia, such as skin or intestine, exhibit rapid cell turnover. From this, it can be seen that the stem cells present must proliferate constitutively. Other epithelia, such as liver or pancreas, exhibit a very slow turnover under normal conditions.

[0223] Intestinal stem cells The self-renewing epithelium of the small intestine is organized into ordered crypts and villi (Gregoreff and Clevers, 2005 Genes Dev 19, 877-90). Along the crypt-villus axis, each cell is polarized, such that cells at the top of the intestinal villi or in the upper part of the colonic crypts are the most differentiated and are continuously lost into the lumen by apoptosis. Stem cells at the base of the crypts continuously proliferate, and the precursor cells in the middle of the crypts proliferate extensively, ensuring that the lost cells are properly replaced. The resulting epithelial turnover time is 5 days in mice. Self-renewing stem cells have long been known to exist near the bottom of the crypts and to give rise to rapidly proliferating transit amplifying (TA) cells that can differentiate into all lineages. The estimated number of stem cells is 4-6 per crypt (Bjerknes and Cheng, 1999 Gastroenterology 116, 7-14). Three differentiated cell types, enterocytes, goblet cells, and enteroendocrine cells, are formed from TA cells and continue to migrate within bands that are closely apposed to each other along the crypt-villus axis. Each villus receives cells from multiple different crypts. The fourth major differentiated cell type, Paneth cells, is present at the bottom of the crypts.

[0224] The colon resembles the small intestine but has a flat surface epithelium instead of villi. Colonic crypts are organized like small intestinal crypts. Paneth cells are not present in colonic crypts. Instead, there are so-called "Deep Crypt Secretory" cells. The flat surface of the epithelium contains differentiated cells (colonocytes and secretory cells). Differentiated goblet cells are also intermingled with TA cells and occur throughout the crypts.

[0225] Isolation of tissue fragments and stem cells Crypts can be isolated from the small intestine and large intestine, including the duodenum, jejunum, ileum, and colon, as well as the pyloric and body regions of the stomach, by protocols known to those skilled in the art. For example, crypts can be isolated by incubating the isolated tissue with a chelating agent that dissociates cells from calcium-dependent and magnesium-dependent interactions with the basement membrane and stromal cell types. After washing the tissue, the epithelial cell layer is separated from the submucosa using a glass slide and incised. Thereafter, incubation is carried out in trypsin, or more preferably EDTA and / or EGTA, and undigested tissue fragments and single cells are separated from the crypts using, for example, filtration and / or centrifugation steps. Other proteolytic enzymes such as collagenase and / or dispase I can be used instead of trypsin. Similar methods are used to isolate pancreatic and gastric fragments. Similar methods may be used to isolate fragments of other tissues described herein. The culture medium of the present invention is suitable for culturing such tissue fragments (see Example 1).

[0226] The culture medium according to the present invention enables the establishment of long-term culture conditions in which a single crypt undergoes crypt multi-division events and at the same time a villus-like epithelial region in which all differentiated cell types are present develops. After culturing the crypts, the cultured crypts undergo dramatic morphological changes. The upper opening of freshly isolated crypts is blocked, this region gradually swells, is filled with apoptotic cells, and resembles and constricts and separates at the villus tip with apoptotic cells. The crypt region undergoes a continuous budding event that creates additional crypts, a process reminiscent of crypt division. In a preferred embodiment of the present invention, the organoids contain villus-like extensions containing all differentiated epithelial cell types including proliferative cells, Paneth cells, enterocytes, and goblet cells. No myofibroblasts or other non-epithelial cells were identified at any stage of the organoids.

[0227] By the expansion of the budding crypt structure, an organoid is created that contains a crypt-like structure surrounding a central lumen covered by a villus-like epithelium and filled with apoptotic cell bodies. The crypt-villus organoid contains a central lumen covered by a villus-like epithelium. The lumens are continuously and intermittently opened to release their contents into the culture medium.

[0228] When a single epithelial stem cell is cultured, a similar crypt-villus organoid structure is formed. After about one week, a structure very similar to the crypt-villus organoid structure obtained using intact crypts is formed.

[0229] Methods for isolating stem cells are known, and the method suitable for use with the present invention can be selected by those skilled in the art depending on the type of stem cell used. For example, isolation of epithelial stem cells may be performed using a compound that binds to Lgr5 and / or Lgr6, which are unique cell surface markers on epithelial stem cells. Examples of such compounds are anti-Lgr5 antibody and anti-Lgr6 antibody.

[0230] In some aspects of the present invention, single Lgr5+ epithelial stem cells, e.g., single Lgr5+ epithelial stem cells derived from the colon, small intestine, or pancreas, can be used to form organoids such as colon organoids, crypt-villus organoids, or pancreatic organoids, respectively.

[0231] In a further example, single Lgr5+ epithelial stem cells derived from the liver, prostate, or stomach can be used to form organoids such as liver organoids, prostate organoids, or stomach organoids, respectively.

[0232] In another aspect, organoids can be obtained using the methods and culture media described herein using tissue fragments containing Lgr5+ stem cells, e.g., cultured crypts derived from the intestinal tract.

[0233] In some embodiments, a single Lgr5+ epithelial stem cell or tissue fragment may be a cancer stem cell or cancer tissue fragment, such as a cancer stem cell or cancer tissue fragment derived from a carcinoma or adenocarcinoma. In some embodiments, a single Lgr5+ epithelial stem cell may also be a stem cell or tissue fragment derived from a neoplastic condition or diseased tissue, such as Barrett's esophagus, cystic fibrosis, or an adenoma. Organoids obtained from cancer starting materials, neoplastic starting materials, or diseased starting materials have characteristics similar to in vivo starting materials and are thus useful as research tools for drug screening, target validation, target discovery, toxicology and toxicity screening, personalized medicine, regenerative medicine, and ex vivo cell / organ models, such as disease models. In one embodiment, the present invention provides an organoid produced or obtained by culturing human stem cells or tissue fragments according to the methods of the present invention. In one embodiment, the present invention provides a crypt-villus organoid or a gastric organoid or a pancreatic organoid or a colonic organoid or a Barrett's esophagus organoid or an adenocarcinoma organoid or a colon carcinoma organoid produced or obtained by culturing human stem cells or tissue fragments according to the methods of the present invention. In one embodiment, the present invention provides a prostate organoid produced or obtained by culturing human stem cells or tissue fragments according to the methods of the present invention. Such populations of organoids produced or obtained by culturing human stem cells or tissue fragments according to the methods of the present invention, such as crypt-villus organoids, gastric organoids, or pancreatic organoids, may each contain more than 10, preferably more than 20, more preferably more than 40 organoids. The organoid collection preferably contains at least 10% viable cells, more preferably at least 20% viable cells, more preferably at least 50% viable cells, more preferably at least 60% viable cells, more preferably at least 70% viable cells, more preferably at least 80% viable cells, more preferably at least 90% viable cells. The viability of the cells can be evaluated using Hoechst staining or propidium iodide staining in FACS.

[0234] The inventors have shown that the culture medium and method of the present invention can be used to culture cancer cell lines including colorectal cancer and adenocarcinoma (see Example 1). As described in Example 1, this culture technique can be widely applied as a research tool for infectious diseases, inflammatory diseases, and neoplastic diseases. Therefore, the stem cells according to the present invention may be cancer stem cells. In some aspects of the present invention, the cancer stem cells can form adenoma organoids or colorectal cancer organoids. In some aspects, these organoids contain Ki67+ cells (Thermo Scientific * Cellomics, Millipore).

[0235] Similarly, the inventors have shown that the culture media and methods of the present invention can be used to culture stem cells having other disease genotypes and / or phenotypes. For example, intestinal stem cells collected from patients with cystic fibrosis can be expanded using the culture media and methods of the present invention. These stem cells maintain the genotype and phenotype of cystic fibrosis. Thus, in some embodiments of the present invention, the stem cells are from a patient having a disease such as cystic fibrosis, inflammatory bowel disease (e.g., Crohn's disease), cancer, adenoma, adenocarcinoma, colon cancer, diabetes (e.g., type I or type II), Barrett's esophagus, Gaucher's disease, α-1-antitrypsin deficiency, Lesch-Nyhan syndrome, anemia, Schwachman-Bodian-Diamond syndrome, polycythemia vera, primary myelofibrosis, glycogen storage disease, familial hypercholesterolemia, Crigler-Najjar syndrome, hereditary hypertyrosinemia, Pompe disease, progressive familial intrahepatic cholestasis, Hurler syndrome, SCID or leaky SCID, Omenn syndrome, cartilage-hair hypoplasia, herpes simplex encephalitis, scleroderma, osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, dyskeratosis congenita, etc. In some embodiments of the present invention, disease organoids can be obtained by culturing stem cells collected from a human or animal having the disease. The disease organoids still have the characteristics of the tissue from which they were obtained. Thus, cystic fibrosis small intestine organoids grown from small intestine crypts are included in the definition of small intestine organoids. Similarly, colon cancer organoids are included in the definition of colon organoids.

[0236] There is some confusion in the literature regarding the definition of cancer stem cells. Here, the inventors follow the consensus view in a recent AACR workshop (Clarke et al., 2006. Cancer Res. 66:9339-44) that cancer stem cells are "tumor cells with the ability of self-renewal and the ability to give rise to heterogeneous cancer cell lineages that constitute the tumor." Thus, cancer stem cells can only be defined experimentally by their ability to recapitulate the development of continuously growing tumors. Other terms in the literature include tumor-initiating cells and tumor-forming cells. Cancer stem cell activity assays need to address the ability of self-renewal and tumor growth. Currently, the gold standard assay is serial xenotransplantation into immunodeficient mice. Furthermore, in the context of the present invention, cancer stem cells typically express Lgr5. However, in some embodiments, cancer-initiating / growing / stem cells that do not express Lgr5 can also be cultured by the culture media and methods of the present invention.

[0237] Genomic and phenotypic integrity of stem cells and organoids containing the stem cells To apply stem cells and their differentiated progeny in clinical and research settings, a reproducible stem cell culture method that provides a cell population of appropriate quality is required. Generally, in vitro expansion of stem cells aims to provide a cell population that closely resembles its in vivo counterpart as much as possible. This property is referred to herein as "genomic and phenotypic integrity" of the cells. Organoids obtained by culturing diseased cells, such as cancer cells or cystic fibrosis cells, also resemble their in vivo counterparts, i.e., they maintain their disease genotype and / or phenotype, and thus, in that sense, also maintain their "genomic and phenotypic integrity", i.e., they maintain the genetic or phenotypic instability characteristic of the disease that evokes the in vivo situation. Thus, in some aspects, the present invention provides "normal" organoids obtained from healthy tissue. In other aspects, the present invention provides "disease" organoids obtained from diseased tissue, such as cancer organoids (e.g., colorectal carcinoma organoids or adenocarcinoma organoids) or cystic fibrosis small intestine organoids.

[0238] For the first time, the inventors have discovered that it is possible to expand human epithelial stem cells in a cultured state for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer, with little loss of genomic and phenotypic integrity (see Example 1). Under the improved culture conditions of the present invention, human intestinal organoids exhibited a budding organoid structure rather than the cystic structure seen under previous culture conditions. Metaphase spreads of organoids over 3 months consistently revealed 46 chromosomes in each of 20 cells taken from three different donors. Furthermore, microarray analysis revealed that the stem cells during culture had a molecular signature similar to that of intestinal crypt cells, including intestinal stem cell genes.

[0239] Thus, in some embodiments, the present invention provides organoids that grow for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer, with little loss of genomic and phenotypic integrity.

[0240] In some embodiments, the present invention provides human intestinal organoids that include budding structures. In some embodiments of the present invention, the human intestinal organoids do not include cystic structures. In some embodiments of the present invention, the human intestinal organoids include more budding structures than cystic structures. The inventors have also demonstrated that human intestinal organoids produced by the culture media and methods of the present invention mimic in vivo cell fate determination in response to external factors. For example, it has previously been shown that Notch inhibition in intestinal stem cells terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. The inventors have also been able to show that the intestinal organoids of the present invention terminate proliferation when treated with a Notch inhibitor, and most cells change to goblet cells within 3 days.

[0241] From these results, it can be seen that the genomic and phenotypic integrity of the stem cells and organoids produced by the methods and media of the present invention is dramatically improved compared to previous methods and media.

[0242] The genomic integrity of the stem cells of the present invention can be confirmed by karyotype analysis. Stem cells and their progeny can be karyotyped using known methods as described in Sato, T et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265, 2009.

[0243] A "normal karyotype" is a karyotype without any noticeable changes and with all chromosomes present (i.e., euploidy). Thus, in a preferred embodiment of the invention, more than 50%; more than 70%; more than 80%; more than 90%; more than 95%; or more than 99% of the stem cells and differentiated cells in the expanded population exhibit a normal karyotype after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, or later. The term "expanded population" includes organoids.

[0244] A "normal phenotype" generally refers to cells that exhibit the same visual characteristics, gene expression, and behavior as average in vivo corresponding cells. In a preferred embodiment of the invention, more than 50%; more than 70%; more than 80%; more than 90%; more than 95%; or more than 99% of the stem cells in the expanded population cultured according to the invention exhibit a normal karyotype after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, or later.

[0245] For example, a normal phenotype may be visually determined by the number of dead cells outside the organoid, the amount of "budding" of the organoid (budding structures are preferred) compared to cyst growth, and the overall integrity of the monolayer consisting of epithelial cells (e.g., columnar squamous phenotype). Additionally, the cell types present may be useful in determining whether the organoid is visually "normal".

[0246] Preferred characteristics of the stem cells and organoids of the invention are outlined below.

[0247] Stem cell markers When mouse genes are referred to herein, the human organoids of the invention may have a similar gene profile, but human gene counterparts are used instead of mouse genes. Thus, human organoids having the gene expression profiles described herein but related to the corresponding human genes are also provided by the invention. Those skilled in the art will be able to readily obtain the human counterparts of the mouse genes listed herein.

[0248] In one aspect, the present invention provides an adult stem cell population characterized by the native expression of Lgr5. In a preferred aspect, the present invention provides an adult stem cell population characterized by the native expression of at least Lgr5, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) of stem cell markers from the group consisting of LGR4, epcam, Cd24a, Cdca7, Axin, CK19, nestin, somatostatin, CXCR4 + , CD133 + , DCAMKL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19, Tnfrsf19. The stem cell markers may be tissue specific. For example, pancreatic stem cells or organoids may have significant levels of CK19, nestin, somatostatin, insulin, glucagon, CXCR4 + , Ngn3, Pdx1, NeuroD, Nkx2.2, Nkx6.1, Pax6, Mafa, Hnf1b, and optionally one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., 1, 2, 3, or 4) of Tnfrsf19. Gastric organoids may be characterized by significant levels of CD133 + , DCAMKL-1, CD44, and optionally one or more (e.g., 1, 2, 3, or 4) of Tnfrsf19. Crypt-villus organoids may be characterized by significant levels of one or more or all (e.g., 1 or 2) of Sord and / or Prss23, or the expression of all genes in Table / Figure 14, e.g., the expression of all genes in Table / Figure 14 at significant levels.

[0249] As used herein in the context of marker expression, the term "significant level" is used synonymously with the term "detectable level" as explained below.

[0250] The small intestine organoid cell population and the stomach organoid cell population also express markers of progenitor cell populations commonly found in the small intestine and stomach, such as one or both of Cd44 and Sox9 (Barker & Huch et al Cell stem cell 2010). These are highly expressed in the stem cells according to the present invention. Cells according to this aspect of the present invention may also upregulate Wnt target genes including, for example, one, two, or all of MMP7, Sp5, Tnfrs19, and axin2. This provides strong evidence that active and strong classical Wnt signaling activity is required to maintain the self-renewal ability of these cultures.

[0251] The inventors observed that the expression of the "stem cell" genes is present in the initial organoids at significantly higher levels than in the differentiated cells that will be the progeny of these stem cells. For example, preferably, the genes LGR5, LGR4, Epcam, CD44, Tnfrsf19, Sox9, Cd24a, Sp5, Prom1 / CD133, Cdca7 are expressed in the organoids of the present invention, but are preferably significantly downregulated when pancreatic organoids, liver organoids, small intestine organoids, and colon organoids differentiate. Furthermore, preferably, the genes RNF43 and ZNRF3 are expressed in the organoids of the present invention.

[0252] "Natural expression" means that the cell has not been manipulated recombinantly in any way, i.e., the cell has not been artificially induced to express these markers or regulate their expression by the introduction of exogenous genetic material, for example, by the introduction of a heterologous (non-natural) promoter or a stronger promoter or other regulatory sequences functionally linked to an endogenous gene or a gene in an exogenously introduced form. Natural expression is expression from genomic DNA within the cell, including introns between exon coding sequences where they exist. Natural expression is not expression from cDNA. Natural expression can be demonstrated by any one of a variety of methods, such as sequencing from within the reading frame of the gene to check for the absence of unrelated heterologous sequences, if necessary. "Adult" means post-embryonic stage. With respect to the stem cells of the present invention, the term "adult stem cell" means that the stem cell has been isolated from the tissue or organ of an animal in a growth stage after the embryonic stage.

[0253] This stem cell population can also be characterized by the absence of natural expression of certain markers at any significant level, and many of these certain markers are associated with cell differentiation. Specifically, the cells of the isolated adult stem cell population do not naturally express at a significant level one or more of Cdl1b, CD13, CD14, AFP, Pdx1, any CYP member (e.g., CYP3A11, CYP11A1). As defined herein, these markers are referred to as negative markers.

[0254] Detection of Markers and Isolation of Cells The term "expressed" is used to describe the presence of a marker within a cell. For a marker to be considered expressed, it must be present at a detectable level. "Detectable level" means that the marker can be detected using one of standard laboratory methods such as PCR, blotting, or FACS analysis. If expression corresponding to an expression level of at least about 100 copies / cell can be reliably detected within the cell after 30 PCR cycles, the gene is considered to be expressed by the cells of the population of the present invention. The terms "expresses" and "expression" have corresponding meanings. At expression levels below this threshold, the marker is considered not to be expressed. Comparison of the expression level of a marker in the cells of the present invention with the expression level of the same marker in another cell, such as an embryonic stem cell, can preferably be done by comparing two cell types isolated from the same species. Preferably, the species is a mammal, more preferably, the species is a human. Such comparison can conveniently be done using a reverse transcriptase polymerase chain reaction (RT-PCR) experiment.

[0255] In some embodiments, a cell population or organoid according to the present invention is considered to express a marker if at least about 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the cells in the cell population or organoid show expression of the marker.

[0256] In some embodiments, a cell expresses a cell marker at a significant level if the cell contains 1x10 2 ~1x10 5 , e.g., 5x10 2 ~1x10 4 or 1x10 3 ~1x10 4 times the mRNA copies of the cell marker code compared to the mRNA copy number of the housekeeping gene GADPH.

[0257] In some embodiments, the gene expression in the organoids or cells of the present invention when cultured in an expansion medium is several-fold (e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold) higher than when the organoids or cells are cultured in a differentiation medium or fully differentiated adult tissue. In some embodiments, the cells or organoids of the present invention when cultured under differentiation conditions show an increase in the expression of genes known as differentiation genes compared to the cells or organoids of the present invention when cultured under expansion conditions, and may also show a decrease in the level of expression of one or more stem cell / progenitor cell genes compared to the cells or organoids of the present invention when cultured in an expansion medium.

[0258] To select the cells of this aspect of the present invention and distinguish them from these other cell types, any one of a number of physical separation methods known in the art may be used. Such physical methods may involve FACS and various immunoaffinity methods based on markers specifically expressed by the cells of the present invention. As mentioned above, three of the cell markers that are highly expressed in the stem cells of the present invention are Lgr5, CD44, and Sox9. Thus, by way of example only, the stem cells of the present invention may be isolated by a number of physical separation methods that rely on the presence of these cell markers.

[0259] In one aspect, the cells of the present invention may be isolated by fluorescence-activated cell sorting (FACS) using an antibody, for example, 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 may be accomplished by a fluorescently labeled antibody or a fluorescently labeled secondary antibody having binding specificity for a primary antibody. Examples of suitable fluorescent labels include, but are not limited to, FITC, Alexa Fluor® 488, GFP, CFSE, CFDA-SE, DyLight488, PE, PerCP, PE-Alexa Fluor® 700, PE-Cy5 (TRI-COLOR®), PE-Cy5.5, PI, PE-Alexa Fluor® 750, and PE-Cy7. This list is provided by way of example only and is not intended to be limiting.

[0260] It will be apparent to those skilled in the art that a purified stem cell population can be obtained by FACS analysis using an anti-Lgr5 antibody. However, in some aspects, it may be preferable to further purify the cell population by performing additional rounds of FACS analysis using one or more other identifiable markers.

[0261] Immunohistochemistry may also be used to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a cell population or organoid. Antibody-antigen interactions can be visualized by a number of methods well known in the art, such as the method described in Barker et al, Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature, 2007 Oct 25;449(7165):1003-7.

[0262] In another aspect, the cells of the present invention may be isolated by immunoaffinity purification, which is a separation method well known in the art. By way of example only, the cells of the present invention may be isolated by immunoaffinity purification against c-kit. As will be apparent to those skilled in the art, this method relies on the immobilization of an antibody on a purification column. The cell sample is then loaded onto the column, whereby the appropriate cells bind to the antibody and thus bind to the column. After a washing step, the cells are eluted from the column using a competitor that preferentially binds to the immobilized anti-c-kit antibody and enables the cells to be released from the column. It will be apparent to those skilled in the art that immunoaffinity purification using an immobilized antibody results in a purified cell population. However, in some aspects, it may be preferred to further purify the cell population by performing additional rounds of immunoaffinity purification using one or more other identifiable markers and to confirm the expression of other relevant intracellular markers using aliquots of the isolated clones.

[0263] It will be apparent to those skilled in the art that prior to the purification of LGR5 or stem cells, any number of purification steps may be performed, such as the purification of epithelium by methods known in the art, such as EDTA purification of epithelium or Epcam FACS sorting.

[0264] It will be apparent to those skilled in the art that consecutive purification steps involving the same physical separation method are not necessarily required. Thus, for example, it is clear that cells may be purified by an immunoaffinity purification step using an SSEA-1 affinity column after a FACS step using an anti-Lgr5 antibody. In certain embodiments, the cells may be cultured for at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days after isolation. In certain aspects, the cells are expanded in culture for longer periods to improve the homogeneity of the cell phenotype of the cell population.

[0265] Microarray analysis, cluster analysis, and comparative gene expression profiling can be used to compare the population phenotype to that of the original parental cell or the appropriate in vivo counterpart (Sato T et al., Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469 415-418).

[0266] Lineage tracing of Lgr5+ stem cells reveals that crypt-villus features are preserved in organoids.

[0267] In another aspect, high-content analysis may be used to evaluate the phenotypic integrity of the stem cells of the present invention. For example, there are numerous high-content screening kits and platforms, such as point scanning 4 color ImageXpress ULTRA (Molecular Devices, Union City, USA), BD Biosciences (formerly Atto Biosciences, Rockville, Maryland)'s spinning disk (nipkow disk) Pathway 855 and 435, Opera (PerkinElmer Inc., Waltham, MA), as well as slit scanning IN Cell 3000 (GE / Amersham Biosciences, Cardiff, UK), Arrayscan VTI (Cellomics (Cellomics)), IN Cell Analyzer 2000 (GE Healthcare Piscataway, New Jersey, USA), Acumen eX3 (TTP LabTech Ltd (Acumen eX3)), Scanalyzer (Scanalyzer LemnaTec, Aachen Germany), as well as ImageXpress MICRO (Molecular Devices, Union City, USA), IN Cell 1000 (GE / Amersham Biosciences Piscataway, New Jersey, USA), Pathway HT (Becton Dickinson Biosciences), as well as ImageXpress MICRO (Molecular Devices, Union City, USA), Scan^R (Olympus Soft Imaging Solutions, Germany).

[0268] Plating density In some aspects of the present invention, as is known in the art, typically, instead of large cell clusters, a single cell suspension or small cell clusters (2 - 50 cells / cluster) are seeded. When such cells divide, they are seeded on a support at a density that promotes cell growth. Typically, when single cells are isolated, a plating density of at least 1 - 500 cells / well is used, and the surface of the well is 0.32 cm 2 . When clusters are seeded, the plating density is preferably 250 - 2500 cells / cm 2 . For replating, a density of about 2500 cells / cm 2 to about 5,000 cells / cm 2 may be used. During replating, as is known in the art, typically, instead of large cell clusters, a single cell suspension or small cell clusters are seeded.

[0269] Further Differentiation In some aspects of the present invention, certain components of the expansion medium can be removed to direct the cell fate of cultured cells towards differentiation. Any components of the culture medium responsible for maintaining the undifferentiated state and / or activating the gene program of stem cells or progenitor cells may be removed from the culture medium.

[0270] In some aspects of the present invention, by removing the inhibitor of the present invention, the cells of the organoid can differentiate into mature cells, for example, mature goblet cells and enteroendocrine cells in the crypt - villus organoid. Thus, in some aspects, the present invention provides a method for further differentiating an organoid using a second culture medium that does not contain the inhibitor of the present invention. See, for example, Example 1.

[0271] For example, in some embodiments, to cause the cells to differentiate, TGF-β inhibitor and / or p38 inhibitor are removed from the cell culture medium. "Removed" from the cell culture medium or "removing" a component from the cell culture medium means that when the cells are replated and the medium is changed, the component is not added to the fresh medium.

[0272] In some embodiments, Wnt is present in the expansion medium but not in the differentiation medium. For example, some embodiments include the step of removing Wnt to differentiate colonic organoids into mature intestinal cells. Wnt may also be removed such that crypt-villus organoids differentiate.

[0273] In some embodiments, R-spondin is present in the expansion medium but not in the differentiation medium. For example, some embodiments include the step of removing R-spondin to differentiate colonic organoids into mature intestinal cells. R-spondin may also be removed such that crypt-villus organoids differentiate. In some embodiments, R-spondin and Wnt may be removed such that crypt-villus organoids differentiate.

[0274] In some embodiments, nicotinamide is present in the expansion medium but not in the differentiation medium. Thus, in some embodiments, nicotinamide and SB202190 (or another p38 inhibitor) are removed from the cell culture medium such that the cells differentiate, for example, into crypt-villus organoids or colonic organoids.

[0275] Thus, a method of obtaining differentiated cells or organoids may include culturing epithelial cells in the culturing method of the present invention (i.e., expansion medium) containing TGF-β and / or p38 inhibitor such that the cells survive and / or proliferate, and then culturing the cells and continuing to replenish the medium. The replenished medium does not contain TGF-β inhibitor and / or p38 inhibitor (i.e., differentiation medium).

[0276] In some embodiments, the differentiation medium contains additional components. For example, in some embodiments, the differentiation medium contains a γ-secretase inhibitor, such as DAPT or DBZ. In some embodiments, the differentiation medium contains RANK ligand (also referred to herein as RANKL). As described above, the addition of a γ-secretase inhibitor can induce the differentiation of intestinal organoid cells, such as small intestinal organoid cells, into secretory cells, such as goblet cells. Adding RANKL to the medium can induce the differentiation of intestinal organoid cells, such as small intestinal organoid cells, into M cells.

[0277] In some embodiments, the present invention provides a culture medium for differentiating stem cells from a tissue of interest, comprising or consisting of components of a culture medium used to expand stem cells from the tissue type of interest, wherein one or more of the following are excluded from the medium for differentiating stem cells: Wnt, R-spondin, BMP inhibitor, TGF-β inhibitor, receptor tyrosine kinase ligand, p38 inhibitor, and nicotinamide.

[0278] Furthermore, the present invention provides a method for expanding a single stem cell or a population of stem cells, preferably for expanding them to generate organoids, the method comprising culturing a single stem cell or a population of stem cells in a culture medium according to the present invention, the method comprising culturing the stem cell, stem cell population, or tissue fragment in a first expansion medium, continuing to culture the stem cell, stem cell population, or tissue fragment, and replenishing the medium with a differentiation medium wherein the differentiation medium does not contain one or more, preferably all, factors selected from TGF-β inhibitor, p38 inhibitor, nicotinamide, and Wnt.

[0279] Generally, when a component is said to be "removed" from the medium, it means that the component is not added when the medium is replenished, i.e., the component is excluded from the replenished medium. When the medium is "replenished", this may mean that the medium is physically removed from the extracellular matrix and then replaced with fresh medium.

[0280] In the case of the colon, liver, and pancreas, there are very few differentiated cells present in the expansion medium. When the expansion medium is replaced with the differentiation medium only once, the cells begin to differentiate. At this stage, the organoids also begin to lose stem cells. Differentiated organoids are suitable for certain applications such as transplantation, drug screening for metabolic diseases, toxicology (e.g., using liver organoids containing hepatocytes), and studying the antibacterial function of the small intestine (but not limited to this). Expanding organoids are generally more suitable for other applications such as regenerative medicine and drug screening, e.g., drug screening for cancer or cystic fibrosis (but not limited to this). Expanding organoids generally have a greater proliferative capacity (and thus a longer lifespan) than differentiated organoids. In some embodiments, colon organoids, liver organoids, and pancreatic organoids do not differentiate further.

[0281] Small intestinal organoids and prostate organoids differ from colonic organoids, liver organoids, and pancreatic organoids in that they are differentiating while maintaining an expanding stem cell population. Small intestinal organoids and prostate organoids need not be cultured in separate differentiation media such that differentiated cell types are present. Small intestinal organoids and prostate organoids can be thought of as having the properties of both expanding and differentiated organoids. However, to achieve complete differentiation of small intestinal organoids, preferably, the small intestinal organoids can be cultured in a separate differentiation medium that does not contain Wnt3a and preferably contains a gamma secretase inhibitor and / or RANK ligand (also referred to herein as RANKL). "Complete" differentiation means that all differentiated cell types are present, including goblet cells, neuroendocrine cells, tuft cells, M cells, enterocytes, and Paneth cells. Some of these differentiated cell types, for example, Paneth cells, are also (sometimes in small amounts) present in the expanding organoids.

[0282] Organoid The aforementioned cells grow into organoids. Therefore, the organoids obtainable by the method of the present invention are a further aspect of the present invention. The organoids described herein are also provided. The organoids are preferably human organoids. As far as the inventors know, this is the first event in which human organoids that function and survive after a long period (i.e., at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer culture; see the examples included herein) have been obtained. The function is preferably characterized by the presence of tissue-specific markers as defined herein and / or the structure of the organoids. Since the final amount of the obtained organoids is correlated with the culture period, those skilled in the art will understand that the present invention is a pioneering invention and potentially opens up new possibilities, for example, in regenerative medicine. Therefore, provided are the organoids described herein that function and survive after at least 3 months (e.g., at least 4 months, 5 months, 6 months, 7 months, 8 months or more) of culture. For example, provided are the organoids described herein that retain at least one or more (e.g., 1, 2, or 3) of their structure, marker expression, and function after at least 3 months (e.g., at least 4 months, 5 months, 6 months, 7 months, 8 months or more) of culture.

[0283] For example, the organoids according to the present invention may comprise a cell population consisting of at least 1x10 3 cells, at least 1x10 4 cells, at least 1x10 5 cells, at least 1x10 6 cells, at least 1x10 7 cells, or more cells. Each organoid contains from about 1x10 3 cells to 5x10 3 cells. The inventors have compared organoids from a single Lgr5+ stem cell with organoids comprising the aforementioned cell population or about 10 4It has been shown that it is possible to grow into an organoid containing a cell population consisting of 4 individual cells. For example, it has now been shown that it is possible to initiate the growth of organoids from a single stem cell in mice. Accordingly, the present invention provides a method for producing organoids from a single stem cell. In some embodiments, the organoid contains about 10

[0284] individual cells. In some embodiments, 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50 organoids may be grown together in one well of a 24-well plate.

[0285] In some embodiments, the present invention provides an organoid or cell population that can survive in a cultured state for at least 3 months, for example, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer when cultured in the culture medium of the present invention.

[0286] Preferably, the cell population or organoid expands at a rate of at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold per week. Thus, in some embodiments, the cell population or organoid expands at a rate of at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold per week.

[0287] The organoids of the present invention can be obtained using cells isolated from any suitable source. Generally, the cells used to produce the organoids are isolated from the same tissue type as the organoids to be produced. The organoids are preferably from a mammal, such as a mouse, cow, pig, or human. Most preferably, the organoids are human.

[0288] In some embodiments, the present invention provides an organoid or cell population that is a normal (healthy) organoid or normal (healthy) cell population, or a diseased organoid or diseased cell population, such as a diseased organoid or diseased cell population obtained by culturing stem cells collected from a human or animal having a disease.

[0289] In some embodiments, the present invention provides an organoid or cell population that is frozen and stored at a temperature lower than -5°C, lower than -10°C, lower than -20°C, lower than -40°C, lower than -60°C, or lower than -80°C, lower than -100°C, lower than -150°C, or lower than about -180°C. The organoids or cell populations of the present invention may be stored in liquid nitrogen. Thus, in some embodiments, the present invention provides an organoid or cell population stored in liquid nitrogen.

[0290] In some embodiments, the present invention provides the organoids of the present invention that are small intestine organoids, colon organoids, stomach organoids, pancreatic organoids, liver organoids, or prostate organoids.

[0291] Organoid structure and morphology The organoids of the present invention that can be obtained by expanding stem cells provide a cell population similar to its in vivo counterpart.

[0292] Image analysis may be used to evaluate cell morphology, cell structure, evidence of apoptosis or cytolysis, and characteristics of cells in culture such as organoid composition and structure. Many types of imaging analysis, such as electron microscopy, confocal microscopy, stereomicroscopy, fluorescence microscopy, are well known in the art. Histological analysis can reveal the basic structure and cell types.

[0293] Examples useful for the description of the organoids produced according to the present invention are shown in the accompanying drawings. It can be seen that the organoids according to the present invention can have a cell layer having at least one bud and a central lumen. Organoids outside the matrigel tend to be larger than those in the center of the matrigel, perhaps because they are more accessible to the necessary growth factors. Structurally, the organoids according to the present invention are often elongated in shape. The organoids according to the present invention may include one or more budding structures that are a single cell epithelial layer having a structure much like a tube or an island. Under confocal microscopy, this structure may be stained keratin positive. The organoids according to the present invention may include cells having a polarized nucleus and a small cytoplasm. The organoid may have a portion formed from multiple layers. Such cells often tend to have a nucleus more centrally located in the cell, that is, a non-polarized nucleus. Cells in the multi-layered portion may self-organize to include a gap, i.e., a lumen, between the cells. In some embodiments, the organoids of the present invention comprise or consist of epithelial cells. In some embodiments, the organoid comprises or consists of a single layer of epithelial cells. In some embodiments, there are no non-epithelial cells in the organoid. In some embodiments, the organoids of the present invention include all the differentiated cell types present in their corresponding in vivo tissue counterparts.

[0294] In some embodiments, the human intestinal organoids exhibited budding organoid structures rather than the cystic structures seen under previous culture conditions. Organoid metaphase spreads over three months consistently revealed 46 chromosomes in each of 20 cells collected from three different donors.

[0295] In some embodiments, the organoids of the invention comprise a single cell monolayer. In some embodiments, the organoids of the invention have portions formed from multiple layers. The multiple cell layers are also referred to herein as "stratified" cell regions. "Stratified" means that there are multiple (more than one) cell layers. In some embodiments, the organoids of the invention comprise a single monolayer that is folded (or invaginated) to form two or more layers. Sometimes, it may be difficult to distinguish between a folded (or invaginated) monolayer and a stratified cell region. In some embodiments, the organoid comprises both a region of stratified cells and a region of folded monolayer. In some embodiments, the organoids of the invention have portions formed from multiple layers and portions comprising a single cell monolayer. Morphologically, the cells appear like their corresponding in vivo tissue counterparts.

[0296] Thus, in some embodiments, the invention provides a three-dimensional organoid comprising epithelial cells surrounding a central lumen, optionally wherein the epithelial cells are present in distinct proliferating and differentiating regions, preferably an organoid obtainable using the culture media and methods of the invention. In some embodiments, the organoids of the invention are three-dimensional organoids comprising epithelial cells arranged in a monolayer, optionally in regions of folded monolayers and regions of stratified cells. In some embodiments, the organoid is free of non-epithelial cells. In some embodiments, all differentiated cell types of normal in vivo tissue are present in the organoid.

[0297] Crypt-villus organoid In the small intestine crypt-villus organoids, the structural arrangement of the organoids closely resembles the in vivo crypt-villus structure. That is, at the bottom of the crypt, Lgr5+ stem cells and their niche cells (Paneth cells) are adjacent, followed by TA cells directly above the bottom of the crypt, and on the side of the villus, and finally, differentiated cells, such as enterocytes that make up the rest of the villus and further differentiate towards the upper part of the villus, follow. It can be seen that the organoids according to the present invention may have a cell layer having at least one bud and a central lumen. Organoids outside the matrigel tend to be larger than those in the center of the matrigel, probably because they are more accessible to the necessary growth factors. Structurally, the organoids according to the present invention are often elongated in shape. Under confocal microscopy, this structure may be stained keratin positive. The organoids according to the present invention may contain cells with polarized nuclei and small cytoplasm. Crypt-villus organoids are generally monolayer.

[0298] In some embodiments, for example, in the case of mouse crypt-villus organoids, the crypt-villus organoid is a three-dimensional organoid comprising a crypt-like region surrounding a central lumen whose inner wall is covered by a villus-like epithelial region. The villus-like epithelial region is an epithelial region containing differentiated cell types. In some embodiments, there are no non-epithelial cells in the organoid.

[0299] In some embodiments, for example, in the case of human crypt-villus organoids, the crypt-villus organoid is a three-dimensional organoid comprising a crypt-like region surrounding a central lumen. In some embodiments, the dividing cells are limited to the budding structure. There are no or very few differentiated cells. Intestinal differentiated cells are formed under differentiation conditions. In some embodiments, there are no non-epithelial cells in the organoid. In some embodiments, when the organoid is expanding, for example, when it is in the expansion culture medium according to the present invention, there are few or no differentiated cells in the organoid.

[0300] In some embodiments, the small intestinal organoids of the present invention cultured in the culture medium of the present invention containing RANKL contain M cells. In some embodiments of the present invention, the small intestinal organoids of the present invention cultured in the culture medium of the present invention containing a γ-secretase inhibitor contain goblet cells. In some embodiments, small intestinal organoids cultured in a differentiation medium (for example, the differentiation medium contains a basal medium, noggin, EGF, a TGF-β inhibitor and a p38 inhibitor, a γ-secretase inhibitor and RANKL) contain, for example, all differentiated cell types including goblet cells, neuroendocrine cells, tuft cells, M cells, enterocytes, and Paneth cells. Some of these differentiated cell types, for example, Paneth cells, are also (sometimes in small amounts) present in the expanding organoids.

[0301] Human intestinal organoids exhibit a budding organoid structure rather than the cystic structure seen under previous culture conditions. The upper opening of freshly isolated crypts is blocked, and this region gradually swells and is filled with apoptotic cells, closely resembling apoptotic cells and constricting and separating at the villus tip. Thus, in some embodiments, the crypt-villus organoids have a crypt-like structure surrounding a central lumen, with an inner wall covered by a villus-like epithelium and filled with apoptotic cell bodies. In some embodiments, the lumen is continuously and intermittently opened to release its contents into the medium.

[0302] In some embodiments, the crypt region undergoes a continuous budding event that creates additional crypts, a process reminiscent of crypt fission.

[0303] The inventors have also demonstrated that human intestinal organoids produced by the media and methods of the present invention mimic in vivo cell fate determination in response to external factors. For example, it has previously been shown that Notch inhibition in intestinal stem cells terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. Thus, in some embodiments, when the crypt-villus organoids of the present invention are treated with a Notch inhibitor, proliferation stops and most cells (e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%) turn into goblet cells within 3 days.

[0304] Metaphase spreads of organoids over 3 months consistently revealed 46 chromosomes in each of 20 cells collected from 3 different donors. Furthermore, microarray analysis revealed that the stem cells in culture had a molecular signature similar to that of intestinal crypt cells, including intestinal stem cell genes.

[0305] Colonic organoid Colonic organoids show a cell composition similar to that of crypt-villus organoids. Therefore, the above comments on crypt-villus organoids shall apply mutatis mutandis to colonic organoids. See, for example, FIGS. 1 and 2.

[0306] Typically, the difference between colonic organoids and small intestinal organoids is that the colonic crypts are shallower, so they somewhat resemble a rugby ball rather than a sphere with protrusions. Both small intestinal organoids and colonic organoids have regions containing stem cells and TA cells, as well as other regions containing differentiating and / or differentiated cells. In the case of small intestinal organoids, the differentiated region is sometimes called "villus-like". The differentiated region of colonic organoids typically has a cell composition similar to that of the "villus-like" region of the small intestine, but the colon itself has no villi.

[0307] The amount of Wnt present can affect the size of the budding structures in the organoid (i.e., the depth of the crypt). The more Wnt there is, the fewer buds there are. The colon produces more Wnt than the small intestine and thus requires less additional Wnt in the medium and typically has shallower crypts than the small intestine. The same difference is seen in organoids.

[0308] In some aspects, the present invention provides colonic organoids. The inventors have discovered that mouse colonic organoids can be obtained by culturing colonic crypts in ENR+Wnt3A (WENR) cell culture medium. Accordingly, in some embodiments, the present invention provides colonic organoids obtained by culturing colonic crypts in WENR medium.

[0309] The inventors have also surprisingly found that human colonic organoids can be maintained using a culture medium containing WENR+gastrin+nicotinamide. In some embodiments, the human colonic organoids of the present invention contain WENR+gastrin+nicotinamide and can be obtained using a medium that also contains a TGFβ inhibitor. For example, in some embodiments, the following cell culture medium may be used to obtain human colonic organoids: WENR+gastrin+nicotinamide+A8301+SB202190. In other embodiments, the following cell culture medium may be used to obtain human colonic organoids: WENR+nicotinamide+A83-01.

[0310] In some embodiments, the maximum diameter of the mouse colonic organoids is about 200 - 700 μm, for example, 250 - 600 μm, 300 - 500 μm, 320 - 450 μm, 340 - 400 μm, 300 - 380 μm, for example, about 360 μm. In some embodiments, the minimum diameter of the colonic organoids is about 100 - 400 μm, for example, 150 - 350 μm, 170 - 300 μm, 190 - 280 μm, 195 - 250 μm, for example, about 235 μm. In further embodiments, the diameter of the organoids may be up to 1 mm. In some embodiments, the maximum diameter of the human colonic organoids is about 300 - 800 μm, for example, 350 - 700 μm, 400 - 600 μm, 450 - 550 μm, 475 - 540 μm, 500 - 530 μm, for example, about 500 μm. In some embodiments, the minimum diameter of the colonic organoids is about 200 - 500 μm, for example, 250 - 450 μm, 300 - 415 μm, 350 - 400 μm, 325 - 380 μm, for example, about 375 μm. In further embodiments, the diameter of the organoids may be up to 1 mm. In some embodiments, the colonic organoids of the present invention include a budding structure. These can be seen by visualizing proliferating cells using EdU staining.

[0311] Human colonic organoids retain their characteristic budding structure under human intestinal stem cell culture ( "HISC") conditions (WENRg + nicotinamide + TGF-β inhibitor (e.g., A83 - 01) + p38 inhibitor (e.g., SB202190)). In some embodiments, the colonic organoids are three - dimensional organoids containing proliferative budding structures and containing stem cells. These stem cell regions surround the central lumen. Dividing cells are generally limited to the budding structures. In some embodiments, there are no or very few differentiated cells. Under differentiation conditions, intestinal differentiated cells, such as mature intestinal cells, are formed. In some embodiments, there are no non - epithelial cells in the organoids.

[0312] Pancreatic organoids The pancreatic organoids of the present invention preferably exhibit budding. In some embodiments, the pancreatic organoids have a diameter of 100 to 1000 micrometers, such as 200 to 900 micrometers, 300 to 1000 micrometers, 400 to 700 micrometers. The pancreatic organoids are preferably monolayer. There are only very early islet structures or ductal structures. The budding structure indicates a healthy growth status and the maintenance of stem cells.

[0313] In some embodiments, for example, when pancreatic organoids are grown in the culture medium of the present invention (in the absence of a TGF-β inhibitor), they mainly consist of cystic structures and there are few budding structures or tubular regions. The cystic structures mainly contain a monolayer, but there may be some multilayer cell regions. The cells express stem cell markers and progenitor cell (duct) markers. There are no differentiated cells such as β cells in this organoid. The cysts are mainly formed by a monolayer, but there are multilayer parts. The cell type resembles stem cells / progenitor cells (duct cell gene expression). There are no differentiated cells (β cells).

[0314] In other embodiments, for example, when pancreatic organoids are grown in the culture medium of the present invention, for example, in the presence of a TGF-β inhibitor such as A83-01, as shown by Krt19 staining, they contain more budding structures / tubular regions (this means that the cells are tubular cells and the structure resembles a duct) (see, for example, Figure 31). A monolayer composed of polarized cells can be identified, but regions with multilayer cells can also be identified.

[0315] Adenocarcinoma organoids and colon cancer organoids Adenocarcinoma organoids and colon cancer organoids generally form cystic structures instead of budding structures. This reminds us that there is no excellent cell niche support. Adenomatous crypts cultured with EFG + noggin show an approximately 16-fold expansion in the first 10 days. Adenoma (adenocarcinoma) organoids and colon cancer organoids can be useful research tools and drug screening models.

[0316] Carcinoma organoids, adenoma organoids, and adenocarcinoma organoids are mainly cystic (see, for example, FIGS. 4 and 9). However, in some embodiments, they may also include structures similar to their normal tissue organoid counterparts.

[0317] Barrett's esophagus (BE) organoids The BE organoids of the present invention include a budding structure (see, for example, FIG. 5). Morphologically, the cells in the organoids of the present invention appear similar to their corresponding in vivo tissue counterparts.

[0318] Barrett's esophagus is a disease characterized by the presence of columnar epithelium replacing normal squamous epithelium as a result of metaplasia in the lower esophagus. A prominent histological feature of Barrett's esophagus is the presence of intestinal goblet cells in the esophagus. Utilizing the similarity between Barrett's esophagus and intestinal epithelium, the inventors have shown that the culture medium and method of the present invention can be used to maintain Barrett's esophagus epithelium for up to 1 month. The inventors have also demonstrated for the first time that adding FGF10 to the culture medium of the present invention can cause Barrett's esophagus organoids to form a budding structure and significantly extend the culture period beyond 3 months. Thus, Barrett's esophagus organoids are an example of the organoids of the present invention. In some embodiments, Barrett's esophagus organoids have a cystic structure. In some embodiments, the Barrett's esophagus organoids of the present invention contain Paneth cells. In some embodiments, the Barrett's esophagus organoids of the present invention express lysozyme.

[0319] Thus, the inventors also describe a culture medium according to the present invention containing FGF10 for culturing Barrett's esophagus epithelium.

[0320] In some embodiments of the present invention, the Barrett's esophageal organoids may be grown using the culture medium according to the present invention, which also contains FGF10. In some embodiments, these Barrett's esophageal organoids express Ki67 and have a minimal number, preferably less than 10%, less than 5%, or less than 1% of PAS-positive cells and mucin-positive cells. In some embodiments, the Barrett's esophageal organoids contain lysozyme-positive Paneth cells.

[0321] Stomach (gastric) organoids (see, e.g., FIG. 46) Mouse stomach organoids grown in the culture medium of the present invention comprise a monolayer epithelium that surrounds a central lumen lined by an epithelial region containing differentiated cell types and includes a gastric gland basal-like region (formed by stem cells and progenitor cells), or consists of the monolayer epithelium. Optionally, there are no non-epithelial cells in the organoids.

[0322] Human stomach organoids grown in the culture medium of the present invention contain cystic structures. The cystic structures are a monolayer consisting of polarized cells. These human stomach organoids grown in the presence of a TGF-β inhibitor closely resemble mouse stomach organoids much more than human organoids grown in the absence of a TGF-β inhibitor.

[0323] Prostate organoids (see FIGS. 41-43) Under culture conditions containing EGF, Noggin, and R-spondin, mouse prostate organoids form three-dimensional cystic structures with lumens. Eventually, the layers fold inward, forming three to four layers consisting of (stratified) epithelial cells. The outer layer consists mainly of CK5+ basal epithelial cells, while the inner layer consists mainly of CK8+ luminal epithelial cells. The stem cell compartment has not been identified. That is, all regions contain dividing cells. Thus, in some embodiments, prostate organoids grown in the absence of testosterone contain a stratified layer of dividing epithelial cells. In a further embodiment, the prostate organoid comprises an outer layer of cells comprising CK5+ basal epithelial cells and an inner layer comprising CK8+ luminal epithelial cells. In some embodiments, prostate organoids grown in the absence of testosterone do not contain stem cells.

[0324] Addition of testosterone to the prostate culture medium The inventors have shown that when (dihydro)testosterone is added to the culture conditions of prostate organoids, the majority of the cells differentiate into CK8+ luminal cells that form a monolayer epithelium folded into two layers. Prostate organoids grown in the presence of testosterone mainly consist of luminal cells with another basal cell layer or luminal cells without another basal cell layer. This structure resembles the in vivo structure. There are differentiated and dividing cells as well as stem and progenitor cells. Thus, in some embodiments, for example, when cultured in a medium containing testosterone, the prostate organoids are three-dimensional organoids containing cystic structures and lumens. In some embodiments, the prostate organoids contain CK8+ luminal cells that form a monolayer epithelium. In some embodiments, the monolayer is folded into two or more layers. In other embodiments, the organoids may contain a stratified cell region. In some embodiments, the prostate organoids contain differentiated cells while maintaining a population of dividing stem cells. In some embodiments, the shape of the organoids is determined by the origin of the cell starting material or tissue starting material (the location within the prostate before isolation). The prostate consists of various lobes or regions that exhibit the various epithelial structures (stratified or folded) described above. After in vitro culture, the organoids appear to maintain to some extent the various macroscopic structures (stratified or folded) of the prostate portion from which the organoids were obtained.

[0325] Liver organoids Structurally, the mouse liver organoids according to the present invention are often elongated in shape. The mouse liver organoids according to the present invention may include one or more budding structures that are a single cell epithelial layer having a structure closely resembling a bile duct. Under confocal microscopy, this structure may be stained keratin positive. The mouse liver organoids according to the present invention may include cells having a polarized nucleus and a small cytoplasm. The organoids may have a portion formed of multiple layers. Such cells often tend to have a nucleus more centrally located in the cell, i.e., a non-polarized nucleus. Cells in the multi-layered portion may self-organize to include a gap or lumen between cells. In some embodiments, the human liver organoids of the present invention generally have a cystic structure.

[0326] In some embodiments, the liver organoid is a three-dimensional organoid having a cystic structure (see, for example, FIG. 30). Under magnified conditions, the organoid may consist of stem cells and progenitor cells, where two regions are defined: (1) a tubular region formed by a single layer of cuboidal epithelium (positive for the duct marker Krt19) and cells covering the inner wall of the central lumen; and (2) a pseudostratified epithelial region where krt19-positive cells and scattered albumin-positive cells are detected. This structure (the region with a single layer epithelium and the region with a pseudostratified epithelium) resembles the embryonic liver bud. Under magnified conditions, fully differentiated cells are not present, but in some embodiments, the expression of hepatocyte / liver bud cell-specific markers can be detected. Depending on the differentiation conditions, the tubular region (single layer epithelium) is lost, and a cystic organoid is formed in which the entire structure becomes a pseudostratified epithelium containing >50% polarized hepatocytes.

[0327] Liver organoids preferably contain hepatocytes and cholangiocytes (although in particular, hepatocytes are seen after differentiation in DM and are not required for expansion). More preferably, at least one of the following markers can be detected: at least one type of hepatocyte marker, such as albumin, transthyretin, B-1 integrin, and glutamine synthetase, and / or at least one of CYP3A11, FAH, tbx3, TAT, and Gck, and / or at least one type of cholangiocyte marker, such as keratin 7 and 19. Those skilled in the art know the methods for detecting each of these markers (i.e., RT-PCR and / or immunofluorescence). Preferably, the expression of each of these markers is evaluated as carried out in the experimental part. Each of these markers is usually expressed after culturing for at least 2 weeks, 3 weeks, or 1 month using the method of the present invention. Microarray analysis of organoids under both culture conditions revealed that liver organoids resemble adult liver tissue.

[0328] Preferably, all cells in the liver organoids express hepatocyte surface markers. For example, in some embodiments, at least 50% (e.g., 50 - 60%), at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% of the cells in the liver organoids express hepatocyte markers. In some embodiments, about 35% of the cells in the liver organoids, e.g., 25 - 45%, 30 - 40%, 33 - 37%, 35% or less, or 15 - 35% of the cells express hepatocyte surface markers. In some embodiments, there are few hepatocytes in the expansion phase, e.g., less than 20%, less than 10%, less than 5%, less than 2%, less than 1% of the cells, preferably 0%. Preferably, the cells and organoids produced according to the present invention also have hepatocyte functions such as expression or positive staining of albumin, B-1 integrin, CK-8, CK-18, transthyretin (TTR), glucose 6P, Met, glutamine synthetase (Glu1), transferrin, Fahd1, Fahd2a, K7, K19, and cytochrome P450 isoform 3A13 (CYP3A13), 51 (CYP51), 2D10 (CYP2D10), 2j6 (CYP2j6), 39A1 (CYP39A1), 4A10 (CYP4A10), 4F13 (CYP4F13), 4F16 (CYP4F16), CYP4B1, and 20A1 (CYP20A1), which are mature liver markers. Also, the fetal liver gene AFP is not detected in either of the two culture conditions in some embodiments, similar to the adult liver. In some embodiments, the expression of alpha-fetoprotein is slightly higher than the background gene expression.

[0329] Also, the liver transcription factors well-known as HNF1a, HNF1b, and HNF4a are highly expressed under both conditions.

[0330] Since the liver and pancreas are closely related organs, the inventors investigated whether their liver cultures also express pancreatic-specific genes. The pancreas is functionally divided into the endocrine pancreas and the exocrine pancreas. The endocrine pancreas is characterized mainly by the expression of insulin, glucagon, and somatostatin. The expression of these hormones is tightly regulated by a set of endocrine pancreas-specific transcription factors, and the most important endocrine pancreas-specific transcription factors are Pdx1 and NeuroD. The exocrine pancreas is formed particularly by the acinar and ductal compartments responsible for the production of digestive enzymes such as amylase, pancreatic lipase, and chymotrypsin. The expression of these genes is also regulated by Ptf1, a specific exocrine pancreas gene.

[0331] The pancreatic-specific genes Ptf1a, pancreatic amylase (Amy2a4), pancreatic lipase (Pnlip), insulin (ins1 and ins2), glucagon (Gcg), chymotrypsin (cela1), Pdx1, and NeuroD are not present in the liver cultures described herein.

[0332] In some embodiments, in liver organoids, one or more or all of the following genes are expressed at levels similar to the corresponding genes in adult liver hepatocytes: Aqpl, Bmp2, Apo3, Apo17a, Sord, C3, Ppara, Pparg, tbx3, lgf1, ll17rb, ll1b, Tgfbi, Apoa1, Apoa4, Apob, Cyp26b1, Cyp27a1, Cyp2b13, Cyp2b9, Cyp2c37, Cyp2f2, Cyp2gl, Cyp2j13, Cyp3a11, Cyp4a10, and Cypf14. See, for example, FIG. 27A.

[0333] In some embodiments, in liver organoids, one or more of the following genes are expressed at levels that are similarly shut down compared to the corresponding genes in adult liver hepatocytes: Ccl2, Osmr, Icam1, and Cxc12.

[0334] In some embodiments, in liver organoids and neonatal livers, one or both of the following genes are differentially expressed: mKi67 and cdkn3. This means that the expression of these genes in the organoids is higher than that in differentiated organoids or whole organs.

[0335] In some embodiments, in liver organoids and neonatal livers, one, two, or all of the following genes are expressed at similar levels: cyp2j6, olfm4, and Lefty1. See, for example, FIG. 27B.

[0336] In some embodiments, the liver organoids of the present invention have a ductal phenotype when cultured in the expansion medium of the present invention (e.g., EM1 or EM2).

[0337] In some embodiments, the liver organoids of the present invention express adult liver markers when cultured in the differentiation medium of the present invention.

[0338] In one embodiment, the liver organoids of the present invention have a gene expression profile as shown in FIG. 27C.

[0339] In a particularly preferred embodiment, the mouse liver cell population or organoids of the present invention have a gene expression profile as shown in FIG. 28. For example, in one preferred embodiment, the mouse liver cell population or organoids of the present invention a) express at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), preferably all, of the following stem cell markers: lgr5, lgr4, epcam, Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1, Cdca7, and Elf3; and / or b) do not express the following stem cell marker: lgr6; and / or c) When grown in the expansion medium of the present invention, it expresses at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), preferably all, of the following hepatocyte markers or cholangiocyte markers: Hnf1a, Hnf1b, Hnf4a, Hhex, Onecut1, Onecut2, Prox1, Cdh1, Foxa2, Gata6, Foxm1, Cebpa, Cebpb, Cebpd, Cebpg, Glu1, Krt7, Krt19, and Met; and / or d) When grown in the expansion medium of the present invention, it does not express at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) of the following genes: afp, Ins1, Ins2, Gcg, Ptf1a, Cela1, Cela2a, Cela3b, Neurod1, Neurod2, Neurog1, Neurog2, Neurog3, Amy2a4, Igflr, Igf2, and Cd34; and / or e) It expresses at least one (e.g., 1, 2, or 3) of the following reprogramming genes: Klf4, Myc, and Pou5f1; and / or f) It does not express the following reprogramming gene: Sox2. Gene expression is preferably detected by measuring expression at the mRNA level, for example, using a microarray.

[0340] More preferably, the mouse liver cell population or organoid of the present invention has all of the above-mentioned characteristics a) to f).

[0341] In some embodiments, the gene expression profile described above for the mouse liver cell population or liver organoid of the present invention is the gene expression profile of a mouse cell population or organoid cultured in the liver expansion medium of the present invention.

[0342] In one aspect, provided is a human liver cell population or organoid of the present invention having the gene expression signature shown in FIG. 29. For example, a human liver cell population or organoid cultured in EM1 of the present invention preferably expresses the genes shown in FIG. 29 as being expressed in the EM1 cell culture medium. For example, a human liver cell population or organoid cultured in EM2 of the present invention preferably expresses the genes shown in FIG. 29 as being expressed in the EM2 cell culture medium. For example, a human liver cell population or organoid cultured in DM of the present invention preferably expresses the genes shown in FIG. 29 as being expressed in the DM cell culture medium.

[0343] For example, in one preferred aspect, the human liver cell population or organoid of the present invention a) expresses at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9), preferably all, of the following stem cell signature genes: LGR4, TACSTD1 / Epcam, CD44, SOX9, SP5, CD24, PROM1, CDCA7, and ELF3; and / or b) expresses at least one (e.g., 1, 2, 3, 4), preferably all, of the following reprogramming genes: KLF4, MYC, POU5F1, and SOX2; and / or c) expresses at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), preferably all, of the following hepatocyte / cholangiocyte-specific genes: HNF1A, HNF1B, HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA, CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19, and MET; and / or d) does not express at least one (e.g., 1, 2, 3, 4, 5, 6), preferably all, of the following hepatocyte / biliary epithelial cell-specific genes: NEUROG2, IGF1R, and CD34, AFP, GCG, and PTF1A, e.g., does not express NEUROG2, IGF1R, and CD34; and / or e) expresses at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18), preferably all, of the following hepatocyte-specific genes: TTR, ALB, FAH, TAT, CYP3A7, APOA1, HMGCS1, PPARG, CYP2B6, CYP2C18, CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2, and SCARB1. Gene expression is preferably detected by measuring expression at the mRNA level, e.g., using a microarray.

[0344] More preferably, the human liver cell population or organoid of the present invention has all of the characteristics a) to e) described above.

[0345] In some embodiments, as shown in FIG. 29, genes in the human liver cell population or organoids of the present invention are upregulated or downregulated relative to the expression of a reference RNA. Preferably, the reference RNA is Universal Human Reference RNA (Stratagene, catalog number 740000). In some embodiments, a gene is upregulated or downregulated relative to the reference RNA if it is shown to be upregulated or downregulated relative to the reference RNA in FIG. 29, but the degree of upregulation or downregulation need not be the same. In other embodiments, the degree of upregulation or downregulation is + / - 35%, + / - 30%, + / - 25%, + / - 20%, + / - 15%, + / - 10%, + / - 5%, + / - 3%, or more preferably + / - 1.5-fold, + / - 2-fold, + / - 3-fold, + / - 5-fold, or approximately the same, as shown in FIG. 29. In other embodiments, as shown in FIG. 29, the absolute expression level of a gene in the human organoids of the present invention is + / - 35%, + / - 30%, + / - 25%, + / - 20%, + / - 15%, + / - 10%, + / - 5%, + / - 3%, or + / - 1.5-fold, + / - 2-fold, + / - 3-fold, + / - 5-fold, or approximately the same.

[0346] The human liver cell population or organoids of the present invention also preferably express Lgr5 and / or Tnfrsf19, preferably both. In some embodiments, the human liver cell population or organoids express Lgr5 and / or Tnfrsf19, preferably both, when cultured in the expansion medium of the present invention. Preferably, the expression of Lgr5 and / or Tnfrsfr19 is detected by RT PCR. In some embodiments, Lgr5 and / or Tnfrsf19 in the organoids or cells when cultured in the differentiation medium are present at significantly lower (e.g., at least 1 / 2, at least 1 / 3, at least 1 / 4, at least 1 / 5, at least 1 / 10, at least 1 / 15) expression levels compared to the expression levels in the organoids or cells when cultured in the expansion medium.

[0347] The liver cells and organoids according to the present invention preferably secrete albumin, for example, at a rate of about 1 μg / hour / 10 6 cells to 10 μg / hour / 10 6 cells, preferably at a rate of 2 μg to 6 μg / hour / 10 6 cells.

[0348] Furthermore, such liver cells and organoids may secrete urea. For example, in a 35 mm cell dish, the activity of urea synthesis may be 1 μg to 50 μg, preferably 5 μg to 30 μg, in 48 hours.

[0349] The liver cells and organoids according to the present invention may, for example, show visible glycogen storage when stained. The ability of the cells and organoids according to the present invention to actively synthesize glycogen can be tested by changing the culture medium from a low glucose differentiation medium to a high glucose DMEM supplemented with 10% FBS and 0.2 μM dexamethasone for 2 days.

[0350] The liver cells and organoids according to the present invention may have inducible cytochrome P450 activity (e.g., CYP1A). Such activity can be tested, for example, using an ethoxyresorufin-O-deethylase (EROD) assay (Cancer Res, 2001, 61:8164-8170). For example, cells or organoids can be exposed to a P450 substrate such as 3-methylcholanthrene, and the EROD activity level can be compared to that of control cells.

[0351] Morphologically, liver organoid cells look like hepatocytes.

[0352] Preferred liver organoids include, or consist of, a cell layer with buds on the outside and a cyst structure with a central lumen as shown in FIG. 30. Such liver organoids may have one or more (e.g., two, three, or all four) of the following characteristics: (a) >5x10 5 cells / cm 3 , preferably >10x10 5 cells / cm 3 of cell density; (b) a thickness corresponding to 2 to 30 cell layers, preferably a thickness corresponding to 2 to 15 cell layers; (c) the cells are in three-dimensional contact with each other, (d) exhibit functions characteristic of healthy liver tissue, (e) have an elongated shape with two defined regions, namely, a monolayer epithelial region where highly polarized cells are detected and keratin markers are expressed (this region resembles the bile duct region), and the other region is formed by a multilayer epithelium with non-polarized cells that may detect albumin expression and constitute the organoid body. It will be apparent to those skilled in the art that such liver organoids are preferably not liver fragments and / or do not contain blood vessels and / or do not contain liver lobules or bile ducts.

[0353] In the context of the present invention, a liver fragment is part of an adult liver, preferably of a human adult liver. Thus, preferably, the liver organoids identified herein are not liver fragments. The liver organoids are preferably obtained using cells derived from an adult liver, preferably epithelial stem cells derived from an adult liver, more preferably epithelial stem cells derived from an adult liver expressing Lgr5. The liver organoids may also be obtained from any cell that expresses Lgr5 upon injury or in culture and thus is a cycling stem cell expressing Lgr5.

[0354] In some embodiments, the liver organoids comprise cells expressing Lgr5. For example, in some embodiments, at least 2%, more preferably at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the cells in the liver organoids express Lgr5. Similarly, the present invention provides cells or cell populations expressing Lgr5 and obtained from the liver organoids of the present invention. Progeny of such cells are also encompassed by the present invention.

[0355] In one embodiment, the liver organoids are liver organoids cultured using the method of the present invention and thus in contact with an extracellular matrix. Preferably, the liver organoids are embedded in a non-mesenchymal extracellular matrix or a mesenchymal extracellular matrix. In the context of the present invention, "in contact with" means physical or mechanical or chemical contact. Physical or mechanical or chemical contact means that a force needs to be used to separate the liver organoid from the extracellular matrix.

[0356] In a preferred embodiment, the liver organoids can be cultured for at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or longer. In some embodiments, the liver organoids are expanded or maintained in culture for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer. Preferably, the liver organoids cultured using the expansion medium of the present invention containing a TGFβ inhibitor can be cultured for at least 4 weeks, more preferably at least 5 weeks, with a 5-fold expansion per week or a population doubling more than twice per week (e.g., at least 10 doublings, at least 20 doublings, more preferably at least 25 doublings, e.g., at least 30 doublings). Preferably, the liver organoids cultured using the expansion medium of the present invention containing a prostaglandin pathway activator in addition to a TGFβ inhibitor can be cultured for at least 7 weeks, more preferably at least 8 weeks, with a doubling more than twice per week (e.g., 2 - 3 doublings) (i.e., at least 15 doublings, at least 25 doublings, at least 30 doublings, at least 32 doublings, at least 35 doublings, e.g., 32 - 40 doublings, or at least 40 doublings, e.g., at least 50 doublings). Thus, preferably, the liver organoids of the present invention, e.g., human liver organoids, are obtained using the expansion medium of the present invention.

[0357] In another preferred embodiment, the liver organoids are derived from a single cell, preferably a single cell expressing Lgr5. More preferably, the single cell contains a nucleic acid construct containing the nucleic acid molecule of interest.

[0358] Organoid composition and gene expression Crypt-villus organoids, colonic crypt organoids, and pancreatic organoids typically contain stem cells and / or progenitor cells. Thus, these organoids share a particular gene expression pattern. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) or all of the following markers can be detected: LGR5, LGR4, EpCAM, CD44, Sox9, CD24a, and CD133 / Prom1, optionally Tnfrsf19. In another embodiment, the expression of one or two or all of the following progenitor cell genes can be detected: Pdx1, Nkx2.2, and Nkx6.1. After differentiation, the gene expression patterns of crypt-villus organoids, colonic crypt organoids, and pancreatic organoids are expected to diverge when the differentiated organoids express tissue-specific adult markers, e.g., insulin in the pancreas.

[0359] Crypt-villus organoid In some embodiments of the invention, the organoid comprises a crypt-villus-like extension containing all differentiated epithelial cell types including proliferative cells, Paneth cells, enterocytes, and goblet cells. In some embodiments, the crypt-villus organoids of the invention do not contain myofibroblasts or other non-epithelial cells. The crypt-villus organoids of the invention preferably contain enterocytes including intestinal absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells within the crypt-villus-like structure. Preferably, at least one (e.g., 2, 3, 4, 5, or 6) of the following markers could be detected (see FIGS. 2e and 14): SMOC2, CDCA7, OLFM4, ASCL2, AXIN2 and / or Lgr5, Tnfrsf19, CD24a, Sox9, CD44, Prom1. In some embodiments, the markers RNF43 and ZNRF3 can be detected. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) or all of SMOC2, CDCA7, OLFM4, ASCL2, AXIN2, and / or Lgr5 are upregulated at least 2-fold, 3-fold, or 4-fold in the crypt, whereas markers that are downregulated to at least 1 / 2, 1 / 3, or 1 / 4 in the crypt include at least one or more (e.g., 1, 2, 3, or 4) or all of ABCG1, ENPP3, CSTE, MUC17, and / or APOA1. In this context, "upregulation" is relative to the upper part of the intestinal villus or colonic crypt. From microarray analysis comparing gene expression of differentiated organoid cells to stem cells, it was revealed that small intestinal crypt-villus organoids and colonic organoids have an equivalent molecular signature of the intestinal crypt including expression of intestinal stem cell genes. Thus, the invention also provides colonic organoids having the molecular signature described above for crypt-villus organoids. Organoids cultured in vitro clearly show an expression profile similar to freshly isolated small intestinal crypts and express known stem cell markers.

[0360] In some embodiments, the mRNA encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) of the genes listed in FIG. 14 (e.g., all of the genes with a minus sign in FIG. 14) that are upregulated in crypt-villus organoids or colonic organoids is, when confirmed by microarray, upregulated in the crypt-villus organoids or colonic organoids of the present invention as compared to freshly isolated small intestinal villi. In some embodiments, the mRNA encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) of the genes (e.g., all of the genes with a minus sign in FIG. 14) that are downregulated in crypt-villus organoids or colonic organoids as listed in FIG. 14 is, when confirmed by microarray, downregulated in the crypt-villus organoids or colonic organoids of the present invention as compared to freshly isolated small intestinal villi. In some embodiments, the fold of upregulation or downregulation is as shown in FIG. 14, + / -25%, e.g., + / -20%, + / -15%, + / -10%, + / -5%, + / -3%, or is approximately the same as that cited in FIG. 14. For example, the crypt-villus organoids of the present invention may have ADORA2B upregulated 9.54-fold + / -25% as compared to freshly isolated small intestinal villi. The same applies to the other genes listed in FIG. 14.

[0361] In some embodiments, the crypt-villus organoids exhibit native expression of Lgr5. In some embodiments, the crypt-villus organoids express at least Lgr5, as well as CK19, nestin, somatostatin, CXCR4 + , CD133 +One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) or all of the natural expression of stem cell markers from the group consisting of DCAMKL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnflα, Hnf4a, Sox9, KRT7, and KRT19 is shown. Further, or alternatively, the crypt-villus organoid may be characterized by the expression of one or more (e.g., 1 or 2) or all of Sord and / or Prss23. Further, or alternatively, the crypt-villus organoid may be characterized by the expression of CD44 and / or Sox9. In another aspect, the crypt-villus organoid shows the expression of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) or all of the markers from the group consisting of lgr5, lgr4, epcam (tacstd1), Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1, and Cdca7.

[0362] In some aspects, the crypt-villus organoid contains Paneth cells that express lysozyme.

[0363] Colonic organoid In some aspects, the colonic organoid contains enteroendocrine cells (e.g., detectable using chromagranin A staining) and goblet cells (detectable using mucin 2 staining). In some aspects, less than 10% (e.g., 0.01 - 5%, 0.1 - 3%) of the cells in the colonic organoid are enteroendocrine cells. In some aspects, less than 30% (e.g., 1 - 25%, 1 - 15%, 5 - 10%) of the cells in the colonic organoid are goblet cells. In some aspects, the distribution of enteroendocrine cells and / or goblet cells is as shown in Figure 1d.

[0364] In some aspects, the colonic organoid contains mature enterocytes (e.g., visualized by alkaline phosphatase staining). In some aspects, less than 10% (e.g., less than 5%, less than 3%, 0.01 - 5%, 0.1 - 3%, 0.1 - 5%) of the cells in the colonic organoid are mature enterocytes.

[0365] In a preferred embodiment, since Paneth cells are not present in the native in vivo colon, the colon organoids do not contain Paneth cells.

[0366] In some embodiments, the colon organoids exhibit native expression of Lgr5.

[0367] In some embodiments, the colon organoids express one or more (e.g., 1, 2, 3, or 4) of villin1, Alpi, ChgA, and Muc2. In some embodiments, the relative amount of villin1 mRNA expressed by the colon organoids of the present invention compared to freshly isolated colon crypts is at least 3% (e.g., at least 5%, at least 8%, at least 10%), e.g., 5 - 15%. In some embodiments, the relative amount of Alpi mRNA expressed by the colon organoids of the present invention compared to freshly isolated colon crypts is at least 0.5% (e.g., at least 1%, at least 2%), e.g., 0.5 - 5%. In some embodiments, the relative amount of ChgA mRNA expressed by the colon organoids of the present invention compared to freshly isolated colon crypts is at least 15% (e.g., at least 20%, at least 22%), e.g., 15 - 30%. In some embodiments, the relative amount of Muc2 mRNA expressed by the colon organoids of the present invention compared to freshly isolated colon crypts is at least 20% (e.g., at least 25%, at least 30%, at least 35%), e.g., 25 - 37%.

[0368] In some embodiments, the human colon organoids of the present invention express known stem cell markers.

[0369] Pancreatic organoids The pancreas contains three cell types: ductal cells, acinar cells, and endocrine cells. Endocrine cells secrete into the bloodstream and produce the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide (PP) that assist in the body's glucose metabolism. Acinar cells are part of the exocrine system that produces digestive enzymes, and ductal cells are derived from the pancreatic ducts that connect acinar cells to the digestive organs. During development, the islets of Langerhans are thought to arise from progenitor endocrine cells that emerge from the pancreatic ducts and aggregate after differentiation to form the islets of Langerhans. The islets of Langerhans contain α cells, β cells, δ cells, and PP cells.

[0370] Pancreatic organoid cells may have an expression pattern similar to ductal cell markers, such as one or more (e.g., 1, 2, or all) of K7, K19, and Hnf1b, and / or one or more general stem cell markers such as Sox9 and / or Onecut1. This is likely to be part of the stem cell signature. Generally, fewer differentiation markers are seen. In some embodiments where cells are isolated from pancreatic ducts to generate the pancreatic organoids of the present invention, the cell type that gives rise to the pancreatic organoids of the present invention is not ductal cells (meaning epithelial cells that form ducts and are positive for keratin 7 and keratin 19), but cells attached to the pancreatic ducts. Cells attached to the pancreatic ducts mean cells that are located in the adjacent cell layer after the duct contacts the pancreatic tissue (i.e., not facing the lumen of the duct). Thus, in embodiments where the cell type that gives rise to the pancreatic organoids is not ductal cells, the pancreatic organoids do not express either K7 or K19. However, such pancreatic organoids more preferably express one or more general stem cell progenitor markers, such as Sox9.

[0371] The pancreatic organoids of the present invention preferably contain α cells, β cells, δ cells, and PP cells. In a more preferred embodiment, the pancreatic organoids contain β cells. For example, the pancreatic organoids may contain more than 1%, more than 5%, more than 10%, more than 15%, or more than 20% β cells. Expression of insulin may be used as a β cell marker.

[0372] In another aspect, the pancreatic organoids include progenitor cell types that can give rise to differentiated cell types when transplanted into a human or animal, optionally progenitor cell types derived from ducts. In a preferred aspect, the progenitor cell type can give rise to insulin-secreting β cells when transplanted into a human or animal. The inventors have shown that human pancreatic organoids grown according to the media and methods of the present invention can be transplanted into mice and stimulate insulin-secreting cells within one month (see Example 4). This can be readily understood to potentially lead to an epoch-making treatment for patients with diabetes and insulin deficiency.

[0373] In some aspects, the pancreatic organoids of the present invention may include duct cells, acinar cells, and endocrine cells. In some aspects, K19 is used as a duct cell marker.

[0374] In some aspects, β cells are present within pancreatic islets or islets of Langerhans. In vivo, one islet generally contains about 1500 cells, for example, 1300 - 1700 cells. In one aspect, the pancreatic organoids contain at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or more islets of Langerhans by mass. In some aspects, the islets of Langerhans in the pancreatic organoids consist of about 65 - 90% β cells, about 15 - 20% α cells, about 3 - 10% δ cells, and about 1% PP cells. However, it is by no means limited to this. For example, in some aspects, it is desirable for the organoids of the present invention to have many β cells. Or, the organoids may contain progenitor cells that can be transplanted to differentiate in vivo.

[0375] In some embodiments, the pancreatic organoids express one, two, or all three of Pdx1, Nkx2.2, and Nkx6.1. The pancreatic organoids may express one, two, three, or all four of NeuroD, Pax6, Pax4, and Mafa. Since Pax4 is a transcription factor essential for the differentiation of insulin-producing cells into endocrine progenitor cells during embryonic development, it serves as a marker for the presence of insulin-producing cells. The pancreatic organoids may express Ngn3.

[0376] In some embodiments, at least one (e.g., one, two, three, four, five) of the following markers can be detected in the pancreatic organoids of the present invention: insulin (ins1 and / or ins2), glucagon (Gcg), somatostatin, Pdx1, and NeuroD. In some embodiments, at least one (e.g., one, two, three, four, five) of the following markers can be detected in the pancreatic organoids of the present invention: insulin (ins1 and / or ins2), glucagon (Gcg), somatostatin, Pdx1, and NeuroD. The following markers are not detected: ptf1a, amy2a4, Pnlip, and cela1. In some embodiments, at least one (e.g., one, two, three, four, five, six, seven, eight, or nine) of the following markers can be detected in the pancreatic organoids of the present invention: Ptf1a, pancreatic amylase (Amy2a4), pancreatic lipase (Pnlip), insulin (ins1 and / or ins2), glucagon (Gcg), somatostatin, chymotrypsin (cela1), Pdx1, and NeuroD.

[0377] In some embodiments, the pancreatic organoids exhibit natural expression of Lgr5. In some embodiments, the pancreatic organoids express at least Lgr5, as well as CK19, nestin, CXCR4 + , CD133 +shows natural expression of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) stem cell markers selected from the group consisting of DCAMKL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19, prom1, Cd24a, Lgr4, epcam. Or, or further, in some embodiments, pancreatic organoids are CK19, nestin, (insulin, glucagon) and CXCR4 + may be characterized by natural expression of one or more (e.g., 1, 2, 3, or 4) thereof.

[0378] In some embodiments, the pancreatic organoids or cells of the invention express somatostatin. Somatostatin is a hormone expressed in differentiated δ cells and may thus serve as a marker for δ cells.

[0379] Or, or further, in some ...

Claims

1. A culture medium for expanding and / or differentiating an adult stem cell population, comprising: i. any one of Rspondin 1-4 and / or an Rspondin mimetic; and ii. One or more inhibitors that directly or indirectly negatively regulate TGF-β signaling.

2. The culture medium of claim 1, wherein the one or more inhibitors bind to and reduce the activity of one or more serine / threonine protein kinases selected from the group consisting of ALK5, ALK4, TGF-β receptor kinase 1, and ALK7.

3. The culture medium of claim 1 or claim 2, wherein the one or more inhibitors that directly or indirectly negatively regulate TGF-β signaling are selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511.

4. The culture medium of any of the preceding claims, further comprising an inhibitor that directly or indirectly negatively regulates p38 signaling.

5. The culture medium of claim 4, wherein the one or more inhibitors that directly or indirectly negatively regulate p38 signaling are selected from the group consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257, and BIRB-796.

6. 2. The culture medium of any of the preceding claims, comprising A83-01 and SB-202190 or A83-01 and SB-203580.

7. 2. The culture medium of any one of the preceding claims, wherein the inhibitors are added at a concentration of 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM, e.g., the total concentration of the one or more inhibitors is 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM.

8. The culture medium of any one of the preceding claims, comprising one or more additional components selected from a BMP inhibitor, a Wnt agonist, a receptor tyrosine kinase ligand, a Rock inhibitor, nicotinamide, and gastrin.

9. The culture medium according to any one of the preceding claims, comprising any one of Rspondins 1 to 4 and / or an Rspondin mimetic, a BMP inhibitor (e.g. Noggin), a TGF-β inhibitor, a receptor tyrosine kinase ligand (e.g. EGF), nicotinamide, a Wnt agonist (e.g. Wnt(3a)), and optionally one or more additional components selected from a p38 inhibitor, gastrin, FGF10, HGF, and a Rock inhibitor.

10. 10. The culture medium of claim 8 or claim 9, wherein the BMP inhibitor is selected from the group consisting of noggin, chordin, chordin-like protein containing a chordin domain, follistatin, follistatin-related protein containing a follistatin domain, DAN, DAN-like protein containing a DAN cysteine ​​knot domain, sclerostin / SOST, and alpha-2 macroglobulin.

11. The culture medium of claim 8 or claim 9, wherein the Wnt agonist is selected from the group consisting of Wnt-3a, a GSK inhibitor (e.g., CHIR99021), Wnt5, Wnt-6a, Norrin, and any other Wnt family protein.

12. The culture medium of claim 8 or claim 9, wherein the receptor tyrosine kinase ligand is a mitogenic growth factor, such as a mitogenic growth factor selected from the growth factor family consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF).

13. 10. The culture medium of claim 8 or claim 9, wherein the Rock inhibitor is selected from the group consisting of R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1071), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152).

14. 2. A culture medium according to any one of the preceding claims, additionally comprising a prostaglandin signalling pathway activator, such as PGE2 and / or AA.

15. 2. A culture medium according to any one of the preceding claims, additionally comprising testosterone, such as (dihydro)testosterone.

16. 16. A culture medium according to any one of claims 1 to 15 for culturing intestinal cells, comprising or consisting of a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin 1 to 4, a TGF-β inhibitor, nicotinamide, and preferably a p38 inhibitor.

17. 16. A culture medium according to any one of claims 1 to 15 for culturing gastric cells, comprising or consisting of a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin 1 to 4, a TGF-β inhibitor, gastrin, nicotinamide, FGF-10, and preferably a p38 inhibitor.

18. 16. A culture medium according to any one of claims 1 to 15 for expanding liver cells, comprising or consisting of a basal medium, any one of Rspondins 1 to 4, Noggin, Nicotinamide, EGF, FGF10, HGF, Gastrin, a TGF-β inhibitor, and PGE2, and preferably Wnt-3a.

19. A culture medium according to any one of claims 1 to 15 for expanding pancreatic cells, comprising or consisting of a basal medium, any one of Rspondin 1 to 4, Noggin, EGF, FGF10, Gastrin, a TGF-β inhibitor, and preferably Exendin 4 and Wnt-3a.

20. A culture medium according to any one of claims 1 to 15 for culturing prostate cells, comprising or consisting of a basal medium, EGF, any one of Rspondins 1 to 4, Noggin, Nicotinamide, a TGF-β inhibitor, and preferably Wnt-3a and FGF-10.

21. 21. A culture medium for culturing prostate cells according to claim 20, further comprising testosterone, such as (dihydro)testosterone.

22. comprising or consisting of components of a culture medium used to culture cells derived from the corresponding non-cancerous tissue type of interest; Optionally, one or more of Wnt-3a, EGF, Noggin, R-spondin, TGF-β inhibitor, p38 inhibitor, nicotinamide, gastrin, FGF10, and HGF are excluded from the medium used to culture non-cancerous cells of the tissue type of interest.

23. A culture medium according to any one of the preceding claims for culturing cancer cells, such as adenocarcinoma cells or carcinoma cells, e.g. cancer stem cells, derived from a tissue type of interest.

23. A culture medium according to any one of the preceding claims for differentiating stem cells derived from a tissue of interest, comprising or consisting of the components of a culture medium used to expand stem cells derived from the tissue type of interest, but excluding one or more of Wnt, Rspondin, BMP inhibitors, TGF-β inhibitors, receptor tyrosine kinase ligands, p38 inhibitors, and nicotinamide.

24. 24. The culture medium of claim 23 for differentiating intestinal cells, comprising or consisting of a basal medium, EGF, Noggin, a TGF-β inhibitor, and a p38 inhibitor.

25. 24. A culture medium for differentiating liver cells according to claim 23, comprising or consisting of a basal medium, Noggin, EGF, Gastrin, a TGF-β inhibitor, a γ-secretase inhibitor such as DAPT or DBZ, and preferably Wnt-3a.

26. 24. A culture medium for differentiating pancreatic cells according to claim 23, comprising or consisting of a basal medium, noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor, and preferably exendin 4.

27. 2. A culture medium according to any one of the preceding claims, in contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as integrins.

28. 28. The culture medium of claim 27, wherein the extracellular matrix is ​​a laminin-containing extracellular matrix, such as Matrigel™ (BD Biosciences).

29. 27. A composition comprising a culture medium according to any one of claims 1 to 26, and an extracellular matrix, or a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as integrins, e.g. a laminin-containing extracellular matrix such as Matrigel™ (BD Biosciences).

30. 13. A hermetically sealed container containing a culture medium or composition according to any one of the preceding claims.

31. Use of a culture medium according to any one of claims 1 to 28 for expanding and / or differentiating stem cells, stem cell populations, tissue fragments or organoids.

32. The use of claim 31, wherein the stem cell, stem cell population, tissue fragment or organoid is selected from the group consisting of one or more of intestinal stem cells, small intestinal crypts, colonic crypts, gastric stem cells, liver stem cells, pancreatic stem cells and prostate stem cells.

33. The use according to claim 31 or claim 32, wherein the stem cells, stem cell populations, tissue fragments or organoids can be obtained from normal tissue.

34. The use according to claim 31 or 32, wherein the stem cell, stem cell population, tissue fragment or organoid can be obtained from diseased tissue, for example adenoma, carcinoma, adenocarcinoma, the intestine of cystic fibrosis patients or the intestine of inflammatory bowel disease patients.

35. A method for expanding single stem cells, stem cell populations or tissue fragments, preferably for expanding them to obtain organoids, comprising culturing single stem cells or stem cell populations in a culture medium according to any one of claims 1 to 28.

36. 36. The method of claim 35, comprising the steps of: Providing stem cells, a stem cell population, or an isolated tissue fragment; Providing a culture medium according to any one of claims 1 to 28; contacting the stem cells with the culture medium; Culturing the cells under appropriate conditions.

37. The method of claim 35, comprising contacting the stem cells, stem cell population, or isolated tissue fragments, and culture medium with an extracellular matrix or a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as integrins, e.g., a laminin-containing extracellular matrix such as Matrigel™ (BD Biosciences).

38. 40. The method of claim 37, wherein the culture medium is diffused into the extracellular matrix.

39. The method of any one of claims 35 to 38, comprising the steps of: Culturing the stem cells, stem cell population, or tissue fragment in a first expansion medium; Continuing to culture the stem cells, stem cell population, or tissue fragments and supplementing the medium with a differentiation medium that does not contain one or more, preferably all, of the factors selected from TGF-β inhibitors, p38 inhibitors, nicotinamide, and Wnt.

40. A method according to any one of claims 35 to 38 for obtaining small intestinal or colonic organoids, comprising the steps of: Expanding small intestinal or colonic stem cells or tissue fragments in a culture medium according to claim 17; and optionally A process for differentiating expanded small intestinal or colonic stem cells or tissue fragments in the culture medium of claim 24.

41. A method according to any one of claims 35 to 39 for obtaining gastric organoids, comprising culturing gastric stem cells or tissue fragments in the culture medium according to claim 16.

42. A method according to any one of claims 35 to 39 for obtaining liver organoids, comprising the steps of: Expanding liver cells or tissue fragments in a culture medium according to claim 19; and optionally, A process for differentiating expanded liver cells or tissue fragments in the culture medium of claim 25.

43. A method according to any one of claims 35 to 39 for obtaining pancreatic organoids, comprising the steps of: Expanding pancreatic cells or tissue fragments in a culture medium according to claim 20; and optionally, 27. A process for differentiating expanded pancreatic cells or tissue fragments in the culture medium of claim 26.

44. A method according to any one of claims 35 to 39 for obtaining prostate organoids, comprising culturing stem cells or tissue fragments of the prostate in the culture medium according to claim 19.

45. A method according to any one of claims 35 to 39 for obtaining an adenocarcinoma or carcinoma organoid, comprising culturing stem cells or tissue fragments of an adenocarcinoma or carcinoma in a culture medium according to claim 22.

46. 40. The method of any one of claims 35-39, wherein the Rock inhibitor is added to the culture medium for the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, optionally every other day.

47. 40. The method of any one of claims 35 to 39, comprising culturing stem cells for 3 months or longer, such as 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer, using a culture medium of any one of claims 1 to 28.

48. Organoid or cell population obtainable by the method according to any one of claims 35 to 47.

49. The organoid or cell population of claim 48, when cultured in a culture medium according to any one of claims 1 to 28, can survive in culture for at least 3 months, for example at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer.

50. 49. The organoid or cell population of claim 48, which has been cultured for at least 3 months, such as at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months, or longer.

51. 51. The organoid or cell population of any one of claims 48-50, which expands at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold per week.

52. 52. The organoid or cell population of any one of claims 48 to 51, which is a human organoid or a human cell population.

53. The organoid or cell population of any one of claims 48 to 52, which is a normal organoid or normal cell population, or a diseased organoid or diseased cell population, such as a diseased organoid or diseased cell population obtained by culturing stem cells taken from a human or animal with a disease.

54. The organoid or cell population of any one of claims 48 to 53, wherein the organoid or cell population is frozen and stored at a temperature below -5°C, below -10°C, below -20°C, below -40°C, below -60°C, below -80°C, below -100°C, or below -150°C, such as at about -180°C.

55. Organoid, preferably according to any one of claims 48 to 54, which is a three-dimensional organoid comprising epithelial cells surrounding a central lumen, optionally said epithelial cells being present in distinct division and differentiation areas.

56. 56. The organoid of claim 55, which is a three-dimensional organoid comprising epithelial cells arranged in a monolayer, optionally in areas of a folded monolayer and areas of stratified cells.

57. The organoid of any one of claims 48 to 56, wherein said organoid is free of non-epithelial cells.

58. The organoid of any one of claims 48 to 57, wherein all differentiated cell types of normal in vivo tissue are present.

59. The organoid of any one of claims 48 to 58, which is a small intestinal organoid, a colonic organoid, a stomach organoid, a pancreatic organoid, a liver organoid, or a prostate organoid.

60. A composition comprising: i) one or more organoids or cell populations according to any one of claims 48 to 59; and ii) A culture medium and / or an extracellular matrix according to any one of claims 1 to 28.

61. An organoid according to any one of claims 48 to 59, or a cell population according to any one of claims 48 to 54, or a composition according to claim 29 or claim 60, for use in drug screening, target validation, target discovery, toxicology, toxicity screening, personalized medicine, regenerative medicine, or an ex vivo cell / organ model, such as for use as a disease model.

62. 62. The organoid, cell population or composition for use according to claim 61, wherein the regenerative or personalized medicine comprises transplantation of the organoid, cell population or composition into a mammal, preferably a human.

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