Medium for stem cells

Abstract

Media and methods for expanding and differentiating stem cell populations and for obtaining organoids. Expanded cell populations and organoids obtained by the methods of the invention and their use in drug screening, toxicity testing and regenerative medicine.

Description

[0001] This application is a divisional application of an application with an application date of June 11, 2012, application number 201810714085.8, and invention name “Culture medium for stem cells”, and an application with an application date of June 11, 2012, application number 201810714085.8, and invention name “Culture medium for stem cells” is a divisional application of an application with an application date of June 11, 2012, application number 201280039061.8, and invention name “Culture medium for stem cells”.

[0002] All documents cited herein are incorporated by reference in their entirety. Technical Field

[0003] The present invention is in the field of stem cell culture media and methods, and in particular, in the field of culture media and methods for expanding stem cell populations, such as human epithelial stem cell populations. Background Art

[0004] There is great concern about 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 for cell testing, drug screening, and toxicity testing. Stem cells also show promise for cell-based treatments, such as in regenerative medicine for treating damaged tissues. They are also used as a source of differentiated cells for transplantation purposes, such as transplanting pancreatic beta cells for the treatment of diabetes, etc. In addition, efficient cell culture media are important for providing and maintaining cell populations for research purposes.

[0005] There is also concern about the culture medium and method for culturing stem cells to form, maintain and amplify organoids, such as intestinal crypt villi, stomach or pancreatic organoids. Organoids include stem cells, such as epithelial stem cells, which maintain their undifferentiated phenotype and self-renewal properties, but also have differentiated offspring that grow into tissue-like structures. Similar to a group of related or identical cells, crypt villi, stomach or pancreatic organoids more closely simulate the basic physiological functions of their organ tissues, and they can be used for toxicity testing or for testing of drugs or food supplements. They can also be used to cultivate pathogens that currently lack suitable tissue cultures or animal models. In addition, such organoids can be used for regenerative medicine, such as for repairing intestinal epithelium after radiation and / or after surgery, or for repairing the intestinal epithelium of patients suffering from inflammatory bowel disease.

[0006] It is clear that there are many clinical and research applications for stem cells and their branched offspring. For all these applications, reproducible stem cell culture methods are extremely important for providing a sufficient amount of cells of suitable quality. For example, for effective drug screening, conditions must be carefully controlled, and the precise culture method for the differentiation and proliferation of the control cells is required so that a group of cells with the same phenotype and karyotype of pure cells can be produced. Similarly, for cell-based treatments in which the cells cultured can be directly provided to the patient, the cell must be reasonably in heredity and phenotype to avoid undesirable immune response or final cell type when provided to the patient.

[0007] Although 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), to date, no long-term culture system has been established that maintains the differentiation potential and phenotypic and genomic integrity of human epithelial stem cells.

[0008] International patent application WO2010 / 090513 discloses a method for culturing epithelial hepatocytes or isolated tissue fragments. The method was optimized to culture human colon and intestinal crypts by adding Wnt-3a to the culture medium. This is the first time that human intestinal stem cell cultures have been cultured for an extended period of time (up to 3 months) and is defined as the first reproducible human intestinal stem cell culture system. However, there is still an urgent need for improved stem cell culture media and methods, particularly human stem cell culture media and methods, which improve the proliferation rate, survival time, and phenotypic and genomic integrity of stem cells grown in culture. Brief description of the invention

[0010] The present invention provides improved culture media and methods for stem cells, especially human epithelial stem cells, and organoids comprising said stem cells, which provide significant advantages over known culture media and methods. The present invention also provides related culture media supplements, compositions and uses.

[0011] Therefore, the present invention provides a culture medium for expanding a stem cell population, wherein the culture medium includes at least one or more inhibitors that bind to one or more serine / threonine protein kinase targets and reduce their activity. It has the effect of continuous growth for at least 3 months at an amplification rate of about five times per week. Serine / threonine protein kinases are preferably selected from: TGFβ receptor kinase 1, ALK4, ALK5, ALK7, p38. Surprisingly, the inventors have found that the inclusion of inhibitors of certain serine / threonine kinases in the culture medium significantly improves the performance of the culture medium in expanding stem cell populations. The stem cell population can be normal (healthy) cells or diseased cells (e.g., cancer stem cells). In particular, inhibitors of p38 and ALK appear to provide the greatest improvement among all tested compounds. This is unexpected because there is no known mechanism to predict how these specific inhibitors work. In fact, a variety of small molecule inhibitors selected for testing and acting in similar pathways have no effect on the method. Therefore, technicians cannot predict that inhibitors of these specific kinases will have such significant improvements to the culture medium. Still further improvement was observed when two inhibitors, for example a p38 inhibitor such as SB202190 and an ALK inhibitor such as A83-01, were co-added to the culture medium.

[0012] To reach this realization, the inventors investigated signal transduction pathways known to be disrupted in certain cancers, such as colorectal cancer. They hypothesized that these pathways that affect the final cell type in cancer also play a role in determining the final cell type in culture conditions. However, it should be emphasized that this hypothesis is completely novel; because the prior art could not predict the effect of any of these additional compounds on the culture medium, and did not specifically expect that any of 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 standard stem cell culture medium. Gastrin and nicotinamide were initially determined to produce significantly improved culture conditions. These factors were incorporated into standard culture conditions and a second screening experiment was performed, in which small molecule inhibitors related to related signal transduction pathways were tested, such as ERK, p38, JNK, PTEN, ROCK and Hedgehog. These pathways were selected because they are known to be destroyed in certain cancers.

[0014] Previous attempts to culture human intestinal stem cells using the aforementioned stem cell culture medium (comprising epithelial growth factor (EGF or "E"), Noggin ("N"), and R-spondin ("R"), also referred to herein as "ENR" medium) optimized by Wnt-3A ("W"), (referred to herein as "WENR" medium) have resulted in the majority of cells metamorphosing within 7 days, with very few cells surviving for more than a month. Such attempts have been met with lengthy proliferation times, chromosomal abnormalities, and morphological changes from budding to cystic structures. "Ccystic" means that the organoids are almost spherical. "Budding" means that the organoids have multiple regions that grow from the underlying structure. Having budding structures is not always an advantage, although budding structures generally have a larger surface area and generally more closely resemble corresponding in vivo structures.

[0015] The inventors showed that the improved method allowed the stem cells to continue growing for at least 7 months.

[0016] The new method also increases the rate at which cells in the expanded population proliferate. This obviously has great use when growing cells for commercial and medical purposes.

[0017] The new method also increases the quality of the cells in the expanded population. This is very advantageous because clinical and research applications of stem cells and their differentiated progeny require reproducible stem cell culture methods that provide high-quality cell populations. Typically, in vitro expansion of stem cells is intended to provide cell populations that are as closely similar as possible to their in vivo counterparts. This property is referred to herein as the "genomic and phenotypic integrity" of the cells.

[0018] The inventors have discovered for the first time 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 exhibit budding organoid structures rather than the cystic structures seen under previous culture conditions. In each of the 20 cells collected from three different donors, metaphase divisions of organoids older than 3 months consistently showed 46 chromosomes. In addition, microarray analysis showed that stem cells in culture had molecular signatures similar to intestinal crypt cells, including intestinal stem cell genes.

[0019] The inventors have also demonstrated that human intestinal organoids produced by the culture medium and methods of the present invention mimic the final cell type decision in vivo in response to external factors. For example, it has been previously 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, when treated with Notch inhibitors, interrupted proliferation and most of the cells transformed 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 to expand stem cells or organoids from other epithelial tissues, such as stomach, pancreas, liver, and prostate (see Examples). The tissue can be normal (healthy) tissue or diseased tissue, such as cancer tissue or tissue exhibiting a cystic fibrosis phenotype.

[0021] These results demonstrate the significant improvement in the genomic and phenotypic integrity of stem cells and organoids produced by the methods and culture media of the present invention compared to previous methods and culture media.

[0022] Therefore, the present invention provides a culture medium for expanding and / or differentiating a population of adult stem cells, wherein the culture medium comprises:

[0023] i. one of Rspondin 1-4 and / or a Rspondin mimetic; and

[0024] ii. One or more inhibitors that directly or indirectly negatively regulate TGF-β signaling.

[0025] The present invention also provides a composition comprising the culture medium of the present invention and an extracellular matrix or a 3D matrix that mimics an extracellular matrix through its interaction with cell membrane proteins such as integrins, for example an extracellular matrix containing laminin, such as Matrigel. TM (BD Biosciences).

[0026] The present invention also provides an airtight container comprising the culture medium or composition of the present invention.

[0027] The present invention also provides use of the culture medium of the present invention for expanding and / or differentiating stem cells, stem cell populations, tissue fragments or organoids.

[0028] The present invention also provides a method for expanding a single stem cell, a stem cell population or a tissue fragment, preferably to produce an organoid, wherein the method comprises culturing a single stem cell or a stem cell population in a culture medium of the present invention.

[0029] The present invention also provides organoids or cell populations obtained by the method of the present invention.

[0030] The present invention also provides an organoid, preferably an organoid obtained by a method according to the present invention, which is a three-dimensional organoid comprising epithelial cells surrounding a central lumen, wherein optionally the epithelial cells are present in different division and differentiation domains.

[0031] The present invention also provides an organoid, preferably an organoid obtained by the method of the present invention, which is a three-dimensional organoid comprising epithelial cells arranged in a monolayer, optionally in regions of folded monolayers, and in regions of stratified cells, and preferably, it is a three-dimensional organoid comprising epithelial cells surrounding a central lumen, wherein optionally, the epithelial cells are present in different division domains and differentiation domains.

[0032] The present invention also provides a composition comprising:

[0033] i) one or more organoids or cell populations of the invention; and

[0034] ii) the culture medium and / or extracellular matrix of the present invention.

[0035] The present invention also provides organoids, cell populations or compositions of the present invention for use in drug screening, target validation, target discovery, toxicology, toxicology screening, personalized medicine, regenerative medicine or in vitro cell / organ models, such as for use as disease models.

[0036] The present invention also provides an organoid, cell population or composition for transplanting the organoid, cell population or composition of the present invention into a mammal, preferably into a human.

[0037] The present invention also provides a stem cell population obtained or obtainable using the culture medium of the present invention or an organoid comprising the stem cell. The stem cell or the organoid comprising the stem cell can be used, for example, for transplantation purposes or other therapeutic applications. For example, the stem cell or the organoid comprising the stem cell can be used for drug screening, target confirmation, target discovery, toxicology and toxicology screening, personalized medicine, regenerative medicine and three-dimensional cell / organ models, such as disease models.

[0038] The present invention also provides a composition comprising the culture medium of the present invention.

[0039] The present invention also provides a culture medium supplement comprising the inhibitor of the present invention.

[0040] The present invention also provides an airtight container comprising the culture medium and / or culture medium supplement of the present invention.

[0041] The specific ingredients of the media, supplements and compositions of the invention can be varied according to particular needs and applications. Likewise, the precise steps of the methods of the invention can be varied according to particular needs and applications.

[0042] The culture medium, supplements, methods, compositions and uses of the present invention can also be optimized by routine experimentation. For example, if the culture medium, supplement or composition does not give the desired level of stem cell expansion, variables such as the amount of each component in the culture medium or supplement, inoculation density, culture conditions, culture period, etc. can be changed in further experiments. The amount of each component of the components described herein can be optimized in a manner independent of other components by routine optimization, or one or more components can be added or removed. The ability of the culture medium to support stem cell expansion can be tested by testing the culture medium with a known culture medium or method or by replacing the culture medium with a known culture medium or method for measurement.

[0043] The culture media, supplements, methods, compositions and uses of the present invention are described in more detail below. Unless otherwise indicated, the practice of the present invention will employ conventional cell culture techniques, molecular biology and microbiology, which are within the scope of those skilled in the art.

[0044] Many textbooks providing guidance on mammalian cell culture media and methods are available, including specialized textbooks on culture media and methods for culturing stem cells. Such textbooks include 'Basic Cell Culture Protocols' (1997) by J. Pollard and JM Walker, 'Mammalian Cell Culture: Essential Techniques' (1997) by A. Doyle and JB Griffiths, 'Culture of Animal Cells: A Manual of Basic Technique' (2005) by RI Freshney, 'Basic Cell Culture Protocols' (2005) by C. Helgason and C. L. Miller, 'Stem Cells: From Bench to Bedside' (2005) by A. Bongso, 'Human Stem Cell Manual: A Laboratory Guide' (2007) by JF Loring, RL Wesselschmidt and PHSchwartz.

[0045] Stem cells and cell culture reagents and equipment for use in the present invention are commercially available, for example, from Cellartis AB ( Sweden), VitroLife AB (Kungsbacka, Sweden), (Invitrogen), Millipore Corporation (Billerica, Massachusetts), (St. Louis, Missouri) and Biomol International LP (Exeter, UK).

[0046] In non-limiting aspects, the present application provides the following exemplary embodiments:

[0047] Embodiment 1. A culture medium for expanding and / or differentiating a population of adult stem cells, wherein the culture medium comprises:

[0048] i. Rspondin 1-4 and / or one of the Rspondin mimetics; and

[0049] ii. One or more inhibitors that directly or indirectly negatively regulate TGF-β signaling.

[0050] Embodiment 2. A culture medium as described in embodiment 1, wherein the one or more inhibitors bind to and reduce the activity of one or more serine / threonine protein kinases selected from the following: ALK5, ALK4, TGF-β kinase inhibitor 1 and ALK7.

[0051] Embodiment 3. A culture medium as described in embodiment 1 or 2, wherein the one or more inhibitors that directly or indirectly negatively regulate TGF-β signal transduction are selected from A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511.

[0052] Embodiment 4. The culture medium of any of the preceding embodiments, wherein the culture medium further comprises an inhibitor that directly or indirectly negatively regulates p38 signal transduction.

[0053] Embodiment 5. The culture medium of embodiment 4, wherein the one or more inhibitors that directly or indirectly negatively regulate p38 signal transduction are selected from SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257 and BIRB-796.

[0054] Embodiment 6. The culture medium of any of the preceding embodiments, wherein the culture medium comprises A83-01 and SB-202190, or A83-01 and SB-20358.

[0055] Embodiment 7. A culture medium as described in any of the preceding embodiments, wherein 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, wherein the total concentration of the one or more inhibitors is 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM.

[0056] Embodiment 8. The culture medium according to any of the preceding embodiments, wherein the culture medium further comprises one or more components selected from the group consisting of a BMP inhibitor, a Wnt agonist, a receptor tyrosine kinase ligand, a Rock inhibitor, nicotinamide, and gastrin.

[0057] Embodiment 9. A culture medium as described in any of the preceding embodiments, wherein the culture medium comprises any one of Rspondin 1-4 and / or Rspondin mimetics, 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 further comprises one or more components selected from the following: a p38 inhibitor, gastrin, FGF10, HGF, and a Rock inhibitor.

[0058] Embodiment 10. A culture medium as described in embodiment 8 or 9, wherein the BMP inhibitor is selected from Noggin, chordin, chordin-like protein including a chordin domain, follistatin, follistatin-related protein including a follistatin domain, DAN, DAN-like protein including a DAN cysteine ​​knot domain, sclerostin / SOST and α-2 macroglobulin.

[0059] Embodiment 11. The culture medium of embodiment 8 or 9, wherein the Wnt agonist is selected from Wnt-3a, a GSK-inhibitor (eg, CHIR99021), Wnt 5, Wnt-6a, Norrin, and any other Wnt family protein.

[0060] Embodiment 12. A culture medium as described in embodiment 8 or 9, wherein the receptor tyrosine kinase ligand is a mitogenic growth factor, for example, selected from the family of growth factors, which consists of 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).

[0061] Embodiment 13. A culture medium as described in embodiment 8 or 9, wherein the Rock inhibitor is selected from R-(+)-trans-4-(l-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(l,4-diazepan-1-ylsulfonyl)isoquinoline (Fasudil or HA1077) and (S)-(+)-2-methyl-l-[(4-methyl-5-isoquinolyl)sulfonyl]-hexahydro-1H-1,4-diazepane dihydrochloride (H-1152).

[0062] Embodiment 14. A culture medium as described in any of the preceding embodiments, wherein the culture medium further comprises a prostaglandin signaling pathway activator, such as PGE2 and / or AA.

[0063] Embodiment 15. A culture medium as described in any of the preceding embodiments, wherein the culture medium further comprises testosterone, such as (dihydro)testosterone.

[0064] Embodiment 16. The culture medium according to any one of embodiments 1 to 15, which is used for culturing intestinal cells, comprises or consists of: a basal medium, any one of Wnt-3a, EGF, Noggin, Rspondin 1-4, a TGF-β inhibitor, nicotinamide, and preferably a p38 inhibitor.

[0065] Embodiment 17. The culture medium according to any one of embodiments 1 to 15, which is used for culturing gastric cells, comprises or consists of: a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin 1-4, a TGF-β inhibitor, gastrin, nicotinamide, FGF-10, and preferably a p38 inhibitor.

[0066] Embodiment 18. A culture medium as described in any one of embodiments 1 to 15, which is used for expanding liver cells, and the culture medium comprises or consists of the following: a basal medium, any one of Rspondin 1-4, Noggin, nicotinamide, EGF, FGF10, HGF, gastrin, a TGF-β inhibitor and PGE2, and preferably Wnt-3a.

[0067] Embodiment 19. A culture medium as described in any one of embodiments 1 to 15, which is used to expand pancreatic cells, and the culture medium comprises or consists of the following: a basal medium, any one of Rspondin 1-4, Noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, and preferably exendin 4 and Wnt-3a.

[0068] Embodiment 20. The culture medium according to any one of embodiments 1 to 15, which is used for culturing prostate cells, comprises or consists of a basal medium, EGF, any one of Rspondin 1-4, Noggin, nicotinamide, a TGF-β inhibitor, and preferably Wnt-3a and FGF-10.

[0069] Embodiment 21. A culture medium for culturing prostate cells as described in Embodiment 20, wherein the culture medium further comprises testosterone, such as (dihydro)testosterone.

[0070] Embodiment 22. A culture medium as described in any of the preceding embodiments, which is used to culture cancer cells, such as cancer stem cells, such as adenocarcinoma or cancer cells from a target tissue type, wherein the culture medium comprises or consists of components of a culture medium for culturing cells from a corresponding non-cancerous target tissue type, optionally wherein the culture medium for culturing non-cancerous cells of the target tissue type does not include one or more of the following: Wnt-3a, EGF, Noggin, Rspondin, TGF-β inhibitor, p38 inhibitor, nicotinamide, gastrin, FGF10 and HGF.

[0071] Embodiment 23. A culture medium as described in any of the preceding embodiments, which is used to differentiate stem cells from a target tissue, wherein the culture medium comprises or consists of components of a culture medium for expanding stem cells from the target tissue type, but wherein the culture medium for differentiating stem cells does not include one or more of the following: Wnt, Rspondin, BMP inhibitors, TGF-β inhibitors, receptor tyrosine kinase ligands, p38 inhibitors and nicotinamide.

[0072] Embodiment 24. The culture medium according to embodiment 23, which is used for differentiating intestinal cells, comprises or consists of the following: a basal medium, EGF, Noggin, a TGF-β inhibitor and a p38 inhibitor.

[0073] Embodiment 25. A culture medium as described in embodiment 23, which is used to differentiate hepatocytes, and the culture medium comprises or consists of the following: a basal medium, Noggin, EGF, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor such as DAPT or DBZ, and preferably Wnt-3a.

[0074] Embodiment 26. The culture medium as described in embodiment 23, which is used for differentiating pancreatic cells, comprises or consists of the following: basal culture medium, Noggin, EGF, FGF10, gastrin, TGF-β inhibitor, γ-secretase inhibitor, and preferably exendin 4.

[0075] Embodiment 27. A culture medium as described in any of the preceding embodiments, wherein the culture medium is in contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix through its interaction with cell membrane proteins such as integrins.

[0076] Embodiment 28. The culture medium according to embodiment 27, wherein the extracellular matrix is ​​an extracellular matrix containing laminin, such as Matrigel. TM (BD Biosciences).

[0077] Embodiment 29. A composition comprising a culture medium according to any one of embodiments 1 to 26 and an extracellular matrix or a 3D matrix that mimics an extracellular matrix through its interaction with cell membrane proteins such as integrins, such as an extracellular matrix containing laminin, such as Matrigel. TM (BD Biosciences).

[0078] Embodiment 30. An airtight container comprising the culture medium or composition described in any one of the preceding embodiments.

[0079] Embodiment 31. Use of the culture medium of any one of embodiments 1 to 28 for expanding and / or differentiating stem cells, stem cell populations, tissue fragments or organoids.

[0080] Embodiment 32. The use as described in embodiment 31, wherein the stem cells, stem cell populations, tissue fragments or organoids are selected from one or more intestinal stem cells, small intestinal crypts, colonic crypts, gastric stem cells, liver stem cells, pancreatic stem cells and prostate stem cells.

[0081] Embodiment 33. The use according to embodiment 31 or 32, wherein the stem cells, stem cell population, tissue fragment or organoid is obtained from normal tissue.

[0082] Embodiment 34. The use as described in embodiment 31 or 32, wherein the stem cells, stem cell population, tissue fragment or organoid is obtained from diseased tissue, such as from an adenoma, a cancer, an adenocarcinoma, the intestine of a patient with cystic fibrosis, or the intestine of a patient with inflammatory bowel disease.

[0083] Embodiment 35. A method for expanding a single stem cell, a stem cell population, or tissue fragments, preferably to obtain organoids, wherein the method comprises culturing the single stem cell or the stem cell population in a culture medium as described in any one of embodiments 1 to 28.

[0084] Embodiment 36. The method of embodiment 35, wherein the method comprises:

[0085] Providing stem cells, stem cell populations or isolated tissue fragments;

[0086] Providing a culture medium according to any one of embodiments 1 to 28;

[0087] contacting the stem cells with the culture medium;

[0088] The cells are cultured under suitable conditions.

[0089] Embodiment 37. A method as described in embodiment 35, wherein the method comprises contacting the stem cells, stem cell populations or isolated tissue fragments and culture medium with an extracellular matrix or a 3D matrix that mimics the extracellular matrix through its interaction with cell membrane proteins such as integrins, the extracellular matrix, for example, an extracellular matrix containing laminin, such as Matrigel TM (BD Biosciences).

[0090] Embodiment 38. A method as described in Embodiment 37, wherein the culture medium diffuses into the extracellular matrix.

[0091] Embodiment 39. The method of any one of embodiments 35 to 38, wherein the method comprises:

[0092] culturing the stem cells, stem cell population, or tissue fragment in a first expansion medium;

[0093] The stem cells, stem cell populations or tissue fragments are continued to be cultured and the culture medium is supplemented with a differentiation medium, wherein the differentiation medium does not include one or more factors selected from the group consisting of: TGF-β inhibitors, p38 inhibitors, nicotinamide and Wnt.

[0094] Embodiment 40. The method according to any one of embodiments 35 to 38, which is used to obtain small intestine or colon organoids, the method comprising:

[0095] Expanding intestinal or colon stem cells or tissue fragments in the culture medium of embodiment 17; and optionally

[0096] Differentiate the expanded intestinal or colonic stem cells or tissue fragments in the culture medium of embodiment 24.

[0097] Embodiment 41. The method of any one of embodiments 35 to 39 for obtaining gastric organoids, the method comprising culturing gastric stem cells or tissue fragments in the culture medium of embodiment 16.

[0098] Embodiment 42. The method of any one of embodiments 35 to 39, for obtaining a liver organoid, the method comprising:

[0099] expanding liver cells or tissue fragments in the culture medium of embodiment 19; and optionally

[0100] Differentiate the expanded liver cells or tissue fragments in the culture medium of embodiment 25.

[0101] Embodiment 43. The method of any one of embodiments 35 to 39, for obtaining a pancreatic organoid, the method comprising:

[0102] expanding pancreatic cells or tissue fragments in the culture medium of embodiment 20; and optionally

[0103] Differentiate the expanded pancreatic cells or tissue fragments in the culture medium of embodiment 26.

[0104] Embodiment 44. The method of any one of embodiments 35 to 39 for obtaining a prostate organoid, the method comprising culturing prostate stem cells or tissue fragments in the culture medium of embodiment 19.

[0105] Embodiment 45. The method according to any one of embodiments 35 to 39, which is used to obtain adenocarcinoma or cancer organoids, the method comprising culturing adenocarcinoma or cancer stem cells or tissue fragments in the culture medium according to embodiment 22.

[0106] Embodiment 46. The method of any one of embodiments 35 to 39, wherein Rock inhibitor is added to the culture medium within the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every other day.

[0107] Embodiment 47. A method as described in any one of embodiments 35 to 39, which comprises culturing the stem cells for 3 months or longer, such as 4, 5, 6, 7, 8, 9 months or longer, using the culture medium described in any one of embodiments 1 to 28.

[0108] Embodiment 48. An organoid or cell population obtained by the method described in any one of embodiments 35 to 47.

[0109] Embodiment 49. The organoid or cell population of embodiment 48, which, when cultured in the culture medium of any one of embodiments 1 to 28, can persist in culture for at least 3 months, e.g., 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.

[0110] Embodiment 50. The organoid or cell population of Embodiment 48, wherein the organoid or cell population is 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.

[0111] Embodiment 51. The organoid or cell population of any one of embodiments 48 to 50, wherein the organoid or cell population expands 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.

[0112] Embodiment 52. The organoid or cell population of any one of embodiments 48 to 51, wherein the organoid or cell population is a human organ or a human cell population.

[0113] Embodiment 53. The organoid or cell population as described in any one of embodiments 48 to 52, wherein the organoid or cell population is a normal organoid or cell population or a diseased organoid or cell population, such as an organoid or cell population obtained by culturing stem cells collected from a human or animal suffering from a disease.

[0114] Embodiment 54. The organoid or cell population of any one of embodiments 48 to 53, which is frozen and stored at 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, e.g., about -180°C.

[0115] Embodiment 55. An organoid, preferably the organoid of any one of embodiments 48 to 54, which is a three-dimensional organoid comprising epithelial cells surrounding a central lumen, wherein optionally, the epithelial cells are present in distinct division and differentiation domains.

[0116] Embodiment 56. The organoid of embodiment 55, which is a three-dimensional organoid comprising epithelial cells arranged in monolayer regions, optionally folded monolayer regions, and stratified cell regions.

[0117] Embodiment 57. The organoid of any one of embodiments 48 to 56, wherein the organoid is devoid of non-epithelial cells.

[0118] Embodiment 58. The organoid of any one of embodiments 48 to 57, wherein said organoid presents all differentiated cell types of normal in vivo tissues.

[0119] Embodiment 59. The organoid of any one of embodiments 48 to 58, wherein the organoid is an intestinal organoid, a colon organoid, a stomach organoid, a pancreatic organoid, a liver organoid, or a prostate organoid.

[0120] Embodiment 60. A composition comprising:

[0121] i) the organoid or cell population of any one or more embodiments 48 to 59; and

[0122] ii) the culture medium and / or extracellular matrix according to any one of embodiments 1 to 28.

[0123] Embodiment 61. An organoid as described in any one of embodiments 48 to 59, or a cell population as described in any one of embodiments 48 to 54, or a composition as described in embodiment 29 or 60, for use in drug screening, target validation, target discovery, toxicology, toxicology screening, personalized medicine, regenerative medicine, or an ex vivo cell / organoid model, for example, as a disease model.

[0124] Embodiment 62. The organoid, cell population or composition of embodiment 61, wherein the regenerative medicine or personalized medicine comprises implanting the organoid, cell population or composition into a mammal, preferably a human. DETAILED DESCRIPTION OF THE INVENTION

[0126] According to the present invention, a culture medium for expanding a stem cell population is provided, wherein the culture medium comprises at least one or more inhibitors that bind to one or more serine / threonine protein kinase targets and reduce their activity, wherein the culture medium has the effect of allowing the stem cell population to grow continuously 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.

[0127] Inhibitors

[0128] The culture medium used in the first aspect of the present invention includes any inhibitor that directly or indirectly negatively regulates TGF-β or p38 signal transduction. In a preferred embodiment, the culture medium of the present invention includes an inhibitor that directly or indirectly negatively regulates TGF-β signal transduction. In some embodiments, the culture medium of the present invention includes an inhibitor that directly or indirectly negatively regulates TGF-β and an inhibitor that directly or indirectly negatively regulates p38 signal transduction. In other embodiments, the culture medium of the present invention also includes Rspondin or a Rspondin mimetic.

[0129] The one or more inhibitors preferably target a serine / threonine protein kinase selected from the group consisting of: TGF-β receptor kinase 1, ALK4, ALK5, ALK7, p38. An inhibitor of any of these kinases is an inhibitor that achieves a reduction in the enzymatic activity of one (or more) of these molecules. Inhibition of ALK and p38 kinases has previously been shown to be associated with B-cell lymphoma ( M Huse K, Hilden VI, Smeland EB, Oksvold MP, "TGF-beta-induced growth inhibition in B-cell lymphoma correlates with Smad1 / 5 signaling and constitutively active p38 MAPK", BMC Immunol. 11: 57, 2010). In this disclosure, it was found that TGF-β-sensitive cell lines expressed higher cell surface levels of ALK-5, and p38 constitutive phosphorylation was confined to TGF-β-sensitive cell lines. Inhibition of p38 MAPK resulted in reduced sensitivity to TGF-β, indicating that phosphorylation of Smad1 / 5 is important for the antiproliferative effect of TGF-β in B-cell lymphoma. This result suggests a role for p38 MAPK in regulating TGF-β-induced antiproliferative effects.

[0130] 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 that act on this pathway at any level (including, for example, by inhibiting Smad1 / 5 signal transduction) would also be beneficial to cell culture. Smad plays a key role in TGF-β signal transduction.

[0131] In some embodiments, the inhibitor of the present invention binds to and reduces the activity of a serine / threonine protein kinase selected from the group consisting of: TGF-β receptor kinase 1, ALK4, ALK5, ALK7, p38.

[0132] In some embodiments of the invention, the culture medium includes a TGF-β inhibitor, meaning any inhibitor that directly or indirectly negatively regulates TGF-β signaling. In some embodiments, the culture medium of the invention includes one or more TGF-β inhibitors that bind to and reduce the activity of one or more serine / threonine protein kinases selected from ALK5, ALK4, TGF-β receptor kinase 1, and ALK7.

[0133] ALK4, ALK5, and ALK7 are all closely related receptors of the TGF-β superfamily. ALK4 has a GI number of 91; ALK5 (also known as TGF-β receptor kinase 1) has a GI number of 7046; and ALK7 has a GI number of 658. In one embodiment, an inhibitor of the present invention binds to ALK4, ALK5 (TGF-β receptor kinase 1) and / or ALK 7 and reduces its activity. In another embodiment, the TGF-β receptor binds to a Smad protein and reduces its activity, such as an R-SMAD or SMAD1-5 (i.e., SMAD 1, SMAD2, SMAD 3, SMAD 4, or SMAD 5). In a preferred embodiment, the culture medium of the present invention includes an ALK5 inhibitor.

[0134] Various methods are known for determining whether a substance is a TGF-β inhibitor. For example, a cell test can be used in which cells are stably transfected with a reporter construct comprising a human PAI-1 promoter or a Smad binding site, driving a luciferase reporter gene. Inhibition of luciferase activity relative 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 a luciferase inhibitor for measuring kinase activity. Phosphosensing assay (Drew AE 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).

[0135] Various TGF-β inhibitors are known in the art (e.g., see Table 1). In some embodiments, the inhibitor that directly or indirectly negatively regulates TGF-β signaling is selected from A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511.

[0136] In some embodiments of the present invention, the culture medium includes a p38 inhibitor, meaning any inhibitor that directly or indirectly negatively regulates p38 signal transduction. In some embodiments, the inhibitor of the present invention binds to p38 (GI No. 1432) and reduces its activity. The p38 protein kinase is part of the mitogen-activated protein kinase (MAPK) family. MAPK is a serine / threonine-specific protein kinase that responds to extracellular stimuli such as environmental stress and inflammatory cytokines and regulates various cell activities such as gene expression, mitosis, differentiation, proliferation, and cell survival / apoptosis. p38 MAPK exists in α, β, β2, γ, and δ subtypes. A p38 inhibitor is an agent that binds to at least one p38 subtype and reduces its activity. Various methods for determining whether a substance is a p38 inhibitor are known and can be used in conjunction with the present invention. Examples include: phosphorylation of Thr180 / Tyr182 with a phospho-specific antibody assay that provides a confirmed measure of cellular p38 activation or inhibition; biochemical recombinant kinase assay; tumor necrosis factor α (TNFα) secretion assay; 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 a variety of p38 activity test kits (e.g., Millipore, Sigma-Aldrich).

[0137] The inventors hypothesize that in some embodiments, high concentrations of p38 (e.g., greater than 100 nM, or greater than 1 μM, greater than 10 μM, or greater than 100 μM) may have an inhibitory effect on TGF-β. However, the inventors do not wish to be bound by this hypothesis and in other embodiments, p38 inhibitors do not inhibit TGF-β signal transduction.

[0138] Various p38 inhibitors are known in the art (e.g., see Table 1). In some embodiments, the inhibitor that directly or indirectly negatively regulates p38 signaling is selected from SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257, and BIRB-796. In other embodiments of the present invention, the culture medium includes both: a) an inhibitor that binds to any one or more kinases selected from ALK4, ALK5, and ALK7 and reduces its activity; and b) an inhibitor that binds to p38 and reduces its activity. In a preferred embodiment, the culture medium includes an inhibitor that binds to ALK5 and reduces its activity and an inhibitor that binds to p38 and reduces its activity.

[0139] In one embodiment, an inhibitor of the invention binds to its target (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% as assessed by a cell-based assay, as described above, examples of cell-based assays for measuring target inhibition are well known in the art.

[0140] The inhibitors of the present invention may have an IC50 value equal to or less than 2000 nM; 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 potency of an inhibitor in inhibiting the biological or biochemical function of its target. The IC50 represents how much of a particular inhibitor is required to inhibit a kinase by 50%. The IC50 value may be calculated according to the test method described above.

[0141] The inhibitors of 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.

[0142] The inhibitors of the present invention can exist in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules, such as natural or synthetic small organic molecules up to 2000Da, preferably up to 800Da or less, mimetic peptides, inorganic molecules, peptides, polypeptides, antisense oligonucleotide aptamers, and structural or functional mimics thereof including small molecules. The inhibitors of the present invention can also be aptamers. As used herein, the term "aptamer" refers to a chain of oligonucleotides (DNA or RNA) that can adopt a highly specific three-dimensional conformation. Aptamers are designed to have high binding affinity and specificity to certain target molecules, including extracellular and intracellular proteins.

[0143] For example, the inhibitor may be a small synthetic molecule having a molecular weight of 50-800 Da, 80-700 Da, 100-600 Da, or 150-500 Da.

[0144] In some embodiments, the small molecule inhibitor comprises a pyridyl imidazole or a 2,4-disubstituted pteridine or quinazoline, for example comprising:

[0145]

[0146] Specific examples of inhibitors that can be used according to the present invention include, but are not limited to, SB-202190, SB-203580, SB-206718, SB-227931, VX-702, VX-745, PD-169316, RO-4402257, BIRB-796, A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208, SJN 2511 (see Table 1). The culture medium of the present invention may include any one or more of the inhibitors listed in Table 1. The culture medium of the present invention may include any combination of one inhibitor with another listed inhibitor. For example, the culture medium of the present invention may include SB-202190 or SB-203580 or A83-01; or the culture medium of the present invention may include SB-202190 and A83-01; or the culture medium of the present invention may include SB-203580 and A83-01. The skilled artisan will recognize that other inhibitors and combinations of inhibitors that bind to the target and reduce its activity may be included in the culture medium or culture medium supplement of the invention.

[0147] The inhibitor of the present invention may be added to the culture medium to a suitable final concentration taking into account the IC50 value of the inhibitor.

[0148] For example, SB-202190 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-202190 can be added to the culture medium at about 10 μM.

[0149] SB-203580 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-203580 can be added to the culture medium at about 10 μM.

[0150] 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, VX-702 may be added to the culture medium at about 5 μM.

[0151] VX-745 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 250 nM to 10 μM. For example, VX-745 can be added to the culture medium at about 1 μM.

[0152] PD-169316 can be added to the culture medium at a concentration of 100 nM to 200 μM, or 200 nM to 100 μM, or 1 μM to 50 μM. For example, PD-169316 can be added to the culture medium at about 20 μM.

[0153] 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, RO-4402257 may be added to the culture medium at about 1 μM.

[0154] BIRB-796 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 500 nM to 10 μM. For example, BIRB-796 can be added to the culture medium at about 1 μM.

[0155] A83-01 can be added to the culture medium at a concentration of 10 nM to 10 μM, or 20 nM to 5 μM, or 50 nM to 1 μM. For example, A83-01 can be added to the culture medium at about 500 nM.

[0156] 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, SB-431542 may be added to the culture medium at about 1 μM.

[0157] SB-505124 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, SB-505124 can be added to the culture medium at about 500 nM.

[0158] SB-525334 can be added to the culture medium at a concentration of 10 nM to 10 μM, or 20 nM to 5 μM, or 50 nM to 1 μM. For example, SB-525334 can be added to the culture medium at about 100 nM.

[0159] LY 36494 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, LY 36494 can be added to the culture medium at about 500 nM.

[0160] Table 1: Exemplary inhibitors of the invention

[0161]

[0162]

[0163]

[0164]

[0165] 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, SD-208 may be added to the culture medium at about 500 nM.

[0166] LY364947 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, LY364947 can be added to the culture medium at about 500 nM.

[0167] SJN 2511 can be added to the culture medium at a concentration of 20 nM to 20 μM, or 40 nM to 10 μM, or 100 nM to 1 μM. For example, SJN 2511 can be added to the culture medium at about 200 nM.

[0168] Thus, in some embodiments, an inhibitor that directly or indirectly negatively regulates TGF-β or p38 signaling is added to the culture medium at a concentration of 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM, for example, wherein the total concentration of one or more inhibitors is 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM.

[0169] In addition to the inhibitor, the cell culture medium generally includes a variety of components, which are necessary for the maintenance and / or expansion of the cells supporting the culture. The cell culture medium of the present invention will therefore generally include other components in addition to the inhibitor of the present invention. In view of the following disclosure, the technician will easily prepare a suitable combination of components. The culture medium of the present invention will generally be a nutrient solution, which includes standard cell culture components, such as amino acids, vitamins, inorganic salts, carbon energy sources and buffers described in more detail below. Other standard cell culture components that can 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" culture medium.

[0170] The culture medium of the present invention can be produced by modifying the existing cell culture medium. The technician will understand the culture medium type that can be used for stem cell culture from common knowledge. Possibly suitable culture medium is commercially available, and includes but is not limited to Dulbecco's modified Eagle's medium (DMEM), minimal essential medium (MEM), knock-out-DMEM (KO-DMEM), Glasgow minimal essential medium (G-MEM), Eagle's basal medium (BME), DMEM / Ham's F12, improved DMEM / Ham's F12, Iscove modified Dulbecco's medium and minimal essential medium (MEM), Ham's F-10, Ham's F-12, Medium199 and RPMI 1640 culture medium. Therefore, in some embodiments, these previously existing cell culture mediums are used as basal medium, to which are added inhibitors of directly or indirectly negatively regulating TGF-β or p38 signal transduction, and optionally to which are added one or more other components described herein.

[0171] In some embodiments, the culture medium of the present invention comprises one or more other 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 Rspondin 1-4 and / or Rspondin mimetics, TGF-β inhibitors, BMP inhibitors (e.g., Noggin) and Wnt agonists (e.g., Wnt (3a)).

[0172] In some embodiments, the culture medium of the present invention includes any one of Rspondin 1-4 and / or Rspondin mimetics, 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 includes one or more other components selected from: p38 inhibitors, gastrin, FGF10, HGF, and Rock inhibitors. Optional other components may be added to optimize the culture medium for culturing cells derived from a specific tissue, as described in more detail later.

[0173] The culture medium of the present invention can include one or more bone morphogenetic protein (BMP) inhibitors. BMP ligands are transduced in dimer form by assembling a transmembrane serine / threonine kinase receptor complex (composed of two type I and two type II receptors) composed of four parts. Complex assembly triggers phosphorylation cascade, activates BMP responsiveness Smad1 / 5 / 8, and causes changes in transcriptional activity. Advantageously, the inventors show that BMP inhibitors promote the expression of Lgr5, so the presence of BMP inhibitors in the culture medium of the present invention will also produce more proliferative organoids (e.g., see Example 3) compared with the absence of BMP inhibitors. Therefore, BMP inhibitors are advantageous components of the amplification culture medium of the present invention. Therefore, when it is desired to culture cells for at least 3 months (e.g., at least 4, 5, 6, 7, 8 or 9 months) in the absence of cell differentiation, it is advantageous to use BMP inhibitors in the use of amplification culture medium.

[0174] Various types of natural BMP binding proteins are known, including Noggin (Peprotech), Chordin and Chordin-like proteins including Chordin domains (R&D Systems), Follistatin and Follistatin-related proteins including Follistatin domains (R&D Systems), DAN and DAN-like proteins including DAN cysteine ​​knot (cystein-knot) domains (R&D Systems), Sclerosis protein / SOST (R&D Systems) and α-2 macroglobulin (R&D Systems). BMP inhibitors are agents that bind to BMP molecules to form complexes, wherein BMP activity is reduced, for example, by preventing or inhibiting the binding of BMP molecules to BMP receptors. Alternatively, the inhibitor can be an agent that binds to BMP receptors and prevents BMP ligands from binding to receptors, such as antibodies that bind to receptors. BMP inhibitors can be proteins or small molecules, and can be naturally occurring, modified and / or partially or completely synthesized. The BMP inhibitors of the culture medium of the present invention can be Noggin, DAN or DAN-like proteins including Cerberus and Gremlin (R&D Systems). These diffusible proteins can bind BMP ligands with different affinities and inhibit their access to signal transduction receptors. The preferred BMP inhibitor for 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 can include about 10ng / ml to about 100ng / ml of Noggin. For example, the culture medium can include at least 10ng / ml of Noggin, at least 20ng / ml of Noggin, at least 50ng / ml of Noggin, at least 100ng / ml of Noggin, about 100ng / ml of Noggin or 100ng / ml of Noggin. In some embodiments, the culture medium can include less than 200ng / ml of Noggin, less than 150ng / ml of Noggin, less than 100ng / ml of Noggin, less than 75ng / ml of Noggin, less than 50ng / ml of Noggin or less than 30ng / ml of Noggin. BMP inhibitors can be added to the culture medium every other day, or every day, or every three days, every four days, every five days, or as needed during the culture period. BMP inhibitors are particularly advantageous components of expansion culture mediums, such as for amplifying pancreas, small intestine, colon, liver, prostate stem cells. However, Noggin has been shown to inhibit some differentiations (e.g., see Example 3). Therefore, in some embodiments, the differentiation culture medium of the present invention does not include BMP inhibitors.

[0175] In some embodiments, cells cultured with BMP inhibitors have upregulated expression of Lgr5 compared to cells cultured without BMP inhibitors. Therefore, adding BMP inhibitors generally results in more proliferative organoids. This is surprising because in the literature, it is described that BMP activity can be used to differentiate pancreatic cells into ductal cells (see keratin 7 and 19 expression) and endocrine cells. Therefore, the technician would expect to include BMP inhibitors such as Noggin to reduce proliferation and increase differentiation. However, the inventors surprisingly found that the use of BMP is advantageous because it leads to more proliferative organoids and higher expression of Lgr5. The culture medium of the present invention may include one or more Wnt agonists. The series of events that occur when Wnt proteins bind to cell surface receptors of members of the frizzled receptor family define the Wnt signal transduction pathway. This leads to the activation of the Dishevelled family protein, which inhibits the protein complex including axin, GSK-3 and protein APC to degrade intracellular β-catenin. The resulting nuclear-enriched β-catenin enhances transcription through the TCF / LEF family transcription factors. Wnt agonists are defined as agents that activate TCF / LEF-mediated transcription in cells. Therefore, Wnt agonists are selected from true Wnt agonists that bind and activate members of the frizzled receptor family, including any one and all of Wnt family proteins, inhibitors of intracellular β-catenin degradation, and activators of TCF / LEF. The Wnt agonist stimulates the Wnt activity in cells to increase 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% (relative to the level of Wnt activity in the absence of the molecule). As known to the skilled person, Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase receptor constructs (Korinek et al., 1997 Science 2751784-1787).

[0176] In some embodiments, Wnt agonists include secreted glycoproteins including Wnt-1 / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D Systems), 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, WnM 1 and Wnt-16. An overview of human Wnt proteins is provided in "THE WNT FAMILY OF SECRETED PROTEINS", R&D Systems Catalog, 2004. Other Wnt agonists include the R-spondin family of secreted proteins, which are involved in the activation and regulation of the Wnt signaling pathway and consist of four members (R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 ((R&D Systems), R-spondin 3 and R-spondin-4) and Norrin (also known as Nome disease protein or NDP) (R&D Systems), which is a secreted regulatory protein that performs the same function as Wnt proteins in that it binds to the Frizzled-4 receptor with high affinity and induces activation of the Wnt signaling pathway (Kestutis Planutis et al., (2007) BMC Cell Biol 8 12). In some embodiments, one or more Wnt agonists used in the present invention are R-spondin mimetics, for example, agonists of Lgr5, such as anti-Lgr5 antibodies. Small molecule agonists of the Wnt signaling pathway that are aminopyrimidine derivatives have recently been identified and are also specifically listed as Wnt agonists (Lm et al. (2005) Angew Chem Int Ed Engl 44, 1987-90).

[0177] In some embodiments, the Wnt agonist is a GSK-inhibitor. Known GSK-inhibitors include small interfering RNA (siRNA, Cell Signaling), lithium (Sigma), kenpaullone (Biomol International, Leost, M et al. (2000) Eur J Biochem 267, 5983-5994), 6-bromoindirubin-30-acetone oxime (Meyer, L et al. (2003) ChemBiol 10, 1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), and FRAT-family members and FRAT-derived peptides that inhibit the interaction of GSK-3 with axin. An overview is provided by 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 the skilled person and include, for example, those described in Liao et al. 2004, Endocrinology, 145(6) 2941-2949.

[0178] In some embodiments, Wnt agonists are inhibitors of RNF43 or ZNRF3. The inventors have found that RNF43 and ZNRF3 are present in the cell membrane and negatively regulate the level of Wnt receptor complexes in the membrane, possibly through ubiquitination of frizzled protein. Therefore, the inventors assume that inhibiting RNF43 or ZNRF3 with antagonistic antibodies, RNAi or small molecule inhibitors will indirectly stimulate the Wnt pathway. RNF43 and ZNRF3 have catalytic annular domains (with ubiquitination activity), which can be used as targets in the design of small molecule inhibitors. Various anti-RNF43 antibodies and various anti-ZNRF3 antibodies are commercially available. In some embodiments, such antibodies are suitable Wnt agonists in the present context.

[0179] In some embodiments, the Wnt agonist is selected from Wnt-3a, a GSK-inhibitor (eg, CHIR99021), Wnt 5, Wnt-6a, Norrin, and any other Wnt family protein.

[0180] In some embodiments, the Wnt agonist comprises or consists of any one of: Rspondin 1, Rspondin 2, Rspondin 3, or Rspondin 4. In preferred embodiments, the Wnt agonist is selected from any one or more of Wnt family members, R-spondin 1-4, Norrin, and GSK-inhibitors. In some embodiments, the Wnt agonist is a GSK-3 inhibitor, such as CHIR99021 (Stemgent 04-0004). In some embodiments, CHIR99021 is added to the culture medium until the final concentration is 50nM to 100μM, such as 100nM to 50μM, 1μM to 10μM, 1μM to 5μM, or 3μM. In some embodiments in which 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 necessary for the proliferation of epithelial stem cells or isolated crypts.

[0181] In other preferred embodiments, 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 added to the basal medium at a concentration of preferably 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, such as 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, or R-spondin 4 ("Rspondin 1-4") are added to the culture medium. Preferably, when two or more of Rspondin 1-4 are added, the final concentration of Rspondin is equal to the above concentrations. When a culture medium described herein is referred to as including "Rspondin 1-4", it means that the culture medium includes any one or more of Rspondin 1, Rspondin 2, Rspondin 3, and Rspondin 4. When a culture medium described herein is referred to as including "Rspondin", it means that the culture medium includes any one or more of Rspondin 1, Rspondin 2, Rspondin 3, Rspondin 4, and Rspondin mimetics.

[0182] During the stem cell culture, the Wnt family member is preferably added to the culture medium every other day, while the culture medium is preferably refreshed every four days.

[0183] In a preferred embodiment, the Wnt agonist is selected from R-spondin, Wnt-3a and Wnt-6. More preferably, both R-spondin and Wnt-3a are used as Wnt agonists. This combination is particularly preferred because it surprisingly has a synergistic effect on organoid formation. Preferred concentrations are about 500 ng / ml or 500 ng / ml for R-spondin and about 100 ng / ml or 100 ng / ml for Wnt3a.

[0184] The culture medium of the present invention may include 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.

[0185] The culture medium of the present invention may include one or more mitogenic growth factors. One or more mitogenic growth factors may be selected from the family of growth factors, including epithelial 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 an effective mitogenic factor for ectoderm and mesoderm cells of various cultures, and has a significant effect on the in vivo and in vitro differentiation of specific cells and the differentiation of some fibroblasts in cell culture. The EGF precursor exists in the form of a membrane-bound molecule, which is proteolytically cleaved to produce a 53-amino acid peptide hormone that stimulates cells. A preferred mitogenic growth factor is EGF. EGF is preferably added to the basal medium at a concentration of 5-500ng / ml or at least 5ng / ml and not higher than 500ng / ml. Preferred concentrations are at least 10, 20, 25, 30, 40, 45 or 50 ng / ml and not more than 500, 450, 400, 350, 300, 250, 200, 150 or 100 ng / ml. A more preferred concentration is at least 50 ng / ml and not more than 100 ng / ml. Even more preferred concentrations are about 50 ng / ml or 50 ng / ml. The same concentration can be used for FGF, preferably for FGF10 or FGF7. If more than one FGF, such as FGF7 and FGF10, are used, the concentration of FGF is as defined above and refers to the total concentration of the FGF used. During stem cell culture, it is preferred to add the mitogenic growth factor to the culture medium every other day, while the culture medium is preferably renewed every four 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 other preferred embodiments, a combination of mitogenic growth factors, such as EGF and KGF or EGF and BDNF, is added to the basal medium. In other preferred embodiments, a combination of mitogenic growth factors, such as EGF and KGF or EGF and FGF10, is added to the basal medium. Mitogenic growth factors are added to the culture medium at a concentration of 5-500 ng / ml or at least 5 ng / ml and not higher than 500 ng / ml, such as at least 10, 20, 25, 30, 40, 45 or 50 ng / ml and not higher than 500, 450, 400, 350, 300, 250, 200, 150 or 100 ng / ml. The mitogenic growth factor can be selected from EGF, TGF-α, KGF, FGF7 and FGF.Preferably, the mitogenic factor is selected from EGF, TGF-α and KGF, or from EGF, TGF-α and FGF7, or from EGF, TGF-α and FGF, or from EGF and KGF, or from EGF and FGF7, or from EGF and FGF, or from TGF-α and KGF, or from TGF-α and FGF7, or from TGF-α and FGF. EGF can be replaced by TGF-α. In some embodiments, the mitogenic growth factor is hepatocyte growth factor (HGF). In some embodiments, HGF is added to the culture medium.

[0186] In some embodiments, the receptor tyrosine kinase ligand is a mitogenic growth factor, for example selected from the family of growth factors consisting of 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).

[0187] A ROCK inhibitor, such as Y-27632 (10 μM; Sigma), may be included in any of the culture media, particularly in the first few days of culture before cell sorting experiments are performed, as it is known to avoid anoikis (a form of programmed cell death induced by anchorage-dependent cells detached from the surrounding extracellular matrix). Thus, any culture medium defined herein may additionally include a ROCK inhibitor in the first few days. In some embodiments, the culture medium of the present invention additionally includes a ROCK inhibitor, such as Y-27632, for example in the first few days of culture before cell sorting experiments are performed.

[0188] Other embodiments of the method of the present invention include culture medium containing ROCK (ρ-kinase) inhibitors. It was found that the addition of Rock inhibitors prevented anoikis, especially when culturing single stem cells. The Rock inhibitor is preferably selected from R-(+)-trans-4-(l-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, Sigma-Aldrich), 5-(l,4-diazacycloheptane-1-ylsulfonyl)isoquinoline (Fasudil or HA1077, Cayman Chemical) and (S)-(+)-2-methyl-l-[(4-methyl-5-isoquinolyl)sulfonyl]-hexahydro-1H-1,4-diazacycloheptane dihydrochloride (H-1152, Tocris Bioschience). During the first seven days of culturing stem cells, the ρ-kinase inhibitor, such as Y-27632, is preferably added to the culture medium every other day. After single cell inoculation or segmentation, in the first few days of culture, for example, in the first 1, 2, 3, 4, 5, 6 or 7 days, a Rock inhibitor is preferably included in the culture medium. Any suitable concentration of Rock inhibitor can be used, for example 1-200 μM, 1-100 μM, 5-50 μM or about 10 μM. The preferred concentration of Y27632 is 10 μM. Therefore, in some embodiments, the present invention provides a method for culturing stem cells and / or a method for obtaining organoids, wherein Rock inhibitors are added to the culture medium within the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every other day. In some embodiments, after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, Rock inhibitors are not added to the culture medium.

[0189] When culturing single stem cells (as described above), i.e. when the starting material of the organoid is a single stem cell, the addition of a Rock inhibitor is particularly important. Thus, in some embodiments, the invention provides a method for obtaining an organoid, wherein the method comprises culturing stem cells, optionally a single stem cell, wherein a Rock inhibitor is added to the culture medium within the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every other day, and optionally no Rock inhibitor is added after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0190] When culturing multiple cells, such as when the starting material for organoids is tissue fragments, Rock inhibitors are less important and sometimes not necessary. Therefore, in some embodiments, the present invention provides a method for obtaining organoids, wherein the method comprises culturing stem cells, optionally tissue fragments, wherein Rock inhibitors are never added or are not added after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0191] When cells are split into multiple cultures, Rock inhibitors are added to the culture medium in the same manner, meaning after splitting, particularly when splitting comprises collecting a single stem cell from a first culture and placing it in a second culture, within 1, 2, 3, 4, 5, 6 or 7 days, optionally every other day. If splitting comprises collecting multiple stem cells from a first culture and placing them in a second culture, adding Rock inhibitors is not so important and sometimes not necessary. Therefore, in some embodiments, wherein the method for obtaining organoids or for culturing stem cells comprises splitting, optionally, when a single cell involves splitting, Rock inhibitors are added to new culture medium within the first 1, 2, 3, 4, 5, 6 or 7 days after splitting, optionally every other day. In some embodiments, wherein the method for obtaining organoids or for culturing stem cells comprises splitting, optionally, when multiple cells involve splitting, Rock inhibitors are never added or are not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0192] In other embodiments, the method of the present invention includes a culture medium further containing a Notch agonist. Notch signaling has been shown to play an important role in final cell type determination and in cell survival and proliferation. Notch receptor proteins can interact with many surface-bound or secreted ligands, including but not limited to δ1, Jagged 1 and 2, and δ-like 1, δ-like 3, and δ-like 4. When the ligand binds, the Notch receptor is activated by a series of cleavage events involving members of the ADAM protease family and intramembrane cleavage regulated by the γ-secretase presenilin. This results in the transport of the intracellular domain of Notch to the nucleus, where it transcriptionally activates downstream genes. Preferred Notch agonists are selected from Jagged 1 and δ1, or active fragments or derivatives thereof. The most preferred Notch agonist is a DSL peptide having the sequence CDDYYYGFGCNKFCRPR (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 not higher than 100 nM. The addition of Notch agonists, especially during the first week of culture, increases the culture efficiency by 2-3 times. Within the first 7 days of culturing stem cells, Notch agonists are preferably added to the culture medium every other day. Therefore, in some embodiments, the present invention provides methods for culturing stem cells and / or methods for obtaining organoids, wherein Notch agonists are added to the culture medium within the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every other day. In some embodiments, after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, Notch agonists are not added to the culture medium.

[0193] The Notch agonist is defined as a molecule that stimulates the Notch activity in the cell to increase 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% (relative to the level of the Notch activity in the absence of the molecule). As known to the technician, Notch activity can be measured by measuring the transcriptional activity of Notch, for example, by the 4xwtCBF1-luciferase reporter construct (Hsieh et al., 1996 Mol Cell.Biol.16,952-959).

[0194] In other embodiments, the cell culture medium is supplemented with a γ-secretase inhibitor, such as DAPT or DBZ. The γ-secretase inhibitor can affect the final cell type decision during differentiation. For example, in some embodiments, the γ-secretase inhibitor can affect the final cell type to secretory cells, such as goblet cells. Any suitable concentration of the γ-secretase inhibitor can be used, such as 1nM to 10μM, 1nM to 1μM, 1nM to 100nM, or preferably 1nM to 20nM. For example, the γ-secretase inhibitor can be added to the culture medium until the final concentration is about 1nM.

[0195] In other embodiments, the cell culture medium is supplemented with gastrin (or a suitable substitute, such as Leu15-gastrin).Gastrin (or a suitable substitute) can be added to the culture medium until the final concentration is 1nM to 10 μM, 1nM to 1 μM, 5nM to 100nM, or preferably 10nM to 50nM.For example, Leu15-gastrin can be added to the culture medium until the final concentration is about 10nM.Gastrin is not necessary for some culture media of the present invention.Therefore, in some embodiments, the culture medium of the present invention does not include gastrin.Especially, gastrin is not necessary for cultivating intestinal stem cells or for obtaining intestinal (crypt-villus or colonic crypt) organoids.However, even when gastrin is not needed, it can still be added to the culture medium without side effects.

[0196] In other embodiments, the culture medium of the present invention is supplemented with nicotinamide. It was found that the addition of nicotinamide improved the culture efficiency and the lifespan of human colon organoids. Nicotinamide is added to the culture medium to a final concentration of 1 mM to 100 mM, 5 mM to 50 mM, or preferably 5 mM to 20 mM. For example, nicotinamide can be added to the culture medium to a final concentration of about 10 mM.

[0197] In a preferred embodiment of the invention, the culture medium is supplemented with nicotinamide and gastrin (or a suitable substitute, such as Leu15-gastrin), wherein nicotinamide and gastrin are added to the culture medium at any of the concentrations described above.

[0198] In some embodiments, the culture medium is supplemented with an activator of the prostaglandin signaling pathway (see Fig.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, the culture medium is supplemented with any one or more of the following compounds: phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, the culture medium is supplemented with PGE2 and / or AA. In some embodiments, PGE2 is added to the culture medium until a final concentration of at least 10 nM, for example 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 culture medium until a final concentration of 50 nM. In some embodiments, AA is added to the culture medium until final concentration is 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 culture medium until final concentration is 10 μg / ml. In the case of culture medium of the present invention, AA and PGE2 are interchangeable. Therefore, when culture medium as herein described is referred to as including PGE2, it can alternatively include AA (with appropriate concentration) rather than PGE2. On the contrary, when culture medium as herein described is referred to as including AA, it can alternatively include PGE2 (with appropriate concentration) rather than AA. In addition, the skilled person will understand that when the culture medium of the present invention includes PGE2 and / or AA, the culture medium may instead include any one or more of the following compounds to replace PGE2 and / or AA or include any one or more of the following compounds in addition to PGE2 and / or AA: phospholipids, prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2) and prostaglandin D2 (PGD2).

[0199] In other embodiments, the culture medium of the present invention is supplemented with RANK ligand (also referred to herein as RANKL). RANK ligand can be used to guide differentiation toward a specific final cell type. For example, when RANK ligand is included in a culture medium for small intestinal cells, preferably in a culture medium for differentiating small intestinal cells, it causes a larger proportion of cells to be differentiated into M cells. Therefore, in some embodiments, the present invention provides a culture medium including RANKL. In particular, the present invention provides a culture medium for culturing, preferably for differentiating small intestinal cells, wherein the culture medium includes RANKL. Any concentration of RANKL can be used, for example 10 ng / ml to 1000 ng / ml, 10 ng / ml to 500 ng / ml or 50 ng / ml to 100 ng / ml. For example, RANKL can be added to the culture medium until the final concentration is about 100 ng / ml.

[0200] A culture medium comprising EGF, Noggin, and R-spondin is referred to herein as "ENR culture medium". A culture medium comprising ENR culture medium and a Wnt agonist such as Wnt-3a is referred to herein as "WENR culture medium". In a preferred embodiment of the present invention, the culture medium comprises WENR culture medium. In a most preferred embodiment of the present invention, the culture medium comprises WENR culture medium supplemented with gastrin and / or nicotinamide (i.e., WENRg or WENR+nicotinamide or WENRg+nicotinamide).

[0201] The pH of the culture medium can be about 7.0 to 7.8, about 7.2 to 7.6, or about 7.4. A buffer can be used to maintain pH. The technician easily selects a suitable buffer. Available buffers include carbonate buffers (e.g., NaHCO3) and phosphates (e.g., NaH2PO4). These buffers are generally used at about 50 mg / l to about 500 mg / l. Other buffers can also be used, such as N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES) and 3-[N-morpholinyl]-propanesulfonic acid (MOPS), generally about 1000 mg / l to about 10,000 mg / l. The culture medium can include a pH indicator such as phenol red to easily monitor the pH state of the culture medium (e.g., about 5 mg / liter to about 50 mg / liter).

[0202] The culture medium used for the present invention may include one or more amino acids. The technician understands the suitable type and amount of amino acids used for stem cell culture medium. The 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 mediums may include all of these amino acids. Typically, each amino acid, when present, is present at about 0.001 g / L to about 1 g / L (typically about 0.01 g / L to about 0.15 g / L) of the culture medium, except for L-glutamine, which is present at about 0.05 g / L to about 1 g / L (typically about 0.1 g / L to about 0.75 g / L). The amino acids may have a synthetic origin.

[0203] The culture medium used for the present invention may include one or more vitamins. The skilled person is aware of the appropriate type and amount of vitamins for stem cell culture medium. The vitamins that may be present include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium 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).

[0204] The culture medium for the present invention may include one or more inorganic salts. The technician understands the appropriate type and amount of inorganic salts for stem cell culture medium. Inorganic salts are generally included in the culture medium to assist in the maintenance of cell osmotic balance and to assist in regulating membrane potential. The inorganic salts that may be present include calcium salts, copper salts, iron salts, magnesium salts, calcium salts, potassium salts, sodium salts, and zinc salts. The salts are generally used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. Specific salts that can be used include CaCl2, CuSO4-5H2O, Fe(NO3)-9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4-H2O, and ZnSO4-7H2O.

[0205] The osmotic pressure of the culture medium can be about 200mOsm / kg to about 400mOsm / kg, about 290mOsm / kg to about 350mOsm / kg, or about 280mOsm / kg to about 310mOsm / kg. The osmotic pressure of the culture medium can be less than about 300mOsm / kg (e.g., about 280mOsm / kg).

[0206] Substratum for the present invention may include a carbon energy source in the form of one or more sugars. The skilled person is aware of suitable types and amounts of sugars for stem cell culture medium. Sugars that may be present include glucose, galactose, maltose, and fructose. Sugars are preferably glucose, particularly D-glucose (dextrose). The carbon energy source will typically be present at about 1 g / L to about 10 g / L.

[0207] The culture medium of the present invention may include serum. Serum obtained from any suitable source may be used, including fetal bovine serum (FBS), goat serum or human serum. Preferably, human serum is used. According to conventional techniques, serum may be used with about 1% by volume to about 30% by volume of the culture medium.

[0208] In other embodiments, the culture medium of the present invention may include a serum replacement. Various serum replacement preparations are commercially available and are known to the skilled person. When a serum replacement is used, it may be used at about 1% to about 30% by volume of the culture medium according to conventional techniques.

[0209] In other embodiments, the culture medium of the present invention can be serum-free and / or serum-free replacement. Serum-free culture medium is a culture medium that does not contain any type of animal serum. Serum-free culture medium can preferably avoid possible exogenous contamination of stem cells. Serum-free replacement culture medium is a culture medium that is not supplemented with any commercial serum replacement preparation.

[0210] In preferred embodiments, the cell culture medium is supplemented with purified, natural, semisynthetic and / or synthetic growth factors and does not include undefined components, such as fetal bovine serum or calf serum. For example, supplements such as B27 (Invitrogen), N-acetylcysteine ​​(Sigma), and N2 (Invitrogen) stimulate proliferation of some cells. In some embodiments, the cell culture medium is supplemented with one or more of these supplements, such as one, any two, or all three of these supplements.

[0211] In other embodiments, the cell culture medium is supplemented with Exendin-4, a 39 amino acid peptide that activates the GLP-1 (glucagon-like peptide-1) receptor to increase intracellular cAMP in pancreatic acinar cells without acting on the VIP (vasoactive intestinal polypeptide) receptor.

[0212] The culture medium used in the present invention may include one or more trace elements, such as ions of 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.

[0213] The culture medium may include a reducing agent, such as β-mercaptoethanol at a concentration of about 0.1 mM.

[0214] The culture medium of the present invention may include one or more additional agents, such as nutrients or growth factors previously reported to improve stem cell culture, such as cholesterol / transferrin / albumin / insulin / progesterone, putrescine, selenite / other factors.

[0215] The culture medium of the present invention can diffuse into the extracellular matrix (ECM). In a preferred method of the present invention, the separated tissue fragments or separated epithelial stem cells are connected to the ECM. ECM is composed of a variety of polysaccharides, water, elastin and glycoproteins, wherein the glycoproteins include collagen, entactin (nidogen), fibronectin and laminin. ECM is secreted by connective tissue cells. Different types of ECM are known, which include different compositions, including different types of glycoproteins and / or different combinations of glycoproteins. ECM is provided by culturing cells that produce ECM, such as fibroblasts, in a container, then removing these cells and adding separated tissue fragments or separated epithelial stem cells. Examples of cells of extracellular matrix-producing cells are: chondrocytes, which mainly produce collagen and proteoglycans; fibroblasts, which mainly produce type IV collagen, laminocytes, interstitial procollagen and fibronectin; and colon myofibroblasts, which mainly produce collagen (types I, III and V), chondroitin sulfate proteoglycans, 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 from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Matrigel TM(BD Biosciences). Synthetic extracellular matrix materials, such as ProNectin (Sigma Z378666) can be used. If desired, a mixture of extracellular matrix materials can be used. The use of ECM for culturing stem cells increases the long-term survival of stem cells and the continued presence of undifferentiated stem cells. In the absence of ECM, stem cell cultures cannot be cultured for a long period of time, and the continued presence of undifferentiated stem cells is not observed. In addition, the presence of ECM allows the culture of three-dimensional tissue organoids, which cannot be cultured in the absence of ECM. Extracellular matrix materials typically sink to the bottom of the culture dish in which the cells are suspended. Typically, when the matrix solidifies at 37°C, culture medium is added and diffused into the ECM. The cells in the culture medium adhere to the ECM by interacting with the surface structures of the ECM, such as interacting with integrins. At about 1 μg / cm 2 , or about 1 μg / cm 2 About 250 μg / cm 2 , or about 1 μg / cm 2 About 150 μg / cm 2 A fibronectin solution (stock solution) of about 1 mg / ml can be used to coat a cell culture vessel. In some embodiments, fibronectin is used at 8 μg / cm 2 Up to 125 μg / cm 2 To coat cell culture vessels.

[0216] Examples of ECMs useful in the methods of the invention include at least one glycoprotein, such as laminin.

[0217] Preferred ECMs for use in the methods of the present invention include at least two different glycoproteins, such as two different types of collagen or collagen and laminin. The ECM may be a synthetic hydrogel extracellular matrix or a naturally occurring ECM. TM Other preferred ECMs are provided by (BD Biosciences), which include laminin, entactin, and collagen IV. In some embodiments, the extracellular matrix is ​​an extracellular matrix containing laminin, such as Matrigel. TM (BD Biosciences).

[0218] In some embodiments, single stem cells, cell populations, or tissue fragments are embedded in matrigel, which is optionally growth factor-reduced and / or phenol red-free.

[0219] In some embodiments, culture medium is placed on top of the ECM. The culture medium is then removed and replenished as needed and when needed. In some embodiments, the culture medium is replenished every 1, 2, 3, 4, 5, 6, or 7 days. If a component is "added" or "removed" from the culture medium, it may in some embodiments mean removing the culture medium itself from the ECM, and then placing new culture medium containing the "added" component or not including the "removed" component on the ECM.

[0220] In some embodiments, the culture medium of the invention is in contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix through its interaction with cell membrane proteins such as integrins.

[0221] In some embodiments, the base medium comprises or consists of modified DMEM / F12 supplemented with penicillin / streptomycin, 10 mM HEPES, Glutamax, 1×N2, 1×B27 (all from Invitrogen), and 1 mM N-acetylcysteine ​​(Sigma).

[0222] Examples of culture media of the present invention

[0223] In one embodiment, the cell culture medium includes a TGF-β 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 embodiment, the cell culture medium includes A83-01 and / or SB202190, preferably A83-01+SB202190. It has been surprisingly found that A83-01+SB202190 used together in the culture medium of the present invention synergistically increases the number of passages of human colon organoids. In one embodiment, the cell culture medium includes WENR+A83-01+SB202190. In one embodiment, the cell culture medium includes WENR+A83-01+SB202190+nicotinamide. In one embodiment, the cell culture medium includes WENRg+nicotinamide+A83-01+SB202190 (wherein "g" is gastrin). In one embodiment, the cell culture medium includes WENR+A83-01+nicotinamide+FGF10. In one embodiment, the cell culture medium includes WENRg+A83-01+nicotinamide+FGF10. In one embodiment, the cell culture medium includes WENRg+A83-01+nicotinamide+FGF10+SB202190. In one embodiment, the cell culture medium is used to obtain colon organoids. Also provided is a colon organoid obtained by culturing epithelial cells using the cell culture medium described in this embodiment.

[0224] For example, in one embodiment, the cell culture medium includes WENRg+A83-01+FGF10, wherein the Wnt agonist is R-spondin, but no other Wnt agonist is present or no nicotinamide is present. For example, in some embodiments, the cell culture medium includes 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 μM), and optionally includes SB202190. These components can be added to a basal medium, such as a DMEM / F12 medium. In some embodiments, the basal medium is further supplemented with any one or more (e.g., 1, 2, 3, 4, or 5) components or all components selected from P / S, Glutamax, 10 nmM Hepes, B27, N2, and N-acetylcysteine. It has been found that the use of such a cell culture medium can be used to obtain pancreatic organoids. Also provided is a pancreatic organoid obtained by culturing epithelial cells using the cell culture medium of this embodiment. In some embodiments, the culture medium does not include gastrin or nicotinamide or gastrin and nicotinamide.

[0225] Tissue-specific culture medium of the present invention

[0226] Particularly preferred culture media are described in the Examples herein.The culture media of the invention may be adapted for use with different tissues, for example as described below.

[0227] Intestinal culture medium

[0228] In some embodiments, the culture medium for small intestinal crypts, such as mouse small intestinal crypts, comprises or consists of a basal medium, such as described above, and further comprises: EGF, such as mouse EGF; BMP inhibitors, such as mouse Noggin; and Rspondin, such as human Rspondin-1 or 4. In some embodiments, this culture medium further comprises 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, such as mouse colonic crypts, comprises or consists of a basal medium, such as described above, and further comprises: a Wnt agonist, such as recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF, such as mouse EGF; BMP inhibitors, such as mouse Noggin; and Rspondin, such as human Rspondin-1 or 4. In some embodiments, this culture medium further comprises a TGF-β inhibitor (e.g., A83-01) and / or a p38 inhibitor (e.g., SB202190).

[0229] In some embodiments, the culture medium for human intestinal stem cells, human small intestinal crypts, or human colonic crypts (also referred to as HISC culture medium) includes or consists of a basal medium, such as described above, and further includes: a Wnt agonist, such as recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as Noggin; Rspondin, such as human Rspondin-1; a TGF-β inhibitor, such as A83-01; a p38 inhibitor, such as SB202190; gastrin; and nicotinamide. In some embodiments, the HISC culture medium may not include a p38 inhibitor and / or gastrin.

[0230] In some embodiments, the present invention provides a culture medium for culturing intestinal cells, which comprises or consists of a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin1-4, a TGF-β inhibitor, nicotinamide and preferably a p38 inhibitor.

[0231] In some embodiments, the culture medium for expanding small intestinal or colon stem cells, such as human small intestinal or colon cells, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM) and glutamine / glutamax), Wnt3A (optionally a conditioned medium), any one of Rspondin1-4 (preferably 1 μg / 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). In other embodiments, the culture medium further comprises a p38 inhibitor, such as SB202190 (preferably 100 nM). In other embodiments, the culture medium further comprises a Rock inhibitor, such as LY2157299.

[0232] 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.

[0233] In some embodiments, the culture medium for differentiating small intestinal or colon stem cells, such as human small intestinal or colon cells, includes or consists of a basal medium (e.g., including modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM) and glutamine / glutamax), Noggin (preferably 50-100 ng / ml), EGF (preferably 10-50 ng / ml), gastrin (preferably 10 nM), TGF-β inhibitors such as A83-01 (preferably 500 nM), and p38 inhibitors such as SB202190 (preferably 100 nM). In some embodiments, the differentiation medium may not include gastrin. In some embodiments, a γ-secretase inhibitor may be added to the differentiation medium (preferably at a concentration of 1 μM). The γ-secretase inhibitor may affect the final cell type determination, such as a tendency toward secretory cells, such as goblet cells. In some embodiments, RANKL may be added to the differentiation medium (e.g., at a concentration of 100 ng / ml). RANKL can influence the final cell type decision during differentiation, such as towards M-cells.

[0234] Cancer Culture Medium

[0235] In some embodiments, the culture medium for colon cancer cells comprises or consists of a basal medium, such as described above, and further comprises: a Wnt agonist, such as recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as Noggin; Rspondin, such as human Rspondin-1; a TGF-β inhibitor, such as A83-01; a p38 inhibitor, such as SB202190; gastrin; and nicotinamide.

[0236] In some embodiments, the culture medium for colon cancer, such as human colon cancer, includes: a basal medium, for example, including modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1mM), primocin and / or P / S (antibiotics) (500x) and hepes), Rspondin (optionally conditioned medium) (preferably 1μg / ml), Noggin (preferably 100ng / ml), nicotinamide (preferably 10mM), EGF (preferably 50ng / ml), gastrin (preferably 50nM), TGF-β inhibitors such as A83-01 (preferably 500nM), p38 inhibitors such as SB202190 (preferably 10μM), optionally including PGE2 (preferably 10nM) and / or Rock inhibitor (preferably 10μM).

[0237] In some embodiments, colon cancer cells can also be grown in HISC culture medium. In some embodiments, colon cancer cells can be cultured in HISC culture medium, wherein the culture medium does not include one or more or all of the following: EGF, Noggin, Rspondin, TGF-β inhibitors, and p38 inhibitors. Cancer cells can have mutations that constitutively activate or inactivate certain growth pathways. For example, many colon cancers produce constitutive activation of the Wnt pathway. In such cases, the culture medium does not require a Wnt agonist. Other mutations can cause the above culture medium to omit other factors. Other epithelial cancers (cancers) can also grow in the culture medium of the present invention. In a preferred embodiment, cancer organoids obtained from cancer stem cells are grown in a culture medium suitable for growing corresponding normal tissue organoids obtained from normal stem cells (the culture medium optionally does not include certain factors). For example, gastric cancer organoids obtained by culturing gastric cancer stem cells can be grown in the same culture conditions as normal gastric organoids obtained by culturing gastric stem cells (the culture medium optionally does not include certain factors). In another example, pancreatic cancer organoids obtained by culturing pancreatic cancer stem cells can be grown in the same culture conditions as normal pancreatic organoids obtained by culturing pancreatic stem cells (the culture medium optionally does not include certain factors). In another example, prostate cancer organoids obtained by culturing prostate cancer stem cells can be grown in the same culture conditions (culture medium optionally does not include certain factors) as normal prostate organoids obtained by culturing prostate stem cells. In another example, liver cancer organoids obtained by culturing liver cancer stem cells can be grown in the same culture conditions (culture medium optionally does not include certain factors) as normal liver organoids obtained by culturing liver stem cells. In many cases, it may be preferred (or at least more convenient) to grow cancer organoids in normal tissue culture medium (without excluding any factor). Normal tissue culture medium should grow cancers with genetic backgrounds without excluding any specific cancer mutations.

[0238] Thus, in some embodiments, the present invention provides a culture medium for culturing cancer cells, e.g., cancer stem cells, such as adenocarcinoma or cancer cells from a target tissue type, wherein the culture medium comprises or consists of culture medium components for culturing cells from a corresponding non-cancerous target tissue type, optionally wherein the culture medium for culturing non-cancerous cells of the target tissue type does not include one or more of: Wnt-3a, EGF, Noggin, Rspondin, TGF-β inhibitors, p38 inhibitors, nicotinamide, gastrin, FGF10, and HGF.

[0239] Adenoma culture medium

[0240] In some embodiments, a culture medium for an intestinal adenoma, such as a mouse intestinal adenoma, comprises a basal medium, such as described above, and further comprises EGF, such as mouse EGF.

[0241] Gastric culture medium

[0242] In some embodiments, the present invention provides a culture medium for culturing gastric cells, which comprises or consists of a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin1-4, a TGF-β inhibitor, gastrin, nicotinamide and preferably a p38 inhibitor.

[0243] In some embodiments, the culture medium for gastric stem cells, such as human gastric stem cells, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM), primocin and / or P / S (antibiotics) (500x), and glutamine / glutamax), any one of Rspondin1-4 (optionally a conditioned medium) (preferably 1 μg / ml), Noggin (optionally a conditioned medium) (preferably 100 ng / ml), Wnt3A (optionally a conditioned medium), nicotinamide (preferably 5 mM), EGF (preferably 50 ng / ml), FGF10 (preferably 200 ng / ml), gastrin (preferably 1 nM), a TGF-β inhibitor such as A83-01 (preferably 2 μM). The culture medium for gastric stem cells optionally further comprises a p38 inhibitor, such as 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).

[0244] In some embodiments, the culture medium for gastric stem cells, such as mouse gastric stem cells, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM), primocin and / or P / S (antibiotics) (500x), and glutamine / glutamax), any one of Rspondin 1-4 (optionally a conditioned medium) (preferably 1 μg / ml), Noggin (optionally a conditioned medium) (preferably 100 ng / ml), Wnt3A (optionally a conditioned medium), EGF (preferably 50 ng / ml), FGF10 (preferably 200 ng / ml), gastrin (preferably 1 nM), and Rock inhibitor (preferably 10 μM). In some embodiments, such a culture medium further comprises a TGF-β inhibitor (e.g., A83-01) and / or a p38 inhibitor (e.g., SB202190).

[0245] Prostate culture medium

[0246] In some embodiments, the culture medium for amplifying prostate stem cells includes testosterone, optionally including dihydrotestosterone (also referred to herein as DHT). Testosterone is a steroid hormone from the androgen group. In humans, most of the testosterone undergoes 5α-reduction to form a more effective androgen, i.e., dihydrotestosterone. Testosterone, dihydrotestosterone, or testosterone mimetics (e.g., molecules that simulate the activity of testosterone bound to androgen receptors) can be added to the culture medium of the present invention. Therefore, when the term testosterone is used, it can also be generally replaced by dihydrotestosterone or testosterone analogs. The inventors have shown that the addition of testosterone to the culture medium for prostate stem cells leads to increased differentiation, but also leads to continuous expansion of stem cell populations (e.g., see Figures 41-45 ). This is very surprising, because the literature teaches that testosterone plays an important role in cell differentiation by inhibiting proliferation and maintaining terminal differentiation (Mirochnik et al., PLoS One, 7 (3), e31052, 2012; Niu et al., Oncogene 29, 3593-3604, 2010). Technicians have expected that adding testosterone to the culture medium for the prostate will result in fully differentiated organoids without further expansion potential. This will be similar to the situation observed when differentiating the colon, pancreas and liver in the differentiation medium. However, on the contrary, the inventors have found that although testosterone increases differentiation, it also causes stem cell expansion to continue. Therefore, the organoids grown in the culture medium including testosterone surprisingly include stem cells and differentiated cells, that is, luminal cells and basal cells.

[0247] In some embodiments, the culture medium for obtaining prostate organoids includes a basal medium and testosterone, optionally including dihydrotestosterone and any one of Rspondin 1-4 or Rspondin mimetics. In some embodiments, the culture medium also includes a BMP inhibitor, such as Noggin. In some embodiments, the culture medium also includes a tyrosine receptor kinase ligand, optionally, wherein 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 includes EGF, Noggin, any one of Rspondin 1-4, and testosterone.

[0248] In a preferred embodiment, the culture medium for prostate cells includes a TGF-β inhibitor. In some embodiments, the culture medium for prostate organoids includes EGF, Noggin, any one of Rspondin 1-4, a TGF-β inhibitor, and testosterone. In some embodiments, the culture medium for prostate cells also includes a p38 inhibitor. In some embodiments, the culture medium for obtaining prostate organoids does not include 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 include testosterone. In some embodiments, the culture medium includes a basal medium, EGF, Noggin, and any one of Rspondin 1-4, and optionally includes a TGF-β inhibitor, and does not include testosterone.

[0249] In some embodiments, the present invention provides a culture medium for culturing prostate cells, which comprises or consists of: a basal medium, EGF, any one of Rspondin 1-4, Noggin, nicotine, a TGF-β inhibitor, and preferably Wnt3a and FGF-10. 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, includes: a basal medium (e.g., including modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM) and glutamine / glutamax), any one of Rspondin 1-4 (optionally a conditioned medium) (preferably 1 μg / ml), Noggin (optionally a 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 also includes a Rock inhibitor (preferably 10 μM). In some embodiments, the culture medium for prostate cells also includes a p38 inhibitor, such as SB202190. In some embodiments in which mouse prostate cells are cultured, the culture medium may not include a TGF-β inhibitor. In other embodiments, the culture medium may not include nicotinamide, FGF10, and / or Rock inhibitor.

[0250] Pancreas culture medium

[0251] In some embodiments, the present invention provides a culture medium for expanding pancreatic cells, which comprises or consists of a basal medium, any one of Rspondin 1-4, Noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, and preferably exendin 4 and Wnt-3a.

[0252] In some embodiments, the culture medium for expanding pancreatic stem cells, such as human pancreatic stem cells, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM), and glutamine / glutamax), any one of Rspondin 1-4 (optionally a conditioned medium) (preferably 1 μg / ml), Noggin (optionally a conditioned medium) (preferably 100 ng / ml), nicotinamide (preferably 10 mM), EGF (preferably 50 ng / ml), FGF10 (preferably 100 ng / ml), gastrin (preferably 100 nM), a TGF-β inhibitor such as A83-01 (preferably 2 μM). In other embodiments, this culture medium further comprises Wnt-3a. In other embodiments, the culture medium further comprises a p38 inhibitor, such as SB202190 (preferably 100 nM). In other embodiments, the culture medium further comprises a Rock inhibitor, such as LY2157299 (preferably 10 μM). In other embodiments, the culture medium further comprises Exendin 4 (preferably 50 ng / ml).

[0253] In some embodiments, the culture medium for expanding pancreatic stem cells, such as mouse pancreatic stem cells, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM), primocin and / or P / S (antibiotics), Hepes, and glutamine / glutamax), any one of Rspondin 1-4 (optionally a conditioned medium) (preferably 1 μg / ml), Noggin (optionally a 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). In other embodiments, such a culture medium further 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.

[0254] In some embodiments, the present invention provides a culture medium for differentiating pancreatic cells, which comprises or consists of a basal medium, Noggin, EGF, FGF10, gastrin, a TGF-β inhibitor, a γ-secretase inhibitor, and preferably exendin4.

[0255] In some embodiments, the culture medium for differentiating pancreatic stem cells, such as human pancreatic stem cells, comprises or consists of a basal medium (e.g., comprising modified 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 other embodiments, such a culture medium further comprises Exendin 4 (preferably 50 ng / ml). In some embodiments, the culture medium for pancreatic cells further comprises a p38 inhibitor, such as SB202190.

[0256] In some embodiments, the culture medium for differentiating pancreatic stem cells, such as mouse pancreatic stem cells, comprises or consists of a basal medium (e.g., comprising modified 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).

[0257] Barrett's esophagus culture medium

[0258] In some embodiments, the culture medium for Barrett's esophagus includes or consists of a basal medium, such as described above, and further includes: a Wnt agonist, such as recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as Noggin; Rspondin, such as human Rspondin-1; a TGF-β inhibitor, such as A83-01; a p38 inhibitor, such as SB202190; gastrin; nicotinamide; and FGF, such as human FGF10 (i.e., HISC+FGF). In some embodiments, this differentiation medium does not include gastrin.

[0259] In some embodiments, the present invention provides a method for obtaining Barrett's esophagus organoids, wherein the method includes culturing epithelium isolated from Barrett's esophagus in a HISC culture medium that optionally also includes FGF10 for 1, 2, 3, 4, 5, 6, 7 days or more; and removing nicotinamide and SB202190 after the initial 1, 2, 3, 4 days or more. In some embodiments, the culture medium also includes a Notich inhibitor, such as DBZ. In some embodiments, Barrett's esophagus organoids cultured in the presence of a Notch inhibitor include little or no proliferating cells, and include more goblet cells compared to organoids cultured in the absence of a Notch inhibitor (see Figure 5).

[0260] Liver culture medium

[0261] In some embodiments, hepatocytes may be grown in a first "expansion" medium (also referred to herein as EM), preferably followed by culturing the cells in a second "differentiation" medium (also referred to herein as DM). However, in some embodiments, the step of differentiating in DM medium is not performed, for example, in some methods, the cells are transplanted and allowed to differentiate in vivo. Similarly, there are expansion medium and differentiation medium for other tissues, such as the pancreas, small intestine, and colon (see above).

[0262] In one embodiment, the liver expansion medium includes EGF, a Wnt agonist, FGF, and nicotinamide. Preferably, the Wnt agonist is R-spondin 1-4 (e.g., any one or more of Rspondin 1, 2, 3, and 4), and thus, the expansion medium is referred to as "ERFNic". A particularly preferred expansion medium also includes HGF and is referred to as "ERFHNic".

[0263] In some embodiments, the liver expansion medium is supplemented with a TGFβ inhibitor. In some embodiments, TGFβ is present at least 5nM, such as at least 50nM, at least 100nM, at least 300nM, at least 450nM, at least 475nM, such as 5nM-500mM, 10nM-100mM, 50nM-700μM, 50nM-10μM, 100nM-1000nM, 350-650nM or more preferably 500nM. The presence of a TGFβ inhibitor in the expansion medium is particularly preferred for embodiments of human cells.

[0264] In some embodiments, the present invention provides a culture medium for expanding hepatocytes, which comprises or consists of a basal medium, any one of Rspondin 1-4, Noggin, nicotinamide, EGF, FGF10, HGF, gastrin, a TGF-β inhibitor and PGE2, and preferably Wnt-3a.

[0265] In some embodiments, the liver expansion medium further comprises a p38 inhibitor.

[0266] In some embodiments, the liver expansion medium is supplemented with an activator of the prostaglandin signaling pathway (also referred to as a prostaglandin pathway activator) (see Fig.24 ). For example, liver expansion medium is supplemented with any one or more of the following compounds: phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, liver expansion medium is supplemented with PGE2 and / or AA. In some embodiments, PGE2 is added to liver expansion medium until the final concentration is at least 10nM, at least 30nM, at least 40nM, at least 45nM, at least 50nM, such as 10nM to 500nM, 10nM to 400nM, 10nM to 300nM, 10nM to 200nM, 10nM to 100nM, 20nM to 50nM. In a preferred embodiment, PGE2 is added to liver expansion medium until the final concentration is 50nM. In some embodiments, AA is added to the liver expansion medium to a final concentration of at least 1 μg / ml, e.g., 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.

[0267] In a preferred embodiment, the liver expansion medium is supplemented with a TGF-β inhibitor and an activator of the prostaglandin signaling pathway (eg, PGE2 and / or AA) and optionally a p38 inhibitor.

[0268] In a preferred embodiment, the liver expansion medium further comprises gastrin.

[0269] In one embodiment, liver differentiation culture medium includes EGF, TGF-β inhibitor, FGF (for example, FGF10, FGF2 or any other suitable FGF family member) and Notch inhibitor. In one embodiment, TGF-β inhibitor is A83-01 and / or Notch inhibitor is DAPT. This differentiation culture medium is referred to as "EAFD" herein and is a preferred differentiation culture medium of the present invention. FGF can be optionally replaced by HGF, or both FGF and HGF can be present or absent in the differentiation culture medium. In some embodiments, EGF can be replaced by HGF or another receptor tyrosine kinase ligand. Dexamethasone can also be added, for example, with a concentration of 10nM to 10μM. Liver differentiation culture medium can optionally include prostaglandin pathway activators, such as PGE2 or AA. However, differentiation culture medium can also not include this component. In some embodiments, oncostatin M can also be added, for example, with a concentration of 1ng / ml to 1mg / ml, so as to assist the differentiation of the final cell type of stem cells.

[0270] In some embodiments, the present invention provides a culture medium for differentiating hepatocytes, which comprises 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.

[0271] In some embodiments, the hepatocytes can be initially cultured in an expansion medium that also includes Wnt and Noggin, such as an "ENRW" medium that includes EGF, Noggin, R-spondin, and Wnt (e.g., Wnt-3A), an optional prostaglandin pathway activator such as PGE2 or AA, an optional TGFβ inhibitor, and optional FGF, HGF, nicotinamide. In a preferred embodiment, the liver expansion medium is supplemented with one, more preferably two, of a TGF-β inhibitor and an activator of the prostaglandin signal transduction pathway (e.g., PGE2 and / or AA).

[0272] In some embodiments, expansion medium for liver includes EGF, Noggin, gastrin, FGF, nicotinamide, TGF-β inhibitors such as A83-01, HGF, Rspondin 1-4 (eg, any one or more of Rspondin 1, 2, 3, or 4), and PGE2.

[0273] In a preferred embodiment, the liver cells can be initially cultured in an expansion medium supplemented with PGE2 and / or AA, the expansion medium including any one of EGF, Noggin, gastrin, FGF10, nicotinamide, A8301, HGF, and Rspondin1-4. Rspondin 1-4 can be provided in the form of Rspo conditioned medium. For example, the expansion medium can include 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 μg / ml) supplemented with PGE2 (50 nM) and / or AA (10 μg / ml). The expansion medium may also include a Rock inhibitor.

[0274] When expanding mouse stem cells, the culture medium may not include one or more of the following: a TGF-β inhibitor (eg, A83-01) and PGE2.

[0275] The inventors have found that this culture medium is optimal for stimulating the initial expansion of cells in the first few days. Therefore, this first expansion culture medium is sometimes referred to as EM1 herein. In some embodiments, after about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more days, such as 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, more than 12 months, more than 14 months, remove Wnt and Noggin. In some embodiments, cells are then expanded in the expansion culture medium of the present invention, as described above, the expansion culture medium does not include Wnt or Noggin. This second expansion culture medium is sometimes referred to as EM2 herein. In some embodiments, in EM2, culture medium cells are cultured for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or longer periods, such as 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks or more weeks. Then, the culture medium can be replaced with an optimized differentiation culture medium, as described above, the culture medium includes a TGF-β inhibitor and a Notch inhibitor. Typically, the differentiation medium does not include Wnt agonists, R-spondin or nicotinamide. In some embodiments, the differentiation medium does not include prostaglandin pathway activators, such as PGE2 or AA. This encourages the differentiation of cells toward mature hepatocytes and cholangiocytes. These cells are suitable for transplantation into humans or animals.

[0276] Expansion Medium for Liver (EM2):

[0277] In one aspect of the present invention, a cell culture medium is provided, which comprises or consists of a basal medium for animal or human cells, to which is added: epidermal growth factor as a mitogenic growth factor, FGF that can bind to FGFR2 or FGFR4, preferably FGF10, nicotinamide, and a preferred Wnt agonist, preferably R-spondin 1-4. This medium is called EM2. This "EM2" medium is preferably used for expanding hepatocytes.

[0278] In some embodiments, the EM2 comprises a prostaglandin pathway activator, such as PGE2 and / or AA.

[0279] In some embodiments, the EM2 comprises a TGF-β inhibitor, such as A83-01.

[0280] Preferably, the Wnt agonist is R-spondin 1-4. The culture medium comprising EGF, R-spondin 1-4, FGF and nicotinamide is referred to herein as ERFNic.

[0281] In some embodiments, the EM2 medium does not include Noggin, and more preferably does not include a BMP-inhibitor. In some embodiments, the EM2 medium does not include a Wnt, such as Wnt-3a.

[0282] In some embodiments, in addition to FGF, HGF is also present. The preferred culture medium that includes HGF in addition to FGF is ERFHNic (EGF + R-spondin (preferably R-spondin 1-4) + FGF (preferably FGF10) + HGF + nicotinamide + prostaglandin pathway inhibitors such as PGE2 and / or AA and TGF-β inhibitors. The inventors have found that ERFHNic culture medium containing TGF-β inhibitors and prostaglandin pathway activators is an optimized culture medium for long-term expansion of cells. In the absence of HGF, cells remain active in the culture medium for no longer than 3 months. In addition, in the absence of HGF, after 10 passages, cells show a growth disadvantage compared to cells cultured in the presence of HGF, as evidenced by a lower proliferation ratio. In particular, at 1 After 5 passages, the cells did not grow organoids at the same rate in the absence of HGF as in the presence of HGF. Thus, HGF was found to be necessary for maintaining a good proliferation rate during long-term culture. Thus, the present invention provides an 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. Months or more, for example, more than 3 years. In practice, some embodiments of the present invention include the use of EM2 for about 20-50 passages of cells. For example, cells can be divided 4-8 times once a week for 7-10 weeks. Preferably, the cells will expand at a rate of about 4-5 times per week or more than 2 times a week. The present invention also provides the ERFHNic medium of the present invention for culturing cells for at least 8 passages, for example, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, At least 60 passages, or 15-35 passages, for example, about 20-30 passages. In some embodiments, TGF-β inhibitors such as A83-01 are also present in EM2 culture medium. This is particularly useful when culturing human cells or organoids. In some embodiments, A83-01 is present at a concentration of 400-600nM, for example 450-550nM, 470-530nM or about 500nM. In embodiments in which a TGF-β inhibitor is present in EM2, a Notch inhibitor is preferably absent. In some embodiments, EM2 also includes a p38 inhibitor.

[0283] Expansion Medium for Liver (EM1):

[0284] In one aspect, the present invention provides a cell culture medium comprising or consisting of a basal culture 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 culture medium is called "ENRW" (EGF, Noggin, R-spondin, and Wnt (e.g., Wnt3A)). This culture medium is called EM1. In some embodiments, EM1 also includes a prostaglandin pathway activator, such as PGE2 and / or AA. In some embodiments, EM1 includes a TGF-β inhibitor, such as A83-01. More preferably, EM1 also includes a prostaglandin pathway activator and a TGF-β inhibitor. The inventors have found that a culture medium containing Wnt and Noggin is ideal for stimulating the initial expansion of cells. Therefore, in some embodiments, only EM1 culture medium is used for 1 passage or 1 week, but it is also expected that EM1 culture medium can be used for about 1 year because it is not harmful to cells. Thus, in some embodiments, EM1 medium is used to culture cells from day 0 to day 10, for example, from day 0-7, day 0-6, day 0-5, day 0-4, day 0-3, day 0-2, day 0-1, wherein day 0 is the day when the cells are separated from the tissue from which they are derived, and day 1 is the next day; or for more than 1 week, for example 2, 3, 4 weeks or more weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer. In some embodiments, EM1 medium is only used for the first day or the first two days of culture. In some embodiments, EM1 medium is used for more than 1 passage, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more passages, for example 20-30 passages, 30-35 passages, 32-40 passages or more. In some embodiments, EM1 medium is used after a freezing step or any other transport step involving medium or temperature changes not coupled with optimal growth. Such "EM1" medium is preferably used for expanding hepatocytes.

[0285] EM1 medium is supplemented with one or more of a compound selected from FGF, HGF, nicotinamide, gastrin, B27, N-acetylcysteine ​​and N2. In the case of starting the culture from frozen raw materials or from a single cell, EM1 medium is preferably supplemented with a ROCK inhibitor. Y27632 is a preferred ROCK inhibitor for use in the present invention.

[0286] Therefore, in one embodiment, there is provided a cell culture medium comprising or consisting of a basal medium for animal or human cells, to which is added: epidermal growth factor, FGF (e.g., FGF capable of binding to FGFR2 or FGFR4), preferably FGF10 and HGF as mitogenic growth factors,

[0287] Prostaglandin pathway activators, such as PGE2 and / or AA;

[0288] TGF-β inhibitors;

[0289] gastrin, niacinamide, B27, N2, and N-acetylcysteine, and preferably;

[0290] A BMP inhibitor, preferably Noggin; and

[0291] Wnt agonist, preferably R-spondin 1 and / or Wnt-3a.

[0292] B27 (Invitrogen), N-acetylcysteine ​​(Sigma) and N2 (Invitrogen), gastrin (Sigma) and nicotinamide (Sigma) can also be added to the above culture medium, and it is believed that cell proliferation is controlled and DNA stability is helped. In the present context, nicotinamide is also referred to as "Nic".

[0293] 'N2 supplement' is available from Invitrogen, Carlsbad, CA; www.invitrogen.com; No. 17502-048; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com; No. F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979. N2 supplement is provided by PAA Laboratories GmbH as a 100× liquid concentrate, which includes 500 μg / ml human transferrin, 500 μg / ml bovine insulin, 0.63 μg / ml prostaglandin, 1611 μg / ml putrescine, and 0.52 μg / ml sodium selenite. N2 supplement can be added to the culture medium as a concentrate, or diluted before addition to the culture medium. It can be used at 1× final concentration or other final concentrations. The use of N2 supplement is a convenient way to incorporate transferrins,insulin,prostaglandins,putrescine and sodium selenite into substratum of the present invention.In some embodiments where substratum comprises B27, it also does not comprise N2.Therefore, when there is B27 as required, embodiments of the present invention may be suitable for not comprising N2.

[0294] 'B27 supplement' (available from Invitrogen, Carlsbad, CA; www.invitrogen.com; current number 17504-044; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com; number F01-002; Brewer et al., J Neurosci Res., 35(5):567-76, 1993) can be used to formulate culture media including biotin, cholesterol, linoleic acid, linolenic acid, prostaglandins, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin. B27 supplement is provided by PAA Laboratories GmbH in the form of liquid 50× concentrate, which also includes biotin, cholesterol, linoleic acid, linolenic acid, prostaglandins, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α tocopherol (vitamin E), albumin, insulin and transferrin. Among these ingredients, at least linolenic acid, retinol, retinyl acetate and triiodothyronine (T3) are nuclear hormone receptor agonists. B27 supplement can be added to culture medium in the form of concentrate, or diluted before being added to culture medium. It can be used at 1× final concentration or other final concentrations. The use of B27 supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, prostaglandins, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α tocopherol (vitamin E), albumin, insulin and transferrin into culture medium of the present invention.

[0295] For example, the cell culture medium may include or consist of a basal medium to which is added: EGF and R-spondin 1 supplemented with FGF10, HGF and nicotinamide; for example, EGF (50 ng / ml) and R-spondin1 (1 μg / ml) supplemented with 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 ug / ml) and TGF-β inhibitors such as A83-01 (500 nM). In some embodiments, the culture medium further comprises a p38 inhibitor. The inventors have found that such a culture medium can be used for long-term expansion of cells. Therefore, such a cell culture medium is preferably used as EM2 of the present invention. The basal culture medium is preferably supplemented with B27, N2 and 200 ng / ml N-acetylcysteine. In some embodiments, the basal culture medium is modified-DMEM / F12. However, any other suitable basal culture medium may be used.

[0296] Another example of a cell culture medium and a method of using such a culture medium comprises or consists of modified-DMEM / F12, preferably supplemented with B27, N2, 200ng / ml N-acetylcysteine, 50ng / ml EGF, 1μg / ml R-spondin, 10nM gastrin, 100ng / ml FGF10, 10mM nicotinamide, 50ng / ml HGF, 50% Wnt conditioned medium, prostaglandin pathway activators such as PGE2 (50nM) and / or AA (10ug / ml) and TGF-β inhibitors such as A83-01 (500nM) and preferably 10-100ng / ml Noggin. Wnt conditioned medium comprises modified DMEM, P / S, B27, N2 and also includes FCS. 293T cells transfected with a Wnt3A expression plasmid produce Wnt. The entire culture medium (ie, with secreted Wnts) was harvested after several days and used as a source of Wnts.

[0297] Therefore, the present invention provides a cell culture medium comprising or consisting of a basal culture medium for animal or human cells, to which is added:

[0298] As mitogenic growth factors, epidermal growth factor, FGF that can bind to FGFR2 or FGFR4, preferably FGF10, and HGF,

[0299] Prostaglandin pathway activators, such as PGE2 and / or AA,

[0300] TGF-β inhibitors,

[0301] Gastrin, nicotinamide, B27, N2 and N-acetylcysteine, and preferably a BMP inhibitor, more preferably Noggin, and

[0302] Wnt agonist, more preferably R-spondin 1 and / or Wnt-3a.

[0303] Therefore, the present invention provides a first preferred culture medium comprising or consisting of a basal culture medium for animal or human cells, to which is added:

[0304] As mitogenic growth factors, epidermal growth factor, FGF10 and HGF,

[0305] Prostaglandin pathway activators, such as PGE2 and / or AA,

[0306] TGF-β inhibitors,

[0307] Gastrin, niacinamide, B27, N2 and N-acetylcysteine,

[0308] A BMP inhibitor, more preferably Noggin, and

[0309] Wnt agonists, more preferably R-spondin 1 and Wnt-3a.

[0310] In some embodiments, a p38 inhibitor is added to the expansion medium.

[0311] This medium can 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 includes all the components of the EM2 medium of the present invention and also includes Wnt-3a and Noggin.

[0312] In embodiments where the basal medium is supplemented with N-acetylcysteine, B27 and N2, the following is preferably added to the medium: EGF, R-spondin 1, gastrin, FGF10, nicotinamide and HGF and Wnt-conditioned medium and prostaglandin pathway activators such as PGE2 and / or AA. Preferably, the basal medium is supplemented with N-acetylcysteine, EGF, R-spondin 1, gastrin, FGF10, nicotinamide and HGF and Wnt-conditioned medium and prostaglandin pathway activators such as PGE2 and / or AA in the amounts described above. Preferably, the TGF-β inhibitor is also present in the amounts described herein.

[0313] For example, in some embodiments, the basal medium can be supplemented with 150 ng / ml to 250 ng / ml of N-acetylcysteine; preferably, the basal medium is supplemented with about 200 ng / ml or precisely 200 ng / ml of N-acetylcysteine. For example, in some embodiments, the basal medium can be supplemented with 40 ng / ml to 60 ng / ml of EGF; preferably, the basal medium is supplemented with about 50 ng / ml or precisely 50 ng / ml of EGF. For example, in some embodiments, the basal medium can be supplemented with 0.5 μg / ml to 1.5 μg / ml of R-spondin 1; preferably, the basal medium is supplemented with about 1 μg / ml or precisely 1 μg / ml of R-spondin 1. For example, in some embodiments, the basal medium can be supplemented with 5 nM to 15 nM of gastrin; preferably, the basal medium is supplemented with about 10 nM or precisely 10 nM of gastrin. For example, in some embodiments, the basal medium can be supplemented with 25 ng / ml to 200 ng / ml of FGF10, such as 70 ng / ml to 130 ng / ml of FGF10; preferably, the basal medium is supplemented with about 100 ng / ml or exactly 100 ng / ml of FGF10. For example, in some embodiments, the basal medium can be supplemented with 5 mM to 15 mM nicotinamide; preferably, the basal medium is supplemented with about 10 mM or exactly 10 mM nicotinamide. For example, in some embodiments, the basal medium can be supplemented with 25 ng / ml to 100 ng / ml of HGF, such as 35 ng / ml to 65 ng / ml of HGF; preferably, the basal medium is supplemented with about 50 ng / ml or exactly 50 ng / ml of HGF. For example, in some embodiments, the basal medium can be supplemented with 35% to 65% of Wnt-conditioned medium; preferably, the basal medium is supplemented with about 50% or exactly 50% of Wnt-conditioned medium.

[0314] For example, in some embodiments, the liver expansion medium is supplemented with an activator of the prostaglandin signaling pathway (see Fig.24). For example, the liver expansion medium is supplemented with any one or more of the following compounds: phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, the liver expansion medium is supplemented with PGE2 and / or AA. In some embodiments, PGE2 is added to the liver expansion medium until the final concentration is at least 10nM, such as 10nM to 500nM, 10nM to 400nM, 10nM to 300nM, 10nM to 200nM, 10nM to 100nM, 20nM to 50nM. In a preferred embodiment, PGE2 is added to the liver expansion medium until the final concentration is 50nM. In some embodiments, AA is added to the liver expansion medium to a final concentration of at least 1 μg / ml, e.g., 1 μg / ml to 100 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.

[0315] In some embodiments, one or both of gastrin and N2 are absent in the cell culture medium.

[0316] Preferably, the basal culture medium is modified-DMEM / F12.

[0317] This first culture medium (e.g., EM1, EM2, or both EM1 and EM2) is preferably used within the first two weeks of the culture method of the present invention. However, it can be used for a shorter period of time, such as 1, 2, 3, 5, 7 or 10 days, or a longer period of time, such as 3, 4, 5, 10, 20 weeks or more, 5 months or more, such as 6, 7, 8, 9, 10, 11, 12 months or more.

[0318] Differentiation Medium (DM) for Liver:

[0319] On the other hand, provide and comprise the basal medium for animal or human cell or the second cell culture medium consisting of this basal medium, add to described basal medium: EGF, TGF-β inhibitor, Notch inhibitor and prostaglandin pathway activator such as PGE2 and / or AA.The inventor has found that this culture medium can be used for differentiated cells.The culture medium for differentiated cells can be referred to as DM herein.

[0320] Preferably, the second cell culture medium further comprises FGF and / or HGF.

[0321] In one embodiment, the second culture medium comprises or consists of a basal culture medium for animal or human cells, to which is added:

[0322] epidermal growth factor, FGF10, and HGF as mitogenic growth factors;

[0323] Notch inhibitors;

[0324] TGF-β inhibitors; and

[0325] Prostaglandin pathway activators, such as PGE2 and / or AA.

[0326] In one embodiment, the TGF-β inhibitor is A83-01 and / or the Notch inhibitor is DAPT. In another embodiment, the DM cell culture medium further comprises dexamethasone. In another embodiment, the DM cell culture medium further comprises oncostatin M. In another embodiment, the DM cell culture medium further comprises gastrin.

[0327] A preferred second cell culture medium and methods of using such a medium are described in the Examples and include or consist of a basal medium to which are added: 50 ng / ml EGF, 100 ng / ml FGF10, 50 nM A8301, and 10 μM DAPT. Modified-DMEM / F12 may be used as a basal medium, as may any other suitable basal medium.

[0328] In some embodiments, the differentiation medium for hepatocytes, e.g., human hepatocytes, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), n-acetylcysteine ​​(1 mM), 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 (DAPT / DBZ) (preferably 10 μM).

[0329] In some embodiments, the differentiation medium for hepatocytes, such as mouse hepatocytes, comprises or consists of a basal medium (e.g., comprising modified DMEM / F12, B27 (50x), 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 (DAPT / DBZ) (preferably 10 μM).

[0330] In some embodiments, the second cell culture medium does not include R-spondin or Wnt. In some embodiments, the second cell culture medium does not include a Wnt agonist. In some embodiments, the second cell culture medium does not include nicotinamide. In some embodiments, the second cell culture medium does not include a BMP inhibitor. In some embodiments, the second cell culture medium does not include a prostaglandin pathway activator, such as PGE2 or AA.

[0331] The inventors have found that both R-spondin1 and nicotinamide inhibit the expression of the mature hepatocyte marker CYP3A11, but promote the expression of the hepatoblast marker albumin. Therefore, to increase the differentiation of cells into more mature liver final cell types, the inventors removed R-spondin and nicotinamide from the cell culture medium. The inventors have also found that the expression of specific bile transcription factors is highly upregulated in expansion medium including R-spondin1, indicating that the culture gene expression is unbalanced and is more inclined towards the bile cell final cell type. The Notch and TGF-β signaling pathways have been implicated in the bile cell final cell type in vivo. In fact, the absence of Rbpj (which is necessary for active Notch signaling) leads to abnormal duct gland occurrence (Zong Y. Development 2009), and the addition of TGF-β to liver explants promotes bile differentiation in vitro (Clotman F. Genes and Development 2005). Because the Notch and TGF-β signaling pathways are highly upregulated in liver cultures ( Fig. 22 ), so the inventors speculated that inhibition of the final cell type of cholangiocytes may trigger differentiation of cells towards a more hepatocyte phenotype. It has been found that the addition of TGF-β inhibitors (e.g., A8301) and Notch inhibitors (e.g., DAPT) to differentiation medium, which preferably does not include R-spondin or Wnt, enhances the expression of mature hepatocyte markers and increases the number of hepatocyte-like cells (e.g., see Example 5).

[0332] Ordinary culture medium

[0333] The cell culture medium of the present invention allows epithelial stem cells or isolated crypts to survive and / or proliferate and / or differentiate on an extracellular matrix. In some embodiments, the cell culture medium of the present invention allows the organoid of the present invention to survive and / or proliferate and / or differentiate on an extracellular matrix, such as crypt villus organoids, colon organoids, pancreatic organoids, gastric organoids, Barrett's esophagus organoids, adenocarcinoma organoids or colon cancer organoids. In some embodiments, the cell culture medium of the present invention allows the organoid of the present invention to survive and / or proliferate and / or differentiate on an extracellular matrix, such as small intestine (crypt villus) organoids, colon organoids, pancreatic organoids, gastric organoids, Barrett's esophagus organoids, adenocarcinoma organoids, cancer organoids, colon cancer organoids, prostate organoids or prostate cancer organoids. Preferably, in embodiments in which there are TGF-β inhibitors and / or p38 inhibitors, the cell culture medium allows the survival and / or proliferation, preferably survival and proliferation, of the cell population or organoid of the present invention. Preferably, embodiments wherein a TGF-β inhibitor and / or a p38 inhibitor is initially present in the cell culture medium but subsequently removed from the culture medium (e.g. by not adding said inhibitor when refreshing the culture medium) allow survival and / or differentiation, preferably survival and differentiation, of the cell populations or organoids of the invention.

[0334] In some embodiments, a p38 inhibitor is added to any of the culture media described herein.

[0335] The term cell culture medium is synonymous with medium, culture medium or cell medium.

[0336] Use of the culture medium of the present invention

[0337] The present invention also provides use of the culture medium of the present invention for expanding and / or differentiating stem cells, stem cell populations, tissue fragments or organoids.

[0338] In some embodiments, the stem cell, stem cell population, tissue fragment, or organoid is selected from 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.

[0339] In some embodiments, stem cells, stem cell populations, tissue fragments, or organoids can be obtained from normal tissue.

[0340] In some embodiments, stem cells, stem cell populations, tissue fragments, or organoids may be obtained from diseased tissue, such as from an adenoma, a carcinoma, an adenocarcinoma, the intestine of a patient with cystic fibrosis, or the intestine of a patient with inflammatory bowel disease.

[0341] Stem cells cultured according to the present invention

[0342] Stem cells are found in many organs of adults and mice. Although there can be significant variation in the precise properties of adult stem cells in each tissue, adult stem cells share at least the following properties: they retain an undifferentiated phenotype; their progeny can differentiate in a form that tends to exist in all lineages in the relevant tissue; they maintain self-sustaining capacity throughout life; and they can regenerate relevant tissues after injury. Stem cells exist in a unique location, the stem cell niche, which provides appropriate cell-to-cell contacts and signals to maintain the stem cell population. The stem cells of the present invention preferably express Lgr5.

[0343] In one embodiment, the present invention provides a cell population or one or more organoids comprising stem cells produced or obtained by culturing stem cells or tissue fragments according to the present invention, wherein the organoids are 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.

[0344] A "population" of cells is greater than 1 cell, preferably at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells, at least 1×10 8 cells or at least 1×10 9 Many cells of a cell.

[0345] The stem cells of the present invention that are cultured according to the present invention may be human stem cells. The stem cells of the present invention that are cultured according to the present invention may be epithelial stem cells.

[0346] In some embodiments, the stem cells of the invention and / or cultured according to the invention are not embryonic stem cells. In some embodiments, the stem cells of the invention and / or cultured according to the invention are not human embryonic stem cells. Preferably, the stem cells of the invention are adult stem cells.

[0347] In a preferred embodiment, the stem cell can be a human epithelial stem cell. Human epithelial stem cells include stem cells of human epithelial tissue origin. These include, but are not limited to, pancreatic tissue, small intestinal tissue, large intestinal tissue, corneal tissue, olfactory tissue, respiratory tissue, gastric tissue, liver and skin, breast tissue and / or prostate tissue, for example, tissue selected from pancreatic tissue, small intestinal tissue, large intestinal tissue, corneal tissue, olfactory tissue and respiratory tissue. Epithelial stem cells can form different cell types composed of epithelium. Some epithelia, such as skin or intestines, show fast cell turnover, indicating that the stem cells present must proliferate continuously. Other epithelia, such as liver or pancreas, show very slow turnover under normal conditions.

[0348] Intestinal stem cells

[0349] The self-renewal epithelium of the small intestine is classified as crypts and villi (Gregoreff and Clevers, 2005Genes Dev19, 877-90). Each cell along the crypt-villus axis is polarized, so that the cells on the top of the intestinal villi or in the upper position of the colonic crypt are extremely differentiated, and are continuously lost into the cavity by apoptosis. The continuous proliferation of stem cells present at the bottom of the crypt and the large-scale proliferation of progenitor cells present in the middle of the crypt ensure that the cells that fall off are properly replaced. The epithelial renewal time produced in mice is 5 days. It has long been known that self-renewal stem cells are present near the bottom of the crypt and produce short-term expansion (TA) cells that can be rapidly proliferated and differentiated in the form of all lineages. The estimated number of stem cells is 4-6 / crypt (Bjerknes and Cheng, 1999Gastroenterology 1 16, 7-14). Three differentiated cell types, enterocytes, goblet cells and enteroendocrine cells, are formed from TA cells and continue their migration in coherent bands along the crypt-villus axis. Each villus receives cells from multiple different crypts. A fourth major differentiated cell type, the Paneth cell, exists at the bottom of the crypt.

[0350] The colon is similar to the small intestine, but has a flat surface epithelium instead of villi. The organization of the colonic crypts is similar to that of the small intestine. Paneth cells are not present in the colonic crypts; instead, so-called "deep crypt secretory" cells are present. The flat surface of the epithelium consists of differentiated cells (colonic epithelial cells and secretory cells). Differentiated goblet cells occur throughout the crypts, also intermixed with transient amplifying cells.

[0351] Isolation of tissue fragments and stem cells

[0352] Crypts can be separated from the small intestine and large intestine, including the duodenum, jejunum, ileum and colon, and the pyloric and body regions of the stomach (according to protocols known to technicians). For example, crypts can be separated by incubating the separated tissue with a chelating agent, which releases cells by its calcium- and magnesium-dependent interactions with basement membranes and matrix types. After washing the tissue, the epithelial cell layer is scraped off from the submucosal layer with a slide and chopped. Afterwards, incubate in trypsin or more preferably in EDTA and / or EGTA, and use, for example, filtration and / or centrifugation steps to separate undigested tissue fragments and single cells from the crypts. Other proteolytic enzymes, such as collagenase and / or dispase I can be used to replace trypsin. Similar methods are used to separate fragments of pancreas and stomach. Similar methods can be used to separate fragments of other tissues described herein. The culture medium of the present invention is suitable for culturing such tissue fragments (see Example 1).

[0353] The culture medium of the present invention allows the establishment of long-term culture conditions under which a single crypt undergoes multiple crypt fission events while simultaneously producing a villus-like epithelial domain in which all differentiated cell types are present. The cultured crypts undergo significant morphological changes after they are introduced into the culture. The upper opening of the newly separated crypt is sealed and the region gradually expands and is filled with apoptotic cells, much like apoptotic cells being pinched off at the tip of the villi. The crypt region undergoes continuous budding events that produce additional crypts, i.e., a process indicating crypt fission. In a preferred embodiment of the present invention, the organoid includes a crypt-like expansion that includes all differentiated epithelial cell types, including proliferating cells, Paneth cells, intestinal epithelial cells, and goblet cells. Confirm the absence of myofibroblasts or other non-epithelial cells at any stage of the organoid.

[0354] The expansion of the budding crypt structure generates an organoid that includes a crypt-like structure surrounding a central cavity lined with villous epithelium and filled with apoptotic cell bodies. Crypt-villous organoids include a central cavity lined with villous epithelium. The cavity is opened at serial time intervals to release the contents into the culture medium.

[0355] Similar crypt-villus organoid structures were formed when single epithelial stem cells were cultured. After about a week, structures very similar to those obtained with intact crypts were formed.

[0356] Methods for isolating stem cells are known, and the skilled person can select a suitable method for use in the present invention depending on the type of stem cell used. For example, compounds that bind to Lgr5 and / or Lgr6 (which are unique cell surface markers on epithelial stem cells) can be used to isolate epithelial stem cells. Examples of such compounds are anti-Lgr5 and anti-Lgr6 antibodies.

[0357] In some embodiments of the invention, a single Lgr5+ epithelial stem cell (eg, from the colon, small intestine, or pancreas) can be used to form organoids, eg, colon, crypt-villus, or pancreatic organoids, respectively.

[0358] In other examples, a single Lgr5+ epithelial stem cell from the liver, prostate, or stomach can be used to obtain organoids, such as liver, prostate, or stomach organoids, respectively.

[0359] In other embodiments, tissue fragments comprising Lgr5+ stem cells, such as cultured crypts from the intestine, can be used to obtain organoids using the methods and culture media described herein.

[0360] In some embodiments, the single Lgr5+ epithelial stem cell or tissue fragment may be a cancer stem cell or cancer tissue fragment, such as from a carcinoma or adenocarcinoma. In some embodiments, the single Lgr5+ epithelial stem cell may be a stem cell or tissue fragment from a tumor pathology or lesion tissue, such as Barrett's esophagus, cystic fibrosis or adenoma. Organoids obtained from cancerous, tumor or lesion starting materials have similar properties to in vivo starting materials and can therefore be used as research tools for drug screening, target confirmation, target discovery, toxicology and toxicology screening, personalized medicine, regenerative medicine, and ex vivo cell / organ models, such as disease models. In one embodiment, the present invention provides organoids 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 crypt-villus organoids or gastric organoids or pancreatic organoids or colon organoids or Barrett's esophagus organoids or adenocarcinoma organoids or colon cancer organoids 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 prostate organoids produced or obtained by culturing human stem cells or tissue fragments according to the methods of the present invention. Such organoid groups, such as crypt-villus, gastric or pancreatic organoids, produced or obtained by culturing human stem cells or tissue fragments according to the method of the present invention may each include more than 10, preferably more than 20, more preferably more than 40 organoids. The collection of organoids preferably includes at least 10% of living cells, more preferably at least 20% of living cells, more preferably at least 50% of living cells, more preferably at least 60% of living cells, more preferably at least 70% of living cells, more preferably at least 80% of living cells, more preferably at least 90% of living cells. Hoechst staining or propidium iodide staining can be used in FACS to evaluate the activity of cells.

[0361] The inventors have shown that the culture medium and methods 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, the culture technique can be widely used as a research tool for infectious, inflammatory and tumor pathologies. Therefore, the stem cells of the present invention can be cancer stem cells. In some embodiments of the present invention, the cancer stem cells can form adenocarcinoma or colon cancer organoids. In some embodiments, the cells included in these organoids are Ki67+ (Thermo Scientific*Cellomics, Millipore).

[0362] Likewise, the inventors have shown that the culture medium and methods of the present invention can be used to culture stem cells with other diseased genotypes and / or phenotypes. For example, the culture medium and methods of the present invention can be used to expand intestinal stem cells collected from patients with cystic fibrosis. These stem cells retain the cystic fibrosis genotype and phenotype. Thus, in some embodiments of the invention, stem cells are collected from a patient suffering from 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, alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome, anemia, Schwachman-Bodian-Diamond syndrome, polycythemia vera, primary myelofibrosis, glycogen storage disease, familial hypercholesterolemia, Crigler-Najjar syndrome, hereditary tyrosinemia, Pompe's disease, progressive familial cholestasis, Hurler syndrome, SCID or leaky SCID, Omenn syndrome, chondro-hair dysplasia, herpes simplex encephalitis, scleroderma, osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, dyskeratosis congenita, and the like. In some embodiments of the invention, diseased organoids can be obtained by culturing stem cells collected from a person or animal with a disease. Diseased organoids still have the characteristics of the tissue from which they were obtained. Thus, cystic fibrosis small intestinal organoids grown from small intestinal crypts are within the definition of small intestinal organoids. Similarly, colon cancer organoids are within the definition of colon organoids.

[0363] There is some confusion in the literature about the definition of cancer stem cells. Here, we follow the consensus reached by the recent AACR seminar (Clarke et al., 2006. Cancer Res. 66: 9339-44), which shows that cancer stem cells "are cells of heterogeneous lineages that are in tumors with self-renewal ability and produce cancer cells including the tumor". Therefore, cancer stem cells can be experimentally defined only by their ability to reproduce the production of continuously growing tumors. Alternative terms in the literature include tumor initiating cells and tumorigenic cells. Tests for cancer stem cell activity need to address the potential for self-renewal and the potential for tumor proliferation. The gold standard test is currently continuous xenotransplantation into immunodeficient mice. In addition, cancer stem cells in the context of the present invention generally express Lgr5. However, in some embodiments, cancer initiation / proliferation / stem cells that do not express Lgr5 can also be cultivated by the culture medium and methods of the present invention.

[0364] Genomic and phenotypic integrity of stem cells and organoids comprising them

[0365] The clinical and research applications for stem cells and their differentiated offspring require reproducible stem cell culture methods that provide cell groups of suitable quality. Generally, the in vitro expansion of stem cells is intended to provide a cell group that is as similar as possible to its in vivo counterpart. This property is referred to herein as the "genomic and phenotypic integrity" of the cell. The organoids obtained by culturing diseased cells, such as cancer cells or cystic fibrosis cells, are also similar to their in vivo counterparts, that is, they maintain their diseased genotypes and / or phenotypes, and therefore also maintain their "genotype and phenotypic integrity", meaning that they maintain the genetic or phenotypic instability characteristics of the disease indicating the in vivo situation. Therefore, in some embodiments, the present invention provides "normal" organoids obtained from healthy tissue. In other embodiments, the present invention provides "disease" organoids obtained from diseased tissue, such as cancer organoids (e.g., colon cancer organoids or adenocarcinoma organoids) or cystic fibrosis small intestine organoids.

[0366] The inventors have discovered for the first time that human epithelial stem cells can be expanded 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 with minimal loss of genotypic and phenotypic integrity (see Example 1). Under the improved culture conditions of the present invention, human intestinal organoids exhibit budding organoid structures rather than cystic structures seen under previous culture conditions. In each of 20 cells collected from three different donors, metaphase divisions of organoids older than 3 months consistently showed 46 chromosomes. In addition, microarray analysis showed that the stem cells in culture had molecular signatures similar to intestinal crypt cells, including intestinal stem cell genes.

[0367] Thus, in some embodiments, the invention provides organoids that are grown 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 minimal loss of genomic and phenotypic integrity.

[0368] In some embodiments, the present invention provides human intestinal organoids comprising 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 also exemplified that the human intestinal organoids produced by the culture medium and methods of the present invention mimic the final cell type determination in vivo in response to external factors. For example, it has been previously shown that Notch inhibition in intestinal stem cells terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. The inventors also showed that the intestinal organoids of the present invention interrupted proliferation when treated with Notch inhibitors and most of the cells were transformed into goblet cells within 3 days.

[0369] These results demonstrate that the methods and culture media of the present invention produce significant improvements in the genomic and phenotypic integrity of stem cells and organoids compared to previous methods and culture media.

[0370] The genomic integrity of the stem cells of the present invention can be confirmed by karyotyping. Stem cells and their progeny can be genomic typed using known methods described in Sato, T et al., Single Lgr5 stem cells build crypt-villus structures in vitro without amesenchymal niche. Nature 459, 262-265, 2009.

[0371] A "normal karyotype" is one in which all chromosomes are present (i.e., euploidy) and there are no significant changes. 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 stem cells and differentiated cells in an expanded population exhibit a normal karyotype after 1, 2, 3, 4, 5, 6, 9, 12 months or more. The term "expanded population" includes organoids.

[0372] "Normal phenotype" refers to cells that exhibit approximately the same visual characteristics, gene expression, and behavior as the average in vivo counterpart. Thus, in preferred embodiments 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 an expanded population cultured according to the invention exhibit a normal phenotype after 1, 2, 3, 4, 5, 6, 9, 12 months or more.

[0373] For example, a visually normal phenotype can be judged by the number of dead cells on the outside of the organoid, the amount of "budding" of the organoid compared to cystic growth (budding structures are preferred), and the overall integrity of the epithelial monolayer (e.g., columnar squamous phenotype). In addition, the cell types present can help judge whether an organoid is visually "normal."

[0374] Preferred properties of the stem cells and organoids of the invention are summarized below.

[0375] Stem cell markers

[0376] When mouse genes are mentioned herein, human organs of the present invention may have similar gene profiles, but with human gene counterparts replacing mouse genes. Thus, the present invention also provides human organs having gene expression profiles as described herein but involving corresponding human genes. A skilled person will readily obtain the human counterparts of the mouse genes listed herein.

[0377] In one embodiment, the invention provides a population of adult stem cells characterized by natural expression of Lgr5. In a preferred embodiment, the invention provides a population of adult stem cells characterized by natural 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) stem cell markers selected 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. Stem cell markers can be tissue specific. For example, pancreatic stem cells or organoids can be characterized by significant levels of natural expression of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, such as 1, 2, 3, or 4) of the following: CK19, nestin, somatostatin, insulin, glucagon, CXCR4 + , Ngn3, Pdx1, NeuroD, Nkx2.2, Nkx6.1, Pax6, Mafa, Hnf1b, optionally Tnfrsf19; gastric organoids can be characterized by significant levels of native expression of one or more (e.g., 1, 2, 3, or 4) of the following: CD133 +, DCAMKL-1, CD44, optionally Tnfrsf19; and the crypt-villus organoids can be characterized by significant levels of expression of one or more or all (e.g., 1 or 2) of the following: Sord and / or Prss23, or by expression / Fig.14 For example, all genes with significant levels of expression were characterized.

[0378] The term "significant level" as used herein in the context of marker expression is used synonymously with the term "detectable level" described below.

[0379] The small intestine and stomach organoid cell populations also express markers of common ancestral populations of 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 of the present invention. The cells of this aspect of the present invention can also upregulate Wnt target genes, including, for example, one, two or all of MMP7, Sp5, Tnfrs19 and Axin 2. This provides strong evidence of the need for an active and robust typical Wnt signaling capability to maintain the self-renewal capacity of these cultures.

[0380] The inventors have observed that expression of "stem cell" genes is present at significantly higher levels in early organoids than in differentiated cells, which become descendants of these stem cells. For example, the genes LGR5, LGR4, Epcam, CD44, Tnfrsf19, Sox9, Cd24a, Sp5, Prom1 / CD133, Cdca7 are preferably expressed in the organoids of the present invention, but are preferably significantly downregulated when pancreatic, liver, small intestine and colon organoids differentiate. In addition, the genes RNF43 and ZNRF3 are preferably expressed in the organoids of the present invention.

[0381] "Natural expression" means that the cell has not been recombinantly processed in any way, that is, the cell is not artificially guided to express these markers or regulate the expression of these markers by introducing exogenous genetic material, such as introducing a heterogeneous (non-natural) or stronger promoter or other regulatory sequence operably linked to an endogenous gene or an exogenous introduction form of the gene. Natural expression comes from the genomic DNA in the cell, including introns between the exons of the coding sequence (when these exist). Natural expression does not come from cDNA. Natural expression can be confirmed by any of a variety of methods if necessary, such as sequencing within the reading frame of the gene to check for the absence of exogenous heterogeneous sequences. "Adult" means post-embryonic. For the stem cells of the present invention, the term "adult stem cell" means that the stem cells are separated from the tissues or organs of the animal at a later growth stage than the embryonic stage.

[0382] This stem cell population is also characterized by the absence of any significant level of natural expression of certain markers, many of which are associated with cell differentiation. Specifically, the cells of the isolated adult stem cell population do not naturally express one or more of the following at significant levels: Cd11b, CD13, CD14, AFP, Pdx1, any CYP member (e.g., CYP3A11, CYP11A1). As defined herein, these markers are referred to as negative markers.

[0383] Detection of markers and isolation of cells

[0384] The term "expressed" is used to describe the presence of a marker within a cell. In order to be considered expressed, a marker must be present at a detectable level. "Detectable level" means that the marker can be detected using one of the standard laboratory methods such as PCR, blotting or FACS analysis. If expression can be reasonably detected after 30 PCR cycles (equivalent to an expression level of about 100 copies / cell in the cell), the gene is considered to be expressed by the cells of the group of the present invention. The terms "express" and "expression" have consistent meanings. When the expression level is below this threshold, the marker is considered not expressed. Comparison between the expression level of a marker in the cells of the present invention and the expression level of the same marker in another cell (e.g., embryonic stem cells) can preferably be performed by comparing two cell types isolated from the same species. Preferably, the species is a mammal, and more preferably, the species is human. Such a comparison can be conveniently performed using a reverse transcriptase-polymerase chain reaction (RT-PCR) experiment.

[0385] In some embodiments, a cell population or organoid of the 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 of the invention exhibit expression of the marker.

[0386] In some embodiments, if the cell comprises 1×10 2 Up to 1×10 5 , for example 5×10 2 Up to 1×10 4 or 1×10 3 Up to 1×10 4 If the number of mRNA copies encoding a cell marker is more than 100 times, the cell expresses the cell marker at a significant level.

[0387] In some embodiments, expression of a gene in an organoid or cell of the invention cultured in expansion medium is several fold higher (e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold) compared to an organoid or cell cultured in differentiation medium or in fully differentiated adult tissue. In some embodiments, a cell or organoid of the invention, when cultured under differentiation conditions, exhibits an increase in expression of genes (which are known to be differentiation genes) compared to a cell or organoid of the invention cultured under expansion conditions, and also exhibits a decrease in the expression level of at least one or more stem cell / progenitor genes compared to a cell or organoid of the invention cultured in expansion medium.

[0388] Any of the many physical separation methods known in the art can be used to select the cells of this aspect of the invention and distinguish them from other cell types. Such physical methods can include FACS and various immune-affinity methods based on markers specifically expressed by the cells of the invention. As described above, Lgr5, CD44 and Sox9 are 3 cell markers expressed at high levels in the stem cells of the invention. Therefore, by way of example only, the stem cells of the invention can be separated by many physical separation methods, which rely on the presence of these markers.

[0389] In one embodiment, cells of the invention can be isolated by FACS using, for example, an antibody against one of these markers. Fluorescence activated cell sorting (FACS) can be used to detect markers characteristic of a specific cell type or lineage. As will be apparent to one skilled in the art, this can be accomplished by fluorescently labeled antibodies, or by fluorescently labeled secondary antibodies that have binding specificity to the primary antibody. Examples of suitable fluorescent labels include, but are not limited to, FITC, Alexa Fluor ... 488, GFP, CFSE, CFDA-SE, DyLight 488, PE, PerCP, PE-Alexa 700、PE-Cy5(TRI- ), PE-Cy5.5, PI, PE-Alexa 750 and PE-Cy7. This list is provided by way of example only and is not intended to be limiting.

[0390] It will be apparent to one skilled in the art that FACS analysis using an anti-Lgr5 antibody will provide a purified stem cell population. However, in some embodiments, it may be preferred to further purify the cell population by performing another FACS analysis using one or more of the other identifiable markers.

[0391] Immunohistochemistry is also used to understand the distribution and location of biomarkers and differentiated expressed proteins in different parts of cell populations or organoids. Visualization of antibody-antigen interactions can be achieved in many ways, which are well known in the art, such as those 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.

[0392] In another embodiment, the cells of the present invention can be separated by immuno-affinity purification, which is a separation method known in the art. By way of example only, the cells of the present invention can be separated by immuno-affinity purification for c-kit. As will be apparent to those skilled in the art, this method relies on the fixation of antibodies on purification columns. Then, the cell sample is loaded onto the column so that suitable cells are bound by antibodies and thus bound to the column. After the washing step, the cells are eluted from the column using a competitor that preferentially binds to the fixed anti-c-kit antibody and allows the cells to be released from the column. It will be apparent to those skilled in the art that immuno-affinity purification using fixed antibodies will provide a purified cell population. However, in some embodiments, it may be preferred to further purify the cell population by performing another immuno-affinity purification using one or more of the other identifiable markers, and to determine the expression of other relevant intracellular markers using aliquots of the isolated clones.

[0393] It will be apparent to those skilled in the art that LGR5 or stem cell purification may be followed by a number of purification steps, such as purification of epithelium using methods known in the art, such as EDTA purification or Epcam FACS sorting of epithelium.

[0394] It will be apparent to one skilled in the art that successive purification steps do not necessarily need to involve the same physical separation method. Thus, it is apparent that, for example, cells can be purified by a FACS step using an anti-Lgr5 antibody followed by an immuno-affinity purification step using an SSEA-1 affinity column. In certain embodiments, the cells can 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 separation. In certain aspects, the cells are expanded in culture for a longer period of time to increase the homogeneity of the cell phenotype in the cell population.

[0395] Microarray analysis, cluster analysis and comparative gene expression profiles can be used to compare population phenotypes with those of the original mother cells or appropriate in vivo counterparts (Sato T et al., Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469 415-418).

[0396] Lineage tracing of Lgr5+ stem cells reveals retention of crypt-villus identity in organoids.

[0397] In another embodiment, high content analysis can be used to assess the phenotypic integrity of the stem cells of the present invention. For example, there are many high-content screening kits and platforms, such as the spot scanning 4-color ImageXpress ULTRA (Molecular Devices, Union City, USA), BD Biosciences' vortex disk (Nipkow disk) Pathway 855 and 435 (formerly Atto Biosciences, Rockville, Maryland), Opera (PerkinElmer Inc., Waltham, MA) and 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 (ScanalyzerLemnaTec, Aachen Germany) and ImageXpress MICRO (Molecular Devices, Union City, USA), IN Cell 1000 (GE / Amersham Biosciences Piscataway, New Jersey, USA), PathwayHT (Becton Dickinson Biosciences) and ImageXpress MICRO (Molecular Devices, Union City, USA), Scan^R (Olympus Soft Imaging Solutions, Germany).

[0398] Coating density

[0399] In some embodiments of the invention, single cell suspensions or small clusters of cells (2-50 cells / cluster) are typically inoculated rather than large clusters of cells, as is known in the art. As they divide, such cells are inoculated onto a support at a density that promotes cell proliferation. Typically, when single cells are isolated, the plating density used is at least 1-500 cells / well with a well surface of 0.32 cm 2 When seeding clusters, the plating density is preferably 250-2500 cells / cm 2 For replating, a density of approximately 2500 cells / cm2 can be used. 2 to approximately 5,000 cells / cm 2 During replating, single cell suspensions or small clusters of cells are typically plated rather than large clusters of cells, as is known in the art.

[0400] Further differentiation

[0401] In some embodiments of the invention, certain components of the expansion medium may be withdrawn to change the final cell type of the cultured cells toward differentiation.Any component of the medium used to maintain the undifferentiated state and / or activate stem cells or histological programs may be withdrawn from the culture medium.

[0402] In some embodiments of the invention, withdrawal of the inhibitors of the invention can allow the cells of the organoids to differentiate into mature cells, such as mature goblet cells and enteroendocrine cells in crypt-villus organoids. Thus, in some embodiments, the invention provides methods for further differentiating organoids using a second culture medium that does not include an inhibitor of the invention. For example, see Example 1.

[0403] For example, in some embodiments, the TGF-β inhibitor and / or p38 inhibitor is withdrawn from the cell culture medium to allow the cells to differentiate. "Withdrawn" or "withdrawal" of a component from a cell culture medium means that when the cells are re-plated and the culture medium is changed, the component is not added to the fresh culture medium.

[0404] In some embodiments, Wnt is present in the expansion medium but not in the differentiation medium. For example, some embodiments include withdrawal of Wnt for differentiation of colon organoids into mature intestinal epithelial cells. Wnt can also be withdrawn to differentiate crypt-villus organoids.

[0405] In some embodiments, Rspondin is present in the expansion medium but not in the differentiation medium. For example, some embodiments include withdrawal of Rspondin for differentiation of colon organoids into mature intestinal epithelial cells. Rspondin can also be withdrawn to differentiate crypt-villus organoids. In some embodiments, Rspondin and Wnt can be withdrawn to differentiate crypt-villus organoids.

[0406] 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 withdrawn from the cell culture medium to differentiate the cells into, for example, crypt-villus organoids or colonic organoids.

[0407] Thus, a method of obtaining differentiated cells or organoids may include culturing epithelial cells in a culture method of the present invention that includes TGF-β and / or a p38 inhibitor to allow cell survival and / or proliferation (i.e., expansion medium), and then continuing to culture the cells and supplementing the culture medium, wherein the supplemented culture medium does not include TGF-β and / or a p38 inhibitor (i.e., differentiation medium).

[0408] In some embodiments, the differentiation medium includes additional components. For example, in some embodiments, the differentiation medium includes a γ secretase inhibitor, such as DAPT or DBZ. In some embodiments, the differentiation medium includes a RANK ligand (also referred to herein as RANKL). As described above, the addition of a γ secretase inhibitor can guide the differentiation of intestinal organoid cells, such as small intestinal organoid cells, toward secretory cells, such as goblet cells. Adding RANKL to the culture medium can guide the differentiation of intestinal organoid cells, such as small intestinal organoid cells, toward M cells.

[0409] In some embodiments, the present invention provides a culture medium for differentiating stem cells from a target tissue, wherein the culture medium comprises or consists of components of a culture medium for expanding stem cells from a target tissue type, but wherein the culture medium for differentiating stem cells does not include one or more of the following: Wnt, Rspondin, BMP inhibitors, TGF-β inhibitors, receptor tyrosine kinase ligands, p38 inhibitors and nicotinamide.

[0410] In addition, the present invention provides a method for expanding single stem cells, stem cell populations or tissue fragments, preferably to produce organoids, wherein the method comprises:

[0411] Culturing stem cells, stem cell populations, or tissue fragments in a first expansion medium;

[0412] The stem cells, stem cell populations or tissue fragments are continued to be cultured and the culture medium is supplemented with a differentiation medium, wherein the differentiation medium does not include one or more factors, preferably does not include all of the factors selected from the group consisting of: TGF-β inhibitors, p38 inhibitors, nicotinamide and Wnt.

[0413] Typically, when a component is described as being "removed" from a culture medium, it means that the component is not added when the culture medium is supplemented, i.e., the supplemented culture medium does not include the component. When the culture medium is "supplemented", it may mean that the culture medium is physically removed from the extracellular matrix and then replaced with fresh culture medium.

[0414] For colon, liver and pancreas, there are very few differentiated cells in the expansion medium. Only when the expansion medium is supplemented with differentiation medium, the cells begin to differentiate. At this stage, the organoid also begins to lose its stem cells. Differentiated organoids may be suitable for certain uses, such as, but not limited to, transplantation, drug screening for metabolic diseases, toxicology (such as using liver organoids including hepatocytes), and for studying the antibacterial function of the small intestine. Amplified organoids may generally be more suitable for other uses, such as, but not limited to, regenerative medicine and drug screening, such as for cancer or cystic fibrosis. Amplified organoids generally have a greater growth potential (therefore, longer life) than differentiated organoids. In some embodiments, colon, liver and pancreatic organoids are not further differentiated.

[0415] Small intestine and prostate organoids are different from colon, liver and pancreatic organoids because they maintain amplified stem cell groups and also differentiate at the same time. They do not need to be cultured in a separate differentiation medium so that differentiated cell types exist. They can be considered to have the properties of amplified and differentiated organoids. However, in order to achieve complete differentiation of small intestine organoids, they can be cultured in a separate differentiation medium, and the differentiation medium preferably does not include Wnt3a and preferably includes a gamma secretase inhibitor and / or RANK ligand (also referred to herein as RANKL). "Complete" differentiation means that there are all differentiated cell types, including goblet cells, neuroendocrine cells, clump cells, M-cells, intestinal epithelial cells and Paneth cells. Some of these differentiated cell types, such as Paneth cells, are also present (sometimes in a smaller amount) in the amplified organoids.

[0416] Organoids

[0417] The above cells grow into organoids. Therefore, the organoids obtained by the method of the present invention are another aspect of the present invention. Organoids as described herein are also provided. Organoids are preferably human organs. To the best of our knowledge, the following is the first time that the human organs obtained are functional and alive after an extended period of time (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 embodiments included herein). Functionality is preferably characterized by the presence of tissue-specific markers and / or by the structure of the organoids defined herein. Because the final amount of the organoids obtained is related to the duration, the technician will understand that the present invention is a groundbreaking invention and may open up new development possibilities in, for example, regenerative medicine. Therefore, organoids as described herein are provided, which are functional and alive after at least 3 months (e.g., at least 4, 5, 6, 7, 8 months or more) of culture. For example, provided are organoids described herein that maintain 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, 5, 6, 7, 8 months, or more) of culture.

[0418] For example, an organoid of the invention may include a population of cells that is at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells or more. Each organoid contains about 1×10 3 cells to 5×10 3 The inventors have shown that organoids can be grown from a single Lgr5+ stem cell to include a population of cells as described above or to include about 10 4 In some embodiments, the organoid comprises about 10 4 In some embodiments, 10-20, or 20-30, or 30-40, or 40-50 organoids can be grown together in one well of a 24-well plate.

[0419] In some embodiments, the invention provides organoids or cell populations that, when cultured in the culture medium of the invention, can persist in culture for at least 3 months, e.g., 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.

[0420] In some embodiments, the invention provides organoids or cell populations, wherein the organoids or cell populations expand 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.

[0421] Preferably, the cell population or organoid will expand at a rate of about 4-5 times per week or more than 2 times per week. Thus, in some embodiments, the cell population or organoid expands 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.

[0422] The organoids of the present invention can be obtained using cells isolated from any suitable source. Typically, the cells used to generate the organoid will be isolated from the same tissue type from which the organoid was generated. The organoid is preferably mammalian, e.g., murine, bovine, porcine or human. Most preferably, the organoid is human.

[0423] In some embodiments, the present invention provides organoids or cell populations, wherein the organoids or cell populations are normal (healthy) organoids or cell populations or diseased organoids or cell populations, such as organoids or cell populations obtained by culturing stem cells collected from a human or animal suffering from a disease.

[0424] In some embodiments, the invention provides organoids or cell populations that are frozen and stored at below -5°C, below -10°C, below -20°C, below -40°C, below -60°C, or below -80°C, below -100°C, below -150°C, or about -180°C. The organoids or cell populations of the invention can be stored in liquid nitrogen. Thus, in some embodiments, the invention provides organoids or cell populations stored in liquid nitrogen.

[0425] In some embodiments, the present invention provides an organoid of the present invention, wherein the organoid is an intestinal organoid, a colon organoid, a stomach organoid, a pancreatic organoid, a liver organoid, or a prostate organoid.

[0426] Organoid structure and morphology

[0427] The organoids of the present invention obtained by expanding stem cells provide a cell population similar to their in vivo counterparts.

[0428] Imaging analysis can be used to evaluate the characteristics of cells in culture, such as cell morphology; cell structure; evidence of apoptosis or cell lysis; and organoid composition and structure. Many types of imaging analysis are known in the art, such as electron microscopy, confocal microscopy, stereomicroscopy, fluorescence microscopy. Histological analysis can reveal basic architecture and cell types.

[0429] Illustrative examples of organoids produced according to the present invention are given in the accompanying drawings. It can be seen that the organoids of the present invention may have a layer of cells with at least one bud and a central cavity. Organoids outside the matrix gel tend to be larger than those in the middle of the matrix gel, probably due to their better access to necessary growth factors. Structurally, the organoids of the present invention are generally elongated in shape. They may include one or more budding structures-a single-cell epithelial layer similar to a duct or an island. Under a confocal microscope, the structure may stain positive for keratin. They may include cells with polarized nuclei and small cytoplasm. Organoids may have sections formed by multiple layers; such cells often tend to have their nuclei more centrally in the cell, i.e., unpolarized. Cells in the multilayered sections may self-organize to include gaps or cavities between cells. In some embodiments, the organoids of the present invention include or consist of epithelial cells. In some embodiments, the organoids include or consist of a single layer of epithelial cells. In some embodiments, non-epithelial cells are absent from the organoids. In some embodiments, the organoids of the present invention include all differentiated cell types present in their corresponding in vivo tissue counterparts.

[0430] In some embodiments, human intestinal organoids exhibited budding organoid structures rather than cystic structures seen in previous culture conditions. Metaphases of organoids older than 3 months consistently displayed 46 chromosomes in each of 20 cells collected from three different donors.

[0431] In some embodiments, the organoids of the present invention include cells of a single monolayer. In some embodiments, the organoids of the present invention have portions formed by multiple layers. Multilayered cells are also referred to herein as "stratified" cell regions. "Stratified" means that there are multiple layers (more than one layer) of cells. In some embodiments, the organoids of the present invention include a single monolayer that is folded (or infolded) to form two or more layers. Sometimes it may be difficult to distinguish between a folded (or infolded) monolayer and a stratified cell region. In some embodiments, the organoids include stratified cell regions and folded monolayer regions. In some embodiments, the organoids of the present invention have portions formed by multiple layers and portions including a single monolayer of cells. Morphologically, the cells appear similar to their corresponding in vivo tissue counterparts.

[0432] Therefore, in some embodiments, the present invention also provides organoids, preferably organoids obtained using the culture medium and method of the present invention, which are three-dimensional organoids including epithelial cells surrounding a central cavity, wherein optionally, the epithelial cells are present in different division domains and differentiation domains. In some embodiments, the organoids of the present invention are three-dimensional organoids including epithelial cells arranged in a monolayer (optionally a folded monolayer) region and a stratified cell region. In some embodiments, the organoids are free of non-epithelial cells. In some embodiments, all differentiated cell types of normal in vivo tissues are present in the organoids.

[0433] Crypt-villus organoids

[0434] In the small intestinal crypt-villus organoids, the structural arrangement of the organoids is very similar to the structure of the crypt-villus in vivo: Lgr5+ stem cells and their niche cells (Paneth cells) are next to each other at the bottom of the crypt, followed by transient expansion cells, which are just above the bottom of the crypt and introduced into the sides of the villus, and finally differentiated cells, such as intestinal epithelial cells that make up the rest of the villus and become increasingly differentiated as they move toward the top of the villus. It can be seen that the organoids of the present invention can have a layer of cells with at least one bud and a central cavity. Organoids outside the matrix gel tend to be larger than those in the middle of the matrix gel, probably due to their better access to essential growth factors. Structurally, the shape of the organoids of the present invention is generally elongated. Under confocal microscopy, the structure can stain positive for keratin. They can include cells with polarized nuclei and small cytoplasm. Crypt-villus organoids are typically single-layered.

[0435] In some embodiments, for example, for mouse crypt-villus organoids, crypt-villus organoids are three-dimensional organoids that include crypt-like domains surrounding a central lumen lined by a villus-like epithelial domain, which is an epithelial domain that includes differentiated cell types. In some embodiments, the organoids are devoid of non-epithelial cells.

[0436] In some embodiments, such as for human crypt-villus organoids, crypt-villus organoids are three-dimensional organoids that include crypt-like domains surrounding a central cavity. In some embodiments, dividing cells are confined to budding structures. There are no or few differentiated cells. Under differentiation conditions, differentiated cells of the intestine are formed. In some embodiments, the organoids are devoid of non-epithelial cells. In some embodiments, when the organoids are expanded, such as when they are in the expansion medium of the present invention, the organoids have few or no differentiated cells.

[0437] In some embodiments, the small intestinal organoids of the present invention cultured in the culture medium of the present invention including RANKL include 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 including a γ-secretase inhibitor include goblet cells. In some embodiments, the small intestinal organoids cultured in a differentiation medium (e.g., wherein the differentiation medium includes a basal medium, Noggin, EGF, a TGF-β inhibitor and a p38 inhibitor, a γ-secretase inhibitor and RANKL) include all differentiated cell types, for example, goblet cells, neuroendocrine cells, plexus cells, M-cells, intestinal epithelial cells and Paneth cells. Some of these differentiated cell types, such as Paneth cells, are also present (sometimes in smaller amounts) in the expanded organoids.

[0438] Human intestinal organoids exhibit budding organoid structures rather than the cystic structures seen under previous culture conditions. The upper opening of the newly isolated crypts is sealed and the area gradually expands and fills with apoptotic cells, much like apoptotic cells are pinched off at the tip of the villi. Thus, in some embodiments, the crypt-villus organoids have a crypt-like structure surrounding a central cavity lined with villus-like epithelium and filled with apoptotic cell bodies. In some embodiments, the cavity is opened at continuous time intervals to release the contents into the culture medium.

[0439] In some embodiments, a crypt region undergoes continuous budding events that produce additional crypts, ie, a process indicative of crypt fission.

[0440] The inventors also exemplified that human intestinal organoids produced by the culture medium and methods of the present invention mimic the final cell type determination in vivo in response to external factors. For example, it has been previously shown that Notch inhibition in intestinal stem cells terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. Therefore, in some embodiments, when the crypt-villus organoids of the present invention are treated with Notch inhibitors, proliferation stops, and within 3 days, most of the cells (e.g., more than 50%, more than 60%, for 70%, more than 80%, more than 90%, for 95%, more than 98%) are transformed into goblet cells.

[0441] In each of 20 cells collected from three different donors, metaphases in organoids older than 3 months consistently showed 46 chromosomes. In addition, microarray analysis showed that the stem cells in culture had a molecular signature similar to that of intestinal crypt cells, including intestinal stem cell genes.

[0442] Colon organoids

[0443] Colon organoids exhibit a similar cellular composition to crypt-villus organoids. Therefore, the above comments on crypt-villus organoids apply mutatis mutandis to colon organoids. For example, see Figures 1 and 2.

[0444] In general, the difference between colon and small intestinal organoids is that the crypts are shallower in the colon, making it look a bit like a "soccer ball" rather than a sphere with protrusions. Both small and colon organoids have domains that include stem cells and transiently expanded (TA) cells, as well as other domains that include differentiating and / or differentiated cells. For small intestinal organoids, the differentiation domain is sometimes called "villus-like". The differentiation domain of colon organoids is generally similar to the "villus-like" domain of the small intestine in terms of cell composition, but the colon itself does not have villi.

[0445] The amount of Wnt present can affect the size of the budding structures in the organoids (i.e., the depth of the crypts). More Wnt reduces budding. The colon produces more Wnt than the small intestine, so it requires less additional Wnt in the culture medium and generally has shallower crypts than the small intestine. The same differences were seen in the organoids.

[0446] In some aspects, the present invention provides colon organoids. The inventors have found that mouse colon organoids can be obtained by culturing colon crypts in ENR+Wnt3A (WENR) cell culture medium. Therefore, in some embodiments, the present invention provides colon organoids obtained by culturing colon crypts in WENR culture medium.

[0447] The inventors have also surprisingly discovered that a culture medium comprising WENR plus gastrin plus nicotinamide can be used to maintain human colon organoids. In some embodiments, the human colon organoids of the present invention are obtained by using a culture medium comprising WENR plus gastrin plus nicotinamide and also comprising a TGFβ inhibitor. For example, in some embodiments, the following cell culture medium can be used to obtain human colon organoids: WENR+gastrin+nicotinamide+A8301+SB202190. In other embodiments, the following cell culture medium can be used to obtain human colon organoids: WENR+nicotinamide+A83-01.

[0448] In some embodiments, the mouse colon organoid has a maximum diameter of about 200-700 μm, such as 250-600 μm, 300-500 μm, 320-450 μm, 340-400 μm, 300-380 μm, such as about 360 μm. In some embodiments, the colon organoid has a minimum diameter of about 100-400 μm, such as 150-350 μm, 170-300 μm, 190-280 μm, 195-250 μm, such as about 235 μm. In other embodiments, the organoid can have a diameter of up to 1 mm. In some embodiments, human colon organoids have a maximum diameter of about 300-800 μm, such as 350-700 μm, 400-600 μm, 450-550 μm, 475-540 μm, 500-530 μm, such as about 500 μm. In some embodiments, colon organoids have a minimum diameter of about 200-500 μm, such as 250-450 μm, 300-415 μm, 350-400 μm, 325-380 μm, such as about 375 μm. In other embodiments, organoids can have a diameter of up to 1 mm. In some embodiments, colon organoids of the present invention include budding structures. These become visible by using EdU staining to visualize proliferating cells.

[0449] Human colon organoids maintain their characteristic budding structures under human intestinal stem cell culture medium ("HISC") conditions (WENRg + nicotinamide + TGF-β inhibitor (e.g., A83-01) + p38 inhibitor (e.g., SB202190)). In some embodiments, colon organoids are three-dimensional organoids that include proliferative and stem cell-containing budding structures. These stem cell domains surround a central cavity. Dividing cells are typically confined to budding structures. In some embodiments, there are no or very few differentiated cells. Under differentiation conditions, differentiated cells of the intestine, such as mature intestinal epithelial cells, are formed. In some embodiments, the organoids are devoid of non-epithelial cells.

[0450] Pancreatic organoids

[0451] The pancreatic organoids of the present invention preferably exhibit budding. In some embodiments, the pancreatic organoids are 100-1000 microns in diameter, such as 200-900 microns, 300-1000 microns, 400-700 microns. The pancreatic organoids are preferably monolayer. Only very early island or duct structures are present. Budding structures indicate a healthy proliferation state and stem cell maintenance.

[0452] In some embodiments, for example, when pancreatic organoids are grown in the culture medium of the present invention (and in the absence of TGF-β inhibitors), pancreatic organoids are mainly cystic structures with few budding structures or ductal domains. The cystic structure mainly includes a single layer, but there may be some stratified cell areas. The cells express stem cell and progenitor (ductal) markers. There are no differentiated cells, such as β-cells, in the organoids. Cysts are mainly formed by a single layer, but there are stratified parts. The cell type is similar to stem cells / progenitor cells (ductal cell gene expression). There are no differentiated cells (β-cells).

[0453] In other embodiments, for example when pancreatic organoids are grown in the presence of a TGF-β inhibitor such as A83-01, for example in a culture medium of the invention, the pancreatic organoids include more budding structures / ductal domains (indicating that the cells are ductal cells rather than structures that resemble ducts), as shown by Krt19 staining (e.g., see Fig.31 ). Monolayers of polarized cells could be identified, but areas with differentiated cells were also identified.

[0454] Adenocarcinoma and Colon Cancer Organoids

[0455] Adenocarcinoma and colon cancer organoids often form cystic structures rather than budding structures. This indicates that there is no good cell niche support. Adenoma crypts cultured with EFG+Noggin showed about 16-fold expansion in the first 10 days. Adenoma (adenocarcinoma) and colon cancer organoids can provide useful research tools and drug screening models.

[0456] Carcinoma, adenoma, and adenocarcinoma organoids are primarily cystic (see, e.g., Figures 4 and 9). However, in some embodiments, they may also include structures similar to their normal tissue organoid counterparts.

[0457] Barrett's esophagus (BE) organoids

[0458] The BE organoids of the present invention include budding structures (eg, see FIG. 5 ).

[0459] Morphologically, cells in the organoids of the invention appear similar to their corresponding in vivo tissue counterparts.

[0460] Barrett's esophagus is a disease marked by the presence of columnar epithelium in the lower esophagus, which replaces normal squamous cell epithelium due to metaplasia. The histological feature of Barrett's esophagus is the presence of intestinal goblet cells in the esophagus. Exploiting the similarities between Barrett's esophagus and intestinal epithelium, the inventors have shown that the culture medium and methods 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 the addition of FGF10 to the culture medium of the present invention enables Barrett's esophagus organoids to form budding structures and significantly extend the duration of culture to more than 3 months. Therefore, Barrett's esophagus organoids are examples of organoids of the present invention. In some embodiments, Barrett's esophagus organoids have cystic structures. In some embodiments, Barrett's esophagus organoids of the present invention include Paneth cells. In some embodiments, Barrett's esophagus organoids of the present invention express lysozyme.

[0461] Therefore, the inventors also describe a culture medium of the present invention comprising FGF10 for culturing Barrett's esophagus epithelium.

[0462] In some embodiments of the invention, Barrett's esophagus organoids can be grown using a culture medium of the invention that also includes FGF10. In some embodiments, the Barrett's esophagus organoids express Ki67 and have a minimal number, such as less than 10%, less than 5%, or less than 1%, of PAS-positive cells and mucin-positive cells. In some embodiments, the Barrett's esophagus organoids include lysozyme-positive Paneth cells.

[0463] Gastric (Stomach) Organoids (See, for example, Figure 46)

[0464] Mouse gastric organoids cultured in the culture medium of the present invention are three-dimensional organoids that include or consist of a single layer of epithelium that includes a gastric gland basal-like domain (formed by stem and progenitor cells) surrounding a central lumen lined by an epithelial domain that includes differentiated cell types, and optionally, wherein the organoid is devoid of non-epithelial cells.

[0465] Human gastric organoids grown in the culture medium of the present invention include cystic structures. The cystic structures are a monolayer of polarized cells. These human gastric organoids grown in the presence of TGF-β are more similar to mouse gastric organoids than human organs grown in the absence of TGF-β.

[0466] Prostate organoids (see Figures 41 to 43 )

[0467] Under culture conditions including EGF, Noggin, and Rspondin, mouse prostate organoids form a three-dimensional cystic structure in the cavity. The layers fold inward in time to form 3-4 layers of (stratified) epithelial cells. The outer layer is mainly composed of CK5+ basic epithelial cells, while the inner layer is mainly composed of CK8+ luminal epithelial cells. It is determined that there is no stem cell compartment; all domains include dividing cells. Therefore, in some embodiments, prostate organoids grown in the absence of testosterone include stratified dividing epithelial cells. In other embodiments, prostate organoids include: an outer layer of cells including CK5+ basic epithelial cells; and an inner layer including CK8+ luminal epithelial cells. In some embodiments, prostate organoids grown in the absence of testosterone do not include any stem cells.

[0468] Adding testosterone to prostate culture medium

[0469] The inventor has shown that adding (dihydro) testosterone to the culture conditions for prostate organoids causes most of the cells to differentiate into CK8+ luminal cells, which form a single layer of epithelium, which is folded on itself to become two layers. The prostate organoids grown in the presence of testosterone are mainly composed of luminal cells with or without a second layer of basal cells. The structure is similar to the structure in vivo. There are cells that differentiate and divide, as well as stem cells and progenitor cells. Therefore, in some embodiments, for example, when cultured in a culture medium including testosterone, prostate organoids are three-dimensional organoids including cystic structures and chambers. In some embodiments, prostate organoids include CK8+ luminal cells that form a single layer of epithelium. In some embodiments, a single layer is folded into two or more layers. In other embodiments, organoids may include the region of stratified cells. In some embodiments, prostate organoids include differentiated cells while maintaining a differentiated cell population. In some embodiments, the shape of the organoid is determined by the source of the cell or tissue starting material (that is, the position in the prostate before separation). The prostate is composed of different leaves or regions showing different above-mentioned epithelial structures (stratification and folding). After in vitro culture, organoids can, to some extent, maintain the distinct macroscopic architecture (divided or folded) of the prostate portion from which they were derived.

[0470] Liver organoids

[0471] Structurally, the mouse liver organoids of the present invention are generally elongated in shape. They may include one or more budding structures, a single-cell epithelial layer with a different structure from the bile duct. Under a confocal microscope, the structure may stain positive for keratin. They may include cells with polarized nuclei and small cytoplasm. Organoids may have sections formed by multiple layers; such cells often tend to have their nuclei more centrally in the cell, i.e., unpolarized. Cells in the multilayered sections may self-organize to include gaps or cavities between cells. In some embodiments, the human liver organoids of the present invention have a roughly cystic structure.

[0472] In some embodiments, the liver organoid is a three-dimensional organoid with a sac-like structure (e.g., see Fig.30 ). Under expansion conditions, organoids can be composed of stem cells and progenitor cells, in which two domains are defined: (1) a ductal domain, which is formed by a single layer of cuboidal epithelium with a cell-lined central lumen (positive for the ductal marker Krt19); and (2) a pseudo-stratified epithelial domain, in which krt19-positive cells and scattered albumin-positive cells are detected. This structure (areas with a single layer of epithelium together with areas of pseudo-stratified epithelium) is similar to embryonic liver buds. Under expansion conditions, there are no fully differentiated cells, although in some embodiments, expression of hepatocyte / hepatoblast-specific markers can be detected. Differentiation conditions lead to the formation of cystic organoids, in which the ductal domain (single layer of epithelium) is lost and the entire structure becomes a pseudo-stratified epithelium including >50% polarized hepatocytes.

[0473] The liver organoid preferably includes hepatocytes and bile duct epithelial cells (although hepatocytes are particularly seen after differentiation in DM, and hepatocytes do not need to be expanded), more preferably, at least one or more of the following markers can be detected: at least one 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 bile duct epithelial cell marker, such as keratin 7 and 19. Technicians know how to detect each of these markers (i.e., RT-PCR and / or immunofluorescence). Preferably, the expression of each of these markers is evaluated as performed in the experimental part. Each of these markers is usually expressed after at least two weeks, three weeks or one month of cultivation using the method of the present invention. Microarray analysis of organoids in two culture conditions showed that liver organoids are similar to adult liver tissue.

[0474] Preferably, all cells in the liver organoid 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 organoid express hepatocyte markers. In some embodiments, about 35% of the cells in the liver organoid express hepatocyte surface markers, such as 25-45%, 30-40%, 33-37%, less than 35%, or 15-35% of the cells. In some embodiments, the expansion phase will have fewer stem cells, such as less than 20%, less than 10%, less than 5% of the cells, less than 2%, less than 1%, preferably 0% of the cells. Preferably, the cells and organoids produced according to the present invention also have hepatocyte function, such as expression or staining positive for mature liver markers: albumin, B-1 integrin, CK-8, CK-18, transthyretin (TTR), glucose 6P, Met, glutamine synthetase (Glul), transferrin, Fahd1, Fahd2a, K7, K19 and cytochrome P450 isoforms 3A13 (CYP3A13), 51 (CYP51) 2D10 (CYP2D10), 2j6 (CYP2j6), 39A1 (CYP39A1), 4A10 (CYP4A10), 4F13 (CYP4F13) 4F16 (CYP4F16), CYP4B1and 20A1 (CYP20A1). In addition, in some embodiments, the embryonic liver gene AFP is not detected in both culture conditions, as in adult liver. In some embodiments, expression of alpha-fetoprotein is just above background gene expression.

[0475] Furthermore, well-known liver transcription factors such as HNF1a, HNF1b, and HNF4a were highly expressed in both conditions.

[0476] Because the liver and pancreas are closely related organs, we investigated whether our liver cultures also expressed pancreas-specific genes. The pancreas is functionally divided into an endocrine pancreas and an exocrine pancreas. The endocrine pancreas is primarily characterized by the expression of insulin, glucagon, and somatostatin. The expression of these hormones is strictly regulated by a group of endocrine pancreas-specific transcription factors, the most important being Pdx1 and NeuroD. The exocrine pancreas is formed by an acinar compartment and a ductal compartment for the production of the digestive enzymes amylase, pancreatic lipase, and chymotrypsin, among others. The expression of these genes is also regulated by specific exocrine pancreatic genes such as Ptf1.

[0477] The pancreatic specific genes Ptf1a, pancreatic amylase (Amy2a4), pancreatic lipase (Pnlip), insulin (ins1 and ins2), glucagon (Gcg), chymotrypsin (celal), Pdx1 and NeuroD are absent in the liver culture medium described herein.

[0478] In some embodiments, one or more or all of the following genes are expressed in liver organoids at levels similar to the corresponding genes in adult liver hepatocytes: Aqpl, Bmp2, Apo3, Apol7a, Sord, C3, Ppara, Pparg, tbx3, lgfl, ll17rb, ll1b, Tgfbi, Apoa1, Apoa4, Apob, Cyp26bl, Cyp27a1, Cyp2bl3, Cyp2b9, Cyp2c37, Cyp2f2, Cyp2gl, Cyp2j13, Cyp3al11, Cyp4a10, and Cypf14. See, e.g., Fig.27A .

[0479] In some embodiments, one or more of the following genes are expressed in liver organoids at similar off levels compared to corresponding genes in adult liver hepatocytes: Ccl2, Osmr, Icam1, and Cxcl2.

[0480] In some embodiments, one or both of the following genes are differentially expressed in liver organoids and neonatal liver: mKi67 and cdkn3, indicating that these genes are more highly expressed in organoids than in differentiated organoids or in whole organs.

[0481] In some embodiments, one, two, or all of the following genes are expressed at similar levels in liver organoids and neonatal livers: cyp2j6, olfm4, and Lefty1. Fig.27B .

[0482] In some embodiments, the hepatic organoids of the invention have a ductal phenotype when cultured in an expansion medium of the invention (eg, EM1 or EM2).

[0483] In some embodiments, the hepatic organoids of the invention express adult liver markers when cultured in the differentiation medium of the invention.

[0484] In one embodiment, the liver organoids of the invention have Fig.27C Gene expression profiles are shown.

[0485] In a particularly preferred embodiment, the mouse liver cell population or organoid of the present invention has Fig.28For example, in a preferred embodiment, the mouse liver cell population or organoid of the present invention:

[0486] a) expressing 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

[0487] b) does not express the following stem cell marker: lgr6; and / or

[0488] 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 or biliary epithelial cell markers when grown in an expansion medium of the invention: Hnf1a, Hnf1b, Hnf4a, Hhex, Onecut1, Onecut2, Prox1, Cdh1, Foxa2, Gata6, Foxm1, Cebpa, Cebpb, Cebpd, Cebpg, Glu1, Krt7, Krt19, and Met; and / or

[0489] d) does not express at least one of the following genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) when grown in the expansion medium of the invention: afp, Ins1, Ins2, Gcg, Ptf1a, Cela1, Cela2a, Cela3b, Neurod1, Neurod2, Neurog1, Neurog2, Neurog3, Amy2a4, Igf1r, Igf2, and Cd34; and / or

[0490] e) expressing at least one (e.g., 1, 2, or 3) of the following reprogramming genes: Klf4, Myc, and Pou5f1; and / or

[0491] f) does not express the following reprogramming genes: Sox2,

[0492] Among them, it is preferred to monitor the expression of genes by measuring the expression at the mRNA level, for example, using a microarray.

[0493] More preferably, the mouse liver cell population or organoid of the present invention has all of the above characteristics a) to f).

[0494] In some embodiments, the gene expression profile of the mouse liver cell population or liver organoid of the present invention is used for the mouse cell population or organoid cultured in the liver expansion medium of the present invention.

[0495] In some embodiments, there is provided a human liver cell population or organoid of the invention having Fig.29 For example, a human liver cell population or organoid cultured in the EM1 of the present invention preferably expresses Fig.29 The genes referred to in are expressed in EM1 cell culture medium. For example, a human liver cell population or organoid cultured in EM2 of the present invention preferably expresses Fig.29 The genes referred to in are expressed in EM2 cell culture medium. For example, human liver cell populations or organoids cultured in the DM of the present invention preferably express Fig.29 The genes indicated in are expressed in DM cell culture medium.

[0496] For example, in a preferred embodiment, the human liver cell population or organoid of the present invention:

[0497] a) expressing at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9), preferably all, of the following stem cell marker genes: LGR4, TACSTD1 / Epcam, CD44, SOX9, SP5, CD24, PROM1, CDCA7, and ELF3; and / or

[0498] b) expressing at least one (e.g., 1, 2, 3, or 4) of the following reprogrammed genes: KLF4, MYC, POU5F1, and SOX2; and / or

[0499] c) expressing 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 / biliary epithelial cell-specific genes: HNF1A, HNF1B, HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA, CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19, and MET; and / or

[0500] 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, for example, it does not express NEUROG2, IGF1R and CD34; and / or

[0501] e) expressing 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 stem cell-specific genes: TTR, ALB, FAH, TAT, CYP3A7, APOA1, HMGCS1, PPARG, CYP2B6, CYP2C18, CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2, and SCARB1;

[0502] Among them, it is preferred to monitor the expression of genes by measuring the expression at the mRNA level, for example, using a microarray.

[0503] More preferably, the human liver cell population or organoid of the present invention has all of the above characteristics a) to e).

[0504] In some embodiments, such as Fig.29 As shown in Figure 1, the expression of genes in the human liver cell population or organoid of the present invention is up-regulated or down-regulated relative to the reference RNA. Preferably, the reference RNA is a universal human reference RNA (Stratagene, No. 740000). In some embodiments, a gene is up-regulated or down-regulated relative to the reference RNA if it is also shown in Figure 1. Fig.29 In other embodiments, the degree of upregulation or downregulation is + / -35%, + / -30%, + / -25%, + / -20%, + / -20%, + / -15%, + / -10%, + / -5%, + / -3%, or more preferably + / -1.5 times, + / -2 times, + / -3 times, + / -5 times, ... Fig.29 In other embodiments, the absolute level of gene expression in the human organ of the invention is + / -35%, + / -30%, + / -25%, + / -20%, + / -15%, + / -10%, + / -5%, + / -3%, or + / -1.5 times, + / -2 times, + / -3 times, + / -5 times, or more. Fig.29 Much the same as shown.

[0505] The human hepatocyte population or organoid of the present invention also preferably expresses Lgr5 and / or Tnfrsf19, preferably both. In some embodiments, when cultured in the expansion medium of the present invention, the human hepatocyte population or organoid expresses Lgr5 and / or Tnfrsf19, preferably both. Preferably, the expression of Lgr5 and / or Tnfrsfr19 is detected by RT PCR. In some embodiments, when cultured in the differentiation medium, Lgr5 and / or Tnfrsf19 are present in the organoid or cell at a lower expression level (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times) compared to their expression levels in the organoid or cell when cultured in the expansion medium.

[0506] Preferably, the liver cells and organoids of the present invention can preferentially secrete albumin, for example, at a concentration of about 1 μg / hour / 10 6 cells to 10 μg / hour / 10 6 cells, preferably 2 μg to 6 μg / hour / 10 6 The rate of cells.

[0507] Furthermore, such cells and organoids can secrete urea. For example, in a 35 mm dish of cells, the activity of urea synthesis can be 1 μg to 50 μg, preferably 5 μg to 30 μg, for 48 hours.

[0508] The liver cells and organoids of the present invention can show obvious glycogen accumulation when stained, for example. The ability of the cells and organoids of the present invention to actively synthesize glycogen can be tested by switching the culture medium from low glucose differentiation medium to high glucose DMEM supplemented with 10% FBS and 0.2 μM dexamethasone within two days.

[0509] The liver cells and organoids of the present invention may have inducible cytochrome P450 activity (such as CYP1A). This activity can be detected, for example, using the ethoxyresorufin-O-deethylase (EROD) test (Cancer Res, 2001, 61: 8164-8170). For example, cells or organoids can be exposed to a P450 substrate such as 3-methylcholanthrene, and the activity level of EROD can be compared with that of control cells.

[0510] Morphologically, liver organoid cells appear hepatocyte-like.

[0511] like Fig.30 As shown, preferred liver organoids comprise or consist of a sac-like structure, outside of which is a cell layer with buds and a central cavity. Such liver organoids may have one or more (e.g., 2, 3, or all 4) of the following characteristics: (a) having a cell density >5×10 5 Cells / cm 3, preferably >10×10 5 Cells / cm 3 ;(b) having a thickness equivalent to 2-30 layers of cells, preferably equivalent to a thickness of 2-15 layers of cells;(c) cells are in contact with each other in three-dimensional space;(d) showing functions inherent to healthy liver tissue;(e) having an elongated shape, having two defined domains, namely a single-layer epithelial domain, in which highly polarized cells can be detected and keratin markers are expressed (this domain is similar to the bile duct domain), and the other domain constitutes the main body of the organoid and is formed by a multilayer epithelium with non-polarized cells, in which albumin expression can be detected. It is obvious to a person 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.

[0512] In the context of the present invention, the liver fragment is a portion of an adult liver, preferably a human adult liver. Thus, preferably, the liver organoid as defined herein is not a liver fragment. The liver organoid is preferably obtained using cells from an adult liver, preferably epithelial stem cells from an adult liver, more preferably epithelial stem cells from an adult liver expressing Lgr5. The liver organoid may also be obtained from any cell that expresses Lgr5 when damaged or cultured and is therefore an Lgr5-expressing circulating stem cell.

[0513] In some embodiments, the liver organoids include 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 invention provides cells or cell populations expressing Lgr5, wherein the cells are obtained from the liver organoids of the invention. The invention also includes the progeny of such cells.

[0514] In an embodiment, the liver organoid is a liver organoid that is still cultured using the method of the present invention and is therefore in contact with an extracellular matrix. Preferably, the liver organoid is embedded in a non-mesenchymal or interstitial extracellular matrix. In the context of the present invention, "contact" means physical or mechanical or chemical contact, which means that a force is required to separate the liver organoid from the extracellular matrix.

[0515] In preferred embodiments, the liver organoids can be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 including a TGFβ inhibitor can be expanded 5 times a week or more than twice a week (e.g., at least 10 doublings, at least 20 doublings, more preferably at least 25 doublings, for example at least 30 doublings) and cultured for at least 4 weeks, most preferably at least 5 weeks. Preferably, liver organoids cultured using the expansion medium of the present invention that includes a prostaglandin pathway activator in addition to a TGFβ inhibitor can be cultured at least 7 weeks (i.e., at least 15 doublings, at least 25 doublings, at least 30 doublings, at least 32 doublings, at least 35 doublings such as 32-40 doublings, for example, at least 50 doublings) at more than 2 doublings per week (e.g., 2-3 doublings). Therefore, preferably, the liver organoids of the present invention, such as human liver organoids, are obtained using the expansion medium of the present invention.

[0516] In another preferred embodiment, the liver organoid is derived from a single cell, preferably a single cell expressing Lgr5, more preferably wherein the single cell comprises a nucleic acid construct comprising a nucleic acid molecule of interest.

[0517] Organoid composition and gene expression

[0518] Crypt-villus, colonic crypts, and pancreatic organoids typically include stem cells and / or progenitor cells, so these organoids share certain gene expression profiles. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) or all of the following markers may be detected: LGR5, LGR4, epcam, Cd44, Sox9, Cd24a, and CD133 / Prom1, and optionally Tnfrsf19. In another embodiment, one or both or all of the following progenitor genes may be detected: Pdx1, Nkx2.2, and Nkx6.1. After differentiation, it is expected that the gene expression profiles of crypt-villus, colonic crypts, and pancreatic organoids are different because the differentiated organoids express tissue-specific adult markers, such as insulin in the pancreas.

[0519] Crypt villus organoids

[0520] In some embodiments of the invention, the organoids include crypt-villus-like extensions that include all differentiated epithelial cell types, including proliferating cells, Paneth cells, enterocytes, and goblet cells. In some embodiments, the crypt-villus organoids of the invention do not include myofibroblasts or other non-epithelial cells. The crypt-villus organoids of the invention preferably include enterocytes in crypt-villus-like structures, including absorptive enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Preferably, at least one of the following markers (e.g., 2, 3, 4, 5, or 6) can be detected: SMOC2, CDCA7, OLFM4, ASCL2, AXIN2, and / or Lgr5, Tnfrsf19, CD24a, Sox9, CD44, Prom1 (see Figure 2e and Fig.14 ). In some embodiments, markers RNF43 and ZNRF3 can be detected. In some embodiments, one or some (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 crypts, and markers that are downregulated at least 2-fold, 3-fold or 4-fold in the crypts include at least one or some (e.g., 1, 2, 3 or 4) or all of ABCG1, ENPP3, CSTE, MUC17 and / or APOA1. In this case, "upregulated" is relative to the villi of the intestine or the top of the colonic crypts. Microarray analysis comparing the gene expression of differentiated organoid cells with stem cells revealed that small intestinal crypt-villus and colonic organoids have comparable small intestinal crypt molecular signatures, including expression of small intestinal stem cell genes. Therefore, the present invention also provides colonic organoids having the molecular signatures of the above-mentioned crypt-villus organoids. In vitro cultured organoids clearly exhibited an expression profile similar to freshly isolated intestinal crypts and expressed known stem cell markers.

[0521] In some embodiments, the encoding Fig.14 mRNA for one or more genes (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) listed in the table above as being upregulated in crypt-villus organoids or colonic organoids, respectively, is upregulated in crypt-villus organoids or colonic organoids of the invention compared to freshly isolated intestinal villi as determined by microarray. In some embodiments, mRNA encoding a gene in Fig.14mRNA of one or more genes (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) listed in the table that are down-regulated in crypt-villus organoids or colonic organoids, respectively, is down-regulated in the crypt-villus organoids or colonic organoids of the invention compared to freshly isolated intestinal villi as determined by microarray. In some embodiments, the up-regulation or down-regulation fold is Fig.14 Multiples shown are + / –25%, such as + / –20%, + / –15%, + / –10%, + / -5%, + / -3% or approximately Fig.14 For example, the crypt-villus organoids of the invention may have ADORA2B upregulated 9.54-fold + / - 25% compared to freshly isolated intestinal villi. Fig.14 Other genes listed.

[0522] In some embodiments, the crypt villus organoids exhibit native expression of Lgr5. In some embodiments, the crypt villus organoids exhibit 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) or all of the following stem cell markers: CK19, nestin, somatostatin, CXCR4 + 、CD133 + , DCAMKL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19. Additionally or alternatively, the crypt-villi organoids may be characterized by expressing one or more or all (e.g., 1 or 2) of the following: Sord and / or Prss23. Additionally or alternatively, the crypt-villi organoids may be characterized by expressing CD44 and / or Sox9. In another embodiment, the crypt-villi organoids exhibit expression of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) or all of the following markers: lgr5, lgr4, epcam (tacstd1), Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1 and Cdca7.

[0523] In some embodiments, the crypt villus organoids include Paneth cells expressing lysozyme.

[0524] Colon organoids

[0525] In some embodiments, colon organoids include enteroendocrine cells (e.g., detected using chromogranin A staining), goblet cells (detected using mucin 2 staining). In some embodiments, less than 10% of the cells in the colon organoids are enteroendocrine cells (e.g., 0.01-5%, 0.1-3%). In some embodiments, less than 30% of the cells in the colon organoids are goblet cells (e.g., 1-25%, 1-15%, 5-10%). In some embodiments, the distribution of enteroendocrine cells and / or goblet cells is as follows Figure 1d shown.

[0526] In some embodiments, the colon organoids include mature intestinal epithelial cells (e.g., as seen by alkaline phosphatase staining). In some embodiments, less than 10% of the cells in the colon organoids are mature intestinal epithelial cells (e.g., less than 5%, less than 3%, 0.01-5%, 0.1-3%, 0.1-5%).

[0527] In preferred embodiments, the colon organoids do not include Paneth cells, as Paneth cells are not present in the naturally occurring in vivo colon.

[0528] In some embodiments, the colon organoids exhibit native expression of Lgr5.

[0529] In some embodiments, the colon organoids express one or more (e.g., 1, 2, 3, or 4) of villin 1, Alpi, ChgA, and Muc2. In some embodiments, the relative amount of villin 1 mRNA expressed by the colon organoids of the present invention is at least 3% (e.g., at least 5%, at least 8%, at least 10%), for example, 5-15%, compared to freshly isolated colon crypts. In some embodiments, the relative amount of Alpi mRNA expressed by the colon organoids of the present invention is at least 0.5% (e.g., at least 1%, at least 2%), for example, 0.5-5%, compared to freshly isolated colon crypts. In some embodiments, the relative amount of ChgA mRNA expressed by the colon organoids of the present invention is at least 15% (e.g., at least 20%, at least 22%), for example, 15-30%, compared to freshly isolated colon crypts. In some embodiments, the relative amount of Muc2 mRNA expressed by the colonic organoids of the invention compared to freshly isolated colonic crypts is at least 20% (eg, at least 25%, at least 30%, at least 35%), such as 25-37%.

[0530] In some embodiments, the human colon organoids of the invention express known stem cell markers.

[0531] Pancreatic organoids

[0532] The pancreas includes three types of cells: duct cells, acinar cells and endocrine cells. Endocrine cells produce the hormones glucagon, insulin somatostatin and pancreatic polypeptide (PP), which are secreted into the blood and help the body regulate sugar metabolism. Acinar cells are part of the exocrine system, which produce digestive enzymes and duct cells from the pancreatic ducts that connect the acinar cells to the digestive organs. During development, pancreatic islets are thought to originate from progenitor endocrine cells that emerge from the pancreatic ducts and aggregate to form islets after differentiation. Pancreatic islets include alpha cells, beta cells, delta cells and PP cells.

[0533] Pancreatic organoid cells may have an expression profile similar to ductal cell markers, such as one or more (e.g., one, two, or all) of K7, K19, and Hnf1b, and / or one or more general stem cell markers such as Sox9 and / or Onecut1. This may be part of its stem cell signature. Typically, fewer differentiation markers are seen. In some embodiments in which cells are isolated from the pancreatic duct to produce the pancreatic organoids of the invention, the cell type from which the pancreatic organoids of the invention are produced is not a ductal cell (i.e., an epithelial cell that is positive for keratin 7 and keratin 19 that form the ductal tube), but it is a cell connected to the pancreatic duct, i.e., a cell located in the next cell layer after the duct contacts the pancreatic tissue (i.e., not toward the lumen of the duct). Therefore, in embodiments in which the cell type from which the pancreatic organoids are produced is not a ductal cell, the pancreatic organoids do not express K7 or K19. However, such pancreatic organoids still preferably express one or more general stem cell progenitor markers, such as Sox9.

[0534] The pancreatic organoids of the present invention preferably include alpha cells, beta cells, delta cells and PP cells. In other preferred embodiments, the pancreatic organoids include beta cells. For example, the pancreatic organoids may include more than 1%, more than 5%, more than 10%, more than 15%, or more than 20% of beta cells. The expression of insulin can be used as a marker for beta cells. In an alternative embodiment, the pancreatic organoids include progenitor cell types that may have ductal origins when transplanted into humans or animals to produce differentiated cell types. In a preferred embodiment, the progenitor cell types can produce insulin-secreting beta cells when implanted in humans or animals. The inventors have shown that human pancreatic organoids grown according to the culture medium and method of the present invention can be implanted into mice and stimulate insulin-secreting cells within one month (see Example 4). It can be easily understood that this will produce a revolutionary treatment for patients with diabetes and insulin deficiency.

[0535] In some embodiments, the pancreatic organoids of the present invention may include ductal cells, acinar cells, and endocrine cells. In some embodiments, K19 is used as a marker for ductal cells.

[0536] In some embodiments, β-cells are present in pancreatic islands or islets of Langerhans. The islands usually include about 1500 cells in vivo, such as 1300-1700 cells. In one embodiment, the pancreatic organoids include 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 by mass. In some embodiments, the islets of pancreatic organoids are composed of about 65-90% β-cells, about 15-20% α-cells, about 3-10% δ cells and about 1% PP cells. However, this is by no means exclusive. For example, in some embodiments, it is desirable to have many β cells in the organoids of the present invention. Alternatively, the organoids may include progenitor cells that can be transplanted to differentiate them in vivo.

[0537] In some embodiments, pancreatic organoids express one, two or all of Pdx1, Nkx2.2 and Nkx6.1. Pancreatic organoids may express one, two, three or all of NeuroD, Pax6, Pax4 and Mafa. Pax4 is used as a marker for the presence of insulin-producing cells because it is an essential transcription factor for differentiation of insulin-producing cells from endocrine progenitor cells during embryonic development. Pancreatic organoids may express Ngn3.

[0538] In some embodiments, at least one of the following markers (e.g., 1, 2, 3, 4, 5) 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 of the following markers (e.g., 1, 2, 3, 4, 5) can be detected in the pancreatic organoids of the present invention: insulin (ins1 and / or ins2), glucagon (Gcg), somatostatin, Pdx1, and NeuroD, and the following markers cannot be detected: ptf1a, amy2a4, Pnlip, and cela1. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of the following markers can be detected in the pancreatic organoids of the invention: Ptf1a, pancreatic amylase (Amy2a4), pancreatic lipase (Pnlip), insulin (ins1 and / or ins2), glucagon (Gcg), somatostatin, chymotrypsin (cela1), Pdx1, and NeuroD.

[0539] In some embodiments, the pancreatic organoids exhibit native expression of Lgr5. In some embodiments, the pancreatic organoids exhibit 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, or 20) of the following stem cell markers: CK19, nestin, CXCR4, + 、CD133 + , DCAM, KL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19, prom1, Cd24a, Lgr4, epcam. Alternatively or additionally, in some embodiments, pancreatic organoids can be expressed by CK19, nestin, (insulin, glucagon) and CXCR4 + The invention can be characterized by the natural expression of one or more (e.g., 1, 2, 3, or 4) of the genes.

[0540] In some embodiments, the pancreatic organoids or cells of the invention express somatostatin. Somatostatin is a hormone expressed in differentiating delta cells and can be used as a marker for delta cells.

[0541] Alternatively or additionally, in some embodiments, the pancreatic organoids exhibit native expression of one or more early endocrine markers, such as at least one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following early endocrine markers: Sox9, Hnf1b, Hnf6, Hnf1a, Nkx2.2, Nkx6.1, and Pdx1.

[0542] Alternatively or additionally, in some embodiments, the pancreatic organoids exhibit native expression of one or more early endocrine markers, such as at least one or more (e.g., 1, 2, 3, 4) of the following early endocrine markers: Foxa2, Hnf6, Hnf1b, and Sox9. In some embodiments, although the pancreatic organoids exhibit native expression of one or more (e.g., 1, 2, 3, or 4) of the following endocrine markers: Foxa2, Hnf6, Hnf1b, and Sox9, they do not exhibit expression of Ngn3.

[0543] Alternatively or additionally, in some embodiments, the pancreatic organoids exhibit native expression of one or more ductal markers, such as keratin 7 and keratin 19, or both. In some embodiments, the pancreatic organoids exhibit significant or detectable levels of native expression of one or more ductal markers. Thus, in some embodiments, the pancreatic organoids have a ductal phenotype. In some embodiments, the pancreatic organoids exhibit expression of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) or all of the following markers selected from the group consisting of: Hnf1A, Hnf1B, Hnf4A, HHEX, ONECUT1, ONECUT2, CDH1, FOXA2, GATA6, CEBPB, CEBPD, CEBPG, Glul, Krt7, Krt19, and MET.

[0544] However, pancreatic organoids can have some ductal features combined with features of insulin-producing precursor cells. For example, they can express one or more Fig. 16B In some embodiments, the pancreatic organoids exhibit ductal markers as shown in Figure 1 relative to adult pancreatic or liver organoids. Fig. 16B For example, in some embodiments, these genes are upregulated or downregulated in pancreatic organoids compared to adult pancreatic liver organoids. Fig. 16B Approximately the same multiple ratio, for example less than + / - 3%, less than + / - 5%, less than + / - 10%, less than + / - 20%.

[0545] In some embodiments, in pancreatic organoids, pancreas-positive cells are visualized from the ductal lining.

[0546] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, or 7), preferably all, of the following genes are upregulated in pancreatic organoids compared to liver organoids: Aaas, Rps4y2, Atp2c2, Akap2, Uts2, Sox17, Agr2. For example, in some embodiments, these genes are upregulated in pancreatic organoids by 1:1 compared to liver organoids. Fig.19 Approximately the same multiple ratio, for example less than + / - 3%, less than + / - 5%, less than + / - 10%, less than + / - 20%.

[0547] In one embodiment, the pancreatic organoids include a total of at least 10 3 At least 10 4 At least 10 5In one embodiment, the pancreatic organoid comprises more than 50%, more than 60%, more than 70%, or more than 80% ductal-like endocrine progenitor cells. However, a lower proportion of ductal-like endocrine progenitor cells is also contemplated.

[0548] Barrett's esophagus (BE) organoids

[0549] The BE organoids of the present invention are Ki67+.

[0550] Preferably, BE organoids have a minimum number (e.g., less than 25%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% of cells) of PAS+ and Mucin+ cells 4 days after withdrawing nicotinamide and SB202190 from the expansion medium to convert it to differentiation medium.

[0551] In some embodiments, the BE organoids include goblet cells. These can be induced by treating the differentiation medium with a γ-secretase inhibitor such as DBZ (eg, 10 μM) for 4 days.

[0552] In some embodiments, the Barrett's esophagus organoids of the invention include Paneth cells.

[0553] In some embodiments, the Barrett's esophagus organoids of the invention express lysozyme.

[0554] Gastric organoids

[0555] In some embodiments, the gastric organoids of the invention exhibit native expression of Lgr5. In some embodiments, the gastric organoids of the invention exhibit 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, or 17) of the following stem cell markers: CK19, nestin, somatostatin, CXCR4, + 、CD133 + , DCAMKL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf, Sox9, KRT7 and KRT19. Alternatively or additionally, in some embodiments, gastric organoids can be expressed by CD133 + , DCAMKL-1, and CD44. Alternatively or additionally, the gastric organoids may be characterized by native expression of one or more (eg, 1, 2, or 3) of CD44 and Sox9.

[0556] Prostate Organoids

[0557] In some embodiments, the prostate organoids of the present invention, such as mouse prostate, show natural expression of Lgr5. In some embodiments, the prostate organoids show natural expression of luminal prostate markers, such as cytokeratin 18 (CK18) and cytokeratin 8 (CK8). In some embodiments, the prostate organoids of the present invention show natural expression of androgen receptor (AR). In some embodiments, the prostate organoids express basal markers, such as p63 and / or cytokeratin 5 (CK5). In some embodiments, when testosterone (e.g., DHT) is added to the culture medium, the expression of basal markers, Lgr5 and Tnfrsf19 is downregulated compared to organoids grown in the absence of testosterone (e.g., DHT). The prostate-specific transcription factor NKX3.1 is expressed under all conditions. Therefore, in some embodiments, the prostate organoids of the present invention show natural expression of the prostate-specific marker Nkx3.1.

[0558] In some embodiments, prostate organoids of the invention, e.g., normal or cancerous human prostate organoids, exhibit native expression of luminal markers, e.g., CK18, CK8, and / or B-MSP. In some embodiments, prostate organoids exhibit native expression of AR. In some embodiments, basal epithelial markers, e.g., CK14, CK5, and / or p63, are expressed. In some embodiments, TNFRSF19 is expressed. The prostate-specific transcription factor NKX3.1 is expressed under all conditions. Thus, in some embodiments, prostate organoids of the invention exhibit native expression of the prostate-specific marker Nkx3.1.

[0559] Testosterone (such as DHT) is added to the culture medium of the present invention to make prostate organoid grow, it maintains stem cell group, allows up to 3 times of faster growth (compared with the situation without testosterone), and also has the differentiated cell type (basal cell and luminal cell) of most (even if not all) of prostate.These conditions allow unlimited cell expansion (so far with 2.5 times population doubling in a week until 9 months).Therefore, in some embodiments, prostate organoid includes all differentiated cell types of prostate, such as basal cell and luminal cell.In a preferred embodiment, prostate organoid includes all differentiated cell types, such as basal cell and luminal cell and stem cell.

[0560] In normal tissue, the addition of testosterone (eg, DHT) increases AR expression in all culture conditions. In some embodiments, prostate organoids have upregulated AR expression compared to prostate cells grown in the absence of testosterone. In tumor tissue, testosterone (eg, DHT) addition does not affect AR expression. Therefore, in some embodiments, prostate cancer organoids do not have increased AR expression compared to prostate cancer cells in vivo. In prostate organoids from normal tissue, stem cell marker LGR5 is expressed under ENRF conditions. In prostate organoids obtained from tumor tissue, LGR5 expression is induced by adding testosterone (eg, DHT). In some embodiments, prostate organoids express LGR5.

[0561] Organoid Function

[0562] In some embodiments, the organoids produced by the culture media and methods of the present invention mimic the final cell type determination in vivo in response to external factors. Preferably, the cells and organoids produced according to the present invention also have tissue-specific functions.

[0563] Pancreatic organoids

[0564] Pancreatic organoids preferably have endocrine and exocrine pancreatic functions, such as expression of one or more (e.g., 1, 2, or all 3) of insulin, glucagon, and somatostatin. The expression of these hormones is strictly regulated by a set of endocrine pancreas-specific transcription factors, most importantly Pdx1 and NeuroD. The exocrine pancreas is formed by acinar and ductal compartments for the production of digestive enzymes such as amylase, pancreatic lipase, and chymotrypsin. The expression of these genes is also regulated by specific exocrine pancreatic genes such as Ptf1a.

[0565] Preferably, the pancreatic cells and organoids of the present invention can preferentially secrete insulin, for example, at about 1 μg / hour / 10 6 cells to 10 μg / hour / 10 6 cells, e.g. 2 μg to 6 μg / hour / 10 6 The rate of cells. The level of insulin secretion can be detected by methods known in the art, such as by immunoblotting compared with a reference or by C-peptide enzyme-linked immunosorbent assay. The preferred method of exemplifying pancreatic organoids can secrete insulin by testing the production of C-peptide. Proinsulin C-peptide is used as an important linker between insulin A-chain and B-chain, and promotes effective assembly and processing of insulin in the endoplasmic reticulum. Then, equimolar amounts of C-peptide and insulin are stored in the secretory granules of pancreatic β cells, and both are eventually released into the portal circulation. Therefore, C-peptide is a preferred marker for insulin secretion.

[0566] Thus, in some embodiments, pancreatic organoids are provided that secrete insulin after in vivo transplantation. In some embodiments, after in vivo transplantation, the pancreatic organoid secretes insulin at about 1 μg / hour / 10 6 cells, for example, at least 2 μg / hour / 10 6 cells, at least 4 μg / hour / 10 6 cells, at least 6 μg / hour / 10 6 cells, at least 8 μg / hour / 10 6 cells or at least 10 μg / hour / 10 6 In some embodiments, the cells in the pancreatic organoids are unable to secrete insulin and / or do not express insulin as a marker when cultured in vitro. However, the cells from the pancreatic organoids of the present invention are preferably able to secrete insulin in vivo when implanted in a patient, for example, in the pancreas of a patient. In some embodiments, the ability to secrete insulin may not be present immediately after transplantation, but may be present within about 1 month after transplantation, for example 6 weeks, 2 months, or 3 months after transplantation.

[0567] In some embodiments, if the enriched endocrine cell sample is obtained from a pancreatic organoid of the present invention, 75-85% of the cells in the enriched endocrine cell sample will be insulin-secreting cells.

[0568] In some embodiments, the present invention provides pancreatic organoids for treating diabetes. In some embodiments, pancreatic organoids are expanded organs, while in other embodiments, they can be differentiated organoids. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, etc.) whole organoids are transplanted into animals or patients, while in other embodiments, cell samples are transplanted into patients.

[0569] Crypt-villus organoids

[0570] The crypt-villus organoid preferably has secretion and self-renewal functions. For example, the crypt-villus organoid preferably secretes mucin, enzyme secretion, and hormone secretion, such as lysozyme, intestinal secretin, secretin, and gastric inhibitory peptide, as well as other glycoproteins.

[0571] Gastric organoids

[0572] The human stomach is structurally and functionally divided into two main regions. The pyloric antrum, which is close to the intestine, mainly produces protective mucus and secretes hormones such as gastrin. The gastric body secretes hydrochloric acid and gastric enzymes such as pepsinogen. The gastric epithelium of both regions is organized in glands called invaginations. These glands harbor gastric stem cells, progenitor cells, and differentiated cells. The exact composition of differentiated cells varies depending on the function of the tissue region. In the pyloric antrum, the gland is mainly composed of mucin 6-producing cells and hormone-producing endocrine cells. In the body, pepsinogen-producing chief cells and acid-secreting parietal cells are scattered between mucus-producing cells and sparse endocrine cells. The surface area between the gastric glands is occupied by mucus-producing cells that mainly produce surface mucus 5.

[0573] Gastric organoids are similar in structure and function to gastric epithelium. Although they are primarily spherical, they may have domains with invaginations that are most likely similar to glandular structures. Staining for mucin and pepsinogen indicated that the most abundant cell types in gastric organoids are mucin 6-producing mucus cells and pepsinogen-producing chief cells (and / or their ancestors). Therefore, RT-PCR indicated the expression of pepsinogen and mucin 6. In addition, the expression of gastrin indicated the presence of endocrine cells, and the expression of Lgr5 indicated the presence of stem cells.

[0574] In some embodiments, the gastric organoids have native expression of one or more (e.g., 1, 2, 3, or 4) of gastrin, pepsinogen, mucin 6, and / or Lgr5. In some embodiments, the gastric organoids include mucin 6-producing mucus cells and pepsinogen-producing chief cells and optionally Lgr5+ stem cells. In some embodiments, the gastric organoids include endocrine cells. In some embodiments, the gastric organoids are primarily spherical, but have domains with invaginations similar to glandular structures.

[0575] Prostate Organoids

[0576] In some embodiments, the prostate organoid comprises or consists of two different epithelial lineages (basal cells and luminal cells). In some embodiments, the basal cells and luminal cells secrete prostatic fluid.

[0577] The prostate epithelium is strongly folded in vivo, ensuring the maximum surface area. Two epithelial lineages form a simple stratified epithelium with basal epithelial cells forming the basal layer / outer layer and strongly polarized luminal epithelial cells forming the inner layer / luminal layer at the top. The luminal compartment is necessary for the secretory function of the prostate. Prostatic fluid is alkaline and composed of a variety of proteins, such as prostate-specific antigen (PSA), human kallikrein 2 (KLK2) and β-microspermogen (β-MSP). The main functions of prostatic fluid are: 1) prepare the environment of the uturus of semen, which is carried out by the alkalinity of the body fluid and the paracrine function of β-MSP; and 2) increase the fluidity of semen, allowing sperm to swim freely, which is carried out by the protease PSA and KLK2 that decomposes seminogelins.

[0578] The expression of secretory proteins is tightly regulated by the androgen receptor (AR), which binds testosterone and subsequently translocates to the nucleus and activates transcription. Disruption of AR function has been shown to strongly downregulate secretory proteins at both the transcriptional and protein levels.

[0579] Fig.43 The stratification of the prostate organoid grown under ENR+ dihydrotestosterone (DHT) conditions is shown, and it is clearly shown that cytokeratin 5+ basal cells form an outer layer of cells and cytokeratin 8+ luminal cells form a strongly polarized inner layer. In some embodiments, the prostate organoid includes cytokeratin 5+ basal cells and cytokeratin 8+ luminal cells, optionally, wherein cytokeratin 5+ basal cells form an outer layer of cells and cytokeratin 8+ luminal cells form a strongly polarized inner layer. In some embodiments, the prostate organoid includes a folded cell layer, optionally including strong folding. Such folding maximizes the surface area of ​​secretory cells, indicating that at the morphological level, the prostate organoid is similar to the prostate in vivo. In some embodiments, the morphology of the prostate organoid is similar to the in vivo morphology of the prostate.

[0580] Prostate organoids cultured in ENR conditions do not secrete prostate fluid into the cavity. On the contrary, adding testosterone (such as DHT) to the culture medium causes secretion of body fluid, such as prostate fluid, in the organoid cavity. This is due to the activation of AR-dependent transcriptional programs by testosterone (such as DHT) in prostate organoids, which causes secretion of body fluid by CK8+ cavity cells. Data show that prostate organoids are similar to prostate epithelium in vivo in both morphology and function.

[0581] Thus, in some embodiments, such as where prostate organoids are cultured in a culture medium comprising testosterone (e.g., DHT), the prostate organoids secrete body fluids, such as prostatic fluid, into the lumen of the organoid. In some embodiments, such as where prostate organoids are cultured in a culture medium comprising testosterone (e.g., DHT), the function of the prostate organoids is similar to the in vivo function of the prostate.

[0582] Tissue fragments

[0583] In the context of the present invention, the tissue fragment is a part of an adult tissue, preferably a human adult tissue, such as a part of a human adult small intestine, colon or pancreas. Other examples of human adult tissues in the context of the present invention include the stomach, liver and prostate. The tissue may be a normal (healthy) tissue or it may be a diseased or infected tissue. Therefore, preferably, the organoid as determined herein is not a tissue fragment. It is preferred to use cells from adult tissue, preferably epithelial stem cells from adult tissue, optionally from adult tissue fragments, more preferably epithelial stem cells from adult tissue or adult tissue fragments expressing Lgr5, to obtain organoids. Therefore, in the context of the present invention, the tissue fragment preferably includes Lgr5+ stem cells.

[0584] In one embodiment, the organoid is an organoid that is still being cultured and is therefore in contact with an extracellular matrix using the method of the present invention (preferably using the culture medium of the present invention). Preferably, the organoid is embedded in a non-mesenchymal extracellular matrix. In the context of the present invention, "contact" means physical or mechanical or chemical contact, which means that force is required to separate the organoid from the extracellular matrix. In some embodiments, the extracellular matrix is ​​a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as matrigel (BD Biosciences). In other embodiments of the present invention, organoids can be removed from the culture and used for transplantation or regeneration purposes. Therefore, the present invention provides organoids of the present invention for transplantation into mammals, preferably into people.

[0585] Survival rate

[0586] The inventors show here for the first time that adding an inhibitor of ALK4, ALK5, ALK7 or p38 kinase to previously described stem cell culture media increases culture plating efficiency by at least 50%, and in some cases by more than 100% (see Table 2). The inventors have shown that including two inhibitors (an ALK inhibitor and a p38 inhibitor, such as A83-01 and SB-202190) in the culture media synergistically extends culture time.

[0587] Thus, in some embodiments of the invention, the stem cells survive 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 more.

[0588] Proliferation rate

[0589] Proliferation rate is evaluated based on the cell population doubling level. The population doubling level refers to the total number of times the cells in the population have doubled since their initial isolation in vitro. The population doubling level can be determined by cell counting. Alternatively, the proliferation rate can be evaluated by cell proliferation tests, such as where specific fluorescent probes are incorporated by BrdU to measure DNA synthesis activity and by Ki67 expression to measure cell proliferation (Thermo Scientific * Cellomics, Millipore).

[0590] Other examples of cell proliferation assays for stem cells are readily available and can be found online or in journals such as Current Protocols. One of many examples is http: / / products.invitrogen.com / ivgn / en / US / adirect / invitrogen? cmd=catDisplayStyle&catKey=101&filterDispName=Cellular Proliferation Assaysfor Stem Cells&filterType=1&OP=filter&filter=ft_1101%2Ff_494303*&_bcs_=H4sIAAAAAAAAAH2NsQrDMAxEv0ZTsEkdKFmzZC70C4IjakFsGVuOfz%2FK0I6F4 x284c48YJxfhffm%0ApQ7gnsMby0ke6x8fRDJMC7hV03u3lE6Swh9M1nNUWUlQq1UFJkXgeIvvotnSbn6Lbl1yPshvQpyq%0ADRIPfQE33RlnKQ21Lvuql7CrAAAA.

[0591] The inventors have observed that using the culture medium of the present invention, cells can be expanded up to an average of 5 times per week. For example, a single cell grown for two weeks will produce an average of about 25 cells. The skilled person will appreciate that the average population doubling time of the stem cells of the present invention can vary depending on many factors, such as the number of passages, culture conditions, seeding density, etc.

[0592] In one embodiment, the average population doubling time may be 6-48 hours, 12-36 hours, 18-30 hours, or about 24 hours. For example, a stem cell population cultured using the medium of the present invention may be expected to double about 4-7 times or about 5 times per week.

[0593] In another embodiment, the average population doubling time is 12-96 hours, 24-72 hours, or about 72 hours. In another embodiment, the cell population is maintained once, more than twice, more than three times, more than four times, or more than five times a week on average.

[0594] Other properties of the organoids of the invention

[0595] In a preferred embodiment, the organoids can be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6 months or longer. In a preferred embodiment, the organoids can be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer. In another preferred embodiment, the organoid is derived from a single cell, preferably a single cell expressing Lgr5, more preferably wherein the single cell comprises a nucleic acid construct comprising a nucleic acid molecule of interest.

[0596] The present invention also provides organoids, which preferably include 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. Hoechst staining or propidium iodide staining can be used in FACS to evaluate the viability of cells.

[0597] The living cells preferably have tissue-specific functions or characteristics of tissue-specific functions, as described above.

[0598] The inventors have shown that organoids produced by the culture media and methods of the present invention can be frozen and stored below -80°C, for example in liquid nitrogen. Frozen organoids can be thawed and placed in culture without loss of their 3D structure and integrity, and without significant cell death. Therefore, in one embodiment, the present invention provides frozen organoids stored below -5°C, below -10°C, below -20°C, below -40°C, below -60°C, or below -80°C.

[0599] The cells and organoids of the present invention are different from any cells and organoids previously prepared (WO2009 / 022907 and WO2010 / 016766) because they have better phenotype (better differentiation spectrum, including goblet cell transformation when adding γ-secretase inhibitor for crypt-villus organoids) and karyotype integrity (as determined by the above method), better survival rate and faster cell proliferation rate. Thus, for the intestinal, colon and pancreatic embodiments, the organoids of the present invention clearly represent human intestinal, colon and pancreatic epithelia with complete preservation of phenotypic and karyotype integrity and maintenance of proliferation and differentiation.

[0600] Uses of stem cells or organoids of the present invention

[0601] The present invention provides uses of the organoids or expanded populations of the present invention for drug screening, (drug) target confirmation, (drug) target discovery, toxicology and toxicology screening, personalized medicine, regenerative medicine and / or as ex vivo cell / organ models such as disease models.

[0602] Cells and organoids cultured according to the culture medium and methods of the present invention are believed to faithfully represent the in vivo situation. This is true for expanded cell populations and organoids grown from normal tissues as well as for expanded cell populations and organoids grown from diseased tissues. Therefore, the organoids or expanded cell populations of the present invention can be used not only as in vitro disease models, but also provide normal in vitro cell / organ models.

[0603] The organoids of the present invention can also be used to culture pathogens and thus can be used as ex vivo infection models. Examples of pathogens that can be cultured using the organoids of the present invention include viruses, bacteria, prions, or fungi that cause disease in their animal hosts. Therefore, the organoids of the present invention can be used as disease models that present an infection state. In some embodiments of the present invention, the organoids can be used for vaccine development and / or production.

[0604] Diseases that can be studied using the organoids of the present invention therefore include genetic diseases, metabolic diseases, pathogenic diseases, inflammatory diseases, and the like, including, for example, but not limited to, 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, alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome, anemia, Schwachman-Bodian-Diamond syndrome, polycythemia vera, primary myelofibrosis, glycogen storage disease, familial hypercholesterolemia, Crigler-Najjar syndrome, hereditary tyrosinemia, Pompe's disease, progressive familial cholestasis, Hurler syndrome, SCID or leaky SCID, Omenn syndrome, chondro-hair dysplasia, herpes simplex encephalitis, scleroderma, osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, dyskeratosis congenita, and the like.

[0605] Typically, cell lines and recent iPS cells have been used as ex vivo cells / organs and / or disease models (e.g., see Robinton et al., Nature 481, 295, 2012). However, these methods are subject to many challenges and disadvantages. For example, cell lines cannot be obtained from all patients (only some biopsies produce successful cell lines), and therefore, cell lines cannot be used for individualized diagnosis and medical treatment. iPS cells generally require certain levels of genetic manipulation to reprogram cells into specific final cell types. Alternatively, they experience culture conditions that affect the integrity of the chromosome karyotype, so the time in the culture must be kept at a minimum (this is also the same for human embryonic stem cells). This shows that iPS cells cannot accurately present the situation in vivo, but attempt to simulate the behavior of cells in vivo. Cell lines and iPS cells also undergo genetic instability.

[0606] On the contrary, the organoid of the present invention provides a genetically stable platform that truthfully presents the situation in vivo. The organoid of the present invention is also continuously expanded, providing a good genetically stable cell source. In particular, the expansion group can be "split", indicating that the organoid is split, and all the cells of the organoid are divided into a new culture dish or culture bottle. Remove the split cells from the organoid, then cultivate and expand itself to produce a new organoid containing a group of further expansion, which is then split again. Split is also referred to as "passage" herein. The organoid of the present invention can be cultured for more than 1 passage, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 times or more, such as 20-30 passages, 30-35 passages, 32-40 passages or more. In some embodiments, the expansion cell group or organoid is split once a month, once every two weeks, once a week, three times a week, four times a week, five times a week, six times a week, or once a day. Therefore, the organoid of the present invention can provide a continuous source of genetically stable cell material. In some embodiments, the expanded organoids of the present invention include all differentiated cell types present in the corresponding in vivo situation. In other embodiments, the organoids of the present invention can be differentiated to provide all differentiated cell types present in vivo. Therefore, the organoids of the present invention can be used to obtain in-depth understanding of the mechanisms of various diseases and treatments for in vitro drug screening, to evaluate possible treatments, to determine possible targets (e.g., proteins) for future novel (drug) treatment development and / or to explore genetic repair in combination with cell replacement therapy.

[0607] The organoids of the present invention can be frozen and thawed, and placed in culture without loss of their genetic integrity or phenotypic properties, and without loss of proliferation capacity. Therefore, the organoids can be easily stored and transported.

[0608] For these reasons, the organoids or expanded cell populations of the invention can be tools for drug screening, target validation, target discovery, toxicology and toxicology screening, and personalized medicine.

[0609] Therefore, in other aspects, the invention provides the use of expanded stem cell populations or organoids, such as the intestinal crypt-villus organoids or pancreatic organoids of the invention, in drug discovery screening, toxicity testing, or medical treatment, such as regenerative medicine. For example, any of the small intestine, colon, pancreas, stomach, liver, or prostate organoids can be used for drug discovery screening, toxicity testing, or for medical treatment, such as regenerative medicine.

[0610] Mucosal vaccines

[0611] Other important uses of organoids are the development of mucosal vaccines. Mucosal vaccines are vaccines that are administered through the mucosa. It can be any mucosal surface, such as through the nose, mouth or rectum. They can be administered through inhalers, nebulizers or other external aids. It has many obvious benefits compared to injection, such as not requiring medical personnel to administer the vaccine, which can be important, for example, in developing countries.

[0612] In the intestine, M cells (or "microfold cells") are cells found in the follicle-associated epithelium of the aggregated lymphoid nodules of the ileum. They transport organisms and particles from the intestinal lumen across the epithelial barrier to immune cells and are therefore important in stimulating mucosal immunity. They have the unique ability to absorb antigens from the lumen of the small intestine by endocytosis or phagocytosis and then deliver them by transcytosis to dendritic cells (antigen presenting cells) and lymphocytes (i.e., T cells) in a unique pouch-like structure located on its basolateral side.

[0613] FIG. 48 shows that mouse organoids can develop into M cells when stimulated with RANK ligand. Fig.49 It was shown that M cells can also be generated in human long organoids. Thus, in some embodiments of the invention, the expanded cell population includes M cells. In some embodiments of the invention, the organoid, such as a small intestinal organoid, includes M cells.

[0614] Mucosal vaccines can significantly increase their efficiency when they target M cells. Therefore, the amplified stem cell populations or organoids of the present invention can be used to test the ability of M cells to absorb pathogens or antigens and present them to the immune system. Therefore, in some embodiments, the present invention provides the use of amplified stem cell populations or organoids of the present invention in drug screening, such as in vaccine development and / or vaccine production. For example, in some embodiments, amplified stem cell populations or organoids can be used to develop or produce vaccines for viral, bacterial, fungal or other parasitic infections, such as (but not limited to) cholera, respiratory syncytial virus (RSV), rotavirus and HIV. In specific embodiments, the present invention provides small intestinal organoids differentiated in a culture medium of the present invention including RANKL for mucosal vaccine development.

[0615] Drug Screening

[0616] For the purpose of preferably high throughput, the expanded stem cell population or organoid of the present invention, such as crypt-villus organoid or pancreatic organoid, is cultured in a multi-well plate, such as a 96-well plate or a 384-well plate. Molecular libraries are used to determine molecules that affect the organoid. Preferred libraries include antibody fragment libraries, peptide phage display libraries, peptide libraries (such as LOPAP TM , SigmaAldrich), lipid libraries (BioMol), synthetic compound libraries (e.g. LOP AC TM , Sigma Aldrich) or natural compound libraries (Specs, TimTec). In addition, gene libraries that induce or inhibit the expression of one or more genes in stem cell offspring can be used. These gene libraries include cDNA libraries, antisense libraries, and siRNA or other non-coding RNA libraries. The cells are preferably exposed to multiple concentrations of the test agent for a period of time. At the end of the exposure period, the culture is evaluated. The term "affect" is used to encompass any changes in the cells, including but not limited to a decrease or loss of proliferation, morphological changes, and cell death. The expanded stem cell populations or organoids of the present invention, such as crypt-villi organoids or pancreatic organoids, can be used to determine drugs that specifically target epithelial cancer cells but do not target the expanded stem cell populations or organoids of the present invention, such as crypt-villi organoids or pancreatic organoids.

[0617] The inventors have shown that biopsies can be collected from the small intestine and expanded for 7-14 days and organoids can be obtained for drug screening. The ability to obtain usable organoids of the present invention in a short period of time indicates that the organoids are highly useful for testing the response of individual patients to specific drugs and adjusting treatment based on the response. In some embodiments, where organoids are obtained from biopsies from patients, the organoids are cultured for less than 21 days, such as less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days (etc.).

[0618] Organoids can also be used for a wide range of drug discovery purposes. For example, Figures 32 to 40 show that small intestinal organoids collected from healthy patients and from cystic fibrosis patients can be used to test drugs for cystic fibrosis. In particular, Figures 32 to 40 show that forskolin-induced swelling of normal small intestinal organoids depends on the cystic fibrosis transmembrane conductance regulator (CFTR), so forskolin-induced swelling can be used as a positive readout to test the correction of CFTR function. Therefore, in some embodiments, the organoids of the present invention can be used to screen for cystic fibrosis drugs. However, it will be appreciated by the skilled artisan that the organoids of the present invention will be widely used as drug screening tools for the following: infectious pathologies, inflammatory pathologies, and tumor pathologies of the human gastrointestinal tract, as well as other diseases of the gastrointestinal tract, as well as infectious pathologies, inflammatory pathologies, and tumor pathologies of other tissues described herein, such as pancreas, liver, and prostate, and other diseases. In some embodiments, the organoids of the present invention can be used to screen for cancer drugs.

[0619] In some embodiments, expanded cell populations, such as organoids of the present invention or organoids obtained using the culture media and methods of the present invention, can be used to test chemical libraries, antibody libraries, natural product (plant extract) libraries, etc. suitable for use as drugs, cosmetics, and / or preventive medicine. For example, in some embodiments, cell biopsies from a target patient, such as tumor cells from a cancer patient, can be cultured using the culture media and methods of the present invention and then treated with a chemical compound or chemical library. It can then be determined which compound is effective in modifying, killing, and / or treating the patient's cells. This allows a specific patient's response to a specific drug to be tested, thereby allowing the treatment to be adjusted for the specific patient. Therefore, this allows for an individualized approach to medicine.

[0620] An additional advantage of using organoids for drug identification in this way is that normal organoids (organoids derived from healthy tissue) can also be screened to examine whether drugs and compounds have minimal effects on healthy tissue. This allows screening for drugs with minimal off-target activity or undesirable side effects.

[0621] In this way, drugs for a variety of diseases can be screened. For example, the organoids of the present invention can be used to screen for drugs for: cystic fibrosis, Barrett's esophagus, cancer, adenocarcinoma, adenoma, inflammatory bowel disease (e.g., Crohn's disease), liver disease, etc. The test parameters depend on the target disease. For example, when screening cancer drugs, cancer cell death is usually the ultimate goal. For cystic fibrosis, measuring the response of organoid expansion to drugs and CFTR stimulants is the goal. In other embodiments, metabolism or gene expression can be evaluated to study the effects of screened drugs and compounds on target cells or organoids.

[0622] Therefore, the present invention provides a method for screening therapeutic drugs or preventive drugs or cosmetics, wherein the method comprises:

[0623] For example, culturing the expanded cell population (e.g., organoid) of the invention using the culture medium of the invention for at least 21 days;

[0624] exposing the expanded cell population (e.g., organoid) of the present invention to a candidate molecule or a library of candidate molecules;

[0625] evaluating any effect of the expanded cell population (e.g., organoid), such as any change in the cells, such as a decrease or loss of proliferation, a change in morphology, and / or cell death;

[0626] Candidate molecules responsible for the described effects are identified as potential drugs or cosmetics.

[0627] In some embodiments, computer- or robotic-assisted culture and data acquisition methods are utilized to increase the throughput of the screen.

[0628] In some embodiments, the expanded cell population (eg, organoid) is obtained from a patient biopsy. In some embodiments, a candidate molecule that produces a desired effect on the cultured expanded cell population (eg, organoid) is administered to the patient.

[0629] Thus, in one aspect, there is provided a method of treating a patient comprising:

[0630] (a) obtaining a biopsy tissue slice from a target lesion tissue of a patient;

[0631] (b) screening suitable drugs using the screening method of the present invention; and

[0632] (c) treating the patient with the medicament obtained in step (b).

[0633] In some embodiments, the drug or cosmetic can be used to treat, prevent or improve the following symptoms: genetic diseases, metabolic diseases, pathogenic diseases, inflammatory diseases, etc., such as, but not limited to, cystic fibrosis, inflammatory bowel disease (such as Crohn's disease), cancer, adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or type II), Barrett's esophagus, Gaucher's disease, alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome, anemia, Schwachman-Bodian-Diamond syndrome, polycythemia vera, primary myelofibrosis, glycogen storage disease, familial hypercholesterolemia, Crigler-Najjar syndrome, hereditary tyrosinemia, Pompe's disease, progressive familial cholestasis, Hurler syndrome, SCID or leaky SCID, Omenn syndrome, cartilage-hair dysplasia, herpes simplex encephalitis, scleroderma, osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, dyskeratosis congenita, etc.

[0634] Target discovery

[0635] In some embodiments, organoids of the invention or cells grown using the culture medium or methods of the invention can be used for target discovery. Cells derived from organoids of healthy or diseased tissue can be used for target validation. The organoids of the present invention can be used for the discovery of drug targets for 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, alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome, anemia, Schwachman-Bodian-Diamond syndrome, polycythemia vera, primary myelofibrosis, glycogen storage disease, familial hypercholesterolemia, Crigler-Najjar syndrome, hereditary tyrosinemia, Pompe disease, progressive familial cholestasis, Hurler syndrome, SCID or leaky SCID, Omenn syndrome, chondro-hair dysplasia, herpes simplex encephalitis, scleroderma, osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, dyskeratosis congenita, etc. Cells and organoids cultured according to the culture medium and methods of the present invention are believed to faithfully represent the in vivo situation. In this regard, they can be tools for the discovery of novel (molecular) targets in specific diseases.

[0636] To study new drug targets, compound libraries (e.g., siRNA) can be used to transduce cells and inactivate specific genes. In some embodiments, cells are transduced with siRNA to inhibit the function of a gene (large) group. Any functional readout of a genome or specific cell function can be used to determine whether a target is relevant to the study. Disease-specific readouts are measured using assays known in the art. For example, cell proliferation is measured to detect genes involved in cancer. For example, the Topflash test described herein can be used to detect changes in Wnt activity caused by siRNA inhibition. When growth reduction or cell death occurs, corresponding siRNA-related genes can be determined by methods known in the art. These genes may be targets for inhibiting the growth of these cells. When determined, the specificity of the determined target for the cell process being studied will need to be determined by methods known in the art. Using these methods, new molecules can be determined as possible drug targets for treatment.

[0637] Target and drug confirmation screening

[0638] Patient-specific organoids derived from diseased and / or normal tissue can be used for target validation of molecules identified in high-throughput screens. The same is true for validation of compounds identified as possible therapeutic drugs in high-throughput screens. The use of original patient material expanded in organoid culture systems can be used to detect false positives from high-throughput drug discovery cell line studies, among other things.

[0639] In some embodiments, expanded stem cell populations (e.g., organoids of the invention), such as crypt-villus organoids or pancreatic organoids, can be used to validate compounds identified as possible drugs or cosmetics in high throughput screens.

[0640] Toxicity testing

[0641] Expanded stem cell populations (e.g., organoids of the invention), such as crypt-villus organoids or pancreatic organoids, can further replace the use of cell lines such as Caco-2 cells in toxicity testing of potential novel drugs or known or novel food supplements.

[0642] Toxicological screens are run in a similar manner to drug screens (described above), but they test the toxicity of the drug rather than its therapeutic effect. Thus, in some embodiments, the effect of the candidate drug is toxic.

[0643] Culture of pathogens

[0644] Furthermore, expanded stem cell populations (e.g., organoids of the invention), such as crypt-villus organoids or pancreatic organoids, can be used to culture pathogens, such as Norwalk virus, for which there is currently a lack of suitable tissue culture or animal models.

[0645] Regenerative medicine and transplantation

[0646] Cultures comprising expanded stem cell populations (e.g., organoids of the invention), such as crypt-villus organoids or pancreatic organoids, can be used in regenerative medicine, such as for post-radiation and / or post-operative repair of intestinal epithelium, for repair of intestinal epithelium in patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and for repair of intestinal epithelium in patients with short bowel syndrome. Other uses are presented in the repair of intestinal epithelium in the alleviation of genetic diseases of the small intestine / colon. Cultures comprising pancreatic organoids can also be used in regenerative medicine, such as for use as a transplant after resection of the pancreas or a portion thereof, and for the treatment of diabetes, such as diabetes I and diabetes II.

[0647] In an alternative embodiment, the expanded epithelial stem cells are reprogrammed into relevant final tissue types such as pancreatic cells including pancreatic β-cells. To date, it has not been possible to regenerate pancreatic cells from adult stem cells. The culture method of the present invention will enable analysis of factors that transdifferentiate the most relevant epithelial stem cells into pancreatic cells including pancreatic β-cells.

[0648] It is obvious to the technician that gene therapy can also be used for methods involving repairing damaged or diseased tissues. For example, the use of genetic information such as DNA and / or RNA for delivering to stem cells can be formed by adenoviral genes or retroviral gene delivery vectors. The technician can replace or repair the specific gene targeted in gene therapy. For example, a normal gene can be inserted into a non-specific position in the genome to replace a non-functional gene. In another example, the abnormal gene sequence can be replaced by a normal gene sequence through homologous recombination. Alternatively, selective reversion mutations can restore the gene to its normal function. Other examples are to change the regulation of a specific gene (to the extent that the gene is turned on or off). Preferably, stem cells are treated in vitro by a gene therapy method and then transferred to a mammal, preferably a person, in need of treatment.

[0649] Because small biopsies collected from adult donors can be amplified without significant limitations or genetic hazards, this technology can be used to generate transplantable epithelia for regenerative purposes. The fact that organoids can be frozen and thawed and placed in culture without loss of their 3D structure and integrity and without significant cell death increases the suitability of organoids for transplantation purposes. In addition, in some embodiments, organoids embedded in or in contact with ECM can be transplanted into mammals, preferably humans. In another embodiment, organoids and ECM can be transplanted into mammals, preferably humans, simultaneously.

[0650] The skilled person will appreciate that ECM can be used as a 3D scaffold to obtain an organized structure including an expanded cell population or organoid of the present invention. Such a structure can then be transplanted into a patient by methods known in the art. ECM scaffolds can be prepared synthetically using ECM proteins, such as collagen and / or laminin, or can be obtained by "decellularizing" separated organs or tissue fragments to leave a scaffold composed of ECM (see, for example, Macchiarini et al., The Lancet, Vol. 372, No. 9655, pp. 2023–2030, 2008). In some embodiments, an ECM scaffold is obtained by decellularizing an organ or tissue fragment, wherein optionally, the organ or tissue fragment is from the pancreas, liver, intestine, stomach, or prostate.

[0651] As described above, the present invention provides an organoid or cell population of the present invention for transplantation into a mammal, preferably into a human. Also provided is a method of treating a patient in need of a transplant, comprising transplanting an organoid or cell population of the present invention into the patient, wherein the patient is a mammal, preferably a human.

[0652] Advantageously, the present invention enables small biopsies to be taken from adult donors and expanded without any significant limitations or genetic hazards, and thus the techniques provided herein can be used to generate implantable epithelia for regenerative purposes.

[0653] Significantly, the inventors have found that when the human pancreatic organoids of the invention are implanted under the renal fascia of mice, these cells differentiate to form insulin-secreting beta cells. This is significant because it suggests that even if the cell populations or organoids of the invention do not secrete insulin at detectable levels and the cells or organoids are cultured in vitro, these cells can be used to implant patients to treat insulin deficiency, such as diabetes.

[0654] Thus, the present invention includes a method of treating an insulin deficiency disorder such as diabetes or a patient having a dysfunctional pancreas comprising transplanting a pancreatic organoid of the present invention or a cell derived from a pancreatic organoid of the present invention into said patient.

[0655] In some embodiments, the cells or organoids do not express or secrete insulin when transplanted into a patient, but differentiate within the patient so that they secrete insulin. For example, the ability to secrete insulin may not be detectable immediately after transplantation, but may be present within about 1 month after transplantation, such as 6 weeks, 2 months, or 3 months after transplantation.

[0656] The patient is preferably a human, but may alternatively be a non-human mammal, such as a cat, dog, horse, cow, pig, sheep, rabbit, or mouse.

[0657] Therefore, the method for treating human or non-human animal patients by cell therapy is included in the scope of the present invention.Such cell therapy includes applying the stem cell or organoid of the present invention to the patient by any suitable means.Specifically, such treatment method includes regenerating damaged tissue.According to the present invention, allogeneic or autologous stem cells or organoids can be used to treat patients. "Autologous" cells are derived from the same organism and are reintroduced into the organism for cell therapy, such as cells for allowing tissue regeneration. However, cells do not have to be separated from the same tissue as the tissue they are introduced into. Autologous cells do not need to be matched with patients to overcome rejection problems. "Allogeneic" cells are derived from cells of individuals different from the individual introduced by the cell (for cell therapy, such as for allowing tissue regeneration), although with the same species. A certain degree of patient matching may still be required to prevent rejection problems.

[0658] Typically, the cells or organoids of the present invention are introduced into the patient's body by injection or transplantation. Typically, the cells will be injected directly into the tissue in which they are intended to function. Alternatively, the cells are injected through the portal vein. A syringe comprising the cells of the present invention and a pharmaceutically acceptable carrier is included within the scope of the present invention. A catheter connected to a syringe comprising the cells of the present invention and a pharmaceutically acceptable carrier is included within the scope of the present invention.

[0659] The skilled artisan will be able to select an appropriate method and route of administration depending on the material being transplanted (ie, cell populations, single cells in a cell suspension, organoids, or organoid fragments) and the organ being treated.

[0660] As mentioned above, the cells of the present invention can be used for regenerating tissue. In order to achieve this function, cells can be directly injected or transplanted into damaged tissue, wherein according to their position in the body, they can breed and eventually differentiate into the desired cell type. Alternatively, organoids can be directly injected or transplanted into damaged tissue. Tissues affected by treatment include all damaged tissues, especially those that can be damaged by disease, i...

Claims

1. A culture medium for culturing an adult epithelial stem cell population, wherein the culture medium comprises: i. Agonist of Lgr5; and ii. p38 inhibitors. 2 . The culture medium according to claim 1 , wherein the agonist of Lgr5 is any one of Rspondin 1-4.

3. The culture medium according to claim 1 or claim 2, wherein the p38 inhibitor is selected from SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257 and BIRB-796.

4. The culture medium according to any one of the preceding claims, wherein the culture medium further comprises a TGF-β inhibitor.

5. The culture medium according to claim 4, wherein the TGF-β inhibitor binds to and reduces the activity of ALK5, ALK4 and / or ALK7.

6. The culture medium of claim 5, wherein the inhibitor that binds to and reduces the activity of ALK5, ALK4 or ALK7 is selected from A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511.

7. A culture medium according to any one of the preceding claims, wherein the culture medium comprises one or more additional components selected from the group consisting of: BMP inhibitors, Wnt agonists, receptor tyrosine kinase ligands, nicotinamide, Rock inhibitors, gastrin, RANKL, GSK3 inhibitors, prostaglandin signaling pathway activators and testosterone.

8. A composition comprising a culture medium as claimed in any one of claims 1 to 7 and an extracellular matrix or a 3D matrix, wherein the 3D matrix simulates an extracellular matrix through its interaction with cell membrane proteins such as integrins, the extracellular matrix, for example, an extracellular matrix containing laminin, such as Matrigel TM (BD Biosciences).

9. An airtight container comprising the culture medium or composition according to any one of the preceding claims.

10. Use of the culture medium according to any one of claims 1 to 7 for expanding or differentiating stem cells, stem cell populations, tissue fragments or organoids.

11. A method for expanding a single epithelial stem cell, a population of epithelial stem cells or tissue fragments, preferably to obtain organoids, wherein the method comprises culturing the single epithelial stem cell or the population of epithelial stem cells in a culture medium according to any one of claims 1 to 7.

12. The method according to claim 11, wherein the method comprises contacting the epithelial stem cells, epithelial stem cell populations or isolated tissue fragments and culture medium with an extracellular matrix or a 3D matrix that mimics an extracellular matrix through its interaction with cell membrane proteins such as integrins, such as an extracellular matrix containing laminin, such as Matrigel. TM (BD Biosciences).

13. The method according to claim 11 or 12, wherein the method comprises: Cultivating the epithelial stem cells, epithelial stem cell population or tissue fragments in the culture medium according to any one of claims 1 to 7; The epithelial stem cells, epithelial stem cell populations or tissue fragments are continued to be cultured and the culture medium is supplemented with a differentiation medium, wherein the differentiation medium does not include one or more factors selected from the group consisting of: TGF-β inhibitors, p38 inhibitors, nicotinamide and Wnt.

14. An organoid or cell population obtainable by the method of any one of claims 11 to 13.

15. A composition comprising: i) one or more organoids or cell populations according to claim 14; and ii) the culture medium and / or extracellular matrix according to any one of claims 1 to 7.

16. Use of the organoid according to claim 14 or the cell population according to claim 14 or the composition according to claim 8 or claim 15 for drug screening, target validation, target discovery, toxicology, toxicology screening or in vitro cell / organ model, such as use as a disease model.

17. An organoid according to claim 14 or a cell population according to claim 14 or a composition according to claim 8 or claim 15 for use in medicine or diagnosis, optionally wherein the medicine is personalized medicine or regenerative medicine.

18. A method for screening therapeutic drugs, preventive drugs or cosmetics, wherein the method comprises: Cultivating the organoid or cell population of claim 14, for example, using the culture medium of any one of claims 1 to 7; exposing the organoid or cell population to a candidate molecule or a library of candidate molecules; evaluating the organoid or cell population for any effect, such as any change in the cells, such as a decrease or loss of proliferation, a change in morphology, and / or cell death; and Candidate molecules responsible for the described effects are identified as potential drugs or cosmetics.

19. A culture medium suitable for expanding a population of adult epithelial stem cells, wherein the culture medium comprises a TGF-β inhibitor and an Lgr5 agonist.

20. A culture medium suitable for expanding hepatic epithelial stem cells, wherein the culture medium comprises: A TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511; R-spondin selected from Rspondin1-4; a prostaglandin signaling pathway activator selected from PGE2 and / or AA; and One or more mitogenic growth factors selected from the group consisting of EGF, HGF and FGF.

21. A culture medium for obtaining prostate organoids, wherein the culture medium comprises a basal medium, and further comprises testosterone or dihydrotestosterone, and further comprises any one of Rspondin 1-4 or a Rspondin mimetic.

22. Pancreatic organoids comprising β-cells.

23. A human organ selected from the group consisting of crypt-villus organoids, colon organoids, pancreatic organoids, gastric organoids, Barrett's esophagus organoids, adenocarcinoma organoids, and colon cancer organoids.

24. Intestinal or crypt-villus organoids, optionally obtained using the culture medium of any of the preceding claims, for use in treating damaged epithelium, such as in patients with microvillus inclusion disease (MVID).

25. A liver culture medium comprising or consisting of a basal medium for animal or human cells, to which is added: one or more receptor tyrosine kinase ligands, such as mitogenic growth factors (such as EGF); nicotinamide; and preferably a Wnt agonist, preferably R-spondin 1-4 and / or CHIR99021, and one or both of the following: a) a prostaglandin pathway activator, such as PGE2 and / or AA, and b) a TGF-β inhibitor, such as A83-01.

26. A method for differentiating small intestinal cells into M cells, wherein the method comprises contacting the small intestinal cells with a RANK ligand.

27. A culture medium suitable for differentiating intestinal cells, comprising or consisting of: Basal medium, EGF, TGF-β inhibitor and p38 inhibitor.

28. A culture medium suitable for differentiating hepatocytes, wherein the culture medium comprises a basal medium, one or more mitogenic growth factors, a TGF-β inhibitor and a Notch inhibitor.

29. A culture medium suitable for differentiating pancreatic cells, comprising or consisting of: Basal medium, EGF, FGF10, gastrin, TGF-β inhibitor and γ-secretase inhibitor.

30. A culture medium suitable for differentiating mouse pancreatic stem cells, wherein the culture medium comprises a basal medium, EGF and a γ-secretase inhibitor, optionally wherein the γ-secretase inhibitor is DAPT or DBZ.

31. A culture medium suitable for culturing prostate cells, wherein the culture medium comprises a basal medium, EGF, Rspondin 1-4, nicotinamide and a TGF-β inhibitor.

32. A culture medium suitable for differentiating Barrett's esophagus epithelial cells, wherein the culture medium comprises a γ-secretase inhibitor, optionally wherein the γ-secretase inhibitor is DBZ, optionally at a final concentration of about 10 μM.

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