Drug for culturing organoid in absence of extracellular matrix

Abstract

Provided are: a drug for forming and proliferating an organoid in the absence of an extracellular matrix, the drug containing an inhibitor for a Hippo signaling pathway as an active ingredient; a proliferation method of an organoid; an organoid proliferated by said proliferation method; a drug for establishing an organoid in the absence of an extracellular matrix; a production method for an organoid; an organoid produced by said production method; a regenerative medicine formulation; and a screening method for a drug with which an organoid can be cultured in the absence of an extracellular matrix.

Description

Agents for culturing organoids in the absence of extracellular matrix

[0001] The present invention relates to agents for culturing organoids in the absence of an extracellular matrix. More specifically, the present invention relates to agents for forming and growing organoids in the absence of an extracellular matrix, methods for growing organoids, organoids grown by the methods, agents for establishing organoids in the absence of an extracellular matrix, methods for producing organoids, organoids produced by the methods, regenerative medicine preparations, and methods for screening agents that enable organoid culture in the absence of an extracellular matrix. This application claims priority based on Japanese Patent Application No. 2022-153698 filed in Japan on September 27, 2022, and Japanese Patent Application No. 2023-090460 filed in Japan on May 31, 2023, the contents of which are incorporated herein by reference.

[0002] Organoid culture technology has been developed, and its application in drug discovery and regenerative medicine is expected. To culture organoids, an extracellular matrix such as Matrigel (registered trademark) is required (see, for example, Patent Document 1, Non-Patent Document 1, etc.). Matrigel (registered trademark) is an animal-derived product, as it is an extract from tumors transplanted into mice. Furthermore, the use of Matrigel (registered trademark) in organoid culture poses a barrier to medical applications due to the possibility of contamination with unknown viruses and its high cost. Furthermore, the use of Matrigel poses challenges from the perspective of animal welfare. Since the emergence of organoid technology, vigorous efforts have been made to develop alternatives to Matrigel (registered trademark) (see, for example, Non-Patent Document 2).

[0003] Japanese Patent Application Laid-Open No. 2016-198033

[0004] S Rezakhani, et al., Extracellular matrix requirements for gastrointestinal organoid cultures, Biomaterials, 276, 121020, 2021. Jeong Hyun Heo, et al., Engineering the Extracellular Matrix for Organoid Culture, Int J Stem Cells, 15 (1), 60-69, 2022.

[0005] Conventionally, synthetic extracellular matrices and collagen have been used as alternatives to Matrigel (registered trademark), but they have not yet achieved sufficient organoid culture efficiency and are not yet widely used.

[0006] Therefore, an object of the present invention is to provide a technique for producing and culturing organoids in the absence of extracellular matrix.

[0007] The present invention includes the following aspects: [1] A drug for forming and growing organoids in the absence of an extracellular matrix, comprising an inhibitor of the Hippo signaling pathway as an active ingredient. [2] The drug for forming and growing organoids in the absence of an extracellular matrix according to [1], wherein the inhibitor of the Hippo signaling pathway is an MST1 / 2 kinase inhibitor or a large tumor suppressor kinase (LATS)1 / 2 kinase inhibitor. [3] The drug for forming and growing organoids in the absence of an extracellular matrix according to [1] or [2], further comprising a cytokine family that binds to gp130. [4] The drug for forming and growing organoids in the absence of an extracellular matrix according to any of [1] to [3], wherein the organoids are epithelial organoids. [5] A method for growing organoids, comprising culturing organoids in the presence of an inhibitor of the Hippo signaling pathway and in the absence of an extracellular matrix. [6] The method for growing organoids according to [5], wherein the organoids are cultured under serum-free conditions. [7] Organoids grown by the method for growing organoids according to [5] or [6]. [8] A drug for establishing organoids in the absence of an extracellular matrix, comprising an inhibitor of the Hippo signaling pathway as an active ingredient. [9] The drug for establishing organoids in the absence of an extracellular matrix according to [8], wherein the inhibitor of the Hippo signaling pathway is an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor.

[10] The drug for establishing organoids in the absence of an extracellular matrix according to [8] or [9], further comprising a cytokine family that binds to gp130.

[11] The drug for establishing organoids in the absence of an extracellular matrix according to any of [8] to

[10] , wherein the organoids are epithelial organoids.

[12] A method for producing organoids, comprising the step of establishing organoids in the presence of an inhibitor of the Hippo signaling pathway and in the absence of extracellular matrix.

[13] The method for producing organoids according to

[12] , wherein the establishment of the organoids is carried out in the presence of a cytokine family that binds to gp130.

[14] The method for producing organoids according to

[12] or

[13] , wherein the establishment of the organoids is carried out under serum-free conditions.

[15] An organoid produced by the production method according to any one of

[12] to

[14] .

[16] A regenerative medicine preparation containing the organoid according to

[15] as an active ingredient.

[17] A method for screening for a drug that enables organoid culture in the absence of an extracellular matrix, the method comprising the steps of culturing organoids in the presence of a test substance and in the absence of an extracellular matrix, and evaluating the proliferation of the organoids, wherein higher proliferation of the organoids compared to that in the absence of the test substance indicates that the test substance is a drug that enables organoid culture in the absence of an extracellular matrix.

[0008] The present invention provides a technique for culturing organoids in the absence of an extracellular matrix.

[0009] FIG. 1 is a schematic diagram illustrating the Hippo signaling pathway. FIG. 2 is a representative photograph and graph showing the results of Experimental Example 1. FIG. 3 is a representative photograph and graph showing the results of Experimental Example 2. FIG. 4 is a representative photograph showing the results of Western blotting in Experimental Example 3. FIG. 5 is a representative photograph showing the results of immunostaining of human small intestine-derived organoids in Experimental Example 4. FIG. 6 is a representative photograph showing the results of immunostaining of human large intestine-derived organoids in Experimental Example 4. FIG. 7 is a graph summarizing the results of FIGS. 5 and 6. FIG. 8 is a graph showing the results of Experimental Example 5. FIG. 9 is a representative photograph showing the results of Experimental Example 6. FIG. 10 is a graph showing the results of Experimental Example 7. FIG. 11 is a representative photograph showing the results of Experimental Example 8. FIG. 12 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 9. FIG. 13 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 10. FIG. 14 is a graph showing the results of Experimental Example 11. 15 is a graph showing the results of Experimental Example 11. FIG. 16 is a graph showing the results of quantitative real-time PCR in Experimental Example 12. FIG. 17 is a graph showing the results of Western blotting in Experimental Example 12. FIG. 18 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 13. FIG. 19 is a representative micrograph of organoids in Experimental Example 13. FIG. 20 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 14. FIG. 21 is a representative micrograph of organoids in Experimental Example 14. FIG. 22 is a graph showing the results of Experimental Example 15. FIG. 23 is a representative micrograph of organoids in Experimental Example 16. FIG. 24 is a graph showing the results of Experimental Example 16. FIG. 25 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 17. FIG. 26 is a graph showing the results of Experimental Example 18. FIG. 27 is a graph showing the results of Experimental Example 19. FIG. 28 is a graph showing the results of Experimental Example 20. Fig. 28 is a micrograph and graph showing the results of Experimental Example 21. Fig. 30 is a schematic diagram outlining Experimental Example 22 and a graph showing the results of Experimental Example 22. Fig. 31 is a schematic diagram outlining Experimental Example 23. Fig. 32 is a photograph showing the results of Experimental Example 23.

[0010] [Drug for Proliferating Organoids in the Absence of Extracellular Matrix] In one embodiment, the present invention provides a drug for forming and proliferating organoids in the absence of extracellular matrix, the drug comprising an inhibitor of the Hippo signaling pathway as an active ingredient.

[0011] As will be described later in the Examples, by adding the agent of this embodiment to the culture medium, it becomes possible to form and grow organoids in the absence of extracellular matrix. Here, "growing" means increasing the number of cells constituting the organoid, and can also be called "culturing". Furthermore, "forming" organoids means establishing organoids, forming organoids again from organoids dissociated into single cells, etc. In one aspect, the agent of this embodiment may be an agent for growing organoids in the absence of extracellular matrix, which contains an inhibitor of the Hippo signaling pathway as an active ingredient.

[0012] Conventionally, it has been impossible to grow organoids in the absence of an extracellular matrix. The agent of this embodiment makes it possible to grow organoids without using any animal-derived products, such as Matrigel (registered trademark), which is an extracellular matrix. Here, animal-derived products refer to substances of unidentified components derived from humans or non-human animals. It is expected that organoids can be applied to regenerative medicine using animal-derived product-free culture techniques. Furthermore, from the standpoints of animal welfare and cost, this method is more advantageous than conventional organoid culture techniques.

[0013] The Hippo signaling pathway is a signaling pathway known to be involved in cell proliferation, apoptosis, stem cell self-renewal, and the like, and is known to be an evolutionarily conserved pathway.

[0014] Figure 1 is a schematic diagram illustrating the Hippo signaling pathway. As shown in Figure 1, the transcription factor TEAD activates the transcription of genes involved in cell proliferation by binding to the coactivator YAP (Yes-associated protein), thereby promoting cell proliferation. YAP exists in phosphorylated and unphosphorylated forms, and unphosphorylated YAP translocates to the nucleus and acts as a transcriptional coactivator for TEAD. On the other hand, phosphorylated YAP binds to the cytoplasmic protein 14-3-3 and cannot translocate to the nucleus, so it cannot function as a coactivator. Therefore, phosphorylation regulation, which determines the nuclear translocation of YAP, is a very important event in cell proliferation.

[0015] Furthermore, as shown in Figure 1, large tumor suppressor kinase (LATS) is known to phosphorylate YAP, localizing it in the cytoplasm and thereby negatively regulating the involvement of YAP in cell proliferation.

[0016] The inhibitor of the Hippo signaling pathway can be any inhibitor that inhibits any step in the above-mentioned signaling pathway, without any particular limitation.

[0017] For example, it may be an inhibitor of MST1 kinase or MST2 kinase (MST1 / 2 kinase inhibitor), or an inhibitor of LATS1 kinase or LATS2 kinase (LATS1 / 2 kinase inhibitor).

[0018] The NCBI accession numbers for the amino acid sequences of human MST1 kinase are NP_001380510.1, NP_001380511.1, NP_001380512.1, NP_001380513.1, NP_001380514.1, NP_066278.3, etc. The NCBI accession numbers for the amino acid sequences of human MST2 kinase are NP_001243241.1, NP_001243242.1, NP_006272.2, etc.

[0019] The NCBI accession numbers for the amino acid sequences of human LATS1 kinase are NP_001257448.1, NP_001337268.1, NP_001337269.1, NP_001337321.1, NP_004681.1, etc. The NCBI accession numbers for the amino acid sequences of human LATS2 kinase are NP_055387.2, etc.

[0020] More specific examples of inhibitors of the Hippo signaling pathway include inhibitors of LATS1 kinase and LATS2 kinase, such as Lats-IN-1 (CAS number: 1424635-83-5, hereinafter sometimes referred to as "Lats-IN" and also referred to as "TRULI"), GA-017 (CAS number: 2351906-74-4), and TDI-011536 (CAS number: 2687970-96-1).

[0021] Examples of extracellular matrices include Matrigel (registered trademark), collagen, fibronectin, proteoglycan, laminin, etc. Conventionally, it has been impossible to grow organoids in the absence of an extracellular matrix.

[0022] Growing organoid in the absence of extracellular matrix means that extracellular matrix is ​​not added from the outside to the culture medium of organoid, and it is acceptable that a trace amount of extracellular matrix produced by organoid itself is mixed into the culture medium, or that a trace amount of extracellular matrix is ​​mixed into the culture medium unintentionally.Here, the trace amount may be within the detection limit.

[0023] In the drug of this embodiment, the organoid includes epithelial organoids, such as small intestine-derived organoids, colon-derived organoids, stomach-derived organoids, biliary tract-derived organoids, pancreatic organoids, mammary gland-derived organoids, hepatocyte organoids, lung organoids, airway organoids, esophageal organoids, salivary gland organoids, and the like.

[0024] Organoid can be the organoid of human origin, or can be the organoid of non-human animal origin.Non-human animal can include mammals, for example, rodents such as mouse, rat, hamster, guinea pig, etc.; ungulates such as pig, cow, goat, horse, sheep, etc.; carnivores such as dog, cat, etc.; primates such as rhesus monkey, cynomolgus monkey, marmoset, orangutan, chimpanzee, etc.

[0025] The drug of this embodiment is used by adding it to an organoid culture medium. The concentration of the drug of this embodiment in the organoid culture medium is preferably about 10 μM or more and 60 μM or less, more preferably about 10 μM or more and 30 μM or less.

[0026] Examples of organoid culture media include those obtained by removing the extracellular matrix from conventional organoid culture media. A preferred organoid culture medium is a basal medium supplemented with at least one of the following components i) to v). While the organoid culture medium may contain serum, it is preferably serum-free, as it is preferable to be free of animal-derived products. i) Wnt agonist; ii) at least one selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), EGF (epidermal growth factor) and epiregulin (EREG), fibroblast growth factor 10 (FGF10), and gastrin-1; iii) bone morphogenetic protein (BMP) inhibitor; iv) transforming growth factor-β (transforming growth factor-β) inhibitor. factor-β, TGF-β inhibitors v) gamma secretase inhibitors

[0027] The organoid culture medium preferably contains at least one selected from the group consisting of IGF1, FGF2, EGF, and epiregulin. The IGF1, FGF2, EGF, and epiregulin to be contained can be selected appropriately. The organoid culture medium preferably contains IGF1 and epiregulin, FGF2 and epiregulin, or IGF1 and FGF2, and more preferably contains IGF1 and FGF2.

[0028] Any serum-free basal cell culture medium can be used as the basal medium. Examples include defined synthetic media buffered to a pH of 7.2 or higher and 7.6 or lower with a carbonate buffer. More specifically, examples include Advanced Dulbecco's Modified Eagle's Medium / Ham's F-12 Mixed Medium (DMEM / F12) supplemented with glutamine, insulin, B27 supplement (Thermo Fisher Scientific), N-acetyl-L-cysteine ​​(Fujifilm Wako Pure Chemical Industries), penicillin, streptomycin, transferrin, etc. Alternatively, RPMI 1640 medium, Advanced RPMI medium, etc. may be used instead of DMEM / F12 medium.

[0029] (Wnt Agonist) A Wnt agonist refers to a drug that activates T-cell factor (hereinafter also referred to as TCF) / lymphoid enhancer factor (hereinafter also referred to as LEF)-mediated transcription in cells. Therefore, Wnt agonists are not limited to Wnt family proteins, but also include Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activators. The Wnt agonist is preferably at least one selected from the group consisting of Wnt proteins, R-spondin, and GSK-3β inhibitors.

[0030] The organoid culture medium preferably contains a Wnt agonist, more preferably a complex of a Wnt protein and its stabilizing substance, afamin, and even more preferably a complex of a Wnt protein and afamin and R-spondin.

[0031] <<Wnt Protein>> The origin of the Wnt protein is not particularly limited, and Wnt proteins derived from various organisms can be used. Among them, Wnt proteins derived from mammals are preferable. Examples of mammals include those similar to those described above. Examples of mammalian Wnt proteins include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In the organoid culture medium, multiple types of Wnt proteins may be used in combination.

[0032] Methods for preparing Wnt proteins include, for example, methods using Wnt protein-expressing cells. The origin of the Wnt protein-expressing cells (e.g., biological species, culture form) is not particularly limited, as long as they stably express Wnt proteins, and they may also be cells that transiently express Wnt proteins. Examples of Wnt protein-expressing cells include L cells (ATCC CRL-2647) that stably express mouse Wnt3a and L cells (ATCC CRL-2814) that stably express mouse Wnt5a. Furthermore, Wnt protein-expressing cells can be produced using known genetic recombination techniques. Specifically, Wnt protein-expressing cells can be produced by inserting DNA encoding a desired Wnt protein into a known expression vector and then introducing the resulting expression vector into an appropriate host cell. The nucleotide sequence of the gene encoding the desired Wnt protein can be obtained from known databases, such as GenBank.

[0033] The Wnt protein expressed by the Wnt protein-expressing cells may be a fragment of the Wnt protein or may contain an amino acid sequence other than that of the Wnt protein, as long as it has Wnt activity. The amino acid sequence other than that of the Wnt protein is not particularly limited, and examples thereof include the amino acid sequence of an affinity tag. Furthermore, the amino acid sequence of the Wnt protein does not need to be completely identical to an amino acid sequence obtainable from a publicly known database such as GenBank; as long as it has Wnt activity, it may be substantially the same as an amino acid sequence obtainable from a publicly known database.

[0034] Examples of amino acid sequences that are substantially identical to the amino acid sequences of Wnt proteins that can be obtained from publicly known databases such as GenBank include amino acid sequences in which one to several amino acids have been deleted, substituted, or added to the amino acid sequences that can be obtained from publicly known databases.

[0035] An amino acid sequence in which one to several amino acids have been deleted, substituted or added means that the number of amino acids that can be deleted, substituted or added (preferably 10 or less, more preferably 7 or less, and even more preferably 6 or less) has been deleted, substituted or added by, for example, a known method for producing mutant peptides such as site-directed mutagenesis.

[0036] Furthermore, examples of substantially identical amino acid sequences include amino acid sequences that have an identity of at least 80% or more, preferably at least 85% or more, more preferably at least 90% or more, even more preferably at least 92% or more, particularly preferably at least 95% or more, and most preferably at least 99% or more with an amino acid sequence that can be obtained from a publicly known database.

[0037] The concentration of Wnt protein is preferably 50 ng / mL or more, more preferably 100 ng / mL to 10 μg / mL or less, even more preferably 200 ng / mL to 1 μg / mL or less, and particularly preferably 300 ng / mL to 1 μg / mL or less.

[0038] R-spondins include the R-spondin family consisting of R-spondin1, R-spondin2, R-spondin3, and R-spondin4. The R-spondin family is a secreted protein known to be involved in the activation and regulation of the Wnt signaling pathway. Multiple types of R-spondin may be used in combination in organoid culture media. Furthermore, as long as they have R-spondin activity, they may be fragments of R-spondin or may contain an amino acid sequence other than the amino acid sequence of R-spondin.

[0039] <<GSK-3β Inhibitors>> Examples of GSK-3β inhibitors include CHIR-99021 (CAS No.: 252917-06-9), CHIR-98014 (CAS No.: 252935-94-7), lithium, Kenpaullone (CAS No.: 142273-20-9), 6-bromoindirubin-30-acetoxime, SB216763 (CAS No.: 280744-09-4), SB415286 (CAS No.: 264218-23-7), FRAT family members that inhibit the interaction between GSK-3 and axin, and FRAT-derived peptides.

[0040] Afamin refers to a glycoprotein belonging to the albumin family, and is known to be present in blood and body fluids. Serum, which is typically added to culture media, contains afamin derived from the animal from which the serum was collected. Because serum contains impurities other than afamin, it is preferable to use afamin alone without serum.

[0041] The origin of afamin contained in the organoid culture medium is not particularly limited, and afamin derived from various organisms can be used. Among these, afamin derived from mammals is preferable. Examples of mammals include those described above. The amino acid sequences of major mammalian afamins and the nucleotide sequences of the genes encoding them can be obtained from publicly known databases such as GenBank. For example, in GenBank, the amino acid sequence of human afamin is registered under the accession number AAA21612, and the nucleotide sequence of the gene encoding it is registered under the accession number L32140, while the amino acid sequence of bovine afamin is registered under the accession number DAA28569, and the nucleotide sequence of the gene encoding it is registered under the accession number GJ060968.

[0042] The afamin contained in the organoid culture medium may be natural afamin contained in serum or the like, purified by a known method, or it may be recombinant afamin, which can be produced by appropriately using known genetic recombination techniques.

[0043] Recombinant afamin can be produced, for example, by inserting DNA encoding afamin into a known expression vector, introducing the resulting expression vector into an appropriate host cell to express the recombinant afamin, and purifying it using a known purification method. The recombinant afamin may also be afamin to which an affinity tag has been added. The affinity tag to be added is not particularly limited, and can be appropriately selected from known affinity tags. The affinity tag is preferably an affinity tag that can be recognized by a specific antibody, and examples include a FLAG tag, a MYC tag, an HA tag, and a V5 tag.

[0044] The Wnt proteins described above are highly hydrophobic because specific serine residues are modified with fatty acids (palmitoleic acid), and therefore Wnt proteins are prone to aggregation or denaturation in aqueous solutions, making them very difficult to purify and store.

[0045] Meanwhile, it has been reported that modification of this specific serine residue with a fatty acid is essential for the physiological activity of Wnt proteins and is involved in binding to members of the Frizzled receptor family.

[0046] It is also known that in aqueous solution, Wnt proteins bind one-to-one with afamin to form a complex, which is solubilized while maintaining high physiological activity. Wnt protein-afamin complexes can be produced by culturing cells that express both Wnt proteins and afamin, or by co-culturing Wnt protein-expressing cells and afamin-expressing cells.

[0047] The concentration of afamin contained in the organoid culture medium is not particularly limited, but is preferably 50 ng / mL to 10 μg / mL, more preferably 100 ng / mL to 1 μg / mL, and even more preferably 300 μg / mL to 1 μg / mL.

[0048] (IGF1) IGF1, also known as somatomedin C, is a factor secreted primarily in the liver in response to stimulation by growth hormone (GH). It is known that most cells in the human body (particularly those of muscle, bone, liver, kidney, nerve, skin, lung, etc.) are affected by IGF1. In addition to its insulin-like effects, IGF1 also functions to regulate cell growth (particularly nerve cells), development, and cellular DNA synthesis.

[0049] The concentration of IGF1 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL or more and 1 μg / mL or less, more preferably 10 ng / mL or more and 1 μg / mL or less, and even more preferably 50 ng / mL or more and 500 ng / mL or less.

[0050] (FGF2) FGF2 is a basic fibroblast growth factor that binds to the fibroblast growth factor receptor (FGFR) and promotes the proliferation and organization of vascular endothelial cells into tubular structures, i.e., angiogenesis. Human FGF2 is known to have two isoforms: low molecular weight (LWL) and high molecular weight (HWL). LWL is primarily present in the cytoplasm and acts via an autocrine mechanism, while HWL is present in the nucleus and is active via an intracellular intracrine mechanism.

[0051] The concentration of FGF2 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL to 500 μg / mL, more preferably 10 ng / mL to 300 μg / mL, and even more preferably 50 ng / mL to 100 μg / mL.

[0052] (FGF10) FGF10 is a molecule secreted mainly from mesenchymal tissues, acts on epithelia via FGFR2b, and is responsible for epithelial-mesenchymal interactions that are important in the formation and repair of various tissues.

[0053] The concentration of FGF10 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL or more and 1 μg / mL or less, more preferably 10 ng / mL or more and 1 μg / mL or less, and even more preferably 30 ng / mL or more and 500 ng / mL or less.

[0054] EGF is a potent mitogen for a variety of cultured ectodermal and mesodermal cells and has a profound effect on the differentiation of specific cells, including some fibroblasts. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53-amino acid peptide hormone that stimulates cells.

[0055] The concentration of EGF contained in the organoid culture medium is preferably 5 ng / mL or more and 500 ng / mL or less, more preferably 10 ng / mL or more and 400 ng / mL or less, and even more preferably 50 ng / mL or more and 200 ng / mL or less.

[0056] (EREG) EREG is an EGF-like growth factor that specifically binds to ErbB1 and ErbB4 of the tyrosine kinase (ErbB) family receptors (ErbB1-4). It is known to stimulate the proliferation of keratinocytes, hepatocytes, fibroblasts, and vascular endothelial cells. EREG is also expressed primarily in malignant tumors of the bladder, lung, kidney, colon, etc., as well as in the placenta and peripheral blood leukocytes.

[0057] The concentration of EREG contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL or more and 1 μg / mL or less, more preferably 10 ng / mL or more and 1 μg / mL or less, and even more preferably 50 ng / mL or more and 500 ng / mL or less.

[0058] (BMP Inhibitors) BMP binds as a dimeric ligand to a receptor complex consisting of two different receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, resulting in activation of the receptor kinase. The type I receptor then phosphorylates specific receptor substrates (SMADs), resulting in transcriptional activity via a signal transduction pathway. Generally, BMP inhibitors are, for example, drugs that bind to BMP molecules to form complexes that neutralize BMP activity, such as drugs that block or inhibit the binding of BMP molecules to BMP receptors. BMP inhibitors are also, for example, drugs that bind to BMP receptors and block or inhibit the binding of BMP molecules to the receptor, acting as antagonists or inverse agonists.

[0059] The BMP inhibitor preferably has an inhibitory activity of 50% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, compared to the BMP activity level in the absence of the inhibitor.

[0060] The BMP inhibitor is preferably a natural BMP binding protein, and examples thereof include chordin-like proteins such as noggin, gremlin, chordin, and chordin domain; follistatin-related proteins such as follistatin and follistatin domain; DAN-like proteins such as DAN and DAN cysteine-knot domain; sclerostin / SOST, decorin, and α-2 macroglobulin.

[0061] Among the BMP inhibitors contained in the organoid culture medium, chordin-like protein or DAN-like protein is preferred, with chordin-like protein being more preferred. As the chordin-like protein, noggin is preferred. Chordin-like protein and DAN-like protein are diffusible proteins that bind to BMP molecules with varying affinities and can inhibit the BMP molecules from approaching signaling receptors. When culturing epithelial stem cells, adding these BMP inhibitors to the organoid culture medium can prevent stem cell loss.

[0062] The concentration of the BMP inhibitor contained in the organoid culture medium is preferably 10 ng / mL or more and 100 ng / mL or less, more preferably 20 ng / mL or more and 100 ng / mL or less, and even more preferably 50 ng / mL or more and 100 ng / mL or less.

[0063] (TGF-β Inhibitors) TGF-β is a type of growth factor that is produced by almost all cells, including those in the kidney, bone marrow, and platelets. There are five subtypes of TGF-β (β1 to β5). TGF-β is known to promote the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, while suppressing the proliferation of epithelial cells and osteoclasts. In general, TGF-β inhibitors are, for example, drugs that block or inhibit the binding of TGF-β to TGF-β receptors, and bind to TGF-β to form a complex that neutralizes TGF-β activity. TGF-β inhibitors are, for example, drugs that bind to TGF-β receptors and block or inhibit the binding of TGF-β to the receptor, and act as antagonists or inverse agonists.

[0064] Examples of TGF-β inhibitors include A83-01 (CAS number: 909910-43-6), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (CAS No.: 1062368-24-4), SB-431542 (CAS No.: 301836-41-9), SB-505124 (CAS No.: 694433-59-5), SD-208 (CAS No.: 627536-09-8), SB-525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), LY2157299 (CAS No.: 700874-72-2), TGF-β RI Kinase Inhibitor II 616452 (CAS No.: 446859-33-2), TGF-β RI Kinase Inhibitor III 616453 (CAS No.: 356559-13-2), TGF-β RI Kinase Inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (CAS No.: 666729-57-3), TGF-β RI Kinase Inhibitor VIII 616459 (CAS number: 356559-20-1), AP12009 (TGF-β2 antisense compound "Trabedersen"), Belagenpumatucel-L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human IgG4 monoclonal antibody that neutralizes TGFβ1)), GC-1008 (anti-TGF-β monoclonal antibody), and the like. Of these, A83-01 is preferred as a TGF-β inhibitor.

[0065] The concentration of the TGF-β inhibitor contained in the organoid culture medium is preferably 100 nM or more and 10 μM or less, more preferably 500 nM or more and 5 μM or less, and even more preferably 500 nM or more and 2 μM or less.

[0066] (γ-Secretase Inhibitors) Examples of γ-secretase inhibitors include BMS299897 (CAS number: 290315-45-6), DAPT (CAS number: 208255-80-5), DBZ (CAS number: 209984-56-5), JLK6 (CAS number: 62252-26-0), L-685458 (CAS number: 292632-98-5), LY411575 (CAS number: 209984-57-6), etc. Among these, LY411575 is preferred.

[0067] The concentration of the gamma-secretase inhibitor contained in the organoid culture medium is preferably 1 nM or more and 10 μM or less, and more preferably 100 nM or more and 1 μM or less.

[0068] (Other Components) The organoid culture medium may further contain a Rho-kinase (Rock) inhibitor. Examples of Rock inhibitors include Y-27632 (CAS number: 129830-38-2), fasudil (HA1077) (CAS number: 103745-39-7), and H-1152 (CAS number: 871543-07-6). When using Y-27632 as a Rock inhibitor, it is preferably added during the first two days of culturing the stem cells dispersed into single cells. The concentration of Y-27632 contained in the organoid culture medium is preferably about 10 μM.

[0069] When the organoid is a hepatocyte organoid, the organoid culture medium preferably further contains a cytokine of the cytokine family that binds to gp130, such as oncostatin M. The concentration of oncostatin M contained in the organoid culture medium may be, for example, 1 ng / mL to 10 μg / mL, for example, 1 ng / mL to 1 μg / mL, or for example, 5 ng / mL to 100 ng / mL.

[0070] The organoid culture medium may further contain gastrin (or a suitable substitute such as Leu15-gastrin). The concentration of gastrin or a suitable substitute contained in the organoid culture medium may be, for example, 1 ng / mL to 10 μg / mL, or may be, for example, 1 ng / mL to 1 μg / mL, or may be, for example, 5 ng / mL to 100 ng / mL.

[0071] The organoid culture medium may further contain at least one amino acid. Examples of amino acids 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. The concentration of L-glutamine contained in the organoid culture medium is 0.05 g / L to 1 g / L (usually 0.1 g / L to 0.75 g / L). The concentration of other amino acids contained in the organoid culture medium is 0.001 g / L to 1 g / L (usually 0.01 g / L to 0.15 g / L). The amino acids may be synthetic.

[0072] The organoid culture medium may further contain at least one vitamin, such as 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), menadione (vitamin K), and the like.

[0073] The organoid culture medium may further comprise at least one inorganic salt.The inorganic salt is for helping to maintain the osmotic balance of cells and for helping to regulate membrane potential.Specific examples of inorganic salt include calcium, copper, iron, magnesium, potassium, sodium, and zinc salts.Salts are usually used in the form of chloride, phosphate, sulfate, nitrate, and bicarbonate.More specific salts include CaCl ２ , CuSO ４ -5H ２ O, Fe(NO ３ ) -9H ２ O, FeSO ４ -7H ２ O, MgCl, MgSO ４ , KCl, NaHCO ３ , NaCl, Na ２ HPO ４ , Na ２ HPO ４ -H ２ O, ZnSO ４ -7H ２ Examples include O.

[0074] The organoid culture medium may further contain at least one sugar that can serve as a carbon energy source. Examples of sugars include glucose, galactose, maltose, and fructose. Among these, glucose is preferred, and D-glucose (dextrose) is particularly preferred. The concentration of sugar contained in the organoid culture medium is preferably 1 g / L or more and 10 g / L or less.

[0075] The organoid culture medium may further contain at least one trace element, such as barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0076] The organoid culture medium may further comprise at least one additional agent, such as a nutrient or growth factor that has been reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite, etc.

[0077] [organoid proliferation method] In one embodiment, the present invention provides the organoid proliferation method, comprising the step of culturing organoid in the presence of the inhibitor of Hippo signaling pathway and in the absence of extracellular matrix.The organoid proliferation method of this embodiment can also be said to be the method for producing organoid.

[0078] As will be described later in Examples, the method of this embodiment can make organoid grow in the absence of extracellular matrix.Here, "grow" means that the number of cells that constitute organoid increases, and can also be called "culture".

[0079] In the method of this embodiment, the inhibitor of Hippo signaling pathway, extracellular matrix, organoid, etc. are the same as those described above. That is, in the method of this embodiment, the inhibitor of Hippo signaling pathway is preferably an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor. In addition, in the method of this embodiment, the organoid is preferably an epithelial organoid.

[0080] In the method of this embodiment, organoid can also be cultured under serum-free conditions.By growing organoid in the absence of extracellular matrix and serum, it is possible to obtain the organoid that is produced without using any animal-derived products.

[0081] [Organoids] In one embodiment, the present invention provides organoids grown by the above-described growth method.

[0082] The organoids of this embodiment are produced without animal-derived products, making them suitable for use in regenerative medicine.

[0083] The organoid of this embodiment may be a gastrointestinal organoid. Conventional gastrointestinal organoids cultured in the presence of Matrigel (registered trademark) are spherical in shape, with a monolayer epithelial cell sheet with apical polarity maintained, with the inside facing apically (apical side).

[0084] In contrast, as described below in the Examples, the organoids of this embodiment have a spherical morphology in which cells are layered, and the apical polarity of the epithelial cells is not maintained, which differs from the morphology of conventional gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).

[0085] The organoid of this embodiment is considered to have differences in gene expression patterns and the like with conventional organoids.However, it is unclear whether it is possible to identify the differences in gene expression patterns and clearly distinguish between conventional organoids and the organoid of this embodiment, and this is not practical.Therefore, it is considered practical to define the organoid of this embodiment by manufacturing method.

[0086] [Drug for establishing organoids in the absence of extracellular matrix] In one embodiment, the present invention provides a drug for establishing organoids in the absence of extracellular matrix, the drug comprising an inhibitor of the Hippo signaling pathway as an active ingredient.

[0087] As mentioned above, the inventors have revealed that by adding the inhibitor of Hippo signaling pathway to culture medium, organoid can be grown in the absence of extracellular matrix.However, just because the organoid that has already been established can be grown, it does not mean that by adding the inhibitor of Hippo signaling pathway to culture medium, organoid can be established from tissue derived from living body.

[0088] In contrast, as described below in the Examples, the inventors have demonstrated that by adding the agent of this embodiment to a culture medium, organoids can be established from tissues derived from living organisms in the absence of an extracellular matrix, and that organoids can also be established from single cells without the use of an extracellular matrix.

[0089] In the drug of this embodiment, the inhibitor of Hippo signaling pathway, extracellular matrix, organoid, etc. are the same as those described above. That is, in the method of this embodiment, the inhibitor of Hippo signaling pathway is preferably an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor. In addition, in the drug of this embodiment, the organoid is preferably an epithelial organoid.

[0090] [the method for producing organoid] In one embodiment, the present invention provides the method for producing organoid, comprising the step of establishing organoid in the presence of the inhibitor of Hippo signaling pathway and in the absence of extracellular matrix.The method for producing organoid of this embodiment can also be said to be the method for establishing organoid.

[0091] As will be described later in Examples, the method of this embodiment can establish organoid from living tissue in the absence of extracellular matrix, and can also establish organoid from single cells without using extracellular matrix.Furthermore, according to the method of this embodiment, organoid can also be established under serum-free conditions.By establishing organoid in the absence of extracellular matrix and serum, it is possible to obtain organoids that are produced without animal-derived products from the time of establishment.

[0092] In the method of this embodiment, the inhibitor of Hippo signaling pathway, extracellular matrix, organoid, etc. are the same as those described above. That is, in the production method of this embodiment, the inhibitor of Hippo signaling pathway is preferably an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor. In addition, in the production method of this embodiment, it is preferable that the organoid is an epithelial organoid.

[0093] [Organoid] In one embodiment, the present invention provides an organoid produced by the above-described production method.

[0094] As described later in the Examples, the inventors have demonstrated that the above-described production method can establish organoids from tissues derived from living organisms in the absence of extracellular matrix, and can also establish organoids from single cells without the use of extracellular matrix. The organoids of this embodiment, particularly when established under serum-free conditions, are free of animal-derived products from the time of establishment. Therefore, they are particularly easy to transplant into living organisms and apply to regenerative medicine. Therefore, in one embodiment, the present invention provides a regenerative medicine preparation containing organoids produced by the above-described production method as an active ingredient.

[0095] The organoid of this embodiment is considered to have differences in gene expression patterns and the like with conventional organoids.However, it is unclear whether it is possible to identify the differences in gene expression patterns and clearly distinguish between conventional organoids and the organoid of this embodiment, and this is not practical.Therefore, it is considered practical to define the organoid of this embodiment by manufacturing method.

[0096] [Method for screening for drugs that enable organoids to be cultured in the absence of extracellular matrix] In one embodiment, the present invention provides a method for screening for drugs that enable organoids to be cultured in the absence of extracellular matrix, the method comprising the steps of culturing organoids in the presence of a test substance and in the absence of an extracellular matrix, and evaluating the proliferation of the organoids, wherein higher proliferation of the organoids compared to that in the absence of the test substance indicates that the test substance is a drug that enables organoids to be cultured in the absence of an extracellular matrix.

[0097] The screening method of this embodiment makes it possible to screen for drugs that enable organoids to be cultured in the absence of extracellular matrix.

[0098] In the screening method of this embodiment, the extracellular matrix, organoids, etc. are the same as those described above.

[0099] In the screening method of this embodiment, the test substance is not particularly limited, and for example, a natural compound library, a synthetic compound library, an existing drug library, etc. can be used.

[0100] The evaluation of organoid proliferation can be carried out by any suitable method, for example, measuring the number of cells based on microscopic observation, measuring the number of cells by measuring ATP, or evaluating the number of cells based on measuring the number of cells using a tetrazolium compound such as MTT. If the proliferation of organoids is higher than that in the absence of the test substance, it can be determined that the test substance is a drug that enables the culture of organoids in the absence of extracellular matrix.

[0101] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0102] Experimental Example 1 (Culturing 1 of organoids in the presence of a LATS kinase inhibitor) Human small intestine-derived organoids and human large intestine-derived organoids were cultured in the presence of Lats-IN-1 (CAS number: 1424635-83-5, hereinafter sometimes referred to as "Lats-IN" or "TRULI"), an inhibitor of LATS1 kinase and LATS2 kinase, and their dynamics were observed.

[0103] Human small intestinal and colonic organoids were established from tissues derived from patients with gastrointestinal tumors who provided informed consent, in accordance with an ethical research plan approved by the Keio University School of Medicine Ethics Committee.

[0104] Each organoid was dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) to obtain a cell suspension. ５ Cells were seeded at 10 cells / well with 25 μL of Matrigel® onto a 48-well plate. After the Matrigel® gelled, 100 μL of medium with the composition shown in Table 1 below was added to each well and cultured at 37°C. 10 mM Lats-IN was added to the medium for the test group. The medium was then replaced every two days.

[0105]

[0106] Figure 2 shows representative photographs and a graph showing the cell counts for each organoid after 7 days of culture. The upper panel of Figure 2 shows the results for organoids derived from human small intestine, and the lower panel of Figure 2 shows the results for organoids derived from human colon. Cell proliferation was measured by measuring adenosine triphosphate (ATP) using a commercially available kit (product name "CellTiter-Glo 3D", Promega).

[0107] The upper left of Figure 2 is a photograph of a control (Ctrl) human small intestine-derived organoid cultured in the absence of Lats-IN, and the upper center of Figure 2 is a photograph of a human small intestine-derived organoid cultured in the presence of Lats-IN. The scale bar is 200 μm. The upper right of Figure 2 is a graph showing the results of measuring the number of cells per well of human small intestine-derived organoids cultured in the control (Ctrl) and in the presence of Lats-IN. The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.

[0108] The bottom left of Figure 2 is a photograph of a control (Ctrl) human colon-derived organoid cultured in the absence of Lats-IN, and the bottom center of Figure 2 is a photograph of a human colon-derived organoid cultured in the presence of Lats-IN. The scale bar is 200 μm. The bottom right of Figure 2 is a graph showing the results of measuring the number of cells per well of human colon-derived organoids cultured in the control (Ctrl) and in the presence of Lats-IN. The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.

[0109] As a result, it was revealed that cell proliferation was significantly activated in both human small intestine-derived organoids and human large intestine-derived organoids by adding a LATS kinase inhibitor to the culture medium.

[0110] [Experimental Example 2] (Culturing organoids in the presence of a LATS kinase inhibitor 2) Conventionally, extracellular matrices such as Matrigel (registered trademark) are essential for organoid culture, and it has been difficult to adequately maintain cell proliferation and differentiation with alternatives to Matrigel (registered trademark).

[0111] Furthermore, when cells are cultured three-dimensionally in the complete absence of extracellular matrix such as Matrigel (registered trademark), they aggregate to form cell masses, but in this state the cell proliferation activity is very low, making permanent subculture completely impossible.

[0112] In this experimental example, human small intestine-derived organoids and human large intestine-derived organoids established in the presence of Matrigel (registered trademark) were cultured in the absence of Matrigel (registered trademark) and in the presence of a LATS kinase inhibitor (final concentration 10 μM), and their dynamics were observed. The experiment was the same as in Experimental Example 1, except that each organoid was cultured in the absence of Matrigel (registered trademark).

[0113] Figure 3 shows representative photographs and graphs showing the cell counts of each organoid after 7 days of culture. The upper panel of Figure 3 shows the results for human small intestine-derived organoids, and the lower panel of Figure 3 shows the results for human colon-derived organoids.

[0114] The upper left of Figure 3 is a photograph of a control (Ctrl) human small intestine-derived organoid cultured in the absence of Lats-IN, and the upper center of Figure 3 is a photograph of a human small intestine-derived organoid cultured in the presence of Lats-IN. The scale bar is 100 μm. The upper right of Figure 3 is a graph showing the results of measuring the cell count of human small intestine-derived organoids cultured in the control (Ctrl) and in the presence of Lats-IN, measured by ATP measurement using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.

[0115] The lower left of Figure 3 is a photograph of a control (Ctrl) human colon-derived organoid cultured in the absence of Lats-IN, and the lower center of Figure 3 is a photograph of a human colon-derived organoid cultured in the presence of Lats-IN. The scale bar is 100 μm. The lower right of Figure 3 is a graph showing the results of measuring the cell number of human colon-derived organoids cultured in the control (Ctrl) and in the presence of Lats-IN. The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.

[0116] As a result, it was revealed that the addition of a LATS kinase inhibitor to the culture medium significantly stimulated cell proliferation in both human small intestine-derived and human large intestine-derived organoids, even in the absence of Matrigel®. It is a surprising result that organoid proliferation in the absence of Matrigel®, which was previously thought to be impossible, has become possible.

[0117] [Experimental Example 3] (Investigation of YAP phosphorylation) Human small intestine-derived organoids were cultured in the absence and presence of Matrigel (registered trademark) and in the absence and presence of a LATS kinase inhibitor (final concentration 10 μM), and YAP phosphorylation was confirmed by Western blotting.

[0118] Figure 4 shows representative photographs showing the results of Western blotting. In Figure 4, "pYAP" indicates phosphorylated YAP, and "YAP" indicates total YAP (the sum of phosphorylated YAP and non-phosphorylated YAP). The antibody used for phosphorylated YAP was phospho-YAP (Ser127) (#4911, Cell Signaling Technology). The antibody used for total YAP was total-YAP (#14074, Cell Signaling Technology).

[0119] As a result, it was confirmed that YAP phosphorylation was significantly suppressed in the presence of Lats-IN compared to its absence, both in the presence and absence of Matrigel (registered trademark).

[0120] [Experimental Example 4] (Study by immunostaining) Human small intestine-derived organoids and human large intestine-derived organoids were cultured in the absence and presence of Matrigel (registered trademark) and in the absence and presence of a LATS kinase inhibitor (final concentration 10 μM) in the same manner as in Experimental Example 2. Subsequently, immunostaining was performed to confirm the localization of YAP. In addition, Ki67, a marker for cell proliferation, was detected to confirm proliferating cells.

[0121] The leftmost column of Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the presence of Matrigel (registered trademark) in the absence of Lats-IN (Ctrl). The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0122] The second column from the left in Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the presence of Matrigel (registered trademark) and Lats-IN. The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row. The scale bar indicates 50 μm.

[0123] The third column from the left in Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the absence of Matrigel (registered trademark) and in the absence of Lats-IN (Ctrl). The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0124] The rightmost column of Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the presence of Lats-IN in the absence of Matrigel (registered trademark). The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0125] The leftmost column of Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the presence of Matrigel (registered trademark) in the absence of Lats-IN (Ctrl). The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0126] The second column from the left in Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the presence of Matrigel (registered trademark) and Lats-IN. The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0127] The third column from the left in Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the absence of Matrigel (registered trademark) and in the absence of Lats-IN (Ctrl). The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0128] The rightmost column of Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the presence of Lats-IN in the absence of Matrigel (registered trademark). The top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc), the second row from the top is an image showing the detection results of Ki67, the third row from the top is an image showing the detection results of YAP, and the fourth row from the top is an image showing the detection results of nuclei (Nuc). The fields of view in the second, third, and fourth rows from the top correspond to the area surrounded by a square in the top row.

[0129] Figure 7 is a graph summarizing the results of Figures 5 and 6. As a result, in both the presence and absence of Matrigel (registered trademark), in the absence of Lats-IN, YAP was localized in the cytoplasm, and when the number of Ki67-positive cells, indicating cell proliferation, was counted, the proportion of Ki67-positive cells was approximately 10% of all cells. In contrast, in both the presence and absence of Matrigel (registered trademark), in the presence of Lats-IN, when the number of cells in which YAP was localized in the nucleus was counted, the proportion of cells in which YAP was localized in the nucleus was approximately 50-80% of all cells, and the proportion of Ki67-positive cells was approximately 50% or more of all cells.

[0130] These results indicate that the addition of Lats-IN inhibits LATS, inhibits YAP phosphorylation, causes YAP to translocate to the nucleus, and activates cell proliferation.

[0131] [Experimental Example 5] (Study of TEAD transcriptional activity) Whether YAP translocated to the nucleus by the addition of Lats-IN supports the transcriptional activity of TEAD was investigated. Specifically, human small intestine-derived organoids and human large intestine-derived organoids were cultured in the absence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 μM), and cell proliferation was observed when TEAD was inhibited using MYF01-37 (CAS number: 2416417-65-5), a TEAD inhibitor.

[0132] Figure 8 is a graph showing the results of measuring the proliferation of human small intestine-derived organoids and human large intestine-derived organoids in the absence of Lats-IN (Ctrl), in the presence of Lats-IN (Lats-IN), in the presence of Lats-IN and MYF01-37 (Lats-IN + MYF), and in the presence of MYF01-37 (MYF). Cell proliferation was measured by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph shows luminescence intensity (relative value) indicating the amount of ATP present.

[0133] As a result, in both organoids, cell proliferation was activated in the group to which Lats-IN was added, but cell proliferation was suppressed in the group to which both Lats-IN and MYF01-37 were added.

[0134] These results indicate that the addition of Lats-IN inhibits LATS function, causing YAP to translocate to the nucleus, and that the nuclear YAP supports the transcriptional activity of TEAD. In other words, the addition of Lats-IN to the culture medium enables cell proliferation in the absence of Matrigel (registered trademark), which was previously impossible, through the pathways of LATS inhibition, YAP nuclear translocation, and TEAD-mediated promotion of the transcriptional activity of cell growth factors.

[0135] [Experimental Example 6] (Investigation of organoid formation from single cells) We investigated whether organoids can be formed from single cells in the absence of Matrigel (registered trademark). Cell-substrate interaction has been thought to be essential for cell survival and proliferation, but cell-substrate interaction does not exist in cultures without Matrigel (registered trademark). This demonstrates that signals from cell-substrate interactions are not involved in the transcriptional regulation of YAP-TEAD by LATS inhibition.

[0136] In addition to cell-substrate interactions, signals from cell-to-cell interactions are also thought to play an important role in cell survival and proliferation.

[0137] Therefore, to investigate whether signals from cell-cell interactions are involved in cell proliferation mediated by transcriptional regulation of YAP-TEAD by LATS inhibition, we dissociated human small intestine-derived organoids into single cells and then observed organoid formation from the single cells.

[0138] Figure 9 is a representative photograph showing the results of organoid formation from single cells in the presence or absence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 μM). In Figure 9, arrowheads indicate cells or organoids. D1, D4, and D7 indicate the first, fourth, and seventh days after the start of culture, respectively. In parentheses at the bottom of Figure 9, the denominator indicates the number of single cells seeded, and the numerator indicates the number of organoids formed. The percentage (%) of cells that formed organoids among the seeded single cells is also shown.

[0139] As a result, it was shown that in the absence of Matrigel (registered trademark) and in the absence of Lats-IN, cells hardly proliferated. In contrast, it was revealed that in the presence of Matrigel (registered trademark), and in the absence of Matrigel (registered trademark) and in the presence of Lats-IN, cells proliferated from single cells and formed organoids. Furthermore, it was shown that in the presence of Matrigel (registered trademark) and Lats-IN, the percentage of cells that formed organoids was higher than in the absence of Matrigel (registered trademark) and in the presence of Lats-IN.

[0140] These results suggest that the transcriptional regulation of YAP-TEAD by LATS inhibition does not involve signals from cell-cell interactions, and that the transcriptional regulation of YAP-TEAD by LATS inhibition activates cell proliferation by itself and is involved in organoid formation.

[0141] [Experimental Example 7] (Establishment of organoids in the absence of Matrigel (registered trademark)) Whether organoids can be established from the human lower gastrointestinal tract in the absence of Matrigel (registered trademark) was examined.

[0142] Crypts were isolated from human small intestine and large intestine tissues and cultured in the absence of Matrigel® in the absence or presence of Lats-IN (final concentration 10 μM).

[0143] Figure 10 is a graph showing the results of measuring the proliferation of crypts isolated from human colon-derived tissue over time. Cell proliferation was measured by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D," Promega). The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present. As shown in Figure 10, in the absence of Lats-IN, isolated crypts increased in number enough to be passaged once, but then gradually underwent apoptosis and completely died after the second passage.

[0144] In contrast, in the presence of Lats-IN, isolated crypts could be passaged almost indefinitely without cell proliferation arrest. Similar results were obtained for crypts isolated from human small intestine-derived tissue.

[0145] These results demonstrate that in the presence of Lats-IN, LATS is inhibited, YAP-mediated cell proliferation occurs, and organoids can be established even in the absence of Matrigel (registered trademark).

[0146] [Experimental Example 8] (Culturing of organoids derived from various organs) Whether it is possible to culture organoids derived from various organs in the absence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 μM) was investigated.

[0147] Figure 11 shows representative photographs showing the results of culturing gastric, biliary, and mammary organoids in the absence of Matrigel (registered trademark) and in the absence of Lats-IN (Ctrl), or in the absence of Matrigel (registered trademark) and in the presence of Lats-IN (Lats-IN). Scale bar: 100 μm.

[0148] As a result, it was shown that cell proliferation was activated in the presence of Lats-IN even in the absence of Matrigel (registered trademark), and that organoids derived from any organ, not just the human lower gastrointestinal tract, could be cultured.

[0149] [Experimental Example 9] (Study of organoid morphology 1) Human lower gastrointestinal organoids established in the presence of Matrigel (registered trademark) were cultured in the absence of Matrigel (registered trademark) and in the presence of Lats-IN (final concentration 10 μM), and their morphology and properties were observed.

[0150] The upper and lower panels of Figure 12 show representative photographs of immunostained human small intestine-derived organoids. The lower panel of Figure 12 shows representative photographs of immunostained human large intestine-derived organoids. The scale bar in Figure 12 indicates 50 μm.

[0151] Immunostaining was performed to detect the basement membrane marker integrin β4 (Intβ4), the apical marker Ezrin, the cell-cell adhesion marker β-catenin (βCat), the Paneth cell marker lysozyme, the germ cell marker Mucin 2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and the nucleus (Nuc). Expression of the stem cell marker Lgr5 was detected by the fluorescence of TdTomato, a fluorescent protein introduced into the Lgr5 locus of the cells.

[0152] Conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark) are spherical, with a single layer of epithelial cell sheet that maintains apical polarity, with the apical side facing inward. Each organoid contains differentiated cells such as stem cells, embryonic cells, and enteroendocrine cells.

[0153] In contrast, as shown in Figure 12, human lower gastrointestinal organoids cultured in the absence of Matrigel (registered trademark) but in the presence of Lats-IN had a spherical morphology with stratified cells, and the apical polarity of the epithelial cells was not maintained, which was significantly different from the morphology of conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).

[0154] However, as shown in Figure 12, human lower gastrointestinal organoids cultured in the absence of Matrigel (registered trademark) but in the presence of Lats-IN were confirmed to express not only the stem cell marker Lgr5, but also the Paneth cell marker Lysozyme, the germ cell marker Mucin2, and the enteroendocrine cell marker Chromogranin A. This indicates that, like conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark), each organoid contains stem cells and differentiated cells.

[0155] [Experimental Example 10] (Study of organoid morphology 2) Human lower gastrointestinal organoids established in the presence of Matrigel (registered trademark) were cultured for seven passages in the absence of Matrigel (registered trademark) and in the presence of Lats-IN (final concentration 10 μM), and then cultured again in the presence of Matrigel (registered trademark), and their morphology and properties were observed.

[0156] The upper and lower panels of Figure 13 are representative photographs of immunostained human small intestine-derived organoids. The lower panel of Figure 13 is a representative photograph of immunostained human large intestine-derived organoids. The scale bar in Figure 13 indicates 50 μm.

[0157] Immunostaining was performed to detect the basement membrane marker integrin β4 (Intβ4), the apical marker Ezrin, the cell-cell adhesion marker β-catenin (βCat), the Paneth cell marker lysozyme, the germ cell marker Mucin 2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and the nucleus (Nuc). Expression of the stem cell marker Lgr5 was detected by the fluorescence of TdTomato, a fluorescent protein introduced into the Lgr5 locus of the cells.

[0158] As a result, as shown in Figure 13, even when human lower gastrointestinal organoids were cultured in the absence of Matrigel (registered trademark) but in the presence of Lats-IN, when they were again cultured in the presence of Matrigel (registered trademark), a single layer of epithelial cell sheets with apical polarity maintained formed a spherical shape with the inside facing apically, and it was revealed that each organoid contained differentiated cells such as stem cells, embryonic cells, and enteroendocrine cells.

[0159] These results demonstrated that the properties of human lower gastrointestinal epithelial cells were maintained even after long-term culture in the absence of Matrigel (registered trademark).

[0160] [Experimental Example 11] (Study of niche factors essential for organoid culture) The inventors previously revealed that the niche factors essential for the culture of human lower gastrointestinal epithelial organoids are Wnt agonists (W), R-spondin (R), EGF (E), BMP inhibitors such as noggin (N), TGF-β inhibitors such as A83-01 (A), IGF1 (I), and FGF2 (F).

[0161] In this experimental example, the relationship between LATS inhibition and niche factors essential for organoid culture was investigated. Figure 14 is a graph showing the results of measuring the proliferation of human small intestine-derived organoids and human large intestine-derived organoids cultured under conditions in which all niche factors of WRENAIF were added and under conditions in which some of these niche factors were removed. As the medium containing all of the niche factors of WRENAIF, a medium with the composition shown in Table 1 above was used. As the medium in which some of these niche factors were removed, a medium with the composition shown in Table 1 above from which the corresponding niche factors were removed was used. Cell proliferation was measured by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.

[0162] In Figure 14, "Ctrl" indicates the results of culturing in the presence of Matrigel (registered trademark), and "Lats-IN" indicates the results of culturing in the absence of Matrigel (registered trademark) but in the presence of Lats-IN (final concentration 10 μM). On the horizontal axis, the niche factors that were added are shown in black, and the niche factors that were not added are shown in light shades.

[0163] The results showed that cells proliferated by adding Lats-IN, even without adding one or two niche factors required for human lower gastrointestinal epithelial organoid culture to the culture medium.

[0164] Figure 15 is a graph showing the results of measuring the proliferation of human small intestine-derived organoids cultured in the absence of Matrigel (registered trademark), in the absence or presence of Lats-IN (final concentration 10 μM), under conditions in which all niche factors of WRENAIF were added, and under conditions in which some of these niche factors were removed. In Figure 15, P0 to P16 indicate passage numbers 0 to 16. In addition, dark colors in the graph indicate that passage was successful, and light colors indicate that passage was not successful.

[0165] As a result, even with the addition of Lat-IN, persistent passage was impossible when cultured in a medium lacking any of the niche factors of WRENAIF. These results demonstrate that Lat-IN does not complement the niche factors essential for organoid culture, but rather cooperates with the niche factors to promote organoid growth.

[0166] [Experimental Example 12] (Investigation of the mechanism by which organoid culture in the absence of Matrigel (registered trademark) becomes possible) We investigated why inhibiting LATS makes it possible to culture organoids in the absence of Matrigel (registered trademark).

[0167] Human small intestine-derived organoids and human large intestine-derived organoids were cultured in the absence or presence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 μM), and the expression level of the axin2 gene, whose expression is regulated by Wnt, was examined by quantitative real-time PCR.

[0168] 16 is a graph showing the results of quantitative real-time PCR. As a result, when organoids were cultured in the presence of Matrigel (registered trademark), axin2 expression was observed, whereas when organoids were cultured in the absence of Matrigel (registered trademark), the expression level of axin2 was significantly reduced. Furthermore, when organoids were cultured in the absence of Matrigel (registered trademark) and in the presence of Lats-IN, the expression level of axin2 was shown to increase to a level equivalent to that in the presence of Matrigel (registered trademark).

[0169] Next, human small intestine-derived organoids were cultured in the absence or presence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 μM), and ERK, which is phosphorylated by EGF signaling, was detected by Western blotting.

[0170] Figure 17 is a graph showing the results of Western blotting. In Figure 17, "pERK" indicates phosphorylated ERK, and "total ERK" indicates total ERK (the sum of phosphorylated ERK and non-phosphorylated ERK). "pYAP" indicates phosphorylated YAP, and "total YAP" indicates total YAP (the sum of phosphorylated YAP and non-phosphorylated YAP). The antibody used for phosphorylated ERK was phospho-ERK (Thr202 / Tyr204) (#4370, Cell Signaling Technology). The antibody used for total ERK was total-ERK (#4695, Cell Signaling Technology). The antibody used for phosphorylated YAP was phospho-YAP (Ser127) (#4911, Cell Signaling Technology). The antibody against total YAP used was total-YAP (#14074, Cell Signaling Technology).

[0171] The results showed that ERK phosphorylation was observed when organoids were cultured in the presence of Matrigel®, whereas ERK phosphorylation was significantly reduced when organoids were cultured in the absence of Matrigel®. Furthermore, when organoids were cultured in the absence of Matrigel® but in the presence of Lats-IN, ERK phosphorylation was elevated to a level comparable to that in the presence of Matrigel®.

[0172] The above results show that Wnt signals and EGF signals, which do not enter cells in the absence of Matrigel (registered trademark), can enter cells even in the absence of Matrigel (registered trademark) by inhibiting LATS, thereby enabling cell proliferation and organoid culture.

[0173] [Experimental Example 13] (Study of Organoid Morphology 3) Human colon-derived organoids were established in the absence of Matrigel (registered trademark) and in the presence of Lats-IN (final concentration 10 μM) in the same manner as in Experimental Example 7, and their morphology and properties were observed. This Experimental Example differed from Experimental Example 9 in that the organoids were Matrigel (registered trademark)-free from the time of establishment.

[0174] Figure 18 shows representative photographs of immunostained human colon-derived organoids on day 7 of culture, after 18 passages since establishment. The scale bar in Figure 18 indicates 50 µm.

[0175] Immunostaining detected the apical marker Ezrin, the basement membrane marker integrin β4 (β4-Integrin), the cell-cell adhesion marker β-catenin (β-Catenin), the germ cell marker Mucin2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and nuclei (Nuc).

[0176] As mentioned above, conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark) are spherical, with a single layer of epithelial cell sheet that maintains apical polarity, with the apical side facing inward, and each organoid contains differentiated cells such as stem cells, embryonic cells, and enteroendocrine cells.

[0177] In contrast, as shown in Figure 18, human colon-derived organoids established in the absence of Matrigel (registered trademark) but in the presence of Lats-IN had a spherical morphology with stratified cells, and the apical polarity of the epithelial cells was not maintained, which was significantly different from the morphology of conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).

[0178] However, as shown in Figure 18, human lower gastrointestinal organoids established in the absence of Matrigel (registered trademark) but in the presence of Lats-IN were confirmed to express Mucin2, an embryonic cell marker, and Chromogranin A, an enteroendocrine cell marker. This suggests that, similar to conventional human lower gastrointestinal organoids established in the presence of Matrigel (registered trademark), each organoid contains both stem cells and differentiated cells.

[0179] Figure 19 shows micrographs of human colon-derived organoids on day 7 of culture, which were established in the absence of Matrigel (registered trademark) and in the presence of Lats-IN and passaged 18 times. The scale bar in the left photograph of Figure 19 is 500 μm, and the scale bar in the right photograph of Figure 19 is 100 μm.

[0180] [Experimental Example 14] (Study of Organoid Morphology 4) As in Experimental Example 7, human colon-derived organoids established in the absence of Matrigel (registered trademark) and in the presence of Lats-IN (final concentration 10 μM) were cultured in the presence of Matrigel (registered trademark) for the first time, and their morphology and properties were observed. This Experimental Example differed from Experimental Example 9 in that the establishment of organoids was performed in the absence of Matrigel (registered trademark).

[0181] Figure 20 shows representative photographs of immunostained human colon-derived organoids established in the absence of Matrigel (registered trademark) but in the presence of Lats-IN, passaged 18 times, and then cultured for 7 days in the presence of Matrigel (registered trademark). The scale bar in Figure 20 indicates 50 μm.

[0182] Immunostaining detected the apical marker Ezrin, the basement membrane marker integrin β4 (β4-Integrin), the cell-cell adhesion marker β-catenin (β-Catenin), the germ cell marker Mucin2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and nuclei (Nuc).

[0183] As a result, as shown in Figure 20, it was revealed that even in human lower gastrointestinal organoids established in the absence of Matrigel (registered trademark) but in the presence of Lats-IN, when cultured in the presence of Matrigel (registered trademark), a single layer of epithelial cell sheets with apical polarity maintained formed a spherical shape with the inside facing apically, and that each organoid contained differentiated cells such as embryonic cells and enteroendocrine cells.

[0184] This result demonstrated that the properties of human lower gastrointestinal epithelial cells were maintained even when established in the absence of Matrigel (registered trademark).

[0185] Figure 21 shows micrographs of human colon-derived organoids established in the absence of Matrigel (registered trademark) but in the presence of Lats-IN, passaged 18 times, and then cultured in the presence of Matrigel (registered trademark) for the first time on day 7. The scale bar in the left photograph of Figure 21 is 500 μm, and the scale bar in the right photograph of Figure 21 is 100 μm.

[0186] [Experimental Example 15] (Culturing of hepatocyte organoids in the absence of Matrigel (registered trademark)) TrypLE Express (Thermo Fisher Scientific) was added to human hepatocyte organoids established from primary human hepatocytes, and the organoids were incubated at 37°C for 5 minutes to dissociate them into single cells.

[0187] The cells were then harvested and suspended in growth medium supplemented with 2, 5, 10, 20, or 50 μM TDI-011536 (CAS No.: 2687970-96-1), and seeded onto an Ultra Low attachment 96-well plate at 2,000 to 4,000 cells / well, preferably 2,000 cells / well. Cells in the growth medium without TDI-011536 were also prepared as a control.

[0188] The growth medium was basal medium (Advanced DMEM / F-12 medium, 10 mM HEPES, 2 mM GlutaMAX, 100 U penicillin, 100 μg / mL streptomycin) supplemented with 10% Afamin-Wnt-3A conditioned medium, 2% R-spondin1 conditioned medium, 2% Noggin conditioned medium, and 20 ng / mL oncostatin M.

[0189] Six days after seeding, the number of viable cells in the human hepatocyte organoids was measured using a commercially available kit (product name: CellTiter-Glo 3D, Promega).

[0190] Figure 22 is a graph showing the results of measuring the number of viable cells. As a result, in the group to which 20 and 50 μM TDI-011536 was added as LATS-IN, the number of viable cells was measured to be 16 and 17 times higher than the control group. The increase in viable cell number was also observed with other LATS-IN, such as TRULI, with an increase in the number of viable cells of about 3 times. It was revealed that in the presence of LATS-IN in the presence of non-Matrigel, an increase in the number of viable cells of human hepatocyte organoids was observed.

[0191] Experimental Example 16 (Establishment of hepatocyte organoids in the absence of Matrigel (registered trademark)) Frozen primary human hepatocytes were suspended in Advanced DMEM / F-12 medium and thawed. Two types of primary human hepatocytes, Line 1 and Line 2, were used.

[0192] Subsequently, each thawed primary human hepatocyte was stained with an APC-labeled mouse anti-human EpCAM antibody, and EpCAM-negative cells were collected using a cell sorter. The collected cells were then suspended in growth medium supplemented with 20 μM TDI-011536 and seeded onto an Ultra Low attachment 96-well plate at 3,000-4,000 cells / well. The cells were then cultured to obtain hepatocyte organoids. For comparison, cells were also prepared using growth medium without TDI-011536 and growth medium without oncostatin M.

[0193] Hepatocyte organoids were passaged in the absence of Matrigel® as follows: First, calcium and magnesium ions were chelated using 1 mM EDTA / phosphate-buffered saline (PBS). Next, TrypLE Express (Thermo Fisher Scientific) was added and the cells were incubated at 37°C for 5 minutes to dissociate into single cells.

[0194] Subsequently, the collected cells were suspended in a growth medium supplemented with 20 μM TDI-011536 and seeded onto an Ultra Low attachment 96-well plate at 2,000 cells / well.

[0195] Figure 23 shows micrographs of each cell taken 1 day and 9 days after the start of culture. The scale bar is 500 μm. Figure 24 is a graph showing the results of measuring changes in cell proliferation over time. In Figure 24, the vertical axis of the graph represents cell proliferation (fold increase), and the horizontal axis represents the number of days of culture. In Figures 23 and 24, "OSM" represents oncostatin M, and "Lats IN" represents a Lats kinase inhibitor (TDI-011536).

[0196] As a result, as shown in Figure 22, an increase in the number of viable cells was observed with the addition of the Lats kinase inhibitor (TDI-011536). Furthermore, as shown in Figure 23, when a growth medium containing both the Lats kinase inhibitor (TDI-011536) and oncostatin M was used, good human hepatocyte organoids were successfully established. The established human hepatocyte organoids could be cultured for at least two months, and the cell number increased to 10 ６ ~10 ８ The cells multiplied by 2 times (Fig. 24).

[0197] [Experimental Example 17] (Study of hepatocyte organoids cultured in the absence of Matrigel (registered trademark)) Hepatocyte organoids cultured in the absence of Matrigel (registered trademark) were subjected to fluorescent immunostaining to confirm that they were hepatocytes. For comparison, fluorescent immunostaining was also performed on hepatocyte organoids cultured in the presence of Matrigel (registered trademark).

[0198] First, human hepatocyte organoids were fixed using 1% formaldehyde / HEPES-buffered saline (HBS). Then, the fixed human hepatocyte organoids were perforated using 1% Triton-X100 / PBS. Then, the human hepatocyte organoids were blocked using 1% BSA / PBS. Subsequently, the hepatocyte marker HNF4α was immunostained using mouse anti-HNF4α antibody (Thermo Fisher Scientific).

[0199] Figure 25 is a micrograph showing the results of fluorescent immunostaining. The scale bar is 50 μm. In Figure 25, "Matrigel (+)" indicates the results for hepatocyte organoids cultured in the presence of Matrigel (registered trademark), and "Matrigel (-)" indicates the results for hepatocyte organoids cultured in the absence of Matrigel (registered trademark). "Nuc" indicates the results of nuclear staining.

[0200] As a result, it was confirmed that the human hepatocyte organoids cultured in the absence of Matrigel (registered trademark) also express the hepatocyte marker HNF4α, similar to the hepatocyte organoids cultured in the presence of Matrigel (registered trademark).This result shows that the human hepatocyte organoids cultured in the absence of Matrigel (registered trademark) are also undoubtedly hepatocyte organoids.

[0201] [Experimental Example 18] (Culturing of organoids in the presence of a LATS kinase inhibitor 3) In the same manner as in Experimental Example 2, human colon-derived organoids and human small intestine-derived organoids were cultured in the absence of Matrigel (registered trademark) and in the presence (final concentration 20 μM) or absence (control) of a LATS kinase inhibitor, and their kinetics were observed.

[0202] The LATS kinase inhibitors used were GA-017 (CAS number: 2351906-74-4), TDI-011536 (CAS number: 2687970-96-1), and TRULI (CAS number: 1424635-83-5, also known as "Lats-IN" or "Lats-IN-1").

[0203] Figure 26 is a graph showing the results of measuring the cell count of each organoid after 6 days of culture by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D," Promega). The vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present. The left side of Figure 26 shows the results for organoids derived from human colon, and the right side of Figure 26 shows the results for organoids derived from human small intestine.

[0204] As a result, it was revealed that regardless of which LATS kinase inhibitor was used, cell proliferation was significantly activated in both human colon-derived organoids and human small intestine-derived organoids, even in the absence of Matrigel (registered trademark).

[0205] [Experimental Example 19] (Study 2 on organoid formation from single cells) Human colon-derived organoids were dissociated into single cells, and then organoid formation from the single cells was observed.

[0206] Figure 27 is a graph showing the results of organoid formation from single cells in the presence (+) or absence (-) of Matrigel (registered trademark), and in the absence (-) or presence (+, final concentration 10 μM) of TRUL1. The parentheses in Figure 27 show the number of single cells seeded in the denominator and the number of organoids formed in the numerator. The vertical axis of the graph shows the percentage (%) of cells that formed organoids among the seeded single cells.

[0207] The results showed that cells hardly proliferated in the absence of Matrigel (registered trademark) and TRUSCO, whereas in the presence of Matrigel (registered trademark) and in the absence of Matrigel (registered trademark) and the presence of TRUSCO, cells proliferated from single cells and formed organoids.

[0208] [Experimental Example 20] (Study of colony size) After dissociating organoids derived from human small intestine into single cells, organoid formation from the single cells was observed. Figure 28 is a graph showing the results of forming organoids from single cells in the presence (+) or absence (-) of Matrigel (registered trademark), and in the absence (-) or presence (+, final concentration 10 μM) of TRUL1, and measuring the colony size. The colony size was measured using software (ImageJ). In Figure 28, the vertical axis represents colony size (mm ２ ) is shown.

[0209] [Experimental Example 21] (Culturing of organoids in the presence of a LATS kinase inhibitor 4) Organoids derived from various human tissues were dissociated into single cells, and then cultured in the absence of Matrigel (registered trademark) and in the presence of a LATS kinase inhibitor, or in the absence of Matrigel (registered trademark) and in the absence of a LATS kinase inhibitor (control), in the same manner as in Experimental Example 2, and their dynamics were observed.

[0210] The LATS kinase inhibitors used were TDI-011536 (CAS number: 2687970-96-1) and TRULI (CAS number: 1424635-83-5, also known as "Lats-IN" or "Lats-IN-1"). The concentration of the LATS kinase inhibitor varied depending on the organoid, ranging from 5 to 20 μM.

[0211] Figure 29 shows bright-field micrographs of each organoid after 6 days of culture, and a graph showing the results of measuring the cell count of each organoid by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph shows luminescence intensity (relative value) indicating the amount of ATP present. In Figure 29, the top left shows the results for human liver-derived organoids, the top center shows the results for human salivary gland-derived organoids, the top right shows the results for human alveolar-derived organoids, the bottom left shows the results for human airway-derived organoids, the bottom second from the left shows the results for human pancreatic-derived organoids, the bottom third from the left shows the results for human stomach-derived organoids, and the bottom right shows the results for human biliary tract-derived organoids. "Control" indicates the results of the control, and "TDI" indicates the results for the group to which TDI-011536 was added.

[0212] As a result, it was revealed that regardless of the use of any of the LATS kinase inhibitors, cell proliferation of organoids derived from various tissues was significantly activated in the absence of Matrigel (registered trademark).

[0213] Experimental Example 22 (Establishment of organoids in the absence of Matrigel (registered trademark)) Crypts were collected from human colons and dissociated into single cells. Organoids were established by culturing them in the presence of Matrigel (registered trademark) or in the absence of Matrigel (registered trademark) and in the presence of TRULI. The cell number of each organoid under these conditions was determined by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega).

[0214] The upper part of Figure 30 is a schematic diagram illustrating the outline of this experimental example. The lower part of Figure 30 is a graph showing the results of measuring the proliferation of organoids. As a result, in the presence of TRUL1, even in the absence of Matrigel (registered trademark), cell proliferation was observed to be comparable to that in the presence of Matrigel (registered trademark).

[0215] [Experimental Example 23] (Orthotopic xenotransplantation of organoids established in the absence of Matrigel (registered trademark)) In Experimental Example 22, human colon-derived organoids established in the absence of Matrigel (registered trademark) and in the presence of TRUL1 were orthotopic xenotransplanted into immunodeficient mice (NOG mice) to examine whether they would survive.

[0216] 31 is a schematic diagram illustrating an outline of this experimental example. Human colon-derived organoids established in the absence of Matrigel (registered trademark) and the presence of TRUL1 were electroporated with a green fluorescent protein (GFP) expression vector to obtain GFP-labeled human colon-derived organoids.

[0217] GFP-labeled human colon-derived organoids were then dissociated into single cells and precultured for 4 days in 2% Matrigel® or 2% collagen gel (50% type IA, 50% type IC). For 2% collagen gel culture, the medium was supplemented with 2.5 μM 16,16-dimethyl prostaglandin E2 (dmPGE2, Cayman Chemical), 50 ng / mL human recombinant hepatocyte growth factor (HGF, PeproTech), 20 ng / mL human recombinant oncostatin M (OSM, PeproTech), and 5 nM human recombinant heregulin β1 (NRG1, PeproTech).

[0218] Next, the colonic epithelium and mucosa of NOG mice were removed, and GFP-labeled human colon-derived organoids were orthotopically xenotransplanted. First, NOG mice fed a normal diet were anesthetized by inhalation of 2-3% isoflurane. The mouse colon was then washed with PBS to remove the luminal contents. A catheter equipped with a small balloon was then inserted into the anus, and the balloon was inflated with air to hold the catheter in place. Next, high-temperature EDTA (250 mM, 50-55°C) was injected into the rectum to remove the colonic mucosa, and Ca, which is necessary for cell-cell adhesion and cell-extracellular matrix adhesion, was removed. ２＋ and Mg ２＋ Chelated.

[0219] Subsequently, the colonic epithelium and mucosa were scraped using a vibrating electric toothbrush (EW-DL22, EW0945, Panasonic). Successful removal of the epithelium and mucosa was confirmed by releasing the isolated colonic epithelium from the vibrating electric toothbrush into a water-filled Petri dish.

[0220] Subsequently, the total number of cells in the pre-cultured GFP-labeled human colon-derived organoids was counted, and the cells were suspended in Advanced DMEM / F12 medium supplemented with 10% (vol / vol) Matrigel® or type IA collagen, and then aliquoted to 1 × 10 cells using a 200 μL pipette. ７ 70 μL of the organoid suspension containing 100 cells was injected into the colon of NOG mice (recipient mice) from which the colonic epithelium and mucosa had been removed. To maintain the colonic engraftment of GFP-labeled human colon-derived organoids, the mouse anus was temporarily sealed with soft paper using adhesive. The feces of the recipient mice were carefully monitored for one week to check for the development of intestinal obstruction.

[0221] The top left of Figure 32 is a bright-field photograph of the colon of a NOG mouse transplanted with GFP-labeled human colon-derived organoids. The second from the left in the top row of Figure 32 is a photograph of GFP fluorescence in the same field as the top left of Figure 32. The third from the left in the top row of Figure 32 is a photograph of the intestine of a mouse with stained damaged areas. The top right of Figure 32 is a micrograph showing the results of immunostaining a section of the colon of a NOG mouse transplanted with GFP-labeled human colon-derived organoids with an anti-human cytokeratin antibody. The scale bar is 200 μm.

[0222] The leftmost photograph in the bottom row of Figure 32 is a fluorescence micrograph showing the results of staining the region surrounded by a dotted line in the top right of Figure 32 by in situ hybridization with mRNA of the human LGR5 gene. The second photograph from the left in the bottom row of Figure 32 is an enlarged photograph of the region surrounded by a dotted line in the top right of Figure 32. The scale bar is 100 μm. The third photograph from the left in the bottom row of Figure 32 is a fluorescence micrograph showing the results of staining the region surrounded by a dotted line in the top right of Figure 32 with an anti-human chromogranin A (CHGA) antibody and an anti-human villin antibody. The scale bar is 100 μm. The rightmost photograph in the bottom row of Figure 32 is a fluorescence micrograph showing the results of staining the region surrounded by a dotted line in the top right of Figure 32 with an anti-human mucin 2 (MUC2) antibody and an anti-human villin antibody. The scale bar is 100 μm.

[0223] These results suggest that human colon-derived organoids established in the absence of Matrigel® and the presence of TRUL1 could be successfully engrafted into immunodeficient mice (NOG mice) by orthotopic xenotransplantation. This indicates that organoids generated in the absence of Matrigel and the presence of TRUL1 can be used in regenerative medicine.

[0224] The present invention provides a technique for culturing organoids in the absence of an extracellular matrix.

Claims

1. A drug for forming and growing organoids in the absence of an extracellular matrix, comprising an inhibitor of the Hippo signaling pathway as an active ingredient.

2. The drug for forming and growing organoids in the absence of an extracellular matrix according to Claim 1, wherein the inhibitor of the Hippo signaling pathway is an MST1 / 2 kinase inhibitor or a Large tumor suppressor kinase (LATS) 1 / 2 kinase inhibitor.

3. The drug for forming and growing organoids in the absence of an extracellular matrix according to Claim 1 or 2, further comprising a cytokine family that binds to gp130.

4. The drug for forming and growing organoids in the absence of an extracellular matrix according to Claim 1 or 2, wherein the organoid is an epithelial organoid.

5. A method for growing organoids, comprising the step of culturing organoids in the presence of an inhibitor of the Hippo signaling pathway and in the absence of an extracellular matrix.

6. The method for growing organoids according to Claim 5, wherein the culturing of the organoids is performed under serum-free conditions.

7. An organoid grown by the growth method according to Claim 5 or 6.

8. A drug for establishing organoids in the absence of an extracellular matrix, comprising an inhibitor of the Hippo signaling pathway as an active ingredient.

9. The drug for establishing organoids in the absence of an extracellular matrix according to Claim 8, wherein the inhibitor of the Hippo signaling pathway is an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor.

10. The drug for establishing organoids in the absence of an extracellular matrix according to Claim 8 or 9, further comprising a cytokine family that binds to gp130.

11. The drug for establishing organoids in the absence of an extracellular matrix according to Claim 8 or 9, wherein the organoid is an epithelial organoid.

12. A method for producing organoids, comprising the step of establishing organoids in the presence of an inhibitor of the Hippo signaling pathway and in the absence of an extracellular matrix.

13. The method for producing organoids according to Claim 12, wherein the establishment of the organoids is performed in the presence of a cytokine family that binds to gp130.

14. The method for producing organoids according to Claim 12 or 13, wherein the establishment of the organoids is performed under serum-free conditions.

15. An organoid produced by the production method according to claim 12 or 13, having a form in which cells are stratified.

16. A regenerative medicine preparation comprising the organoid according to claim 15 as an active ingredient.

17. A method for screening a drug that enables culturing of an organoid in the absence of an extracellular matrix, comprising: a step of culturing the organoid in the presence of a test substance and in the absence of an extracellular matrix; a step of evaluating the growth of the organoid, and a method, wherein the growth of the organoid being higher as compared to in the absence of the test substance indicates that the test substance is a drug that enables culturing of the organoid in the absence of an extracellular matrix.