Medicament for culturing organoids in absence of extracellular matrix

Abstract

A drug for forming and proliferating an organoid in the absence of an extracellular matrix, a method for proliferating the organoid, the organoid proliferated by the proliferation method, a drug for establishing the organoid in the absence of an extracellular matrix, a method for producing the organoid, and a method for producing the organoid, which contain a Hippo signaling pathway inhibitor as an active ingredient. An organoid produced by the production method; a regenerative medical preparation; and a method for screening a drug capable of culturing the organoid in the absence of an extracellular matrix.

Description

Technical Field

[0001] The present invention relates to a drug for culturing organoids in the absence of an extracellular matrix. In more detail, the present invention relates to a drug for forming and proliferating organoids in the absence of an extracellular matrix, a method for proliferating organoids, an organoid proliferated by the proliferating method, a drug for establishing organoids in the absence of an extracellular matrix, a method for manufacturing organoids, an organoid manufactured by the manufacturing method, a regenerative medical preparation, and a screening method for a drug that can cultivate organoids in the absence of an extracellular matrix. This application claims priority to 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, and its contents are cited here. Background Art

[0002] Organoid culture technology has been developed and is expected to be used in drug development and regenerative medicine. Cultivating organoids requires extracellular matrices such as Matrigel (registered trademark) (for example, refer to Patent Document 1, Non-Patent Document 1, etc.). Matrigel (registered trademark) is an extract of tumors transplanted into mice and is therefore an animal-derived product. In addition, the use of Matrigel (registered trademark) in the cultivation of organoids is an obstacle to medical applications due to the possibility of mixing with unknown viruses and high prices. Moreover, the use of Matrigel is also a problem from the perspective of animal protection. Since the emergence of organoid technology, the development of alternative substances to Matrigel (registered trademark) has been actively carried out (for example, refer to Non-Patent Document 2).

[0003] Related technical literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2016-198033

[0006] Non-patent literature

[0007] Non-patent literature 1: S Rezakhani, et al., Extracellular matrix requirements for gastrointestinal organoid cultures, Biomaterials, 276, 121020, 2021.

[0008] Non-patent document 2: Jeong Hyun Heo, et al., Engineering the Extracellular Matrix for Organoid Culture, Int J Stem Cells, 15(1), 60-69, 2022. Summary of the invention

[0009] [Problems to be solved by the invention]

[0010] In the past, synthetic extracellular matrix, collagen, etc. have been used as alternatives to Matrigel (registered trademark), but they have not been widely used because the culture efficiency of organoids is not high enough.

[0011] Therefore, an object of the present invention is to provide a technology for preparing and culturing organoids in the absence of an extracellular matrix.

[0012] [Solutions to solve the problem]

[0013] The present invention includes the following aspects.

[0014] [1] A drug for forming and growing organoids in the absence of an extracellular matrix, wherein the active ingredient of the drug is a Hippo signaling pathway inhibitor.

[0015] [2] The drug for forming and proliferating organoids in the absence of an extracellular matrix according to [1], wherein the Hippo signaling pathway inhibitor is an MST1 / 2 kinase inhibitor or a large tumor suppressor kinase (LATS) 1 / 2 kinase inhibitor.

[0016] [3] The drug for forming and growing organoids in the absence of an extracellular matrix according to [1] or [2], further comprising a family of cytokines that bind to gp130.

[0017] [4] The agent for forming and growing an organoid in the absence of an extracellular matrix according to any one of [1] to [3], wherein the organoid is an epithelial organoid.

[0018] [5] A method for growing organoids, comprising the step of culturing the organoids in the presence of a Hippo signaling pathway inhibitor and in the absence of an extracellular matrix.

[0019] [6] The method for growing an organoid according to [5], wherein the organoid is cultured under serum-free conditions.

[0020] [7] An organoid, wherein the organoid is proliferated using the proliferative method described in [5] or [6].

[0021] [8] A drug for establishing organoids in the absence of an extracellular matrix, wherein the active ingredient of the drug is a Hippo signaling pathway inhibitor.

[0022] [9] The drug for establishing organoids in the absence of an extracellular matrix according to [8], wherein the Hippo signaling pathway inhibitor is a MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor.

[0023]

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

[0024]

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

[10] , wherein the organoid is an epithelial organoid.

[0025]

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

[0026]

[13] The method for producing an organoid according to

[12] , wherein the organoid is established in the presence of a family of cytokines that bind to gp130.

[0027]

[14] The method for producing an organoid according to

[12] or

[13] , wherein the organoid is established under serum-free conditions.

[0028]

[15] An organoid, wherein the organoid is produced by the production method described in any one of

[12] to

[14] .

[0029]

[16] A regenerative medicine preparation, wherein the active ingredient of the regenerative medicine preparation is the organoid described in

[15] .

[0030]

[17] A method for screening a drug that can culture organoids in the absence of an extracellular matrix, comprising: a step of culturing organoids in the presence of a test substance and in the absence of an extracellular matrix; and a step of evaluating the proliferation of the organoids; wherein, if the proliferation of the organoids is higher than that in the absence of the test substance, it indicates that the test substance is a drug that can culture organoids in the absence of an extracellular matrix.

[0031] [Effects of the invention]

[0032] The present invention can provide a technology for culturing organoids in the absence of an extracellular matrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram illustrating the Hippo signaling pathway.

[0034] Figure 2 Representative photographs and graphs of the results of Experimental Example 1.

[0035] Figure 3 Representative photographs and graphs of the results of Experimental Example 2.

[0036] Figure 4 These are representative photographs of the results of Western blotting in Experimental Example 3.

[0037] Figure 5 Representative photographs of the immunostaining results of human small intestine-derived organoids in Experimental Example 4.

[0038] Figure 6 These are representative photographs of the immunostaining results of human colon-derived organoids in Experimental Example 4.

[0039] Figure 7 For the summary Figure 5 , Figure 6 Result graph.

[0040] Figure 8 The results of Experimental Example 5 are shown.

[0041] Fig. 9 Representative photos of the results of Experimental Example 6.

[0042] Fig.10 The results of Experimental Example 7 are shown.

[0043] Fig.11 These are representative photos of the results of Experimental Example 8.

[0044] Fig.12 Representative photos of the immunostaining results of organoids in Experimental Example 9.

[0045] Fig.13 Representative photos of the immunostaining results of organoids in Experimental Example 10.

[0046] Fig.14 The results of Experimental Example 11 are shown.

[0047] Fig.15 The results of Experimental Example 11 are shown.

[0048] Fig.16 The results of quantitative real-time PCR in Experimental Example 12 are shown.

[0049] Fig.17 The results of Western blotting in Experimental Example 12 are shown.

[0050] Fig.18 Representative photos of the immunostaining results of organoids in Experimental Example 13.

[0051] Fig.19 Representative microscopic photographs of organoids in Experimental Example 13.

[0052] Fig. 20 Representative photos of the immunostaining results of organoids in Experimental Example 14.

[0053] Fig.21 Representative microscopic photographs of organoids in Experimental Example 14.

[0054] Fig. 22 The results of Experimental Example 15 are shown.

[0055] Fig.23 Representative microscopic photographs of organoids in Experimental Example 16.

[0056] Fig.24 The results of Experimental Example 16 are shown.

[0057] Fig.25 Representative photos of the immunostaining results of organoids in Experimental Example 17.

[0058] Fig.26 The results of Experimental Example 18 are shown.

[0059] Fig. 27 The results of Experimental Example 19 are shown.

[0060] Fig.28 The results of Experimental Example 20 are shown.

[0061] Fig.29 These are microscope photographs and graphs showing the results of Experimental Example 21.

[0062] Fig.30 A schematic diagram for explaining the outline of Experimental Example 22 and a diagram showing the results of Experimental Example 22.

[0063] Fig.31 This is a schematic diagram for explaining the outline of Experimental Example 23.

[0064] Fig.32 This is a photograph of the results of Experimental Example 23. DETAILED DESCRIPTION

[0065] [Drugs for growing organoids in the absence of extracellular matrix]

[0066] In one embodiment, the present invention provides a drug for forming and proliferating organoids in the absence of an extracellular matrix, wherein the active ingredient of the drug is a Hippo signaling pathway inhibitor.

[0067] As described later in the examples, by adding the drug of the present embodiment to the culture medium, organoids can be formed and proliferated in the absence of an extracellular matrix. Among them, "proliferation" refers to an increase in the number of cells that make up the organoid, which can also be said to be "culture". In addition, "forming" organoids refers to establishing organoids, forming organoids again from organoids dissociated into single cells, etc. In one embodiment, the drug of the present embodiment can be a drug whose active ingredient is a Hippo signaling pathway inhibitor, which is used to proliferate organoids in the absence of an extracellular matrix.

[0068] In the past, it was impossible to proliferate organoids in the absence of an extracellular matrix. With the medicine of this embodiment, organoids can be proliferated without using animal-derived products such as extracellular matrix Matrigel (registered trademark). Among them, animal-derived products refer to substances whose components are not identified from humans or non-human animals. Organoids are expected to be applied to regenerative medicine through culture technology without animal-derived products. In addition, from the perspective of animal protection and cost, it is also more advantageous than conventional organoid culture technology.

[0069] The Hippo signaling pathway is a signaling pathway known to be involved in cell proliferation, apoptosis, and stem cell self-renewal, and is an evolutionarily conserved pathway.

[0070] Figure 1 Figure 2 is a schematic diagram illustrating the Hippo signaling pathway. Figure 1 As shown, it is known that 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 has phosphorylated and non-phosphorylated forms. Non-phosphorylated YAP is transferred to the nucleus and acts as a transcriptional coactivator of TEAD. On the other hand, phosphorylated YAP binds to the cytoplasmic protein 14-3 and cannot be transferred to the nucleus, so it cannot function as a coactivator. Therefore, phosphorylation regulation that determines the nuclear transfer of YAP is a very important event in cell proliferation.

[0071] In addition, if Figure 1 As shown, large tumor suppressor kinase (LATS) is known to phosphorylate YAP, localizing YAP in the cytoplasm, thereby negatively regulating YAP to participate in cell proliferation.

[0072] As the Hippo signaling pathway inhibitor, any substance that inhibits any stage of the above-mentioned signaling pathway can be used without particular limitation.

[0073] For example, it may be an inhibitor of MST1 kinase or MST2 kinase (MST1 / 2 kinase inhibitor), or it may be an inhibitor of LATS1 kinase or LATS2 kinase (LATS1 / 2 kinase inhibitor).

[0074] The NCBI accession numbers of the amino acid sequence 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 of the amino acid sequence of human MST2 kinase are NP_001243241.1, NP_001243242.1, NP_006272.2, etc.

[0075] The NCBI accession numbers of the amino acid sequence of human LATS1 kinase are NP_001257448.1, NP_001337268.1, NP_001337269.1, NP_001337321.1, NP_004681.1, etc. The NCBI accession number of the amino acid sequence of human LATS2 kinase is NP_055387.2, etc.

[0076] As more specific examples of Hippo signaling pathway inhibitors, for example, Lats-IN-1 (CAS No.: 1424635-83-5, hereinafter may be referred to as "Lats-IN". Also referred to as "TRULI"), which is an inhibitor of LATS1 kinase and LATS2 kinase, GA-017 (CAS No.: 2351906-74-4), TDI-011536 (CAS No.: 2687970-96-1), etc. can be cited.

[0077] Examples of the extracellular matrix include Matrigel (registered trademark), collagen, fibronectin, proteoglycan, laminin, etc. Conventionally, it has been impossible to grow organoids in the absence of an extracellular matrix.

[0078] Proliferating organoids in the absence of an extracellular matrix means not adding an extracellular matrix to the culture medium of the organoid from the outside, allowing trace amounts of extracellular matrix produced by the organoid itself to mix into the culture medium, or allowing trace amounts of extracellular matrix to accidentally mix into the culture medium. Here, trace amounts can refer to the detection limit.

[0079] In the drug of this embodiment, as organoids, epithelial organoids can be listed, for example, small intestine-derived organoids, large intestine-derived organoids, stomach-derived organoids, bile duct-derived organoids, pancreatic organoids, mammary gland-derived organoids, hepatocyte organoids, lung organoids, respiratory tract organoids, esophageal organoids, salivary gland organoids, etc.

[0080] The organoids may be human-derived or non-human-derived. Examples of non-human animals include mammals, such as rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cattle, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as common macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees.

[0081] The drug of the present embodiment is added to the medium for organoid culture and used. The concentration of the drug of the present embodiment in the medium for organoid culture is preferably 10 μM or more and 60 μM or less, and more preferably 10 μM or more and 30 μM or less.

[0082] As the medium for organoid culture, there can be cited a medium after the extracellular matrix is ​​removed from the conventional medium for organoid culture. As the medium for organoid culture, it is preferably a medium in which at least one of the following i) to v) components is added to the basic medium. The medium for organoid culture may also contain serum, but from the viewpoint of preferably being free of animal-derived products, it is preferably serum-free.

[0083] i) Wnt agonists

[0084] 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), epidermal growth factor (EREG), fibroblast growth factor 10 (FGF10), and gastrin-1

[0085] iii) Bone morphogenetic protein (BMP) inhibitors

[0086] iv) Transforming growth factor-β (TGF-β) inhibitors

[0087] v) γ-secretase inhibitors

[0088] The medium for organoid culture preferably contains at least one selected from the group consisting of IGF1, FGF2, EGF and epiregulin. Which one of IGF1, FGF2, EGF and epiregulin is contained can be appropriately selected. The medium for organoid culture preferably contains IGF1 and epiregulin, FGF2 and epiregulin, or IGF1 and FGF2, more preferably IGF1 and FGF2.

[0089] As the basic culture medium, any serum-free basic culture medium for cell culture can be used. For example, synthetic culture medium buffered to a pH of more than 7.2 and less than 7.6 with a carbonate buffer can be cited. More specifically, advanced-Double's modified Eagle 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, Ltd.), penicillin, streptomycin, transferrin, etc. can be cited. In addition, RPMI1640 culture medium, advanced RPMI culture medium, etc. can also be used instead of DMEM / F12 culture medium.

[0090] (Wnt agonist)

[0091] Wnt agonists refer to drugs that activate 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, and include Wnt agonists activated by binding to Frizzled receptor family members, intracellular β-catenin degradation inhibitors, and TCF / LEF activating substances. Wnt agonists are preferably at least one selected from the group consisting of Wnt proteins, R-spondin and GSK-3β inhibitors.

[0092] The medium for organoid culture preferably contains a Wnt agonist. As the Wnt agonist, it is more preferable to contain a complex of a Wnt protein and its stabilizing substance, Afamin, and it is even more preferable to contain a complex of a Wnt protein and Afamin and R-spondin.

[0093] 《Wnt Protein》

[0094] As Wnt protein, its source is not particularly limited, and various biological source Wnt proteins can be used. Among them, mammalian source Wnt protein is preferred. As mammals, the same mammals as mentioned above can be listed. As mammalian Wnt proteins, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, etc. can be listed. In the medium for organoid culture, a variety of Wnt proteins can be used in combination.

[0095] As a method for preparing Wnt protein, for example, a method for preparing using Wnt protein expressing cells can be cited. In Wnt protein expressing cells, the cell source (biological species, culture form, etc.) is not particularly limited, as long as it is a cell that stably expresses Wnt protein, it can also be a cell that transiently expresses Wnt protein. As Wnt protein expressing cells, for example, L cells (ATCC CRL-2647) that stably express mouse Wnt3a, L cells (ATCC CRL-2814) that stably express mouse Wnt5a, etc. can be cited. In addition, Wnt protein expressing cells can be made using known gene recombination techniques. That is, the DNA encoding the desired Wnt protein is inserted into a known expression vector, and the obtained expression vector is introduced into a suitable host cell, so that Wnt protein expressing cells can be made. For example, the base sequence of the gene encoding the desired Wnt protein can be obtained from known databases such as GenBank.

[0096] The Wnt protein expressed by the Wnt protein-expressing cell may be a fragment of the Wnt protein as long as it has Wnt activity, or may contain an amino acid sequence other than the amino acid sequence of the Wnt protein. There is no particular limitation on the amino acid sequence other than the amino acid sequence of the Wnt protein, and for example, the amino acid sequence of an affinity tag may be cited. In addition, the amino acid sequence of the Wnt protein does not need to be completely identical to the amino acid sequence that can be obtained from a known database such as GenBank, and may be an amino acid sequence substantially identical to the amino acid sequence that can be obtained from a known database as long as it has Wnt activity.

[0097] Examples of amino acid sequences substantially identical to the amino acid sequences of Wnt proteins available from known databases such as GenBank include amino acid sequences in which one to several amino acids are deleted, substituted, or added to amino acid sequences available from known databases.

[0098] An amino acid sequence in which 1 to several amino acids are deleted, substituted or added refers to the deletion, substitution or addition of 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) by, for example, a known mutant peptide preparation method such as site-directed mutagenesis.

[0099] In addition, examples of substantially identical amino acid sequences include amino acid sequences having an identity of at least 80% or more, preferably at least 85% or more, more preferably at least 90% or more, further 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 known database.

[0100] The concentration of the Wnt protein is preferably 50 ng / mL or more, more preferably 100 ng / mL or more and 10 μg / mL or less, further preferably 200 ng / mL or more and 1 μg / mL or less, and particularly preferably 300 ng / mL or more and 1 μg / mL or less.

[0101] R-spondin

[0102] As R-spondin, the R-spondin family consisting of R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4 can be cited. The R-spondin family is known to be a secreted protein and is related to the activation and regulation of the Wnt signaling pathway. In the culture medium for organoid culture, a variety of R-spondins can be used in combination. In addition, as long as it has R-spondin activity, it can be a fragment of R-spondin, and it can also contain an amino acid sequence other than the amino acid sequence of R-spondin.

[0103] 《GSK-3β inhibitors》

[0104] Examples of GSK-3β inhibitors include CHIR-99021 (CAS No.: 252917-06-9), CHIR-98014 (CAS No.: 252935-94-7), lithium, kemperolone (CAS No.: 142273-20-9), 6-bromoindirubin-30-acetone oxime, SB216763 (CAS No.: 280744-09-4), SB415286 (CAS No.: 264218-23-7), FRAT family members that inhibit the interaction between GSK-3 and axin, FRAT-derived peptides, and the like.

[0105] Afamin

[0106] Afamin is a glycoprotein belonging to the albumin family and is present in blood or body fluids. The serum added to the culture medium generally contains afamin from the animal from which the serum was collected. Since serum contains impurities other than afamin, it is preferred to use afamin alone without using serum.

[0107] The source of Afamin contained in the medium for organoid culture is not particularly limited, and various biological sources of Afamin can be used. Among them, mammalian source Afamin is preferred. As mammals, the same mammals as mentioned above can be listed. For example, the amino acid sequence of Afamin of major mammals and the base sequence encoding its gene can be obtained from known databases such as GenBank. For example, in GenBank, the accession number of the amino acid sequence of human Afamin is AAA21612, and the accession number of the base sequence encoding its gene is L32140, and the accession number of the amino acid sequence of bovine Afamin is DAA28569, and the accession number of the base sequence encoding its gene is GJ060968.

[0108] Afamin contained in the medium for organoid culture may be obtained by purifying natural afamin contained in serum or the like by a known method, or may be recombinant afamin. Recombinant afamin may be produced by appropriately using known gene recombination techniques.

[0109] The method for producing recombinant Afamin is, for example, the following production method: DNA encoding Afamin can be inserted into a known expression vector, the obtained expression vector is introduced into an appropriate host cell to express the recombinant Afamin, and the recombinant Afamin is purified using a known purification method. The recombinant Afamin can be Afamin to which an affinity tag is added. The added affinity tag is not particularly limited, and can be appropriately selected from known affinity tags for use. The affinity tag is preferably an affinity tag recognized by a specific antibody, for example, a FLAG tag, a MYC tag, a HA tag, a V5 tag, etc. can be listed.

[0110] The Wnt protein has strong hydrophobicity due to modification of a specific serine residue with a fatty acid (palmitoleic acid), and therefore, it is known that the Wnt protein is easily aggregated or denatured in an aqueous solution, making purification and storage very difficult.

[0111] On the other hand, it has been reported that modification of this specific serine residue with fatty acids is essential for the physiological activity of Wnt proteins and is involved in their binding to members of the Frizzled receptor family.

[0112] In addition, it is known that in aqueous solution, Wnt protein and Afamin are bound one-to-one to form a complex, which is soluble while maintaining high physiological activity. The Wnt protein-Afamin complex can be produced by culturing cells expressing both Wnt protein and Afamin, or by co-culturing Wnt protein-expressing cells and Afamin-expressing cells.

[0113] 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 further preferably 300 μg / mL to 1 μg / mL.

[0114] (IGF1)

[0115] IGF1, also known as growth factor C, is a factor that is mainly secreted in the liver by the stimulation of growth hormone (GH). It is known that most cells in the human body (especially muscle, bone, liver, kidney, nerve, skin and lung cells) are affected by IGF1. In addition to its insulin-like effects, IGF1 also has the function of regulating cell (especially nerve cell) growth, development, and cell DNA synthesis.

[0116] The concentration of IGF1 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and further preferably 50 ng / mL to 500 ng / mL.

[0117] (FGF2)

[0118] FGF2 is a basic fibroblast growth factor that binds to the fibroblast growth factor receptor (FGFR) and has the function of promoting the proliferation of vascular endothelial cells and their organization into tubular structures, that is, promoting angiogenesis. In addition, it is known that human FGF2 has two isoforms, low molecular weight type (LWL) and high molecular weight type (HWL). LWL is mainly present in the cytoplasm and exerts its effects through self-secretion (autocrine), while HWL is located in the nucleus and shows activity in the intracellular secretory mechanism that exerts its effects in the cell.

[0119] 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 further preferably 50 ng / mL to 100 μg / mL.

[0120] (FGF10)

[0121] FGF10 is mainly secreted from the interstitial tissue and acts on the epithelium through FGFR2b. It is a molecule that plays an important role in epithelial-mesenchymal interaction in the formation and repair of various tissues.

[0122] The concentration of FGF10 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and further preferably 30 ng / mL to 500 ng / mL.

[0123] (EGF)

[0124] EGF is a strong mitotic factor for various cultured ectoderm and mesoderm cells, and has a significant effect on the specific cell differentiation of certain fibroblasts. The EGF precursor exists in the form of a membrane-bound molecule, which is cut off by protein degradation to generate a 53-amino acid peptide hormone that stimulates cells.

[0125] The concentration of EGF contained in the organoid culture medium is preferably 5 ng / mL to 500 ng / mL, more preferably 10 ng / mL to 400 ng / mL, and even more preferably 50 ng / mL to 200 ng / mL.

[0126] (EREG)

[0127] EREG is an EGF-like growth factor that specifically binds to ErbB1 and ErbB4 in the tyrosine kinase (ErbB) family receptors (ErbB1-4). It is known to stimulate the proliferation of keratinocytes, hepatocytes, fibroblasts, and vascular endothelial cells. In addition, EREG is mainly expressed in malignant tumors of the bladder, lung, kidney, colon, etc., placenta, and peripheral blood leukocytes.

[0128] The concentration of EREG contained in the organoid culture medium is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and further preferably 50 ng / mL to 500 ng / mL.

[0129] (BMP inhibitors)

[0130] BMP, as a dimeric ligand, binds to a receptor complex consisting of two different receptor serine / threonine kinases, type I and type II receptors. Type II receptors phosphorylate type I receptors, thereby activating the receptor kinase. The type I receptor then phosphorylates specific receptor substrates (SMADs), thereby directing transcriptional activity through signaling pathways. Typically, BMP inhibitors, for example, are drugs that prevent or inhibit the binding of BMP molecules to BMP receptors, and are drugs that bind to BMP molecules in order to form a complex that neutralizes BMP activity. In addition, BMP inhibitors, for example, are drugs that bind to BMP receptors to prevent or inhibit the binding of BMP molecules to receptors, and are drugs that act as antagonists or inverse agonists.

[0131] The BMP inhibitor preferably has an inhibitory activity of 50% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, compared to the BMP activity level in the absence of the inhibitor.

[0132] BMP inhibitors are preferably natural BMP-binding proteins, for example, 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 cystine knot domain; Sclerostin / SOST, Decorin, α-2 macroglobulin, etc.

[0133] The BMP inhibitor contained in the medium for organoid culture is preferably a Chordin-like protein or a DAN-like protein, and more preferably a Chordin-like protein. The Chordin-like protein is preferably Noggin. Chordin-like protein and DAN-like protein are diffusible proteins that can bind to BMP molecules with various affinities and inhibit BMP molecules from approaching signal transduction receptors. When culturing epithelial stem cells, by adding these BMP inhibitors to the medium for organoid culture, stem cell loss can be prevented.

[0134] The concentration of the BMP inhibitor contained in the organoid culture medium is preferably 10 ng / mL to 100 ng / mL, more preferably 20 ng / mL to 100 ng / mL, and even more preferably 50 ng / mL to 100 ng / mL.

[0135] (TGF-β inhibitor)

[0136] TGF-β is a type of growth factor produced by almost all cells such as the kidney, bone marrow, and platelets. There are 5 subtypes of TGF-β (β1 to β5). In addition, it is known that TGF-β promotes osteoblast proliferation and the synthesis and proliferation of connective tissues such as collagen, and inhibits epithelial cell proliferation and osteoclasts. Generally, TGF-β inhibitors are drugs that, for example, prevent or inhibit the binding of TGF-β to TGF-β receptors, and are drugs that bind to TGF-β in order to form a complex that neutralizes the activity of TGF-β. In addition, TGF-β inhibitors are drugs that, for example, bind to TGF-β receptors to prevent or inhibit the binding of TGF-β to receptors, and are drugs that act as antagonists or inverse agonists.

[0137] Examples of TGF-β inhibitors include A83-01 (CAS No. 909910-43-6), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinone)-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), and TGF-βRI kinase inhibitor II 616452 (CAS No.: 446859-33-2), TGF-βRI kinase inhibitor III 616453 (CAS No.: 356559-13-2), TGF-βRI kinase inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-βRI kinase inhibitor VII 616458 (CAS No.: 666729-57-3), TGF-βRI kinase inhibitor VIII 616459 (CAS No.: 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 (CAT Metelimumab (human IgG4 monoclonal antibody neutralizing TGFβ1), GC-1008 (anti-TGF-β monoclonal antibody), etc. The TGF-β inhibitor is preferably A83-01.

[0138] The concentration of the TGF-β inhibitor contained in the organoid culture medium is preferably 100 nM to 10 μM, more preferably 500 nM to 5 μM, and even more preferably 500 nM to 2 μM.

[0139] (γ-secretase inhibitors)

[0140] Examples of γ-secretase include BMS299897 (CAS No.: 290315-45-6), DAPT (CAS No.: 208255-80-5), DBZ (CAS No.: 209984-56-5), JLK6 (CAS No.: 62252-26-0), L-685458 (CAS No.: 292632-98-5), and LY411575 (CAS No.: 209984-57-6). Among them, LY411575 is preferred.

[0141] The concentration of the γ-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.

[0142] (Other ingredients)

[0143] The medium for organoid culture may also contain a Rho-kinase (Rock) inhibitor. Examples of Rock inhibitors include Y-27632 (CAS No.: 129830-38-2), Fasudil (HA1077) (CAS No.: 103745-39-7), and H-1152 (CAS No.: 871543-07-6). When Y-27632 is used as a Rock inhibitor, it is preferably added within the first two days of culturing stem cells dispersed into single cells. The Y-27632 contained in the medium for organoid culture is preferably about 10 μM.

[0144] When the organoid is a hepatocyte organoid, the medium for organoid culture 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 medium for organoid culture may be, for example, 1 ng / mL or more and 10 μg / mL or less, for example, 1 ng / mL or more and 1 μg / mL or less, for example, 5 ng / mL or more and 100 ng / mL or less.

[0145] The medium for organoid culture may further contain gastrin (or a suitable substitute such as Leu15-gastrin). The concentration of gastrin or a suitable substitute contained in the medium for organoid culture may be, for example, 1 ng / mL or more and 10 μg / mL or less, for example, 1 ng / mL or more and 1 μg / mL or less, for example, 5 ng / mL or more and 100 ng / mL or less.

[0146] The culture medium for organoid culture may further include at least one amino acid. As amino acids, for example, 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 can be cited. The concentration of L-glutamine contained in the culture medium for organoid culture is more than 0.05g / L and less than 1g / L (usually more than 0.1g / L and less than 0.75g / L). Other amino acids contained in the culture medium for organoid culture are more than 0.001g / L and less than 1g / L (usually more than 0.01g / L and less than 0.15g / L). Amino acids can also be synthetic.

[0147] The medium for organoid culture may further include at least one vitamin. Examples of vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0148] The medium for organoid culture may further include at least one inorganic salt. Inorganic salts help maintain the osmotic pressure balance of cells and help regulate membrane potential. As specific examples of inorganic salts, calcium salts, copper salts, iron salts, magnesium salts, potassium salts, sodium salts, and zinc salts can be listed. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specific salts can include CaCl2, CuSO4-5H2O, Fe(NO3)-9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4-H2O, ZnSO4-7H2O, etc.

[0149] The medium for organoid culture may further contain at least one sugar that can serve as a carbon energy source. As sugars, for example, glucose, galactose, maltose, and fructose can be cited. Among them, glucose is preferred, and D-glucose (dextrose) is particularly preferred. The concentration of sugar contained in the medium for organoid culture is preferably 1 g / L or more and 10 g / L or less.

[0150] The medium for organoid culture may further contain at least one trace element. Examples of trace elements include barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0151] The medium for organoid culture may further contain at least one additional drug. Examples of the drug include nutrients or growth factors reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite, and the like.

[0152] [Organoid Proliferation Method]

[0153] In one embodiment, the present invention provides a method for growing an organoid, comprising the step of culturing the organoid in the presence of a Hippo signaling pathway inhibitor and in the absence of an extracellular matrix. The method for growing an organoid of this embodiment can also be referred to as a method for producing an organoid.

[0154] As described later in the Examples, the method of this embodiment can proliferate organoids in the absence of an extracellular matrix. Here, "proliferate" means that the number of cells constituting the organoid increases, and can also be referred to as "culture".

[0155] In the method of this embodiment, the Hippo signaling pathway inhibitor, the extracellular matrix, the organoid, etc. are the same as described above. That is, in the method of this embodiment, the Hippo signaling pathway inhibitor 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.

[0156] In the method of this embodiment, organoids can also be cultured under serum-free conditions. By growing organoids in the absence of an extracellular matrix and serum, organoids produced without using any animal-derived products can be obtained.

[0157] [Organoids]

[0158] In one embodiment, the present invention provides an organoid proliferated by the above-mentioned proliferative method.

[0159] The organoids of this embodiment are produced without animal-derived products, and therefore can be easily applied to regenerative medicine.

[0160] The organoid of this embodiment may be a digestive tract organoid. Conventional digestive tract organoids cultured in the presence of Matrigel (registered trademark) are spherical in shape, with the inner side of a single-layer epithelial cell sheet maintaining apical polarity being the apical side.

[0161] In contrast, as described later in the Examples, the organoid of the present embodiment has a spherical morphology of multilayered cells and does not maintain the apical polarity of epithelial cells, which is different from the morphology of conventional gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).

[0162] It is believed that the organoid of the present embodiment is different from conventional organoids in terms of gene expression patterns, etc. However, it is unknown and unrealistic whether the difference in gene expression patterns can be confirmed and whether it is a conventional organoid or an organoid of the present embodiment can be clearly determined. Therefore, it is believed that it is practical to define the organoid of the present embodiment by its production method.

[0163] [Drugs for establishing organoids in the absence of extracellular matrix]

[0164] In one embodiment, the present invention provides a drug for establishing an organoid in the absence of an extracellular matrix, wherein the active ingredient of the drug is a Hippo signaling pathway inhibitor.

[0165] As described above, the inventors have found that organoids can be grown in the absence of an extracellular matrix by adding a Hippo signaling pathway inhibitor to the culture medium. However, although the established organoids can be grown, it cannot be said that by adding a Hippo signaling pathway inhibitor to the culture medium, organoids can be established from biological source tissues.

[0166] In contrast, as described later in the Examples, the inventors have found that by adding the drug of the present embodiment to the culture medium, organoids can be established from biological source tissues in the absence of an extracellular matrix, and further, organoids can be established from single cells without using an extracellular matrix.

[0167] In the medicine of this embodiment, the Hippo signaling pathway inhibitor, the extracellular matrix, the organoid, etc. are the same as described above. That is, in the method of this embodiment, the Hippo signaling pathway inhibitor is preferably an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor. In addition, in the medicine of this embodiment, the organoid is preferably an epithelial organoid.

[0168] [Method for producing organoids]

[0169] In one embodiment, the present invention provides a method for producing an organoid, comprising the step of establishing an organoid in the presence of a Hippo signaling pathway inhibitor and in the absence of an extracellular matrix. The method for producing an organoid of this embodiment can also be referred to as a method for establishing an organoid.

[0170] As described later in the examples, by the method of this embodiment, organoids can be established from biological tissues in the absence of an extracellular matrix, and organoids can also be established from single cells without using an extracellular matrix. Further, according to the method of this embodiment, organoids can also be established under serum-free conditions. By establishing organoids in the absence of an extracellular matrix and serum-free conditions, organoids made in an animal-free product manner from the time of establishment can be obtained.

[0171] In the method of this embodiment, the Hippo signaling pathway inhibitor, the extracellular matrix, the organoid, etc. are the same as described above. That is, in the production method of this embodiment, the Hippo signaling pathway inhibitor is preferably an MST1 / 2 kinase inhibitor or a LATS1 / 2 kinase inhibitor. In addition, in the production method of this embodiment, the organoid is preferably an epithelial organoid.

[0172] [Organoids]

[0173] In one embodiment, the present invention provides an organoid produced by the above production method.

[0174] As described later in the examples, the inventors have found that, by the above-mentioned manufacturing method, organoids can be established from biological source tissues in the absence of an extracellular matrix, and organoids can also be established from single cells without using an extracellular matrix. The organoids of this embodiment, especially when established under serum-free conditions, are obtained from the time of establishment in an animal-free product-free manner. Therefore, it is particularly easy to transplant into an organism and apply to regenerative medicine. Therefore, in one embodiment, the present invention provides a regenerative medicine preparation whose active ingredient is an organoid manufactured by the above-mentioned manufacturing method.

[0175] It is believed that the organoid of the present embodiment is different from conventional organoids in terms of gene expression patterns, etc. However, it is unknown and unrealistic whether the difference in gene expression patterns can be confirmed and whether it is a conventional organoid or an organoid of the present embodiment can be clearly determined. Therefore, it is believed that it is practical to define the organoid of the present embodiment by its production method.

[0176] [Screening method for drugs that can culture organoids in the absence of extracellular matrix]

[0177] In one embodiment, the present invention provides a method for screening drugs that can culture organoids in the absence of an extracellular matrix, comprising: a step of culturing organoids in the presence of a test substance and in the absence of an extracellular matrix; and a step of evaluating the proliferation of the organoids; wherein, a higher proliferation of the organoids than in the absence of the test substance indicates that the test substance is a drug that can culture organoids in the absence of an extracellular matrix.

[0178] The screening method of this embodiment can screen out drugs that can culture organoids in the absence of an extracellular matrix.

[0179] In the screening method of this embodiment, the extracellular matrix, organoid, etc. are the same as described above.

[0180] In the screening method of this embodiment, the substance to be tested is not particularly limited, and for example, a natural compound library, a synthetic compound library, an existing drug library, etc. can be used.

[0181] The proliferation of organoids can be evaluated by appropriate methods, such as cell number measurement based on microscopic observation, cell number measurement based on ATP measurement, evaluation based on cell number measurement of tetrazolium compounds such as MTT, etc. When the proliferation of organoids is higher than that in the absence of the test substance, it can be judged that the test substance is a drug that can culture organoids in the absence of extracellular matrix.

[0182] [Example]

[0183] The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to the following Examples.

[0184] [Experimental Example 1]

[0185] (Organoid culture in the presence of LATS kinase inhibitors 1)

[0186] Human small intestine-derived organoids and human large intestine-derived organoids were cultured in the presence of Lats-IN-1 (CAS No.: 1424635-83-5, hereinafter referred to as "Lats-IN". Also referred to as "TRULI"), an inhibitor of LATS1 kinase and LATS2 kinase, and their dynamics were observed.

[0187] Human small intestine-derived organoids and human large intestine-derived organoids were established from tissues derived from patients with digestive organ tumors who had given their instructions and consent, according to an ethical research plan approved by the Ethics Committee of the Keio University School of Medicine.

[0188] TrypLE Express (Thermo Fisher Scientific) was used to dissociate various organs into single cells to obtain cell suspension. 5 Cells / well were inoculated with 25 μL Matrigel (registered trademark) in a 48-well plate. After the Matrigel (registered trademark) gelled, 100 μL of the culture medium shown in Table 1 below was added to each well and cultured at 37°C. 10 mM Lats-IN was added to the culture medium of the test group. After that, the culture medium was replaced every 2 days.

[0189] [Table 1]

[0190]

[0191] Figure 2 Representative photographs of various organs after 7 days of culture and graphs showing cell numbers. Figure 2 The upper part shows the results of human small intestine-derived organoids. Figure 2 The lower part shows the results of human colon-derived organoids. Cell proliferation was measured by measuring adenosine triphosphate (ATP) using a commercially available kit (product name "CellTiter-Glo 3D", Promega).

[0192] Figure 2 The left side of the upper part is a photo of the control (Ctrl) human small intestine-derived organoid cultured without Lats-IN. Figure 2 The upper center is a photograph of human small intestine-derived organoids cultured in the presence of Lats-IN. The scale bar is 200 μm. Figure 2 The upper right side shows 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 represents the luminescence intensity (relative value), which represents the amount of ATP present.

[0193] Figure 2 The left side of the lower part is a photo of the control (Ctrl) human colon-derived organoid cultured without Lats-IN. Figure 2 The lower center is a photograph of human colon-derived organoids cultured in the presence of Lats-IN. The scale bar is 200 μm. Figure 2 The right side of the lower part shows 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 represents the luminescence intensity (relative value), which represents the amount of ATP present.

[0194] The results showed that by adding LATS kinase inhibitors to the culture medium, the cell proliferation of human small intestine-derived organoids and human large intestine-derived organoids was significantly activated.

[0195] [Experimental Example 2]

[0196] (Organoid culture in the presence of LATS kinase inhibitors 2)

[0197] Conventionally, an extracellular matrix such as Matrigel (registered trademark) is required for organoid culture, but it is difficult to sufficiently maintain cell proliferation and differentiation using substitutes for Matrigel (registered trademark).

[0198] Furthermore, when cells are cultured three-dimensionally without an extracellular matrix such as Matrigel (registered trademark), the cells aggregate to form cell masses. In this state, the cell proliferation activity is extremely low, making long-term passaging completely impossible.

[0199] In this experimental example, human small intestine-derived organoids and human large intestine-derived organoids established under the condition of Matrigel (registered trademark) were cultured in the absence of Matrigel (registered trademark) and LATS kinase inhibitor (final concentration 10 μM) to observe their dynamics. The same as Experimental Example 1 except that each organ was cultured under the condition of Matrigel (registered trademark) was not present.

[0200] Figure 3 Representative photographs of various organs after 7 days of culture and graphs showing cell numbers. Figure 3 The upper part shows the results of human small intestine-derived organoids. Figure 3 The lower part shows the results for human colon-derived organoids.

[0201] Figure 3 The left side of the upper part is a photo of the control (Ctrl) human small intestine-derived organoid cultured without Lats-IN. Figure 3 The upper center is a photograph of human small intestine-derived organoids cultured in the presence of Lats-IN. The scale bar is 100 μm. Figure 3 The upper right side shows the results of the cell count of human small intestine-derived organoids cultured in the control (Ctrl) and in the presence of Lats-IN, which was determined by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph represents the luminescence intensity (relative value), which represents the amount of ATP present.

[0202] Figure 3 The left side of the lower part is a photo of the control (Ctrl) human colon-derived organoid cultured without Lats-IN. Figure 3 The lower center is a photograph of human colon-derived organoids cultured in the presence of Lats-IN. The scale bar is 100 μm. Figure 3The lower right side shows the results of measuring the number of cells of human colon-derived organoids cultured in the control (Ctrl) and in the presence of Lats-IN. The vertical axis of the graph represents the luminescence intensity (relative value), which represents the amount of ATP present.

[0203] The results showed that adding LATS kinase inhibitors to the culture medium significantly activated cell proliferation in human small intestine-derived organoids and human intestinal-derived organoids even without Matrigel (registered trademark). It was previously thought that organoid proliferation without Matrigel (registered trademark) was impossible, but now it is possible, which is a surprising result.

[0204] [Experimental Example 3]

[0205] (Study on YAP phosphorylation)

[0206] Human small intestine-derived organoids were cultured in the absence or presence of Matrigel (registered trademark) and in the absence or presence of a LATS kinase inhibitor (final concentration 10 μM), and YAP phosphorylation was confirmed by Western blotting.

[0207] Figure 4 Representative photographs of Western blot results are shown. Figure 4 In the table, "pYAP" indicates phosphorylated YAP, and "YAP" indicates total YAP (the sum of phosphorylated YAP and non-phosphorylated YAP). Phospho-YAP (Ser127) (#4911, Cell Signaling Technology) was used as an antibody against phosphorylated YAP. Total-YAP (#14074, Cell Signaling Technology) was used as an antibody against total YAP.

[0208] The results confirmed that, regardless of the presence or absence of Matrigel (registered trademark), in the presence of Lats-IN, phosphorylation of YAP was significantly inhibited compared to the absence of Lats-IN.

[0209] [Experimental Example 4]

[0210] (Immunostaining study)

[0211] In the absence or presence of Matrigel (registered trademark) and the absence or presence of LATS kinase inhibitor (final concentration 10 μM), human small intestine-derived organoids and human large intestine-derived organoids were cultured as in Experimental Example 2. Next, immunostaining was performed to determine the localization of YAP. In addition, Ki67, a cell proliferation marker, was detected to confirm proliferating cells.

[0212] Figure 5The leftmost column is a photo of the immunostaining results of human small intestine-derived organoids cultured under conditions with Matrigel (registered trademark) and without Lats-IN (Ctrl). The top part is an image of the combined results of Ki67, YAP, and nuclear (Nuc). The second part from the top shows the results of Ki67, the third part from the top shows the results of YAP, and the fourth part from the top shows the results of nuclear (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0213] Figure 5 The second column from the left is a photo of the immunostaining results of human small intestine-derived organoids cultured under conditions of Matrigel (registered trademark) and Lats-IN. The top part is an image of the combined results of Ki67, YAP, and nucleus (Nuc). The second part from the top shows the results of Ki67, the third part from the top shows the results of YAP, and the fourth part from the top shows the results of nucleus (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part. The scale bar represents 50 μm.

[0214] Figure 5 The third column from the left is a photo of the immunostaining results of human small intestine-derived organoids cultured without Matrigel (registered trademark) and Lats-IN (Ctrl). The top part is an image of the combined results of Ki67, YAP, and nuclear (Nuc). The second part from the top shows the results of Ki67, the third part from the top shows the results of YAP, and the fourth part from the top shows the results of nuclear (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0215] Figure 5 The rightmost column shows a photo of the immunostaining results of human small intestine-derived organoids cultured without Matrigel (registered trademark) and with Lats-IN. The top part is an image of the combined results of Ki67, YAP, and nuclear (Nuc), with the second part from the top showing the results of Ki67, the third part from the top showing the results of YAP, and the fourth part from the top showing the results of nuclear (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0216] Figure 6The leftmost column is a photo of the immunostaining results of human colon-derived organoids cultured under conditions of Matrigel (registered trademark) and without Lats-IN (Ctrl). The top part is an image of the combined results of Ki67, YAP, and nuclear (Nuc), with the second part from the top showing the results of Ki67, the third part from the top showing the results of YAP, and the fourth part from the top showing the results of nuclear (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0217] Figure 6 The second column from the left is a photo of the immunostaining results of human colon-derived organoids cultured under conditions of Matrigel (registered trademark) and Lats-IN. The top part is an image that merges the detection results of Ki67, YAP, and nucleus (Nuc). The second part from the top shows the detection results of Ki67, the third part from the top shows the detection results of YAP, and the fourth part from the top shows the detection results of nucleus (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0218] Figure 6 The third column from the left is a photo of the immunostaining results of human colon-derived organoids cultured without Matrigel (registered trademark) and Lats-IN (Ctrl). The top part is an image of the combined results of Ki67, YAP, and nuclear (Nuc). The second part from the top shows the results of Ki67, the third part from the top shows the results of YAP, and the fourth part from the top shows the results of nuclear (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0219] Figure 6 The rightmost column is a photo of the immunostaining results of human colon-derived organoids cultured without Matrigel (registered trademark) and with Lats-IN. The top part is an image of the combined results of Ki67, YAP, and nuclear (Nuc). The second part from the top shows the results of Ki67, the third part from the top shows the results of YAP, and the fourth part from the top shows the results of nuclear (Nuc). The fields of view of the second, third, and fourth parts from the top correspond to the area surrounded by the quadrilateral in the top part.

[0220] Figure 7 It is a summary Figure 5 , Figure 6The results are shown in Figure 2. The results show that, regardless of the presence or absence of Matrigel (registered trademark), in the absence of Lats-IN, YAP is localized in the cytoplasm, and the results of counting the number of Ki67-positive cells indicating cell proliferation show that the proportion of Ki67-positive cells is about 10% of all cells. On the contrary, regardless of the presence or absence of Matrigel (registered trademark), in the presence of Lats-IN, the results of counting the number of cells in which YAP is localized in the nucleus show that the proportion of cells in which YAP is localized in the nucleus is about 50-80% of all cells, and the proportion of Ki67-positive cells is more than about 50% of all cells.

[0221] The above results indicate that by adding Lats-IN, LATS was inhibited, YAP phosphorylation was suppressed, YAP translocated to the nucleus, and cell proliferation was activated.

[0222] [Experimental Example 5]

[0223] (Study on the transcriptional activity of TEAD)

[0224] We investigated whether YAP transferred to the nucleus by adding Lats-IN assists the transcriptional activity of TEAD. Specifically, we cultured human small intestine-derived organoids and human large intestine-derived organoids without Matrigel (registered trademark) and without or with Lats-IN (final concentration 10 μM), and observed cell proliferation when TEAD was inhibited using the TEAD inhibitor MYF01-37 (CAS number: 2416417-65-5).

[0225] Figure 8 The results of measuring the proliferation of human small intestine-derived organoids and human large intestine-derived organoids without Lats-IN (Ctrl), with Lats-IN (Lats-IN), with Lats-IN and MYF01-37 (Lats-IN+MYF), and with MYF01-37 (MYF) are shown. Cell proliferation was measured by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the figure represents the luminescence intensity (relative value), which characterizes the amount of ATP present.

[0226] The results showed that, in any organoid, cell proliferation was activated in the group to which Lats-IN was added, but cell proliferation was inhibited in the group to which both Lats-IN and MYF01-37 were added.

[0227] This result shows that the addition of Lats-IN inhibits LATS function, YAP is transferred to the nucleus, and YAP transferred to the nucleus assists the transcriptional activity of TEAD. In other words, this result shows that the addition of Lats-IN to the culture medium makes it possible to proliferate cells without Matrigel (registered trademark) through the pathway of LATS inhibition, YAP nuclear transfer, and promotion of cell growth factor transcriptional activity through TEAD, which was previously impossible.

[0228] [Experimental Example 6]

[0229] (Study on the formation of organoids from single cells)

[0230] We studied whether organoids can be formed from single cells without Matrigel (registered trademark). It has been believed that cell-matrix interactions are necessary for cell survival and proliferation, but there are no cell-matrix interactions in culture without Matrigel (registered trademark). Therefore, it can be seen that signals from cell-matrix interactions are not involved in YAP-TEAD transcriptional regulation based on LATS inhibition.

[0231] In addition to cell-matrix interactions, cell-to-cell (cell-to-cell) signals are also believed to play an important role in cell survival and proliferation.

[0232] Therefore, in order to investigate whether cell-cell interaction signals are involved in cell proliferation through YAP-TEAD transcriptional regulation based on LATS inhibition, human small intestine-derived organoids were dissociated into single cells and the formation of organoids from single cells was observed.

[0233] Fig. 9 Representative photographs are shown of 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). Fig. 9 In the figure, arrows indicate cells or organoids. In addition, D1, D4, and D7 indicate the first day, fourth day, and seventh day after the start of culture, respectively. Fig. 9 In the brackets at the bottom, the denominator indicates the number of seeded single cells, and the numerator indicates the number of organoids formed. In addition, the ratio (%) of cells that formed organoids among the seeded single cells is also shown.

[0234] The results showed that cells hardly proliferated in the absence of Matrigel (registered trademark) and Lats-IN. On the contrary, the results showed that cells proliferated from single cells to form organoids in the presence of Matrigel (registered trademark) and in the absence of Matrigel (registered trademark) and in the presence of Lats-IN. In addition, it was shown that the proportion of cells that formed organoids was higher in the presence of Matrigel (registered trademark) and Lats-IN than in the absence of Matrigel (registered trademark) and in the presence of Lats-IN.

[0235] Based on the above results, it can be seen that the signals of cell-to-cell interaction are not involved in the YAP-TEAD transcriptional regulation based on LATS inhibition. The YAP-TEAD transcriptional regulation based on LATS inhibition activates cell proliferation alone through its signal and is related to organoid formation.

[0236] [Experimental Example 7]

[0237] (Organoid establishment without Matrigel (registered trademark))

[0238] The study investigated whether organoids could be established from the human lower gastrointestinal tract without Matrigel (registered trademark).

[0239] Crypts were isolated from human small intestine-derived tissue and human large intestine-derived tissue, and the isolated crypts were cultured without Matrigel (registered trademark) and without or with Lats-IN (final concentration 10 μM).

[0240] Fig.10 The figure shows the results of the proliferation of crypts isolated from human colon tissue measured 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 figure represents the luminescence intensity (relative value), which represents the amount of ATP present. Fig.10 As shown, in the absence of Lats-IN, the isolated crypts increased enough to survive one passage, but then gradually became apoptotic and completely died after the second passage.

[0241] In contrast, in the presence of Lats-IN, the isolated crypts did not stop proliferating and could be passaged almost permanently. The same results were obtained in crypts isolated from human small intestine tissue.

[0242] These results indicate that in the presence of Lats-IN, LATS is inhibited, cell proliferation is induced through YAP, and organoids can be established even in the absence of Matrigel (registered trademark).

[0243] [Experimental Example 8]

[0244] (Cultivation of organoids from various organs)

[0245] We investigated whether organoids derived from various organs could be cultured without Matrigel (registered trademark) and without Lats-IN or with Lats-IN (final concentration 10 μM).

[0246] Fig.11 Representative photographs of the culture results of gastric organoids, biliary organoids, and mammary gland organoids without Matrigel (registered trademark) and Lats-IN (Ctrl) or without Matrigel (registered trademark) and with Lats-IN (Lats-IN) are shown. The scale bar is 100 μm.

[0247] The results showed that even in the absence of Matrigel (registered trademark), cell proliferation was activated in the presence of Lats-IN, and that not only organoids derived from the human lower gastrointestinal tract but also organoids derived from any organ could be cultured.

[0248] [Experimental Example 9]

[0249] (Study on organoid morphology 1)

[0250] 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.

[0251] Fig.12 The upper part shows representative photos of immunostaining of human small intestine-derived organoids. Fig.12 The lower part shows representative photos of immunostaining of human colon-derived organoids. Fig.12 In the figure, the scale bar indicates 50 μm.

[0252] In immunostaining, basement membrane marker integrin β4 (Intβ4), apical marker Ezrin, intercellular adhesion marker β-catenin (βCat), Paneth cell marker Lysozyme, embryonic cell marker Mucin2 (MUC2), intestinal endocrine cell marker ChromograninA (CHGA), and nucleus (Nuc) were detected. In addition, the expression of Lgr5, a stem cell marker, was detected by fluorescence of the fluorescent protein TdTomato introduced into the Lgr5 locus of the cells.

[0253] Conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark) are spherical in shape, with the inner side of a single-layer epithelial cell sheet that maintains apical polarity being the apical side, and one organoid contains differentiated cells such as stem cells, embryonic cells, and intestinal endocrine cells.

[0254] On the contrary, Fig.12 As shown, human lower gastrointestinal organoids cultured in the presence of Lats-IN and without Matrigel (registered trademark) have a multilayered spherical morphology and do not maintain the apical polarity of epithelial cells, which is very different from the morphology of conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).

[0255] However, if Fig.12 As shown, human lower gastrointestinal organoids cultured in the presence of Lats-IN and without Matrigel (registered trademark) confirmed the expression of not only Lgr5, a stem cell marker, but also Lysozyme, a Paneth cell marker, Mucin2, an embryonic cell marker, and ChromograninA, an intestinal endocrine cell marker. Like conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark), one organoid contains stem cells and differentiated cells.

[0256] [Experimental Example 10]

[0257] (Study on organoid morphology 2)

[0258] Human lower gastrointestinal organoids established in the presence of Matrigel (registered trademark) were subcultured for 7 generations 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) to observe their morphology and properties.

[0259] Fig.13 The upper part shows representative photos of immunostaining of human small intestine-derived organoids. Fig.13 The lower part shows representative photos of immunostaining of human colon-derived organoids. Fig.13 In the figure, the scale bar indicates 50 μm.

[0260] In immunostaining, basement membrane marker integrin β4 (Intβ4), apical marker Ezrin, intercellular adhesion marker β-catenin (βCat), Paneth cell marker Lysozyme, embryonic cell marker Mucin2 (MUC2), intestinal endocrine cell marker ChromograninA (CHGA), and nucleus (Nuc) were detected. In addition, the expression of Lgr5, a stem cell marker, was detected by fluorescence of the fluorescent protein TdTomato introduced into the Lgr5 locus of the cells.

[0261] The results show that if Fig.13 As shown, even human lower gastrointestinal organoids cultured in the absence of Matrigel (registered trademark) and in the presence of Lats-IN, when cultured again in the presence of Matrigel (registered trademark), still have a spherical shape, with the inner side of the single-layer epithelial cell sheet maintaining apical polarity as the apical side, and one organoid contains differentiated cells such as stem cells, embryonic cells, and intestinal endocrine cells.

[0262] This result indicates that the properties of human lower gastrointestinal epithelial cells remain unchanged even when cultured for a long period of time without Matrigel (registered trademark).

[0263] [Experimental Example 11]

[0264] (Study on the microenvironmental factors necessary for organoid culture)

[0265] The inventors have previously clarified that the microenvironmental factors required 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).

[0266] In this experimental example, the association between LATS inhibition and microenvironmental factors essential for organoid culture was investigated. Fig.14 The results of the measurement of the proliferation of human small intestine-derived organoids and human large intestine-derived organoids cultured under the conditions of adding all the microenvironmental factors of WRENAIF and removing some of these microenvironmental factors are shown. The culture medium of the composition shown in Table 1 above is used as the culture medium for adding all the microenvironmental factors of WRENAIF, and the culture medium in which the corresponding microenvironmental factors are removed from the culture medium of the composition shown in Table 1 above is used as the culture medium for removing some of these microenvironmental factors. Cell proliferation is measured by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the figure represents the luminescence intensity (relative value), which characterizes the amount of ATP present.

[0267] Fig.14 In the figure, "Ctrl" indicates the result of culturing with Matrigel (registered trademark), and "Lats-IN" indicates the result of culturing without Matrigel (registered trademark) and with Lats-IN (final concentration 10 μM). In addition, in the horizontal axis, added microenvironmental factors are indicated in black, and unadded microenvironmental factors are indicated in light colors.

[0268] The results showed that even without adding one or two microenvironmental factors necessary for the culture of human lower gastrointestinal epithelial organoids to the culture medium, the cells would proliferate by adding Lats-IN.

[0269] Fig.15 The results of measuring the proliferation of human small intestine-derived organoids cultured without Matrigel (registered trademark) and without or with Lats-IN (final concentration 10 μM), with all microenvironmental factors of WRENAIF added, and with some of these microenvironmental factors removed are shown. Fig.15 In the figure, P0 to P16 represent passage number 0 to passage number 16. In addition, dark colors in the figure indicate that passage is possible, and light colors indicate that passage is impossible.

[0270] The results showed that even if Lats-IN was added, if the culture medium lacked any of the microenvironmental factors in WRENAIF, the cells could not be subcultured for a long time. From the above results, it can be seen that Lat-IN cannot supplement the microenvironmental factors necessary for organoid culture, but rather promotes the proliferation of organoids in synergy with microenvironmental factors.

[0271] [Experimental Example 12]

[0272] (Study on the mechanism that enables organoid culture without Matrigel (registered trademark))

[0273] The researchers studied why it is possible to culture organoids without Matrigel (registered trademark) by inhibiting LATS.

[0274] 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 regulated by Wnt expression was studied by quantitative real-time PCR.

[0275] Fig.16The results of quantitative real-time PCR are shown. The results show that compared to the AXIN2 expression observed when culturing organoids under the condition of Matrigel (registered trademark), the expression level of AXIN2 is significantly reduced when culturing organoids under the condition of no Matrigel (registered trademark). In addition, it is also shown that if organoids are cultured under the condition of no Matrigel (registered trademark) and Lats-IN, the expression level of AXIN2 rises to the same level as under the condition of Matrigel (registered trademark).

[0276] 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 phosphorylated ERK induced by EGF signaling was detected by Western blotting.

[0277] Fig.17 The results of Western blotting are shown. Fig.17 In the table, "pERK" represents phosphorylated ERK, and "total ERK" represents total ERK (the sum of phosphorylated ERK and non-phosphorylated ERK). In addition, "pYAP" represents phosphorylated YAP, and "total YAP" represents total YAP (the sum of phosphorylated YAP and non-phosphorylated YAP). As an antibody against phosphorylated ERK, phospho-ERK (Thr202 / Tyr204) (#4370, Cell Signaling Technology) was used. As an antibody against total ERK, total-ERK (#4695, Cell Signaling Technology) was used. As an antibody against phosphorylated YAP, phospho-YAP (Ser127) (#4911, Cell Signaling Technology) was used. As an antibody against total YAP, total-YAP (#14074, Cell Signaling Technology) was used.

[0278] The results showed that ERK phosphorylation was significantly reduced when organoids were cultured without Matrigel (registered trademark) compared to the ERK phosphorylation observed when organoids were cultured with Matrigel (registered trademark). In addition, it was shown that if organoids were cultured without Matrigel (registered trademark) and with Lats-IN, ERK phosphorylation increased to the same level as in the presence of Matrigel (registered trademark).

[0279] The above results show that Wnt signals and EGF signals, which cannot enter cells in the absence of Matrigel (registered trademark), can enter cells even in the absence of Matrigel (registered trademark) by inhibiting LATS. This result shows that since cell proliferation becomes possible, organoid culture becomes possible.

[0280] [Experimental Example 13]

[0281] (Study on organoid morphology 3)

[0282] As in Experimental Example 7, human colon-derived organoids were established without Matrigel (registered trademark) and with Lats-IN (final concentration 10 μM), and the morphology and properties were observed. This Experimental Example differs from Experimental Example 9 in that Matrigel (registered trademark) was not used from the time of organoid establishment.

[0283] Fig.18 Representative photos of immunostaining of human colon-derived organoids seven days after establishment, passage 18, and culture. Fig.18 In the figure, the scale bar indicates 50 μm.

[0284] In immunostaining, the apical marker Ezrin, the basement membrane marker β4-Integrin, the intercellular adhesion marker β-catenin, the embryonic cell marker Mucin2 (MUC2), the intestinal endocrine cell marker Chromogranin A (CHGA), and the nucleus (Nuc) were detected.

[0285] As described above, conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark) are spherical in shape, with the inner side of a single-layer epithelial cell sheet maintaining apical polarity being the apical side, and one organoid contains differentiated cells such as stem cells, embryonic cells, and intestinal endocrine cells.

[0286] On the contrary, Fig.18 As shown, the human colon-derived organoids established in the absence of Matrigel (registered trademark) and with Lats-IN have a multilayered spherical morphology and do not maintain the apical polarity of epithelial cells, which is very different from the morphology of conventional human lower gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).

[0287] However, if Fig.18As shown, human lower gastrointestinal organoids established in the absence of Matrigel (registered trademark) and with Lats-IN have been shown to express the embryonic cell marker Mucin2 and the intestinal endocrine cell marker ChromograninA. Therefore, it is believed that, like conventional human lower gastrointestinal organoids established in the presence of Matrigel (registered trademark), one organoid contains stem cells and differentiated cells.

[0288] Fig.19 This is a microscopic photograph of human colon-derived organoids that were established without Matrigel (registered trademark) and cultured in the presence of Lats-IN for 7 days after 18 passages. Fig.19 The scale bar on the left photo is 500 μm. Fig.19 The scale bar in the photo on the right is 100 μm.

[0289] [Experimental Example 14]

[0290] (Study on organoid morphology 4)

[0291] As in Experimental Example 7, human colon-derived organoids established without Matrigel (registered trademark) and with Lats-IN (final concentration 10 μM) were initially cultured in the presence of Matrigel (registered trademark) to observe morphology and properties. This experimental example differs from Experimental Example 9 in that organoids were established without Matrigel (registered trademark).

[0292] Fig. 20 Representative photographs of immunostaining of human colon-derived organoids established without Matrigel (registered trademark) and in the presence of Lats-IN, passaged 18 times, and then cultured for the first time for 7 days in the presence of Matrigel (registered trademark). Fig. 20 In the figure, the scale bar indicates 50 μm.

[0293] In immunostaining, the apical marker Ezrin, the basement membrane marker β4-Integrin, the intercellular adhesion marker β-catenin, the embryonic cell marker Mucin2 (MUC2), the intestinal endocrine cell marker Chromogranin A (CHGA), and the nucleus (Nuc) were detected.

[0294] The results show that if Fig. 20As shown, even human lower gastrointestinal organoids established without Matrigel (registered trademark) and with Lats-IN are spherical when cultured with Matrigel (registered trademark), with the inner side of the single-layer epithelial cell sheet maintaining apical polarity as the apical side, and one organoid contains differentiated cells such as embryonic cells and intestinal endocrine cells.

[0295] This result indicates that the properties of human lower gastrointestinal epithelial cells remain unchanged even when established under Matrigel (registered trademark)-free conditions.

[0296] Fig.21 This is a microscopic photograph of human colon-derived organoids established without Matrigel (registered trademark) but with Lats-IN, passaged 18 times, and then cultured for the first time for 7 days in the presence of Matrigel (registered trademark). Fig.21 The scale bar on the left photo is 500 μm. Fig.21 The scale bar in the photo on the right is 100 μm.

[0297] [Experimental Example 15]

[0298] (Cultivation of hepatocyte organoids without Matrigel (registered trademark))

[0299] TrypLE Express (Thermo Fisher Scientific) was added to human hepatocyte organoids established from primary human hepatocytes and incubated at 37°C for 5 minutes to dissociate into single cells.

[0300] Next, the cells were collected, suspended in a proliferation medium supplemented with 2 μM, 5 μM, 10 μM, 20 μM, or 50 μM TDI-011536 (CAS No.: 2687970-96-1), and seeded at 2,000 cells / well (preferably 2,000 to 4,000 cells / well) in an UltraLow ​​attachment 96-well plate. In addition, cells in a proliferation medium not supplemented with TDI-011536 were used as a control.

[0301] The proliferation medium is a medium prepared by adding 10% Afamin-Wnt-3A conditioned medium, 2% R-spondin1 conditioned medium, 2% Noggin conditioned medium, and 20 ng / mL oncostatin M to the basal medium (advanced DMEM / F-12 medium, 10 mM HEPES, 2 mM GlutaMAX, 100 U penicillin, 100 μg / mL streptomycin).

[0302] On the sixth day after seeding, the number of viable cells in human hepatocyte organoids was measured using a commercially available kit (product name "CellTiter-Glo 3D", Promega).

[0303] Fig. 22 The results of the determination of the number of viable cells are shown. As a result, in the groups with 20 μM TDI-011536 (LATS-IN) and 50 μM TDI-011536, the number of viable cells was measured to be 16 and 17 times higher than that of the control group. An increase in the number of viable cells was also observed in other LATS-INs (such as TRULI), and the number of viable cells increased by about 3 times, indicating that the number of viable cells in human hepatocyte organoids increased under the conditions of LATS-IN without Matrigel.

[0304] [Experimental Example 16]

[0305] (Establishment of hepatocyte organoids without Matrigel (registered trademark))

[0306] The frozen primary human hepatocytes were suspended and thawed in advanced DMEM / F-12 medium. Line 1 and Line 2 were used as primary human hepatocytes.

[0307] Next, each thawed primary human hepatocyte was stained with APC-labeled mouse anti-human EpCAM antibody, and EpCAM-negative cells were recovered using a cell sorter. The recovered cells were then suspended in a proliferation medium supplemented with 20 μM TDI-011536 and seeded in an Ultra Low attachment 96-well plate at 3,000 to 4,000 cells / well. The cells were then cultured to obtain hepatocyte organoids. In addition, for comparison, cells cultured in a proliferation medium without TDI-011536 and a proliferation medium without oncostatin M were also prepared.

[0308] Passaging of hepatocyte organoids without Matrigel (registered trademark) was performed 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 incubated at 37°C for 5 minutes to dissociate into single cells.

[0309] Next, the recovered cells were suspended in a proliferation medium supplemented with 20 μM TDI-011536, and seeded at 2,000 cells / well in an Ultra Low attachment 96-well plate.

[0310] Fig.23 These are microscope photos of each cell taken 1 day and 9 days after the start of culture. The scale bar is 500 μm. Fig.24 The results of measuring cell proliferation over time are shown. Fig.24 In the figure, the vertical axis represents cell proliferation (fold), and the horizontal axis represents the number of days in culture. Fig.23 and Fig.24 In the table, “OSM” means oncostatin M, and “Lats IN” means Lats kinase inhibitor (TDI-011536).

[0311] Results, such as Fig. 22 As shown in Figure 2, an increase in the number of viable cells was observed with the addition of a Lats kinase inhibitor (TDI-011536). Fig.23 As shown in Figure 2, human hepatocyte organoids were successfully established using a proliferation medium supplemented with both Lats kinase inhibitor (TDI-011536) and oncostatin M. The established human hepatocyte organoids can be cultured for at least 2 months, with the number of cells proliferating from the establishment to 10 6 ~10 8 times( Fig.24 ).

[0312] [Experimental Example 17]

[0313] (Study on hepatocyte organoids cultured without Matrigel (registered trademark))

[0314] Fluorescence immunostaining of hepatocyte organoids cultured without Matrigel (registered trademark) confirmed that they were hepatocytes. Fluorescence immunostaining of hepatocyte organoids cultured with Matrigel (registered trademark) was also performed for comparison.

[0315] First, human hepatocyte organoids were fixed using 1% formaldehyde / HEPES buffered saline (HBS). Then, fixed human hepatocyte organoids were perforated using 1% Triton-X100 / PBS. Then, human hepatocyte organoids were blocked using 1% BSA / PBS. Next, the hepatocyte marker HNF4α was immunostained using mouse anti-HNF4α antibody (Thermo Fisher Scientific).

[0316] Fig.25 The microscope photographs of the fluorescent immunostaining results are shown. The scale bar is 50 μm. Fig.25 In the table, "Matrigel (+)" shows the results of hepatocyte organoids cultured in the presence of Matrigel (registered trademark), and "Matrigel (-)" shows the results of hepatocyte organoids cultured in the absence of Matrigel (registered trademark). In addition, "Nuc" indicates the results of nuclear staining.

[0317] The results showed that human hepatocyte organoids cultured without Matrigel (registered trademark) expressed the hepatocyte marker HNF4α, just like hepatocyte organoids cultured with Matrigel (registered trademark). The results showed that human hepatocyte organoids cultured without Matrigel (registered trademark) were undoubtedly hepatocyte organoids.

[0318] [Experimental Example 18]

[0319] (Organoid culture in the presence of LATS kinase inhibitors 3)

[0320] As in Experimental Example 2, human large intestine-derived organoids and human small intestine-derived organoids were cultured in the absence of Matrigel (registered trademark) and with a LATS kinase inhibitor (final concentration 20 μM) or without a LATS kinase inhibitor (control), and their dynamics were observed.

[0321] GA-017 (CAS No.: 2351906-74-4), TDI-011536 (CAS No.: 2687970-96-1) and TRULI (CAS No.: 1424635-83-5, also known as "Lats-IN", "Lats-IN-1") were used as LATS kinase inhibitors.

[0322] Fig.26 The results show the number of cells in each organ after 6 days of culture, which was determined by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph represents the luminescence intensity (relative value), which represents the amount of ATP present. Fig.26 On the left is the result of human colon-derived organoids. Fig.26 On the right are the results of human small intestine-derived organoids.

[0323] The results showed that when any LATS kinase inhibitor was used, cell proliferation of human large intestine-derived organoids and human small intestine-derived organoids was significantly activated even in the absence of Matrigel (registered trademark).

[0324] [Experimental Example 19]

[0325] (Study on the formation of organoids from single cells 2)

[0326] Human colon-derived organoids were dissociated into single cells and then the formation of organoids from single cells was observed.

[0327] Fig. 27The 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 TRULI are shown. Fig. 27 In the brackets of , the denominator represents the number of seeded single cells, and the numerator represents the number of formed organoids. In addition, the vertical axis of the graph represents the ratio (%) of cells that formed organoids among the seeded single cells.

[0328] The results showed that cells hardly proliferated under conditions without Matrigel (registered trademark) and TRULI. On the contrary, cells proliferated from single cells to form organoids under conditions with Matrigel (registered trademark) and under conditions without Matrigel (registered trademark) and TRULI.

[0329] [Experimental Example 20]

[0330] (Study of colony size)

[0331] After human small intestine-derived organoids were dissociated into single cells, the formation of organoids from single cells was observed. Fig.28 The diagram shows the results of colony size measurement when organoids were formed from single cells in the presence (+) or absence (-) of Matrigel (registered trademark) and in the absence (-) or presence (+, final concentration 10 μM) of TRULI. The colony size was measured using software (Image J). Fig.28 The vertical axis represents the colony size (mm 2 ).

[0332] [Experimental Example 21]

[0333] (Organoid culture in the presence of LATS kinase inhibitors 4)

[0334] After dissociating various human tissue-derived organoids into single cells, they were cultured in the presence of a LATS kinase inhibitor without Matrigel (registered trademark) or in the absence of Matrigel (registered trademark) and a LATS kinase inhibitor (control) as in Experimental Example 2, and their dynamics were observed.

[0335] TDI-011536 (CAS No.: 2687970-96-1) and TRULI (CAS No.: 1424635-83-5, also known as "Lats-IN" and "Lats-IN-1") were used as LATS kinase inhibitors. The concentration of LATS kinase inhibitor used was 5 μM to 20 μM depending on the organoid.

[0336] Fig.29This is a bright field microscope photo of each organ after 6 days of culture and shows the results of measuring the number of cells in each organ by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega). The vertical axis of the graph represents the luminescence intensity (relative value), which represents the amount of ATP present. Fig.29 In the figure, the upper left part shows the results of human liver-derived organoids, the upper center part shows the results of human salivary gland-derived organoids, the upper right part shows the results of human alveolar-derived organoids, the lower left part shows the results of human respiratory tract-derived organoids, the lower second from the left part shows the results of human pancreas-derived organoids, the lower third from the left part shows the results of human stomach-derived organoids, and the lower right part shows the results of human bile duct-derived organoids. In addition, "Control" indicates the results of the control group, and "TDI" indicates the results of the group to which TDI-011536 was added.

[0337] The results showed that regardless of which LATS kinase inhibitor was used, cell proliferation of organoids from various tissues was significantly activated in the absence of Matrigel (registered trademark).

[0338] [Experimental Example 22]

[0339] (Organoid establishment without Matrigel (registered trademark))

[0340] Crypts were extracted from human colon and dissociated into single cells, and then cultured in the presence of Matrigel (registered trademark) or in the absence of Matrigel (registered trademark) and in the presence of TRULI to establish organoids. In addition, the number of cells in each organ under the conditions was determined by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega).

[0341] Fig.30 The upper part is a schematic diagram for explaining the outline of this experimental example. Fig.30 The lower part shows the results of measuring the growth of organoids. As a result, under the condition of TRULI, even without Matrigel (registered trademark), the same level of cell growth as under the condition of Matrigel (registered trademark) was observed.

[0342] [Experimental Example 23]

[0343] (Orthotopic xenotransplantation of organoids established without Matrigel (registered trademark))

[0344] We investigated whether the human colon-derived organoids established in Experimental Example 22 without Matrigel (registered trademark) and with TRULI functioned normally when orthotopically xenotransplanted into immunodeficient mice (NOG mice).

[0345] Fig.31 This is a schematic diagram for explaining the outline of this experimental example. A green fluorescent protein (GFP) expression vector was electroporated into human colon-derived organoids established in the absence of Matrigel (registered trademark) and in the presence of TRULI to obtain GFP-labeled human colon-derived organoids.

[0346] Next, after dissociating GFP-labeled human colon-derived organoids into single cells, they were pre-cultured for 4 days in a culture medium supplemented with 2% Matrigel (registered trademark) or 2% collagen gel (50% type IA, 50% type IC). During the culture in 2% collagen gel, 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) were added to the culture medium.

[0347] Next, the colon epithelium and mucosa of NOG mice were removed, and GFP-labeled human colon-derived organoids were xenotransplanted in situ. First, NOG mice fed a normal diet were anesthetized by inhalation of 2-3% isoflurane. Next, the mouse colon was washed with PBS and the luminal contents were removed. Then, a catheter equipped with a small balloon was inserted into the anus, and the balloon was inflated with air to maintain the catheter. Next, high-temperature EDTA (250mM, 50℃~55℃) was injected into the rectum to remove the colon mucosa and chelate Ca necessary for cell-cell adhesion and cell-extracellular matrix adhesion. 2+ and Mg 2+ .

[0348] Next, the colon epithelium and mucosa were rubbed using a vibrating electric toothbrush (EW-DL22, EW0945, Panasonic). The separated colon epithelium was released from the vibrating electric toothbrush into a petri dish filled with water to confirm that the epithelium and mucosa were successfully removed.

[0349] Next, after counting the total number of cells in the pre-cultured GFP-labeled human colon-derived organoids, the cells were suspended in advanced DMEM / F12 medium supplemented with 10% (vol / vol) Matrigel (registered trademark) or type IA collagen, and 70 μL containing 1×10 7The organoid suspension of 10 cells was injected into the colon of NOG mice (recipient mice) from which the colon epithelium and mucosa had been removed. Then, in order to maintain the function of GFP-labeled human colon-derived organoids in the colon, the anus of the mouse was temporarily blocked with soft paper using an adhesive. The feces of the recipient mice were carefully observed for one week to check for the occurrence of intestinal obstruction.

[0350] Fig.32 The leftmost portion of the upper portion is a bright field photograph of the colon of a NOG mouse transplanted with GFP-labeled human colon-derived organoids. Fig.32 The second one from the left in the upper part is taken with Fig.32 The upper leftmost panel shows the GFP fluorescence image of the same field of view. Fig.32 The third from the left in the upper part is a photograph of the mouse intestine with the injured part stained. Fig.32 The upper right part shows a microscopic photograph of the results of immunostaining with anti-human cytokeratin antibody on a colon section of a NOG mouse transplanted with GFP-labeled human colon-derived organoids. The scale bar is 200 μm.

[0351] Fig.32 The leftmost part of the lower part shows the in situ hybridization with the mRNA of the human LGR5 gene. Fig.32 The fluorescence microscopy photograph of the staining results of the area surrounded by the dotted line on the right side of the upper part. Fig.32 The second one from the left in the lower part is Fig.32 The enlarged photo of the area surrounded by the dashed line on the right side of the upper part. The scale bar is 100 μm. Fig.32 The third from the left in the lower part shows the use of anti-human chromogranin A (CHGA) antibody and anti-human chorionic villus (Villin) antibody to Fig.32 Fluorescence micrograph of the staining result of the area surrounded by the dashed line on the right side of the upper part. The scale bar is 100 μm. Fig.32 The lower right part shows the use of anti-human mucin 2 (MUC2) antibody and anti-human villin (Villin) antibody to Fig.32 Fluorescence micrograph of the staining result of the area surrounded by the dashed line on the right side of the upper part. The scale bar is 100 μm.

[0352] The above results show that human colon-derived organoids established in the absence of Matrigel (registered trademark) and with TRUL1 can function normally after orthotopic xenotransplantation into immunodeficient mice (NOG mice). This shows that organoids created in the absence of Matrigel and with TRUL1 can be used for regenerative medicine.

[0353] [Industrial Application Possibility]

[0354] According to the present invention, a technique for culturing organoids in the absence of an extracellular matrix can be provided.

Claims

1. A drug for forming and growing organoids in the absence of an extracellular matrix, wherein: The active ingredient of the drug is a Hippo signaling pathway inhibitor.

2. The medicament for forming and growing organoids in the absence of an extracellular matrix according to claim 1, wherein The Hippo signaling pathway inhibitor 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, wherein: The drugs also include a family of cytokines that bind 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 organoids are epithelial organoids.

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

6. The method for growing organoids according to claim 5, wherein: The organoids were cultured under serum-free conditions.

7. An organoid, wherein: The organoid is proliferated by the proliferating method of claim 5 or 6.

8. A drug for establishing organoids in the absence of an extracellular matrix, wherein: The active ingredient of the drug is a Hippo signaling pathway inhibitor.

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

10. The medicament for establishing an organoid in the absence of an extracellular matrix according to claim 8 or 9, wherein: The drugs also include a family of cytokines that bind to gp130.

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

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

13. The method for producing an organoid according to claim 12, wherein: The organoids were established in the presence of a family of cytokines that bind to gp130.

14. The method for producing an organoid according to claim 12 or 13, wherein: The organoids were established under serum-free conditions.

15. An organoid, wherein: The organoid is produced by the production method according to claim 12 or 13.

16. A regenerative medicine preparation, wherein: The active ingredient of the regenerative medicine preparation is the organoid according to claim 15.

17. A method for screening drugs capable of culturing organoids in the absence of an extracellular matrix, comprising: A step of culturing the organoids in the presence of a test substance and in the absence of an extracellular matrix; as well as a step of evaluating the proliferation of the organoid; Among them, the proliferation of the organoid is higher than that in the presence of the test substance, indicating that the test substance is a drug that can culture organoids in the absence of extracellular matrix.

Images