Method for producing organoids, culture medium for producing organoids, method for evaluating organoids and test substances

Abstract

Provided is an organoid production method comprising culturing a human stem cell in a culture medium that contains a cyclic peptide having an amino acid sequence indicated by formula (1), or a pharmaceutically acceptable salt thereof. [In formula (1), X1-X6 each indicate a specific modified amino acid, X7 indicates an arbitrary amino acid residue, R either does not exist or indicates a C-terminal modification group, n indicates an integer of 0 or 1, PeG indicates N-(2-phenylethyl)-glycine, and Nal1 indicates β-(1-naphthyl)-L-alanine.]　[Formula 1]

Description

[Technical Field]

[0001] The present invention relates to a method for producing organoids, a culture medium for producing organoids, organoids, and a method for evaluating test substances. This application claims priority based on Japanese Patent Application No. 2021-160758, filed in Japan on September 30, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] TGF-β signaling is known to be involved in the inhibitory effect on proliferation and the accumulation of extracellular matrix in many cells, including epithelial cells, endothelial cells, hematopoiesis, and lymphocytes. Regulation of TGF-β signaling is necessary for organoid production, and in particular, downregulation of TGF-β signaling is required to promote cell proliferation.

[0003] As a method for downregulating TGF-β signaling, TGF-β receptor antagonists have been used, which bind to and inhibit the TGF-β receptor, one of the receptors involved in TGF-β signaling (see Patent Document 1 and Non-Patent Document 1). However, it has been reported that when antagonists such as A83-01 are used, off-target (receptors other than the TGF-β receptor) are also inhibited, and this effect is a concern (see Non-Patent Document 2). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent Application Publication No. 2014 / 0243227 [Non-patent literature]

[0005] [Non-Patent Document 1] Sato T., et al., Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium, Gastroenterology, 141 (5), 1762-1772, 2011. [Non-Patent Document 2] Vogt J., et al., The specificities of small molecule inhibitors of the TGF-beta and BMP pathways, Cell Signal., 23 (11), 1831-1842, 2011. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide an excellent method for producing organoids, a culture medium used in the production, organoids obtained by the production method, and a method for evaluating a test substance using organoids. [Means for solving the problem]

[0007] The present invention includes the following embodiments. [1] A method for producing organoids, comprising culturing human stem cells in a culture medium containing a cyclic peptide having the amino acid sequence described in formula (1) below (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof (hereinafter also referred to as the "cyclic peptide group"). [ka] [In formula (1), X 1 This represents Tyr, W6N, W7N, or Nal1, and X 2 This represents Val, Lys, KCOp, or KCOm, and X 3 This represents Tyr, or 4Py, and X 4 This represents Asp, Phe, KCOp, Glu, or KCOm, and X 5represents Tyr or 4Py, and X 6 represents Val, Glu, KCOp, or KCOm, and X 7 represents any amino acid residue, R represents none or a C-terminal modifying group, n represents an integer of 0 or 1, PeG is N-(2-phenylethyl)-glycine, Nal1 is β-(1-naphthyl)-L-alanine, W6N is (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid, W7N is (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid, KCOp is N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine, KCOm is N6-(methyl((2S,3R,4R,5R)-2,3,4,5,6-)pentahydroxyhexyl)carbamoyl)-L-lysine, and 4Py is 4-pyridyl-L-alanine.] [2] The combination of X 1 ~X 7 , R, and n in the formula (1) is the combination shown in Table 1 below, The method for producing an organoid according to [1].

Table 1

[10] A method for producing an organoid according to any one of [1] to [9], wherein the culture medium further contains a Rho kinase (ROCK) signaling inhibitor.

[11] A method for producing an organoid according to any one of [1] to

[10] , wherein the culture medium substantially does not contain an antagonist of ALK5, ALK4, or ALK7, or a combination thereof.

[12] A method for producing an organoid according to any one of [1] to

[11] , wherein the human stem cells are cultured in contact with an extracellular matrix.

[13] A culture medium for organoid production containing a cyclic peptide having the amino acid sequence (SEQ ID NO: 1) described in formula (1) below, or a pharmaceutically acceptable salt thereof. [ka] [In formula (1), X 1 This represents Tyr, W6N, W7N, or Nal1, and X 2 This represents Val, Lys, KCOp, or KCOm, and X 3 This represents Tyr, or 4Py, and X 4 This represents Asp, Phe, KCOp, Glu, or KCOm, and X 5 This represents Tyr, or 4Py, and X 6 This represents Val, Glu, KCOp, or KCOm, and X 7represents any amino acid residue, R represents either no residue or a C-terminal modification group, n represents an integer of 0 or 1, PeG is N-(2-phenylethyl)-glycine, Nal1 is β-(1-naphthyl)-L-alanine, W6N is (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridine-3-yl)propanoic acid, and W7N is (S)-2-amino-3 -(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid, KCOp is N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine, KCOm is N6-(methyl((2S,3R,4R,5R)-2,3,4,5,6-)pentahydroxyhexyl)carbamoyl)-L-lysine, and 4Py is 4-pyridyl-L-alanine. An organoid obtained by the organoid manufacturing method described in any of

[14] [1] to

[12] . A method for evaluating a test substance, comprising the steps of bringing the test substance into contact with an organoid described in

[15]

[14] , and evaluating the effect of the test substance on the organoid. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a method for producing organoids with excellent growth properties, a culture medium used in the production method, organoids obtained by the production method, and a method for evaluating a test substance using the organoids. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a microscopic image showing the results of Experimental Example 2. [Figure 2] Figure 2 is a graph showing the results of Experiment Example 3. [Figure 3] Figure 3 is a graph showing the results of Experiment Example 3. [Figure 4] Figure 4 is a graph showing the results of Experiment Example 4. [Figure 5] Figure 5 is a microscopic image showing the results of Experiment Example 5. [Figure 6]Figure 6 is a microscopic image showing the results of Experimental Example 5. [Figure 7] Figure 7 is a microscopic image showing the results of Experimental Example 6. [Modes for carrying out the invention]

[0010] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited in any way to the following embodiments.

[0011] Each component exemplified herein, for example, a component contained in a culture medium, can be used individually or in combination of two or more, unless otherwise specified.

[0012] A, B, a, and b represent arbitrary numerical values, and in the relationship A>B>a>b, the notation "A~B" etc. which represents a numerical range is synonymous with "A or greater, B or less" in this specification. Also, the notation "A~B, preferably a~b" etc. which represents a numerical range is synonymous with "A or greater, B or less", "A or greater, b or less", "a or greater, B or less", and "a or greater, b or less", and "a or greater, b or less", respectively.

[0013] In this specification, "culture medium containing substance X" and "in the presence of substance X" mean a culture medium to which exogenous substance X has been added, a culture medium containing exogenous substance X, or the presence of exogenous substance X. That is, if cells or tissues present in the culture medium endogenously express, secrete, or produce substance X, endogenous substance X is distinguished from exogenous substance X, and a culture medium that does not contain exogenous substance X does not fall under the category of "culture medium containing substance X" even if it contains endogenous substance X.

[0014] [Method for manufacturing organoids] In one embodiment, the present invention provides a method for producing organoids, comprising culturing human stem cells in a culture medium containing a cyclic peptide having the amino acid sequence described in formula (1) below or a pharmaceutically acceptable salt thereof.

[0015] When human stem cells are cultured in the above-mentioned culture medium, the human stem cells proliferate, and organoids containing both stem cells and differentiated cells are formed.

[0016] In this specification, amino acids include natural and unnatural amino acids. Hereinafter, "cyclic peptides having the amino acid sequence described in formula (1) below (SEQ ID NO: 1) or pharmaceutically acceptable salts thereof" may be referred to as the "cyclic peptide group."

[0017] [ka]

[0018] In formula (1), X 1 This represents Tyr, W6N, W7N, or Nal1, and X 2 This represents Val, Lys, KCOp, or KCOm, and X 3 This represents Tyr, or 4Py, and X 4 This represents Asp, Phe, KCOp, Glu, or KCOm, and X 5 This represents Tyr, or 4Py, and X 6 This represents Val, Glu, KCOp, or KCOm, and X 7 represents any amino acid residue, R represents either no residue or a C-terminal modification group, n represents an integer of 0 or 1, PeG is N-(2-phenylethyl)-glycine, Nal1 is β-(1-naphthyl)-L-alanine, W6N is (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridine-3-yl)propanoic acid, and W7N is (S)-2-amino-3 -(1H-pyrrolo[2,3-b]pyridine-3-yl)propanoic acid, KCOp is N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine, KCOm is N6-(methyl((2S,3R,4R,5R)-2,3,4,5,6-)pentahydroxyhexyl)carbamoyl)-L-lysine, and 4Py is 4-pyridyl-L-alanine.

[0019] As will be described later in the examples, the organoid manufacturing method of this embodiment allows for the culture of human stem cells without using TGF-β receptor antagonists such as A83-01. Many antagonists are known to act on multiple receptors; for example, A83-01 is known to act strongly not only on the TGF-β receptor ALK5, but also on VEGFR, RIPK2, MINK1, p38α MAPK, PKD1, and FGF-R1. On the other hand, since cyclic peptides act on ligands of TGF-β receptors, they can eliminate the off-target effects caused by TGF-β receptor antagonists. Here, TGF-β receptor ligands refer to ligands that have agonist activity. Furthermore, since cyclic peptides are presumed to have virtually no off-target effects, human stem cells can be cultured with cyclic peptides in the culture medium at a lower concentration than the required concentration of TGF-β receptor antagonists.

[0020] It is presumed that the cyclic peptide group can bind, preferably specifically, to exogenous or endogenous TGF-β receptor ligands.

[0021] The organoid production method of this embodiment allows for the culture of human stem cells at a lower concentration than the required concentration of TGF-β receptor antagonists by using a group of cyclic peptides instead of TGF-β receptor antagonists, which was previously used in cell culture using TGF-β receptor antagonists such as A83-01.

[0022] In the organoid manufacturing method of this embodiment, human stem cells refer to human cells or cells derived from humans that possess self-renewal and differentiation capabilities. Examples of human stem cells include human somatic stem cells such as human epithelial stem cells, human mesenchymal stem cells, human vascular cells, and human cancer cells; human pluripotent stem cells such as human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs); and human progenitor cells, which are cells in the intermediate stage of differentiation from human stem cells into specific somatic cells or germ cells and possess the ability to differentiate into specific tissues and organs. In the culture method of this embodiment, a cell population including human stem cells can be used as the human stem cells.

[0023] In the organoid manufacturing method of this embodiment, if human stem cells form cell aggregates, a dispersion treatment is usually performed. Dispersion refers to separating cells into cell populations of 100 or fewer, preferably 50 or fewer, and more preferably single cells, by dispersion treatment such as enzymatic treatment or physical treatment. Examples of dispersion treatments include mechanical dispersion treatment; and treatment using a cell dispersion solution such as trypsin, collagenase, papain, or ethylenediaminetetraacetic acid. Before performing the dispersion treatment, the cells may be treated with a cytoprotective agent such as heparin, a ROCK inhibitor, or insulin-like growth factor to prevent cell death.

[0024] The cyclic peptide is characterized by containing Asn-Val-Tyr-Asp (SEQ ID NO: 2) or Asn-Val-4Py-Asp in sequences 5-8 of formula (1-1) (SEQ ID NO: 1), and Val-Nal1-Tyr-His or Val-Nal1-4Py-His in sequences 11-14. Peptides having these amino acid sequences are active as ligands for the TGF-β receptor.

[0025] [ka]

[0026] Sequences 2, 4, 9, and 15 are each composed of residues selected from the group consisting of hydrophilic amino acids, hydrophobic amino acids, and aromatic amino acids. By appropriately adjusting the combination of these residues, the solubility in the culture medium can be controlled while maintaining the higher-order structure of the cyclic peptide.

[0027] In formula (1) or formula (1-1) above, X 7 X is a linker at the C-terminus of a peptide and can be one or more arbitrary amino acid residues. 7 It is used in translation synthesis systems for binding with puromycin, small molecule compounds, other peptides, and proteins. 7 Examples include those containing Gly and Gly-Lys. Also, n is 0 or 1.

[0028] Furthermore, R is either absent or a modifying group for the C-terminal carboxyl group. Preferably, the modifying group forms a -COOR structure with the C-terminal carboxyl group and removes the negative charge from the carboxyl group and adds a positive charge. Examples of such modifying groups include amino groups, amide groups, aminoethyl groups, pyrazolidine groups, piperidine groups, imidazolidine groups, and piperazine groups. R can also contain a polyethylene glycol chain.

[0029] As the optimal amino acid sequence for a cyclic peptide, X of the cyclic peptide 1 ~X 6 The combination is preferably one of the combinations shown in Table 1 above, X 1 is Nal1, X 2 Val, X 4 Asp, X 5 Tyr, X 6 A combination of Val or KCOp is more preferable.

[0030] In this specification, cyclic peptides can be produced by known manufacturing methods such as chemical synthesis methods including liquid-phase methods, solid-phase methods, hybrid methods combining liquid-phase and solid-phase methods, and genetic recombination methods.

[0031] In the solid-phase method, cyclic peptides are synthesized, for example, by the following method. First, the hydroxyl group of a resin containing a hydroxyl group is esterified with the carboxyl group of the first amino acid (usually the C-terminal amino acid of the target peptide), whose α-amino group is protected by a protecting group. As an esterification catalyst, known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used.

[0032] Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid, in which all functional groups except the carboxyl group of the main chain are protected, is added. The carboxyl group is then activated to bond the first and second amino acids. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid, in which all functional groups except the carboxyl group of the main chain are protected, is added. The carboxyl group is then activated to bond the second and third amino acids. This process is repeated until a peptide of the desired length is synthesized, at which point all functional groups are deprotected.

[0033] Examples of resins used in the solid-phase process include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins can be used after being washed with a solvent (such as dimethylformamide (DMF), 2-propanol, or methylene chloride).

[0034] Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, and allyloxycarbonyl (Alloc) group. The Cbz group can be deprotected by hydrofluoric acid or hydrogenation, the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine.

[0035] For α-carboxyl group protection, methyl esters, ethyl esters, benzyl esters, tert-butyl esters, cyclohexyl esters, etc., can be used.

[0036] Other functional groups of amino acids include the hydroxyl groups of serine and threonine, which can be protected with benzyl or tert-butyl groups, and the hydroxyl group of tyrosine, which can be protected with 2-bromobenzyloxycarbonyl or tert-butyl groups. The amino groups of the lysine side chains, and the carboxyl groups of glutamic acid and aspartic acid, can be protected in the same way as α-amino and α-carboxyl groups.

[0037] The carboxyl group can be activated using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxidehexafluorophosphate (HBTU).

[0038] Peptide chains can be cleaved from resin by treatment with acids such as TFA and hydrogen fluoride (HF).

[0039] The production of cyclic peptides by genetic recombination (translational synthesis system) can be carried out using nucleic acids that encode the target cyclic peptide. These nucleic acids can be DNA or RNA.

[0040] Nucleic acids encoding cyclic peptides can be prepared by known methods. For example, they can be synthesized using an automated synthesis apparatus. Restriction enzyme recognition sites may be added to insert the obtained DNA into a vector, or a base sequence encoding an amino acid sequence for cleaving the synthesized peptide chain with an enzyme may be incorporated.

[0041] To suppress degradation by host-derived proteases, a chimeric protein expression method can be used, in which the target peptide is expressed as a chimeric peptide with another peptide. In this case, the nucleic acids used are those encoding the target peptide and the peptide that binds to it.

[0042] Next, an expression vector containing a nucleic acid encoding the peptide is prepared. The nucleic acid encoding the peptide can be inserted downstream of the promoter in the expression vector either as is, or after digestion with restriction enzymes or the addition of a linker. Examples of vectors include E. coli plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e-phage, M13 phage, etc.), viruses (retroviruses, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and cosmids.

[0043] The promoter can be appropriately selected depending on the type of host. If the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or CMV (cytomegalovirus) can be used. If the host is E. coli, a promoter such as the trp promoter, T7 promoter, or lac promoter can be used.

[0044] Expression vectors can also incorporate nucleic acids that encode DNA replication origins (ori), selection markers (antibiotic resistance, nutritional requirements, etc.), enhancers, splicing signals, polyalpha alpha addition signals, and tags (FLAG, HA, GST, GFP, etc.).

[0045] Next, suitable host cells are transformed with the expression vector described above. The host can be appropriately selected in relation to the vector, and examples include Escherichia coli, Bacillus subtilis, Bacillus species, yeast, insect organisms, insect cells, and animal cells. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Transformation can be carried out by known methods such as lipofection, calcium phosphate, electroporation, microinjection, and particle gun, depending on the type of host. By culturing the transformed cells according to standard methods, the target peptide is expressed.

[0046] In the purification of peptides from cultured transformed cells, the cultured cells are first collected, suspended in a suitable buffer, and then destroyed by methods such as sonication or freeze-thaw cycles. A crude extract is then obtained by centrifugation or filtration. If peptides are secreted into the culture medium, the supernatant is collected.

[0047] Peptides can also be purified from crude extracts or culture supernatants by known methods or similar methods (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reverse-phase high-performance liquid chromatography, etc.).

[0048] The obtained peptide can be converted from a free form to a salt, or from a salt to a free form, by known or equivalent methods.

[0049] Translation synthesis systems include cell-free translation systems. Cell-free translation systems include, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factors (IF), elongation factors (EF), termination factors (RF), ribosomal regeneration factors (RRF), and other factors necessary for translation. E. coli extracts or wheat germ extracts can be added to increase expression efficiency. Rabbit red blood cell extracts or insect cell extracts can also be added. In cell-free translation systems, for example, mRNA display can be performed, in which case puromycin, which is responsible for binding between template mRNA and translated peptides, can be linked via a linker.

[0050] By continuously supplying energy to a system containing these components using dialysis, peptides can be produced in concentrations ranging from several hundred μg to several mg / mL. A system containing RNA polymerase can be used to simultaneously perform transcription from gene DNA. Commercially available cell-free translation systems include Roche Diagnostics' RTS-100 (registered trademark) and PGI's PURESYSTEM (registered trademark) from E. coli, and systems using wheat germ extract from companies such as Zoigene and CellFree Science. Cell-free translation systems allow for the acquisition of highly purified expression products without the need for purification.

[0051] In cell-free translation systems, artificial aminoacyl-tRNAs can be used instead of aminoacyl-tRNAs synthesized by natural aminoacyl-tRNA synthetases, by cylating (acylating) non-natural amino acids or hydroxy acids to tRNAs. Such aminoacyl-tRNAs can be synthesized using artificial ribozymes such as flexizyme.

[0052] By using tRNA to which unnatural amino acids or hydroxy acids are linked, it is possible to translate a desired codon in association with the unnatural amino acid or hydroxy acid.

[0053] Table 2 below shows the unnatural amino acids in cyclic peptides. The three-dimensional structures of the chemical formulas are omitted in Table 2.

[0054] [Table 2]

[0055] Peptide cyclization is performed by forming a thioether bond between the thiol group of Cys in seq. 16 and, for example, the chloroacetyl group of chloroacetylated Phe in seq. 1 (Cl-CH2-CO-NH-C(Bzl)-COO-) or Cl-R-CO-NH-C(Bzl)-COO- (where R is an alkanediyl group) through a cyclization reaction. In this case, the bond between Phe and Cys in the amino acid sequence shown in formula (1) above is a bond via a thioether bond. For cyclization, a complex of chloroacetylated Phe and tRNA can be generated by flexizyme and used in the translation synthesis system.

[0056] The thioether bond formed between the chloroacetyl group and the thiol is resistant to decomposition even under reducing conditions, resulting in a long half-life.

[0057] The cyclic peptide has the ability to bind to the ligand of the TGF-β receptor. The TGF-β receptor can be ALK5, ALK4, or ALK7, or a combination thereof. The NCBI accession numbers for human ALK5 are NP_001124388.1, NP_001293139.1, NP_004603.1, etc. The NCBI accession numbers for human ALK4 are NP_004293.1, NP_064732.3, NP_064733.3, etc. The NCBI accession numbers for human ALK7 are NP_001104501.1, NP_001104502.1, NP_001104503.1, NP_660302.2, etc.

[0058] Examples of TGF-β receptor ligands to which the above-mentioned cyclic peptides bind include TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 being preferred. The NCBI accession number for human TGF-β1 is NP_000651.3, etc. The NCBI accession numbers for human TGF-β2 are NP_001129071.1, NP_003229.1, etc. The NCBI accession numbers for human TGF-β3 are NP_001316867.1, NP_001316868.1, NP_003230.1, etc.

[0059] The above-mentioned cyclic peptides may also be derivatives. Examples of derivatives of cyclic peptides include: those obtained by adding a polyethylene glycol chain or the like to any of the amino acids constituting the cyclic peptide; those obtained by converting any of the amino acids constituting the cyclic peptide to the D-isomer; and complexes to which low-molecular-weight compounds, other peptides, or proteins are bound via a linker.

[0060] Examples of pharmaceutically acceptable salts of cyclic peptides include sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, salts with trimethylamine, salts with pyridine, salts with ethanolamine, salts with tartaric acid, salts with citric acid, salts with succinic acid, and salts with malic acid. pharmaceutically acceptable salts of cyclic peptides can be produced by salt transfection of the cyclic peptide. A pharmaceutically acceptable salt includes a bioacceptable salt.

[0061] In the organoid production method of this embodiment, the concentration of cyclic peptides in the culture medium (the total concentration of cyclic peptides and their derivatives, as well as their pharmaceutically acceptable salts) is typically 0.1 nM to 10 μM, for example, 0.1 nM to 1,000 μM, and is adjusted as appropriate depending on the cell type.

[0062] In the organoid production method of this embodiment, the culture medium is usually prepared by adding a specific cyclic peptide to a basal medium. In addition, various components as shown below may be added to the culture medium as appropriate. Examples of basal media include Dulbecco's modified Eagle medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Eagle Minimum Essential Medium (BME), DMEM / F12, Advanced DMEM / F12, Iskov modified Dulbecco medium, Ham F-10, Ham F-12, 199 medium, and RPMI1640 medium. In this specification, basal medium refers to a medium containing amino acids, antioxidants, minerals, and a carbon source such as glucose, and optionally containing the minimum components necessary for culture, such as serum, fatty acids, and proteins such as insulin and transferrin.

[0063] The culture medium may contain a Wnt signaling enhancer. Examples of Wnt signaling enhancers include Wnt family members, GSK inhibitors, or Lgr5 agonists, or combinations thereof.

[0064] Wnt family members include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Wnt family members can also be found in complexes with afamin, a stabilizing substance.

[0065] Examples of GSK inhibitors include GSK-3β inhibitors, such as CHIR99021 (CAS number: 252917-06-9), Kenpaullone (CAS number: 142273-20-9), and 6-Bromoindirubin-3'-oxime (BIO, CAS number: 667463-62-9).

[0066] Examples of Lgr5 agonists include the R-spongin family. Examples of the R-spongin family include R-spongin 1, R-spongin 2, R-spongin 3, R-spongin 4, etc., with R-spongin 1 being preferred among them.

[0067] When CHIR99021 is used as a Wnt signal enhancer, the concentration of CHIR99021 in the culture medium is typically 0.1 μM to 30 μM, preferably 0.2 μM to 20 μM.

[0068] When Wnt3a is used as a Wnt signal enhancer, the concentration of Wnt3a in the culture medium is typically 0.1 ng / mL to 20 ng / mL, preferably 0.2 ng / mL to 10 ng / mL.

[0069] The culture medium may contain epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), or hepatocyte growth factor (HGF), or a combination thereof.

[0070] The concentration of EGF in the culture medium is typically 5 ng / mL to 500 ng / mL, preferably 25 ng / mL to 300 ng / mL, and more preferably 50 to 100 ng / mL.

[0071] Examples of FGF include FGF2, FGF4, FGF7, and FGF10, with FGF10 being preferred among these. The concentration of FGF in the culture medium is typically 20 ng / mL to 500 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 100 to 500 ng / mL.

[0072] IGF1 is preferred as the IGF. The concentration of IGF in the culture medium is usually 5 ng / mL to 1,000 ng / mL, preferably 10 ng / mL to 1,000 ng / mL, and more preferably 50 ng / mL to 1,000 ng / mL.

[0073] The concentration of HGF in the culture medium is typically 1 ng / mL to 100 ng / mL, preferably 10 ng / mL to 100 ng / mL, more preferably 20 ng / mL to 100 ng / mL, and even more preferably 40 ng / mL to 100 ng / mL.

[0074] The culture medium may contain a bone morphogenetic factor (BMP) inhibitor. The BMP inhibitor is preferably Noggin. The concentration of the BMP inhibitor in the culture medium is usually 10 to 100 ng / mL, preferably 20 to 100 ng / mL, and more preferably 50 to 100 ng / mL.

[0075] The culture medium may contain ROCK signaling inhibitors. Examples of ROCK signaling inhibitors include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), Azabenzimidazole-aminofurazans (CAS number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and Blebbisstatin (CAS number: 856925-71-8).

[0076] The concentration of ROCK signaling inhibitors in the culture medium is typically 1 μM to 20 μM, preferably 5 μM to 15 μM.

[0077] In addition to the components mentioned above, the culture medium may contain, for example, gastrin (or a suitable substitute such as Leu15-gastrin I); p38 inhibitors such as SB202190 (CAS number: 152121-30-7) and Doramapimod (CAS number: 285983-48-4); antibacterial agents such as penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, and tetracycline antibiotics; IL-6 family cytokines such as interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), leukemia suppressor factor (LIF), cardiotropin-1 (CT-1), and ciliary neurotrophic factor (CNTF); retinoic acid; nicotinamide; tumor cell growth factor-α (TGF-α) such as forskolin; and DAPT (γ-secretase It may contain: inhibitors; dimethyl sulfoxide (DMSO); dexamethasone (Dex); fatty acids such as lauric acid, myristic acid, linoleic acid, linolenic acid, and oleic acid; and combinations thereof.

[0078] The culture medium may contain supplements such as B27 supplements, glutamic acid-containing supplements such as the GlutaMax series, amino acid aqueous solutions such as MEM Non-Essential Amino Acids Solution, and N2 supplements, which are sold by Thermo Fisher Scientific and other companies.

[0079] In the organoid production method of this embodiment, it is preferable that the culture medium substantially does not contain any TGF-β receptor antagonists, namely ALK5, ALK4, ALK7, or any combination thereof. Here, "substantially contained" means that the active ingredient is not intentionally mixed in, or that unintentional mixing to an extent that is negligible is permitted. Specifically, the concentration of TGF-β receptor antagonists contained in the culture medium is 1 nM or less, preferably 0.1 nM or less, and more preferably 0.01 nM or less.

[0080] Examples of TGF-β receptor antagonists include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2). Examples of TGF-β receptor ligands include TGF-β1, TGF-β2, and TGF-β3.

[0081] In the organoid manufacturing method of this embodiment, it is preferable to culture the human stem cells while in contact with the extracellular matrix.

[0082] Methods for culturing human stem cells in contact with the extracellular matrix include embedding the stem cells in the extracellular matrix (ECM), mixing the ECM into the culture medium, and coating the culture vessel surface with ECM. Examples of ECM include components of the basement membrane and glycoproteins present in the intercellular spaces. Examples of components of the basement membrane include type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. Examples of glycoproteins present in the intercellular spaces include collagen, laminin, entactin, fibronectin, and heparin sulfate. Commercially available products containing ECM can be used as ECM. Examples of commercially available products containing ECM include Matrigel (registered trademark, Corning Corporation) and human-type laminin (Sigma-America).

[0083] A culture medium mixed with ECM can be prepared by mixing in an amount of ECM such that, relative to the volume of other components in the medium, the volume of ECM is typically 1% or more, preferably 5% to 100% or more, and more preferably 10% to 90% or more. One method for mixing in ECM is pipetting on an ice bath. Mixing means that the ECM is not visible to the naked eye in the culture medium.

[0084] The culture medium can be changed approximately once every 1 to 5 days. Culturing is usually carried out under temperature conditions of 30°C to 50°C, preferably 32°C to 48°C, and more preferably 34°C to 46°C. Culturing is carried out in an atmosphere with a carbon dioxide content of 1% to 15% by volume, preferably 2% to 14% by volume, and more preferably 3% to 13% by volume or less.

[0085] [Organoid Manufacturing Medium] In one embodiment, the present invention provides a culture medium for organoid production containing a cyclic peptide having the amino acid sequence described in formula (1) above, or a pharmaceutically acceptable salt thereof.

[0086] The culture medium for organoid production in this embodiment can also be called an organoid culture medium. Human stem cells can be cultured well using the culture medium of this embodiment to produce organoids.

[0087] The culture medium is prepared by adding various components to the basal culture medium. The basal culture medium and various components are the same as those described above.

[0088] [Organoid] In one embodiment, the present invention provides an organoid obtained by the organoid manufacturing method described above. In this specification, "organoid" means a self-organizing three-dimensional cell culture obtained by culturing stem cells in vitro. The cell culture may have a structure similar to an organ. The organoid of this embodiment can be cultured in the absence of a TGF-β receptor antagonist. Therefore, unlike organoids obtained using conventional TGF-β receptor antagonists, it is not affected by off-target inhibition by TGF-β receptor antagonists and is considered to better reflect the state of cells in vivo.

[0089] Examples of organoids in this embodiment include colon organoids, small intestine organoids, liver organoids, bile duct organoids, brain organoids, ovarian cancer organoids, retinal organoids, and the like.

[0090] It is believed that the organoids of this embodiment differ in gene expression profiles and other characteristics from organoids obtained by other manufacturing methods. However, identifying such differences and determining whether an organoid is from this embodiment or obtained by another manufacturing method based on differences in gene expression profiles is extremely difficult and impractical. Therefore, it is more practical to determine the organoids by their manufacturing method.

[0091] [Method for evaluating test substances] In one embodiment, the present invention provides a method for evaluating a test substance, which includes the steps of bringing the test substance into contact with the organoid described above (hereinafter also referred to as "step 1") and evaluating the effect of the test substance on the organoid (hereinafter also referred to as "step 2").

[0092] Examples of test substances include libraries of natural compounds, synthetic compounds, existing drugs, and metabolites. New drugs can also be used as test substances.

[0093] In step 1, a cell-based assay system such as a Transwell-type assay system, or a biomimetic system such as Organ-on-a-Chip, can be constructed using the organoid to bring the test substance into contact with it.

[0094] In step 2, the effect of the test substance on the organoid can be evaluated by Western blotting, ELISA, immunohistochemistry, etc. [Examples]

[0095] The present invention will be described in more detail below with reference to experimental examples, but the present invention is not limited in any way to the following experimental examples. In the following experimental examples, sterilized equipment was used in all experiments.

[0096] [Experimental Example 1] (Preparation of culture medium for colon epithelial stem cells) The culture medium was prepared by adding each component to the basal medium to the concentrations shown in Table 3 below. In Table 3, "CM" stands for Conditioned Medium. Advanced DMEM / F12 was used as the basal medium. Peptides #7401 and #2392 were used (hereinafter, they may be referred to as "#7401 peptide" and "#2392 peptide," respectively). For peptide #7401, the chemical formula is shown in formula (2-1) and the amino acid sequence (SEQ ID NO: 3) is shown in formula (2-2). Similarly, for peptide #2392, the chemical formula is shown in formula (3-1) and the amino acid sequence (SEQ ID NO: 4) is shown in formula (3-2).

[0097] [Table 3]

[0098] [ka]

[0099] [ka]

[0100] [ka]

[0101] [ka]

[0102] [Experimental Example 2] (Culture of human colon epithelial cells) Human colon epithelial cells (including human colon stem cells) were dispersed in Matrigel (registered trademark, manufactured by BD Biosciences). Subsequently, the dispersion was seeded into each well of a 48-well tissue culture plate and incubated at 37°C for 10 minutes to polymerize Matrigel (registered trademark).

[0103] Next, the culture medium prepared in Experimental Example 1 (300 μL / well) was layered into each well, and the cultures were incubated at 37°C in the presence of 5 volume% CO2 for 7 days. The culture medium was changed every 2 or 3 days. Subsequently, the contents of the wells after 7 days of incubation were observed with a light microscope. Bright-field images are shown in Figure 1. In Figure 1, "ND" indicates that data was not acquired.

[0104] As a result, it was revealed that culturing human colon stem cells using a medium supplemented with peptides #2392 and #7401 could produce organoids with an efficiency equal to or greater than that achieved when A83-01 was added.

[0105] [Experimental Example 3] (Culture of human liver cells) Human primary frozen suspension hepatocytes (BIOPRRIDIC, HEP187-S) were thawed in a 37°C water bath and suspended in a 50 mL tube containing serum-free medium (Advanced DMEM / F12 with HEPES, GLUTAMAX, and PENICILIN / STEREPTOMYCIN). The cells were then centrifuged. After centrifugation, the supernatant was removed, and the cells were resuspended in serum-free medium to prepare a human liver cell suspension. 20,000 human liver cells were taken from this suspension, mixed with 50 μL of Matrigel® (BD Biosciences), seeded in a 24-well tissue culture plate, and incubated at 37°C for 10 minutes until Matrigel® was completely polymerized. After polymerization, culture media (500 μL / well) prepared by adding each component to the basal medium at the concentrations shown in Table 4 below were overlaid and cultured at 37°C in the presence of 5 volume % CO2. Advanced DMEM / F12 was used as the basal medium. The culture medium was replaced every two or three days.

[0106] [Table 4]

[0107] Figure 2 is a graph showing the results of measuring cell proliferation. In Figure 2, "PDi" shows the results when 5 μM of #7401 peptide was added to the culture medium, "A+" shows the results when 5 μM of A83-01 was added to the culture medium, and "A-" shows the results when neither #7401 peptide nor A83-01 was added to the culture medium.

[0108] The results in Figure 2 show that when 5 μM of A83-01 was added to the culture medium, no cell proliferation was observed after 35 days. This result suggests that A83-01 may have been affected by metabolism by hepatocytes.

[0109] Figure 3 is a graph showing the results of measuring cell proliferation at low concentrations of #7401 peptide. In Figure 3, "5μM PDi" shows the results when 5μM of #7401 peptide is added to the culture medium, "500nM PDi" shows the results when 500nM of #7401 peptide is added to the culture medium, "50nM PDi" shows the results when 50nM of #7401 peptide is added to the culture medium, "A+" shows the results when 5μM of A83-01 is added to the culture medium, and "A-" shows the results when neither #7401 peptide nor A83-01 is added to the culture medium.

[0110] The results shown in Figure 3 demonstrate that cells can proliferate sufficiently even at a concentration of 50 nM of peptide #7401 in the culture medium.

[0111] [Experimental Example 4] (Specificity evaluation of peptide #7401 using SMAD binding element (SBE) reporter assay) Reporter cells (TGF / SMAD Signaling Pathway SBE Reporter, Luciferase, HEK293 Recombint Cell Line, BPS Bioscience Inc.) were stimulated with 0.5 nM of various TGF-β (TGF-β1 to TGF-β3) and A83-01 or #7401 peptide at the concentrations shown in Figure 4, and ONE-Glo was observed after 20 hours. TM The signal was detected using the Luciferase Assay System. In this assay system, when the TGF-β signal is transmitted to the nucleus, the reporter gene luciferase is expressed, and the signal is detected. The results are shown in Figure 4.

[0112] [Experimental Example 5] (Examination of EGFR signaling and p38 MAPK signaling) Upregulation of EGFR signaling is known to induce cell proliferation. Therefore, we investigated cell proliferation in the absence of EGFR signaling. Furthermore, A83-01 has been reported to inhibit p38 MAPK signaling (Non-Patent Literature 2), and downregulation of p38 MAPK signaling is known to be effective for long-term intestinal culture (Non-Patent Literature 1). Therefore, we investigated culture with and without p38 MAPK signaling.

[0113] The culture medium was prepared by adding each component to the basal medium (Advanced DMEM / F12) to the concentrations shown in Table 5 below. Human small intestinal epithelial cells (including human small intestinal stem cells) were dispersed in Matrigel (registered trademark, BD Biosciences). Subsequently, the dispersion was seeded into each well of a 48-well tissue culture plate and incubated at 37°C for 10 minutes to polymerize Matrigel (registered trademark).

[0114] [Table 5]

[0115] Next, 300 μL / well of culture medium was added as a top layer, and the cells were incubated at 37°C in the presence of 5 volume % CO2 for 10 days. The culture medium was changed every 2 or 3 days. Cells in the medium containing EGF were incubated for 10 days, and cells in the medium without EGF were incubated for 14 days. Subsequently, bright-field microscopy of the cells in the wells and an Lgr5-tdTomato reporter assay were performed. Figure 5 shows the culture results in the medium containing EGF (50 ng / mL), and Figure 6 shows the culture results in the medium without EGF. In Figures 5 and 6, "#3-a_A500S" indicates the culture result in a medium containing 500 nM of A83-01 and 10 μM of SB202190, "#7_A500" indicates the culture result in a medium containing 500 nM of A83-01 but without SB202190, "#8_A5000" indicates the culture result in a medium containing 5000 nM of A83-01 but without SB202190, "#9_P50" indicates the culture result in a medium containing 50 nM of #7401 peptide but without SB202190, and "#10_P500" indicates the culture result in a medium containing 500 nM of #7401 peptide but without SB202190.

[0116] Figure 5, "#3-a_A500S," shows the results indicating inhibition of cell proliferation and colony formation due to downregulation of p38 MAPK signaling. Furthermore, the results for "#7_A500" and "#8_A5000" in Figure 5, similar to the results for "#3-a_A500S," show that cell proliferation and colony formation were inhibited in a concentration-dependent manner when A83-01 was used. Therefore, these results suggest the potential for inhibiting p38 MAPK signaling by A83-01.

[0117] The results for "#3-a_A500S" in Figure 6 support the results of Non-Patent Literature 1, which shows that downregulation of p38 MAPK signaling is effective for long-term intestinal culture. "#7_A500" and "#8_A5000" in Figure 6 show higher stem cell characteristics than "#9_P50" and "#10_P500," and furthermore, "#8_A5000" shows higher stem cell characteristics than "#7_A500," suggesting the possibility of p38 MAPK signaling inhibition by A83-01.

[0118] [Experimental Example 6] (Formation of cerebral cortical organoids) hiPSCs (PChiPS771 strain, Lot.A01QM28, Reprocell, Inc.) were cultured without feeders. Specifically, the hiPSCs were washed with phosphate-buffered saline (PBS) and then dispersed into single cells using TrypLE Select (Thermo Fisher Scientific). Subsequently, the dispersed hiPSCs were seeded into plastic culture dishes coated with a human recombinant laminin fragment containing only the active site of laminin-511 (product name "iMatrix-511", Nippi Corporation), and cultured without feeders in StemFit AK02N medium (Ajinomoto Co., Inc.) in the presence of Y27632 (ROCK inhibitor, 10 μM). When a 60 mm dish (Iwaki Corporation, for cell culture) was used as the plastic culture dish, the number of seeded hiPSCs dispersed into single cells was 3 × 10⁶. 4 It was set to one per dish.

[0119] One day after seeding, the culture medium was replaced with StemFit AK02N medium that does not contain Y27632. Thereafter, the medium was replaced with StemFit AK02N medium that does not contain Y27632 every 1-2 days. Subsequently, the cells reached 80% confluence 6 days after seeding.

[0120] Next, the enlarged hiPSC cultures were treated with a cell dispersion using TrypLE Select (Thermo Fisher Scientific product name) and then dispersed into single cells by pipetting. The dispersed hiPSC enlarged cultures were then placed in a medium (100 μL / well) prepared by adding each component to basal medium ("DMEM / F-12, HEPES", Thermo Fisher Scientific product name) at the concentrations shown in Table 6 below, and then transferred to a 96-well culture plate (product name "PrimeSurface 96V bottom plate", Sumitomo Bakelite Corporation) at a rate of 1 × 10⁶ 4 Cells were seeded to a cell density of 1 cell / well and cultured in suspension in an incubator (37°C, 1 atm, CO2 concentration 5v / v%) for 7 days to obtain aggregates of hiPSCs.

[0121] [Table 6]

[0122] Next, 230 μL / well of culture medium was removed from the wells. Then, 150 μL / well of culture medium prepared by adding each component to the basal medium ("DMEM / F-12, HEPES," Thermo Fisher Scientific product name) to the concentrations shown in Table 7 below was added, and the cultures were incubated in a suspension culture in an incubator (37°C, 1 atm, CO2 concentration 5 v / v%) with stirring for 7 days.

[0123] [Table 7]

[0124] Next, the contents of the wells were transferred to a 50 mL Falcon® conical tube (Corning) containing 10 mL of PBS. Then, the mixture was inverted and mixed five times, the supernatant was removed, and the cell aggregates were collected. Thirty of the collected cell aggregates, along with a culture medium (30 ml / well) prepared by adding each component to the basal medium ("DMEM / F-12, HEPES," Thermo Fisher Scientific product name) to the concentrations shown in Table 8 below, were added to a single-use bioreactor (ABLE) and cultured in suspension with stirring. The culture medium was changed every four days. Twelve weeks after the start of suspension culture, the cell aggregates (cerebral cortex organoids) in the single-use bioreactor were collected and transferred to a 96-well culture plate. Figure 7 is an optical microscope image showing the state of the cell aggregates in the 96-well culture plate after 12 weeks of suspension culture. The lower part of Figure 7 is an image of the entire well, and the upper part of Figure 7 is a magnified image. The scale bar is 500 μm.

[0125] [Table 8] [Industrial applicability]

[0126] According to the present invention, it is possible to provide a method for producing organoids with excellent growth properties, a culture medium used for producing the organoids, organoids obtained by the culture, and a method for evaluating a test substance using the organoids.

Claims

1. A method for producing organoids, comprising culturing human stem cells in a culture medium containing a cyclic peptide whose chemical formula is represented by the following formula (2-1) or the following formula (3-1), or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 【Chemistry 2】

2. The method for producing an organoid according to claim 1, wherein the total concentration of the cyclic peptide and its pharmaceutically acceptable salt contained in the culture medium is 0.1 nM to 10 μM.

3. The method for producing an organoid according to claim 1 or claim 2, wherein the culture medium further contains a Wnt signal enhancer.

4. The method for producing an organoid according to claim 1 or claim 2, wherein the culture medium further contains epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), or hepatocyte growth factor (HGF), or a combination thereof.

5. The method for producing an organoid according to claim 1 or claim 2, wherein the culture medium further contains a bone morphogenetic factor (BMP) inhibitor.

6. The method for producing an organoid according to claim 1 or claim 2, wherein the culture medium further contains a Rho kinase (ROCK) signaling inhibitor.

7. A method for producing an organoid according to claim 1 or 2, wherein the culture medium substantially does not contain an antagonist of ALK5, ALK4, or ALK7, or a combination thereof.

8. A method for producing an organoid according to claim 1 or claim 2, wherein the human stem cells are cultured while in contact with an extracellular matrix.

9. A culture medium for producing organoids, comprising a cyclic peptide whose chemical formula is represented by the following formula (2-1) or the following formula (3-1), or a pharmaceutically acceptable salt thereof. 【Transformation 3】 【Chemistry 4】

10. A method for evaluating a test substance, comprising the steps of: obtaining an organoid by the method for manufacturing an organoid described in claim 1 or claim 2; contacting the organoid with a test substance; and evaluating the effect of the test substance on the organoid.

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