Method for producing brain organoids containing aggregated tau protein

Abstract

This method for producing a brain organoid including aggregated tau protein comprises: a step (a) for culturing pluripotent stem cells in the presence of SMAD inhibitor to form embryoid bodies; a step (b) for embedding the embryoid bodies in an extracellular matrix and three-dimensionally culturing the embryoid bodies in the presence of SMAD inhibitor and GSK3β inhibitor to form an organoid that includes neural precursor cells; a step (c) for removing the organoid from the extracellular matrix and suspension-culturing the organoid in the presence of LIF to form a brain organoid; a step (d) for forcing the brain organoid to express a modified MAPT gene; and a step (e) for further suspension-culturing the brain organoid after step (d) to obtain a brain organoid having an aggregated tau protein.

Description

Technical Field

[0001] The present invention relates to a method for producing a brain organoid having aggregated tau protein. More specifically, the present invention relates to a method for producing a brain organoid having aggregated tau protein, a brain organoid, and a method for screening a prophylactic or therapeutic agent for tauopathy. This application claims priority based on Japanese Patent Application No. 2021-046480 filed in Japan on March 19, 2021, the content of which is incorporated herein by reference.

Background Art

[0002] An organoid is a small organ formed by the aggregation of cells and has a structure and function similar to those of an organ in vivo. In recent years, research on producing various organoids from pluripotent stem cells has been actively conducted. For example, brain organoids, intestinal organoids, liver organoids, kidney organoids, etc. have been produced.

[0003] Neurodegenerative diseases in which inclusion bodies of fibrillated tau protein are observed in nerve cells and glial cells as pathological images are collectively referred to as tauopathy. Tauopathy includes Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, etc. It is known that abnormal accumulation of phosphorylated tau protein is observed intracellularly in the brains of tauopathy patients.

[0004] Alzheimer's disease model mice have been developed for the development of therapeutic agents for Alzheimer's disease. However, the structures of the brains of humans and mice are different, and the findings obtained from Alzheimer's disease model mice may not be applicable to humans. Therefore, an experimental system that can more faithfully reproduce the pathological condition of Alzheimer's disease and can be used in vitro is required.

[0005] For example, Patent Document 1 describes the production of a brain organoid having amyloid plaques from iPS cells derived from Alzheimer's disease patients. In addition, Non-Patent Documents 1 and 2 describe tau aggregation model cells. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2021 / 006107 [Non-patent literature]

[0007] [Non-Patent Document 1] Choi SH, et al., A three-dimensional human neural cell culture model of Alzheimer's disease, Nature, 515 (7526), ​​274-278, 2014. [Non-Patent Document 2] Matsumoto G., et al., Tau Fibril Formation in Cultured Cells Compatible with a Mouse Model of Tauopathy, Int. J. Mol. Sci., 19, 1497, 2018. [Overview of the project] [Problems that the invention aims to solve]

[0008] However, no brain organoids that can be used as a tau aggregation model are known. Therefore, the present invention aims to provide a technology for efficiently producing brain organoids having aggregated tau protein. [Means for solving the problem]

[0009] The present invention includes the following embodiments. [1] A method for producing brain organoids having aggregated tau protein, comprising the steps of: (a) culturing pluripotent stem cells in the presence of a SMAD inhibitor to form germ cells; (b) embedding the germ cells in an extracellular matrix and culturing them in three dimensions in the presence of a SMAD inhibitor and a glycogen synthase kinase 3β (GSK3β) inhibitor to form organoids containing neural progenitor cells; (c) removing the organoids from the extracellular matrix and culturing them in suspension in the presence of Leukemia Inhibitory Factor (LIF) to form brain organoids; (d) forcing the expression of a mutant Microtubule Associated Protein Tau (MAPT) gene in the brain organoids; and (e) further culturing the brain organoids after step (d) in suspension to obtain brain organoids having aggregated tau protein. [2] The method for producing a brain organoid according to [1], wherein the mutant MAPT gene is a gene encoding a tau protein having a P301L mutation. [3] A method for producing a brain organoid according to [1] or [2], wherein the mutant MAPT gene is forcibly expressed by gene transfer using adeno-associated virus (AAV). [4] A method for producing a brain organoid according to [3], wherein the mutant MAPT gene is forcibly expressed by injecting the AAV into the brain organoid. [5] The manufacturing method according to any one of [1] to [4], wherein at least a portion of step (c) is carried out in the presence of more than 20 volume percent of oxygen. [6] A manufacturing method according to any one of [1] to [5], wherein step (c) is performed for three weeks or more. [7] A manufacturing method according to any one of [1] to [6], wherein step (e) is performed for five weeks or more. [8] A manufacturing method according to any one of [1] to [7], further comprising the step of culturing the pluripotent stem cells in the presence of less than 100 ng / mL of Fibroblast Growth Factor-2 (FGF2) before step (a). [9] A method for culturing the pluripotent stem cells in a feeder-free manner, as described in any of [1] to [8].

[10] A brain organoid in which, in a cross-section, the average ratio of the area of ​​the region containing phosphorylated tau protein to the total area of ​​the cross-section is 2% or more, or in a cross-section, the average ratio of the area of ​​the region containing aggregated tau protein to the total area of ​​the cross-section is 1% or more.

[11] A method for screening for a therapeutic agent for tauopathy, comprising the steps of culturing a brain organoid described in

[10] in the presence of a test substance, and measuring the amount of phosphorylated tau protein or aggregated tau protein in the brain organoid, wherein a decrease in the amount of phosphorylated tau protein or aggregated tau protein compared to the absence of the test substance indicates that the test substance is a therapeutic agent for tauopathy.

[12] A step of culturing pluripotent stem cells in the presence of a SMAD inhibitor to form germ cells (a); a step of embedding the germ cells in an extracellular matrix and culturing them in three dimensions in the presence of a SMAD inhibitor and a GSK3β inhibitor to form organoids containing neural progenitor cells (b); a step of removing the organoids from the extracellular matrix and culturing them in suspension in the presence of LIF to form brain organoids (c); a step of forcing the expression of a mutant MAPT gene in the brain organoids (d); and a step of further culturing the brain organoids after step (d) in suspension for five weeks or more (e). A method for screening for a preventive or therapeutic agent of tauopathy, comprising: and a step (f) during or after step (e) of measuring the amount of phosphorylated tau protein or aggregated tau protein present in the brain organoid, wherein at least a portion of steps (c) to (e) is performed in the presence of the test substance, and a decrease in the amount of phosphorylated tau protein or aggregated tau protein measured in step (f) compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent of tauopathy. [Effects of the Invention]

[0010] According to the present invention, a technique for efficiently producing brain organoids containing aggregated tau protein can be provided.

Brief Description of the Drawings

[0011] [Figure 1] Figure 1 is a diagram showing the schedule of producing brain organoids in Experimental Example 1. [Figure 2] Figure 2 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 1. [Figure 3] Figure 3 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 1. [Figure 4] Figure 4 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 1. [Figure 5A] Figure 5A is a photograph showing the results of observing the brain organoids prepared from PS2-2 cells in Experimental Example 1 under an electron microscope. [Figure 5B] Figure 5B is a photograph showing the results of observing the brain organoids prepared from PS2-2 cells in Experimental Example 1 under an electron microscope. [Figure 6] Figure 6 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 2. [Figure 7] Figure 7 is a graph showing the results of quantifying the results of Figure 6. [Figure 8] Figure 8 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 2. [Figure 9] Figure 9 is a graph showing the results of quantifying the results of Figure 8. [Figure 10] Figure 10 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 3. [Figure 11] Figure 11 is a photograph showing the results of Western blotting in Experimental Example 4. [Figure 12] Figure 12 is a graph showing the results of quantifying the results of Figure 11. [Figure 13] Figure 13 is a graph in which gene expression was analyzed based on the results of RNA-seq analysis in Experimental Example 5, and the clustering results were plotted two-dimensionally using UMAP. [Figure 14]Figure 14 is a graph showing the expression analysis results for the CNP gene, PLP1 gene, and MBP gene in Experiment Example 5. [Figure 15] Figure 15 is a graph showing the expression analysis results of the OLIG1 and OLIG2 genes in Experimental Example 5. [Figure 16] Figure 16 is a graph showing the expression analysis results for the LINGO1 and APOD genes in Experimental Example 5. [Figure 17] Figure 17 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 5. [Modes for carrying out the invention]

[0012] [Method for producing brain organoids containing aggregated tau protein] In one embodiment, the present invention provides a method for producing brain organoids having aggregated tau protein, comprising the steps of: (a) culturing pluripotent stem cells in the presence of a SMAD inhibitor to form germ cells; (b) embedding the germ cells in an extracellular matrix and culturing them in three dimensions in the presence of a SMAD inhibitor and a GSK3β inhibitor to form organoids containing neural progenitor cells; (c) removing the organoids from the extracellular matrix and culturing them in suspension in the presence of LIF to form brain organoids; (d) forcing the brain organoids to express a mutant MAPT gene; and (e) further culturing the brain organoids after step (d) in suspension to obtain brain organoids having aggregated tau protein.

[0013] As will be described later in the examples, the manufacturing method of this embodiment makes it possible to efficiently produce brain organoids having aggregated tau protein.

[0014] In this specification, examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). It is preferable that the pluripotent stem cells are human cells. Furthermore, the pluripotent stem cells may be wild-type cells, cells with mutations in Alzheimer's disease-related genes, or cells possessing the ApoE4 / 4 isoform, which is an Alzheimer's disease risk factor.

[0015] Alzheimer's disease-related genes include the Presenilin 1 (PS1) gene, the Presenilin 2 (PS2) gene, and the β-amyloid precusor protein (APP) gene.

[0016] The NCBI accession number for the human PS1 gene genomic DNA is NC_000014.9. The NCBI accession number for the human PS2 gene genomic DNA is NC_000001.11. The NCBI accession number for the human APP gene genomic DNA is NC_000021.9. The NCBI accession number for the human APOE gene genomic DNA is NC_000019.10.

[0017] Pluripotent stem cells with mutations in Alzheimer's disease-related genes include pluripotent stem cells that have mutations in these Alzheimer's disease-related genes that lead to the development of Alzheimer's disease.

[0018] Mutations that can lead to the development of Alzheimer's disease include, but are not limited to, a gene mutation in the PS1 gene that causes an amino acid mutation (A246E) at the 246th amino acid of the PS1 protein, from alanine to glutamic acid (N141I) at the 141st amino acid of the PS2 gene, and duplication of the APP gene.

[0019] The mutations or isoforms that lead to the development of Alzheimer's disease may be mutations or isoforms that have been artificially introduced by genome editing or the like. Alternatively, pluripotent stem cells produced from cells derived from Alzheimer's disease patients may be used as pluripotent stem cells that have mutations in Alzheimer's disease-related genes or pluripotent stem cells that have isoforms related to Alzheimer's disease.

[0020] The following describes each step of the manufacturing method of this embodiment. First, in step (a), pluripotent stem cells form germ layers in the presence of a SMAD inhibitor. It is preferable to carry out step (a) for about 7 days. This step allows for differentiation of pluripotent stem cells into neural cells. It is preferable to use a combination of a BMP inhibitor and a TGF-β inhibitor as the SMAD inhibitor.

[0021] Examples of BMP inhibitors that can be used include Dorsomorphin (CAS number: 866405-64-3), DMH1 (CAS number: 1206711-16-1), and LDN-193189 (CAS number: 1062368-24-4). These may be used individually or in combination of two or more. The amount of BMP inhibitor added to the culture medium is, for example, about 1 to 3 μM.

[0022] In addition, TGF-β inhibitors such as A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), and RepSox (CAS number: 446859-33-2) can be used. These may be used individually or in combination of two or more. The amount of TGF-β inhibitor added to the culture medium is, for example, about 1 to 3 μM.

[0023] Any serum-free cell culture medium can be used as the basic culture medium. For example, standard synthetic media buffered with a carbonate-based buffer to a pH of 7.2 to 7.6 are examples. More specifically, examples include Advanced Dulbecco's Modified Eagle Medium / Ham F-12 mixed medium (DMEM / F12), Neurobasal medium (Thermo Fisher Scientific), Neurobasal Plus medium (Thermo Fisher Scientific), RPMI1640 medium, Advanced RPMI medium, etc. Pluripotent stem cells can be cultured by adding SMAD inhibitors and other additives to the basic medium.

[0024] In the manufacturing method of this embodiment, it is preferable to carry out all steps without using feeder cells, in a feeder-free manner. That is, it is preferable that the pluripotent stem cells are cultured in a feeder-free manner, and it is also preferable that steps (a) to (c) be carried out in a feeder-free manner. This simplifies the culture operation and prevents the contamination of brain organoids with feeder cells.

[0025] During the last 1-2 days of step (a), SMAD inhibitors and GSK3β inhibitors may be added to the culture medium. The SMAD inhibitors and GSK3β inhibitors are the same as those used in step (b) described later.

[0026] Prior to step (a), a further step may be performed in which pluripotent stem cells are cultured in the presence of less than 100 ng / mL of Fibroblast Growth Factor-2 (FGF2). The amount of FGF2 added to the culture medium is preferably less than 100 ng / mL, and may be, for example, 50 ng / mL or 10 ng / mL.

[0027] FGF2 may be derived from humans or mice, but it is preferable that it be derived from humans.

[0028] When culturing pluripotent stem cells in a feeder-free environment, it is common practice to add 100 ng / mL of FGF2 to the culture medium. In contrast, the inventors have demonstrated that reducing the amount of FGF2 added to the culture medium increases the efficiency of producing brain organoids containing aggregated tau protein.

[0029] If the amount of FGF2 added is too low, pluripotent stem cells may differentiate prematurely. Conversely, if the amount of FGF2 added is too high, it tends to be difficult to obtain neuroepithelial-like structures.

[0030] Furthermore, for example, if the amount of FGF2 added to the culture medium is 10 ng / mL, it becomes difficult to maintain pluripotent stem cells if the culture is continued for more than about 3 weeks. For this reason, when culturing pluripotent stem cells in a feeder-free environment in the presence of 10 ng / mL of FGF2, it is preferable to limit the culture time to 4 weeks or less.

[0031] Next, in step (b), the germ cells from step (a) are embedded in the extracellular matrix and cultured in three dimensions in the presence of a SMAD inhibitor and a GSK3β inhibitor to form organoids. Here, it is preferable to embed the germ cells in the extracellular matrix without dispersing them. If the germ cells are dispersed, the method of dispersion is not particularly limited and can be physical or enzymatic, but enzymatic methods are preferred from the viewpoint of not damaging cells. Enzymes that can be used in the enzymatic method include TrypLE Express (Thermo Fisher Scientific), TrypLE Select (Thermo Fisher Scientific), trypsin, collagenase, dispase I, etc. It is preferable to carry out step (b) for about 7 days.

[0032] Examples of extracellular matrix materials include type IV collagen, laminin, heparan sulfate proteoglycans, and entactin. Commercially available extracellular matrix materials such as Matrigel (registered trademark, manufactured by Corning) may also be used.

[0033] As the SMAD inhibitor, it is preferable to use the TGF-β inhibitors mentioned above. In step (a), it is preferable to use a combination of two or more SMAD inhibitors, but in step (b), one type may be used alone. In particular, it is preferable to use SB-431542 as the SMAD inhibitor in step (b). The amount of SMAD inhibitor added to the culture medium is, for example, about 1 to 5 μM.

[0034] Examples of GSK3β inhibitors include CHIR99021 (CAS number: 252917-06-9), Kenpaullone (CAS number: 142273-20-9), and 6-Bromoindirubin-3'-oxime (CAS number: 029-16241). The amount of GSK3β inhibitor added to the culture medium is, for example, about 1 to 5 μM. Among these, CHIR99021 is preferred.

[0035] Next, in step (c), the organoids from step (b) are removed from the extracellular matrix and cultured in suspension in a culture medium. Suspension culture is preferably performed using a bioreactor. A commercially available bioreactor can be used.

[0036] It is preferable to add Leukemia Inhibitory Factor (LIF) to the culture medium in at least part of step (c). The amount of LIF to add is, for example, about 5 to 50 ng / mL. The addition of LIF to the culture medium may be performed in only part of step (c) or in the entirety of step (c). LIF may be derived from humans or mice, but it is preferable that it be derived from humans. The inventors have shown that the efficiency of producing brain organoids containing aggregated tau protein is increased by performing suspension culture in the presence of LIF.

[0037] It is preferable to carry out step (c) for three weeks or more. As will be described later in the examples, it was found that carrying out step (c) for three weeks or more (five weeks or more from the start of step (a)), preferably six weeks or more (eight weeks or more from the start of step (a)), and more preferably eleven weeks or more (thirteen weeks or more from the start of step (a)) significantly increases the aggregated tau protein in brain organoids.

[0038] The culture medium in step (c) may contain additives in addition to LIF. Examples of additives include N2 supplement (Thermo Fisher Scientific) and B27 supplement (Thermo Fisher Scientific). Hereinafter, the culture medium containing LIF, N2 supplement, and B27 supplement may be referred to as the "differentiation induction medium."

[0039] Furthermore, in the latter half of step (c), additional additives may be added to the culture medium. Examples of additives include, in addition to the LIF, N2 supplement, and B27 supplement mentioned above, Brian-derived neurotrophic factor (BDNF), Glial-Derived Neurotrophic Factor (GDNF), and cAMP. Hereinafter, a culture medium containing LIF, N2 supplement, B27 supplement, BDNF, GDNF, and cAMP may be referred to as a "neuromaturation medium."

[0040] It is preferable to carry out at least a portion of step (c) in the presence of more than 20% by volume of oxygen. More than 20% by volume of oxygen is an oxygen concentration higher than the oxygen concentration in normal air (approximately 20% by volume). The oxygen concentration in step (c) is preferably more than 20% by volume, and may be, for example, 30% by volume, 40% by volume, or 50% by volume. The inventors have shown that carrying out suspension culture under a higher oxygen concentration than usual increases the efficiency of producing brain organoids having aggregated tau protein.

[0041] The period during which the oxygen concentration is increased may be, for example, from approximately 7 days after the start of process (c) until the end of culture (the end of process (e) described later), from approximately 14 days after the start of process (c) until the end of culture, or from approximately 21 days after the start of process (c) until the end of culture.

[0042] Next, in step (d), the mutant Microtubule Associated Protein Tau (MAPT) gene is forced to be expressed in the brain organoid after step (c).

[0043] For tau protein, isoforms 4R0N, 4R1N, and 4R2N can be used. Examples of mutant MAPT genes include genes encoding tau protein with the P301L mutation (amino acid sequence shown in SEQ ID NO: 1) and tau protein with the P301S mutation (amino acid sequence shown in SEQ ID NO: 2).

[0044] Forced expression of the mutant MAPT gene is preferably performed by viral gene transfer, and particularly preferably by adeno-associated virus (AAV). Gene transfer can be performed by creating an AAV vector into which the mutant tau protein gene linked to the CAG promoter, SYN1 promoter, EF1α promoter, CMV promoter, etc., is inserted. Examples of AAVs that can be used include AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ / 8, AAV-PHP.B, AAV-PHP.eB, etc.

[0045] Furthermore, gene transfer using AAV is preferably performed by inserting a glass tube into the brain organoid and injecting AAV. As described later in the examples, it was found that injecting viral particles (viral particle suspension) into the brain organoid resulted in a significantly greater increase in aggregated tau protein than adding viral particles to the culture medium of the brain organoid.

[0046] For injecting the virus particles, for example, an electric microinjector can be used. The injection rate of the virus particle suspension into the brain organoid is preferably, for example, 50 to 200 nL / min, and can be, for example, about 100 nL / min.

[0047] Next, in step (e), the brain organoids after step (d) are further cultured in suspension to obtain brain organoids containing aggregated tau protein. Suspension culture is preferably carried out in the presence of more than 20% by volume of oxygen.

[0048] Step (e) is preferably performed for 5 weeks or more. As will be described later in the examples, performing step (e) for 5 weeks or more tends to significantly increase the aggregated tau protein in the brain organoids.

[0049] [Brain organoids containing aggregated tau protein] In one embodiment, the present invention provides a brain organoid having aggregated tau protein, wherein, in a cross-section, the average ratio of the area of ​​the region containing phosphorylated tau protein to the total area of ​​the cross-section is 2% or more, or the average ratio of the area of ​​the region containing aggregated tau protein to the total area of ​​the cross-section is 1% or more. The brain organoid of this embodiment only needs to include cross-sections that satisfy the above area ratios; it is not necessary for all cross-sections to satisfy the above area ratios.

[0050] The brain organoids of this embodiment are preferably of human origin. Conventionally, no technology for efficiently producing aggregated tau protein has been reported. In contrast, the brain organoids of this embodiment can be produced efficiently and can therefore be used as a tau aggregation model. For example, they are useful as a model for studying the pathogenesis of tauopathy, or as a model for studying prevention and treatment methods. The brain organoids of this embodiment can be produced by the manufacturing method described above.

[0051] The brain organoid of one embodiment has aggregated tau protein, and in a cross-section, the average ratio of the area of ​​the region containing phosphorylated tau protein to the total area of ​​the cross-section is 2% or more, preferably 3% or more, and more preferably 5% or more. The brain organoid of this embodiment only needs to include cross-sections that satisfy the above area ratio; it is not necessary for all cross-sections to satisfy the above area ratio.

[0052] The region containing phosphorylated tau protein can be detected, for example, by immunostaining using an antibody against phosphorylated tau protein. Examples of antibodies against phosphorylated tau protein include anti-phosphorylated tau antibody (clone AT8, Fujirebio Inc.) and PHF-1 anti-phospho tau antibody (courtesy of P. Davies). Other antibodies that can detect phosphorylated tau protein can also be used.

[0053] The brain organoid of one embodiment has aggregated tau protein, and in a cross-section, the average ratio of the area of ​​the region containing aggregated tau protein to the total area of ​​the cross-section is 1% or more, preferably 2% or more, and more preferably 5% or more. The brain organoid of this embodiment only needs to include cross-sections that satisfy the above area ratio; it is not necessary for all cross-sections to satisfy the above area ratio.

[0054] The region containing aggregated tau protein (misfolded tau protein, tau protein that has undergone structural changes) can be detected, for example, by immunostaining using an antibody against aggregated tau protein. Examples of antibodies against aggregated tau protein include monoclonal antibodies (clone MC1) and monoclonal antibodies (clone Alz50). It is believed that aggregated tau protein is phosphorylated.

[0055] As will be described later, the brain organoids of this embodiment can be used for screening for preventive or therapeutic drugs for tauopathy. Therefore, in one embodiment, the present invention provides a screening kit for preventive or therapeutic drugs for tauopathy, comprising brain organoids having aggregated tau protein. The brain organoids having aggregated tau protein are the same as those described above.

[0056] [Screening methods for tauopathies] In one embodiment, the present invention provides a screening method for therapeutic drugs for tauopathy, comprising the steps of culturing a brain organoid having aggregated tau protein as described above in the presence of a test substance, and measuring the amount of phosphorylated tau protein or aggregated tau protein present in the brain organoid, wherein a decrease in the amount of phosphorylated tau protein or aggregated tau protein compared to the absence of the test substance indicates that the test substance is a therapeutic drug for tauopathy.

[0057] There are no particular restrictions on the test substances, and examples include libraries of natural compounds, synthetic compounds, existing drugs, and metabolites.

[0058] The amount of phosphorylated tau protein can be measured, for example, by subjecting brain organoids to immunostaining, Western blotting, ELISA, etc., using a phosphorylated tau antibody.

[0059] The amount of aggregated tau protein can be measured, for example, by subjecting brain organoids to immunostaining using antibodies against aggregated tau protein, Western blotting, ELISA, etc.

[0060] For example, if the amount of phosphorylated tau protein or aggregated tau protein in brain organoids cultured in the presence of the test substance decreases compared to the control, then the test substance can be considered a therapeutic agent for tauopathy. Here, the control can be brain organoids cultured in the absence of the test substance.

[0061] The screening method of this embodiment allows for the screening of therapeutic drugs for tauopathy. The therapeutic drugs for tauopathy obtained by the screening method of this embodiment can be described as drugs that, when administered after the formation of aggregated tau protein, reduce or eliminate the amount of aggregated tau protein.

[0062] [Screening methods for preventive or therapeutic drugs for tauopathies] In one embodiment, the present invention provides a method comprising: (a) culturing pluripotent stem cells in the presence of a SMAD inhibitor to form germ cells; (b) embedding the germ cells in an extracellular matrix and culturing them in three dimensions in the presence of a SMAD inhibitor and a GSK3β inhibitor to form organoids containing neural progenitor cells; (c) removing the organoids from the extracellular matrix and culturing them in suspension in the presence of LIF to form brain organoids; (d) forcing the brain organoids to express a mutant MAPT gene; and further culturing the brain organoids after step (d) in suspension for five weeks or more. The present invention provides a screening method for a preventive or therapeutic agent for tauopathy, comprising (e) and (f) measuring the amount of phosphorylated tau protein or aggregated tau protein present in the brain organoid during or after step (e), wherein at least a portion of steps (c) to (e) is performed in the presence of the test substance, and a decrease in the amount of phosphorylated tau protein or aggregated tau protein measured in step (f) compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for tauopathy.

[0063] In the screening method of this embodiment, steps (a) to (e) are the same as steps (a) to (e) in the method for producing brain organoids having aggregated tau protein described above, but differ from the production method described above in that at least a part of steps (c) to (e) is performed in the presence of the test substance.

[0064] The test substances are the same as those described above for screening methods for the treatment of tauopathy.

[0065] In the screening method of this embodiment, step (f) measures the amount of phosphorylated tau protein or aggregated tau protein present in the brain organoid. The amount of phosphorylated tau protein and aggregated tau protein can be measured by the same method as described above.

[0066] In step (f), if the amount of phosphorylated tau protein or aggregated tau protein in brain organoids cultured in the presence of the test substance decreases compared to the control, then the test substance can be said to be a preventive or therapeutic agent for tauopathy. Here, the control can be brain organoids cultured in the absence of the test substance.

[0067] The prophylactic agent for tauopathy obtained by the screening method of this embodiment can be described as a drug that suppresses or prevents the formation of aggregated tau proteins when administered before the formation of aggregated tau proteins. Furthermore, the therapeutic agent for tauopathy obtained by the screening method of this embodiment can be described as a drug that reduces or eliminates the amount of aggregated tau proteins when administered after the formation of aggregated tau proteins. [Examples]

[0068] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0069] [Experimental Example 1] (Preparation of brain organoids containing aggregated tau protein 1) First, pluripotent stem cells were cultured to create brain organoids. As pluripotent stem cells, we used 201B7, a wild-type human iPS cell line, and PS1-2 and PS2-2, human iPS cell lines derived from Alzheimer's disease patients.

[0070] It has been revealed that PS1-2 cells have a gene mutation in the PS1 gene that causes an amino acid mutation (A246E) at the 246th amino acid of the PS1 protein, changing from alanine to glutamic acid. Furthermore, it has been revealed that PS2-2 cells have a gene mutation in the PS2 gene that causes an amino acid mutation (N141I) at the 141st amino acid of the PS2 protein, changing from asparagine to isoleucine.

[0071] Figure 1 shows the schedule for brain organoid production. Each pluripotent stem cell was cultured in a feeder-free manner without the use of feeder cells. First, each cell was cultured for 1 to 3 weeks in a medium containing 10 ng / mL of FGF2.

[0072] Next, on Day 0 of culture, each cell, which had been dissociated into single cells, was suspended in iPS medium containing 2 μM Dorsomorphin (Sigma-A), 2 μM A83-01 (Tocris), and 10 μM Y27632 (Nacalai Tesque), but without FGF2, and seeded into a 96-well plate. On Day 1 of culture, the same amount of medium as above, but without Y27632, was added. On Day 3 of culture, half of the medium was replaced. In Figure 1, "Dorso" refers to Dorsomorphin and "A83" refers to A83-01. As a result, embryoid bodies were formed.

[0073] Next, on days 5-6 of culture, half of the culture medium was replaced with nerve induction medium. The nerve induction medium consisted of DMEM / F12, GlutaMAX (Thermo Fisher Scientific), 1 μM CHIR99021 (Ceragen Technologies), 1 μM SB-431542 (Ceragen Technologies), 1 × N2 supplement (Thermo Fisher Scientific), 1 × NEAA (Thermo Fisher Scientific), and 1 × penicillin / streptomycin. In Figure 1, "CHIR" refers to CHIR99021 and "SB" refers to SB-431542.

[0074] Next, on day 7 of culture, each cell was embedded in Matrigel (BD Biosciences) and cultured for another 6 days in nerve induction medium. Half of the medium was replaced every other day.

[0075] Next, on day 14 of culture, the organoids formed by mechanically dissociating Matrigel using a 5 mL pipette were extracted. Subsequently, the organoids were suspended in differentiation induction medium and cultured in suspension in a bioreactor (Able). The differentiation induction medium consisted of DMEM / F12, GlutaMAX (Thermo Fisher Scientific), 1 × N2 supplement (Thermo Fisher Scientific), 1 × B27 supplement (Thermo Fisher Scientific), 2.5 μg / mL insulin (Sigma-A), 0.1 mM 2-mercaptoethanol, 1 × NEAA (Thermo Fisher Scientific), 1 × penicillin / streptomycin, and 10 ng / mL LIF (Merck Millipore). The medium was changed every 2-3 days. In Figure 1, "N2" indicates the N2 supplement, "B27" indicates the B27 supplement, and "LIF" indicates LIF.

[0076] From day 28 of culture, the oxygen concentration in the incubator was set to 40% by volume.

[0077] From day 71 of culture, the differentiation induction medium was replaced with neuronal maturation medium, and suspension culture was continued. The neuronal maturation medium consisted of Neurobasal Plus (Thermo Fisher Scientific), 1x B27 supplement (Thermo Fisher Scientific), 20 ng / mL BDNF (Peprotec), 20 ng / mL GDNF (Peprotec), 0.5 mM cAMP (Sigma-A), 1x GlutaMAX (Thermo Fisher Scientific), 0.2 mM ascorbic acid (Sigma-A), 1x Antibiotic-Antimycotic (Thermo Fisher Scientific), and 10 ng / mL LIF (Merck Millipore). The medium was changed every 2-3 days. In Figure 1, "B27" represents the B27 supplement, "BDNF" represents BDNF, "GDNF" represents GDNF, and "cAMP" represents cAMP. As a result, brain organoids were formed.

[0078] A mutant MAPT gene was introduced into each brain organoid on day 84 (12 weeks) of culture. The mutant MAPT gene used was the gene encoding the tau protein with the P301L mutation (amino acid sequence shown in SEQ ID NO: 1). More specifically, an AAV virus expression plasmid incorporating the mutant MAPT gene downstream of the CAG promoter was introduced into packaging cells to prepare AAV virus particles (hereinafter sometimes referred to as "AAV(PHP.B)-CAG-Tau(1N4R)-P301L").

[0079] For comparison, we also prepared AAV virus particles expressing green fluorescent protein (EGFP) instead of the mutant MAPT gene (hereinafter sometimes referred to as "AAV(PHP.B)-CAG-EGFP").

[0080] The titer of the virus particle (AAV(PHP.B)-CAG-Tau(1N4R)-P301L) is 1.32 × 10⁻¹⁴ 13The concentration was VG / mL (where "VG" stands for "Vector Genome"). The titer of the virus particle (AAV(PHP.B)-CAG-EGFP) was 2 × 10⁻¹⁰. 13 It was VG / mL.

[0081] Each viral particle was introduced either by adding 0.5, 2, or 10 μL of each to the culture medium of the brain organoids, or by inserting a glass tube into the brain organoid and injecting the viral suspension. Injection was performed using an electric microinjector (product name "BJ-110", BEX Co., Ltd.) at an introduction rate of 100 nL / min, delivering 2 μL of viral particles per brain organoid.

[0082] Subsequently, the brain organoids were cultured in suspension, and on day 120 of the culture (Day 120, 106 days after the start of suspension culture), each brain organoid was removed and fixed with 4% paraformaldehyde. Then, the fixed organoids were embedded in an embedding medium (product name "OCT Compound", model number 45833, Sakura FineTech Japan Co., Ltd.), and sections were prepared.

[0083] Next, each section was immunostained using an anti-tau antibody (clone RTM38, Fujifilm Wako Pure Chemical Corporation), an anti-phosphorylated tau antibody (clone AT8, Fujirebio Inc.), and an antibody against aggregated tau protein (clone MC1), respectively, to examine tau protein aggregation. The nuclei were also stained with Hoechst33342.

[0084] Figures 2 to 4 are fluorescence micrographs showing the results of immunohistochemistry. Figure 2 shows the results of brain organoids prepared from 201B7 cells, and Figure 3 shows the results of brain organoids prepared from PS2-2 cells.

[0085] In Figures 2 and 3, "Injection(+)Tau" indicates the result of introducing AAV(PHP.B)-CAG-Tau(1N4R)-P301L by injection, and (-) indicates that no virus particles were introduced. "Injection(+)GFP" indicates the result of introducing AAV(PHP.B)-CAG-EGFP by injection. "MC1" indicates the result of staining with MC1 antibody, "AT8" indicates the result of staining with anti-phosphorylated tau antibody, "Tau" indicates the result of staining with anti-tau antibody, "GFP" indicates the result of detecting EGFP fluorescence, "Hoechst" indicates the result of staining the nucleus with Hoechst33342, and "Merge" indicates the result of combining photographs.

[0086] As a result, it was revealed that in brain organoids into which AAV(PHP.B)-CAG-Tau(1N4R)-P301L was injected, phosphorylated tau protein and aggregated tau protein were significantly increased.

[0087] Figure 4 is a fluorescence micrograph showing the results of adding 0.5, 2, and 10 μL of AAV(PHP.B)-CAG-Tau(1N4R)-P301L to the culture medium of brain organoids prepared from PS1-2 cells.

[0088] In Figure 4, "MC1" indicates the result of staining with MC1 antibody, "Tau" indicates the result of staining with anti-tau antibody, "Hoechst" indicates the result of staining the nucleus with Hoechst33342, and "Merge" indicates the result of combining photographs. In addition, "0.5μL", "2μL", and "10μL" indicate the results of adding 0.5, 2, and 10μL of AAV(PHP.B)-CAG-Tau(1N4R)-P301L, respectively.

[0089] The results showed that injecting viral particles into brain organoids resulted in a significantly greater increase in aggregated tau protein than adding viral particles to the culture medium of brain organoids.

[0090] Figures 5A and 5B show images of brain organoids prepared from PS2-2 cells, injected with AAV(PHP.B)-CAG-Tau(1N4R)-P301L, fixed after 5 weeks, and observed by immunoelectron microscopy analysis using MC1 antibody. The scale bar is 100 nm. As a result, tau filament-like structures were observed. This result indicates that the brain organoids in this experiment can be used as a tau aggregation model.

[0091] [Experimental Example 2] (Preparation of brain organoids containing aggregated tau protein 2) Pluripotent stem cells were cultured and brain organoids were created in the same manner as in Experimental Example 1. The wild-type human iPS cell line 201B7 was used as the pluripotent stem cells.

[0092] Mutant MAPT genes were introduced into brain organoids at three different stages of culture: days 35-41 (5 weeks), days 56-62 (8 weeks), and days 91-97 (13 weeks). The mutant MAPT gene was introduced by injecting the aforementioned viral particles (AAV(PHP.B)-CAG-Tau(1N4R)-P301L) using a gas injector. For comparison, a group was also prepared in which AAV viral particles expressing EGFP (AAV(PHP.B)-CAG-EGFP) were injected instead of the mutant MAPT gene.

[0093] Subsequently, each brain organoid was cultured in suspension, and five weeks after the introduction of the virus particles, each brain organoid was removed and fixed with 4% paraformaldehyde. Then, the fixed organoids were embedded in resin and sections were prepared.

[0094] Next, each section was immunostained using an anti-tau antibody (clone RTM38, Fujifilm Wako Pure Chemical Corporation), an anti-phosphorylated tau antibody (clone AT8, Fujirebio Inc.), and an antibody against aggregated tau protein (clone MC1), respectively, to examine tau protein aggregation. The nuclei were also stained with Hoechst33342.

[0095] Figure 6 is a fluorescence micrograph showing the results of immunohistochemistry. Figure 7 is a graph showing the quantified results of Figure 6. In Figures 6 and 7, "5w," "8w," and "13w" indicate the results of injecting viral particles into brain organoids at 35-41 days (5 weeks), 56-62 days (8 weeks), and 91-97 days (13 weeks), respectively. "GFP" indicates the result of introducing AAV(PHP.B)-CAG-EGFP, and "Tau(P301L)" indicates the result of introducing AAV(PHP.B)-CAG-Tau(1N4R)-P301L.

[0096] In Figure 6, "AT8" indicates the result of staining with anti-phosphorylated tau antibody, "Tau" indicates the result of staining with anti-tau antibody, "Hoechst" indicates the result of staining the nucleus with Hoechst33342, and "Merge" indicates the result of combining photographs. Additionally, "w / o AAV injection" indicates the result without injection of AAV virus particles.

[0097] In Figure 7, the vertical axis represents the percentage (%) of the area of ​​the region containing phosphorylated tau protein (the region stained with AT8 antibody) relative to the total area of ​​the cross-section of the brain organoid. The numerical values ​​represent the mean ± standard deviation, "**" indicates a statistically significant difference at P<0.004, and "***" indicates a statistically significant difference at P<0.001.

[0098] As a result, it was revealed that in brain organoids cultured at 35-41 days (5 weeks), 56-62 days (8 weeks), and 91-97 days (13 weeks), the introduction of AAV(PHP.B)-CAG-Tau(1N4R)-P301L by injection resulted in an average ratio of the area of ​​phosphorylated tau protein-containing regions (regions stained with AT8 antibody) to the total area of ​​the cross-section of the brain organoids exceeding 2%.

[0099] In particular, it was revealed that when AAV(PHP.B)-CAG-Tau(1N4R)-P301L was injected into brain organoids cultured at 91-97 days (13 weeks), the ratio of the area of ​​the region containing phosphorylated tau protein (region stained with AT8 antibody) to the total area of ​​the cross-section of the brain organoid significantly increased.

[0100] Figure 8 is a fluorescence micrograph showing the results of immunohistochemistry. Figure 9 is a graph showing the quantified results of Figure 8. In Figures 8 and 9, "5w," "8w," and "13w" indicate the results of injecting viral particles into brain organoids at 35-41 days (5 weeks), 56-62 days (8 weeks), and 91-97 days (13 weeks), respectively. "GFP" indicates the result of introducing AAV(PHP.B)-CAG-EGFP, and "Tau(P301L)" indicates the result of introducing AAV(PHP.B)-CAG-Tau(1N4R)-P301L.

[0101] In Figure 8, "MC1" indicates the result of staining with MC1 antibody, "Tau" indicates the result of staining with anti-tau antibody, "Hoechst" indicates the result of staining the nucleus with Hoechst33342, and "Merge" indicates the result of combining photographs. Also, "w / o AAV injection" indicates the result of not injecting AAV virus particles.

[0102] In Figure 9, the vertical axis represents the percentage (%) of the area of ​​the region containing aggregated tau protein (the region stained with MC1 antibody) relative to the total area of ​​the cross-section of the brain organoid. The numerical values ​​represent the mean ± standard deviation, "*" indicates a statistically significant difference at P<0.004, and "**" indicates a statistically significant difference at P<0.006.

[0103] As a result, it was revealed that in brain organoids cultured at 35-41 days (5 weeks), 56-62 days (8 weeks), and 91-97 days (13 weeks), the introduction of AAV(PHP.B)-CAG-Tau(1N4R)-P301L by injection resulted in an average ratio of the area of ​​aggregated tau protein (areas stained with MC1 antibody) to the total area of ​​the cross-section of the brain organoids exceeding 1%.

[0104] In particular, it was revealed that when AAV(PHP.B)-CAG-Tau(1N4R)-P301L was injected into brain organoids cultured at 91-97 days (13 weeks), the ratio of the area of ​​aggregated tau protein (areas stained with MC1 antibody) to the total area of ​​the cross-section of the brain organoid significantly increased.

[0105] These results further support the possibility of using the brain organoids from this experiment as a tau aggregation model.

[0106] [Experimental Example 3] (Analysis of brain organoids containing aggregated tau protein 1) Pluripotent stem cells were cultured and brain organoids were created in the same manner as in Experimental Example 1. The wild-type human iPS cell line 201B7 was used as the pluripotent stem cells.

[0107] Mutant MAPT genes were introduced into brain organoids cultured for 91-97 days (13 weeks). The mutant MAPT gene was introduced by injecting the aforementioned viral particles (AAV(PHP.B)-CAG-Tau(1N4R)-P301L) using a gas injector. For comparison, a group was also prepared in which AAV viral particles expressing EGFP (AAV(PHP.B)-CAG-EGFP) were injected instead of the mutant MAPT gene.

[0108] Subsequently, suspension culture of each brain organoid was continued, and five weeks after the introduction of the virus particles, each brain organoid was removed and fixed with 4% paraformaldehyde. Next, the fixed organoids were embedded in resin and sections were prepared. For comparison, sections of brain tissue from Alzheimer's disease patients and brain tissue from healthy individuals were also prepared.

[0109] Next, each section was immunostained using thioflavin S and an anti-phosphorylated tau antibody (clone AT8, Fujirebio Inc.) to examine the aggregation of tau protein. The nuclei were also stained with Hoechst33342. Thioflavin S is a compound that specifically binds to the β-sheet structure of proteins and emits fluorescence.

[0110] Figure 10 shows fluorescence micrographs illustrating the results of immunohistochemistry. In Figure 10, "Control Organoids" indicates the results of brain organoids introduced with AAV(PHP.B)-CAG-EGFP, "Tau-P301L Organoids" indicates the results of brain organoids introduced with AAV(PHP.B)-CAG-Tau(1N4R)-P301L, "Control Brain" indicates the results of brain tissue from healthy individuals, and "AD Brain" indicates the results of brain tissue from Alzheimer's disease patients. Additionally, "Thioflavin S" indicates staining with thioflavin S, "AT8" indicates staining with antiphosphorylated tau antibody, "Hoechst" indicates staining of the nucleus with Hoechst33342, and "Merge" indicates a composite image.

[0111] As a result, in brain organoids into which AAV(PHP.B)-CAG-Tau(1N4R)-P301L was introduced, regions containing β-sheet structures (regions stained with thioflavin S) and regions containing phosphorylated tau protein (regions stained with AT8 antibody) were detected, similar to brain tissue derived from Alzheimer's disease patients. Furthermore, it was revealed that these stained regions overlapped.

[0112] These results indicate that brain organoids into which AAV(PHP.B)-CAG-Tau(1N4R)-P301L has been introduced contain aggregates that form β-sheet structures containing phosphorylated tau, similar to those found in brain tissue derived from Alzheimer's disease patients.

[0113] These results further support the possibility of using the brain organoids from this experiment as a tau aggregation model.

[0114] [Experimental Example 4] (Analysis of brain organoids containing aggregated tau protein 2) Pluripotent stem cells were cultured and brain organoids were created in the same manner as in Experimental Example 1. The wild-type human iPS cell line 201B7 was used as the pluripotent stem cells.

[0115] Mutant MAPT genes were introduced into brain organoids at 56-62 days (8 weeks) and 91-97 days (13 weeks) of culture. The mutant MAPT gene was introduced by injecting the aforementioned viral particles (AAV(PHP.B)-CAG-Tau(1N4R)-P301L) using a gas injector. For comparison, a group was also prepared in which AAV viral particles expressing EGFP (AAV(PHP.B)-CAG-EGFP) were injected instead of the mutant MAPT gene.

[0116] Subsequently, suspension culture of each brain organoid was continued, and five weeks after the introduction of the virus particles, each brain organoid was removed, homogenized, and a sarcosyl-insoluble fraction was prepared.

[0117] Next, the presence or absence of phosphorylated tau in the prepared sarcosyl-insoluble fraction was analyzed by Western blotting using an anti-tau antibody (clone HT7, Fujirebio Inc.) and an anti-phosphorylated tau antibody (pS396, Thermo Fisher Scientific Inc.). The anti-tau antibody (clone HT7) detects all tau, while the anti-phosphorylated tau antibody (pS396) detects phosphorylated tau.

[0118] Figure 11 is a photograph showing the results of Western blotting. Figure 12 is a graph showing the quantitative results of Figure 11. In Figures 11 and 12, "8w" and "13w" indicate the results of injecting viral particles into brain organoids at 56-62 days (8 weeks) and 91-97 days (13 weeks), respectively.

[0119] In Figure 11, "GFP" indicates the result of introducing AAV(PHP.B)-CAG-EGFP, and "Tau(P301L)" indicates the result of introducing AAV(PHP.B)-CAG-Tau(1N4R)-P301L. Also in Figure 11, "HT7" indicates the result of staining with anti-tau antibody, and "pS396" indicates the result of staining with anti-phosphorylated tau antibody. The arrowheads indicate tau and phosphorylated tau bands.

[0120] In Figure 12, "control" indicates the result of introducing AAV(PHP.B)-CAG-EGFP, and "Tau-P301L" indicates the result of introducing AAV(PHP.B)-CAG-Tau(1N4R)-P301L. The vertical axis in Figure 12 shows the intensity (relative value) of the phosphorylated tau band in the sarcosyl-insoluble fraction. Also, "*" indicates a statistically significant difference at p<0.05.

[0121] As a result, it was revealed that phosphorylated tau was present in the sarcosyl-insoluble fraction of brain organoids into which AAV(PHP.B)-CAG-Tau(1N4R)-P301L had been introduced. In particular, it was revealed that the amount of phosphorylated tau in the sarcosyl-insoluble fraction increased significantly when AAV(PHP.B)-CAG-Tau(1N4R)-P301L was introduced into brain organoids cultured 91 to 97 days (13 weeks).

[0122] These results further support the possibility of using the brain organoids from this experiment as a tau aggregation model.

[0123] [Experimental Example 5] (Analysis of brain organoids containing aggregated tau protein 3) Pluripotent stem cells were cultured and brain organoids were created in the same manner as in Experimental Example 1. The wild-type human iPS cell line 201B7 was used as the pluripotent stem cells.

[0124] Mutant MAPT genes were introduced into brain organoids cultured for 91-97 days (13 weeks). The mutant MAPT gene was introduced by injecting the aforementioned viral particles (AAV(PHP.B)-CAG-Tau(1N4R)-P301L) using a gas injector. For comparison, a group was also prepared in which AAV viral particles expressing EGFP (AAV(PHP.B)-CAG-EGFP) were injected instead of the mutant MAPT gene.

[0125] Subsequently, suspension culture of each brain organoid was continued, and five weeks after the introduction of the virus particles, each brain organoid was removed and single-cell RNA-seq analysis was performed. Figure 13 is a graph plotting the clustering results, which were obtained by analyzing gene expression based on the RNA-seq analysis results, using UMAP in a two-dimensional manner.

[0126] In Figure 13, "Control-FBO" represents the results of brain organoids introduced with AAV(PHP.B)-CAG-EGFP, and "Tau-P301L-FBO" represents the results of brain organoids introduced with AAV(PHP.B)-CAG-Tau(1N4R)-P301L. As a result, it became clear that in Tau-P301L-FBO, a cluster was present at the location indicated by the arrow in Figure 13, which was not present in Control-FBO.

[0127] Figures 14-16 are graphs showing the results of gene expression analysis for each gene shown in the figure. We investigated published papers and extracted genes expressed in neuronal clusters that are present only in Alzheimer's disease patients, and then analyzed the expression of those genes.

[0128] In Figures 14-16, "cont" indicates the results of brain organoids introduced with AAV(PHP.B)-CAG-EGFP, and "tau" indicates the results of brain organoids introduced with AAV(PHP.B)-CAG-Tau(1N4R)-P301L.

[0129] As a result, it was revealed that the clusters that appeared in Tau-P301L-FBO showed increased gene expression of LINGO1, OLIG1, OLIG2, CNPase (CNP), and other genes.

[0130] Next, the expression and localization of CNP at the protein level were analyzed by immunohistochemistry. Control-FBO and Tau-P301L-FBO were fixed with 4% paraformaldehyde. Subsequently, the fixed organoids were embedded in resin and sections were prepared. For comparison, sections of brain tissue from Alzheimer's disease patients and brain tissue from healthy individuals were also prepared.

[0131] Next, each section was immunostained using anti-CNPase antibody, anti-tau antibody (RTM38, Fujifilm Wako Pure Chemical Corporation), and anti-HuC / HuD antibody, respectively. The nuclei were also stained with Hoechst33342.

[0132] Figure 17 shows fluorescence micrographs illustrating the results of immunohistochemistry. In Figure 17, "Control-FBO" indicates the results of brain organoids into which AAV(PHP.B)-CAG-EGFP was introduced, "Tau-P301L-FBO" indicates the results of brain organoids into which AAV(PHP.B)-CAG-Tau(1N4R)-P301L was introduced, "Control Brain" indicates the results of brain tissue from a healthy individual, and "AD Brain" indicates the results of brain tissue from an Alzheimer's disease patient. Additionally, "CNPase" indicates the results of staining with anti-CNPase antibody, "Tau" indicates the results of staining with anti-tau antibody, "Hoechst" indicates the results of nucleus staining with Hoechst33342, "HuC / D" indicates the results of staining with anti-HuC / HuD antibody, and "Merge" indicates the results of image synthesis.

[0133] As a result, co-localization of tau protein and CNPase was confirmed in brain organoids into which AAV(PHP.B)-CAG-Tau(1N4R)-P301L was introduced. Furthermore, co-localization of tau protein and CNPase was also confirmed in brain tissue derived from Alzheimer's disease patients.

[0134] These results further support the possibility of using the brain organoids from this experiment as a tau aggregation model. [Industrial applicability]

[0135] According to the present invention, a technique for efficiently producing brain organoids containing aggregated tau protein can be provided.

Claims

1. (a) A step in which human pluripotent stem cells are cultured in the presence of a SMAD inhibitor to form germ layers, (b) The germ layer is embedded in an extracellular matrix and cultured in three dimensions in the presence of an SMAD inhibitor and a glycogen synthase kinase 3β (GSK3β) inhibitor to form an organoid containing neural progenitor cells. (c) Steps include removing the organoid from the extracellular matrix and culturing it in suspension in the presence of Leukemia Inhibitory Factor (LIF) to form brain organoids, (d) A step of forcing the expression of a mutant Microtubule Associated Protein Tau (MAPT) gene in the brain organoid, The process includes step (e), which involves further suspension culture of the brain organoid after step (d) to obtain a brain organoid having aggregated tau protein, A method for producing a human brain organoid having aggregated tau protein, comprising performing step (c) above for three weeks or more.

2. The manufacturing method according to claim 1, wherein the mutant MAPT gene is a gene encoding a tau protein having a P301L mutation.

3. The manufacturing method according to claim 1 or 2, wherein the forced expression of the mutant MAPT gene is performed by gene transfer using adeno-associated virus (AAV).

4. The manufacturing method according to claim 3, wherein the forced expression of the mutant MAPT gene is performed by injecting the AAV into the brain organoid.

5. The manufacturing method according to any one of claims 1 to 4, wherein at least a portion of step (c) is carried out in the presence of more than 20% by volume of oxygen.

6. The manufacturing method according to any one of claims 1 to 5, wherein step (e) is performed for five weeks or more.

7. The method for producing a product according to any one of claims 1 to 6, further comprising the step of culturing the pluripotent stem cells in the presence of less than 100 ng / mL of Fibroblast Growth Factor-2 (FGF2) before step (a).

8. The manufacturing method according to any one of claims 1 to 7, wherein the culture of the pluripotent stem cells is performed in a feeder-free manner.

9. In a cross-section of a human brain organoid, the ratio of the area of ​​the region containing phosphorylated tau protein to the total area of ​​the cross-section is 2% or more, or in a cross-section, the ratio of the area of ​​the region containing aggregated tau protein to the total area of ​​the cross-section is 1% or more. The ratio of the area of ​​the region containing phosphorylated tau protein is the ratio of the area of ​​the immunostained phosphorylated tau protein region to the total area of ​​the cross-section of the fixed human brain organoid section. The ratio of the area of ​​the region where aggregated tau protein is present is the ratio of the area of ​​the immunostained region where tau protein is present to the total area of ​​the cross-section of the fixed human brain organoid.

10. A step of culturing the human brain organoid described in claim 9 in the presence of a test substance, The process includes measuring the amount of phosphorylated tau protein or aggregated tau protein present in the aforementioned human brain organoid, A method for screening for a therapeutic agent for tauopathy, wherein a decrease in the amount of phosphorylated tau protein or aggregated tau protein compared to the absence of the test substance indicates that the test substance is a therapeutic agent for tauopathy.

11. (a) A step in which human pluripotent stem cells are cultured in the presence of a SMAD inhibitor to form germ layers, (b) The germ layer is embedded in an extracellular matrix and cultured in three dimensions in the presence of a SMAD inhibitor and a GSK3β inhibitor to form an organoid containing neural progenitor cells, (c) Steps include removing the organoid from the extracellular matrix and culturing it in suspension in the presence of LIF to form brain organoids, (d) A step of forcing the expression of the mutant MAPT gene in the brain organoid, The brain organoid after step (d) is further cultured in suspension for five weeks or more (e), The process includes, during or after step (e), step (f) measuring the amount of phosphorylated tau protein or aggregated tau protein in the brain organoid, At least a portion of the above steps (c) to (e) is carried out in the presence of the test substance. A method for screening for a preventive or therapeutic agent for tauopathy, wherein the amount of phosphorylated tau protein or aggregated tau protein measured in step (f) above decreases compared to the absence of the test substance, indicating that the test substance is a preventive or therapeutic agent for tauopathy.

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