Method for producing nerve cell in which TDP-43 aggregation is induced in cytoplasm, and application thereof

Abstract

A purpose of the present invention is to provide: a method for producing nerve cells in which TDP-43 aggregation is induced in cytoplasm, and a method for inducing TDP-43 aggregation in the cytoplasm of nerve cells that does not induce cell death; a cell population obtainable by said method for producing nerve cells; and a method for evaluating a test substance and a method for screening for a preventive and / or therapeutic drug for neurodegenerative diseases, using said method. The present invention provides a method for producing nerve cells in which TDP-43 aggregation is induced in cytoplasm, the method including a step A of culturing nerve cells, which have been induced to differentiate from human-derived pluripotent stem cells, in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

Description

Method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, and its application

[0001] The present invention relates to a method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, and a method for inducing TDP-43 aggregation in the cytoplasm of nerve cells. The present invention further relates to a cell population obtained by the method for producing nerve cells described above. The present invention further relates to a method for evaluating a test substance using the method described above, and a method for screening for the prevention and / or treatment of neurodegenerative diseases.

[0002] Amyotrophic lateral sclerosis (ALS) is a disease characterized by progressive damage to motor nerve cells. It has a very poor prognosis, with death typically occurring within an average of 3 to 5 years due to respiratory failure, pneumonia, suffocation, and other complications. There is currently no drug that can fundamentally cure ALS, and research and development of new drugs are actively underway. Approximately 10% of ALS cases are familial, involving disease-related gene mutations, while the remaining 90% are classified as sporadic (Non-Patent Literature 1). It is known that 95-99% of ALS patients experience aggregation of a protein called TDP-43 in the cytoplasm, which is thought to be one of the factors causing cell damage (Non-Patent Literature 2). TDP-43 is normally localized in the cell nucleus and functions broadly in RNA splicing, localization regulation, and translation (Non-Patent Literature 3). Research is being conducted to reproduce TDP-43 aggregation in the cytoplasm to elucidate the pathological mechanisms of these diseases and to develop new therapeutic drugs. For example, there are reports of reproducing TDP-43 aggregation by forcibly expressing mutated TDP-43 (Non-Patent Literature 4). There are also reports of inducing TDP-43 aggregation by chemical stimulation; for example, there are reports of aggregation being induced in about 30 minutes by stimulation with arsenite (Non-Patent Literature 5). In addition, methods for inducing TDP-43 aggregation using oxidative stress inducers such as hydrogen peroxide (Non-Patent Literature 6), protease inhibitors such as MG-132 (Non-Patent Literature 7), and endoplasmic reticulum stress inducers such as puromycin and thapsigargin have been reported (Non-Patent Literature 8). Puromycin is an antibiotic often used in gene editing to select cells that have undergone editing, and by introducing a puromycin resistance gene along with the target gene, gene-edited cells can be efficiently obtained. During selection, puromycin is treated at a concentration of 2-5 μg / mL to damage cells that do not possess the resistance gene (Non-Patent Literature 9). Furthermore, using HeLa cells, after stimulating with 2.5 μg / mL Puromycin for 20 minutes, 300 μM H was used. ２ O ２ It has been reported that stimulating the area for 30 minutes induces TDP-43 aggregation (Non-Patent Document 10).

[0003] It has been reported that TDP-43 aggregation is reversible and degradable in the early stages of diseases such as ALS, but as the disease progresses, it becomes an irreversible, rigid aggregation that is difficult to degrade (Non-Patent Literature 11). Furthermore, it has been reported that this irreversible, rigid aggregation exhibits high cytotoxicity (Non-Patent Literature 12).

[0004] Richard J Mead et al., Nat Rev Drug Discov, Vol. 22, pp. 185-212, 2023. Lassi Koski et al., Int J Mol Sci, Vol. 22, paper number 12193, 2021. Non-Nuoc Tran et al., Front Cell Dev Biol, Vol. 10, paper number 931968, 2022. Kotaro Oiwa et al., Sci Adv, Vol. 9, paper number eadf6895, 2023. Brian D Freibaum et al., J Cell Biol, Vol. 223, paper number 202308083, 2024. Xinxin Zuo et al., Nat Adam K. Walker et al., Struct Mol Biol, Vol. 28, pp. 132-142, 2021; PLOS One, Vol. 8, paper number e81170, 2013; Mark Y. Fang et al., Neuron, Vol. 103, pp. 802-819, 2019; Alesandro T. Caputo et al., Sci Rep, Vol. 11, paper number 5247, 2021; Juan Carlos Rengifo-Gonzalez et al., ELife, Vol. 10, paper number e67605, 2021; Terry R. Suk et al., Mol Neurodegenerator, Vol. 15, p. 45, 2020; Anukool A Bhopatkar et al., J Biol Chem, Vol. 295, pp. 2506-2519, 2020.

[0005] Non-patent document 8 describes how 24-hour stimulation with 5 μg / mL puromycin induces TDP-43 aggregation in iPS cell-derived neurons. However, puromycin concentrations of 2-5 μg / mL are used to injure cells lacking puromycin resistance genes during selection, and are concentrations that injure many cells. While TDP-43 is thought to induce cytotoxicity after aggregation, it is not possible to distinguish between puromycin-induced injury and TDP-43 aggregation-induced injury, making it impossible to evaluate motor neuron cell death occurring in ALS and frontotemporal lobar degeneration (FTLD).

[0006] The present invention aims to provide a method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, and a method for inducing TDP-43 aggregation in the cytoplasm of nerve cells without inducing cell death. The present invention further aims to provide a cell population obtained by the above-described method for producing nerve cells. The present invention further aims to provide a method for evaluating a test substance using the above-described method, and a method for screening for the prevention and / or treatment of neurodegenerative diseases.

[0007] As a result of diligent research to solve the above problems, the inventors of this invention have found that nerve cells differentiated from human-derived pluripotent stem cells can be cultured in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death, thereby producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm. This invention was completed based on these findings.

[0008] In other words, the present invention provides the following inventions: <1> A method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, comprising step A of culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. <2> The method according to <1>, wherein the protein synthesis inhibitor is at least one selected from the group consisting of aminonucleoside antibiotics, peptidyl nucleoside antibiotics, aminoglycoside antibiotics, and glutarimide antibiotics. <3> The method according to <2>, wherein the aminonucleoside antibiotic is Puromycin. <4> The method according to <2>, wherein the peptidyl nucleoside antibiotic is Blasticidin S. <5> The method according to <2>, wherein the aminoglycoside antibiotic is at least one selected from the group consisting of G-418 and Hygromycin B. <6> The manufacturing method according to <2>, wherein the glutarimide antibiotic is Cycloheximide. <7> The manufacturing method according to <1>, wherein the protein synthesis inhibitor is Puromycin, and the concentration that does not induce cell death is greater than 5 μg / mL to 400 μg / mL. <8> The manufacturing method according to <1>, wherein the protein synthesis inhibitor is Blasticidin S, and the concentration that does not induce cell death is greater than 1 μg / mL to 400 μg / mL. <9> The manufacturing method according to <1>, wherein the protein synthesis inhibitor is Hygromycin B, and the concentration that does not induce cell death is greater than 300 μg / mL to 20 mg / mL. <10> The manufacturing method according to <1>, wherein the protein synthesis inhibitor is G-418, and the concentration that does not induce cell death is greater than 600 μg / mL to 20 mg / mL. <11> The method for producing cells according to <1>, wherein the protein synthesis inhibitor is Cycloheximide, and the concentration that does not induce cell death is greater than 10 μmol / L to 400 μmol / L. <12> The method for producing cells according to any one of <1> to <11>, wherein the culture time for step A is 2 hours to 72 hours. <13> The method for producing cells according to any one of <1> to <12>, wherein step A includes step A1, in which an oxidative stress inducer is added to the culture medium and then cultured.<14> The manufacturing method according to any one of <1> to <13>, wherein the oxidative stress inducer is at least one selected from the group consisting of hydrogen peroxide and ferroptosis inducers. <15> The manufacturing method according to any one of <1> to <14>, wherein step A includes step A1 of adding hydrogen peroxide to the culture medium at a concentration of more than 0.01 mmol / L to 5.0 mmol / L, and then culturing. <16> The manufacturing method according to any one of <1> to <15>, wherein step A includes step A1 of adding hydrogen peroxide to the culture medium at a concentration of 0.5 mmol / L to 2.0 mmol / L, and then culturing. <17> The manufacturing method according to any one of <13> to <15>, wherein the culturing time for step A1 is 1 minute to 360 minutes. <18> The manufacturing method according to any one of <13> to <15>, wherein the culturing time for step A1 is 30 minutes to 60 minutes. <19> The method for producing a neuron according to any one of <1> to <18>, wherein the neuron is a motor neuron, a cerebral cortical excitatory neuron, or a substantia nigra neuron. <20> The method for producing a neuron according to any one of <1> to <19>, further comprising step B of co-culturing the neuron with at least one selected from the group consisting of astrocytes and microglia. <21> A method for inducing TDP-43 aggregation in the cytoplasm of neurons, comprising step A of culturing neurons differentiated from human pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. <22> A cell population obtained by the method of any one of <1> to <20>, comprising neurons satisfying at least one of the following (i) to (ii): (i) positive for a cytoplasmic stress granule marker; (ii) positive for a cytoplasmic TDP-43 marker; and having a cell viability of 20% or more compared to an untreated cell population. <23> A cell population comprising nerve cells obtained by any one of the methods of <1> to <20>, wherein at least one of the following (i) to (ii): (i) positive for cytoplasmic stress granule markers; (ii) positive for cytoplasmic TDP-43 marker; and wherein at least 50% of the living cells included in the cell population satisfy at least one of (i) and (ii).<24> A method for evaluating a test substance, comprising step B of culturing nerve cells differentiated from human-derived pluripotent stem cells in the presence of a test substance, and step C of culturing the nerve cells differentiated from the human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. <25> The evaluation method according to <24>, wherein step B is performed first, followed by step C, steps B and C are performed simultaneously, or step C is performed first, followed by step B. <26> A screening method for the prevention and / or treatment of neurodegenerative diseases, comprising step B of culturing nerve cells differentiated from human-derived pluripotent stem cells in the presence of a test substance, and step C of culturing the nerve cells differentiated from the human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. <27> The screening method according to <26>, wherein step B is performed first, followed by step C, steps B and C are performed simultaneously, or step C is performed first, followed by step B. <28> The screening method according to <26> or <27>, comprising the steps of: culturing nerve cells that have been in contact with the test substance and control nerve cells that have not been in contact with the test substance; measuring the localization amount of TDP-43 aggregation in the cytoplasm of the nerve cells; and selecting a test substance that suppresses the localization of TDP-43 aggregation in the cytoplasm compared with a control that has not been in contact with the test substance, as a candidate for the prevention and / or treatment of neurodegenerative diseases.<29> The above neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), familial frontotemporal dementia parkinsonism linked to chromosome 17 (FTDP-17), Paget's disease of bone and inclusion body myopathy with frontotemporal dementia (IBMPFD), Perry syndrome, Parkinson's disease (PD), Parkinson's dementia complex (PDC), familial British dementia (FBD), and Huntington's disease (HD). A screening method according to any one of <26> to <28>, wherein the selected condition is at least one selected from the group consisting of ), Machado Joseph disease (MJD / SCA3), Alzheimer's disease (AD), Lewy body dementia (DLB), corticobasal degeneration (CBD), argyrophilic granule disease / argyrophilic granule dementia (AGD), diffuse neurofibrillary tangle disease with calcification (DNTC), and progressive supranuclear palsy (PSP).

[0009] In this invention, TDP-43 aggregation is induced by a protein synthesis inhibitor at a concentration that does not induce cell death, allowing for the evaluation of a large number of nerve cells. In this invention, since nerve damage caused by stimulation with the protein synthesis inhibitor is minimal, it is possible to evaluate nerve damage originating from TDP-43 aggregation.

[0010] Figure 1 shows images of unstimulated nerve cells (Hoechst33342, TDP-43, G3BP1). Figure 2 shows images of nerve cells stimulated with Puromycin (Hoechst33342, TDP-43, G3BP1). Figure 3 shows the quantitative results of the number of viable cells in nerve cells stimulated with Puromycin. Figure 4 shows the quantitative results of TDP-43 aggregation in nerve cells stimulated with Puromycin. Figure 5 shows the quantitative results of the number of viable cells in nerve cells stimulated with Blasticidin S. Figure 6 shows the quantitative results of TDP-43 aggregation in nerve cells stimulated with Blasticidin S. Figure 7 shows the quantitative results of the number of viable cells in nerve cells stimulated with G-418. Figure 8 shows the quantitative results of TDP-43 aggregation in nerve cells stimulated with G-418. Figure 9 shows the quantitative results of the number of viable cells in nerve cells stimulated with Hygromycin B. Figure 10 shows the quantitative results of TDP-43 aggregation in nerve cells stimulated with Hygromycin B. Figure 11 shows the quantitative results of the number of viable cells in nerve cells stimulated with Cycloheximide. Figure 12 shows the quantitative results of TDP-43 aggregation in nerve cells stimulated with Cycloheximide. Figure 13 shows images of unstimulated nerve cells (TDP-43, G3BP1, Hoechst33342). Figure 14 shows images of nerve cells stimulated with Puromycin and hydrogen peroxide (TDP-43, G3BP1, Hoechst33342). Figure 15 shows the results of evaluating the effect of ketoconazole on the induction of TDP-43 aggregation by puromycin stimulation using wild-type neurons and GRN heterozygous knockout cells (quantification of the percentage of cells having granular co-localization signals of TDP-43 and G3BP1 outside the nuclear region). Figure 16 shows the results of evaluating the effect of ketoconazole on the induction of TDP-43 aggregation by puromycin stimulation using wild-type neurons and GRN heterozygous knockout cells (quantification of the number of granular co-localization signals of TDP-43 and G3BP1 outside the nuclear region per cell). Figure 17 shows images of neurons (TDP-43) immediately after stimulation removal and 4 hours after stimulation with no stimulation, puromycin stimulation, or puromycin and hydrogen peroxide stimulation.Figure 18 shows the quantitative results of TDP-43 aggregation in nerve cells over time from 0 to 4 hours after no stimulation, puromycin stimulation, or stimulation with puromycin and hydrogen peroxide. Figure 19 shows the treatment conditions for puromycin and hydrogen peroxide stimulation. Figure 20 shows the quantitative results of TDP-43 aggregation in nerve cells at 0 and 3 hours after no stimulation, puromycin stimulation, or stimulation with puromycin and hydrogen peroxide. Figure 21 shows the treatment conditions for puromycin and hydrogen peroxide stimulation (1, 5, and 240 minutes). Figure 22 shows the quantitative results of TDP-43 aggregation in nerve cells at 0 and 3 hours after no stimulation, puromycin stimulation, or stimulation with puromycin and hydrogen peroxide (1, 5, and 240 minutes). Figure 23 shows the quantitative results of TDP-43 aggregation in neurons at 0 and 3 hours after unstimulated, puromycin-stimulated, or puromycin and RSL3-stimulated cells. Figure 24 shows images of neuronal / astrocyte co-culture systems (Hoechst33342, TDP-43, G3BP1) under unstimulated, puromycin-stimulated, or puromycin and hydrogen peroxide-stimulated conditions. Figure 25 shows images of neuronal / astrocyte / microglia tripartite co-culture systems (Hoechst33342, TDP-43, G3BP1) under unstimulated or puromycin and hydrogen peroxide-stimulated conditions.

[0011] The embodiments of the present invention will be described in detail below.

[0012] <Method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, and method for inducing TDP-43 aggregation in the cytoplasm of nerve cells> The present invention relates to a method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, comprising step A of culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. The present invention further relates to a method for inducing TDP-43 aggregation in the cytoplasm of nerve cells, comprising step A of culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

[0013] According to the present invention, it is possible to induce TDP-43 aggregation in the cytoplasm of nerve cells differentiated from human-derived pluripotent stem cells, which is a characteristic feature of the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Nerve cells in which TDP-43 aggregation has been induced in the cytoplasm can be used for the development of new therapeutic drugs for neurodegenerative diseases, elucidation of disease mechanisms, and biomarker discovery.

[0014] Examples of human-derived pluripotent stem cells include pluripotent stem cells derived from samples without mutations in disease-related genes that cause neurological diseases, pluripotent stem cells derived from samples collected from healthy individuals (healthy subjects) without neurological diseases, pluripotent stem cells derived from samples collected from patients with diseases, or pluripotent stem cells derived from samples with mutations in disease-related genes. "Without mutations in disease-related genes" means that the cells do not have mutations in disease-related genes that cause neurological diseases. In other words, even if there is a mutation in a gene, if it is a mutation that does not cause disease, it will be interpreted as having no mutations in disease-related genes. Furthermore, in the case of "having mutations in disease-related genes," the mutation may be an endogenous gene mutation that the patient originally possesses, or an exogenous gene mutation that has been artificially introduced. It is envisioned that by producing the nerve cells of the present invention using pluripotent stem cells derived from samples collected from patients with diseases, or pluripotent stem cells derived from samples with mutations in disease-related genes, it may be possible to mimic the pathogenesis of diseases derived from the sample or diseases caused by mutations in disease-related genes.

[0015] Examples of human-derived pluripotent stem cells include human iPS cells (human-induced pluripotent stem cells), human embryonic stem cells (human ES cells), and human mesenchymal stem cells. Human iPS cells are preferred, but the study is not limited to them. Human iPS cells are iPS cells created from human cells.

[0016] ES cells can be established, for example, by culturing early embryos before implantation, the inner cell mass that makes up the aforementioned early embryo, or single blastomeres (Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, JA et al., Science, 282, 1145-1147 (1998)). Early embryos may be produced by nuclear transfer of somatic cell nuclei (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Iritani et al. (Protein Nucleic Acid Enzymes, 44, 892 (1999)), Baguisi et al. (Nature Biotechnology, 17, 456 (1999)), Wakayama et al. (Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc.Natl.Acad.Sci.USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000)), Tachibana et al. (Human Embryonic Stem Cells) Derived by Somatic Cell Nuclear Transfer, Cell (2013) in press. Parthenogenesis embryos may also be used as early embryos (Kim et al. (Science, 315, 482-486 (2007)), Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al. (Cloning Stem Cells, 9, 432-449 (2007)), Revazova et al. (Cloning Stem Cells, 10, 11-24 (2008)).In addition to the above-mentioned paper, regarding the production of ES cells, Strelchenko N., et al. Reprod Biomed Online. 9: 623-629, 2004; Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al. Stem Cells 24: 2669-2676, 2006; Wassarman, P.M. et al. Methods in Enzymology, Vol. 365, 2003, etc. are useful for reference. In addition, fusion ES cells obtained by cell fusion of ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

[0017] Among ES cells, there are those available from preservation institutions or those commercially available. For example, for human ES cells, they are available from the Institute of Medical Science, the University of Tokyo (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0018] iPS cells refer to cells having pluripotency (multidifferentiability) and proliferation ability, which are produced by reprogramming somatic cells by introducing reprogramming factors. iPS cells show properties similar to ES cells. The somatic cells used for the production of iPS cells are not particularly limited and may be differentiated somatic cells or undifferentiated stem cells. iPS cells can be produced by known methods, etc. Naturally, it is also assumed that iPS cell production methods to be developed in the future will be applied.

[0019] The most basic method for generating iPS cells involves introducing four transcription factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into cells using a virus (Takahashi K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi K, et al: Cell 131 (5), 861-72, 2007). For human iPS cells, there are reports of establishment by introducing four factors: Oct3 / 4, Sox2, Lin28, and Nanog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). The establishment of iPS cells by introducing three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), two factors Oct3 / 4 and Klf4 (Kim JB, et al: Nature 454 (7204), 646-650, 2008), or Oct3 / 4 only (Kim JB, et al: Cell 136 (3), 411-419, 2009) has also been reported. Furthermore, methods for introducing proteins, which are the expression products of genes, into cells have also been reported (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al: Cell Stem Cell 4, 472-476, 2009). On the other hand, there are reports that the efficiency of synthesis and the number of introduced factors can be improved by using inhibitors against histone methyltransferase G9a such as BIX-01294, or histone deacetylase inhibitors such as valproic acid (VPA) or BayK8644 (Huangfu D, et al: Nat.Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat.Biotechnol. 26 (11), 1269-1275, 2008; Silva J, et al: PLoS.Biol. 6 (10), e253, 2008).Studies on gene transfer methods have also progressed. In addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903), 945-949, 2008), plasmids (Okita K, et al: Science322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat.Methods 6, 363-369, 2009), or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been developed for gene transfer.

[0020] Cells in which transformation, i.e., reprogramming (reinitialization) into iPS cells has occurred can be selected using as indicators the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct3 / 4, Fgf-4, Esg-1, and Cript. The selected cells can be recovered as iPS cells.

[0021] As a method for producing iPS cells, in addition to the method of production by direct reprogramming by gene expression, iPS cells can also be induced from somatic cells by adding compounds, etc. (Hou P et al: Science 341 (6146), 651-654, 2013).

[0022] iPS cells can also be obtained, for example, from FUJIFILM Cellular Dynamics, Inc. (FCDI), Kyoto University, National University Corporation, or the BioResource Research Center, RIKEN, an Independent Administrative Institution.

[0023] Methods for obtaining nerve cells differentiated from human-derived pluripotent stem cells include, for example, induction from somatic cells collected from healthy individuals (healthy subjects) without disease-related gene mutations that cause neurological diseases, or from somatic cells collected from patients with neurological diseases; induction from established human iPS cell lines; and induction from human iPS cells that have disease-related gene mutations that cause neurological diseases.

[0024] The nerve cells differentiated from human-derived pluripotent stem cells are not particularly limited, but are preferably motor neurons, cerebral cortical excitatory neurons, or substantia nigra neurons, and are particularly preferably motor neurons or cerebral cortical excitatory neurons.

[0025] There are no particular limitations on the methods for inducing differentiation of human-derived pluripotent stem cells into nerve cells, but these include methods that involve generating neural stem cells from pluripotent stem cells using small molecule compound treatment and then inducing them into nerve cells, and methods that involve directly inducing them into nerve cells through gene expression, etc.

[0026] Methods for inducing differentiation of pluripotent stem cells into nerve cells include, for example: (1) culturing in serum-free medium to form embryoid bodies (cell aggregates containing neural progenitor cells) and then differentiating them (SFEB method: Watanabe K., et al, Nat. Neurosci., 8: 288-296, 2005; SFEBq method: Wataya T., et al Proc. Natl. Acad. Sci. USA., 105: 11796-11801, 2008); (2) culturing on stromal cells and then differentiating them (SDIA method: Kawasaki H., et al, Neuron, 28: 31-40, 2000); (3) culturing on Matrigel with added drugs and then differentiating them (Chambers SM, et al, Nat. Biotechnol., 27: 275-280, (2009); (4) A method of differentiation by culturing in a medium containing small molecule compounds as cytokine substitutes (U.S. Patent No. 5,843,780); (5) A method of differentiation by introducing and expressing neuroinducible factors (such as the gene encoding neurogenin2 (NGN2) protein) into pluripotent stem cells (WO2014 / 148646; and Zhang Y., et al, Neuron, 78: 785-98, 2013); (6) A method of differentiation by introducing and expressing miR-9 / 9*-124 into pluripotent stem cells; and combinations of these methods.

[0027] Of the above, (5) the method of introducing a gene encoding the NGN2 protein into pluripotent stem cells and expressing it is preferred because it can produce mature nerve cells in a short period of time and with high efficiency.

[0028] Neurogenein 2 protein is a transcription factor known to promote differentiation into nerve cells during development, and its amino acid sequence is exemplified by NP_076924 in humans and NP_033848 in mice. The neurogenein 2 gene (Official full name: neurogenein 2, Official symbol: NEUROG2, also called the Ngn2 gene) is the DNA that encodes the neurogenein 2 protein, and examples include DNA having the nucleotide sequences of NM_009718 (mouse) or NM_024019 (human), which are registered as standard sequences, or their transcription variants. Alternatively, it may be DNA having sufficient complementarity to hybridize under stringent conditions to nucleic acids having the above standard sequences and transcription variants.

[0029] As excitatory neurons of the cerebral cortex differentiated from human iPS cells, it is preferable to use cells produced by forcibly expressing the Ngn2 gene from human iPS cells.

[0030] As nerve cells differentiated from human-derived pluripotent stem cells, commercially available nerve cells may be used, for example, iCell® motor nerve cells (FCDI, C1050, C1048).

[0031] As substantia nigra neurons differentiated from human-derived pluripotent stem cells, commercially available neurons may be used, for example, iCell® dopamine neurons (FCDI, C1087, C1028).

[0032] The nerve cells are preferably cells that express at least one nerve cell-specific marker gene consisting of β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2), and that have β-III tubulin-positive processes (hereinafter referred to as neurites).

[0033] The expression level of a marker gene can usually be analyzed by the amount of the transcript corresponding to the gene, or the amount of its translation product, activity, etc. The measurement of the expression level can be performed by measuring the mRNA, which is the transcript of the gene, or the protein, which is the translation product of the gene. However, it is preferably performed by measuring the mRNA or its reverse transcription product, cDNA. The detection or measurement of the expression of the translation product (protein) can be performed by immunocytochemical staining using an antibody to detect the protein in the cell.

[0034] Step A can be performed by seeding cells in a medium and starting the culture. The lower limit value of the cell density at the time of seeding the cells is not particularly limited. For example, it is preferably ４ cells / cm ２ or more, more preferably ４ cells / cm ２ or more, still more preferably ４ cells / cm ２ or more, and even more preferably ４ cells / cm ２ or more. The upper limit value of the cell density is not particularly limited. For example, it may be ４ cells / cm ２ or less, preferably ４ cells / cm ２ less, more preferably ４ cells / cm ２ or less, still more preferably ４ cells / cm ２ or less. The cell density is preferably ４ cells / cm ２ or more and ４ cells / cm ２ or less, more preferably ４ cells / cm ２ or more and ４ cells / cm ２ or less, and still more preferably ４ cells / cm ２ or more and ４ cells / cm ２The following, and more preferably 12 × 10 ４ cells / cm ２ The above 20 x 10 ４ cells / cm ２ The following applies:

[0035] In step A, nerve cells differentiated from human-derived pluripotent stem cells are cultured in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. As a concentration that does not induce cell death, for example, if the number of viable cells is 20% or more compared to the unstimulated condition, it can be determined that cell death has not been induced, and it is preferable to use a concentration that can reproduce such conditions.

[0036] Examples of protein synthesis inhibitors include at least one selected from the group consisting of aminonucleoside antibiotics, peptidyl nucleoside antibiotics, aminoglycoside antibiotics, and glutarimide antibiotics. Among these, aminonucleoside antibiotics and peptidyl nucleoside antibiotics are preferred.

[0037] Examples of aminonucleoside antibiotics include puromycin. Examples of peptidyl nucleoside antibiotics include blasticidin S, nikkomycin Z, and polyoxin D. Examples of aminoglycoside antibiotics include G-418, hydroxycin B, kanamycin, gentamycin, and streptomycin. Examples of glutarimide antibiotics include cycloheximide and actiketal.

[0038] When the protein synthesis inhibitor is puromycin, the concentration that does not induce cell death is preferably greater than 5 μg / mL to 400 μg / mL, more preferably greater than 5 μg / mL to 100 μg / mL, even more preferably greater than 5 μg / mL to 40 μg / mL, and even more preferably between 10 μg / mL and 15 μg / mL. A concentration of 400 μg / mL or less is preferable from the viewpoint of cytotoxicity.

[0039] When the protein synthesis inhibitor is Blasticidin S, the concentration that does not induce cell death is preferably greater than 1 μg / mL to 400 μg / mL, more preferably greater than 1 μg / mL to 90 μg / mL, even more preferably greater than 1 μg / mL to 40 μg / mL, and even more preferably between 10 μg / mL and 40 μg / mL. A concentration of 400 μg / mL or less is preferable from the viewpoint of cytotoxicity.

[0040] When the protein synthesis inhibitor is hydroxycin B, the concentration that does not induce cell death is preferably greater than 300 μg / mL to 20 mg / mL, more preferably greater than 300 μg / mL to 2000 μg / mL, and even more preferably between 600 μg / mL and 1500 μg / mL. A concentration of 20 mg / mL or less is preferable from the viewpoint of cytotoxicity.

[0041] When the protein synthesis inhibitor is G-418, the concentration that does not induce cell death is preferably greater than 600 μg / mL to 20 mg / mL, more preferably greater than 600 μg / mL to 2000 μg / mL, and even more preferably between 1500 μg / mL and 2000 μg / mL. A concentration of 20 mg / mL or less is preferable from the viewpoint of cytotoxicity.

[0042] When the protein synthesis inhibitor is cycloheximide, the concentration that does not induce cell death is preferably greater than 10 μmol / L to 400 μmol / L, more preferably greater than 10 μmol / L to 40 μmol / L, and even more preferably between 15 μmol / L and 20 μmol / L. A concentration of 400 μmol / L or less is preferable from the viewpoint of cytotoxicity.

[0043] The culture time in step A (i.e., the culture time in the culture medium containing the protein synthesis inhibitor) is preferably 2 to 72 hours, more preferably 6 to 60 hours, even more preferably 12 to 50 hours, and particularly preferably 24 to 48 hours. From the viewpoint of cytotoxicity, a culture period of 72 hours or less in step A is preferable.

[0044] The culture medium can be selected from known media or commercially available media. The culture medium used for cultivation can be prepared by adding additives to the basal medium. Here, the basal media are, for example, DMEM (Dulbeccoo's Modified Eagle Medium), DMEM / F12, BrainPhys® Neuronal Medium, Neurobasal®-A Medium, Neurobasal® Medium, Neurobasal® Plus Medium, Neuroal Progenitor Basal Medium, NS-A Basal Medium, Basal Medium Eagle (BME), BGJb Medium, CMRL 1066 Medium, Glasgow Minimum Essential Medium (MEM), Improved Examples include MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle MEM, αMEM, Ham's F12 Medium, RPMI 1640 Medium, and Fischer's Medium. Furthermore, the culture medium may be a single medium or a combination of two or more mediums.

[0045] Preferred culture media include Neurobasal® Medium, Neurobasal®-A Medium, Neurobasal® Plus Medium, DMEM / F12, and DMEM, with Neurobasal® Medium being particularly preferred.

[0046] Additives may be added if they do not exhibit substantially antioxidant activity, or if added at concentrations that do not exhibit substantially antioxidant activity. Specifically, examples include, but are not limited to, serum, retinoic acid, Wnt, BMP, bFGF, EGF, HGF, Sonic hedgehog (Shh), interleukins, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B-27® supplement (antioxidant-free), and ITS-supplement. A preferred additive is B-27® supplement (antioxidant-free).

[0047] According to one embodiment of the present invention, it is possible to induce irreversible aggregation of TDP-43 that is difficult to decompose. Methods for reproducing the aggregation of TDP-43 that is difficult to decompose, and methods for evaluating the effect of drugs on it, are extremely useful for developing therapeutic drugs for diseases caused by TDP-43 aggregation, such as ALS.

[0048] To confirm whether the aggregation of TDP-43, which is resistant to degradation, has been induced, one can, for example, evaluate whether the percentage of cells showing TDP-43 aggregation changes after applying a stimulus that induces TDP-43 aggregation and then removing the stimulus. Although not particularly limited, for example, if the percentage of cells showing TDP-43 aggregation is detected at 30% or more both immediately after the stimulus is removed (e.g., 0 hours after stimulus removal) and after a certain period of time has elapsed since the stimulus was removed (e.g., 3 hours after stimulus removal), it can be determined that the aggregation of TDP-43, which is resistant to degradation, has been induced.

[0049] In one embodiment of the present invention, step A may include step A1, in which an oxidative stress inducer is added to the culture medium and then cultured.

[0050] Oxidative stress inducers are chemical substances that cause oxidative stress by increasing reactive oxygen species (ROS) in the body or inhibiting the body's antioxidant system. Examples of oxidative stress inducers include paraquat, menadione, rotenone, tert-butyl hydroperoxide (tBHP), hydrogen peroxide, and ferroptosis inducers, with hydrogen peroxide or ferroptosis inducers being preferred, and hydrogen peroxide being more preferred.

[0051] In one embodiment of the present invention, step A may include step A1, in which hydrogen peroxide is added to the culture medium at a concentration of greater than 0.01 mmol / L to 5.0 mmol / L (preferably 0.05 mmol / L to 5.0 mmol / L, more preferably 0.08 mmol / L to 2.0 mmol / L, and even more preferably 0.1 mmol / L to 1 mmol / L), and then cultured. The lower limit of the concentration of hydrogen peroxide added in step A1 is not particularly limited, but it is preferably greater than 0.01 mmol / L, more preferably 0.05 mmol / L or higher, even more preferably 0.08 mmol / L or higher, and particularly preferably 0.1 mmol / L or higher. A concentration greater than 0.01 mmol / L is preferable from the viewpoint of inducing aggregation of TDP-43, which is difficult to decompose. Furthermore, while there is no particular upper limit to the concentration of hydrogen peroxide added in step A1, it is preferably 5.0 mmol / L or less, more preferably 3.0 mmol / L or less, even more preferably 2.0 mmol / L or less, even more preferably 1.0 mmol / L or less, and particularly preferably 0.5 mmol / L. A concentration of 5.0 mmol / L or less is preferred from the viewpoint of cytotoxicity. The culture time in step A1 is preferably 1 to 360 minutes, preferably 10 to 120 minutes, more preferably 30 to 90 minutes, and even more preferably 30 to 60 minutes or 45 to 90 minutes.

[0052] In another embodiment of the present invention, step A may include step A1, in which a ferroptosis-inducing agent is added to the culture medium, followed by culturing. Examples of ferroptosis-inducing agents include elastin, FIN56 (Ferroptosis-inducing 56), sorafenib, BSO (L-Buthionine-(S,R)-Sulfoximine), and RSL3 (Ras-selective lethal small molecule 3), with elastin, FIN56, or RSL3 being preferred, and RSL3 being more preferred.

[0053] For culturing nerve cells, you can simply select general cell culture conditions: 37°C, 5% CO2. ２Examples of conditions include the following. During cultivation, the culture medium may be changed at appropriate intervals (preferably once every 1 to 7 days, more preferably once every 2 to 3 days), but it is preferable not to change the culture medium during the cultivation period.

[0054] Nerve cells are preferably cultured in two dimensions. Cell culture vessels such as plates, dishes, cell culture inserts, and cell culture flasks can be used for cell culture.

[0055] One embodiment of the present invention may further include step B, in which nerve cells differentiated from human-derived pluripotent stem cells are co-cultured with at least one selected from the group consisting of human-derived astrocytes and human-derived microglia.

[0056] According to one embodiment of the present invention, a method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm is provided, comprising the steps of: adding the nerve cells and astrocytes to a culture vessel; co-culturing the nerve cells and astrocytes in the culture vessel to produce a co-culture product; and culturing the co-culture product, which includes nerve cells differentiated from human-derived pluripotent stem cells, in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

[0057] Furthermore, according to another embodiment of the present invention, a method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm is provided, comprising the steps of: adding the nerve cells, astrocytes, and microglia to a culture vessel; co-culturing the nerve cells, astrocytes, and microglia in the culture vessel to produce a co-culture; and culturing the co-culture, which includes nerve cells differentiated from human-derived pluripotent stem cells, in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

[0058] In step B, the co-culture may be either a two-dimensional or three-dimensional culture. The three-dimensional culture may be in the form of a spheroid.

[0059] Furthermore, when adding the nerve cells, astrocytes, or microglia to the culture vessel in step B, the timing of adding each cell may be simultaneous or separate.

[0060] In step B, when preparing a two-dimensional co-culture containing the nerve cells, astrocytes, and microglia, it is preferable to pre-culture the astrocytes in a culture vessel, and then add the nerve cells and microglia to the culture vessel containing the cultured astrocytes.

[0061] In step B, when preparing a three-dimensional culture containing the nerve cells, astrocytes, and microglia, it is preferable to add the nerve cells, astrocytes, and microglia to the culture vessel simultaneously prior to co-culturing the cells.

[0062] "Prior to co-culturing cells" means that if, for example, 1 to 24 hours have passed since adding one or two types of cells, co-culturing will not be considered to have begun.

[0063] Specifically, for example, when preparing a three-dimensional culture, the three types of cells described above may be placed in a single container, suspended and mixed in a culture medium, and then added to the culture vessel. In this case, the step of adding the nerve cells, astrocytes, and microglia to the culture vessel includes the step of suspending the nerve cells, astrocytes, and microglia in a culture medium, and the step of simultaneously adding the culture medium containing the nerve cells, astrocytes, and microglia obtained above to the culture vessel.

[0064] Alternatively, each of the three types of cells described above may be suspended in a separate culture medium and added simultaneously to the same culture vessel. Alternatively, the three types of frozen cells, each previously cryopreserved separately, may be thawed and added simultaneously to the same culture vessel. However, the method of adding the nerve cells, astrocytes, and microglia to the culture vessel simultaneously is not limited to the above.

[0065] In this invention, two or three types of cells (astrocytes, nerve cells, and / or microglia) can be co-cultured in any proportion. Furthermore, by co-culturing two or three types of cells (astrocytes, nerve cells, and microglia), it is possible to better reflect and mimic the human brain than with conventional culture systems, which is useful for studying the brain's inherent functions and pathogenesis mechanisms. By inducing TDP-43 aggregation using the above co-culture, it is expected that this can be utilized in studying pathogenesis mechanisms that better reflect the human brain.

[0066] In the present invention, the astrocytes and microglia used in co-culture with the nerve cells are preferably differentiated from human-derived pluripotent stem cells.

[0067] Examples of human-derived pluripotent stem cells include human iPS cells, human ES cells, and human mesenchymal stem cells. Human iPS cells are preferred, but the study is not limited to them. Human iPS cells are iPS cells created from human cells.

[0068] Examples of human-derived pluripotent stem cells include, but are not limited to, pluripotent stem cells derived from a sample that does not have mutations in disease-related genes, or pluripotent stem cells derived from a sample that does have mutations in disease-related genes. However, it is preferable, for example, that the pluripotent stem cells derived from a sample that does not have mutations in disease-related genes. "Does not have mutations in disease-related genes" means that the sample does not have mutations in disease-related genes that cause neurological diseases. In other words, even if there is a mutation in a gene, if the mutation is not one that causes disease, it will be interpreted as not having a mutation in a disease-related gene.

[0069] According to the present invention, nerve cells in which TDP-43 aggregation is induced in the cytoplasm can be produced. Whether or not TDP-43 aggregation is induced in the cytoplasm can be evaluated, for example, by performing immunofluorescence staining and cell nucleus staining as described below, and then acquiring images of the cells. For example, the aggregation of TDP-43 in nerve cells can be analyzed by using image analysis software of a confocal quantitative image cytometer (e.g., Yokogawa Electric Corporation, CellVoyager CQ1) to recognize granular signals of TDP-43 and stress granule markers (e.g., G3BP1), and evaluating the degree of aggregation.

[0070] <Cell Population> The present invention further relates to a cell population comprising nerve cells obtained by a method for producing nerve cells according to the present invention, which satisfy at least one of the following (i) to (ii): (i) positive for cytoplasmic stress granule markers; (ii) positive for cytoplasmic TDP-43 markers, wherein the cell viability is 20% or more (preferably 30% or more, more preferably 40% or more) compared to an untreated cell population.

[0071] The present invention further relates to a cell population comprising nerve cells obtained by a method for producing nerve cells according to the present invention, which satisfy at least one of the following (i) to (ii): (i) positive for cytoplasmic stress granule markers; (ii) positive for cytoplasmic TDP-43 markers; wherein 50% or more (preferably 60% or more, more preferably 70% or more) of the living cells included in the cell population satisfy at least one of (i) and (ii).

[0072] The nerve cells that satisfy at least one of the above conditions (i) to (ii) are not particularly limited, but are preferably motor nerve cells, cerebral cortical excitatory nerve cells, or substantia nigra nerve cells.

[0073] Examples of cytoplasmic stress granule markers include G3BP1, G3BP2, ATXN2, ATXN2L, UBAP2, UBAP2L, TIA1, TIAR, CARPIN1, USP10, TRIM25, TRIM56, DDX3X, FMR1, CSDE1, PRRC2C, NUFI2, HDAC6, etc., with G3BP1 and TIA1 being preferred.

[0074] The TDP-43 marker, known as the TAR DNA-binding protein of 43 kDa, is a nuclear-localized RNA-binding protein. TDP-43 is involved in the transcription and splicing of various genes.

[0075] Whether a marker is positive or negative can usually be analyzed by measuring the production level of the transcript or translation product of the gene, or its activity. Expression levels can be measured by measuring mRNA, which is the transcript of the gene, or protein, which is the translation product of the gene, but it is preferable to measure mRNA or its reverse transcript, cDNA. The expression of translation products (proteins) can be detected or measured by immunohistochemistry, which uses antibodies to detect proteins within cells.

[0076] Whether or not the marker is positive can preferably be analyzed by immunofluorescence staining using an anti-TDP-43 antibody and an antibody that recognizes stress granule markers.

[0077] As an anti-TDP-43 antibody, any antibody capable of binding to TDP-43 is acceptable. As an antibody that recognizes stress granule markers, any antibody capable of binding to stress granule markers is acceptable.

[0078] The anti-TDP-43 antibody and the antibody that recognizes stress granule markers may be commercially available or prepared appropriately by conventional methods, and may be either monoclonal or polyclonal antibodies. The anti-TDP-43 antibody and the antibody that recognizes stress granule markers can be prepared by methods utilizing cell fusion technology or genetic recombination technology.

[0079] Antibodies that recognize anti-TDP-43 antibodies and stress granule markers are Fab, Fab', and F(ab') of the antibody. ２ Fv, Fd, single-stranded Fv (scFv), disulfide-bonded Fv (sdFv), V Ｌ , V Ｈ , Diamond Body ((V Ｌ -V Ｈ ) ２ Or (V Ｈ -V Ｌ ) ２ ), Triabody (trivalent antibody), Tetrabody (tetravalent antibody), Minibody ((scF Ｖ -C Ｈ 3) ２ ), IgG-delta-CH2, scFv-Fc, (scFv) ２ -Fc fragments, etc., may also be used.

[0080] Examples of commercially available anti-TDP-43 antibodies include the anti-TDP-43 antibody (Proteintech, 10782-2-AP). Examples of commercially available antibodies that recognize stress granule markers include the anti-G3BP1 antibody (Proteintech, 66486-1-Ig).

[0081] Specific examples of immunofluorescence staining include the following methods: An anti-TDP-43 antibody is used as the primary antibody. A labeled secondary antibody, labeled with a fluorescent substance capable of detecting the above anti-TDP-43 antibody, is used as the secondary antibody. Alternatively, an anti-TDP-43 antibody and an antibody that recognizes stress granule markers are used. As secondary antibodies, a labeled secondary antibody labeled with a first fluorescent substance capable of detecting the above anti-TDP-43 antibody, and a labeled secondary antibody labeled with a second fluorescent substance capable of detecting the antibody that recognizes the above stress granule markers are used, respectively. Different fluorescent substances are used for the first and second fluorescent substances.

[0082] As fluorescent substances, you can use fluorescent substances such as HiLyte dyes, fluorescein, dansyl, fluorescein camine, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, rhodamine X isothiocyanate, sulforodamine 101, Lucifer Yellow, acridine, acridine isothiocyanate, riboflavin, or derivatives thereof. Specific examples of fluorescent substances include HiLyte-based dyes such as HiLyte Fluor 647, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 680, HiLyte Fluor 750 (all trade names of HiLyte Bioscience, Inc.), Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye 488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555, Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye 700, and Alexa Fluor Dye Alexa-based dyes such as 750 (all trade names of Molecular Probes, Alexa Fluor is a registered trademark), and CyDye-based dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 (all trade names of Amersham Biosciences) can be used.

[0083] Nerve cells are reacted with a primary antibody and a labeled secondary antibody to generate a complex of TDP-43, the primary antibody, and the labeled secondary antibody, as well as a complex of a stress granule marker, the primary antibody, and the labeled secondary antibody.

[0084] When determining whether a marker is positive or negative by immunofluorescence staining, it is preferable to perform a step to distinguish between the cytoplasm and nucleus of nerve cells. Distinguishing between the cytoplasm and nucleus of nerve cells can be done by staining with a reagent that can stain the cell nucleus. Examples of reagents that can stain the cell nucleus include Hoechst® 33342 (Dojindo, 346-07951).

[0085] In one example of the present invention, after performing immunofluorescence staining and cell nucleus staining as described above, images of cells can be acquired. For example, by using image analysis software of a confocal quantitative image cytometer (e.g., Yokogawa Electric Corporation, CellVoyager CQ1) to recognize granular signals of the TDP-43 marker and stress granule marker (e.g., G3BP1), it is possible to determine whether the cytoplasmic TDP-43 marker is positive and whether the cytoplasmic stress granule marker is positive.

[0086] <Method for evaluating test substances, and method for screening for the prevention and / or treatment of neurodegenerative diseases> The nerve cells obtained according to the present invention, in which TDP-43 aggregation is induced in the cytoplasm, can be used for screening new drugs useful for neurodegenerative diseases in which TDP-43 aggregation is known to play a significant role in the onset and progression. The nerve cells obtained according to the present invention, in which TDP-43 aggregation is induced in the cytoplasm, are particularly useful for research on chronic neurodegenerative diseases, the development of therapeutic drugs for them, the identification of disease biomarkers, and the development of diagnostic agents. In the present invention, chronic neurodegenerative diseases can be evaluated by using nerve cells produced from pluripotent stem cells and culturing the nerve cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

[0087] The present invention provides a method for evaluating a test substance, comprising step B of culturing nerve cells differentiated from human-derived pluripotent stem cells in the presence of a test substance, and step C of culturing the nerve cells differentiated from the above-mentioned human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

[0088] In the above method for evaluating the test substance, step B may be performed first, followed by step C. In the above method for evaluating the test substance, steps B and C may be performed simultaneously. In the above method for evaluating the test substance, step C may be performed first, followed by step B.

[0089] The present invention further provides a method for screening for the prevention and / or treatment of neurodegenerative diseases, comprising step B of culturing nerve cells differentiated from human-derived pluripotent stem cells in the presence of a test substance, and step C of culturing the above-mentioned nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

[0090] In the above screening method, step B may be performed first, followed by step C. In the above screening method, steps B and C may be performed simultaneously. In the above screening method, step C may be performed first, followed by step B.

[0091] In one embodiment of the screening method of the present invention, the steps of culturing nerve cells that have been in contact with the test substance and control nerve cells that have not been in contact with the test substance are performed; measuring the localization amount of TDP-43 aggregation in the cytoplasm of the nerve cells; and selecting a test substance that suppresses the localization of TDP-43 aggregation in the cytoplasm compared with a control that has not been in contact with the test substance, as a candidate for the prevention and / or treatment of neurodegenerative diseases.

[0092] Examples of test substances include proteins, peptides, antibodies, nucleic acids (gene expression vectors, siRNA, antisense oligonucleotides, mRNA), viral vectors (AAV, lentivirus, adenovirus, etc.), non-peptide compounds, synthetic compounds, synthetic small molecule compounds, natural compounds, cell extracts, extracellular vesicles, plant extracts, animal tissue extracts, plasma, extracts derived from marine organisms, cell culture supernatants, and microbial fermentation products.

[0093] Furthermore, test substances can be obtained using any of the many combinatorial library methods known in the art, including (1) the biological library method, (2) the synthetic library method using deconvolution, (3) the one-bead-one-compound library method, and (4) the synthetic library method using affinity chromatography sorting. The biological library method using affinity chromatography sorting is mainly limited to peptide libraries, but the other approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of molecular library synthesis methods can be found in the art (DeWitt et al. (1993) Proc.Natl.Acad.Sci.USA 90: 6909-13; Erb et al. (1994) Proc.Natl.Acad.Sci.USA 91: 11422-6; Zuckermann et al. (1994) J.Med.Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew.Chem.Int.Ed.Engl. 33: 2059; Carell et al. (1994) Angew.Chem.Int.Ed.Engl. 33: 2061; Gallop et al. (1994) J.Med.Chem. 37: 1233-51).Compound libraries can be prepared in solution (see Houghten (1992) Bio / Techniques 13: 412-21), beads (Lam (1991) Nature 354: 82-4), tips (Fodor (1993) Nature 364: 555-6), bacteria (US Patent No. 5,223,409), spores (US Patent Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc.Natl.Acad.Sci.USA 89: 1865-9), or phages (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990)). It can be manufactured as Proc.Natl.Acad.Sci.USA 87: 6378-82; Felici (1991) J.Mol.Biol. 222: 301-10; U.S. Patent Application Publication No. 2002 / 0103360).

[0094] The process of culturing nerve cells in the presence of the test substance may be carried out by adding the test substance to the nerve cell culture medium. The culture time is not particularly limited as long as a change in the indicator can be confirmed, but for example, it may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. The concentration of the added test substance can be adjusted as appropriate depending on the type of compound (solubility, toxicity, etc.).

[0095] The culture medium used when culturing nerve cells in the presence of a test substance is not particularly limited, as long as it is a culture medium capable of culturing nerve cells.

[0096] The culture temperature when culturing nerve cells in the presence of the test substance is not particularly limited, but is about 30 to 40°C, preferably about 37°C, CO ２ The culture is carried out in an atmosphere containing CO ２ The concentration is preferably about 2-5%.

[0097] Neurodegenerative diseases include, but are not limited to, at least one selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), familial frontotemporal dementia-parkinsonism linked to chromosome 17 (FTDP-17), Paget's disease of bone and inclusion body myopathy with frontotemporal dementia (IBMPFD), Perry syndrome, Parkinson's disease (PD), Parkinson's dementia complex (PDC), familial British dementia (FBD), Huntington's disease (HD), Machado Joseph disease (MJD / SCA3), Alzheimer's disease (AD), Lewy body dementia (DLB), corticobasal degeneration (CBD), argyrophilic grain disease / argyrophilic grain dementia (AGD), diffuse neurofibrillary tangle disease with calcification (DNTC), and progressive supranuclear palsy (PSP).

[0098] Preferred neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Huntington's disease (HD), or Alzheimer's disease (AD), with amyotrophic lateral sclerosis (ALS) being more preferred.

[0099] The present invention will be specifically described by the following examples, but the present invention is not limited to the scope of these examples.

[0100] Test Example 1: Reproduction of TDP-43 aggregation by various protein synthesis inhibitors <Plate coating> iMatrix-511 silk (Matrixome, 892021) was diluted 166.7 times with PBS and added at a rate of 70 μL / well to a PDL-coated 96-well plate (Corning, 356640), and incubated at 37°C for 1 day.

[0101] <Cell Seeding> The following culture medium was prepared. Frozen stocks of nerve cells, produced by forced expression of the Ngn2 gene in iPS cells, were thawed in a 37°C warm bath. After thawing, the cells were added to the culture medium and centrifuged at 600 × g at room temperature for 5 minutes. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the number of cells was counted. Then, the cells were diluted with culture medium and rehydrated in 100 μL / well (5 × 10⁻⁶). ４ Seeds were sown in cells / well, at 37°C and 5% CO2. ２ The cells were cultured under specific conditions.

[0102]

[0103] <Stimulation with protein synthesis inhibitors and cell fixation> Three days after culturing nerve cells, various protein synthesis inhibitors were diluted in the culture medium and the cells were treated. Twenty-four hours after treatment, the cells were fixed by treating them for 30 minutes with a formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555).

[0104] <Immunofluorescence Staining> After fixing the cells, they were washed with PBS(-) and then blocked and permeabilized by treating them for 30 minutes with 1% and 0.2% solutions of BSA (Sigma Aldrich, A2153) and Triton X-100 solution (BioVision, 2104-100) diluted with PBS(-) (1% BSA / 0.2% Triton-X). Subsequently, as a primary antibody reaction, the cells were treated with 1000-fold diluted anti-TDP-43 antibody (Proteintech, 10782-2-AP) and 1000-fold diluted anti-G3BP1 antibody (Proteintech, 66486-1-Ig) in 1% BSA / 0.2% Triton-X, and left to stand overnight at 4°C.

[0105] The following day, after washing with PBS(-), the samples were treated with 1000-fold diluted Goat anti-rabbit Alexa Fluor® 488 (Thermo Fisher Scientific, A11008), 1000-fold diluted Goat anti-rabbit Alexa Fluor® 594 (Thermo Fisher Scientific, A11005), and 1000-fold diluted Hoechst® 33342 (Dojindo, 346-07951) as secondary antibody reactions, and left to stand at room temperature for 60 minutes. After washing with PBS(-), images were acquired using a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1).

[0106] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized, and the percentage of cells with signals co-localized by both signals was quantified. In addition, the number of viable cells was quantified by counting the Hoechst 33342 signal. In unstimulated neurons, granular TDP-43 signals and G3BP1 signals were hardly observed in the cytoplasm (Figure 1). On the other hand, in neurons stimulated with purocycin, an aminonucleoside-based protein synthesis inhibitor, at 10 μg / mL, granular signals of TDP-43 co-localized with G3BP1 signals were observed extranuclear (Figure 2).

[0107] Figure 3 shows the number of viable neurons stimulated with puromycin. Figure 4 shows the quantified percentage of neurons with signals exhibiting co-localization of TDP-43 and G3BP1 among the viable cells. Strong cell damage was observed with puromycin treatment at concentrations of 2 and 5 μg / mL (Figure 3). Compared to the untreated group (no stimulation), the viability rate was 5.1% in the 2 μg / mL stimulated group and 9.6% in the 5 μg / mL stimulated group. The viability rate in the 10 μg / mL stimulated group was 20.4%. Furthermore, strong TDP-43 aggregation was induced with puromycin treatment at concentrations of 10–40 μg / mL (Figure 4).

[0108] Figures 5 and 6 show the number of viable neurons stimulated with Blasticidin S, a peptidyl nucleoside-based protein synthesis inhibitor, and the percentage of viable neurons exhibiting signals with co-localization of TDP-43 and G3BP1. No strong cytotoxicity was observed at any concentration (Figure 5). Furthermore, treatment with Blasticidin S at concentrations of 10–20 μg / mL strongly induced TDP-43 aggregation (Figure 6).

[0109] Figures 7 and 8 show the number of viable neurons stimulated with G-418, an aminoglycoside-based protein synthesis inhibitor, and the percentage of viable neurons exhibiting signals with co-localization of TDP-43 and G3BP1. No strong cytotoxicity was observed at any concentration (Figure 7). Furthermore, weak TDP-43 aggregation was induced by G-418 treatment at concentrations of 1500–2000 μg / mL (Figure 8).

[0110] Figures 9 and 10 show the number of viable neurons stimulated with Hygromycin B, an aminoglycoside-based protein synthesis inhibitor, and the percentage of viable neurons exhibiting signals with co-localization of TDP-43 and G3BP1. No strong cytotoxicity was observed at any concentration (Figure 9). Furthermore, weak TDP-43 aggregation was induced by treatment with Hygromycin B at concentrations of 600–2000 μg / mL (Figure 10).

[0111] Figures 11 and 12 show the number of viable neurons stimulated with Cycloheximide, a glutarimide-based protein synthesis inhibitor, and the percentage of viable neurons exhibiting signals with co-localization of TDP-43 and G3BP1. No strong cytotoxicity was observed at any concentration (Figure 11). Furthermore, treatment with Cycloheximide at concentrations of 15–40 μmol / L induced weak TDP-43 aggregation (Figure 12).

[0112] Test Example 2: Reproduction of TDP-43 aggregation induced by protein synthesis inhibitors and hydrogen peroxide stimulation. Plate coating and cell seeding were performed in the same manner as in Test Example 1.

[0113] <Induction of TDP-43 Aggregation and Cell Fixation> Three days after culturing nerve cells, puromycin was added to the culture medium to a concentration of 10 μg / mL. Twenty-three hours after treatment, hydrogen peroxide was added to a final concentration of 1 mM. One hour after hydrogen peroxide treatment, cells were fixed by treating with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555) for 30 minutes. Immunofluorescence staining was performed in the same manner as in Test Example 1.

[0114] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized, and aggregation was observed. In addition, the nuclear region was recognized using the signal of Hoechst 33342, and the portion of the TDP-43 signal region excluding the nuclear region was recognized as cytoplasm. In unstimulated (non-stimulated) neurons, granular TDP-43 signals in the cytoplasm were hardly observed (Figure 13, left center, bottom left). Also, no strong signal of G3BP1 was observed (Figure 13, center top). On the other hand, in neurons stimulated with puromycin and hydrogen peroxide, many granular TDP-43 signals were observed extranuclear (Figure 14, bottom left white arrow). Also, many granular G3BP1 signals were observed extranuclear (Figure 14, center bottom white arrow). Furthermore, numerous granular signals of TDP-43 co-localized with G3BP1 outside the nucleus were observed (white arrow in the lower right of Figure 14). It was confirmed that the amount of granular signals of TDP-43 differed significantly depending on whether stimulation was present or not.

[0115] Test Example 3: Evaluation of the effect of compounds on TDP-43 aggregation induction. Plate coating and cell seeding were carried out in the same manner as in Test Example 1. The cells used were wild-type and nerve cells produced by forcing the expression of the Ngn2 gene in iPS cells heterozygously knocked out of the GRN gene.

[0116] <Stimulation with the evaluation compound> Ketoconazole (Tokyo Chemical Industries, Ltd., K0045) was added as the evaluation compound at concentrations of 1, 3, and 10 μM 30 minutes before stimulation with the protein synthesis inhibitor. Stimulation with the protein synthesis inhibitor, cell fixation, and immunofluorescence staining were performed in the same manner as in Test Example 1.

[0117] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized, and the percentage of cells with signals co-localized with both signals outside the nuclear region, and the number of co-localized signals per cell were quantified. As a result, it was confirmed that in both wild-type neurons and GRN hetero-knockout neurons, stimulation with a protein synthesis inhibitor increased the percentage of cells with signals in the cytoplasm and the number of extranuclear signals per cell, and that treatment with ketoconazole suppressed these increases in a concentration-dependent manner (Figures 15 and 16). This indicates that this evaluation system can be used to evaluate and screen for drugs that suppress the localization of TDP-43 aggregation, and that it is possible to select candidate drugs for the prevention and / or treatment of neurodegenerative diseases.

[0118] Test Example 4: Reproduction of TDP-43 aggregation that is resistant to degradation by protein synthesis inhibitors and hydrogen peroxide stimulation. Plate coating and cell seeding were carried out in the same manner as in Test Example 1.

[0119] <Induction of TDP-43 Aggregation and Cell Fixation> Three days after culturing nerve cells, puromycin was added to the culture medium and treated for 18 hours. In the group treated with hydrogen peroxide, hydrogen peroxide was added 17 hours after puromycin treatment to a final concentration of 1 mM and treated for 1 hour. After that, the stimulus was removed and the culture medium was added, and the cells were fixed after 0 to 4 hours. Cell fixation was performed by treating with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555) for 30 minutes. Immunofluorescence staining was performed in the same manner as in Test Example 1.

[0120] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized, and the percentage of cells with signals co-localized by both signals was quantified. In addition, the number of viable cells was quantified by counting the Hoechst 33342 signal. In samples fixed immediately after stimulation removal, the percentage of cells with granular signals of TDP-43 co-localized with G3BP1 signals extranuclear increased compared to unstimulated samples, whether stimulated with puromycin or puromycin and hydrogen peroxide (Figure 17 upper white arrow, Figure 18). In samples fixed 4 hours after stimulus removal, the granular signal of TDP-43 co-localized with the extranuclear G3BP1 signal disappeared in the group stimulated with Puromycin, but the signal remained in the group stimulated with Puromycin and hydrogen peroxide (Figure 17, lower white arrow; Figure 18). When the percentage of cells with granular signals of TDP-43 co-localized with the extranuclear G3BP1 signal was quantified 0 to 4 hours after stimulus removal, the percentage decreased over time in the group stimulated with Puromycin, while many cells in the group stimulated with Puromycin and hydrogen peroxide still possessed granular signals even after 4 hours (Figure 18). These results indicate that TDP-43 aggregation, which is less susceptible to degradation, can be induced by stimulation with a protein synthesis inhibitor and hydrogen peroxide.

[0121] Test Example 5: Treatment conditions for hydrogen peroxide to induce TDP-43 aggregation that is resistant to degradation. Plate coating and cell seeding were carried out in the same manner as in Test Example 1.

[0122] <Induction of TDP-43 Aggregation and Cell Fixation> Three days after culturing nerve cells, 20 μg / mL Puromycin was applied for 20 hours, or 0.01–3 mM hydrogen peroxide was applied simultaneously with Puromycin for 10–180 minutes, under the conditions shown in Figure 19. After removing the stimulus, culture medium was added, and cells were fixed at 0 and 3 hours. Cell fixation was performed by treating with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555) for 30 minutes. Immunofluorescence staining was performed in the same manner as in Test Example 1.

[0123] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized. Cells exhibiting co-localized signals of both signals outside the nuclear region were identified as cells in which TDP-43 aggregation was induced, and their proportion was quantified (Figures 20A-E). As a result, in the group that received neither puromycin nor hydrogen peroxide stimulation (no stimulation), there were almost no cells exhibiting TDP-43 aggregation. On the other hand, in the group stimulated with puromycin alone (0 mM), TDP-43 aggregation was observed in more than 40% of cells under all conditions at 0 hours after stimulation removal, but at 3 hours after stimulation removal, the proportion of cells showing TDP-43 aggregation decreased to less than 30% under all conditions (Figures 20A-E). On the other hand, under conditions where hydrogen peroxide was treated for 10 to 60 minutes (Figures 20A-C), the percentage of cells showing TDP-43 aggregation 3 hours after removal of stimulation was 30% or more, even under conditions where the concentration exceeded 0.01 mM. This indicates that TDP-43 aggregation, which is resistant to degradation, can be induced by stimulating cells with puromycin and hydrogen peroxide at concentrations exceeding 0.01 mM. Under conditions where hydrogen peroxide was treated for 120 to 180 minutes (Figures 20D, E), the percentage of cells showing TDP-43 aggregation was 30% or more when stimulated with puromycin and hydrogen peroxide at concentrations exceeding 0.01 mM. In other words, it was confirmed that TDP-43 aggregation, which is resistant to degradation, can be induced by stimulating cells with puromycin and hydrogen peroxide. On the other hand, high concentrations of hydrogen peroxide caused cytotoxicity, resulting in a decrease in the percentage of cells showing aggregation 3 hours after removal of stimulation (Figures 20D, E).

[0124] Test Example 6: Treatment conditions with hydrogen peroxide to induce TDP-43 aggregation that is resistant to degradation (1, 5, 240 minutes) Plate coating and cell seeding were performed in the same manner as in Test Example 1.

[0125] <Induction of TDP-43 Aggregation and Cell Fixation> Three days after culturing nerve cells, 20 μg / mL Puromycin was applied for 21 hours, or 0.01–3 mM hydrogen peroxide was applied simultaneously with Puromycin for 1–240 minutes, under the conditions shown in Figure 21. After removing the stimulus, culture medium was added, and cells were fixed at 0 and 3 hours. Cell fixation was performed by treating with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555) for 30 minutes. Immunofluorescence staining was performed in the same manner as in Test Example 1.

[0126] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized. Cells exhibiting co-localized signals of both signals outside the nuclear region were identified as cells in which TDP-43 aggregation was induced, and their proportion was quantified (Figures 22A-C). As a result, in the group that received neither puromycin nor hydrogen peroxide stimulation (no stimulation), there were almost no cells exhibiting TDP-43 aggregation. On the other hand, in the group stimulated with puromycin alone (0 mM), TDP-43 aggregation was observed in more than 40% of cells under all conditions at 0 hours after stimulation removal, but at 3 hours after stimulation removal, the proportion of cells showing TDP-43 aggregation decreased to less than 30% under all conditions (Figures 22A-C). On the other hand, under conditions where hydrogen peroxide was treated for 1 minute (Figure 22A), at concentrations exceeding 0.5 mM, the percentage of cells showing TDP-43 aggregation 3 hours after removal of stimulation was 30% or more. Under conditions where hydrogen peroxide was treated for 5 minutes (Figure 22B), at concentrations exceeding 0.5 mM, the percentage of cells showing TDP-43 aggregation 3 hours after removal of stimulation was 30% or more. Under conditions where hydrogen peroxide was treated for 240 minutes (Figure 22C), at concentrations of 0.05 mM, 0.1 mM, and 0.5 mM, the percentage of cells showing TDP-43 aggregation 3 hours after removal of stimulation was 30% or more. These results indicate that stimulating with hydrogen peroxide in addition to puromycin can induce TDP-43 aggregation that is less susceptible to degradation.

[0127] Test Example 7: Induction of TDP-43 aggregation that is resistant to degradation by puromycin and RSL3 stimulation, plate coating, and cell seeding were performed in the same manner as in Test Example 1.

[0128] <Induction of TDP-43 Aggregation and Cell Fixation> Three days after culturing nerve cells, they were treated with 20 μg / mL Puromycin for 20 hours, or with 0.3 μM RSL3 for 180 minutes to induce oxidative stress simultaneously with Puromycin, under the conditions shown in Figure X. After that, the stimulation was removed and culture medium was added, and the cells were fixed at 0 and 3 hours. Cell fixation was performed by treating with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555) for 30 minutes. Immunofluorescence staining was performed in the same manner as in Test Example 1.

[0129] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized. Cells exhibiting co-localized signals of both signals outside the nuclear region were identified as cells in which TDP-43 aggregation was induced, and their proportion was quantified (Figure 23). As a result, in the group that did not receive stimulation (no stimulation), there were almost no cells exhibiting TDP-43 aggregation. On the other hand, in the group stimulated with puromycin alone, TDP-43 aggregation was observed in more than 40% of cells at 0 hours after stimulation removal, but at 3 hours after stimulation removal, the proportion of cells showing TDP-43 aggregation decreased to less than 30% (Figure 23). On the other hand, under conditions where cells were treated with 0.3 μM RSL3 in addition to puromycin for 180 minutes, the percentage of cells showing TDP-43 aggregation was over 30% three hours after stimulation removal. This indicates that stimulating cells with RSL3 in addition to puromycin can induce TDP-43 aggregation that is less susceptible to degradation.

[0130] Test Example 8: Reproduction of TDP-43 aggregation using nerve cell / astrocyte co-culture system and nerve cell / astrocyte / microglia ternary co-culture system <Preparation of nerve cell / astrocyte co-culture system and nerve cell / astrocyte / microglia ternary co-culture system> Matrigel basement membrane matrix (Corning, 356234), diluted 120-fold with DMEM / F12 (Life Technologies, 11320-033), was added to a 96-well plate at 65 μL / well and left to stand at 4°C. After 24 hours, it was replaced with progenitor cell medium and used. Astrocyte progenitor cells (XCell Science, XCS-AP-001-1V) were added to the 96-well plate in a 5 × 10⁶ layer. ４ Sow at a density of cells / well, at 37°C and 5% CO2. ２ The cells were cultured under specific conditions. The following day, the culture medium was replaced with differentiation induction medium, and the cells were cultured for a further 5 days to induce differentiation into astrocytes. For nerve cells, we used nerve cells that were produced by forcing the expression of the Ngn2 gene from iPS cells and then cryopreserved. The cryopreserved cells were thawed in a 37°C bath, and after thawing, the cells were added to co-culture medium and centrifuged at 600 x g at room temperature for 5 minutes. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of co-culture medium, and the number of cells was counted. For microglia, we used iPS cell-derived microglia (iCell microglia, FCDI, C1110). The cryopreserved cells were thawed in a 37°C bath, and after thawing, the cells were added to co-culture medium and centrifuged at 600 x g at room temperature for 5 minutes. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of co-culture medium, and the number of cells was counted. Nerve cells and microglia were mixed in a 3:1 ratio, and 6 x 10⁶ cells were added. ４ Seeds were sown on astrocytes to achieve a cells / well ratio. 37°C, 5% CO2 ２ The cells were cultured under specific conditions, and the culture medium was changed once a week.

[0131]

[0132] <Induction of TDP-43 Aggregation and Cell Fixation> After two weeks of culture, puromycin was diluted in culture medium and added to the cells for 18 hours, after which the cells were fixed. In the group treated with hydrogen peroxide, hydrogen peroxide was added 17 hours after puromycin treatment to a final concentration of 1 mM, and the cells were fixed after 1 hour. Cell fixation was performed by treating the cells with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555) for 30 minutes. Immunofluorescence staining was performed in the same manner as in Test Example 1.

[0133] <Evaluation of TDP-43 Aggregation> When nerve cell / astrocyte co-culture systems were stimulated with puromycin, or puromycin and hydrogen peroxide, cells with granular TDP-43 signals co-localized with G3BP1 signals extranuclear were observed (Figure 24, white arrow). Similarly, in nerve cell / astrocyte / microglia tripartite co-culture systems, stimulation with puromycin and hydrogen peroxide also allowed for the observation of cells with granular TDP-43 signals co-localized with G3BP1 signals extranuclear (Figure 25, white arrow). These findings demonstrate that TDP-43 aggregation can be induced in nerve cell / astrocyte co-culture systems and nerve cell / astrocyte / microglia tripartite co-culture systems by stimulating them with protein synthesis inhibitors, or with protein synthesis inhibitors and hydrogen peroxide.

Claims

1. A method for producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm, comprising step A, of culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

2. The method for producing a protein according to claim 1, wherein the protein synthesis inhibitor is at least one selected from the group consisting of aminonucleoside antibiotics, peptidyl nucleoside antibiotics, aminoglycoside antibiotics, and glutarimide antibiotics.

3. The manufacturing method according to claim 2, wherein the aminonucleoside antibiotic is puromycin.

4. The manufacturing method according to claim 2, wherein the peptidyl nucleoside antibiotic is Blasticidin S.

5. The manufacturing method according to claim 2, wherein the aminoglycoside antibiotic is at least one selected from the group consisting of G-418 and Hygromycin B.

6. The manufacturing method according to claim 2, wherein the glutarimide antibiotic is cycloheximide.

7. The method for producing a protein synthesis inhibitor according to claim 1, wherein the protein synthesis inhibitor is puromycin, and the concentration that does not induce cell death is greater than 5 μg / mL and 400 μg / mL.

8. The method for producing the protein synthesis inhibitor according to claim 1, wherein the protein synthesis inhibitor is blasticidin S, and the concentration that does not induce cell death is greater than 1 μg / mL and 400 μg / mL.

9. The method for producing the protein synthesis inhibitor according to claim 1, wherein the protein synthesis inhibitor is hygromycin B, and the concentration that does not induce cell death is greater than 300 μg / mL and 20 mg / mL.

10. The method for producing the protein synthesis inhibitor according to claim 1, wherein the protein synthesis inhibitor is G-418, and the concentration that does not induce cell death is greater than 600 μg / mL and less than 20 mg / mL.

11. The method for producing the protein synthesis inhibitor according to claim 1, wherein the protein synthesis inhibitor is cycloheximide, and the concentration that does not induce cell death is greater than 10 μmol / L and less than 400 μmol / L.

12. The manufacturing method according to any one of claims 1 to 11, wherein the culture time in step A is 2 hours to 72 hours.

13. The manufacturing method according to any one of claims 1 to 11, wherein step A includes step A1 of adding an oxidative stress inducer to the culture medium and then culturing.

14. The manufacturing method according to any one of claims 1 to 11, wherein the oxidative stress inducer is at least one selected from the group consisting of hydrogen peroxide and ferroptosis inducers.

15. The manufacturing method according to any one of claims 1 to 11, wherein step A includes step A1, in which hydrogen peroxide is added to the culture medium at a concentration of more than 0.01 mmol / L to 5.0 mmol / L, and then cultured.

16. The manufacturing method according to any one of claims 1 to 11, wherein step A includes step A1 of adding hydrogen peroxide to the culture medium at a concentration of 0.5 mmol / L to 2.0 mmol / L and then culturing.

17. The manufacturing method according to claim 13, wherein the culture time in step A1 is 1 minute to 360 minutes.

18. The manufacturing method according to claim 13, wherein the culture time in step A1 is 30 minutes to 60 minutes.

19. The manufacturing method according to any one of claims 1 to 11, wherein the nerve cells are motor nerve cells, cerebral cortical excitatory nerve cells, or substantia nigra nerve cells.

20. The manufacturing method according to any one of claims 1 to 11, further comprising step B of co-culturing the nerve cells with at least one selected from the group consisting of astrocytes and microglia.

21. A method for inducing TDP-43 aggregation in the cytoplasm of nerve cells, comprising step A, of culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

22. A cell population obtained by the method of any one of claims 1 to 11, comprising nerve cells satisfying at least one of the following (i) to (ii): (i) positive for cytoplasmic stress granule markers; (ii) positive for cytoplasmic TDP-43 markers; wherein the cell viability is 20% or more compared to an untreated cell population.

23. A cell population comprising nerve cells obtained by the method of any one of claims 1 to 11, wherein at least one of the following (i) to (ii): (i) positive for cytoplasmic stress granule markers; (ii) positive for cytoplasmic TDP-43 markers; wherein 50% or more of the living cells included in the cell population satisfy at least one of (i) and (ii).

24. A method for evaluating a test substance, comprising step B of culturing nerve cells differentiated from human-derived pluripotent stem cells in the presence of a test substance, and step C of culturing the nerve cells differentiated from the human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

25. The evaluation method according to claim 24, wherein step B is performed, and then step C is performed, step B and step C are performed simultaneously, or step C is performed, and then step B is performed.

26. A method for screening for the prevention and / or treatment of neurodegenerative diseases, comprising step B of culturing nerve cells differentiated from human-derived pluripotent stem cells in the presence of a test substance, and step C of culturing the nerve cells differentiated from the human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.

27. The screening method according to claim 26, wherein step B is performed, and then step C is performed, step B and step C are performed simultaneously, or step C is performed, and then step B is performed.

28. The screening method according to claim 26 or 27, comprising the steps of: culturing nerve cells that have been in contact with the test substance and control nerve cells that have not been in contact with the test substance; measuring the localization of TDP-43 aggregation in the cytoplasm of the nerve cells; and selecting a test substance that suppresses the localization of TDP-43 aggregation in the cytoplasm compared with a control that has not been in contact with the test substance, as a candidate for the prevention and / or treatment of neurodegenerative diseases.

29. The screening method according to claim 26 or 27, wherein the neurodegenerative disease is at least one selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, familial frontotemporal dementia parkinsonism linked to chromosome 17, Paget's disease of bone and inclusion body myopathy with frontotemporal dementia, Perry syndrome, Parkinson's disease, Parkinson's dementia complex, familial British dementia, Huntington's disease, Machado-Joseph disease, Alzheimer's disease, Lewy body dementia, corticobasal degeneration, argyrophilic granule disease / argyrophilic granule dementia, diffuse neurofibrillary tangle disease with calcification, and progressive supranuclear palsy.

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