Method for producing nerve cells damaged by oxidative stress, and use thereof

Abstract

The present invention addresses the problem of providing: a method for producing nerve cells damaged by oxidative stress from human-derived pluripotent stem cells; a method for culturing cells capable of producing nerve cells damaged by oxidative stress from human-derived pluripotent stem cells; damaged nerve cells subjected to oxidative stress; a method for evaluating a test substance using the nerve cells; a method for screening a drug for preventing and / or treating neurodegenerative diseases; a method for screening a necroptosis inhibitor; and a method for screening a ferroptosis inhibitor. The present invention provides a method for producing nerve cells damaged by oxidative stress, the method comprising: a step a for inoculating nerve cells induced by differentiation from human-derived pluripotent stem cells at a cell density of 20.0 * 104 cells / cm2 or less using a culture medium that substantially does not contain an antioxidant and substantially does not contain an oxidant; and a step b for culturing using a culture medium that substantially does not contain an antioxidant and substantially does not contain an oxidant.

Description

Technical Field

[0001] The present invention relates to a method for producing and culturing neural cells damaged by oxidative stress. The present invention also relates to neural cells damaged by oxidative stress. The present invention also relates to a method for evaluating a test substance using the neural cells, a method for screening drugs for preventing and / or treating neurodegenerative diseases, a method for screening necroptosis inhibitors, and a method for screening ferroptosis inhibitors. Background Art

[0002] Neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are diseases in which nerve cells are gradually damaged and fall off (non-patent documents 1 and 2), and oxidative stress is known to play a major role in their pathological mechanisms (non-patent documents 3 and 4). In in vitro studies of human neurodegenerative diseases, it is difficult to obtain human central nervous system cells, so rat or mouse cells have been used in the past. However, in recent years, since the invention of artificial pluripotent stem cells (iPS cells), human central nervous system cells containing nerve cells can be obtained relatively easily, which is of great help to the study of human neurodegenerative diseases (non-patent documents 5 to 7).

[0003] The effects of oxidative stress on neurodegenerative diseases are being studied using iPS cell-derived neurons. For example, in a study by Feng-Lan Chiu et al., oxidative stress was applied to iPS cell-derived neurons by treating them with hydrogen peroxide for 6 hours, reproducing the neuronal cell damage of neurodegenerative diseases (Non-Patent Document 8). In addition, Patent Document 1 describes the use of arsenite for 16 hours to treat motor nerves made from iPS cells derived from familial ALS patients to induce damage. Patent Document 2 describes the use of a short exposure (transient treatment) of hydrogen peroxide to motor nerve cells made from human iPS cells to cause cell damage. However, neurodegenerative diseases are diseases in which nerve cells gradually fall off over a long period of time. In contrast, under the conditions of using oxidants such as hydrogen peroxide, due to the induction of nerve damage by a short period of oxidative stress stimulation, it is possible that the actual neurodegenerative disease that occurs in vivo may not be reproduced.

[0004] Therefore, there is the research (non-patent literature 9) of reproducing the nerve cell damage of neurodegenerative disease by chronically applying weak oxidative stress. In this study, it is believed that only when applying weak oxidative stress and knocking out the gene related to the disease, nerve damage will be induced, therefore familial neurodegenerative disease is reproduced. In addition, in patent documentation 3 and non-patent literature 10, it is recorded that the cerebral cortex nerve cells made by familial AD patient source iPS cells are enhanced by cultivating in a culture medium without antioxidants, thereby inducing the content of nerve cell damage.

[0005] However, it is known that 95% of neurodegenerative diseases, such as AD, are sporadic (Non-Patent Document 11) and 90% of ALS are sporadic (Non-Patent Document 12), and that the majority of patients do not have mutations in disease-related genes. Therefore, there has been a demand for an evaluation system that can easily replicate the course of sporadic neurodegenerative diseases, but sufficient progress has been made.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application No. 2015-506905

[0009] Patent Document 2: Japanese Patent Application Publication No. 2019-037245

[0010] Patent Document 3: International Publication No. WO2013 / 140327

[0011] Non-patent literature

[0012] Non-patent literature 1: Brittany N Dugger et al., Cold Spring Harbor Perspectives in Biology, Vol. 9, Article No. a028035, 2017

[0013] Non-patent document 2: Hao Chi et al., International Journal of Molecular Sciences, Vol. 19, Article No. 3082, 2018

[0014] Non-patent literature 3: Giovanna Cenini et al., Oxidative Medicine and Cellular Longevity, Vol. 2019, Article No. 2105607, 2019

[0015] Non-patent literature 4: Vanessa Castelli et al., Frontiers in Molecular Neuroscience, Vol. 12, Article No. 132, 2019

[0016] Non-patent document 5: Okano et al., Molecular Brain, Vol. 7, No. 22, pp. 1-12, 2014

[0017] Non-patent document 6: Valadez-Barba et al., Regenerative Therapy, Vol. 15, pp. 332-339, 2020

[0018] Non-patent document 7: Zeng et al., STEM CELLS TRANSLATIONAL MEDICINE, Vol. 3, pp. 1418-1428, 2014

[0019] Non-patent document 8: Feng-Lan Chiu et al., Human Molecular Genetics, Vol. 24, pp. 6066-6079, 2015

[0020] Non-patent literature 9: Ruilin Tian et al., Nature Neuroscience, Vol. 24, pp. 1020-1034, 2021

[0021] Non-Patent Document 10: Kondo et al., Cell Stem Cell, Vol. 12, pp. 487-496, 2013

[0022] Non-patent document 11: Jitin Bali et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, pp. 15307-15311, 2012

[0023] Non-patent document 12: Francois Gros-Louis et al., Biochimica et Biophysica Acta, Vol. 1762, pp. 956-972, 2006 Summary of the Invention

[0024] Technical issues to be solved by the invention

[0025] The present invention aims to provide a method for producing oxidatively damaged neural cells from human pluripotent stem cells, and a cell culture method capable of producing oxidatively damaged neural cells from human pluripotent stem cells. The present invention also aims to provide neural cells damaged by oxidative stress. The present invention also aims to provide a method for evaluating a test substance using the aforementioned neural cells, a method for screening drugs for preventing and / or treating neurodegenerative diseases, a method for screening necroptosis inhibitors, and a method for screening ferroptosis inhibitors.

[0026] Means for solving technical problems

[0027] The present inventors have conducted intensive research to solve the above problems and have found that the use of a culture medium containing substantially no antioxidant and substantially no oxidant at 20.0×10 4 cells / cm 2 The present invention was completed based on this finding by seeding neural cells differentiated from human pluripotent stem cells at the following cell density and culturing them in a culture medium that contains substantially no antioxidants and substantially no oxidants, thereby producing neural cells damaged by oxidative stress.

[0028] That is, according to the present invention, the following inventions can be provided.

[0029] <1> A method for producing nerve cells damaged by oxidative stress, comprising: step a, using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant at 20.0×10 4 cells / cm 2 The neural cells differentiated from human pluripotent stem cells are seeded at the following cell density; and in step b, culture is performed using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant.

[0030] <2> The production method according to <1>, wherein

[0031] The cell density was 0.2×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 the following.

[0032] <3> The production method according to <1> or <2>, wherein

[0033] Human pluripotent stem cells are pluripotent stem cells that do not have mutations in disease-related genes.

[0034] <4> The production method according to any one of <1> to <3>, wherein

[0035] The above-mentioned nerve cells are motor nerve cells, cerebral cortex excitatory nerve cells or substantia nigra nerve cells.

[0036] <5> The production method according to any one of <1> to <4>, wherein

[0037] The nerve cells damaged by oxidative stress satisfy at least one of the following (i) to (iv):

[0038] (i) Markers related to oxidative stress were positive;

[0039] (ii) neurite retraction;

[0040] (iii) induced necroptosis; and

[0041] (iv) Induced ferroptosis.

[0042] <6> A cell culture method comprising: step a, using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant at 20.0×10 4 cells / cm 2 The neural cells differentiated from human pluripotent stem cells are seeded at the following cell density; and in step b, culture is performed using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant.

[0043] <7> The culture method according to <6>, wherein

[0044] The cell density is 0.2×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 the following.

[0045] <8> The culture method according to <6> or <7>, wherein

[0046] Human pluripotent stem cells are pluripotent stem cells that do not have mutations in disease-related genes.

[0047] <9> The culture method according to any one of <6> to <8>, wherein

[0048] The above-mentioned nerve cells are motor nerve cells, cerebral cortex excitatory nerve cells or substantia nigra nerve cells.

[0049] <10> A neural cell obtained by the production method described in <1>, which satisfies at least one of the following (i) to (iv):

[0050] (i) Markers related to oxidative stress were positive;

[0051] (ii) neurite length retraction;

[0052] (iii) induced necroptosis; and

[0053] (iv) Induced ferroptosis.

[0054] <11> The cell according to <10>, wherein

[0055] The above-mentioned nerve cells are motor nerve cells, cerebral cortex excitatory nerve cells or substantia nigra nerve cells.

[0056] <12> A method for evaluating a test substance, comprising the step of contacting the neural cell according to <10> with the test substance.

[0057] <13> The method for evaluating a test substance according to <12>, further comprising the step of producing the neural cell according to <10> by the method according to <1>, and contacting the neural cell with the test substance.

[0058] <14> A method for screening a drug for preventing and / or treating a neurodegenerative disease, comprising the step of contacting the neural cell according to <10> with a test substance.

[0059] <15> The screening method according to <14>, further comprising the step of producing the neural cell according to <10> by the method according to <1>, and contacting the neural cell with a test substance.

[0060] <16> The screening method according to <14>, wherein

[0061] The neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), spinocerebellar atrophy, frontotemporal lobar degeneration (FTLD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lewy body disease, Huntington's disease, and Niemann-Pick disease.

[0062] <17> The screening method according to <14>, wherein

[0063] After the nerve cells described in <10> are brought into contact with the test substance, the following steps are included:

[0064] (A) Culturing neural cells exposed to the test substance and control neural cells not exposed to the test substance;

[0065] (B) measuring nerve damage in the nerve cells; and

[0066] (C) A test substance that suppresses the above-mentioned nerve damage compared to a control not exposed to the test substance is selected as a candidate for a preventive and / or therapeutic drug for a neurodegenerative disease.

[0067] <18> The screening method according to <17>, wherein

[0068] The step (B) is a step of measuring the number of nerve cells and / or the length of neurites obtained in the step (A).

[0069] <19> The method according to <17> or <18>, wherein

[0070] The step (C) is a step of selecting a test substance whose cell number and / or neurite length in neurons contacted with the test substance is higher than that of the control as a candidate for a preventive and / or therapeutic drug for a neurodegenerative disease.

[0071] <20> A method for screening a necroptosis inhibitor, comprising the following steps:

[0072] (1) contacting the nerve cells described in <10> with a test substance;

[0073] (2) culturing the neural cells contacted with the test substance in the step (1) and the neural cells of the control group that have not been contacted with the test substance;

[0074] (3) measuring nerve damage in the nerve cells; and

[0075] (4) A test substance that inhibits the above-mentioned nerve damage compared to a control not exposed to the test substance is selected as a candidate necroptosis inhibitor.

[0076] <21> The screening method according to <20>, further comprising the step of producing the neural cell according to <10> by the method according to <1>, and contacting the neural cell with a test substance.

[0077] <22> A method for screening ferroptosis inhibitors, comprising the following steps:

[0078] (1) contacting the nerve cells described in <10> with a test substance;

[0079] (2) culturing the neural cells contacted with the test substance in the step (1) and the neural cells of the control group that have not been contacted with the test substance;

[0080] (3) measuring nerve damage in the nerve cells; and

[0081] (4) A test substance that inhibits the above-mentioned nerve damage compared to a control that has not been exposed to the test substance is selected as a candidate ferroptosis inhibitor.

[0082] <23> The screening method according to <22>, further comprising the step of producing the neural cell according to <10> by the method according to <1>, and contacting the neural cell with a test substance.

[0083] Effects of the Invention

[0084] According to the present invention, neural cells damaged by oxidative stress can be produced from human pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 The graph shows the results of quantification over time of neurite length of neurons in which the medium was replaced after 48 hours of culture.

[0086] Figure 2 The graph shows the results of quantification over time of neurite length of neurons whose culture medium was replaced after 72 hours of culture.

[0087] Figure 3 The graph shows the results of quantification over time of neurite length of neurons whose culture medium was replaced after 96 hours of culture.

[0088] Figure 4 Indicates the 4 cells / cm 2 The results of quantification of neurite length over time at the time of seeding at different cell seeding densities.

[0089] Figure 5 Indicates the 4 cells / cm 2 The results of quantification of neurite length over time at the time of seeding at different cell seeding densities.

[0090] Figure 6 Indicates that 1.9×10 4 cells / cm 2 The results of quantification of neurite length over time at the time of seeding at different cell seeding densities.

[0091] Figure 7 Indicates that 4.7×10 4 cells / cm 2 The results of quantification of neurite length over time at the time of seeding at different cell seeding densities.

[0092] Figure 8 Indicates that 9.4×10 4 cells / cm 2 The results of quantification of neurite length over time at the time of seeding at different cell seeding densities.

[0093] Figure 9Indicates the ratio of 12.5×10 4 cells / cm 2 The results of quantification of neurite length over time at the time of seeding at different cell seeding densities.

[0094] Figure 10 The graph shows the results of quantification over time of the neurite length of neurons cultured using BrainPhys medium as a basal medium.

[0095] Figure 11 The graph shows the results of quantification over time of the neurite length of neurons cultured using Neurobasal medium as a basal medium.

[0096] Figure 12 The results show the quantitative results of the LDH (lactate dehydrogenase) levels in the culture medium from the 3rd to the 6th day of culture.

[0097] Figure 13 The graph shows the results of quantification of ROS (reactive oxygen species) in nerve cells after culturing for 6 days.

[0098] Figure 14 Shown are the results of time-dependent quantification of neurite length in neurons treated with a necroptosis inhibitor (necrostatin-1) and images of representative cells. Figure 14 (A) shows the temporal changes in neurite length. Figure 14 (B) to Figure 14 (D) shows the image on the 6th day of culture. Figure 14 (B) indicates Stress(-), Figure 14 (C) indicates Stress(+), Figure 14 (D) indicates 20 μM necrostatin-1.

[0099] Figure 15 The graph shows the results of quantification of neurite length over time in neurons treated with a ferroptosis inhibitor (ferrostatin-1).

[0100] Figure 16 The graph shows the results of quantification of neurite length over time in neurons treated with a ferroptosis inhibitor (lipostatin-1).

[0101] Figure 17 The graph shows the results of quantification of neurite length over time in neurons treated with a ferroptosis inhibitor (UAMC-3203).

[0102] Figure 18 The graph shows the results of calculating the area under the curve (AUC) from the 14-day time curve of neurite length in nerve cells after treatment with various compounds of the compound library.

[0103] Figure 19 The graph shows the results of quantification of neurite length over time in neurons after treatment with an ALS therapeutic drug (edaravone). DETAILED DESCRIPTION

[0104] Hereinafter, embodiments of the present invention will be described in detail.

[0105] The present invention relates to a method for producing nerve cells damaged by oxidative stress, comprising: step a, using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant at a temperature of 20.0×10 4 cells / cm 2 The invention relates to a method for seeding neural cells differentiated from human pluripotent stem cells at the following cell density; and step b) culturing the cells using a culture medium that is substantially free of antioxidants and substantially free of oxidants. Specifically, the invention relates to a method for producing neural cells damaged by oxidative stress by chronically subjecting neural cells produced from human pluripotent stem cells to weak oxidative stress.

[0106] The present invention also relates to a cell culture method comprising: step a, using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant at a temperature of 20.0×10 4 cells / cm 2 The invention relates to a method for seeding neural cells differentiated from human pluripotent stem cells at the following cell density; and step b) culturing the cells using a culture medium that is substantially free of antioxidants and substantially free of oxidants. Specifically, the invention relates to a method for culturing neural cells damaged by oxidative stress by chronically applying weak oxidative stress to the neural cells differentiated from human pluripotent stem cells.

[0107] In the method for producing neural cells and the method for culturing cells according to the present invention, the cell density at the time of seeding is only 20.0×10 4 cells / cm 2 The following is sufficient. Conventionally, inducing neural damage caused by oxidative stress required the addition of exogenous oxidants or the use of neural cells derived from patients harboring mutations in disease-related genes. The present invention discovered that even neural cells generated from iPS cells derived from subjects without mutations in disease-related genes can achieve a cell density sufficient to induce cellular damage caused by chronic oxidative stress.

[0108] From the perspective of using the cells obtained by the production method and cell culture method of the present invention for evaluating a test substance, the cell density at the time of seeding is preferably set within a range that allows determination of the presence or absence of cell damage caused by chronic oxidative stress. The lower limit of the cell density is not particularly limited, but is preferably 0.2 × 10 4cells / cm 2 More than 0.3×10 4 cells / cm 2 More preferably, 1.0×10 4 cells / cm 2 More than 2.0×10 4 cells / cm 2 Above, particularly preferably 3.0×10 4 cells / cm 2 More than, most preferably 4.0×10 4 cells / cm 2 The upper limit of cell density can be, for example, 20.0×10 4 cells / cm 2 Below, preferably less than 20.0×10 4 cells / cm 2 , more preferably 12.5×10 4 cells / cm 2 Below, more preferably 12.0×10 4 cells / cm 2 Below, more preferably 11.0×10 4 cells / cm 2 Below, particularly preferably 10.0×10 4 cells / cm 2 The cell density is preferably 0.2×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 Below, more preferably 0.3×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 Below, more preferably 1.0×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 Below, more preferably 3.0×10 4 cells / cm 2 Above and 12.5×10 4 cells / cm 2 Below, particularly preferably 4.0×10 4 cells / cm 2 Above and 10.0×10 4 cells / cm 2 the following.

[0109] Examples of human pluripotent stem cells include human iPS cells (human induced pluripotent stem cells), human ES cells (human embryonic stem cells), and human mesenchymal stem cells. Human iPS cells are preferred, but are not particularly limited. Human iPS cells are iPS cells produced from human cells.

[0110] Human pluripotent stem cells are preferably those without 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 a gene has a mutation, as long as the mutation is not a cause of the disease, it is interpreted as the absence of a disease-related gene mutation.

[0111] ES cells can be established, for example, by culturing early preimplantation embryos, inner cell masses constituting the early embryos, single blastomeres, and the like (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press: Mouse Embryo Manipulation Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, (1994); Thomson, JA et al.: Science, 282, 1145-1147 (1998)). As early embryos, those produced by nuclear transfer of somatic cell nuclei can be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Iritani et al. (Protein Nuclease, 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)). As early embryos, parthenogenetic embryos can be used (Kim et al. (Science, 315, 482-486 (2007)), Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cells, 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 papers published above, for the production of ES cells, please refer to 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, PM et al., Methods in Enzymology, Vol. 365, 2003, etc.

[0112] Furthermore, fused 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.

[0113] ES cells can also be obtained from storage institutions or commercially available. For example, human ES cells can be obtained from the Institute of Regenerative Medicine, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0114] iPS cells are cells with pluripotency (multiple differentiation capabilities) and proliferation capacity produced by reprogramming somatic cells through the introduction of reprogramming factors. iPS cells exhibit properties similar to ES cells. The somatic cells used in the production of iPS cells are not particularly limited and can be differentiated somatic cells or undifferentiated stem cells. iPS cells can be produced using known methods, etc. Furthermore, it is also conceivable that future iPS cell production methods will be applicable.

[0115] The most basic method for producing iPS cells is to use viruses to introduce four transcription factors, namely Oct3 / 4, Sox2, Klf4, and c-Myc, into cells (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi K et al.: Cell 131(5), 861-72, 2007). Regarding human iPS cells, there have been reports of the establishment of cells by introducing four factors, namely Oct4, Sox2, Lin28, and Nonog (Yu J et al.: Science 318(5858), 1917-1920, 2007). There are also reports on the establishment of iPS cells by introducing three factors other than c-Myc (Nakagawa M et al.: Nat. Biotechnol. 26(1), 101-106, 2008), by introducing two factors, Oct3 / 4 and Klf4 (Kim JB et al.: Nature 454(7204), 646-650, 2008), or by introducing only Oct3 / 4 (Kim JB et al.: Cell 136(3), 411-419, 2009). In addition, there are also reports on methods for introducing a protein, which is a gene expression product, into cells (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 also reports that the production efficiency can be improved or the introduced factors can be reduced by using BIX-01294, an inhibitor of histone methyltransferase G9a, or valproic acid (VPA) or BayK8644, an inhibitor of histone deacetylase (Huangfu D et al.: Nature Biotechnology 26 (7), 795-797, 2008; Huangfu D et al.: Nature Biotechnology 26 (11), 1269-1275, 2008; Silva J et al.: PLoS.Biol. (Plos Biology) 6 (10), e253, 2008).Gene transfer methods have also been studied. In addition to technologies using retroviruses for gene transfer, methods using 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.: Science 322 (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 et al.: Nature 458, 771-775, 2009) have also been developed. K, Smuga-Otto K, Tian S et al.: Science 324, 797-801, 2009) technology used for gene introduction.

[0116] Cells that have undergone transformation (reprogramming) into iPS cells can be selected using the expression of pluripotent stem cell markers (undifferentiation markers) such as Fbxo15, Nanog, Oct3 / 4, Fgf-4, Esg-1, and Cript as indicators. The selected cells can be recovered as iPS cells.

[0117] As a method for producing iPS cells, in addition to methods of producing them by direct activation of gene expression, iPS cells can also be induced from somatic cells by adding compounds or the like (Hou P et al.: Science 341(6146), 651-654, 2013).

[0118] iPS cells can also be provided from, for example, FUJIFILM Cellular Dynamics, Inc. (FCDI), Kyoto University, or the RIKEN BioResource Center.

[0119] Methods for obtaining neural cells differentiated and induced from human pluripotent stem cells include, for example, induction of human iPS cells produced from somatic cells collected from healthy individuals (healthy individuals) with no neurological disease or mutations in disease-related genes that cause neurological diseases, or from patients with neurological diseases, induction of human iPS cells from established human iPS cell lines, and the like.

[0120] The neural cells differentiated and induced from human pluripotent stem cells are not particularly limited, but are preferably motor neurons, cerebral cortex excitatory neurons, or substantia nigra neurons, and particularly preferably motor neurons or cerebral cortex excitatory neurons.

[0121] The method for inducing differentiation of neural cells from human pluripotent stem cells is not particularly limited, but includes methods of producing neural stem cells from pluripotent stem cells using treatment with low molecular weight compounds, and then inducing them into neural cells, and methods of directly inducing them into neural cells by gene expression, etc.

[0122] Examples of methods for inducing differentiation of neural cells from pluripotent stem cells include:

[0123] (1) Methods of forming embryoid bodies (cell clusters containing neural progenitor cells) by culturing in serum-free medium and then differentiating them (SFEB method: Watanabe K. et al., Nat. Neurosci., 8:288-296, 2005; SFEBq method: Wataya T. et al., Proc. Nat. Acad. Sci. USA, 105:11796-11801, 2008);

[0124] (2) Methods of culturing and differentiating cells on stromal cells (SDIA method: Kawasaki H. et al., Neuron, 28:31-40, 2000);

[0125] (3) A method of culturing and differentiating cells on Matrigel supplemented with drugs (Chambers SM et al., Nature Biotechnology, 27: 275-280, 2009);

[0126] (4) A method of culturing and differentiating cells in a medium containing low-molecular-weight compounds as cytokine substitutes (U.S. Patent No. 5,843,780);

[0127] (5) Methods for differentiating pluripotent stem cells by introducing neural induction factors (e.g., neurogenin 2 (Ngn2)) into them and expressing them (WO2014 / 148646; and Zhang Y. et al., Neuron, 78:785-98, 2013);

[0128] (6) A method for differentiating pluripotent stem cells by introducing miR-9 / 9*-124 and expressing it in the cells;

[0129] and combinations of these methods.

[0130] Among the above methods, (5) the method of introducing and expressing neurogenin 2 in pluripotent stem cells is preferred because mature neural cells can be obtained efficiently in a short time.

[0131] The neurogenin 2 protein is known to be a transcription factor that promotes differentiation into neural cells during development. Its amino acid sequence is exemplified by NP_076924 in humans and NP_033848 in mice. The neurogenin 2 gene (Official full name: neurogenin 2, Official symbol: NEUROG2, also known as the Ngn2 gene) is DNA encoding the neurogenin 2 protein. Examples include DNA having the nucleotide sequence of NM_009718 (mouse) or NM_024019 (human), which are registered as standard sequences, or transcript variants thereof.

[0132] Furthermore, the DNA may be complementary to a degree that allows hybridization under stringent conditions with nucleic acids having the sequences of the aforementioned standard sequence and transcript variants.

[0133] As cerebral cortical excitatory neurons induced to differentiate from human iPS cells, cells prepared by forced expression of the Ngn2 gene from human iPS cells are preferably used.

[0134] As the neural cells differentiated and induced from human pluripotent stem cells, commercially available neural cells can be used, for example, iCell (trademark) motor neuron cells (FCDI, C1050, C1048) can be used.

[0135] As substantia nigra neurons induced to differentiate from human pluripotent stem cells, commercially available neurons can be used, for example, iCell (trademark) dopamine neurons (FCDI, C1087, C1028) and the like can be used.

[0136] The neural cells are preferably cells that express at least one of the neural cell-specific marker genes consisting of β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2), and have β-III tubulin-positive processes (hereinafter referred to as neurites).

[0137] The expression level of the marker gene can usually be analyzed by measuring the amount of the transcription product corresponding to the gene or the amount of its translation product, activity, etc. The expression level can be determined by measuring the gene's transcription product, i.e., mRNA, or the gene's translation product, i.e., protein, but is preferably determined by measuring mRNA or its reverse transcription product, i.e., cDNA. The expression of the translation product (protein) can be detected or measured by immunohistochemical staining using antibodies to detect proteins in cells.

[0138] The culture of neural cells in the present invention may be carried out by selecting a culture medium, temperature, and other conditions appropriate for the neural cells to be used.

[0139] In the present invention, a culture medium that contains substantially no antioxidant and substantially no oxidant is used.

[0140] Examples of the antioxidant include vitamin A, glutathione, vitamin E or its derivatives, superoxide dismutase (SOD), and catalase.

[0141] Examples of the oxidizing agent include hydrogen peroxide, arsenite, and sodium nitroprusside.

[0142] “Substantially free of” means that the antioxidant or oxidant is not contained in an amount that can exert its function.

[0143] The culture medium may contain any components or additives such as factors that are suitable for the purpose of culture, as long as they do not hinder the culture of neural cells.

[0144] The culture medium can be selected from known culture media or commercially available culture media. The culture medium used for the culture can be prepared by adding additives to a basal culture medium. Here, examples of the basal medium include DMEM, DMEM (Dulbecco's Modified Eagle Medium) / F12 BrainPhys Neuronal Medium, Neurobasal Medium-A, Neurobasal Medium, Neural Progenitor Basal Medium, NS-A Basal Medium, Basal Medium Eagle (BME), BGJb Medium, CMRL-1066 Medium, Glasgow Minimum Essential Medium (MEM), Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle MEM, αMEM, and Ham's F12 Medium. Medium), RPMI 1640 Medium, and Fischer's Medium. DMEM / F12 or BrainPhys Neuronal Medium are preferred, with DMEM / F12 being more preferred. A single medium or a combination of two or more mediums may be used as the culture medium.

[0145] Additives may be added as long as they do not substantially exhibit an antioxidant effect and at a concentration that does not substantially exhibit an antioxidant effect. Specifically, examples include serum, retinoic acid, Wnt, BMP, bFGF, EGF, HGF, Sonic Hedgehog (Shh), interleukins, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B-27 (trademark) supplement (does not contain antioxidants), and ITS-supplements, but are not particularly limited. A preferred additive is B-27 (trademark) supplement (does not contain antioxidants).

[0146] As the culture conditions for neural cells, general cell culture conditions may be selected. Examples include conditions of 37°C and 5% CO2. The culture medium may be replaced at appropriate intervals during the culture period (preferably every 1 to 7 days, more preferably every 2 to 3 days), but preferably, the culture medium is not replaced during the culture period.

[0147] Neurons are preferably cultured in two dimensions. Cell culture vessels such as culture plates, culture dishes, cell culture inserts, and cell culture flasks can be used for cell culture.

[0148] The neural cells damaged by oxidative stress produced by the method for producing neural cells of the present invention preferably satisfy at least one (preferably two, more preferably all three) of the following (i) to (iv):

[0149] (i) Markers related to oxidative stress were positive;

[0150] (ii) neurite retraction;

[0151] (iii) induced necroptosis; and

[0152] (iv) induced ferroptosis. According to the present invention, there is provided a nerve cell that satisfies at least one of the above (i) to (iv) manufactured by the method for manufacturing a nerve cell of the present invention. The nerve cell of the present invention is not particularly limited, and is preferably a motor nerve cell, a cerebral cortex excitatory nerve cell or a substantia nigra nerve cell, more preferably a motor nerve cell or a cerebral cortex excitatory nerve cell.

[0153] Examples of markers related to oxidative stress include oxidative stress markers and secondary markers caused by oxidative stress.

[0154] Oxidative stress markers include reactive oxygen species (ROS), in vivo products generated by reactive oxygen species, antioxidant enzymes, and antioxidant substances. ROS include superoxide anion radicals (O2· - ), hydrogen peroxide (H2O2), hydroxyl radical (·OH), singlet oxygen ( 1 Examples of the active oxygen species include oxygen (O2), nitric oxide (NO·), nitrogen dioxide (NO2·), ozone (O3), and lipid peroxides (LOOH). Examples of in vivo products generated by active oxygen include 8-hydroxydeoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), and lipid peroxides. Examples of antioxidant enzymes include SOD, catalase, and GPx. Examples of antioxidant substances include glutathione (GSH / GSSG), bilirubin, and vitamins.

[0155] Examples of secondary markers caused by oxidative stress include phosphorylation, accumulation, and aggregation of TAR DNA-binding protein 43 (TDP-43), β-amyloid protein, Tau protein, and α-synuclein.

[0156] The marker associated with oxidative stress is preferably ROS, 8-OHdG, 8-OHG, lipid peroxides, glutathione, TDP-43 protein, β-amyloid protein, tau protein, or α-synuclein, and more preferably ROS.

[0157] A positive result for reactive oxygen species (ROS) indicates an elevated level of ROS compared to the level of ROS in cells cultured in a culture medium containing antioxidants. Intracellular ROS levels can be quantified using commercially available reagents such as CellROX (trademark) Probe for Oxidative Stress Detection (Green) (Thermo Fisher Scientific, C10444).

[0158] Neurite retraction can be confirmed by measuring neurite length. For example, neurite length can be quantified using Neurotrack software (Sartorius, 9600-0010) for the IncuCyte S3.

[0159] Regarding whether necroptosis is induced, for example, the degree of damage caused by oxidative stress is evaluated for cells treated with a necroptosis inhibitor (e.g., necrostatin-1) and for cells not treated with a necroptosis inhibitor (e.g., necrostatin-1). When the degree of damage is reduced when the cells are treated with a necroptosis inhibitor (e.g., necrostatin-1), it can be determined that necroptosis is induced. In addition, by quantifying the phosphorylation of receptor interacting protein kinase 1 (RIPK1), receptor interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL), which are known markers associated with necroptosis, it is also possible to determine whether necroptosis is induced.

[0160] Regarding whether ferroptosis is induced, for example, the degree of damage caused by oxidative stress is evaluated for the case where cells are treated with a ferroptosis inhibitor (e.g., ferroptosis inhibitor-1, lipostatin-1, or UAMC-3203, etc.) and the case where cells are not treated with a ferroptosis inhibitor (e.g., ferroptosis inhibitor-1, lipostatin-1, or UAMC-3203, etc.). When the degree of damage is reduced when cells are treated with a ferroptosis inhibitor (e.g., ferroptosis inhibitor-1, lipostatin-1, or UAMC-3203, etc.), it can be determined that ferroptosis is induced. In addition, by quantifying the amount of divalent iron or lipid peroxide (4-hydroxynonanal or malondialdehyde) in cells, which are known to be markers associated with ferroptosis, it can also be determined whether ferroptosis is induced.

[0161] According to the present invention, it is possible to manufacture the nerve cells subjected to the damage of oxidative stress. About whether being subjected to the evaluation of the nerve damage caused by oxidative stress, it is possible to carry out by evaluating cell death (cell number of nerve cells), measuring the mark relevant to oxidative stress, evaluating cell damage, measuring the mark relevant to nerve damage or measuring neurite length, but is not particularly limited. In the above, it is preferred to evaluate cell death (cell number of nerve cells) or measure neurite length.

[0162] Cell death can be detected using live cell detection reagents such as Cell Titer Glo (Promega) or Cell counting kit-8 (Tong Ren Tang) or cell death detection reagents such as propidium iodide or NucGreen Dead (Thermo Fisher Scientific).

[0163] Cell damage can be evaluated by evaluation methods such as LDH detection, WST detection, or ATP detection. For example, kits such as Cytotoxicity Detection Kit PLUS (LDH) (Sigma aldrich) can be used for LDH detection.

[0164] Examples of markers associated with nerve damage include Enolase 2 (Neuron-Specific Enolase), which can be measured by the ELISA method.

[0165] The neural cells damaged by oxidative stress according to the present invention can be used to screen for new drugs useful for neurodegenerative diseases for which oxidative stress is known to be significantly associated with the onset and progression of the disease. The neural cells damaged by oxidative stress according to the present invention are particularly useful for the study of sporadic chronic neurodegenerative diseases or the development of therapeutic drugs for them, the identification of disease biomarkers, or the development of diagnostic drugs. In the present invention, it is preferred to use neural cells produced from pluripotent stem cells that do not have mutations in disease-related genes, and to chronically impart weak oxidative stress to the cells using a culture medium that is substantially free of antioxidants and substantially free of oxidants, thereby enabling the evaluation of sporadic chronic neurodegenerative diseases.

[0166] In the present invention, cell death is observed after 2 days of culture initiation, allowing evaluation of the drug within approximately 2 weeks. Furthermore, since the present invention can reproduce cell death caused by necroptosis, it is possible to screen for necroptosis inhibitors. Furthermore, since the present invention can reproduce cell death caused by ferroptosis, it is possible to screen for ferroptosis inhibitors.

[0167] That is, according to the present invention, there is provided a method for evaluating a test substance, comprising the step of contacting the nerve cells damaged by oxidative stress of the present invention with a test substance.

[0168] The present invention further provides a method for screening a preventive and / or therapeutic drug for a neurodegenerative disease, comprising the step of contacting the neural cells of the present invention with a test substance.

[0169] According to the present invention, a method for screening a necroptosis inhibitor is further provided, comprising the following steps:

[0170] (1) contacting the neural cells of the present invention with a test substance;

[0171] (2) culturing the neural cells contacted with the test substance in the step (1) and the neural cells of the control group that have not been contacted with the test substance;

[0172] (3) measuring nerve damage in the nerve cells; and

[0173] (4) A test substance that inhibits the above-mentioned nerve damage compared to a control not exposed to the test substance is selected as a candidate necroptosis inhibitor.

[0174] According to the present invention, a method for screening ferroptosis inhibitors is further provided, which comprises the following steps:

[0175] (1) contacting the neural cells of the present invention with a test substance;

[0176] (2) culturing the neural cells contacted with the test substance in the step (1) and the neural cells of the control group that have not been contacted with the test substance;

[0177] (3) measuring nerve damage in the nerve cells; and

[0178] (4) A test substance that inhibits the above-mentioned nerve damage compared to a control that has not been exposed to the test substance is selected as a candidate ferroptosis inhibitor.

[0179] The above-mentioned methods for evaluating test substances, screening methods for preventing and / or treating neurodegenerative diseases, screening methods for necroptosis inhibitors, and screening methods for ferroptosis inhibitors may also include producing the neural cells of the present invention by the method for producing neural cells damaged by oxidative stress of the present invention.

[0180] 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 low molecular weight compounds, natural compounds, cell extracts, extracellular vesicles, plant extracts, animal tissue extracts, plasma, extracts from marine organisms, cell culture supernatants, and microbial fermentation products.

[0181] Furthermore, the test substance can be obtained using any of a variety of combinatorial library methods known in the art, including (1) biological library methods, (2) synthetic library methods using deconvolution, (3) one-bead one-compound library methods, and (4) synthetic library methods using affinity chromatography sorting. The biological library method using affinity chromatography screening is limited to peptide libraries, but other methods can be applied to low molecular weight compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing molecular libraries 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), on beads (Lam (1991) Nature 354:82-4), on chips (Fodor (1993) Nature 364:555-6), in bacteria (U.S. Pat. No. 5,223,409), in spores (U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,403,484, and U.S. Pat. No. 5,223,409), on plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9), or on phages (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application Publication No. 2002 / 0103360).

[0182] The nerve cells are brought into contact with the test substance by adding the test substance to the culture medium of the nerve cells. The contact time is not particularly limited as long as the change in the indicator can be confirmed, and is, for example, 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 appropriately adjusted according to the type of compound (solubility, toxicity, etc.).

[0183] The culture medium for neural cells used when bringing the neural cells into contact with the test substance is not particularly limited as long as it is a medium capable of culturing neural cells.

[0184] The culture temperature when the test substance is brought into contact with the nerve cells is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and culture is performed in an atmosphere of air containing CO2, with a CO2 concentration of preferably about 2 to 5%.

[0185] Examples of neurodegenerative diseases include Alzheimer's disease (AD), spinocerebellar atrophy, frontotemporal lobar degeneration (FTLD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lewy body disease, Huntington's disease, and Niemann-Pick disease. Preferred neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Niemann-Pick disease, and more preferably amyotrophic lateral sclerosis (ALS).

[0186] Examples of diseases involved in necroptosis include neurodegenerative diseases, acute kidney injury, alcoholic liver injury, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, acute lung injury, acute respiratory distress syndrome, systemic inflammatory response syndrome, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, heart failure, arteriosclerosis, aortic aneurysm, psoriasis, rheumatoid arthritis, acute myeloid leukemia, chronic lymphocytic leukemia, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, colon cancer, cervical cancer, malignant melanoma, glioblastoma, lung cancer, breast cancer, pancreatic cancer, etc. Diseases involved in necroptosis are preferably neurodegenerative diseases, preferably multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and more preferably amyotrophic lateral sclerosis (ALS).

[0187] Examples of diseases involving ferroptosis include neurodegenerative diseases, age-related macular degeneration, Fuchsian corneal endothelial dystrophy, chronic obstructive pulmonary disease, radiation-induced lung injury, acute lung injury, asthma, pulmonary fibrosis, tuberculosis, Pseudomonas aeruginosa infection, paraquat poisoning, ischemia-reperfusion injury, alcoholic liver injury, autoimmune hepatitis, non-alcoholic steatohepatitis, acetaminophen-induced liver injury, liver fibrosis, liver transplantation, acute pancreatitis, diabetes, islet transplantation, hemochromatosis, transfusion-related immunomodulation, hemolytic anemia, and radiation-induced hematopoietic disorders. , periventricular leukomalacia, hemorrhagic / ischemic stroke, hemorrhagic dementia, traumatic brain injury, epilepsy, ischemic reperfusion injury, doxorubicin cardiomyopathy, iron overload cardiomyopathy, myocardial infarction / fibrosis, atherosclerosis, heart transplantation, acute kidney injury, polycystic kidney disease, kidney transplantation, Crohn's disease, ulcerative colitis, pregnancy-induced hypertensive nephropathy, endometriosis, infertility, neuroblastoma, glioblastoma, colon cancer, lung cancer, head and neck cancer, gastric cancer, pancreatic ductal adenocarcinoma, breast cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, Burkitt's lymphoma, etc. As the disease involved in ferroptosis, neurodegenerative diseases are preferred, preferably Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease or Niemann-Pick disease, more preferably amyotrophic lateral sclerosis (ALS).

[0188] In one embodiment of the present invention, after the neural cells of the present invention are brought into contact with a test substance, the following steps can be performed:

[0189] (A) Cultivation of neural cells exposed to a test substance and control neural cells not exposed to the test substance;

[0190] (B) measuring nerve damage in the nerve cells; and

[0191] (C) A test substance that suppresses the above-mentioned nerve damage compared to a control not exposed to the test substance is selected as a candidate for a preventive and / or therapeutic drug for a neurodegenerative disease.

[0192] Preferably, step (B) is a step of measuring the number of nerve cells and / or the length of neurites obtained in step (A).

[0193] Preferably, step (C) is a step of selecting a test substance whose cell number and / or neurite length in neurons contacted with the test substance are higher than those of the control as a candidate for a preventive and / or therapeutic drug for a neurodegenerative disease.

[0194] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the scope of the examples.

[0195] Example

[0196] Test Example 1: Evaluation of Neurite Length of Neurons Cultured in a Medium Containing No Antioxidants from the Time of Cell Seeding and Cultured in a Medium Containing No Antioxidants by Medium Exchange After Cell Seeding

[0197] <Culture plate coating>

[0198] iMatrix-511silk (Matrixome, 892021) diluted 16.7-fold with PBS (phosphate-buffered saline) was added to a 96-well culture plate coated with PDL (poly-D-lysine) (Corning, 356461) at 70 μL / well and incubated at 37°C for 3 to 72 hours.

[0199] <Preparation of culture medium>

[0200] The antioxidant-containing medium used a medium obtained by adding DMSO (dimethyl sulfoxide) to a +AO medium to obtain 0.1 volume % (v / v) (Stress(-)), and the antioxidant-free medium used a medium obtained by adding DMSO to a -AO medium to obtain 0.1 volume % (v / v) (Stress(+)).

[0201] [Table 1]

[0202] +AO medium

[0203] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0204] -AO medium

[0205] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 9.8 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.2

[0206] <Cell seeding>

[0207] The neural cells (considered to be cerebral cortical excitatory neural cells) produced by forced expression of the Ngn2 gene (Neurogenin2 gene) from iPS cells (Chao Wang. et al., Stem Cell Reports., 9: 1221-1233, 2017) were thawed in a 37°C warm bath. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in the culture medium, and the number of cells was counted. The cells were then diluted with culture medium at 200 μL / well (9.4×10 4 cells / cm 2) and cultured at 37°C, 5% CO2. Images were acquired every 6 hours using an Incucyte S3 (Sartorius, Incucyte S3). The culture medium was replaced after 48, 72, or 96 hours.

[0208] <Evaluation of protrusion length>

[0209] The neurite length was quantified using the Neurotrack software (Sartorius, 9600-0010) of IncuCyte S3. After 14 days of culture, under the conditions of Stress (+) cultured in a culture medium without antioxidants, the neurite length at any time after the process retraction (the sum of the neurite lengths per unit area) became less than half of the maximum value of the neurite length of the Stress (-) group cultured in a culture medium containing antioxidants, and was judged to be process retraction. The results of quantifying the neurite length are shown in Figures 1 to 3 .

[0210] Figure 1 It is the result of quantifying the length of neurites of nerve cells whose culture medium was replaced after 48 hours of culture. The vertical axis represents the sum of the length of neurites per unit area, and the horizontal axis represents the number of culture days. As a result, in the Stress(+) group (▲) cultured in a culture medium that did not contain antioxidants from the beginning, the retraction of neurites (nerve damage) was confirmed after 4 days of culture. On the other hand, in the Stress(-) group (■) cultured in a culture medium containing antioxidants from the beginning, the group (◆) cultured in a culture medium containing the same antioxidants after 48 hours of initial culture, and the group (▼) cultured in a culture medium containing antioxidants after 48 hours of initial culture, and then replaced with a culture medium without antioxidants. After 72 hours of culture ( Figure 2 ) or cells whose medium was changed after 96 hours ( Figure 3), only in the Stress(+) group (▲) cultured in a culture medium without antioxidants from the beginning could the retraction of neurites (nerve damage) be confirmed after 4 days of culture, while in the Stress(-) group (■) cultured in a culture medium containing antioxidants from the beginning, the group (◆) cultured in a culture medium containing antioxidants and the culture medium was replaced in the same culture medium, and the group (▼) cultured in a culture medium containing antioxidants and the culture medium was replaced in a culture medium without antioxidants, no retraction of neurites (nerve damage) was confirmed. This shows that in order to induce nerve damage under weak oxidative stress (when cultured in a culture medium without antioxidants), it is important to culture in a culture medium without antioxidants from the time of cell inoculation.

[0211] Experimental Example 2: Evaluation of the Effect of Cell Seeding Density on Oxidative Stress-Induced Neuronal Cell Damage

[0212] <Culture plate coating>

[0213] iMatrix-511silk (Matrixom, 892021) diluted 16.7-fold with PBS was added to a PDL-coated 96-well culture plate (Corning, 356461) at 70 μL / well and incubated at 37° C. for 3 to 72 hours.

[0214] <Preparation of culture medium>

[0215] The antioxidant-containing medium used a medium obtained by adding DMSO to +AO medium to obtain 0.1 volume % (v / v) (Stress(-)), and the antioxidant-free medium used a medium obtained by adding DMSO to -AO medium to obtain 0.1 volume % (v / v) (Stress(+)).

[0216] [Table 2]

[0217] +AO medium

[0218] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0219] -AO medium

[0220] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 9.8 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.2

[0221] <Cell seeding>

[0222] The same method as in Experimental Example 1 was used to thaw the neural cells (considered to be cerebral cortical excitatory neural cells) produced by forced expression of the Ngn2 gene from iPS cells in a 37°C warm bath. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in the culture medium, and the number of cells was counted. The cells were then diluted with culture medium at 200μL / well (0.3 to 37.5×10 4 cells / cm 2 ) were inoculated and cultured at 37°C under 5% CO 2 . After culture, images were acquired every 6 hours using an Incucyte S3 (Sartorius, Incucyte S3).

[0223] <Evaluation of protrusion length>

[0224] The neurite length was quantified using the Neurotrack software (Sartorius, 9600-0010) for IncuCyte S3. The results of the neurite length quantification are shown in Figures 4 to 9 The results of cell seeding density and the detectability of neuronal damage are summarized in Table 3. In addition, regarding the detectability of neuronal damage, whether the neurite length retracted was evaluated using the same criteria as in Test Example 1, and the case where retraction was determined was considered detectable damage.

[0225] [Table 3]

[0226] <![CDATA[Cell seeding density (×10 4 cells / cm 2 )]]> Can damage be detected? 0.3 Detectable damage 0.9 Detectable damage 1.9 Detectable damage 3.1 Detectable damage 4.7 Detectable damage 9.4 Detectable damage 10.9 Detectable damage 12.5 Detectable damage 25.0 Undetectable damage 37.5 Undetectable damage

[0227] The cells were seeded at a density of 0.3, 0.9, 1.9, 4.7, 9.4, or 12.5 × 10 4 cells / cm 2 The temporal changes in neurite length at the time of inoculation are shown in Figures 4 to 9 The vertical axis represents the total length of neurites per unit area, and the horizontal axis represents the number of days of culture. As a result, the cell seeding density was 0.3 to 12.5 cells / cm 2 In the stress (+) group (▲) cultured in a medium without antioxidants, neurite retraction (nerve damage) was observed. 4 cells / cm 2 The experimental results of inoculation with different inoculation densities are shown in Table 3. When the inoculation density becomes 25.0×10 4 cells / cm 2 Above this, the cell density is too high, and therefore the retraction of neurites (nerve damage) cannot be detected. 4 cells / cm 2It is important to seed and culture cells at the following cell densities.

[0228] Test Example 3: Evaluation of the effect of basal culture medium on oxidative stress-induced neuronal cell damage

[0229] <Culture plate coating>

[0230] iMatrix-511silk (Matrixom, 892021) diluted 16.7-fold with PBS was added to a PDL-coated 96-well culture plate (Corning, 356461) at 70 μL / well and incubated at 37° C. for 3 to 72 hours.

[0231] <Preparation of culture medium>

[0232] The antioxidant-containing medium used a medium obtained by adding DMSO to +AO medium to obtain 0.1 volume % (v / v) (Stress(-)), and the antioxidant-free medium used a medium obtained by adding DMSO to -AO medium to obtain 0.1 volume % (v / v) (Stress(+)).

[0233] [Table 4]

[0234] +AO medium

[0235]

[0236] -AO medium

[0237]

[0238] <Cell seeding>

[0239] The same method as in Experimental Example 1 was used to thaw the neural cells (considered to be cerebral cortical excitatory neural cells) produced by forced expression of the Ngn2 gene from iPS cells in a 37°C warm bath. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in the culture medium, and the number of cells was counted. The cells were then diluted with culture medium and centrifuged at 200 μL / well (4.7×10 4 cells / cm 2 ) were inoculated and cultured at 37°C under 5% CO 2 . After culture, images were acquired every 6 hours using an Incucyte S3 (Sartorius, Incucyte S3).

[0240] <Evaluation of protrusion length>

[0241] The neurite length was quantified using the Neurotrack software (Sartorius, 9600-0010) of the IncuCyte S3. The results of the neurite length quantification are shown in Figure 10 and Figure 11 The retraction of neurites was evaluated using the same criteria as in Test Example 1.

[0242] The vertical axis represents the total length of neurites per unit area, and the horizontal axis represents the number of days of culture. Figure 10 ) and Neurobasal medium ( Figure 11 ) in either medium, neurite retraction (nerve damage) was observed in the Stress (+) group (▲) cultured in antioxidant-free medium, whereas no neurite retraction was observed in the Stress (-) group (■) cultured in antioxidant-containing medium. This suggests that culturing in BrainPhys medium or Neurobasal medium without antioxidants can induce nerve damage.

[0243] Experimental Example 4: Quantitative evaluation of oxidative stress-induced neuronal damage

[0244] <Culture plate coating>

[0245] A solution of iMatrix-511silk (Matrixome, 892021) diluted 16.7-fold with PBS was added to a PDL-coated flask (Greiner Bio-one, 661940) at 20 mL / flask and incubated at 37° C. for 3 to 72 hours.

[0246] <Preparation of culture medium>

[0247] The antioxidant-containing medium used a medium obtained by adding DMSO to +AO medium to obtain 0.1 volume % (v / v) (Stress(-)), and the antioxidant-free medium used a medium obtained by adding DMSO to -AO medium to obtain 0.1 volume % (v / v) (Stress(+)).

[0248] [Table 5]

[0249] +AO medium

[0250] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0251] -AO medium

[0252] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 29.4 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.6

[0253] <Cell seeding>

[0254] The same method as in Experimental Example 1 was used to thaw the neural cells (considered to be cerebral cortical excitatory neural cells) produced by forced expression of the Ngn2 gene from iPS cells in a 37°C warm bath. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the number of cells was counted. The cells were then diluted with culture medium and inoculated into 6.9×10 4 cells / cm 2 The cells were plated at a density of 100 μg / mL and cultured at 37°C under 5% CO2. The culture medium was collected on the 3rd, 4th, 5th, and 6th days after culture.

[0255] LDH test

[0256] The cytotoxicity detection kit PLUS (LDH) (Sigma aldrich, 4744934001) was used for the quantification of LDH. According to the attached document, the quantification of LDH was performed in the culture medium from the 3rd to the 6th day of culture. The quantitative results are shown in Figure 12 .

[0257] Compared to the culture medium of the neural cells of the Stress(-) group cultured in a medium containing antioxidants (■), the culture medium of the neural cells of the Stress(+) group cultured in a medium without antioxidants (▲) showed increased LDH levels on the 5th and 6th days of culture, indicating that cell damage occurred in the Stress(+) group.

[0258] Experimental Example 5: Quantification of ROS Signaling

[0259] <Culture plate coating>

[0260] iMatrix-511silk (Matrixome, 892021) diluted 16.7-fold with PBS was added to a PDL-coated 96-well culture plate (Corning, 356640) at 70 μL / well and incubated at 37° C. for 3 to 72 hours.

[0261] <Preparation of culture medium>

[0262] The antioxidant-containing medium used a medium obtained by adding DMSO to +AO medium to obtain 0.1 volume % (v / v) (Stress(-)), and the antioxidant-free medium used a medium obtained by adding DMSO to -AO medium to obtain 0.1 volume % (v / v) (Stress(+)).

[0263] [Table 6]

[0264] +AO medium

[0265] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0266] -AO medium

[0267] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 9.8 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.2

[0268] <Cell seeding>

[0269] Frozen cells (iCell motor neurons, FCDI, C1048) were thawed in a 37°C incubator. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the cell number was counted. The cells were then diluted with culture medium at 200 μL / well (4.7×10 4 cells / cm 2 ) were inoculated and cultured at 37°C and 5% CO2 for 6 days.

[0270] Quantification of ROS Signaling

[0271] The quantification of ROS signals was performed using the CellROX (trademark) oxidative stress detection probe (green) (Thermo Fisher Scientific, C10444). Following the implementation of the attached document, the intracellular ROS signals of the neural cells after 6 days of culture were quantified using a confocal quantitative image cytometer (CQ1, Yokogawa Electric Corporation). The results of quantification of the average fluorescence intensity of the nuclear region are shown in FIG. Figure 13 .

[0272] Compared with the neural cells cultured in the medium containing antioxidants (Stress(-) group), a stronger signal of ROS was detected in the neural cells cultured in the medium without antioxidants (Stress(+) group).

[0273] Test Example 6: Evaluation of compounds against oxidative stress-induced neuronal damage (necroptosis inhibitors)

[0274] <Culture plate coating>

[0275] iMatrix-511silk (Matrixom, 892021) diluted 16.7-fold with PBS was added to a PDL-coated 96-well culture plate (Corning, 356461) at 70 μL / well and incubated at 37° C. for 3 to 72 hours.

[0276] <Preparation of test plate>

[0277] The antioxidant-containing medium used a medium in which DMSO was added to +AO medium to a concentration of 0.1% (v / v) (Stress(-)), the antioxidant-free medium used a medium in which DMSO was added to -AO medium to a concentration of 0.1% (v / v) (Stress(+)), and the drug evaluation group used a medium in which DMSO-dissolved compounds (0.01 to 20 μM) were added to -AO medium at a concentration of 0.1% (v / v).

[0278] [Table 7]

[0279] +AO medium

[0280] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0281] -AO medium

[0282] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 9.8 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.2

[0283] <Cell seeding>

[0284] Frozen cells (iCell motor neurons, FCDI, C1048) were thawed in a 37°C incubator. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the cell number was counted. The cells were then diluted with culture medium at 200 μL / well (4.7×10 4 cells / cm 2 ) were inoculated and cultured at 37°C under 5% CO 2 . After culture, images were acquired every 6 hours using an Incucyte S3 (Sartorius, Incucyte S3).

[0285] <Evaluation of protrusion length>

[0286] Neurite length was quantified using Neurotrack software (Sartorius, 9600-0010) for IncuCyte S3. The results of neurite length quantification and images of representative cells are shown in Figure 14 .

[0287] Neurons cultured in a medium without antioxidants (Stress(+) group) (▲) were damaged after 4 days of culture, and the total length of neurites decreased. On the other hand, in the group treated with necrostatin-1 (●), a known necroptosis inhibitor, the decrease in total neurite length was suppressed in a concentration-dependent manner. This indicates that the nerve damage in this evaluation system is caused by necroptosis.

[0288] Test Example 7: Evaluation of compounds against oxidative stress-induced neuronal damage (ferroptosis inhibitors)

[0289] <Culture plate coating>

[0290] iMatrix-511silk (Matrixom, 892021) diluted 16.7-fold with PBS was added to a PDL-coated 96-well culture plate (Corning, 356461) at 70 μL / well and incubated at 37° C. for 3 to 72 hours.

[0291] <Preparation of test plate>

[0292] The antioxidant-containing medium used a medium in which DMSO was added to +AO medium to a concentration of 0.1% (v / v) (Stress(-)), the antioxidant-free medium used a medium in which DMSO was added to -AO medium to a concentration of 0.1% (v / v) (Stress(+)), and the drug evaluation group used a medium in which DMSO-dissolved compounds (0.004 to 20 μM) were added to -AO medium at a concentration of 0.1% (v / v).

[0293] [Table 8]

[0294] +AO medium

[0295] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0296] -AO medium

[0297] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 9.8 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.2

[0298] <Cell seeding>

[0299] Neural cells (thought to be cerebral cortical excitatory neurons) produced by forced expression of the Ngn2 gene from iPS cells were thawed in a 37°C incubator. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the number of cells was counted. The cells were then diluted with culture medium and centrifuged at 200 μL / well (4.7×10 4 cells / cm 2 ) were inoculated and cultured at 37°C under 5% CO 2 . After culture, images were acquired every 6 hours using an Incucyte S3 (Sartorius, Incucyte S3).

[0300] <Evaluation of protrusion length>

[0301] The neurite length was quantified using the Neurotrack software (Sartorius, 9600-0010) of the IncuCyte S3. The results of the neurite length quantification are shown in Figures 15-17 .

[0302] Neurons cultured in a medium without antioxidants (Stress(+) group) (▲) were damaged after one day of culture, and the total length of neurites decreased. On the other hand, in the group treated with ferroptosis inhibitors (●), all compounds inhibited the reduction in the total length of neurites in a concentration-dependent manner. This shows that the nerve damage in this evaluation system is caused by ferroptosis.

[0303] Test Example 8: Compound Screening Using an Oxidative Stress-Induced Neuronal Damage Evaluation System

[0304] <Culture plate coating>

[0305] iMatrix-511silk (Matrixom, 892021) diluted 16.7-fold with PBS was added to a PDL-coated 96-well culture plate (Corning, 356461) at 70 μL / well and incubated at 37° C. for 3 to 72 hours.

[0306] <Preparation of test plate>

[0307] The compound library used was StemSelect Library compounds (Merck, 569774). The antioxidant-containing medium used a medium prepared by adding 0.1% (v / v) DMSO to +AO medium (Stress(-)), the antioxidant-free medium used a medium prepared by adding 0.1% (v / v) DMSO to -AO medium (Stress(+)), and the drug evaluation group used a medium prepared by adding 0.1% (v / v) DMSO dissolved in -AO medium.

[0308] [Table 9]

[0309] +AO medium

[0310] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 4.9 B-27 Supplement (50X), serum-free (Thermo Fisher Scientific, 17504-044) 0.1

[0311] -AO medium

[0312] mL DMEM / F12 (Thermo Fisher Scientific, 11320-033) 9.8 B-27 Supplement (50X), without antioxidant (Thermo Fisher Scientific, 10889-038) 0.2

[0313] <Cell seeding>

[0314] Frozen cells (iCell motor neurons, FCDI, C1048) were thawed in a 37°C incubator. After thawing, the cells were added to the culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the cell number was counted. The cells were then diluted with culture medium at 200 μL / well (4.7×10 4 cells / cm 2 ) were inoculated and cultured at 37°C under 5% CO 2 . After culture, images were acquired every 6 hours using an Incucyte S3 (Sartorius, Incucyte S3).

[0315] <Evaluation of protrusion length>

[0316] The neurite length was quantified using the Neurotrack software (Sartorius, 9600-0010) of the IncuCyte S3. The area under the curve (AUC) was calculated based on the time curve of the neurite length over 14 days, and the effects of the drugs were compared. That is, a large AUC indicates that the retraction of the neurite is suppressed (suppression of nerve damage). The values ​​of each compound and AUC are shown in Figure 18 . Compared with the neural cells cultured in the medium containing antioxidants (Stress(-) group) (■), the AUC was reduced in the neural cells cultured in the medium without antioxidants (Stress(+) group) (▲). In the compound-treated group (○), CD437 / AHPN, Simvastatin, Mevastatin, Reversine, KB-R7943, Licochalcone-A, Telomerase Inhibitor IX, Purmorphamine, LXRα / β agonist, Mifepristone, AY 9944, γ-Secretase Inhibitor XXI, ATRA-BA hybrid and Mn-cpx 3 strongly inhibited the reduction of AUC.

[0317] Test Example 9: Evaluation of ALS therapeutic drug (edaravone) for oxidative stress-induced neuronal damage

[0318] The effect of edaravone, a therapeutic drug for ALS, on oxidative stress-induced neuronal damage was evaluated by the same method as in Test Example 7. The results of quantification of neurite length are shown in FIG. Figure 19Neurons cultured in a medium without antioxidants (Stress (+) group) (▲) were damaged after 4 days of culture, and the total length of neurites decreased. In the group treated with edaravone (●), the decrease in total neurite length was suppressed in a concentration-dependent manner. This demonstrates that this evaluation system can be used to screen for candidate drugs for the treatment of ALS.

Claims

1. A method for producing nerve cells damaged by oxidative stress, comprising: Step a: using a culture medium containing substantially no antioxidant and substantially no oxidant at 20.0×10 4 cells / cm 2 The neural cells differentiated from human pluripotent stem cells are seeded at the following cell density; and in step b, culture is performed using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant.

2. The manufacturing method according to claim 1, wherein The cell density was 0.2×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 the following.

3. The manufacturing method according to claim 1 or 2, wherein: Human pluripotent stem cells are pluripotent stem cells that do not have mutations in disease-related genes.

4. The manufacturing method according to claim 1 or 2, wherein: The nerve cells are motor nerve cells, cerebral cortex excitatory nerve cells or substantia nigra nerve cells.

5. The manufacturing method according to claim 1 or 2, wherein: The nerve cells damaged by oxidative stress satisfy at least one of the following (i) to (iv): (i) Markers related to oxidative stress were positive; (ii) neurite retraction; (iii) induced necroptosis; and (iv) Induced ferroptosis.

6. A cell culture method comprising: Step a: using a culture medium containing substantially no antioxidant and substantially no oxidant at 20.0×10 4 cells / cm 2 The neural cells differentiated from human pluripotent stem cells are seeded at the following cell density; and in step b, culture is performed using a culture medium that does not substantially contain an antioxidant and does not substantially contain an oxidant.

7. The culture method according to claim 6, wherein The cell density was 0.2×10 4 cells / cm 2 Above and 20.0×10 4 cells / cm 2 the following.

8. The culture method according to claim 6 or 7, wherein Human pluripotent stem cells are pluripotent stem cells that do not have mutations in disease-related genes.

9. The culture method according to claim 6 or 7, wherein The nerve cells are motor nerve cells, cerebral cortex excitatory nerve cells or substantia nigra nerve cells.

10. A neural cell obtained by the production method according to claim 1, which satisfies at least one of the following (i) to (iv): (i) Markers related to oxidative stress were positive; (ii) neurite length retraction; (iii) induced necroptosis; and (iv) Induced ferroptosis.

11. The cell according to claim 10, wherein The nerve cells are motor nerve cells, cerebral cortex excitatory nerve cells or substantia nigra nerve cells.

12. A method for evaluating a test substance, comprising the step of contacting the neural cell according to claim 10 with a test substance.

13. The method for evaluating a test substance according to claim 12, further comprising the step of producing the neural cell according to claim 10 by the method according to claim 1, and contacting the neural cell with the test substance.

14. A method for screening a drug for preventing and / or treating a neurodegenerative disease, comprising the step of contacting the neural cell according to claim 10 with a test substance.

15. The screening method according to claim 14, further comprising the step of producing the neural cell according to claim 10 by the method according to claim 1, and contacting the neural cell with a test substance.

16. The screening method according to claim 14, wherein The neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), spinocerebellar atrophy, frontotemporal lobar degeneration (FTLD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lewy body disease, Huntington's disease, and Niemann-Pick disease.

17. The screening method according to claim 14, wherein After the nerve cells according to claim 10 are brought into contact with a test substance, the method further comprises the following steps: (A) culturing neural cells exposed to the test substance and control neural cells not exposed to the test substance; (B) measuring nerve damage in the nerve cells; as well as (C) A test substance that suppresses the nerve damage compared to a control not exposed to the test substance is selected as a candidate for a preventive and / or therapeutic drug for a neurodegenerative disease.

18. The screening method according to claim 17, wherein The step (B) is a step of measuring the number of nerve cells and / or the length of neurites obtained in the step (A).

19. The method according to claim 17 or 18, wherein The step (C) is a step of selecting a test substance whose cell number and / or neurite length values ​​of neurons contacted with the test substance are higher than those of the control as a candidate for a preventive and / or therapeutic drug for a neurodegenerative disease.

20. A method for screening a necroptosis inhibitor, comprising the following steps: (1) contacting the neural cell according to claim 10 with a test substance; (2) culturing the neural cells contacted with the test substance in step (1) and control neural cells not contacted with the test substance; (3) determining nerve damage in the nerve cells; and (4) A test substance that inhibits the nerve damage compared to a control not exposed to the test substance is selected as a candidate necroptosis inhibitor.

21. The screening method according to claim 20, further comprising the step of producing the neural cell according to claim 10 by the method according to claim 1, and contacting the neural cell with a test substance.

22. A method for screening ferroptosis inhibitors, comprising the following steps: (1) contacting the neural cell according to claim 10 with a test substance; (2) culturing the neural cells contacted with the test substance in step (1) and control neural cells not contacted with the test substance; (3) determining nerve damage in the nerve cells; and (4) A test substance that inhibits the nerve damage compared to a control that has not been exposed to the test substance is selected as a candidate ferroptosis inhibitor.

23. The screening method according to claim 22, further comprising the step of producing the neural cell according to claim 10 by the method according to claim 1, and contacting the neural cell with a test substance.

Images