Test substance evaluation method and test substance evaluation kit

Abstract

The present invention addresses the problem of providing a test substance evaluation method and a test substance evaluation kit with which it is possible to distinguish between damage caused by suppression or deletion of a function of a gene product and damage caused by stimulation in human-derived pluripotent stem cells in which a function of a gene product involved in cholesterol metabolism regulation has been suppressed or deleted or cells differentiated from said human-derived pluripotent stem cells, and to evaluate the effect of the test substance only on damage caused by stimulation. The present invention provides a test substance evaluation method, the method comprising: a step (a) for culturing human-derived pluripotent stem cells in which a function of a gene product involved in cholesterol metabolism regulation has been suppressed or deleted or cells differentiated from said human-derived pluripotent stem cells in a culture medium substantially free of serum and containing sterols; and a step (b) for bringing the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells into contact with a test substance.

Description

Test substance evaluation method and test substance evaluation kit

[0001] The present invention relates to a method for evaluating a test substance using human-derived pluripotent stem cells in which the function of a gene product involved in the regulation of cholesterol metabolism is suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells.The present invention further relates to a kit for evaluating a test substance, comprising human-derived pluripotent stem cells in which the function of a gene product involved in the regulation of cholesterol metabolism is suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells.

[0002] Recently, research using gene-edited cells has reported that 7-dehydrocholesterol reductase (DHCR7), an enzyme in the cholesterol synthesis pathway, is involved in ferroptosis (Non-Patent Documents 1 and 2). Ferroptosis is known to be deeply involved in cancer, neurodegenerative diseases, and other conditions (Non-Patent Documents 3 and 4). Understanding the relationship between cholesterol synthesis enzymes and ferroptosis is crucial for elucidating the mechanisms of these diseases and developing therapeutic agents. Non-Patent Document 1 reports that DHCR7 deficiency increases its substrate, 7-dehydrocholesterol (7-DHC), which suppresses ferroptosis. Meanwhile, it has been reported that deficiency of enzymes upstream of DHCR7 reduces the amount of 7-DHC produced downstream, suggesting that these enzymes are also deeply involved in ferroptosis. Non-Patent Document 5 describes that in cells in which DHCR7 function is suppressed, 7-DHC increases, which acts as a substitute for cholesterol, and therefore suppression of DHCR7 function does not result in suppression of cell proliferation. Furthermore, Non-Patent Document 6 reports that pluripotency markers are reduced in DHCR7-deficient cells, and that the reduction in pluripotency markers is restored by adding cholesterol to the culture medium.

[0003] Yaxu Li et al., Nature, Vol. 626, pp. 411-418, 2024; Florencio Porto Freitas et al., Nature, Vol. 626, pp. 401-410, 2024; Chen Zhang et al., Mol Cancer, Vol. 21, Paper No. 47, 2022; Mengmeng Ou et al., Mol Metab, Vol. 61, Paper No. 101502, 2022; Carlos Fernandez et al., J Lipid Res, Vol. 46, pp. 920-929, 2005; Kevin R Francis et al., Nat Med, Vol. 22, pp. 388-396, 2016

[0004] An object of the present invention is to provide a method and kit for evaluating a test substance that can distinguish between damage caused by the suppression or deletion of the function of a gene product involved in regulating cholesterol metabolism and damage caused by a stimulus in human-derived pluripotent stem cells in which the function of the gene product is suppressed or deleted, or in cells differentiated from the human-derived pluripotent stem cells, and that can evaluate the effect of the test substance only on damage caused by the stimulus.

[0005] As a result of intensive research to achieve the above object, the present inventors have found that the above object can be achieved by culturing human-derived pluripotent stem cells, or cells differentiated from such human-derived pluripotent stem cells, in which the function of a gene product involved in regulating cholesterol metabolism has been suppressed or deleted, in a medium that is substantially free of serum and contains sterols. The present invention was completed based on these findings.

[0006] That is, the present invention provides the following inventions. <1> A method for evaluating a test substance, comprising the steps of: (a) culturing human-derived pluripotent stem cells, in which the function of a gene product involved in regulating cholesterol metabolism has been suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells, in a medium that is substantially free of serum and contains sterols; and (b) contacting the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells with the test substance. <2> The evaluation method according to <1>, in which the sterols include at least one selected from the group consisting of cholesterol, 7-dehydrocholesterol, and desmosterol. <3> The evaluation method according to <1> or <2>, in which the gene is a gene for sterol-C5-desaturase in the cholesterol biosynthetic pathway or an enzyme upstream thereof. <4> The evaluation method according to <3>, in which a substrate of the enzyme in the cholesterol biosynthetic pathway is a substance that does not suppress ferroptosis. <5> The evaluation method according to <3> or <4>, wherein the sterol is cholesterol or desmosterol. <6> The evaluation method according to any one of <1> to <5>, wherein the gene is an EBP cholestenol delta-isomerase gene. <7> The evaluation method according to any one of <1> to <6>, wherein the cells differentiated from the human-derived pluripotent stem cells are neural cells or neural cell precursor cells. <8> The evaluation method according to any one of <1> to <7>, wherein the evaluation method is a method for evaluating cytotoxicity caused by oxidative stress in the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells. <9> The evaluation method according to any one of <1> to <8>, wherein the evaluation method is a method for evaluating cytotoxicity caused by ferroptosis in the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells. <10> The evaluation method according to any one of <1> to <9>, wherein the test substance is a ferroptosis inhibitor.<11> The evaluation method according to any one of <1> to <10>, comprising, between the steps (a) and (b), a step (c) of inducing ferroptosis-mediated cell damage in the cells cultured in the step (a), and, after the step (b), a step (d) of culturing the cells contacted with the test substance in the step (b) and control cells not contacted with the test substance, a step (e) of measuring nerve damage in the cells, and a step (f) of selecting, as a candidate for a ferroptosis inhibitor, a test substance that suppresses the nerve damage compared to a control not contacted with the test substance. <12> The evaluation method according to <11>, wherein the step (c) of inducing ferroptosis-mediated cell damage is a step of culturing the cells in a culture medium that is substantially free of antioxidants and substantially free of oxidants. <13> The evaluation method according to <11>, wherein the step (c) of inducing cell damage by ferroptosis is a step of treating the cells with a ferroptosis inducer. <14> A kit for evaluating a test substance to be used in the evaluation method according to any one of <1> to <13>, comprising: human-derived pluripotent stem cells in which the function of a gene product involved in cholesterol metabolism regulation is suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells; and a medium that is substantially free of serum and contains sterols.

[0007] According to the present invention, in human-derived pluripotent stem cells in which the function of a gene product involved in regulating cholesterol metabolism is suppressed or deleted, or in cells differentiated from the above-mentioned human-derived pluripotent stem cells, it is possible to distinguish between damage caused by the suppression or deletion of the function of the gene product and damage caused by stimulation, and to evaluate the effect of a test substance only on damage caused by stimulation.

[0008] Figure 1 shows the results of evaluating ferroptosis inhibitors in an oxidative stress-induced nerve injury assay model using wild-type neurons under cholesterol-free conditions. Figure 2 shows the results of evaluating neurite length in wild-type neurons and EBP knockout neurons under cholesterol-free conditions in an oxidative stress-induced nerve injury assay model. Figure 3 shows the results of evaluating neurite length in wild-type neurons and EBP knockout neurons under cholesterol-containing conditions in an oxidative stress-induced nerve injury assay model. Figure 4 shows the results of evaluating ferroptosis inhibitors in an oxidative stress-induced nerve injury assay model using EBP knockout neurons under cholesterol-containing conditions. Figure 5 shows the results of evaluating the effect of desmosterol on EBP knockout neurons in an oxidative stress-induced nerve injury assay model. Figure 6 shows the results of evaluating ferroptosis inhibitors in an oxidative stress-induced nerve injury assay model using EBP knockout neurons under desmosterol-containing conditions.

[0009] Hereinafter, embodiments of the present invention will be described in detail.

[0010] <Method for evaluating a test substance> The present invention relates to a method for evaluating a test substance, comprising the steps of: (a) culturing human-derived pluripotent stem cells, in which the function of a gene product involved in regulating cholesterol metabolism has been suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells, in a medium that is substantially free of serum and contains sterols; and (b) contacting the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells with the test substance.

[0011] The present invention relates to a method for evaluating a test substance against cell damage caused by various stimuli using human-derived pluripotent stem cells in which the function of a gene product involved in regulating cholesterol metabolism has been suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells, in which the effect of the test substance is evaluated by distinguishing between damage caused by the suppression or deletion of the function of the gene product and damage caused by various stimuli by adding sterols to the culture medium.

[0012] According to the present invention, by adding sterols to the culture medium of human-derived pluripotent stem cells in which the function of a gene product involved in cholesterol metabolism regulation is suppressed or deleted, or cells differentiated from the human-derived pluripotent stem cells, it is possible to distinguish between damage caused by the suppression or deletion of the gene product function and damage caused by various stimuli. Furthermore, by deleting the gene, it is also possible to verify the effect of a test substance on a cholesterol synthesis enzyme related to ferroptosis. This clarifies the relationship between enzymes in the cholesterol synthesis pathway and damage caused by various stimuli. The present invention can be used to develop novel therapeutic agents for diseases associated with damage caused by various stimuli.

[0013] In Non-Patent Document 1, ferroptosis was induced in cells in which the enzyme gene in the cholesterol synthesis pathway upstream of DHCR7 (sterol-C5-desaturase or further upstream) was knocked out, and cell damage was evaluated. In the present invention, it was found that by suppressing the function of cholesterol synthase upstream of DHCR7 (sterol-C5-desaturase or further upstream), ferroptosis-independent damage occurs due to cholesterol deficiency. In other words, in cells in which the function of cholesterol synthase upstream of DHCR7 is suppressed, the effect of cholesterol synthase on ferroptosis alone cannot be evaluated. Therefore, Non-Patent Document 1 does not take into consideration damage caused by suppression of enzyme function, and therefore is unable to distinguish between the effects of suppression of enzyme function and ferroptosis.

[0014] According to the present invention, it is possible to evaluate the effect of cholesterol synthetic enzymes on ferroptosis alone, and furthermore, it is possible to analyze the mechanism of drugs that target these enzymes to control ferroptosis. According to the present invention, it is possible to evaluate the relationship between enzymes involved in cholesterol metabolic regulation and ferroptosis, and the present invention is very useful for understanding the pathology of cancer, neurodegenerative diseases, etc., and for developing new therapeutic agents.

[0015] Examples of human-derived 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.

[0016] ES cells can be established, for example, by culturing pre-implantation early embryos, the inner cell mass constituting the above-mentioned early embryos, single blastomeres, etc. (Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, J.A. et al., Science, 282, 1145-1147 (1998)). As the early embryo, an early embryo produced by nuclear transfer of a nucleus from a somatic cell may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Iritani A. et al. (Protein, Nucleic Acid, Enzyme, 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). Parthenogenetic 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 papers, references for the production of ES cells include 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. Note that 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.

[0017] Some ES cells are available from collection institutions or commercially available. For example, human ES cells are available from the Institute of Medical and Biological Sciences, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0018] iPS cells refer to cells that have pluripotency (multipotency) and proliferation ability, and are produced by reprogramming somatic cells through the introduction of reprogramming factors. iPS cells exhibit properties similar to ES cells. The somatic cells used to produce iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. iPS cells can be produced by known methods. It is also naturally expected that iPS cell production methods developed in the future will also be applied.

[0019] The most basic method for generating iPS cells is to introduce four transcription factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into cells using viruses (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi, K, et al: Cell 131(5), 861-72, 2007). Human iPS cells have been reported to be established by introducing four factors, Oct4, Sox2, Lin28, and Nanog (Yu J, et al: Science 318(5858), 1917-1920, 2007). The establishment of iPS cells has also been reported 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 only Oct3 / 4 (Kim JB, et al.: Cell 136(3), 411-419, 2009). Techniques for introducing proteins, 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, it has been reported that the use of histone methyltransferase G9a inhibitor BIX-01294, or histone deacetylase inhibitors such as valproic acid (VPA) or BayK8644 can improve production efficiency and reduce the amount of factors introduced (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).Gene transfer methods have also been investigated, and 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 that have been transformed into iPS cells, i.e., reprogrammed, can be selected using the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct3 / 4, Fgf-4, Esg-1, and Cript as indicators. The selected cells can be recovered as iPS cells.

[0021] In addition to direct reprogramming through gene expression, iPS cells can also be induced from somatic cells by adding compounds (Hou P et al: Science 341(6146), 651-654, 2013).

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

[0023] In the human-derived pluripotent stem cells of the present invention, the function of a gene product involved in the regulation of cholesterol metabolism is suppressed or deleted.

[0024] Methods for suppressing or eliminating the function of a gene product involved in regulating cholesterol metabolism include gene disruption (gene knockout), reduction of gene expression (gene knockdown), and gene mutation.

[0025] Gene disruption (gene knockout) is a method of completely suppressing the expression or function of a target gene by disrupting the target gene itself. Examples of gene disruption (gene knockout) methods include gene targeting, artificial nucleases, and CRISPR / Cas systems (e.g., CRISPR / Cas9s systems).

[0026] Gene targeting is a technique that uses homologous recombination to modify endogenous genes, and can be used to delete genes, remove exons, introduce genes, or introduce point mutations.

[0027] Artificial nucleases are proteins that combine a DNA-binding domain designed to bind to any base sequence with the cleavage domain of a DNA cleavage enzyme, making them capable of cleaving any base sequence. Two types of artificial nucleases are commonly used: zinc finger nucleases (ZFNs), which recognize DNA sequences using the DNA-binding domain of zinc fingers, and TALENs, which recognize DNA sequences using the DNA-binding domain of TALEs. In cells transfected with artificial nucleases, specific DNA undergoes double-strand breaks (DSBs), which are repaired by the non-homologous end joining (NHEJ) mechanism. Repair errors, such as base pair deletions and insertions, frequently occur, resulting in frameshifts and gene knockouts.

[0028] The CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein) system is an immune mechanism used by bacteria against viruses and other pathogens. Genome editing technology using the CRISPR / Cas9 system allows for the cleavage of any desired base sequence by introducing a single molecule of guide RNA (gRNA) containing crRNA and tracrRNA with the desired base sequence into cells along with Cas9 mRNA or protein. The cleaved DNA frequently undergoes repair errors, such as base pair deletions and insertions, similar to artificial nucleases, resulting in gene knockout.

[0029] Methods for reducing gene expression (gene knockdown) include gene targeting, antisense oligonucleotides, and RNA interference.

[0030] Reduction of gene expression level by gene targeting (gene knockdown) can be achieved by deleting an expression control region such as a polyA addition sequence in the gene or by introducing a mutation into the expression control region.

[0031] Reducing gene expression levels using antisense oligonucleotides (gene knockdown) is a technique in which short nucleic acids (antisense oligonucleotides) with a base sequence complementary to the mRNA sequence of a target gene are introduced from outside the cell, where they bind to the target mRNA and inhibit translation.

[0032] RNA interference is a phenomenon in which double-stranded RNA called short interfering RNA (siRNA), consisting of pairs of 21-23 bases, binds to mRNA with a complementary sequence in the body and degrades that mRNA. Introducing siRNA into cells can induce gene expression suppression.

[0033] The gene involved in cholesterol metabolic regulation is preferably a gene for sterol-C5-desaturase or an enzyme upstream thereof in the cholesterol biosynthetic pathway, and the substrate of the enzyme in the cholesterol biosynthetic pathway is preferably a substance that does not suppress ferroptosis.

[0034] Specific examples of genes involved in cholesterol metabolism regulation include DHCR24, LSS, CYP51A1, LBR, TM7SF2, SC4MOL, NSDHL, HSD17B7, EBP, and SC5DL. The gene involved in cholesterol metabolism regulation is preferably a gene for EBP or an enzyme upstream thereof, more preferably EBP or SC5DL, and more preferably EBP.

[0035] DHCR24 refers to 24-dehydrocholesterol reductase. Other names for DHCR24 include: delta(24)-sterol reductase 3, beta-hydroxysterol delta 24-reductase 3, desmosterol-to-cholesterol enzyme diminuto / dwarf1 homolog seladin 1, and selective AD indicator 1.

[0036] LSS means lanosterol synthase. Other names for LSS are listed below: lanosterol synthase 2,3-epoxysqualene-lanosterol cyclase epididymis secretory sperm binding protein hOSC lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) oxidosqualene-lanosterol cyclase

[0037] CYP51A1 means sterol 14-alpha-demethylase. Other names for CYP51A1 are listed below: lanosterol 14-demethylase, ergosterol 14-demethylase, 14α-methylenelanosterol 14-demethylase, lanosterol C14-demethylase, sterol 14α-demethylase, sterol 14(15)-reductase, and cytochrome P450 lanosterol 14α-demethylase.

[0038] LBR stands for lamin B receptor. Other names for LBR are listed below: delta(14)-sterol reductase LBR 3-beta-hydroxysterol Delta (14)-reductase C-14 sterol reductase delta-14-SR integral nuclear envelope inner membrane protein; sterol C14-reductase tudor domain containing 18

[0039] TM7SF2 stands for transmembrane 7 superfamily member 2. Other names for TM7SF2 are listed below: delta(14)-sterol reductase TM7SF2 3-beta-hydroxysterol Delta (14)-reductase C-14 sterol reductase; another new gene 1 protein; delta-14-SR putative sterol reductase SR-1 sterol C14-reductase

[0040] SC4MOL means methylsterol monooxygenase 1. Other names for SC4MOL are listed below: methylsterol monooxygenase 1 C-4 methylsterol oxidase sterol-C4-methyl oxidase

[0041] NSDHL stands for NAD(P)-dependent steroid dehydrogenase-like. Other names for NSDHL are listed below: sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating NAD(P)-dependent steroid dehydrogenase-like protein transcript epididymis secretory sperm binding protein protein H105e3 short chain dehydrogenase / reductase family 31E, member 1

[0042] HSD17B7 means hydroxysteroid 17-beta dehydrogenase 7. Other names for HSD17B7 are listed below: 3-keto-steroid reductase / 17-beta-hydroxysteroid dehydrogenase 7 17 beta-hydroxysteroid dehydrogenase type VII 17-beta-HSD 7 17-beta-hydroxysteroid dehydrogenase 7 17beta hydroxysteroid dehydrogenase 3-keto-steroid reductase dihydrotestosterone oxidoreductase estradiol 17-beta-dehydrogenase 7 short chain dehydrogenase / reductase family 37C member 1

[0043] EBP stands for EBP cholestenol delta-isomerase. Other names for EBP include: 3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase, 3-beta-hydroxysteroid-delta-8,delta-7-isomerase, Chondrodysplasia punctata-2, X-linked dominant (Happle syndrome), D8-D7 sterol isomerase, cholestenol Delta-isomerase, delta(8)-Delta(7) sterol isomerase, emopamil binding protein (sterol isomerase), and emopamil-binding protein sterol 8-isomerase.

[0044] SC5DL means sterol-C5-desaturase. Other names for SC5DL are listed below: lathosterol oxidase, 3beta-hydroxysteroid-delta5-desaturase, C-5 sterol desaturase, delta(7)-sterol 5(6)-desaturase, delta(7)-sterol 5-desaturase, delta(7)-sterol C5(6)-desaturase, fungal ERG3, delta-5-desaturase-like, lathosterol 5-desaturase, lathosterol dehydrogenase, sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, S. cerevisiae)-like.

[0045] In the present invention, cells differentiated from human-derived pluripotent stem cells may be used. Cells differentiated from human-derived pluripotent stem cells refer to any cells that have been subjected to differentiation induction treatment to differentiate from human-derived pluripotent stem cells into a specific type of cell. Specific examples of differentiated cells include, but are not limited to, muscle cells such as cardiomyocytes and skeletal myoblasts; nervous system cells such as neurons, astrocytes, microglia, oligodendrocytes, motor neurons, and dopamine-producing cells; retinal cells such as retinal pigment epithelial cells; hematopoietic cells such as blood cells and bone marrow cells; immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells; organ-constituting cells such as hepatocytes, pancreatic beta cells, and kidney cells; chondrocytes; germ cells; and progenitor cells and somatic stem cells that differentiate into these cells. Typical examples of progenitor cells and somatic stem cells include mesenchymal stem cells in cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in nervous system cells, and hematopoietic stem cells and lymphoid stem cells in hematopoietic system cells and immune-related cells.

[0046] The cells differentiated from human-derived pluripotent stem cells are preferably neural cells or neural cell precursor cells.

[0047] The differentiation of human-derived pluripotent stem cells can be induced using any known technique.

[0048] As an example, we will explain the differentiation induction of human-derived pluripotent stem cells into neurons. Methods for obtaining neurons induced to differentiate from human-derived pluripotent stem cells include, for example, deriving them from somatic cells collected from healthy individuals (healthy subjects) who do not have a mutation in a disease-related gene that causes a nervous system disease or a nervous system disease, or from human iPS cells produced from somatic cells collected from patients with a nervous system disease, or from established human iPS cell lines.

[0049] Methods for inducing differentiation of human-derived pluripotent stem cells into neurons include, but are not limited to, methods in which neural stem cells are produced from pluripotent stem cells using treatment with low molecular weight compounds, etc., and then the cells are induced to differentiate into neurons, and methods in which the cells are directly induced to differentiate into neurons through gene expression, etc.

[0050] Methods for inducing differentiation of pluripotent stem cells into neural cells include, for example, (1) a method in which cells are cultured in a serum-free medium to form embryoid bodies (cell masses containing neural precursor cells) and then differentiated (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) a method in which cells are cultured on stromal cells to differentiate (SDIA method: Kawasaki H., et al., Neuron, 28: 31-40, 2000); and (3) a method in which cells are cultured on drug-added matrigel to differentiate (Chambers SM, et al., Nat. Biotechnol., 27: 275-280, (2009); (4) a method of differentiation by culturing in a medium containing a low molecular weight compound as a substitute for cytokines (U.S. Patent No. 5,843,780); (5) a method of differentiation by introducing and expressing a neural induction factor (such as neurogenin 2 (Ngn2)) 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.

[0051] Of the above, (5) the method of introducing and expressing neurogenin 2 into pluripotent stem cells is preferred because it allows for the production of mature neurons in a short period of time and with high efficiency.

[0052] Neurogenin2 protein is a transcription factor known to promote differentiation into neurons during development, and its amino acid sequence is exemplified by NP_076924 in humans and NP_033848 in mice. The neurogenin2 gene (official full name: neurogenin2, official symbol: NEUROG2, also known as the Ngn2 gene) is DNA encoding the Neurogenin2 protein, including, for example, DNA having the nucleotide sequence of NM_009718 (mouse) or NM_024019 (human), registered as standard sequences, or their transcript variants. Furthermore, the DNA may be sufficiently complementary to nucleic acids having the above-mentioned standard sequences and the sequences of their transcript variants to the extent that it can hybridize under stringent conditions.

[0053] As the nerve cells induced to differentiate from human-derived pluripotent stem cells, commercially available nerve cells may be used, such as iCell™ motor nerve cells (FCDI, C1050, C1048).

[0054] As the substantia nigra neurons induced to differentiate from human-derived pluripotent stem cells, commercially available neurons may be used, such as iCell™ dopamine neurons (FCDI, C1087, C1028).

[0055] Neurons are preferably cells that express at least one or more neuron-specific marker genes, including 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).

[0056] The expression level of a marker gene can usually be analyzed by the amount of transcription product corresponding to the gene, or the amount or activity of its translation product. The expression level can be measured by measuring mRNA, which is a transcription product of the gene, or protein, which is a translation product of the gene, but is preferably measured by measuring mRNA or cDNA, which is a reverse transcription product of the gene. The expression of the translation product (protein) can be detected or measured by immunocytostaining, which uses an antibody to detect intracellular proteins.

[0057] In step (a) of the present invention, cells are cultured in a medium that is substantially free of serum and contains sterols.

[0058] The present invention provides a method for evaluating the effect of a test substance by adding sterols to the culture medium of human-derived pluripotent stem cells in which the function of gene products involved in regulating cholesterol metabolism has been suppressed or deleted, or cells differentiated from the above-mentioned human-derived pluripotent stem cells, thereby distinguishing between damage caused by the suppression of cholesterol synthesis enzyme function and damage caused by various stimuli.

[0059] The sterols may be any sterols that are downstream of the enzyme whose function is inhibited, and preferably do not affect ferroptosis.

[0060] Specific examples of sterols include 14-demethyllanosterol, zymosterol, 5α-cholesta-7,24-dien-3β-ol, 7-dehydrodesmosterol, desmosterol, 24,25-dihydrolanosterol, 4,4-dimethyl-5α-cholest-8-en-3β-ol, zymostenol, lathosterol, 7-dehydrocholesterol, and cholesterol.

[0061] The sterols preferably include at least one selected from the group consisting of cholesterol, 7-dehydrocholesterol, lathosterol, and desmosterol. The sterols more preferably include at least one selected from the group consisting of cholesterol, 7-dehydrocholesterol, and desmosterol. The sterols are particularly preferably cholesterol or desmosterol.

[0062] The content of sterols in the medium is not particularly limited, but is generally 100 to 20,000 ng / mL, preferably 200 to 10,000 ng / mL, more preferably 500 to 5,000 ng / mL, and even more preferably 1,000 to 3,000 ng / mL.

[0063] The medium used in step (a) is substantially serum-free. "Substantially serum-free" means that the serum content in the medium is 1% by volume or less, and preferably 0% by volume.

[0064] The medium can be selected from known or commercially available media. The medium used for culture can be a basal medium to which additives have been added. Here, examples of the basal medium include Dulbecco's Modified Eagle Medium (DMEM), DMEM / 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), and Improved MEM. Examples of the medium include 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. A single medium may be used, or two or more types of medium may be used in combination.

[0065] The culture medium may contain any additives, such as factors suitable for the purpose of the culture, as long as they do not interfere with the cell culture.

[0066] Specific examples of additives include, but are not limited to, serum, retinoic acid, Wnt, bone morphogenetic protein (BMP), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), Sonic hedgehog (Shh), interleukins, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B-27™ supplement (antioxidant-free), ITS supplement, etc. A preferred additive is B-27™ supplement (antioxidant-free).

[0067] The cell culture conditions may be selected from general cell culture conditions: 37°C, 5% CO ２ During the culture, the 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 medium during the culture period.

[0068] The cells may be cultured in either two-dimensional or three-dimensional culture. Cell culture vessels such as plates, dishes, cell culture inserts, and cell culture flasks can be used for cell culture.

[0069] In step (b), human-derived pluripotent stem cells or cells differentiated from human-derived pluripotent stem cells are contacted with a test substance.

[0070] Examples of test substances include proteins, peptides, antibodies, nucleic acids (gene expression vectors, siRNA, antisense oligonucleotides, mRNA, miRNA), 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 derived from marine organisms, cell culture supernatants, and microbial fermentation products.

[0071] Test substances can also be obtained using any of the many approaches to combinatorial library technology known in the art, including (1) biological library technology, (2) synthetic library technology using deconvolution, (3) one-bead one-compound library technology, and (4) synthetic library technology using affinity chromatography selection. While the biological library technology using affinity chromatography selection is limited to peptide libraries, other approaches can be applied to small molecule libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of 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 stored in solution (see Houghten (1992) Bio / Techniques 13: 412-21) or on beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. 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) Proc. Natl. Acad. Sci. USA 87: 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application Publication No. 2002 / 0103360).

[0072] The test substance is preferably a ferroptosis inhibitor.

[0073] Contacting human-derived pluripotent stem cells or cells differentiated from human-derived pluripotent stem cells with a test substance may be carried out by adding the test substance to a cell culture medium. The contact time is not particularly limited as long as a change in an indicator can be confirmed, and may be, 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 test substance to be added can be adjusted appropriately depending on the type of compound (solubility, toxicity, etc.).

[0074] The cell culture medium used when contacting the cells with the test substance is not particularly limited as long as it is a medium that can culture the cells.

[0075] The culture temperature when contacting the cells with the test substance is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and CO ２ The culture was carried out under an atmosphere of CO ２ The concentration is preferably about 2-5%.

[0076] The method for evaluating a test substance of the present invention is preferably a method for evaluating cytotoxicity due to oxidative stress in human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells.The method for evaluating a test substance of the present invention is preferably a method for evaluating cytotoxicity due to ferroptosis in human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells.

[0077] More preferably, the ferroptosis is ferroptosis induced by oxidative stress.

[0078] Whether or not cells are under oxidative stress can be evaluated by measuring a marker associated with oxidative stress, preferably ROS, 8-OHdG, 8-OHG, lipid peroxide, glutathione, TDP-43 protein, amyloid β protein, tau protein, or α-synuclein.

[0079] A positive result for reactive oxygen species (ROS) indicates elevated levels of ROS compared to the levels of ROS in cells cultured in a culture medium containing antioxidants. Quantification of intracellular ROS levels can be performed using commercially available reagents such as CellROX™ Green Reagent for oxidative stress detection (Thermo Fisher Scientific, C10444).

[0080] In one example, the method for evaluating a test substance of the present invention includes, between steps (a) and (b), a step (c) of inducing cell damage by ferroptosis in the cells cultured in step (a); and, after step (b), a step (d) of culturing the cells contacted with the test substance in step (b) and control cells not contacted with the test substance; a step (e) of measuring nerve damage in the cells; and a step (f) of selecting, as a candidate ferroptosis inhibitor, a test substance that suppresses the nerve damage compared to the control not contacted with the test substance.

[0081] In one example, the step (c) of inducing cell damage by ferroptosis is preferably a step of culturing the cells using a culture medium that is substantially free of antioxidants and substantially free of oxidants.

[0082] Antioxidants include vitamin A, glutathione, vitamin E or a derivative thereof, superoxide dismutase (SOD), and catalase.

[0083] Oxidizing agents include hydrogen peroxide, arsenite, and sodium nitroprusside.

[0084] By "substantially free" it is meant that no antioxidants or oxidizing agents are present in amounts that would allow their function to be achieved.

[0085] In another example, the step (c) of inducing cell damage by ferroptosis is preferably a step of treating the cells with a ferroptosis inducer.

[0086] Examples of ferroptosis inducers that can be used include RSL3, ML-162, ML-210, FIN56, L-Buthione-(S,R)-Sulfoximine, Acetaminophen, Erastin, Sulfasalazine, Sorafenib, Artemisinin, and Artesunate.

[0087] In the step (c) of inducing ferroptosis-induced cell damage, whether ferroptosis-induced cell damage has been induced can be determined, for example, by evaluating the degree of damage caused by oxidative stress when cells are treated with a ferroptosis inhibitor (e.g., ferrostatin-1, liproxstatin-1, or UAMC-3203, etc.) and when cells are not treated with a ferroptosis inhibitor (e.g., ferrostatin-1, liproxstatin-1, or UAMC-3203, etc.), and determining that ferroptosis-induced cell damage has been induced if the degree of damage is reduced when cells are treated with a ferroptosis inhibitor (e.g., ferrostatin-1, liproxstatin-1, or UAMC-3203, etc.). It should be noted that whether ferroptosis has been induced can also be determined by quantifying the amount of intracellular divalent iron or lipid peroxides (4-hydroxynonenal or malondialdehyde), which are known as markers associated with ferroptosis.

[0088] Step (d) of culturing the cells contacted with the test substance in step (b) and the control cells not contacted with the test substance can be carried out under ordinary cell culture conditions.

[0089] In step (e), neuronal damage in cells is measured. Measurement of neuronal damage can be performed by, but is not limited to, assessing cell death (neuronal cell number), measuring a marker related to oxidative stress, assessing cell damage, measuring a marker related to neuronal damage, or measuring neurite length. Among the above, assessing cell death (neuronal cell number) or measuring neurite length is preferred.

[0090] Cell death can be detected using live cell detection reagents such as Cell Titer Glo (Promega) or Cell counting kit-8 (Dojindo), or cell death detection reagents such as Propidium Iodide or NucGreen Dead (Thermo Fisher Scientific).

[0091] The marker associated with oxidative stress is preferably ROS, 8-OHdG, 8-OHG, lipid peroxide, glutathione, TDP-43 protein, amyloid β protein, tau protein, or α-synuclein.

[0092] Cytotoxicity can be evaluated using an evaluation method such as an LDH assay, a WST assay, or an ATP assay. For example, a kit such as Cytotoxicity Detection Kit PLUS (LDH) (Sigma-Aldrich) can be used for the LDH assay.

[0093] Markers for nerve damage include Enolase 2 (Neuron-Specific Enolase), which can be measured using the ELISA method.

[0094] Neurite length can be quantified, for example, using Neurotrack software on an IncuCyte S3 (Sartorius, 9600-0010).

[0095] In step (f), a test substance that suppresses the above-mentioned nerve damage compared to a control that has not been contacted with the test substance is selected as a candidate for a ferroptosis inhibitor.

[0096] The selected ferroptosis inhibitors are useful as candidates for preventive and / or therapeutic agents for diseases involving ferroptosis.

[0097] Diseases in which ferroptosis is involved include neurodegenerative diseases, age-related macular degeneration, Fuchs' intracorneal dystrophy, chronic obstructive pulmonary disease, radiation pneumonitis, 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, pancreatic islet transplantation, hemochromatosis, transfusion-associated immunomodulation, hemolytic anemia, radiation-induced hematopoietic disorders, and periventricular hyperplasia. These include leukomalacia, hemorrhagic / ischemic stroke, hemorrhagic dementia, traumatic brain injury, epilepsy, ischemia-reperfusion injury, doxorubicin-induced cardiomyopathy, iron overload cardiomyopathy, myocardial infarction / fibrosis, atherosclerosis, heart transplant, acute kidney injury, polycystic kidney disease, kidney transplant, Crohn's disease, ulcerative colitis, preeclampsia, endometriosis, infertility, neuroblastoma, glioblastoma, colorectal cancer, lung cancer, head and neck cancer, gastric cancer, pancreatic ductal adenocarcinoma, breast cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, and Burkitt's lymphoma.

[0098] The disease associated with ferroptosis is preferably a neurodegenerative disease. Examples of neurodegenerative diseases include Alzheimer's disease (AD), spinocerebellar degeneration, frontotemporal lobar degeneration (FTLD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Lewy body disease, Huntington's disease, and Niemann-Pick disease, among which Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease, or Niemann-Pick disease is preferred, and amyotrophic lateral sclerosis (ALS) is more preferred.

[0099] <Test substance evaluation kit> According to the present invention, there is provided a test substance evaluation kit for use in the evaluation method of the present invention, which comprises human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells in which the function of a gene product involved in cholesterol metabolism regulation is suppressed or deleted, and a medium that is substantially free of serum and contains sterols.

[0100] Details and preferred embodiments of the "human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells in which the function of a gene product involved in regulating cholesterol metabolism is suppressed or deleted" and the "medium that is substantially serum-free and contains sterols" are as described above in this specification.

[0101] The test substance evaluation kit of the present invention may optionally contain a reagent for measuring nerve damage. The test substance evaluation kit of the present invention may further contain instructions for carrying out the evaluation method of the present invention.

[0102] The present invention will be specifically explained by the following examples, but the present invention is not limited to the scope of the examples.

[0103] Test Example 1: Oxidative Stress-Induced Nerve Damage Assay <Plate Coating> iMatrix-511 silk (Matrixome, 892021) was diluted 166.7-fold with PBS and added to a PDL-coated 96-well plate (Corning, 356461) at 70 μL / well, followed by incubation at 37° C. for 3 to 72 hours.

[0104] <Preparation of assay plate> The medium containing antioxidants was +AO medium to which solvent was added at 0.1% by volume (v / v) (Stress (-)), the medium without antioxidants was -AO medium to which solvent was added at 0.1% by volume (v / v) (Stress (+)), and the medium for drug evaluation was -AO medium to which a compound was added at a concentration of 0.1% by volume (v / v). In experiments in which cholesterol was added, cholesterol was added to a final concentration of 1500 ng / mL in all groups.

[0105]

[0106] <Cell seeding> iPS cells used were those described in the report by Takuya Kondo et al. (Nature aging, Vol. 2, pp. 125-139, 2022). EBP knockout iPS cells were generated by utilizing a repair error in the NHEJ mechanism after DNA cleavage. Frozen stocks of neurons generated by forced expression of the Ngn2 gene from wild-type and EBP knockout iPS cells were thawed in a warm bath at 37°C. After thawing, the cells were added to culture medium and centrifuged at 600 x 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 placed at 200 μL / well (4.7 x 10 ４ cells / cm ２ ) and incubated at 37°C and 5% CO ２ After the culture, images were taken every 6 hours using an IncuCyte S3 (Sartorius, Incucyte S3) and neurite length was quantified to evaluate cell damage.

[0107] <Evaluation of Neurite Length> Neurite length was quantified using Neurotrack software (Sartorius, 9600-0010) on an IncuCyte S3.

[0108] Figure 1 shows the results of a test using neurons generated from wild-type iPS cells without added cholesterol. The vertical axis of the graph represents the total neurite length per field, and the horizontal axis represents the date of measurement. Neurons cultured in antioxidant-free medium (Stress (+) group) (▲: black) began to suffer damage and the total neurite length decreased after approximately three days of culture. Treatment with 1 μM of the ferroptosis inhibitors ferrostatin-1 (●: gray), riproxistatin-1 (▼: gray), and UAMC-3203 (◆: gray) prevented the decrease in the total neurite length. These results demonstrate that oxidative stress-induced neuronal injury in this evaluation system is ferroptosis.

[0109] Figure 2 shows the results of a comparison of neurons generated from wild-type iPS cells and EBP knockout iPS cells in the absence of added cholesterol. When compared under conditions without ferroptosis induction (Stress (-)), the total neurite length was reduced in the EBP knockout cells (●: gray) compared to the wild-type cells (■: black), confirming poor growth due to cholesterol deficiency. This indicates that the decrease in total neurite length in the EBP knockout cells in which ferroptosis was induced (▼: gray) was due to both damage caused by ferroptosis and poor growth due to cholesterol deficiency.

[0110] Figure 3 shows the results of a comparison between neurons generated from wild-type iPS cells and EBP knockout iPS cells under cholesterol-supplemented conditions. Under conditions where ferroptosis was not induced by adding cholesterol (Stress (-)), there was almost no difference between the wild-type (■: black) and EBP knockout (●: gray) cells. This indicates that the addition of cholesterol suppressed the growth defect caused by cholesterol deficiency. This indicates that when ferroptosis was induced in EBP knockout cells (▼: gray), the reduction in total neurite length was caused solely by the influence of ferroptosis.

[0111] Figure 4 shows the results of evaluating ferroptosis inhibitors in neurons generated from EBP knockout iPS cells in the presence of cholesterol. Neurons cultured in antioxidant-free medium (Stress (+) group) (▼: gray) began to show damage after about one day of culture, resulting in a decrease in total neurite length. As in Figure 3, the addition of cholesterol restored total neurite length in antioxidant-containing medium to the same level as wild-type neurons, suggesting that the cell damage observed in antioxidant-free medium with cholesterol addition is due to ferroptosis. Under these conditions, the ferroptosis inhibitors ferrostatin-1 (◆: black), riproxistatin-1 (▲: black), and UAMC-3203 (▼: black) suppressed the loss of neurite length, confirming the effectiveness of ferroptosis inhibitors against ferroptosis damage in EBP knockout neurons.

[0112] Test Example 2: Oxidative stress-induced nerve injury assay under desmosterol-added conditions

[0113] Plate coating, assay plate preparation, cell seeding, and neurite length evaluation were performed in the same manner as in Test Example 1. In the experiment in which desmosterol was added, desmosterol was added to all groups to a final concentration of 1500 ng / mL.

[0114] Figure 5 shows the results of evaluating the effects of desmosterol on neurons generated from EBP knockout iPS cells. Test Example 1 showed that EBP knockout cells were affected by ferroptosis and by poor growth due to cholesterol deficiency. It was confirmed that the addition of desmosterol to the medium increased the total neurite length compared to when desmosterol was not added. This indicates that the addition of desmosterol can suppress poor growth due to cholesterol deficiency and enable highly specific evaluation of the effects of ferroptosis.

[0115] Figure 6 shows the results of evaluating ferroptosis inhibitors in neurons generated from EBP knockout iPS cells in the presence of desmosterol. Neurons cultured in antioxidant-free medium (Stress (+) group) (◆: black) were damaged after approximately 30 hours of culture, resulting in a decrease in total neurite length. Under these conditions, the ferroptosis inhibitor ferrostatin-1 (▲: black) suppressed the decrease in neurite length, confirming the effect of ferroptosis inhibitors on ferroptosis injury in EBP knockout neurons.

Claims

1. A method for evaluating a test substance, comprising the steps of: (a) culturing human-derived pluripotent stem cells, or cells differentiated from said human-derived pluripotent stem cells, in which the function of a gene product involved in regulating cholesterol metabolism has been suppressed or deleted, in a medium that is substantially free of serum and contains sterols; and (b) contacting said human-derived pluripotent stem cells or cells differentiated from said human-derived pluripotent stem cells with the test substance.

2. The evaluation method according to claim 1, wherein the sterols include at least one selected from the group consisting of cholesterol, 7-dehydrocholesterol, and desmosterol.

3. The evaluation method according to claim 1 or 2, wherein the gene is a gene encoding sterol-C5-desaturase or an enzyme upstream thereof in the cholesterol biosynthetic pathway.

4. The evaluation method according to claim 3, wherein the substrate of the enzyme in the cholesterol biosynthetic pathway is a substance that does not suppress ferroptosis.

5. The evaluation method according to claim 3, wherein the sterol is cholesterol or desmosterol.

6. The evaluation method according to claim 1 or 2, wherein the gene is an EBP cholestenol delta-isomerase gene.

7. The evaluation method according to claim 1 or 2, wherein the cells differentiated from the human-derived pluripotent stem cells are neural cells or precursor cells of neural cells.

8. The evaluation method according to claim 1 or 2, which is a method for evaluating cell damage caused by oxidative stress in the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells.

9. The evaluation method described in claim 1 or 2, which is a method for evaluating cell damage caused by ferroptosis in the human-derived pluripotent stem cells or cells differentiated from the human-derived pluripotent stem cells.

10. The evaluation method described in claim 1 or 2, wherein the test substance is a ferroptosis inhibitor.

11. The evaluation method according to claim 1 or 2, comprising a step (c) between step (a) and step (b) of inducing cell damage by ferroptosis in the cells cultured in step (a); and a step (d) after step (b) of culturing the cells contacted with the test substance in step (b) and control cells not contacted with the test substance; a step (e) of measuring nerve damage in the cells; and a step (f) of selecting, as a candidate ferroptosis inhibitor, a test substance that suppresses the nerve damage compared to a control not contacted with the test substance.

12. The evaluation method described in claim 11, wherein the step (c) of inducing cell damage by ferroptosis is a step of culturing the cells in a culture medium that is substantially free of antioxidants and substantially free of oxidants.

13. The evaluation method according to claim 11, wherein the step (c) of inducing cell damage by ferroptosis is a step of treating the cells with a ferroptosis inducer.

14. A test substance evaluation kit for use in the evaluation method described in claim 1 or 2, comprising: human-derived pluripotent stem cells or cells differentiated from said human-derived pluripotent stem cells in which the function of a gene product involved in regulating cholesterol metabolism is suppressed or deleted; and a medium that is substantially free of serum and contains sterols.

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