Method for evaluating intercellular interaction of neuroinflammation

Abstract

The present invention addresses the problem of providing a method for evaluating intercellular interaction in neuroinflammation by using a co-culture containing human-derived neural cells capable of mimicking the function of a human brain. The present invention provides a method for evaluating intercellular interaction of neuroinflammation, the method comprising: a step for producing a co-culture containing at least two cells selected from the group consisting of human-derived astrocytes, human-derived neurons, human-derived microglia, and human-derived oligodendrocytes; a step for providing an inflammatory stimulus to a first cell contained in the co-culture; a step for detecting at least one selected from the group consisting of inflammatory reaction markers, cellular neuronal activity, and cell morphology in a cell contained in the co-culture; and a step for evaluating at least one change selected from the group consisting of the inflammatory reaction marker, the neuronal activity, and the cell morphology over time in the first cell and in a second cell included in the co-culture and different from the first cell.

Description

Methods for assessing cellular interactions in neuroinflammation

[0001] The present invention relates to a method for evaluating cell-cell interactions in neuroinflammation using co-cultures containing human-derived nervous system cells.

[0002] The central nervous system is composed of neurons and glial cells (astrocytes, microglia, and oligodendrocytes), and these cells interact to maintain normal brain function (Non-Patent Document 1). In addition to normal brain functions such as axonal conduction, synaptic transmission, and information processing (Non-Patent Document 2), intercellular interactions have also been reported to be important during pathological conditions. For example, it has been reported that microglia are activated by aging and inflammation, affecting astrocytes, and that these astrocytes in turn damage neurons, thereby contributing to the pathological mechanisms of neurodegenerative diseases (Non-Patent Documents 3 and 4). Therefore, methods capable of assessing intercellular interactions between neurons and glial cells are extremely useful for evaluating the intrinsic function of the human brain and studying pathological mechanisms.

[0003] A study has evaluated the effects of intercellular interactions between neurons and glial cells using various coculture systems (Non-Patent Document 5). This study reported that various cell combinations, including a triple coculture of neurons, astrocytes, and microglia, altered the amount of cytokines in the culture medium after stimulation with lipopolysaccharide (LPS). These results suggest that different cells influence each other. However, this method does not allow for the evaluation of which cells are activated and when, or with which other cells. To study the effects of intercellular interactions on normal brain function and pathological mechanisms, a method that can assess when and which cells interact with which other cells would be useful.

[0004] Patent Document 1 describes the results of microglia-dependent astrocyte C3 release when a co-culture system of astrocytes, neurons, and microglia is stimulated with LPS. Patent Document 2 describes the results of LPS stimulation of a co-culture system of astrocytes, neurons, and microglia, followed by morphological observation by immunostaining and measurement of cytokines in the culture medium.

[0005] Francesca Cerbai et al., PLoS One, Vol. 7, Article No. e45250, 2012; R. Douglas Fields et al., Science, Vol. 298, pp. 556-562, 2002; Shane A. Liddelow et al., Nature, Vol. 541, pp. 481-487, 2017; Laura E. Clarke et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, pp. E1896-E1905, 2018; Sudha R. Guttikonda et al., Nature Neuroscience, Vol. 24, pp. 343-354, 2021

[0006] JP 2022-511385 A International Publication No. WO2023 / 039567 A

[0007] The objective of the present invention is to provide a method for evaluating cell-cell interactions in neuroinflammation using a co-culture containing human-derived nervous system cells that can mimic the functions of the human brain.

[0008] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that it is possible to evaluate the intercellular interactions of neuroinflammation by applying an inflammatory stimulus to a first cell contained in a co-culture containing at least two cells selected from astrocytes, neurons, microglia, and oligodendrocytes, and evaluating changes in inflammatory response markers over time in the first cell and the second cell.The present invention was completed based on these findings.

[0009] That is, the present invention provides the following: <1> A method for evaluating cell-cell interactions in neuroinflammation, comprising the steps of: preparing a co-culture containing at least two cells selected from the group consisting of human astrocytes, human neurons, human microglia, and human oligodendrocytes; applying an inflammatory stimulus to a first cell contained in the co-culture; detecting at least one selected from the group consisting of an inflammatory response marker in a cell contained in the co-culture, cellular neural activity, and cell morphology; and evaluating over time changes in at least one selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cell and a second cell contained in the co-culture, the second cell being different from the first cell. <2> The method described in <1>, wherein the astrocytes, neurons, microglia, and oligodendrocytes are differentiated from human pluripotent stem cells. <3> The method described in <2>, wherein the human pluripotent stem cells are human iPS cells. <4> The method according to any one of <1> to <3>, wherein the first cell is a human microglia, astrocyte, or neuron. <5> The method according to any one of <1> to <4>, wherein, when the first cell is a human astrocyte, the second cell is at least one selected from the group consisting of a human neuron and a microglia. <6> The method according to any one of <1> to <4>, wherein, when the first cell is a human neuron, the second cell is at least one selected from the group consisting of astrocyte and microglia. <7> The method according to any one of <1> to <4>, wherein, when the first cell is a human microglia, the second cell is at least one selected from the group consisting of astrocyte and neuron. <8> The method according to any one of <1> to <7>, wherein the co-culture is a two-dimensional culture or a three-dimensional culture. <9> The method according to any one of <1> to <8>, wherein the inflammatory stimulus is a chemical stimulus.<10> The method according to <9>, wherein the chemical stimulus is at least one substance selected from the group consisting of lipopolysaccharide, rotenone, amyloid β protein, tau protein, α-synuclein, and TDP-43. <11> The method according to any one of <1> to <8>, wherein the inflammatory stimulus is a stimulus obtained by adding cells different from the cells in the co-culture, or by adding a culture supernatant obtained by culturing cells different from the cells in the co-culture. <12> The method according to any one of <1> to <8>, wherein the inflammatory stimulus is induced by aging, genetic mutation, or gene transfer of the first cell in the co-culture. <13> The method according to <12>, wherein the protein produced by genetic mutation of the first cell in the co-culture or by gene transfer to the first cell in the co-culture is at least one selected from the group consisting of amyloid β protein, tau protein, α-synuclein, and TDP-43. <14> The method according to any one of <1> to <8>, wherein the inflammatory stimulus that stimulates the first cells is induced by a substance secreted by cells different from the first cells in the co-culture. <15> The method according to any one of <1> to <14>, wherein the inflammatory response marker is at least one selected from the group consisting of NF-κB, TNF-α, and STAT1. <16> The method according to any one of <1> to <15>, wherein the step of detecting the inflammatory response marker in cells contained in the co-culture is a step of visualizing the inflammatory marker protein by immunostaining. <17> The method according to any one of <1> to <16>, wherein the step of detecting the inflammatory response marker in cells contained in the co-culture is a step of visualizing the mRNA expression level of the inflammatory marker gene by fluorescent in situ hybridization. <18> The method according to any one of <1> to <14>, wherein the neural activity is at least one selected from the group consisting of intracellular calcium flux and cellular action potential. <19> The method according to any one of <1> to <14>, wherein the step of detecting intracellular neural activity in the cells contained in the co-culture is a step of visualizing intracellular calcium flux.<20> The method according to <19>, wherein the step of visualizing the flow of intracellular calcium is calcium imaging. <21> The method according to any one of <1> to <14>, wherein the step of detecting intracellular neural activity in the cells contained in the co-culture is a step of measuring an action potential of the cells.

[0010] The method for evaluating cell-cell interactions in neuroinflammation according to the present invention can be used to develop therapeutic drugs and discover biomarkers for various neurodegenerative diseases involving neuroinflammation.

[0011] Figure 1 shows the time course of NF-κB localization in co-cultures after LPS stimulation. Figure 2 shows staining for NF-κB and IBA1, a microglial marker, 0.5 hours after LPS stimulation. Figure 3 shows staining for NF-κB and IBA1, a microglial marker, 3 hours after LPS stimulation. Figure 4 shows the time course of NF-κB localization in neuronal and astrocyte co-cultures after LPS stimulation. Figure 5 shows the time course of NF-κB localization in R-7050-treated co-cultures after LPS stimulation. Figure 6 shows staining for NF-κB and IBA1, a microglial marker, in R-7050-treated co-cultures after 1, 3, and 6 hours after LPS stimulation. Figure 7 shows the time course of STAT1 localization in ternary cocultures of neurons, astrocytes, and microglia after stimulation with LPS. Figure 8 shows staining for STAT1 and the microglial marker IBA1 in ternary cocultures of neurons, astrocytes, and microglia 1 and 6 hours after stimulation with LPS. Figure 9 shows the time course of STAT1 localization in binary cocultures of neurons and astrocytes after stimulation with LPS. Figure 10 shows the time course of NF-κB localization in ternary cocultures of neurons, astrocytes, and microglia containing approximately 0.11% microglia after stimulation with LPS. Figure 11 shows staining for NF-κB and the microglial marker IBA1 in ternary cocultures of neurons, astrocytes, and microglia containing approximately 0.11% microglia 1 and 3 hours after stimulation with LPS. Figure 12 shows staining of NF-κB, the astrocyte marker GFAP, and the neuronal marker NeuN in neuron-astrocyte cocultures after TNF-α stimulation. Figure 13 shows calcium imaging evaluation of BPM (peak calcium oscillations per minute).

[0012] Hereinafter, embodiments of the present invention will be described in detail.

[0013] The method for evaluating cell-cell interactions in neuroinflammation according to the present invention comprises the steps of: preparing a co-culture containing at least two cells selected from the group consisting of human-derived astrocytes, human-derived neurons, human-derived microglia, and human-derived oligodendrocytes; applying an inflammatory stimulus to a first cell contained in the co-culture; detecting at least one selected from the group consisting of an inflammatory response marker in a cell contained in the co-culture, cellular neural activity, and cell morphology; and evaluating over time changes in at least one selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cell and a second cell contained in the co-culture that is different from the first cell.

[0014] In the present invention, neuroinflammation is induced in a co-culture containing at least two cells selected from the group consisting of astrocytes, neurons, microglia, and oligodendrocytes by stimulation with LPS or by introducing mutations into the cells or by forced gene expression. Then, the localization and expression level of inflammatory response markers are evaluated over time by immunostaining or fluorescence in situ hybridization (FISH). This makes it possible to visualize which of multiple cells was activated and at what timing, and to evaluate intercellular interactions by observing changes in activation over time.

[0015] Furthermore, in the present invention, in a co-culture containing at least two cells selected from the group consisting of astrocytes, neurons, microglia, and oligodendrocytes, neuroinflammation is induced by stimulation with LPS or the like, or by introducing mutations into the cells or by forced gene expression. Then, the neural activity of the cells is evaluated over time using intracellular calcium flux and cellular action potentials as indicators, thereby making it possible to visualize which of multiple cells was activated and at what timing, and to evaluate intercellular interactions by observing changes in activation over time.

[0016] Furthermore, in the present invention, in a co-culture containing at least two cells selected from the group consisting of astrocytes, neurons, microglia, and oligodendrocytes, neuroinflammation is induced by stimulation with LPS or the like, or by introducing mutations into the cells or by forced gene expression. Then, by evaluating changes in cell morphology over time using cell morphology (cell morphology) as an indicator, it is possible to visualize which of multiple cells was activated and at what timing, and to evaluate intercellular interactions by observing changes in activation over time.

[0017] The astrocytes, neurons, microglia, and oligodendrocytes are preferably differentiated from human-derived pluripotent stem cells.

[0018] 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.

[0019] Examples of human-derived pluripotent stem cells include pluripotent stem cells derived from specimens lacking mutations in disease-related genes that cause nervous system diseases, pluripotent stem cells derived from specimens collected from healthy individuals (healthy individuals) without nervous system diseases, pluripotent stem cells derived from specimens collected from patients with diseases, and pluripotent stem cells derived from specimens with mutations in disease-related genes. "No mutations in disease-related genes" refers to the absence of mutations in disease-related genes that would cause nervous system diseases. That is, even if a gene has a mutation, if the mutation does not cause the disease, it is interpreted as the absence of a mutation in the disease-related gene. Furthermore, "mutations in disease-related genes" may refer to endogenous gene mutations inherent in the patient or exogenous gene mutations artificially introduced into the patient. It is anticipated that the co-culture of the present invention can mimic the pathology of a disease originating from the specimen or a disease caused by a mutation in a disease-related gene by preparing the co-culture of the present invention using pluripotent stem cells derived from a specimen collected from a patient with a disease or pluripotent stem cells derived from a specimen with a mutation in a disease-related gene.

[0020] ES cells can be established, for example, by culturing pre-implantation early embryos, inner cell masses constituting the above-mentioned early embryos, single blastomeres, and the like (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; Akira Iritani et al.: Protein Nucleic Acid Enzymes, 44, 892, 1999; Baguisi et al.: Nature Biotechnology, 17, 456, 1999; Wakayama et al.: Nature, 394, 369, 1998; Nature Genetics, 22, 127, 1999; Proc. Natl. Acad. Sci. USA, 96, 14984, 1999; Rideout III et al. al: Nature Genetics, 24, 109, 2000, Tachibana et al: Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell, 2013, in press). As the early embryo, a parthenogenetic embryo may be used (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 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, P. M. et al: Methods in Enzymology, Vol. 365, 2003, etc. are useful for the production of ES cells. Fusion ES cells obtained by cell fusion between ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

[0021] Some ES cells are available from collection institutions or commercially available. For example, human ES cells are available from the Institute for Frontier Medical Sciences, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0022] 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.

[0023] The most basic method for generating iPS cells is to introduce four transcription factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into cells using a virus (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi K, et al.: Cell 131(5), 861-72, 2007). There have been reports of the establishment of human iPS cells by introducing four factors, Oct4, Sox2, Lin28, and Nanog (Yu J, et al.: Science 318(5858), 1917-1920, 2007). It has also been reported that iPS cells can be established 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 J B, et al: Nature 454(7204), 646-650, 2008), or only Oct3 / 4 (Kim J B, et al: Cell 136(3), 411-419, 2009). Furthermore, a method for introducing a protein, which is an expression product of a gene, into a cell has 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 an inhibitor of histone methyltransferase G9a, such as BIX-01294, or a histone deacetylase inhibitor, such as valproic acid (VPA) or BayK8644, can improve the production efficiency and reduce the amount of factors to be 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).Methods for gene transfer 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: 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. 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.

[0024] 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.

[0025] 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).

[0026] iPS cells can also be obtained from, for example, FUJIFILM Cellular Dynamics, Inc. (FCDI), Kyoto University, or the RIKEN BioResource Center.

[0027] Examples of human-derived astrocytes include astrocytes induced to differentiate from astrocyte precursor cells (e.g., XCell Science, XCS-AP-001-1V), iCell (registered trademark) astrocytes (FUJIFILM Cellular Dynamics, C1037), etc., with astrocytes induced to differentiate from astrocyte precursor cells being preferred. When inducing differentiation from astrocyte precursor cells into astrocytes, it is preferable to induce differentiation in the absence of serum.

[0028] Methods for obtaining human-derived nerve 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 a patient with a nervous system disease, or from an established human iPS cell line.

[0029] The nerve cells induced to differentiate from human-derived pluripotent stem cells are not particularly limited, but are preferably motor nerve cells, cerebral cortical excitatory nerve cells, or substantia nigra nerve cells.

[0030] Methods for inducing differentiation of human-derived pluripotent stem cells into neurons include, but are not limited to, a method in which neural stem cells are produced from pluripotent stem cells using treatment with low molecular weight compounds, etc., and then the differentiation into neurons is induced directly by gene expression, etc.

[0031] Methods for inducing differentiation of pluripotent stem cells into neural cells include, for example, (1) a method of culturing in a serum-free medium to form embryoid bodies (cell masses containing neural precursor cells) and then differentiating them (SFEB method: Watanabe K, et al., Nat. Neurosci., 8: 288-296, 2005; SFEBq method: Wataya T., et al., Proc. Natl. Acad. Sci. USA., 105: 11796-11801, 2008); (2) a method of culturing on stromal cells to differentiate them (SDIA method: Kawasaki H, et al., Neuron, 28: 31-40, 2000); (3) a method of culturing on drug-added matrigel to differentiate them (Chambers S.M., et al., J. Immunol. 2010, 11: 11796-11801, 2000); 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. Pat. No. 5,843,780); (5) a method of differentiation by introducing and expressing a neural induction factor (such as a gene encoding Neurogenin 2 (NGN2) protein) into pluripotent stem cells (WO2014 / 148646; and Zhang Y, et al, Neuron, 78:785-98, 2013); (6) a method of differentiation by introducing and expressing miR-9 / 9*-124 into pluripotent stem cells; and combinations of these methods.

[0032] Of the above, (5) the method of introducing a gene encoding the NGN2 protein into pluripotent stem cells and expressing it is preferred because it allows mature neurons to be obtained in a short period of time and with high efficiency.

[0033] Examples of human-derived neurons include induced neurons differentiated from iPS cells by forced expression of NGN2, iCell (registered trademark) glutamatergic neurons (FUJIFILM Cellular Dynamics, C1033), iCell (registered trademark) GABAergic neurons (FUJIFILM Cellular Dynamics, C1008), iCell (registered trademark) dopaminergic neurons (FUJIFILM Cellular Dynamics, C1028), and iCell (registered trademark) motor neurons (FUJIFILM Cellular Dynamics, C1048), of which induced neurons differentiated from iPS cells by forced expression of NGN2 and iCell (registered trademark) glutamatergic neurons are preferred.

[0034] Examples of human-derived microglia include iCell (registered trademark) microglia (FUJIFILM Cellular Dynamics, C1110) and microglia (Axol Bioscience, AX0664), with iCell (registered trademark) microglia being preferred.

[0035] Human-derived oligodendrocytes include induced oligodendrocytes differentiated from iPS cells by forced expression of SOX10 (SRY-box transcription factor 10), OLIG2 (Oligodendrocyte lineage transcription factor 2), and NKX6-2 (NK6 homeobox 2); induced oligodendrocytes differentiated from iPS cells by forced expression of SOX10 and OLIG2; induced oligodendrocytes differentiated from iPS cells by forced expression of SOX10; and oligodendrocytes induced to differentiate from iPS cells by adding a substance that promotes oligodendrocyte induction to the culture medium (Panagiotis Douvaras, et al., Nat. Protocols., 10:1143-54, 2015; Tomoko Yamashita, et al., PLoS One., 12: e0171847.), and induced oligodendrocytes differentiated from iPS cells by forced expression of SOX10, OLIG2, and NKX6-2 are preferred.

[0036] Astrocytes are cells that express at least one astrocyte-specific marker gene, which includes GFAP (glial fibrillary acidic protein), S100β (S100 calcium binding protein B), KCNJ10 (potassium inwardly rectifying channel subfamily J member 10), AQP4 (aquaporin-4), and SLC1A3 (solute carrier family 1 member 3).

[0037] 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).

[0038] Microglia are cells that express at least one of the microglia-specific marker genes, including IBA1 (ionized calcium-binding adapter molecule 1), CD33, CD45, TREM2 (triggering receptor expressed on myeloid cells 2), P2RY12 (purinergic receptor P2Y, G-protein coupled, 12), TMEM119 (transmembrane protein 119), and CX3CR1 (CX3C-chemokine receptor 1).

[0039] Oligodendrocytes are cells that express at least one or more of the oligodendrocyte-specific marker genes OLIG2, O4 (oligodendrocyte marker O4), and MBP (myelin basic protein).

[0040] 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.

[0041] In the present invention, a co-culture is prepared containing at least two cells selected from the group consisting of human astrocytes, human neurons, human microglia, and human oligodendrocytes. The cell combinations used include, but are not limited to, astrocytes and neurons, astrocytes and microglia, astrocytes and oligodendrocytes, neurons and microglia, neurons and oligodendrocytes, microglia and oligodendrocytes, astrocytes, neurons and microglia, astrocytes, neurons and oligodendrocytes, astrocytes, microglia and oligodendrocytes, neurons, microglia and oligodendrocytes, and astrocytes, neurons, microglia and oligodendrocytes.

[0042] For example, when astrocytes, neurons, and microglia are used, the proportion of astrocytes among all cells used is 30% or more, preferably 35% or more, more preferably 40% or more, and even more preferably 45% or more. The lower limit of the proportion of astrocytes among all cells used is not particularly limited, but it is sufficient that at least one cell is present in the culture vessel, and is generally 0.1% or more, preferably 1% or more, more preferably 5% or more, and even more preferably 10% or more. The upper limit of the proportion of astrocytes among all cells used is not particularly limited, but it is generally 80% or less, preferably 70% or less. The proportion of microglia among all cells used is preferably 50% or less, more preferably 40% or less, and even more preferably 30% or less. The lower limit of the proportion of microglia among all cells used is not particularly limited, but it is sufficient that at least one cell is present in the culture vessel, and is generally 0.1% or more, preferably 1% or more, more preferably 2% or more, even more preferably 5% or more, and even more preferably 10% or more. The upper limit of the proportion of microglia among all cells used is not particularly limited, but is generally 65% ​​or less, preferably 60% or less. The proportion of neurons among all cells used is preferably 10% or more, more preferably 15% or more, even more preferably 20% or more, and particularly preferably 25% or more. The lower limit of the proportion of neurons among all cells used is not particularly limited, but it is sufficient that there is at least one cell in the culture vessel, and is generally 0.1% or more, preferably 1% or more, and more preferably 5% or more. The upper limit of the proportion of neurons among all cells used is not particularly limited, but is generally 65% ​​or less, preferably 60% or less.

[0043] In the present invention, the above-mentioned cells are co-cultured in a culture vessel. Examples of culture vessels that can be used include well-equipped plates, dishes, cell culture inserts, and cell culture flasks, with well-equipped plates being preferred. Specific examples include ViewPlate (PerkinElmer), 96-well microplates (Greiner), 96-well polystyrene microplates (Corning), PrimeSurface® plates (Sumitomo Bakelite), and cell-repellent plates (Greiner Bio One). ViewPlate (PerkinElmer) and PrimeSurface® plates (Sumitomo Bakelite) are preferred.

[0044] The shape of the culture vessel may be, but is not limited to, a flat bottom, a round bottom, a U-bottom, or a V-bottom.

[0045] When the co-culture is a three-dimensional culture, the surface in contact with the medium in the container is preferably non-cell-adhesive. This allows the cells to be cultured in a suspended state, facilitating the formation of spheroids. Alternatively, only certain areas of the surface inside the container may be cell-adhesive, while the remaining areas may be non-cell-adhesive. In this case, cells can gather at the cell-adhesive areas and form spheroids.

[0046] When the co-culture is a two-dimensional culture, the lower limit of the cell density (the cell density is the total cell density of two or more cells used; the same applies below) when seeded into the culture vessel is not particularly limited, but is, for example, 2.5 × 10 ４ cells / cm ２ It is preferable that the ratio is 5.0×10 or more. ４ cells / cm ２ More preferably, it is 10.0 x 10 ４ cells / cm ２ More preferably, it is 15.0 × 10 or more. ４ cells / cm ２ It is even more preferable that the value is 20.0 × 10 or more. ４ cells / cm ２It is particularly preferable that the value is 25.0×10 or more. ４ cells / cm ２ The upper limit of the cell density at the time of seeding into the culture vessel is not particularly limited, but for example, it is preferably 60.0 × 10 ４ cells / cm ２ or less, 55.0 × 10 ４ cells / cm ２ Preferably, less than 50.0 x 10 ４ cells / cm ２ More preferably, 45.0 x 10 ４ cells / cm ２ More preferably, 40.0 x 10 ４ cells / cm ２ Even more preferably, the following is 35.0 x 10 ４ cells / cm ２ The following are particularly preferred:

[0047] When the co-culture is a two-dimensional culture, the cell density at the time of seeding into the culture vessel is preferably 5.0 × 10 ４ cells / cm ２ Above 55.0 x 10 ４ cells / cm ２ or less, more preferably 10.0 × 10 ４ cells / cm ２ Above 50.0 x 10 ４ cells / cm ２ More preferably, it is 15.0 × 10 ４ cells / cm ２ Above 45.0 x 10 ４ cells / cm ２ and even more preferably 20.0 × 10 ４ cells / cm ２ Above 40.0 x 10 ４ cells / cm ２ It is particularly preferably 25.0 × 10 ４ cells / cm ２ 35.0 x 10 ４ cells / cm ２ The following is the result.

[0048] When the co-culture is a three-dimensional culture, the lower limit of the number of cells (the number of cells is the total number of two or three types of cells; the same applies below) seeded onto a 96-well plate is not particularly limited, but is, for example, 0.7 × 10 ４ Preferably, the number of cells / well is 1.0 x 10 ４ More preferably, 1.2 x 10 cells / well or more ４ More preferably, 1.4 x 10 cells / well or more ４ Even more preferably, 1.6 x 10 cells / well or more ４ More than 1.8 x 10 cells / well is particularly preferred. ４ The upper limit of the cell density at the time of seeding into the culture vessel is not particularly limited, but is, for example, 8.0 × 10 ４ cells / well or less, 8.0 x 10 ４ Preferably less than 5.0 x 10 cells / well ４ cells / well or less, more preferably 3.0 x 10 ４ More preferably, 2.5 x 10 cells / well or less ４ Even more preferably, 2.3 x 10 cells / well or less ４ Cells / well or less is particularly preferred.

[0049] When the co-culture is a three-dimensional culture, the number of cells seeded onto a 96-well plate is preferably 1.0 x 10 ４ Cells / well > 8.0 x 10 ４ cells / well or less, more preferably 1.2 x 10 ４ 5.0 x 10 cells / well or more ４ cells / well or less, more preferably 1.4 x 10 ４ Cells / well > 3.0 x 10 ４ cells / well or less, and even more preferably 1.6 x 10 ４ 2.5 x 10 cells / well or more ４ cells / well or less, and particularly preferably 1.8 x 10 ４ Cells / well > 2.3 x 10 ４ cells / well or less.

[0050] 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, the basal medium may be, for example, DMEM, DMEM (Dulbecco's Modified Eagle Medium) / F12, BrainPhys (registered trademark) Neuronal Medium, Neurobasal (trademark) Medium-A, Neurobasal (trademark) 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 Examples of the medium include 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.

[0051] Specific examples of additives that can be added to the culture medium include serum, retinoic acid, Wnt, bone morphogenetic protein (BMP) (e.g., BMP-4), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (Shh), insulin-like growth factor 1 (IGF-1), Activin A, Heregulin β-1, interleukins, 8-Br-cAMP, heparin, heparan sulfate, laminin, collagen, fibronectin, progesterone, selenite, B-27 (registered trademark) supplement, N2 supplement with transferrin (Apo), N2 Examples of such supplements include, but are not limited to, Supplement with Transferrin, GlutaMAX™, L(+)-ascorbic acid, ITS-Supplement, MEM Non-Essential Amino Acid, etc. Furthermore, antibiotics (penicillin, streptomycin, etc.) may also be added.

[0052] The culture conditions may be selected from those for general cell culture: 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 not necessary to change the medium during the culture period.

[0053] The co-culture may be either a two-dimensional culture or a three-dimensional culture, which may be in the form of a spheroid.

[0054] In the present invention, a step of applying an inflammatory stimulus to first cells contained in the co-culture is carried out. The first cells are preferably human microglia, human astrocytes, or human neurons. The first cells may be one type or two or more types, and are not particularly limited, but are preferably one type, for example.

[0055] In the present invention, even if an inflammatory stimulus to a first cell induces a change in an inflammatory response marker in a second cell different from the first cell, if the change can be distinguished from the change in the inflammatory response marker in the second cell induced by the first cell to which the inflammatory stimulus has been applied, the second cell different from the first cell is not considered to have been stimulated by the inflammatory stimulus to the first cell.

[0056] An example of the inflammatory stimulus is a chemical stimulus, such as, but not limited to, at least one substance selected from the group consisting of lipopolysaccharide, rotenone, amyloid β protein, tau protein, α-synuclein, and TDP-43 (TAR DNA-binding protein 43).

[0057] Another example of an inflammatory stimulus is stimulation by adding cells different from the cells in the co-culture, or by adding a culture supernatant obtained by culturing cells different from the cells in the co-culture.

[0058] Another example of an inflammatory stimulus is one induced by senescence, genetic mutation, or gene transfer of the first cell in the co-culture. For example, if the first cell in the co-culture is an astrocyte, senescent astrocytes can be used as the inflammatory stimulus. For one induced by genetic mutation or gene transfer, genetically engineered cells can be used as the inflammatory stimulus.

[0059] The protein produced by genetic mutation of the first cells in the co-culture or by gene transfer into the first cells in the co-culture can include, but is not limited to, at least one selected from the group consisting of amyloid β protein, tau protein, α-synuclein, and TDP-43. The inflammatory stimulus that stimulates the first cells may be induced by a substance secreted by a cell different from the first cells in the co-culture.

[0060] Further examples of inflammatory stimuli include physical stimuli, such as ultraviolet (UV) irradiation and mechanical stimuli (e.g., pipetting using a culture pipette), but are not limited thereto.

[0061] The step of applying an inflammatory stimulus to the first cells contained in the co-culture can be carried out for, for example, 10 minutes or more, preferably 20 minutes or more, more preferably 30 minutes or more, even more preferably 1 hour or more, even more preferably 2 hours or more, and even more preferably 3 hours or more. The step of applying an inflammatory stimulus to the first cells contained in the co-culture can be carried out for, for example, 120 hours or less, preferably 96 hours or less, more preferably 72 hours or less, and even more preferably 48 hours or less.

[0062] In the present invention, a step of detecting at least one selected from the group consisting of an inflammatory response marker in cells contained in a co-culture, cellular neural activity, and cell morphology, and a step of evaluating changes in at least one selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology over time in a first cell and a second cell contained in the co-culture that is different from the first cell, are carried out.

[0063] In the present invention, in evaluating the intercellular interaction between the first cell and a second cell different from the first cell, an inflammatory stimulus may be applied to the first cell, resulting in activation or inhibition of the second cell, and then it may be evaluated whether the activation or inhibition of the second cell affects the function of the first cell. In this case, it is envisioned that a step of evaluating, over time, at least one change selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cell is carried out.

[0064] As a first example, when the first cell is a human astrocyte, the second cell is preferably at least one selected from the group consisting of a human neuron and a human microglia. As a second example, when the first cell is a human neuron, the second cell is preferably at least one selected from the group consisting of a human astrocyte and a human microglia. As a third example, when the first cell is a human microglia, the second cell is preferably at least one selected from the group consisting of a human astrocyte and a human neuron.

[0065] In the present invention, in addition to evaluating the cell-cell interaction between the first cell and the second cell, a step of evaluating over time in a third cell different from the first cell and the second cell in at least one change selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology may be performed. In this case, it is possible to evaluate whether application of an inflammatory stimulus to the first cell results in activation or inhibition of the second cell different from the first cell, and further whether the second cell affects the function of the third cell different from the first cell and the second cell.

[0066] When evaluating interactions between three different types of cells, for example, as a first example, when the first cell is a human astrocyte, the second cell is a human neuron, and the third cell is a human microglia. Alternatively, as another embodiment, when the first cell is a human astrocyte, the second cell is a human microglia, and the third cell is a human neuron. As a second example, when the first cell is a human neuron, the second cell is a human astrocyte, and the third cell is a human microglia. Alternatively, as another embodiment, when the first cell is a human neuron, the second cell is a human microglia, and the third cell is astrocyte. As a third example, when the first cell is a human microglia, the second cell is a human astrocyte, and the third cell is a human neuron. In another embodiment, when the first cell is a human microglia, the second cell is a human neuron, and the third cell is a human astrocyte. When evaluating interactions between three different types of cells, it is preferable that when the first cell is a human microglia, the second cell is a human astrocyte, and the third cell is a human neuron.

[0067] In the present invention, cell-cell interaction can be evaluated by stimulating the first cells in a co-culture and then observing an inflammatory response marker over time. The inflammatory response marker is preferably at least one selected from the group consisting of NF-κB, TNF-α, and STAT1, more preferably NF-κB or STAT1, and particularly preferably NF-κB.

[0068] In the present invention, the inflammatory response marker of the second cell is an inflammatory response marker induced by the first cell to which an inflammatory stimulus has been applied, from the viewpoint of evaluating whether or not an intercellular interaction occurs between the first cell and the second cell.

[0069] In the present invention, the inflammatory response marker of a third cell is an inflammatory response marker induced by a second cell that has interacted with a first cell, from the perspective of evaluating whether intercellular interaction occurs between the second cell and the third cell.

[0070] In the present invention, various markers such as mRNA of NF-κB, STAT1, or TNF-α can be selected as inflammatory response markers, allowing a wide range of activation and the accompanying intercellular interactions to be evaluated. Furthermore, because of changes in gene expression and protein localization, intercellular interactions can be evaluated by capturing changes in the early stages of the response.

[0071] Specifically, it is preferable to observe changes in the localization of NF-κB, changes in the localization of STAT1, changes in the expression level of TNF-α mRNA, etc. The step of detecting an inflammatory response marker in cells contained in the co-culture product may be, for example, a step of visualizing an inflammatory marker protein by immunostaining, or a step of visualizing the expression level of mRNA of an inflammatory marker gene by fluorescence in situ hybridization (FISH).

[0072] The step of evaluating the change in the inflammatory response marker over time preferably involves continuously measuring the change in the visualized inflammatory response marker, for example, from 0 hour to 7 days, preferably from 0 hour to 3 days, more preferably from 0 hour to 48 hours, and even more preferably from 0 hour to 24 hours. By using immunostaining or FISH, it is possible to evaluate intercellular interactions by visualizing in which cells the inflammatory response marker has changed and when.

[0073] In the present invention, cell-cell interactions can be evaluated by stimulating the first cell in the co-culture and then observing changes in neural activity over time. The neural activity is preferably at least one selected from the group consisting of intracellular calcium flux and cellular action potentials, with intracellular calcium flux being more preferred.

[0074] In the present invention, the neural activity of the second cell is the neural activity induced by the first cell to which an inflammatory stimulus has been applied, from the viewpoint of evaluating whether intercellular interaction occurs between the first cell and the second cell.

[0075] In the present invention, the neural activity of a third cell is the neural activity induced by a second cell that interacts with a first cell, from the perspective of evaluating whether intercellular interaction occurs between the second cell and the third cell.

[0076] In the present invention, various indicators of neural activity, such as intracellular calcium flux or cellular action potential, can be selected, allowing a wide range of activation and the accompanying intercellular interactions to be evaluated.

[0077] Specifically, it is preferable to observe changes in intracellular calcium flux or changes in cellular action potential.

[0078] The step of detecting intracellular neural activity in the co-culture may be, for example, a step of visualizing intracellular calcium flux. A method for visualizing intracellular calcium flux includes calcium imaging. Furthermore, by visualizing intracellular calcium flux using calcium imaging and measuring calcium oscillations, the number of calcium oscillation peaks per minute (Beats Per Minute, BPM) can be evaluated, for example.

[0079] The step of detecting neural activity in cells contained in the co-culture may also be, for example, a step of measuring the action potential of the cells, which may involve measuring changes in the action potential of individual cells using a microelectrode array (MEA), or measuring changes in the number of network bursts or peak intervals between cells.

[0080] In the step of evaluating changes in neural activity over time, it is preferable to continuously measure the detected changes in neural activity, for example, from 0 hour to 7 days, preferably from 0 hour to 3 days, more preferably from 0 hour to 48 hours, and even more preferably from 0 hour to 24 hours. The MEA method makes it possible to evaluate intercellular interactions by visualizing in which cells changes in neural activity and when they occurred.

[0081] In the present invention, cell-cell interactions can be evaluated by stimulating the first cell in the co-culture and then observing changes in cell morphology over time.

[0082] In the present invention, the change in morphology of the second cell is a change in cell morphology induced by the first cell to which an inflammatory stimulus has been applied, from the viewpoint of evaluating whether or not an intercellular interaction occurs between the first cell and the second cell.

[0083] In the present invention, the change in morphology of a third cell is a change in cell morphology induced by a second cell that has interacted with a first cell, from the perspective of evaluating whether intercellular interaction occurs between the second cell and the third cell.

[0084] The step of detecting the morphology of cells contained in the co-culture may include, for example, a step of visualizing the cells contained in the co-culture by immunostaining a marker protein specific to each cell. Further, for example, a step of visualizing cells sampled over time by immunostaining, taking images of the cells, and analyzing the obtained images to measure changes in cell morphology may be included.

[0085] In the step of evaluating changes in cell morphology over time, it is preferable to continuously evaluate the detected changes in cell morphology, for example, from 0 hour to 7 days, preferably from 0 hour to 3 days, more preferably from 0 hour to 48 hours, and even more preferably from 0 hour to 24 hours. By analyzing the changes over time in the obtained images, it is possible to visualize which cells changed in cell morphology and when, and evaluate intercellular interactions.

[0086] The present invention will be specifically explained by the following examples, but the present invention is not limited to the scope of the examples.

[0087] Test Example 1: Evaluation of cell-cell interactions by immunostaining <1> Preparation of two-dimensional co-culture of astrocytes, neurons, and microglia Matrigel basement membrane matrix (Corning, 356234) diluted 120-fold with DMEM / F12 (Life technologies, 11320-033) was added to a 96-well plate at 65 μL / well and allowed to stand at 4° C. After 24 hours, the medium was replaced with progenitor cell medium and used.

[0088] Astrocyte precursor cells (XCell Science, XCS-AP-001-1V) were cultured in a 96-well plate at 5 × 10 ４ The cells were seeded at a density of 1000 cells / well and incubated at 37°C, 5% CO ２ The next day, the medium was replaced with a differentiation-inducing medium, and the cells were cultured for an additional 5 days to induce differentiation into astrocytes.

[0089] The neurons used were cryopreserved neurons generated by forced expression of the Ngn2 gene from iPS cells (WO2014 / 148646; and Zhang Y, et al., Neuron, 78:785-98, 2013). The cryopreserved cells were thawed in a 37°C warm bath. After thawing, the cells were added to co-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 co-culture medium, and the cell number was counted.

[0090] iPS cell-derived microglia (iCell® Microglia, FCDI, C1110) were used as microglia. Cryopreserved cells were thawed in a 37°C warm bath. After thawing, the cells were added to co-culture medium and centrifuged at 600 × g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0091] Neurons and microglia were mixed at a ratio of 3:1, and 4 × 10 ４ The cells were seeded on top of the astrocytes at 100 cells / well. ２ The cells were cultured under these conditions, and half of the medium was replaced three times a week.

[0092] <2> Microglia activation and cell fixation. Microglia were specifically stimulated by adding LPS (Fujifilm Wako Pure Chemical Industries, Ltd., 124-05151) to 100 ng / mL of 2D co-cultures cultured for 4 weeks. Cells were fixed by 30-minute treatment with formaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd., 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., 166-23555) at 0.5, 1, 3, 6, and 24 hours after treatment.

[0093] In addition, 2D co-cultures cultured for 19 days were treated with 100 ng / mL LPS (Fujifilm Wako Pure Chemical Industries, Ltd., 124-05151) and 10 μmol / L of the TNFR1 inhibitor R-7050 (Cayman Chemical Company, 16870). The effect of TNF-α on cell-cell interactions induced by LPS stimulation was evaluated. Cells were fixed for 30 minutes with formaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd., 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., 166-23555) at 0.5, 1, 3, 6, and 24 hours after treatment.

[0094] <3> Evaluation of NF-κB Localization The fixed cells were washed with PBS(-) and then blocked and permeabilized by treatment for 30 minutes with a solution of BSA (Sigma Aldrich, A4161) and Triton® X-100 (BioVision, 2104-100) diluted to 1% and 0.2% in PBS(-) (1% BSA / 0.2% Triton X-100). Subsequently, as the primary antibody reaction, the cells were treated with an anti-IBA antibody (Fujifilm Wako Pure Chemical Industries, 011-27991) diluted 800-fold with 1% BSA / 0.2% Triton X-100, and an anti-NF-κB antibody (Cell Signaling, 8242) diluted 400-fold, and then left to stand overnight at 4°C.

[0095] The next day, after washing with PBS(-), the sections were treated with 1000-fold diluted Donkey anti-goat Alexa Fluor® 488 (Thermo Fisher Scientific, A11055), 1000-fold diluted Donkey anti-rabbit Alexa Fluor® 594 (Thermo Fisher Scientific, A32754), or 1000-fold diluted Hoechst® 33342 (Dojindo, 346-07951) as a secondary antibody reaction, and then left to stand at room temperature for 60 minutes.

[0096] After washing with PBS(-), the cells were photographed using a fluorescence microscope (Nikon, ECLIPSE Ti) and images were acquired. Figure 1 shows the time-dependent changes in the localization of NF-κB after LPS stimulation in two-dimensional co-cultures. In the absence of R-7050 treatment, nuclear translocation of NF-κB was observed only in some cells 0.5 and 1 hour after LPS treatment, but in most cells 3 and 6 hours later.

[0097] Figures 2 and 3 show staining for NF-κB and the microglial marker IBA1. At 0.5 hours after LPS stimulation, NF-κB localized in the nucleus colocalized with the microglial marker IBA1. Meanwhile, at 3 hours after LPS stimulation, NF-κB was localized in the nucleus in cells other than IBA1-positive cells. This indicates that at 0.5 hours after LPS stimulation, microglia were activated, resulting in NF-κB translocation to the nucleus, and subsequently, other cells were activated, resulting in NF-κB translocation to the nucleus. Figure 4 shows the time-dependent change in NF-κB localization in a neuron-astrocyte coculture after LPS stimulation. No nuclear translocation of NF-κB was observed at any time point. These results confirm that microglial activation activated other cells and that cell-cell interactions occurred.

[0098] On the other hand, as shown in Figure 5, under the R-7050 treatment condition, NF-κB nuclear translocation was confirmed only in a portion of cells at 1, 3, and 6 hours after LPS stimulation. Furthermore, as shown in Figure 6, the NF-κB nuclear signal at 1, 3, and 6 hours after LPS stimulation all colocalized with the microglial marker IBA1, confirming that NF-κB nuclear translocation occurred in microglia. These results indicated that treatment with a TNFR1 inhibitor suppressed the influence of microglial activation on other cells (cell-cell interaction). Furthermore, it was shown that the cell-cell interaction between microglia and other cells is mediated by TNF-α.

[0099] <4> Evaluation of STAT1 Localization The fixed cells were washed with PBS(-) and then blocked and permeabilized by treatment with a solution of BSA (Sigma Aldrich, A4161) and Triton® X-100 (BioVision, 2104-100) diluted to 1% and 0.2% in PBS(-) (1% BSA / 0.2% Triton X-100) for 30 minutes. Subsequently, as the primary antibody reaction, the cells were treated with an anti-IBA antibody (Fujifilm Wako Pure Chemical Industries, 011-27991) diluted 800-fold in 1% BSA / 0.2% Triton X-100, or an anti-STAT1 antibody (Cell Signaling, 14994S) diluted 400-fold, and then left to stand overnight at 4°C.

[0100] The next day, after washing with PBS (-), the secondary antibody reaction was performed using 1000-fold diluted Donkey anti-goat Alexa Fluor (registered trademark) 488 (Thermo Fisher Scientific, A11055), 1000-fold diluted Donkey anti-rabbit Alexa Fluor (registered trademark) 594 (Thermo Fisher Scientific, A32754), and 1000-fold diluted Hoechst (registered trademark) 33342 (Dojindo, 346-07951), and left to stand at room temperature for 60 minutes. After washing with PBS (-), the image was taken with a fluorescence microscope (Nikon, ECLIPSE Ti) to obtain the image.

[0101] STAT1 localization was evaluated using the method described in <4> of Test Example 1. Figure 7 shows the time-dependent change in STAT1 localization after LPS stimulation in a triple coculture system. At 1 hour and 3 hours after LPS stimulation, STAT1 was localized to the nucleus in some cells, but at 6 hours after stimulation, STAT1 was localized to the nucleus in many cells. Figure 8 shows staining of STAT1 and IBA1, a microglial marker. STAT1 localized to the nucleus at 1 hour after LPS stimulation colocalized with the microglial marker IBA1. Meanwhile, at 6 hours after LPS stimulation, STAT1 was localized to the nucleus in cells other than IBA1-positive cells. In other words, it was confirmed that at 1 hour and 3 hours after LPS stimulation, only microglia were activated, resulting in STAT1 nuclear translocation, and subsequently, other cells were also activated, resulting in STAT1 nuclear translocation. Figure 9 shows the time course of STAT1 localization after LPS stimulation in a co-culture of neurons and astrocytes. As a result, no nuclear translocation of STAT1 was observed at any time point. These results confirm that microglial activation activated other cells and that cell-cell interactions occurred.

[0102] Test Example 2: Evaluation of intercellular interactions by immunostaining using a three-dimensional ternary co-culture system <1> Preparation of three-dimensional co-culture Human iPS cell-derived astrocyte precursor cells (XCell Science, XCS-AP-001-1V) were seeded in a flask coated with Matrigel basement membrane matrix (Corning, 354234) and cultured for 5 days (37°C, 5% CO) using a differentiation-inducing medium. ２ ) induces differentiation into astrocytes.

[0103]

[0104] The differentiated astrocytes were detached using TrypLE™ Select (Thermo Fisher Scientific, 12563-029) and centrifuged at 600 x g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0105] Neurons generated by forced expression of the Ngn2 gene from iPS cells are thawed in a 37°C warm bath. After thawing, the cells are added to co-culture medium and centrifuged at 600 x g at room temperature for 5 minutes. After centrifugation, the supernatant is removed, and the cells are suspended in 1 mL of co-culture medium and counted.

[0106] iPS cell-derived microglia (iCell® Microglia, FCDI, C1110) are thawed in a 37°C water bath. After thawing, the cells are added to co-culture medium and centrifuged at 600 x g for 5 minutes at room temperature. After centrifugation, the supernatant is removed, and the cells are suspended in 1 mL of co-culture medium and counted.

[0107] Astrocytes, neurons, and microglia were mixed in a ratio of 10:6:3, and 1.9 × 10 ４ The cells were seeded onto a 96-well plate (PrimeSurface (registered trademark) Plate 96U, Sumitomo Bakelite, MS-9096U) at 37°C, 5% CO ２ The cells were cultured under the conditions described above, and half of the medium was replaced three times a week.

[0108]

[0109] <2> Cell-cell interactions during neuroinflammation Three-dimensional co-cultures cultured for three weeks were treated with lipopolysaccharide (LPS) from E. coli O127 (Fujifilm Wako Pure Chemical Industries, Ltd., 124-05151) to a final concentration of 100 ng / mL, and fixed for 30 minutes with paraformaldehyde phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd., 161-20141) diluted to 3% with PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd., 166-23555) after 0, 0.5, 1, 3, 6, and 24 hours. After washing with PBS (-), the cells were permeabilized by treating with Triton (registered trademark) X-100 (BioVision, Ltd., 2104-100) diluted to 0.4% with PBS (-) for 15 minutes. After permeabilization, the sections were blocked by treating them with a 4% BSA solution (Sigma Aldrich, A416) prepared in PBS(-) for 30 minutes. Then, as the primary antibody reaction, they were treated with a 1000-fold diluted anti-IBA1 antibody (Fujifilm Wako Pure Chemical Industries, Ltd., 011-27991) and an 800-fold diluted anti-NF-κB p65 antibody (Cell Signaling, 8242), and left to stand overnight at 4°C.

[0110] The next day, after washing with PBS(-), the sections were treated with 1000-fold diluted Donkey anti-goat Alexa Fluor® 488 (Thermo Fisher Scientific, A11055), 1000-fold diluted Donkey anti-rabbit Alexa Fluor® 594 (Thermo Fisher Scientific, A32754), and 1000-fold diluted Hoechst® 33342 solution (Dojindo, H342) as secondary antibody reactions, and then allowed to stand at room temperature for 60 minutes. After washing with PBS(-), images are taken using a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation, CellVoyager (registered trademark) CQ1) to obtain images.

[0111] Test Example 3: Evaluation of the amount of microglia required to induce cell-cell interactions The amount of cells required for activated microglia to affect other cells was examined. Two-dimensional ternary co-cultures of astrocytes, neurons, and microglia were prepared using the same method as in <1> of Test Example 1, so that the microglia ratios were approximately 11%, 1.1%, and 0.11%. Furthermore, LPS stimulation and cell fixation were performed using the same method as in <1> of Test Example 1. Furthermore, NF-κB localization was evaluated using the same method as in <1> of Test Example 1.

[0112] The time course of NF-κB localization in a three-component coculture in which the proportion of microglia was approximately 0.11% is shown in Figure 10. As a result, nuclear translocation of NF-κB was confirmed only in some cells 1 hour after LPS treatment, but in many cells 3 and 6 hours after treatment.

[0113] Figure 11 shows staining for NF-κB and the microglial marker IBA1. One hour after LPS stimulation, the NF-κB signal localized in the nucleus co-localized with the microglial marker IBA1. Meanwhile, three hours after LPS stimulation, NF-κB was localized in the nucleus in cells other than IBA1-positive cells. This means that even in a three-way co-culture where the proportion of microglia was approximately 0.11%, it was confirmed that microglia were activated first after LPS stimulation, followed by activation of other cells, and that cell-cell interactions were occurring.

[0114] Test Example 4: Evaluation of cell-cell interactions by calcium imaging <1> Preparation of binary co-cultures of astrocytes and neurons, and ternary co-cultures of astrocytes, neurons, and microglia Matrigel basement membrane matrix (Corning, 356234) diluted 120-fold with DMEM / F12 (Life technologies, 11320-033) was added to a 96-well plate at 65 μL / well and allowed to stand at 4° C. After 24 hours, the medium was replaced with progenitor cell medium and used.

[0115] Astrocyte precursor cells (XCell Science, XCS-AP-001-1V) were cultured in a 96-well plate at 6 × 10 ４The cells were seeded at a density of 1000 cells / well and cultured at 37°C and 5% CO2. After the medium was replaced with a differentiation-inducing medium, the cells were cultured for an additional 5 days to induce differentiation into astrocytes.

[0116] The neurons were generated by forced expression of the Ngn2 gene from iPS cells and cryopreserved. The cryopreserved cells were thawed in a 37°C warm bath. After thawing, the cells were added to co-culture medium and centrifuged at 600 x g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0117] Microglia derived from iPS cells (iCell® Microglia, FCDI, C1110) were used. Cryopreserved cells were thawed in a 37°C warm bath. After thawing, the cells were added to co-culture medium and centrifuged at 600 × g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0118] The two-way coculture contained 3 × 10 neurons. ４ For the triple co-culture, neurons and microglia were seeded on top of the astrocytes at 3 × 10 cells / well. ４ The cells were seeded on top of the astrocytes at 100 cells / well. ２ The cells were cultured under these conditions, and half of the medium was replaced once a week.

[0119] <2> Activation of microglia or astrocytes and evaluation of neural activity by calcium imaging After 4 weeks of co-culture, microglia and astrocytes were stimulated by adding LPS (Fujifilm Wako Pure Chemical Industries, Ltd., 124-05151) to a concentration of 100 ng / mL or TNF-α (Cell signaling technology, 8902SC) to a concentration of 7.5 ng / mL.

[0120] Three hours after stimulation with TNF-α, the cells were fixed by the method described in Test Example 1 <1>, and the localization of NF-κB was evaluated by the same method as in Test Example 1 <1>.

[0121] Furthermore, 24 hours after stimulation with LPS or TNF-α, neural activity was evaluated by calcium imaging. Working solution and recording solution were prepared by adding the reagents shown in Tables 3 and 4 to HBSS(-).

[0122]

[0123]

[0124] 50 μL of medium was removed from the wells containing 100 μL of medium, and 50 μL / well of working solution warmed to 37°C was added. ２ After incubation for 1 hour under these conditions, the medium was replaced with Recording solution warmed to 37°C, and fluorescence was measured for 10 minutes using FDSS® / μcell (Hamamatsu Photonics, C13299). Neuronal activity was evaluated by quantifying the number of calcium oscillation peaks using Wave Checker Software (Hamamatsu Photonics).

[0125] Figure 12 shows the results of staining for NF-κB, GFAP, and NeuN when two-dimensional co-cultures of neurons and astrocytes were stimulated with TNF-α. When two-dimensional co-cultures of neurons and astrocytes were stimulated with TNF-α, NF-κB translocation into the nucleus occurred in GFAP-positive cells, an astrocyte marker, three hours after stimulation, confirming astrocyte activation. On the other hand, NF-κB translocation into the nucleus was not observed in NeuN-positive cells, a neuronal marker. In other words, it was shown that TNF-α stimulated only astrocytes in the co-cultures.

[0126] Figure 13 shows calcium imaging results for two-dimensional cocultures of neurons and astrocytes, and two-dimensional ternary cocultures of neurons, astrocytes, and microglia. Calcium imaging was performed 24 hours after stimulation of astrocytes with TNF-α. Increased BPM (peak calcium oscillations per minute) was observed in both the two- and three-way cocultures compared to controls, indicating enhanced neural activity. The increased BPM observed in the two-way cocultures also confirmed that astrocytes stimulated by TNF-α enhanced neural activity, indicating the occurrence of cell-cell interactions between astrocytes and neurons. Furthermore, stimulation of the two-way cocultures with LPS did not alter BPM, but stimulation of the three-way cocultures with LPS resulted in an increase in BPM compared to controls. In the triple co-culture, Test Example 1 showed that LPS stimulation activated microglia, releasing TNF-α, which then activated astrocytes. These results showed that in the triple co-culture stimulated with LPS, microglia were first activated as the first type of cell, followed by activation of astrocytes as the second type of cell via TNF-α, which in turn enhanced the activity of neurons as the third type of cell, confirming that cell-to-cell interactions occurred among the three types of cell.

Claims

1. A method for evaluating cell-cell interactions in neuroinflammation, comprising: preparing a co-culture containing at least two cells selected from the group consisting of human astrocytes, human neurons, human microglia, and human oligodendrocytes; providing an inflammatory stimulus to a first cell contained in the co-culture; detecting at least one selected from the group consisting of an inflammatory response marker, cellular neural activity, and cell morphology in a cell contained in the co-culture; and evaluating over time at least one change selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cell and a second cell contained in the co-culture that is different from the first cell.

2. The method according to claim 1, wherein the astrocytes, the neurons, the microglia, and the oligodendrocytes are differentiated from human-derived pluripotent stem cells.

3. The method according to claim 2, wherein the human-derived pluripotent stem cells are human iPS cells.

4. The method of any one of claims 1 to 3, wherein the first cell is a human microglia, a human astrocyte, or a human neuron.

5. A method according to any one of claims 1 to 3, wherein when the first cell is a human astrocyte, the second cell is at least one selected from the group consisting of a human neuron and a human microglia.

6. A method according to any one of claims 1 to 3, wherein when the first cell is a human-derived neuron, the second cell is at least one selected from the group consisting of a human-derived astrocyte and a human-derived microglia.

7. A method according to any one of claims 1 to 3, wherein when the first cell is a human-derived microglia, the second cell is at least one selected from the group consisting of a human-derived astrocyte and a human-derived neuron.

8. The method according to any one of claims 1 to 3, wherein the co-culture is a two-dimensional culture or a three-dimensional culture.

9. The method according to any one of claims 1 to 3, wherein the inflammatory stimulus is a chemical stimulus.

10. The method according to claim 9, wherein the chemical stimulation is performed with at least one substance selected from the group consisting of lipopolysaccharide, rotenone, amyloid β protein, tau protein, α-synuclein, and TDP-43.

11. The method according to any one of claims 1 to 3, wherein the inflammatory stimulus is a stimulus obtained by adding cells different from the cells in the co-culture, or by adding a culture supernatant obtained by culturing cells different from the cells in the co-culture.

12. The method of any one of claims 1 to 3, wherein the inflammatory stimulus is induced by senescence, genetic mutation, or gene introduction of a first cell in the co-culture.

13. The method according to claim 12, wherein the protein produced by a genetic mutation in the first cell in the co-culture or by gene introduction into the first cell in the co-culture is at least one selected from the group consisting of amyloid β protein, tau protein, α-synuclein, and TDP-43.

14. A method according to any one of claims 1 to 3, wherein the inflammatory stimulus that stimulates the first cell is induced by a substance secreted from another cell different from the first cell in the co-culture.

15. The method according to any one of claims 1 to 3, wherein the inflammatory response marker is at least one selected from the group consisting of NF-κB, TNF-α and STAT1.

16. A method according to any one of claims 1 to 3, wherein the step of detecting an inflammatory response marker in cells contained in the co-culture is a step of visualizing the inflammatory marker protein by immunostaining.

17. A method according to any one of claims 1 to 3, wherein the step of detecting an inflammatory response marker in cells contained in the co-culture is a step of visualizing the mRNA expression level of the inflammatory marker gene by fluorescent in situ hybridization.

18. The method according to any one of claims 1 to 3, wherein the neural activity is at least one selected from the group consisting of intracellular calcium flux and cellular action potential.

19. A method according to any one of claims 1 to 3, wherein the step of detecting neural activity in the cells contained in the co-culture is a step of visualizing intracellular calcium flux.

20. The method according to claim 19, wherein the step of visualizing the flow of intracellular calcium is calcium imaging.

21. The method according to any one of claims 1 to 3, wherein the step of detecting neural activity in cells contained in the co-culture is a step of measuring action potentials of the cells.