Human astrocyte population, cell population culture, method for producing human astrocyte population, and method for evaluating test substance

Abstract

The present invention addresses the problem of providing: a human astrocyte population obtained by differentiation induction from astrocyte precursor cells derived from human iPS cells; a method for producing the human astrocyte population; and a method for evaluating a test substance using the human astrocyte population. According to the present invention, provided is a human astrocyte population obtained by differentiation induction from astrocyte precursor cells derived from human iPS cells, the human astrocyte population comprising at least 90% of human astrocytes, the human astrocytes comprising at least 90% of the human astrocytes, and the human astrocytes comprising at least 90% of the human astrocytes, the human astrocytes comprising at least 90% of the human astrocytes, and the human astrocytes comprising at least 90% of the human astrocytes. A) CDKN2A is positive, b) at least one gene marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1, and HLA-DRB5 is positive, and c) the C3 expression level standardized with GAPDH of a reference gene is 0.05 copy number / copy number or less.

Description

Technical Field

[0001] The present invention relates to a human astrocyte population, a cell population culture, and a method for producing a human astrocyte population. In addition, the present invention relates to an evaluation method for a test substance using the produced human astrocyte population. In addition, the present invention relates to a method for producing a co-culture comprising the produced human astrocyte population, human-derived neural cells, and human-derived microglia, and the co-culture. Background Art

[0002] Astrocytes, a type of glial cells constituting the brain, play various roles in maintaining the stability of the central nervous system, such as supplying nutrients to neurons and forming or removing synapses, and have attracted attention from the viewpoints of disease mechanism elucidation and drug development.

[0003] The cells of the central nervous system composed of nerve cells or glial cells are difficult to obtain from the human brain, and most studies of the central nervous system use cells from rats or mice. However, in recent years, the invention of iPS cells has made it relatively easy to obtain human nerve cells or glial cells, and these cells are very useful in the study of human neurodegenerative diseases (Non-Patent Documents 1 to 3).

[0004] When conducting research on neurodegenerative diseases, it is very important to consider the effects of cell aging associated with aging, which is the biggest risk factor for the disease (non-patent document 4). It is known that cell aging of astrocytes is also associated with neurodegenerative diseases (non-patent documents 5-7), and research on reproducing aging astrocytes is underway (non-patent document 8). However, since aging is reset by reprogramming in iPS cells (non-patent documents 9-10), it is considered difficult to reproduce aging.

[0005] In Non-Patent Document 11, regarding iPS cell-derived astrocytes, a study was conducted to reproduce aging by producing astrocytes from iPS cells derived from patients with mutations in disease-related genes. However, in the method of Non-Patent Document 11, long-term culture is performed after complete differentiation induction into astrocytes, so it is unclear whether senescent astrocytes can be obtained.

[0006] Furthermore, the cell culture method of astrocytes is usually cultured in a medium containing serum, but it has recently been known that astrocytes are irreversibly activated when exposed to serum (Non-Patent Document 12). Astrocytes in the brain are not exposed to serum due to the presence of the blood-brain barrier (Non-Patent Document 13), and it is believed that it is important to culture them under conditions that do not contain serum when studying the functions of astrocytes (Non-Patent Document 14).

[0007] Patent Documents 1 and 2 describe methods for culturing astrocytes under serum-free conditions. However, the method of Patent Document 1 describes differentiation into neurons or oligodendrocytes other than astrocytes, and it is unclear whether the further differentiated astrocytes are senescent. Furthermore, the method of Patent Document 2 describes that A1 astrocytes with neurodamage can be obtained, but it is unclear whether senescent astrocytes without neurodamage can be obtained.

[0008] Patent Document 3 describes a method for preparing astrocyte-like cells from human cells. However, in the method of Patent Document 3, differentiation into astrocytes takes a short period of time, and therefore it is unclear whether aged astrocytes can be obtained.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: International Publication No. 2021 / 045217 Pamphlet

[0012] Patent Document 2: International Publication No. 2019 / 235576 Pamphlet

[0013] Patent Document 3: International Publication No. 2017 / 057523 Pamphlet

[0014] Non-patent literature

[0015] Non-patent document 1: Okano et al., Molecular Brain, Vol. 7, No. 22, pp. 1-12, 2014

[0016] Non-patent document 2: Valadez-Barba et al., Regenerative Therapy, Vol. 15, pp. 332-339, 2020

[0017] Non-patent document 3: Zeng et al., STEM CELLS TRANSLATIONAL MEDICINE, Vol. 3, pp. 1418-1428, 2014

[0018] Non-patent document 4: Kritsilis et al., International Journal of Molecular Sciences, Vol. 19, No. 10, 2937, pp. 1-37, 2018

[0019] Non-patent document 5: Turnquist et al., Cell Death and Differentiation, Vol. 23, pp. 1515-1528, 2016

[0020] Non-patent literature 6: Han et al., Frontiers in Aging Neuroscience, Vol. 12, No. 148, 2020

[0021] Non-patent literature 7: Vazquez-Villasenor et al., Neuropathology and Applied Neurobiology, Vol. 46, pp. 171-185, 2020

[0022] Non-patent document 8: Simmnacher et al., Experimental Neurology, Vol. 334, No. 113466, pp. 1-13, 2020

[0023] Non-patent document 9: Lapasset et al., GENES & DEVELOPMENT, Vol. 25, No. 21, pp. 2248-2253, 2011

[0024] Non-patent document 10: Miller et al., Cell Stem Cell, Vol. 13, No. 6, pp. 691-705, 2013

[0025] Non-patent literature 11: Birger et al., EBioMedicine (Lancet), Vol. 50, pp. 274-289, 2019

[0026] Non-patent document 12: Foo et al., Neuron, Vol. 71, No. 5, pp. 799-811, 2011

[0027] Non-patent document 13: Stokum et al., Neurochemical Research, Vol. 40, pp. 317-328, 2015

[0028] Non-patent literature 14: Jia et al., Journal of Neuroscience Methods, Vol. 307, pp. 240-247, 2018 Summary of the invention

[0029] Technical issues to be solved by the invention

[0030] From the perspective of drug development research, human senescent astrocytes are of great value, and a method for easily obtaining human senescent astrocytes is required. As mentioned above, some results have been achieved in inducing differentiation into astrocytes, but a method for reliably inducing senescent human astrocytes from astrocyte precursor cells derived from human iPS cells has not yet been found.

[0031] Therefore, the problem to be solved by the present invention is to provide a human astrocyte population comprising senescent human astrocytes induced from astrocyte precursor cells derived from human iPS cells. The problem to be solved by the present invention is also to provide a cell population culture comprising the above-mentioned human astrocyte population and a method for producing the above-mentioned human astrocyte population. The problem to be solved by the present invention is also to provide an evaluation method for a test substance using the above-mentioned human astrocyte population. The problem to be solved by the present invention is also to provide a method for producing a co-culture and a co-culture, which co-culture comprises the above-mentioned human astrocyte population, human-derived neural cells and human-derived microglia.

[0032] Means for solving technical problems

[0033] The present inventors have conducted intensive studies to solve the above problems and have found that human iPS cell-derived astrocyte precursor cells can be induced to become senescent human astrocytes by senescent human iPS cell-derived astrocyte precursor cells under proliferation conditions. The present invention was completed based on this finding.

[0034] That is, according to the present invention, the following inventions can be provided.

[0035] <1> A human astrocyte population obtained by differentiation-induced astrocyte precursor cells derived from human iPS cells,

[0036] The above human astrocyte population comprises at least 90% human astrocytes,

[0037] In the above human astrocytes,

[0038] a) CDKN2A is positive,

[0039] b) at least one marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1 and HLA-DRB5 is positive,

[0040] c) The expression level of C3 normalized with reference gene GAPDH was less than 0.05 copies / copies.

[0041] <2> The human astrocyte population according to <1>, wherein

[0042] In the human astrocytes, the expression level of CDKN2A normalized with the reference gene GAPDH was 0.004 copies / copy or more.

[0043] <3> The human astrocyte population according to <1> or <2>, wherein:

[0044] In the human astrocytes, the expression level of IGFBP5 normalized with reference gene GAPDH was 0.1 copies / copy or more.

[0045] <4> The human astrocyte population according to <1> or <2>, wherein

[0046] In the human astrocytes, the expression level of NNMT normalized with reference gene GAPDH was 0.005 copies / copy or more.

[0047] <5> The human astrocyte population according to <1> or <2>, wherein

[0048] In the human astrocytes, the expression level of HLA-DRB5 normalized with reference gene GAPDH was 0.1 copies / copy or more.

[0049] <6> The human astrocyte population according to <1> to <5>, wherein

[0050] At least one marker selected from the group consisting of γH2AX and SA-β-GAL is positive.

[0051] <7> The cell population according to any one of <1> to <6>, wherein

[0052] The human iPS cell-derived astrocyte precursor cells are astrocyte precursor cells produced from human iPS cells derived from a healthy human.

[0053] <8> A cell population culture comprising the human astrocyte population according to any one of <1> to <7> and a medium substantially free of serum.

[0054] <9> The cell population culture according to <8>, further comprising at least one factor selected from the group consisting of BMP4 (bone morphogenetic factor) and CNTF (ciliary neurotrophic factor).

[0055] <10> The method for producing an astrocyte population according to any one of <1> to <7>, comprising:

[0056] A step of proliferating astrocyte precursor cells derived from human iPS cells; and

[0057] The process of inducing the differentiation of astrocyte precursor cells derived from human iPS cells after proliferation.

[0058] <11> A method for evaluating a test substance, comprising the step of contacting the human astrocyte population according to any one of <1> to <7> with a test substance.

[0059] <12> A method for producing a co-culture, comprising: a step of adding the human astrocyte population, human-derived neural cells, and human-derived microglia described in any one of <1> to <7> to a culture container; and a step of co-culturing the human astrocyte population, the neural cells, and the microglia in the culture container.

[0060] <13> The manufacturing method according to <12>, wherein

[0061] The above-mentioned neural cells and microglial cells are cells obtained by differentiation induction from human-derived pluripotent stem cells.

[0062] <14> The production method according to <12> or <13>, wherein

[0063] The above-mentioned pluripotent stem cells derived from humans are human iPS cells.

[0064] <15> The production method according to any one of <12> to <14>, wherein

[0065] The above co-culture is a two-dimensional culture or a three-dimensional culture.

[0066] <16> A co-culture comprising the human astrocyte population according to any one of <1> to <7> obtained by the production method according to any one of <12> to <15>, human-derived neural cells, and human-derived microglial cells.

[0067] Effects of the Invention

[0068] According to the present invention, a population of aged human astrocytes can be produced from astrocyte precursor cells derived from human iPS cells. The population of human astrocytes of the present invention and the method for evaluating a test substance of the present invention are useful in drug development research and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Images showing fluorescent immunostaining of senescent human astrocytes using anti-GFAP antibody.

[0070] Figure 2The graph shows the results of quantification of the GFAP-positive rate of aged human astrocytes using flow cytometry.

[0071] Figure 3 The results show the quantitative results of the expression levels of senescence-related markers in senescent human astrocytes and non-senescent human astrocytes using digital PCR.

[0072] Figure 4 The graph shows the results of quantification of C3 expression levels in senescent human astrocytes and non-senescent human astrocytes using digital PCR.

[0073] Figure 5 The graph shows the results of fluorescent immunostaining of senescent human astrocytes and non-senescent human astrocytes using anti-γH2AX antibody.

[0074] Figure 6 The graph shows the results of SA-β-GAL staining of senescent human astrocytes and non-senescent human astrocytes.

[0075] Figure 7 The results of quantifying the CDKN2A expression levels in non-senescent human astrocytes, cells cultured for a long time with non-senescent human astrocytes, and senescent human astrocytes using digital PCR. The triangles represent cells cultured in a precursor cell culture medium for 43 days, which were then replaced with a differentiation induction medium to induce differentiation into astrocytes ((1)). The quadrilaterals represent cells cultured in a non-senescent astrocyte culture medium for 42 days ((2)). The circles represent cells cultured in a precursor cell culture medium for 85 days, which were then replaced with a differentiation induction medium to induce differentiation into astrocytes ((3)).

[0076] Figure 8 Images showing fluorescent immunostaining of two-dimensional co-cultures created using aged human astrocytes, neurons, and microglia.

[0077] Fig. 9 Images showing fluorescent immunostaining of three-dimensional co-cultures created using aged human astrocytes, neurons, and microglia. DETAILED DESCRIPTION

[0078] The embodiments of the present invention are described in detail below.

[0079] CDKN2A stands for cyclin-dependent kinase inhibitor 2A.

[0080] IGFBP5 stands for Insulin-like Growth Factor Binding Protein 5.

[0081] NNMT stands for Nicotinamide N-Methyltransferase.

[0082] HLA-DRB1 stands for Human Leukocyte Antigen-DRB1.

[0083] HLA-DRB5 stands for Human Leukocyte Antigen-DRB5.

[0084] C3 represents the complement molecule C3.

[0085] GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase.

[0086] BMP4 stands for Bone Morphogenetic Protein 4.

[0087] CNTF stands for ciliary neurotrophic factor.

[0088] GFAP stands for glial fibrillary acidic protein.

[0089] γH2AX indicates phosphorylated histone H2AX.

[0090] SA-β-GAL stands for Senescence-associated beta-galactosidase.

[0091] The present invention relates to a human astrocyte population induced by differentiation from astrocyte precursor cells derived from human iPS cells.

[0092] The human astrocyte population of the present invention comprises at least 90% of human astrocytes, among which,

[0093] a) CDKN2A is positive,

[0094] b) at least one marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1 and HLA-DRB5 is positive,

[0095] c) The expression level of C3 normalized with reference gene GAPDH was less than 0.05 copies / copy.

[0096] <Human iPS cells>

[0097] “Human iPS cells” refer to artificial pluripotent stem cells (induced pluripotent stem cells) produced from human cells.

[0098] In this specification, "iPS cells" means cells with pluripotency (multi-differentiation ability) and proliferation ability produced by initializing (reprogramming) somatic cells through the introduction of initialization factors. iPS cells show properties close to ES cells (Embryonic Stem cells: embryonic stem cells). The somatic cells used in the production of iPS cells are not particularly limited, and can be differentiated somatic cells or undifferentiated stem cells. In addition, their source is not particularly limited, and it is preferred to use somatic cells of mammals (for example, primates such as humans or chimpanzees, rodents such as mice or rats), and more preferably human somatic cells. iPS cells can be produced by known methods, etc. And, of course, it is also conceivable to apply iPS cell production methods developed in the future.

[0099] The most basic method for producing iPS cells is to use viruses to introduce four transcription factors, namely Oct3 / 4, Sox2, Klf4 and c-Myc, into cells (Takahashi K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007). Regarding human iPS cells, there are reports that they were established by introducing four factors, namely Oct4, Sox2, Lin28 and Nanog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). There are also reports on the establishment of iPS cells by introducing three factors other than c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), introducing two factors Oct3 / 4 and Klf4 (Kim JB, et al: Nature 454 (7204), 646-650, 2008), or introducing only Oct3 / 4 (Kim JB, et al: Cell 136 (3), 411-419, 2009). In addition, there are also reports on methods for introducing a gene expression product, i.e., a protein, into cells (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al: Cell Stem Cell 4, 472-476, 2009).

[0100] As a method for producing iPS cells, in addition to a method of producing by direct induction of gene expression, iPS cells can also be induced from somatic cells by adding a compound or the like (Hou P et al: Science 341 (6146), 651-654, 2013).

[0101] Furthermore, established artificial pluripotent stem cells can also be obtained, for example, from Kyoto University, iPS Academia Japan, Inc., or the RIKEN Bioresource Research Center.

[0102] Cells that have undergone transformation into iPS cells, i.e., initialized, can be selected using the expression of pluripotent stem cell markers (undifferentiation markers) such as Nanog, Oct / 4, Fgf-4, Esg-1, and Cript as indices, and the selected cells can be recovered as iPS cells.

[0103] <Astrocyte precursor cells derived from human iPS cells>

[0104] “Astrocyte precursor cells derived from human iPS cells” refers to astrocyte precursor cells produced from human iPS cells.

[0105] In this specification, "astrocyte precursor cell" means a cell having the ability to differentiate into astrocytes. The presence of astrocyte precursor cells can be determined by observing a marker significantly expressed in astrocyte precursor cells. As markers of astrocyte precursor cells, for example, NFIA, NFIB, SOX9, HEY1, HEY2, FABP7 and ZBTB20 can be cited.

[0106] As a method for obtaining the "astrocyte precursor cells" used in the present invention, for example, there can be cited separation from the cerebral cortex or spinal cord obtained from a patient by surgery, induction from cells capable of differentiating into human astrocytes, induction from human pluripotent stem cells, etc. These astrocyte precursor cells can all be used as the "astrocyte precursor cells" in the method of the present invention.

[0107] As the "astrocyte precursor cells" used in the present invention, human astrocyte precursor cells induced from human pluripotent stem cells are preferably used.

[0108] Examples of human pluripotent stem cells include human iPS cells (human induced pluripotent stem cells), human ES cells (human embryonic stem cells), and human mesenchymal stem cells, but are not particularly limited. Human iPS cells are preferably used.

[0109] That is, according to the present invention, there is provided a human astrocyte population obtained by inducing differentiation from astrocyte precursor cells.

[0110] The above human astrocyte population comprises at least 90% human astrocytes,

[0111] In the above human astrocytes,

[0112] a) CDKN2A is positive;

[0113] b) at least one marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1 and HLA-DRB5 is positive; and

[0114] c) The expression level of C3 normalized with reference gene GAPDH was less than 0.05 copies / copy.

[0115] As a method for obtaining "astrocyte precursor cells derived from human iPS cells", for example, it can be obtained by induction from human iPS cells made from somatic cells collected from healthy persons (healthy persons) who do not have mutations in disease-related genes that cause neurological diseases or neurological diseases, or from somatic cells collected from patients with neurological diseases, or from human iPS cells that have been strained, etc. The above-mentioned neurological diseases are not particularly limited, and for example, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, Rett syndrome, etc. can be cited.

[0116] All of these human iPS cell-derived astrocyte precursor cells can be used as the starting cells, ie, "human iPS cell-derived astrocyte precursor cells" in the present invention.

[0117] As the "astroglial precursor cells derived from human iPS cells" used in the present invention, it is preferred to use astrocyte precursor cells induced from human iPS cells derived from healthy subjects, astrocyte precursor cells induced from cultured human iPS cells, or astrocyte precursor cells induced from human iPS cells with gene mutations derived from diseases, and it is more preferred to use astrocyte precursor cells produced from human iPS cells derived from healthy subjects.

[0118] Astrocyte precursor cells derived from human iPS cells can be used in the present invention in an isolated state or in a state mixed with other cells. Furthermore, it is also conceivable that differentiation induction from human iPS cells to astrocyte precursor cells and differentiation induction from astrocyte precursor cells to human astrocytes are performed continuously.

[0119] <Human astrocytes>

[0120] "Human astrocytes" refers to human astrocytes.

[0121] In this specification, "astrocyte" refers to a kind of glial cell present in the central nervous system, and it is believed that by structurally supporting the function of neurons or regulating neurotransmitters, energy and extracellular ions, it contributes to the regulation of neurotransmitters. In addition, it is believed that oligodendrocytes, which are also a kind of glial cells, are supplied with substances that promote myelination, and are cells that play important functions in nervous tissue together with neurons and oligodendrocytes. It can be identified by markers specifically expressed in astrocytes, as such markers, for example, GFAP, S100B (S100calcium binding protein B: calcium binding protein B), KCNJ10 (potassium inwardly rectifying channel subfamily J member 10: potassium inward rectifying channel subfamily J member 10), AQP4 (Aquaporin-4: water channel protein-4) and SLC1A3 (solute carrier family 1 member 3: solute carrier family 1 member 3, also known as GLAST or EAAT1) and the like. For example, by analyzing using an immunohistological method, cells expressing at least one of the above-mentioned mRNA, sugar chain, or protein can be identified as astrocytes.

[0122] <Human astrocyte population>

[0123] "Human astrocyte population" refers to a cell population comprising human astrocytes.

[0124] In this specification, "cell group" refers to a group containing at least one type of cell, and may also contain two or more types of arbitrary cells. In addition, the cell group may be in a state of being dispersed (floating) in a medium such as a culture medium, in a state of being attached to the bottom surface of a culture container, in a state of a cell aggregate (spherical cell group) formed by agglutination of multiple cells, or in a layered cell group, and is not particularly limited.

[0125] The "human astrocyte population" of the present invention preferably comprises at least 90% of human astrocytes, more preferably at least 95% of human astrocytes, further preferably at least 99% of human astrocytes, and particularly preferably 100% of human astrocytes.

[0126] The ratio of human astrocytes in the human astrocyte population can be determined, for example, by using an anti-GFAP antibody as an antibody that specifically recognizes human astrocytes and quantifying the GFAP positive rate by flow cytometry.

[0127] Examples of cells other than human astrocytes included in the human astrocyte population of the present invention include, but are not particularly limited to, astrocyte precursor cells, neural stem cells, oligodendrocyte precursor cells, neural cells, and oligodendrocytes.

[0128] <Aging human astrocytes>

[0129] “Senescent human astrocytes (human senescent astrocytes)” refers to human astrocytes that are positive for the expression of senescence-related markers characteristic of cellular senescence.

[0130] As aging-related markers, for example, but not particularly limited, there can be mentioned γH2AX, which is considered to be a DNA damage response (DDR) marker; CDKN2A (p16INK4a and p14ARF), p21, and p53, which are considered to be tumor suppressors or cell cycle regulators; senescence-associated β-galactosidase (SA-β-GAL), which is considered to be a lysosome-related protein; inflammatory cytokines such as IL-6, IL-8, and vascular endothelial growth factor (VEGF), which are considered to be markers of cellular senescence-associated secretory phenotype (SASP).

[0131] In the present specification, "senescent human astrocytes" are, for example, preferably positive for at least one of the senescence-related markers selected from the group including CDKN2A, γH2AX, SA-β-GAL and SASP markers, more preferably positive for at least one of the senescence-related markers selected from the group including CDKN2A, γH2AX and SA-β-GAL, and even more preferably positive for CDKN2A.

[0132] In this specification, "marker" refers to a substance present in a cell and can identify or distinguish the type or property of the cell based on its presence or amount. Specific examples of markers include mRNA, proteins and sugar chains encoded by the mRNA, and fragments thereof.

[0133] In the present specification, “positive for a marker” means that the expression level of the marker in human astrocytes contained in the human astrocyte population of the present invention as the final product is higher than the expression level of the marker in the starting cells.

[0134] In the case where the marker is a gene, the expression level of the marker represents the expression level (expression amount) of the gene, and can usually be analyzed by the production amount of the transcription product corresponding to the gene or the production amount, activity, etc. of its translation product. The expression level can be measured by measuring the transcription product of the gene, i.e., mRNA, or the translation product of the gene, i.e., protein, but preferably by measuring mRNA or its reverse transcription product, i.e., cDNA. The detection or measurement of the expression of the translation product (protein) can be performed by immunostaining of proteins in cells using antibodies.

[0135] The NCBI accession number of each gene is shown in Table 1. According to the following NCBI accession number, the sequence information of each gene can be obtained and the expression level of the gene can be measured.

[0136] [Table 1]

[0137] Gene name NCBI accession Number CDKN2A NM_000077,NM_058197,NM_058195,NM_001195132,NM_001363763 IGFBP5 NM_000599 NNMT NM_006169, NM_001372045, NM_001372046, NM_001372047 HLA-DRB1 NM_002124, NM_001243965, NM_001359193, NM_001359194 HLA-DRB5 NM_002125 C3 NM_000064

[0138] The method for determining the expression level of the gene expressed by the human astrocytes contained in the human astrocyte group of the present invention is not particularly limited, for example, it can be determined by quantitative RT-PCR. RT-PCR is a method for synthesizing cDNA using the mRNA to be measured as a template, and amplifying the cDNA by PCR using the cDNA as a template. As a quantitative RT-PCR, for example, a primer formed by combining a quencher fluorescent dye and a reporter fluorescent dye can be used to perform PCR and quantify the amount of amplified product in each cycle, and a method for determining the amount of template DNA in the sample according to the number of cycles in which the detected fluorescence intensity increases sharply (real-time PCR), a method for dispersing the limiting dilution sample DNA in micropartitions for PCR proliferation, and directly counting the number of micropartitions containing the target gene, thereby absolutely quantifying the concentration of the target gene in the sample (digital PCR), etc. As a method for dispersing the sample DNA in micropartitions by digital PCR, a method for making droplets, a method for dispersing on a chip, etc. can be cited, but it is not particularly limited. The method of quantitative RT-PCR is well known in the art and can also be implemented using a commercially available kit. According to quantitative RT-PCR, the expression level or copy number of a gene can be measured as a relative value relative to the expression level or copy number of a reference gene (e.g., GAPDH gene) used as a control. In addition, the determination of the mRNA of a gene can also be performed by gel electrophoresis of the amplification product obtained by amplifying mRNA by conventional RT-PCR, etc., and measuring the band intensity after staining. Alternatively, a DNA chip can also be used to detect or quantify the mRNA or cDNA of a gene. The determination of the expression level of the gene expressed by human astrocytes can also be performed using a next-generation sequencer. In addition, the cells that become the object of measurement can be obtained by extracting a portion from the culture process.

[0139] As a numerical value representing the expression level, for example, when the expression level is measured by real-time PCR, the Ct (Cycle threshold) value can be used. The Ct value refers to the number of cycles when the PCR amplification product reaches a specified amount. The number of amplification cycles is plotted on the horizontal axis, and the amount of PCR product is plotted on the vertical axis to create an amplification curve. When a threshold is set on the value of the PCR product amount, the number of cycles at the point where the threshold intersects the amplification curve becomes the Ct value. When the expression level is measured using a fluorescently labeled probe, the fluorescence intensity can also be used.

[0140] In the case of measuring expression by digital PCR, the copy number (copy number / μL) can be used. The number of micropartitions containing the target gene (positive) and the number of micropartitions not containing the target gene (negative) are counted, and the copy number is calculated based on the proportion of negatives. The number of target genes contained in the positive can be calculated by using Poisson distribution, and the copy number can be corrected.

[0141] <CDKN2A>

[0142] “CDKN2A”, also known as Cyclin dependent kinase inhibitor 2A, is a tumor suppressor gene encoding p16INK4a (p16) and p14ARF (p14).

[0143] The human astrocytes included in the human astrocyte population of the present invention are preferably CDKN2A-positive.

[0144] In the present invention, when CDKN2A is positive, in the above-mentioned human astrocytes, the expression level of CDKN2A standardized with the reference gene GAPDH is preferably 0.002 copies / copy number or more, more preferably 0.003 copies / copy number or more, further preferably 0.004 copies / copy number or more, further preferably 0.005 copies / copy number or more, and particularly preferably 0.006 copies / copy number or more.

[0145] In the above human astrocytes, the upper limit of the expression level of CDKN2A normalized with the reference gene GAPDH is not particularly limited, and it is assumed that the higher the expression level, the more aged the human astrocytes included in the human astrocyte population of the present invention.

[0146] The human astrocytes contained in the human astrocyte population of the present invention may be positive for at least one marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1 and HLA-DRB5 in addition to the above-mentioned CDKN2A. Preferably, at least one marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1 and HLA-DRB5 is positive, more preferably, at least one marker selected from the group consisting of IGFBP5, NNMT and HLA-DRB5 is positive, further preferably, at least one marker selected from the group consisting of IGFBP5 and NNMT is positive, and even more preferably, IGFBP5 is positive.

[0147] <IGFBP5>

[0148] "IGFBP5", also known as insulin-like growth factor binding protein 5, is one of the proteins that exists in conjunction with insulin-like growth factor (IGF) in the blood or tissues. It is believed that IGF regulates its affinity with the receptor, distribution (local release), and metabolism (stabilization / decomposition) by binding to insulin-like growth factor binding protein (IGFBP). IGFBPs bind to IGF-I and IGF-II circulating in body fluids to regulate activity (promotion / inhibition), distribution (local release), and metabolism (stabilization / decomposition). In addition to IGF-BPs with high affinity for IGFs, IGFBPrP (IGF-BP related Protein) with similar structure / function but low affinity has also been identified. IGFBP5 is mainly produced by vascular smooth muscle cells and exists locally in bone tissue, promoting the effects of IGF-I on smooth muscle cells, fibroblasts, or osteoblasts. Also, it is known that it forms a trimer with ALC (Acidic-labile subunit) similarly to IGFBP3, and is considered to be a regulator of the senescence-associated secretory phenomenon (SASP).

[0149] The human astrocytes contained in the human astrocyte population of the present invention may be positive for IGFBP5, and preferably are positive for IGFBP5.

[0150] In the present invention, when IGFBP5 is positive, in the above-mentioned human astrocytes, the expression level of IGFBP5 standardized with the reference gene GAPDH is preferably above 0.08 copies / copy number, more preferably above 0.09 copies / copy number, further preferably above 0.1 copies / copy number, further preferably above 0.15 copies / copy number, and particularly preferably above 0.2 copies / copy number.

[0151] In the human astrocytes, the upper limit of the expression level of IGFBP5 normalized with reference gene GAPDH is not particularly limited, and it is presumed that the higher the expression level, the more aged the human astrocytes included in the human astrocyte population of the present invention.

[0152] <NNMT>

[0153] "NNMT", also known as nicotinamide N-methyltransferase, is an enzyme that catalyzes the methylation of nicotinamide and similar compounds using S-adenosylmethionine as a methyl donor to generate S-adenosyl-L-homocysteine ​​and 1-methylnicotinamide. It is also known to be a contributing factor required for various metabolic and epigenetic processes including the regulation of aging, cellular stress response, and weight gain.

[0154] The human astrocytes included in the human astrocyte population of the present invention may be NNMT-positive, and preferably NNMT-positive.

[0155] In the present invention, when NNMT is positive, in the above-mentioned human astrocytes, the expression level of NNMT standardized with the reference gene GAPDH is preferably above 0.003 copies / copy number, more preferably above 0.004 copies / copy number, further preferably above 0.005 copies / copy number, further preferably above 0.01 copies / copy number, and especially preferably above 0.015 copies / copy number.

[0156] In the above human astrocytes, the upper limit of the expression level of NNMT normalized with reference gene GAPDH is not particularly limited, and it is presumed that the higher the expression level, the more aged the human astrocytes included in the human astrocyte population of the present invention.

[0157] <HLA-DRB1 and HLA-DRB5>

[0158] "HLA-DRB1" is also called human leukocyte antigen-DRB1, major histocompatibility complex class IIDRβ1, and is one of the haplotypes of human leukocyte antigen (HLA, Human Leukocyte Antigen) that is considered to function as a histocompatibility antigen. HLA-DRB1 is a protein-coding gene located in the HLA class II region on the short arm of chromosome 6. HLA class II molecules are known to be heterodimers composed of α (DRA) chains and β (DRB) chains, all of which are fixed to the membrane and present peptides derived from extracellular proteins, thereby playing a central role in the immune system.

[0159] Diseases associated with HLA-DRB1 include multiple sclerosis and sarcoidosis 1, and pathways associated with them include D28 signaling in helper T cells, etc. In addition, HLA-DRB5 is known as an important paralogous gene of the HLA-DRB1 gene.

[0160] "HLA-DRB5" is also called human leukocyte antigen-DRB5 (Human Leukocyte Antigen-DRB5) and major histocompatibility complex class IIDRβ5, and is one of the haplotypes of HLA like HLA-DRB1.

[0161] Among the diseases associated with HLA-DRB5, pityriasis rosea, an inflammatory keratosis (scaly rash) of the skin common in young adults, and multiple epiphyseal dysplasia caused by abnormalities in collagen 9 are known, and the pathways associated therewith include CD28 signaling in helper T cells, etc. In addition, HLA-DRB1 is known as an important paralogous gene of the HLA-DRB5 gene.

[0162] In the human astrocytes contained in the human astrocyte population of the present invention, at least one marker selected from the group including HLA-DRB1 and HLA-DRB5 may be positive, preferably at least one marker selected from the group including HLA-DRB1 and HLA-DRB5 is positive, and more preferably HLA-DRB5 is positive.

[0163] In the present invention, when HLA-DRB5 is positive, in the above-mentioned human astrocytes, the expression level of HLA-DRB5 standardized with the reference gene GAPDH is preferably 0.08 copies / copy number or more, more preferably 0.09 copies / copy number or more, further preferably 0.1 copies / copy number or more, further preferably 0.15 copies / copy number or more, particularly preferably 0.2 copies / copy number or more, and even more preferably 0.25 copies / copy number or more.

[0164] In the above human astrocytes, the upper limit of the expression level of HLA-DRB5 normalized with the reference gene GAPDH is not particularly limited, and it is assumed that the higher the expression level, the more aged the human astrocytes included in the human astrocyte population of the present invention.

[0165] In the human astrocytes included in the human astrocyte population of the present invention, in addition to being positive for CDKN2A, C3 may be negative, and the expression level of C3 normalized with reference gene GAPDH may be 0.05 copies / copy or less.

[0166] <C3>

[0167] "C3" is also called complement molecule C3, and is one of the complements present in the serum of mammals. It is also considered to be a specific marker of human A1 astrocytes that show neurological damage. Examples of specific markers of human A1 astrocytes include GBP2, SERPING1, and C3, but are not limited to these.

[0168] In the human astrocytes contained in the human astrocyte population of the present invention, C3 is preferably negative.

[0169] In the present invention, when C3 is negative, in the above-mentioned human astrocytes, the expression level of C3 standardized with the reference gene GAPDH is less than 0.05 copies / copy number, more preferably less than 0.02 copies / copy number, further preferably less than 0.01 copies / copy number, further preferably less than 0.001 copies / copy number, particularly preferably less than 0.0001 copies / copy number, and most preferably less than the detection limit.

[0170] In the human astrocytes, the lower limit of the expression level of C3 normalized with the reference gene GAPDH is not particularly limited, and it is assumed that the higher the expression level, the more different the cell has properties from the human astrocytes included in the human astrocyte population of the present invention.

[0171] The human astrocytes included in the human astrocyte population of the present invention may be positive for at least one marker selected from the group consisting of γH2AX and SA-β-GAL, and preferably at least one marker selected from the group consisting of γH2AX and SA-β-GAL is positive.

[0172] <γH2AX>

[0173] "γH2AX" is also called phosphorylated histone H2AX and is considered one of the DNA damage markers. When double-strand breaks occur due to DNA damage, H2AX, a type of histone, is rapidly and widely phosphorylated. γH2AX is not only used to evaluate the genotoxicity and carcinogenicity of chemical substances, active oxygen, ultraviolet rays, radiation, etc., but has also been considered an indicator for evaluating cell senescence in recent years.

[0174] <SA-β-GAL>

[0175] "SA-β-GAL" is one of the enzymes also called senescence-associated beta-galactosidase. When β-galactosidase is stained with X-gal as a substrate under weakly acidic conditions (pH 6), senescent cells are stained blue, while proliferating cells are not stained. Therefore, it is widely used as a marker that can easily detect senescent cells that cause cell senescence.

[0176] As another embodiment, the present invention relates to a cell population culture comprising a human astrocyte population induced by differentiation from astrocyte precursor cells derived from human iPS cells.

[0177] <Cell Population Culture>

[0178] A "cell population culture" refers to a culture comprising a population of cells.

[0179] In the present specification, the "cell population culture" may include a cell population containing at least one type of cell and arbitrary components such as a culture medium.

[0180] <Cultivation of human astrocytes>

[0181] The culture of human astrocytes in the present invention can be carried out by selecting a culture medium, temperature, and other conditions corresponding to the source and state of the human astrocytes used. As long as it does not hinder the culture of human astrocytes in the present invention, the culture medium may contain any components such as factors that are consistent with the purpose of culture. The culture medium can be selected from a known culture medium or a commercially available culture medium. For example, it can be used by adding appropriate components (serum, protein, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.) to MEM (minimum essential medium), DMEM (Dulbecco's modified Eagle medium), DMEM / F12, or culture media modified therefrom, which are general culture media.

[0182] As the culture medium used in the present invention, astrocytes in the brain are not exposed to serum due to the presence of the blood-brain barrier. In order to study the functions of astrocytes and make the environment closer to that in vivo, it is preferred to use a culture medium that does not substantially contain serum (serum-free culture medium).

[0183] In the present invention, "serum-free medium" means a medium that does not contain unadjusted or unpurified serum. In this specification, even a medium mixed with components derived from purified blood or components derived from animal tissue (e.g., growth factors) is included in the serum-free medium as long as it does not contain unadjusted or unpurified serum.

[0184] The cell population culture in the present invention preferably comprises a human astrocyte population containing human senescent astrocytes and a medium substantially free of serum.

[0185] The culture medium used in the present invention may contain any components such as factors necessary for inducing differentiation of human astrocyte precursor cells into human astrocytes.

[0186] The cell population culture of the present invention aims to efficiently differentiate human astrocyte precursor cells into human astrocytes, and preferably further comprises at least one factor selected from the group consisting of BMP4 (bone morphogenetic factor 4) and CNTF (ciliary neurotrophic factor).

[0187] <Method for producing human astrocyte population>

[0188] As another embodiment, the present invention relates to a method for producing a cell population containing senescent human astrocytes.

[0189] According to the present invention, there is provided a method for producing a human astrocyte population, comprising: a step of proliferating astrocyte precursor cells derived from human iPS cells; and a step of differentiating and inducing the proliferated astrocyte precursor cells derived from human iPS cells.

[0190] As the culture conditions in the manufacturing method of the present invention, general cell culture conditions can be selected. Examples include conditions of 37°C and 5% CO2. During the culture process, the culture medium is preferably replaced at appropriate intervals (preferably once every 1 to 7 days, more preferably once every 2 to 3 days). In the case of preparing the cell population of the present invention using human astrocyte precursor cells derived from iPS cells as materials, human senescent astrocytes can be prepared by inducing differentiation into human astrocytes after culturing under proliferation conditions for more than 50 days. As the period of culture under proliferation conditions, it is preferably more than 50 days, more preferably more than 70 days, and further preferably more than 80 days.

[0191] Furthermore, from the viewpoint of applying stress that induces senescence, human astrocyte precursor cells are preferably cultured two-dimensionally.

[0192] In the culture of cells, cell culture containers such as culture plates, culture bowls, cell culture inserts, cell culture flasks, and cell culture bags can be used. In addition, as cell culture bags, preferably, containers with air permeability are used. When a large number of cells are required, large culture tanks can be used. Cultivation can be carried out in either an open system or a closed system.

[0193] <Human astrocyte population and method for evaluating a test substance using the same>

[0194] According to the present invention, a cell population comprising senescent human astrocytes is provided.

[0195] The human astrocyte population of the present invention can also be used to screen candidate drug compounds that act on human senescent astrocytes or evaluate the safety of candidate drug compounds.

[0196] According to the present invention, a method for evaluating a test substance is provided, which includes the step of contacting a cell group containing human astrocytes of the present invention with a test substance. For example, in the process of culturing astrocyte precursor cells for a long time to make them age, by contacting with the test substance, the anti-aging effect can be evaluated. In addition, for example, by contacting a cell group containing human astrocytes of the present invention with a test substance, the test substance that controls the adverse effects of aging, such as the effect of inhibiting the secretory phenomenon associated with cell senescence (SASP, Senescence-Associated Secretory Phenotype), can be evaluated. In addition, it is also possible to search for test substances that show the effect of rejuvenating aging cells.

[0197] <Method for producing a co-culture comprising a human astrocyte population, human-derived neural cells, and human-derived microglial cells>

[0198] According to the present invention, there is provided a method for producing a co-culture, comprising:

[0199] The steps of adding the human astrocyte group, human-derived neural cells and human-derived microglial cells to a culture container; and co-culturing the human astrocyte group, the neural cells and the microglial cells in the culture container.

[0200] In the present invention, the co-culture may be a two-dimensional culture or a three-dimensional culture. As a three-dimensional culture, it may be in the shape of a sphere.

[0201] In the present invention, when the human astrocyte population, neural cells, and microglial cells of the present invention are added to a culture container, the timing of adding each cell may be simultaneous or may be separate.

[0202] In the present invention, when preparing a two-dimensional co-culture, the human astrocyte population is preferably cultured in advance in a culture container, and neurons and microglia can be added to the culture container containing the cultured human astrocyte population.

[0203] In the present invention, when preparing a three-dimensional culture, it is preferred that the above-mentioned human astrocyte group, neural cells, and microglia cells be added simultaneously to a culture container before co-culturing of cells.

[0204] “Before co-culture of cells” means that co-culture is not considered to have started within 1 to 24 hours after one or two types of cells have been added.

[0205] Specifically, for example, in the case of preparing a three-dimensional culture, the three types of cells can be placed in one container, suspended and mixed in a culture medium, and then added to the culture container. In this case, the step of adding the human astrocyte group, human-derived neural cells, and human-derived microglia to the culture container includes the step of suspending the human astrocyte group, human-derived neural cells, and human-derived microglia in a culture medium and the step of simultaneously adding the culture medium containing the human astrocyte group, the neural cells, and the microglia obtained above to the culture container.

[0206] Alternatively, the three types of cells may be suspended in different culture media and added to the same culture container at the same time. Alternatively, the three types of frozen cells that have been cryopreserved may be melted and added to the same culture container at the same time.

[0207] However, the method of simultaneously adding the human astrocyte group, neural cells, and microglial cells to the culture container is not limited to the above.

[0208] In the present invention, three kinds of cells (astrocytes, neurons and microglia) can be co-cultured in any ratio. In addition, by co-culturing three kinds of cells (astrocytes, neurons and microglia), it is possible to better reflect and simulate the human brain than previous culture systems, which is useful in studying the original function of the brain or the pathological formation mechanism. In the present invention, owing to the use of three kinds of cells, it is possible to evaluate the cell-cell interaction in brain function or pathological formation.

[0209] The neural cells and microglial cells used for co-culture with the above-mentioned human astrocyte group are preferably cells induced to differentiate from human-derived pluripotent stem cells.

[0210] Examples of human-derived pluripotent stem cells include human iPS cells, human ES cells, and human mesenchymal stem cells. Human iPS cells are preferred, but are not particularly limited. Human iPS cells refer to iPS cells produced from human cells.

[0211] As pluripotent stem cells derived from humans, pluripotent stem cells derived from a subject in which disease-related genes do not have mutations or pluripotent stem cells derived from a subject in which disease-related genes have mutations can be cited, although not particularly limited, but preferably pluripotent stem cells derived from a subject in which disease-related genes do not have mutations. "No mutation in disease-related genes" means that there is no mutation in disease-related genes that is the cause of nervous system diseases. That is, even if there is a mutation in a gene, as long as it is a mutation that will not be the cause of the disease, it is interpreted as no mutation in the disease-related gene.

[0212] ES cells can be established, for example, by culturing early pre-implantation embryos, inner cell masses constituting the early embryos, single blastomeres, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press 1994, Thomson, JA et al: Science, 282, 1145-1147, 1998). As early embryos, those produced by nuclear transplantation of somatic cell nuclei can be used (Wilmut et al: Nature, 385, 810, 1997; Cibelli et al: Science, 280, 1256, 1998; Iritani et al: Protein Nuclease, 44, 892, 1999; Baguisi et al: Nature Biotechnology, 17, 456 (, 1999; Wakayama et al: Nature, 394, 369, 1998; Nature Genetics, 22, 127, 1999; Proc. Natl. Acad. Sci. USA, 96, 14984, 1999; Rideout III et al: Nature Genetics, 24, 109, 2000; Tachibana et al: Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transplantation). Transfer (Human Embryonic Stem Cells Derived from Somatic Cell Nuclear Transfer), Cell, 2013, in press (Cell, 2013, to be published). As early embryos, parthenogenetic embryos can be used (Kim et al: Science, 315, 482-486, 2007, Nakajima et al: Stem Cells, 25, 983-985, 2007, Kim et al: Cell Stem 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, for the preparation of ES cells, reference may be made to Strelchenko N, et al: Reprod Biomed Online. (Reproductive Biomed Online Journal) 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 (Enzymology Methods Journal), Vol. 365, 2003, etc. In addition, 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.

[0213] ES cells can also be obtained from storage institutions or commercially available. For example, human ES cells can be obtained from the Institute of Regenerative Medicine, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0214] As a method for obtaining human-derived neural cells, for example, there can be mentioned induction from somatic cells collected from a healthy person (healthy person) who does not have a mutation in a disease-related gene that causes a nervous system disease or a nervous system disease, or from somatic cells collected from a patient who has a mutation in a disease-related gene that causes a nervous system disease or a nervous system disease, or induction from established human iPS cells, etc.

[0215] The neural cells induced to differentiate from human-derived pluripotent stem cells are not particularly limited, but are preferably motor neurons, cerebral cortex excitatory neurons, or substantia nigra neurons.

[0216] The method for inducing differentiation of neural cells from human-derived pluripotent stem cells is not particularly limited, but there are methods of preparing neural stem cells from pluripotent stem cells using low molecular weight compound treatment and then inducing neural cells, and methods of directly inducing neural cells by gene expression and the like.

[0217] Examples of methods for inducing differentiation of neural cells from pluripotent stem cells include:

[0218] (1) A method of forming embryoid bodies (cell clusters containing neural progenitor cells) by culturing in a serum-free medium and then differentiating them (SFEB method: Watanabe K, et al, Nat. Neurosci., 8, 288-296, 2005; SFEBq method: Wataya T, et al, Proc. Natl. Acad. Sci. USA, 105, 11796-11801, 2008);

[0219] (2) Method of culturing and differentiating cells on stromal cells (SDIA method: Kawasaki H, et al, Neuron, 28, 31-40, 2000);

[0220] (3) A method of culturing and differentiating cells on a matrix gel supplemented with drugs (Chambers SM, et al, Nat. Biotechnol., 27, 275-280, 2009);

[0221] (4) A method of culturing and differentiating in a medium containing a low molecular weight compound as a substitute for cytokines (U.S. Patent No. 5,843,780);

[0222] (5) A method for differentiating pluripotent stem cells by introducing neural induction factors (Ngn2, Neurogenin 2, etc.) into the pluripotent stem cells and causing them to express (WO2014 / 148646; and Zhang Y, et al, Neuron, 78, 785-98, 2013);

[0223] (6) A method for differentiating pluripotent stem cells by introducing miR-9 / 9*-124 into the pluripotent stem cells and expressing the miR-9 / 9*-124;

[0224] and combinations of these methods.

[0225] Among the above, the method (5) of introducing and expressing Ngn2 in pluripotent stem cells is preferred because mature neural cells can be obtained in a short period of time and with high efficiency.

[0226] Examples of human-derived neural cells include induced neural cells differentiated from iPS cells by forced expression of Ngn2, iCell glutamatergic neurons (FUJIFILM Cellular Dynamics, C1033), iCell GABAergic neurons (FUJIFILM Cellular Dynamics, C1008), iCell dopamine neurons (FUJIFILM Cellular Dynamics, C1028), iCell motor neurons (FUJIFILM Cellular Dynamics, C1048), etc. Preferred are induced neural cells differentiated from iPS cells by forced expression of Ngn2 and iCell glutamatergic neurons.

[0227] Examples of human-derived microglial cells include iCell microglial cells (FUJIFILM Cellular Dynamics, C1110) and microglial cells (Axol Bioscience, AX0664). Among them, iCell microglial cells are preferred.

[0228] The neural cells are preferably cells that express at least one neural cell-specific marker gene consisting of β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2) and have β-III tubulin-positive processes (hereinafter referred to as neurites).

[0229] Microglia are cells that express at least one microglia-specific marker gene consisting of 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).

[0230] In the present invention, the above-mentioned cells are co-cultured in a culture container.

[0231] As a culture container, a culture plate, a culture bowl, a cell culture insert or a cell culture flask with holes can be used, preferably a culture plate with holes. Specifically, ViewPlate (PerkinElmer), 96-well microplate (Greiner), 96-well polystyrene microplate (Corning), PrimeSurface (trademark) plate (SumitomoBakelite Co., Ltd.), Cell-Repellent plate (Greiner Bio one) and the like can be cited, preferably ViewPlate (PerkinElmer), PrimeSurface (trademark) plate (Sumitomo Bakelite Company Limited).

[0232] Examples of the shape of the culture container include a flat bottom, a round bottom, a U-shaped bottom, a V-shaped bottom, and the like, but the shape is not particularly limited.

[0233] In the case where the co-culture is a three-dimensional culture, the property of the surface contacted by the culture medium in the container is preferably cell non-adhesive. Thus, the cells are cultured in a floating state and easily form spheroids. Alternatively, only a certain part of the surface inside the container can be set as cell adhesive, and the other parts can be set as cell non-adhesive. In this case, the cells can gather at the cell adhesive part to form a spheroid.

[0234] When the co-culture is a two-dimensional culture, the lower limit of the cell density (cell density refers to the total cell density of two or more cells used. The same applies hereinafter) when inoculated into the culture container is not particularly limited, and is preferably 2.5×10 4 Cells / cm 2 More preferably, 5.0×10 4 Cells / cm 2 More preferably, 10.0×10 4 Cells / cm 2 More preferably, 15.0×10 4 Cells / cm 2 More than 20.0×10 4 Cells / cm 2 Above, the most preferred is 25.0×10 4 Cells / cm 2 The upper limit of the cell density when inoculated into the culture container is not particularly limited, and may be, for example, 60.0×10 4 Cells / cm 2 Below, preferably less than 55.0×10 4Cells / cm 2 , more preferably 50.0×10 4 Cells / cm 2 Below, more preferably 45.0×10 4 Cells / cm 2 Below, more preferably 40.0×10 4 Cells / cm 2 Below, particularly preferably 35.0×10 4 Cells / cm 2 the following.

[0235] When the co-culture is a two-dimensional culture, the cell density when seeded into the culture vessel is preferably 5.0×10 4 Cells / cm 2 Above and 55.0×10 4 Cells / cm 2 Below, more preferably 10.0×10 4 Cells / cm 2 Above and 50.0×10 4 Cells / cm 2 Below, more preferably 15.0×10 4 Cells / cm 2 Above and 45.0×10 4 Cells / cm 2 Below, more preferably 20.0×10 4 Cells / cm 2 Above and 40.0×10 4 Cells / cm 2 Below, particularly preferably 25.0×10 4 Cells / cm 2 Above and 35.0×10 4 Cells / cm 2 the following.

[0236] When the co-culture is a three-dimensional culture, the lower limit of the number of cells (the number of cells refers to the total number of cells of the three types. The same applies hereinafter) when seeded in a 96-well plate is not particularly limited, and is preferably 0.7×10 4 cells / well or more, more preferably 1.0×10 4 cells / well or more, more preferably 1.2×10 4 cells / well or more, more preferably 1.4×10 4 cells / well or more, particularly preferably 1.6×10 4 cells / well or more, preferably 1.8×10 4The upper limit of the cell density when inoculated into the culture container is not particularly limited, and can be, for example, 8.0×10 4 Cells / well or less, preferably less than 8.0×10 4 cells / well, preferably 5.0×10 4 cells / well or less, more preferably 3.0×10 4 cells / well or less, more preferably 2.5×10 4 cells / well or less, particularly preferably 2.3×10 4 cells / well.

[0237] When the co-culture is a three-dimensional culture, the number of cells seeded in a 96-well plate is preferably 1.0 × 10 4 Cells / well or more and 8.0×10 4 cells / well or less, preferably 1.2×10 4 Cells / well or more and 5.0×10 4 cells / well or less, more preferably 1.4×10 4 Cells / well or more and 3.0×10 4 cells / well or less, more preferably 1.6×10 4 Cells / well or more and 2.5×10 4 cells / well or less, particularly preferably 1.8×10 4 Cells / well or more and 2.3×10 4 cells / well.

[0238] The culture medium can be selected from known culture media or commercially available culture media. The culture medium used for culture can be used by adding additives to a basal culture medium. Here, examples of the basal medium include DMEM, DMEM (Dulbecco's Modified Eagle Medium) / F12, BrainPhys Neuronal Medium, Neurobasal Medium-A, Neurobasal Medium, Neural Progenitor Basal Medium, NS-A Basal Medium, Basal Medium Eagle (BME), BGJb Medium, CMRL1066 Medium, Glasgow Minimum Essential Medium (GMEM) (MEM), Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle MEM, αMEM, and Ham's F12. Medium (Ham's F12 medium), RPMI 1640 Medium (RPMI 1640 medium), Fischer's Medium (Fischer's medium), etc. Also, a single medium may be used as the medium, or two or more mediums may be used in combination.

[0239] Specific examples of additives that can be added to the culture medium include serum, retinoic acid, Wnt, BMP (such as BMP-4), CNTF, GDNF (Glial cell line-derived neurotrophic factor), bFGF (basic fibroblast growth factor), EGF (Epidermal growth factor), HGF (Hepatocyte growth factor), SHH (Sonic hedgehog), IGF-1 (Insulin-like Growth Factor 1), Activin A, Heregulin β-1, interleukins, 8-Br-cAMP, heparin, heparin sulfate, laminin, collagen, fibronectin, progesterone, selenite, B-27 (trademark) supplement, N2 Supplement with Transferrin (Apo), N2 Supplement with Transferrin, GlutaMAX, L(+)-ascorbic acid, ITS-supplement, MEM Non-Essential Amino Acid (essential amino acids), etc., but not particularly limited. In addition, antibiotics (penicillin, streptomycin, etc.) may be added.

[0240] According to the present invention, there is provided a co-culture comprising the above-mentioned human astrocyte population, human-derived neural cells, and human-derived microglial cells obtained by the above-mentioned method for producing a co-culture of the present invention.

[0241] The present invention will be specifically described by the following examples, but the present invention is not limited to the scope of the examples.

[0242] Example

[0243] Experimental Example 1: Induction of human senescent astrocytes from astrocyte precursor cells derived from human iPS cells

[0244] (1) Senescence induction based on long-term culture of astrocyte precursor cells

[0245] Astrocyte precursor cells derived from human iPS cells were purchased from Axol Bioscience (ax0083), Applied Stem Cell (ASE-9322P), or XCell Science (XCS-AP-001-1V) and used.

[0246] 15 mL of iMatrix-511silk (Matrixome, 892021) diluted 167-fold with PBS (-) (FUJIFILM Wako Pure Chemical Corporation, 166-23555) was added to a flask (CELLC OAT (registered trademark), PDL, 650 ml, flask, filter cap, Greiner Bio One, 661940) and allowed to stand at 4° C. After 24 hours, the medium was replaced with the following precursor cell medium and used.

[0247] Composition of precursor cell culture medium

[0248] The reagents shown in Table 2 were added to DMEM / F12 (Life technologies, 11320-033).

[0249] [Table 2]

[0250]

[0251] Astrocyte precursor cells derived from human iPS cells were suspended in precursor cell culture medium at a concentration of 30 × 10 4 The cells were inoculated into flasks at a density of 10 cells / flask and cultured at 37°C and 5% CO 2 . The cells were subcultured every 2 weeks and culture was continued for 84 days.

[0252] (2) Differentiation induction into human senescent astrocytes

[0253] Matrigel basement membrane matrix (Corning, 356234) diluted 120-fold with DMEM / F12 (Life technologies, 11320-033) was added to a 6-well plate at 1.5 mL / well and allowed to stand at 4° C. After 24 hours, the plate was replaced with a precursor cell culture medium.

[0254] Astrocyte precursor cells derived from human iPS cells cultured for 84 days were detached by TrypLE Select (Thermo Fisher Scientific, 12563-029) and cultured at 30 × 10 4 Cells were seeded into 6-well plates at a density of 1.54 × 10 / well and incubated at 37°C with 5% CO. 2 The next day, the medium was replaced with the following differentiation induction medium and the cells were cultured for 5 more days to induce differentiation into human senescent astrocytes.

[0255] Composition of differentiation induction medium

[0256] The reagents shown in Table 3 were added to DMEM / F12 (Life technologies, 11320-033).

[0257] [Table 3]

[0258]

[0259] (3) Expression of astrocyte markers

[0260] The expression of GFAP, a marker of astrocytes, was confirmed by immunostaining. For human astrocytes prepared from aged human astrocyte precursor cells and human astrocytes prepared from non-senescent human astrocyte precursor cells, a formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, 061-00416) diluted 10 times with PBS (-) (FUJIFILM Wako Pure Chemical Corporation, 166-23555) was added and fixed by standing at room temperature for 30 minutes. After washing three times with PBS (-), a solution (1% BSA) prepared by adding 1% bovine serum albumin (BSA) to PBS (-) was added and blocked. The blocking solution was removed, and a solution in which the primary antibody (Millipore, MAB3402) was diluted 3000 times with 1% BSA was added, and it was left to stand at 4°C overnight. After washing with PBS (-) three times, a solution of secondary antibody (Thermo Fisher Scientific, A11005) diluted 1000 times with 1% BSA was added and treated at room temperature for 1 hour. After washing with PBS (-) three times, images were acquired using IncuCyte (registered trademark) S3 (Essen BioScience, 4647). The results are shown in Figure 1 .

[0261] (4) Quantification of human astrocyte cell number by flow cytometry

[0262] Regarding human astrocytes prepared from aged human astrocyte precursor cells, the GFAP positive rate was quantified by flow cytometry. Regarding live cells and dead cells, they were labeled using the LIVE / DEAD Aqua Fixable Dead Cell Staining Kit (Thermo Fisher Scientific, L34957) according to the attached instruction manual. 2% formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, 061-00416) was added to the cells and fixed by standing for 15 minutes. After washing with Perm / Wash Buffer (BD Bioscience, 554723), the cells were labeled with GFAP monoclonal antibody (131-17719), Alexa Fluor (registered trademark) 488 (Thermo Fisher Scientific, A-21294) or Alexa Fluor (registered trademark) 488 Mouse IgG1, κ Isotype Ctrl (FC) Antibody (Biolegend, 400129), and then only live cells were selected using an Attune NxT flow cytometer (Thermo Fisher Scientific) using the signal of the LIVE / DEAD Aqua fixable dead cell stain as an index. The fluorescence signal intensity when irradiated with 488 nm laser and the number of cells with the signal intensity (count) were calculated ( Figure 2 ). For cells treated with GFAP monoclonal antibody, Alexa Fluor 488, cells showing fluorescence signal intensity stronger than that when treated with negative control Alexa Fluor 488 Mouse IgG1, κIsotype Ctrl (FC) Antibody that does not react to a specific endogenous antigen were defined as GFAP positive, and the ratio of GFAP positive cells to the total number of living cells was calculated as the GFAP positive rate.

[0263] Among human astrocytes produced from aged human astrocyte precursor cells, 99% were GFAP positive.

[0264] (5) Quantification of marker expression by digital PCR

[0265] Total RNA was extracted from human aged astrocytes. RNeasy (registered trademark) Plus Mini Kit (Qiagen, 74136) was used for the extraction of Total RNA according to the attached instruction manual. PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, RR047A) was used for the synthesis of cDNA from Total RNA according to the attached instruction manual.

[0266] The markers were quantified using droplet digital PCR, ddPCR EvaGreen Supermix (BioRad, 1864034). Samples were prepared with the composition shown in Table 4, and marker expression levels were absolutely quantified using the QX200 AutoDG Droplet Digital PCR System (Bio Rad, 1864100J3).

[0267] [Table 4]

[0268]

[0269] As primers, the primers listed in the following Table 5 were used.

[0270] [Table 5]

[0271]

[0272] PCR reaction was performed under the conditions shown in Table 6 below, and the expression level was calculated by the expression level of the target gene relative to the expression level of GAPDH (copy number / copy number).

[0273] [Table 6]

[0274]

[0275] The results are shown in Figure 3 and Figure 4 . It was confirmed that the expression levels of CDKN2A, IGFBP5, NNMT and HLA-DRB5 in aged human astrocytes were higher than those in non-aged human astrocytes. In addition, it was confirmed that aged human astrocytes and non-aged human astrocytes hardly expressed C3 compared with human A1 astrocytes. C3 was not detected in all 6 cases of non-aged human astrocytes. C3 was not detected in 5 of 7 cases of aged human astrocytes. That is, it was confirmed that aged human astrocytes and non-aged human astrocytes are not human A1 astrocytes.

[0276] (6) Staining of senescence marker (γH2AX)

[0277] The focus formation of γH2AX, one of the senescence markers, was evaluated by immunostaining. A formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, 061-00416) diluted 10-fold with PBS(-) (FUJIFILM Wako Pure Chemical Corporation, 166-23555) was added to human astrocytes prepared from senescent human astrocyte precursor cells and human astrocytes prepared from non-senescent human astrocyte precursor cells, and the cells were fixed by standing at room temperature for 30 minutes. After washing three times with PBS(-), a solution prepared by adding 1% BSA to PBS(-) (1% BSA) was added for blocking, and a solution prepared by diluting the primary antibody (Millipore, 05-636-I) 3000-fold with 1% BSA was added, and the cells were left standing at 4°C overnight. After washing three times with PBS(-), a solution of secondary antibody (ThermoFisher Scientific, A11029) diluted 1000 times with 1% BSA was added and treated at room temperature for 1 hour. After washing three times with PBS(-), images were captured and acquired using IncuCyte (registered trademark) S3 (Essen BioScience, 4647). The number of foci (point signals) was quantified using Image J. The fluorescent signal was binarized (threshold: 70), and the number of foci (points) was quantified using "Analyze Particles". The results are shown in Figure 5 middle.

[0278] It was confirmed that more foci of γH2AX, one of the senescence markers, were formed in senescent human astrocytes compared with non-senescent human astrocytes.

[0279] (7) Staining of senescence markers (SA-β-GAL)

[0280] The expression of SA-β-GAL, one of the aging markers, was evaluated. For human astrocytes produced from aged human astrocyte precursor cells and human astrocytes produced from non-aging human astrocyte precursor cells, formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, 061-00416) diluted 10 times with PBS (-) (FUJIFILM Wako Pure Chemical Corporation, 166-23555) was added and fixed by standing at room temperature for 30 minutes. The expression of SA-β-GAL was measured using Cellular Senescence Detection Kit-SPIDER-βGal (DOJINDO LABORATORIES., SG03) according to the attached instruction manual. Images were acquired by photographing using IncuCyte (registered trademark) S3 (Essen BioScience, 4647), and the positive area (Total area: total area) and fluorescence intensity (Total integrated intensity: total integrated intensity) were quantified using Basic Analysis software. The results are shown in Figure 6 .

[0281] It was confirmed that SA-β-GAL was more strongly expressed in aged human astrocytes than in non-aged human astrocytes.

[0282] Experimental Example 2: Comparison of senescence markers in long-term culture of human astrocyte precursor cells and human astrocytes

[0283] Astrocyte precursor cells derived from human iPS cells purchased from Axol Bioscience (ax0083), Applied Stem Cell (ASE-9322P), or XCellScience (XCS-AP-001-1V) were used.

[0284] Astrocyte precursor cells derived from human iPS cells were cultured in a precursor cell medium for 43 days, and then replaced with a differentiation induction medium and differentiated into human astrocytes ( Figure 7 In addition, the cells obtained in (1) were cultured for 42 days ( Figure 7 (2)) and astrocyte precursor cells derived from human iPS cells were cultured in a precursor cell culture medium for 85 days and then replaced with a differentiation induction medium to induce differentiation into human astrocytes ( Figure 7Total RNA was extracted using RNeasy (registered trademark) Plus Mini Kit (Qiagen, 74136) according to the attached instruction manual. The expression of CDKN2A, a marker of aging, was absolutely quantified by the same method as the quantification of marker expression by digital PCR in Experimental Example 1 (5). The results are shown in Figure 7 .

[0285] The expression level of CDKN2A in human astrocytes prepared from astrocyte precursor cells derived from human iPS cells cultured for 85 days was 0.0109 copies / copy number, while the expression level of CDKN2A in human astrocytes prepared from astrocyte precursor cells derived from human iPS cells cultured for 43 days and further cultured for 42 days was 0.0025 copies / copy number. It can be seen from this that in order to prepare aged human astrocytes, long-term culture in the state of astrocyte precursor cells is important.

[0286] Experimental Example 3: Preparation of two-dimensional co-culture of aged astrocytes, neurons and microglia

[0287] (1) Senescence induction based on long-term culture of astrocyte precursor cells and conversion to human senescent astrocytes Differentiation induction

[0288] Astrocyte precursor cells derived from human iPS cells were purchased from Axol Bioscience (ax0083), Applied Stem Cell (ASE-9322P), or XCell Science (XCS-AP-001-1V) and used.

[0289] Matrigel basement membrane matrix (Corning, 356234) diluted 120 times 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, it was replaced with a precursor cell culture medium. In addition, a precursor cell culture medium was prepared with the same composition as in Table 2 of Test Example 1.

[0290] Astrocyte precursor cells derived from human iPS cells cultured for 84 days were cultured at 5×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured at 37°C and 5% CO2. The next day, the culture medium was replaced with a differentiation induction medium and cultured for 5 more days to induce differentiation into senescent astrocytes. In addition, a differentiation induction medium was prepared with the same composition as in Table 3 of Test Example 1.

[0291] (2) Preparation of two-dimensional co-culture

[0292] Neural cells were produced from iPS cells by forced expression of the Ngn2 gene (Chao Wang, et al, Stem Cell Reports., 9: 1221-1233, 2017), and cryopreserved neural cells were used. The cryopreserved cells were thawed in a 37°C warm bath, and after melting, the cells were added to the co-culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of co-culture medium, and the number of cells was counted.

[0293] Composition of co-culture medium

[0294] The reagents shown in Table 7 were added to DMEM / F12 (Life technologies, 11320-033).

[0295] [Table 7]

[0296]

[0297] Microglia derived from iPS cells (iCell microglia, FCDI, C1110) were used as microglia. The cryopreserved cells were thawed in a 37°C warm bath, and after melting, the cells were added to the co-culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of co-culture medium, and the number of cells was counted.

[0298] Neurons and microglia were expressed at 3×10 4 The cells were inoculated onto senescent astrocytes at 1 cell / well and cultured at 37°C and 5% CO2. The culture medium was replaced three times a week, with half of the medium replaced each time.

[0299] (3) Cell fixation

[0300] The two-dimensional co-cultures cultured for 3 weeks were fixed by treating with a formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, 061-00416) diluted 10-fold with PBS(-) (FUJIFILM Wako Pure Chemical Corporation, 166-23555) for 30 minutes.

[0301] (4) Immunostaining

[0302] The fixed cells were washed with PBS(-), and then treated for 30 minutes in a solution of BSA (Sigma Aldrich, A4161) and Triton X-100 (BioVision, 2104-100) diluted with PBS(-) to 1% and 0.2% (1% BSA / 0.2% Triton X-100) for blocking and permeabilization. Thereafter, as a primary antibody reaction, anti-MAP2 antibody (Novus Biologicals, NB300-213) diluted 800-fold, anti-GFAP antibody (Millipore, MAB3402) diluted 3000-fold, and anti-IBA antibody (FUJIFILM WakoPure Chemical Corporation, 011-27991) diluted 800-fold were treated with 1% BSA / 0.2% Triton X-100, and left to stand overnight at 4°C.

[0303] The next day, after washing with PBS (-), as a secondary antibody reaction, Goat anti-rabbit Alexa Fluor 488 (Thermo fisher scientific, A11008) diluted 1000 times, Goat anti-chicken Alexa Fluor 594 (Thermo fisher scientific, A11042) diluted 1000 times, Goat anti-mouse Alexa Fluor 647 (Thermo fisher scientific, A32728) diluted 1000 times and Hoechst 33342 (Dojindo, 346-07951) diluted 1000 times were treated and left to stand at room temperature for 60 minutes. After washing with PBS (-), images were obtained by taking pictures with a fluorescence microscope (Nikon, ECLIPSE Ti). Figure 8 The acquired images are shown in . (A) is the nucleus (Hoechst), (B) is the microglia (IBA1), (C) is the nerve cell (MAP2), and (D) is the stained astrocyte. Signals can be detected in all cells.

[0304] Experimental Example 4: Preparation of three-dimensional co-culture of aged astrocytes, neurons and microglia

[0305] (1) Senescence induction based on long-term culture of astrocyte precursor cells and conversion to human senescent astrocytes Differentiation induction

[0306] Astrocyte precursor cells derived from human iPS cells cultured for 84 days (XCell Science, XCS-AP-001-1V) were seeded into a flask coated with a matrigel basement membrane matrix (Corning, 354234) and cultured for 5 days (37°C, 5% CO2) using a differentiation induction medium to induce differentiation into astrocytes. In addition, a differentiation induction medium was prepared with the same composition as in Table 3 of Test Example 1.

[0307] (2) Preparation of three-dimensional co-culture

[0308] The astrocytes induced by differentiation were detached by TrypLE Select (Thermo Fisher Scientific, 12563-029) and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of 3D co-culture medium, and the number of cells was counted.

[0309] Neural cells prepared from iPS cells by forced expression of the Ngn2 gene in the same manner as in Experimental Example 3 were thawed in a 37°C warm bath. After thawing, the cells were added to a 3D co-culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of a 3D co-culture medium, and the number of cells was counted.

[0310] iPS cell-derived microglia (iCell microglia, FCDI, C1110) were thawed in a 37°C warm bath. After thawing, the cells were added to 3D co-culture medium and centrifuged at 600×g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of 3D co-culture medium, and the number of cells was counted.

[0311] Astrocytes, neurons, and microglia were mixed in a ratio of 10:6:3 to obtain 1.9×10 4 Cells were seeded into 96-well plates (PrimeSurface (trademark) plates 96U, Sumitomo Bakelite Company Limited, MS-9096U) at a rate of cells / well and cultured at 37°C and 5% CO2. The culture medium was replaced three times a week, with half of the medium replaced each time.

[0312] Composition of 3D co-culture medium

[0313] The reagents shown in Table 8 were added to DMEM / F12 (Life technologies, 11320-033).

[0314] [Table 8]

[0315]

[0316] (3) Cell fixation and immunostaining

[0317] The three-dimensional co-cultures cultured for 3 weeks were fixed by treating in 4% paraformaldehyde / phosphate buffer (FUJIFILM WakoPure Chemical Corporation, 161-20141) for 30 minutes. After washing with PBS (-), the cells were permeabilized by treating with Triton X-100 (BioVision, 2104-100) diluted to 0.4% in PBS (-) for 15 minutes. After the permeabilization, the cells were blocked by treating with a BSA solution (Sigma Aldrich, A416) prepared in PBS (-) at a concentration of 4% for 30 minutes. Thereafter, as a primary antibody reaction, anti-MAP2 antibody (Novus, NB300-213) diluted 600-fold, anti-IBA1 antibody (FUJIFILM Wako Pure Chemical Corporation, 019-19741) diluted 600-fold, and anti-GFAP antibody (Merck, MAB3402) diluted 3000-fold were treated and left to stand at 4°C overnight.

[0318] On the next day, after washing with PBS (-), as secondary antibody reaction, Goat anti-rabbit Alexa Fluor 488 (goat anti-rabbit Alexa Fluor 488) (Thermo fisher scientific, A11008) diluted 1000 times, Goat anti-chicken Alexa Fluor 594 (goat anti-chicken Alexa Fluor594) (Thermo fisher scientific, A11042) diluted 1000 times, Goat anti-Mouse AlexaFluor 647 (goat anti-mouse AlexaFluor 647) (Thermo fisher scientific, A32728) diluted 1000 times and Hoechst 33342 solution (Hoechst 33342 staining solution) (Dojindo, H342) diluted 1000 times were treated and left to stand at room temperature for 60 minutes. After washing with PBS (-), images were acquired by photographing using a confocal quantitative imager CQ1 (YOKOGAWA ELECTRIC CORPORATION, CellVoyager CQ1). Fig. 9 The acquired images are shown in . (A) is the nucleus (Hoechst), (B) is the microglia (IBA1), (C) is the nerve cell (MAP2), and (D) is the stained astrocyte. All signals were detected.

Claims

1. A human astrocyte population obtained by differentiation and induction of astrocyte precursor cells derived from human iPS cells, The human astrocyte population comprises at least 90% human astrocytes, In the human astrocytes, a) CDKN2A is positive, b) at least one gene marker selected from the group consisting of IGFBP5, NNMT, HLA-DRB1 and HLA-DRB5 is positive, c) The expression level of C3 normalized with reference gene GAPDH was less than 0.05 copies / copy.

2. The human astrocyte population according to claim 1, wherein In the human astrocytes, the expression level of CDKN2A normalized with reference gene GAPDH was 0.004 copies / copy or more.

3. The human astrocyte population according to claim 1 or 2, wherein: In the human astrocytes, the expression level of IGFBP5 normalized with reference gene GAPDH is 0.1 copies / copy or more.

4. The human astrocyte population according to claim 1 or 2, wherein: In the human astrocytes, the expression level of NNMT normalized with reference gene GAPDH was 0.005 copies / copy or more.

5. The human astrocyte population according to claim 1 or 2, wherein: In the human astrocytes, the expression level of HLA-DRB5 normalized with reference gene GAPDH is 0.1 copies / copy or more.

6. The human astrocyte population according to claim 1 or 2, wherein: Furthermore, at least one gene marker selected from the group consisting of γH2AX and SA-β-GAL is positive.

7. The cell population according to claim 1 or 2, wherein The human iPS cell-derived astrocyte precursor cells are astrocyte precursor cells produced from human iPS cells derived from a healthy human. 8 . A cell population culture comprising the human astrocyte population according to claim 1 or 2 and a culture medium substantially free of serum. 9 . The cell population culture according to claim 8 , further comprising at least one factor selected from the group consisting of BMP4 and CNTF.

10. The method for producing an astrocyte population according to claim 1 or 2, comprising: A step of proliferating astrocyte precursor cells derived from human iPS cells; and The process of inducing the differentiation of astrocyte precursor cells derived from human iPS cells after proliferation. 11 . A method for evaluating a test substance, comprising the step of contacting the human astrocyte population according to claim 1 or 2 with a test substance.

12. A method for producing a co-culture, comprising: A step of adding the human astrocyte population, human-derived neural cells, and human-derived microglial cells according to claim 1 or 2 to a culture container; and a step of co-culturing the human astrocyte population, the neural cells, and the microglial cells in the culture container.

13. The manufacturing method according to claim 12, wherein: The neural cells and the microglial cells are cells differentiated and induced from human-derived pluripotent stem cells.

14. The manufacturing method according to claim 12 or 13, wherein: The human-derived pluripotent stem cells are human iPS cells.

15. The manufacturing method according to claim 12 or 13, wherein: The co-culture is a two-dimensional culture or a three-dimensional culture. 16 . A co-culture comprising the human astrocyte population according to claim 1 or 2 obtained by the production method according to claim 12 or 13, human-derived neural cells, and human-derived microglial cells.

Images