Method for producing cell aggregate containing glial progenitor cells

Abstract

This method for producing a cell aggregate including glial progenitor cells comprises: (1) a step for forming a cell aggregate by subjecting pluripotent stem cells to a suspension culture for 5-10 days in an embryoid body-forming medium containing at least one SMAD signaling inhibitor and at least one Wnt signaling activator, in the absence of feeder cells; (2) a step for subjecting the cell aggregate obtained in (1) to a suspension culture in an embryoid body-forming medium containing retinoic acid; (3) a step for subjecting the cell aggregate obtained in (2) to a suspension culture in an embryoid body-forming medium or nerve and glia-proliferating medium, which contains retinoic acid and at least one SHH signaling activator; and (4) a step for subjecting the cell aggregate obtained in (3) to a suspension culture in a nerve and glia-proliferating medium which does not contain retinoic acid and contains at least one SHH signaling activator.

Description

Technical Field

[0001] The present invention relates to cell aggregates containing glial progenitor cells derived from pluripotent stem cells, a method for producing the same, and the like.

Background Art

[0002] Demyelinating diseases including spinal cord injury and diseases based on glial cell disorders rarely heal naturally, and in most cases, not only the loss of glial cells that form myelin but also axonal degeneration and cell body degeneration are caused, exacerbating the condition. In particular, although spinal cord injury shows some rehabilitation effects in some patients, there is no fundamental treatment method for regenerating lost nerve axons and glial cells, and it is a disease with high unmet medical needs. As a treatment strategy for nerve regeneration and remyelination, cell replenishment by transplantation is regarded as promising.

[0003] Many research results have been reported to realize the treatment of transplanting neural progenitor cells, and in recent years, methods for inducing the differentiation of neural progenitor cells from pluripotent stem cells are known (for example, Non-Patent Documents 1 and 2). In addition, as a method for culturing nervous system cells, a method of forming embryoid bodies in a serum-free medium and performing suspension culture is also known (for example, Non-Patent Document 3).

[0004] Since the loss of myelin is prominent in spinal cord injury, it has been considered important to transplant cells containing a large number of oligodendrocytes among glial cells for its treatment (Non-Patent Documents 4, 6, and 7), but which cell types and to what extent are required to show a therapeutic effect has not been clarified so far.

[0005] Furthermore, for the practical application of cell transplantation, it is essential that undifferentiated pluripotent stem cells do not remain in order to prevent tumorigenesis of the transplanted cells, and it is desirable that no components derived from heterologous cells are included in order to avoid contamination with infectious agents. However, cell aggregates containing oligodendrocyte progenitor cells reported so far (Non-Patent Documents 4, 5 and Patent Document 1) are produced in the presence of feeder cells such as mouse cells, so there is a possibility that they contain feeder cells or components derived from heterologous cells derived from feeder cells. Therefore, it is necessary to establish a manufacturing method that is more suitable for clinical application and does not contain components derived from heterologous cells.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention aims to provide a cell aggregate that is useful for regenerative medicine, does not contain components derived from heterologous cells, and has a high content rate of glial progenitor cells, and to provide a method for producing a cell aggregate containing glial progenitor cells in which there is no possibility of contamination with components derived from heterologous cells.

Means for Solving the Problems

[0009] The present inventors focused on the decrease in the interaction between myelin - neurons - astrocytes in the lesion, and considered that it is important that the cells used for transplantation contain all of oligodendrocyte progenitor cells, neural progenitor cells, and astrocyte progenitor cells. They investigated a method for inducing differentiation of such cell aggregates from iPS cells and a differentiation induction method more suitable for clinical application.

[0010] The present inventors first examined the method described in Non - Patent Document 5, which is a conventional differentiation induction method. In the method described in Non - Patent Document 5, iPS cells are maintained and cultured on mouse cells, which are feeder cells. That is, since iPS cells are induced to differentiate using a medium containing components derived from heterologous cells, the obtained cell aggregates containing oligodendrocyte progenitor cells may contain components derived from heterologous cells. On the other hand, for clinical application, it has been desired to exclude components derived from heterologous cells as much as possible. Therefore, the present inventors intensively studied to develop a method for inducing differentiation of cell aggregates containing oligodendrocyte progenitor cells from feeder - free iPS cells, that is, using a medium free of components derived from heterologous cells.

[0011] The present inventors attempted differentiation induction with reference to the method described in Non - Patent Document 5 using feeder - free iPS cells cultured in the absence of feeder cells in an undifferentiated maintenance medium not containing components derived from heterologous cells. As a result, depending on the cell line of iPS cells, there were cases where embryoid bodies (hereinafter sometimes abbreviated as "EB") could not be formed, and it was found that the probability of finally being induced into cell aggregates without the ability to differentiate into oligodendrocytes was high, suggesting that this differentiation induction method is unstable.

[0012] Therefore, as a result of investigating methods for forming EBs, it was found that by changing the culturing method to serum-free embryoid body quick (hereinafter sometimes abbreviated as the "SFEBq method") and by adopting a combination of a specific medium and a differentiation inducer during the process of differentiation induction, it was possible to stably form EBs and obtain cell aggregates (neurospheres) containing glial progenitor cells that have the ability to differentiate into oligodendrocytes. It was also found that these cell aggregates exhibit a therapeutic effect when transplanted into a disease model animal. That is, the present inventors have for the first time successfully established a production method capable of mass-producing cell aggregates containing glial progenitor cells that do not contain components derived from heterologous cells.

[0013] That is, the present invention relates to the following. [1] A method for producing a cell aggregate containing glial progenitor cells, comprising the following steps: (1) A step of forming cell aggregates by culturing pluripotent stem cells in suspension for 5 to 10 days in an embryoid body formation medium containing one or more SMAD signal transduction inhibitors and one or more Wnt signal transduction activators in the absence of feeder cells; (2) A step of culturing the cell aggregates obtained in (1) in suspension in an embryoid body formation medium containing retinoic acid; (3) A step of culturing the cell aggregates obtained in (2) in suspension in an embryoid body formation medium containing retinoic acid and one or more SHH signal transduction activators or a medium for nerve / glia proliferation; and (4) A step of culturing the cell aggregates obtained in (3) in suspension in a medium for nerve / glia proliferation that does not contain retinoic acid and contains one or more SHH signal transduction activators. The method further comprising the following step: (5) A step of culturing the cell aggregates obtained in (4) in suspension in a medium for nerve / glia proliferation that does not contain both retinoic acid and an SHH signal transduction activator. The production method may include this step. [2] The production method according to [1], wherein in the step (1), the pluripotent stem cells are cultured using a culture vessel having a plurality of pores of uniform shape. [3] In the step (1), compared with the start of the step (1), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA of SOX1, PAX6, HES4, and HES5 has increased by 100-fold or more; 2) Obtaining cell aggregates in which the expression level of OCT3 / 4 RNA has decreased to 1 / 200 or less; and 3) Obtaining cell aggregates in which the expression level of NANOG RNA has decreased to 1 / 400 or less The production method according to [1] or [2], wherein the step (2) is started after the step (1) is continued until at least one of the above is satisfied. [4] In the step (2), compared with the start of the step (2), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA of ASCL1, DCX, HEY1, ZBTB20, βIII tubulin, ELAVL3, and SLIT1 has increased by 5-fold or more; and 2) Obtaining cell aggregates in which the expression level of at least one RNA of HOXB3, HOXA4, HOXB4, HOXB6, and HOXB8 has increased by 5-fold or more The production method according to any one of [1] to [3], wherein the step (3) is started after the step (2) is continued until at least one of the above is satisfied. [5] The production method according to any one of [1] to [4], wherein the step (2) is performed for 4 to 11 days. [6] The production method according to any one of [1] to [5], wherein the oxygen concentration is 3% to 10% in the steps (1) and (2). [7] In the step (3), compared with the start of the step (3), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA of HEY2, NKX6.2, and NKX2.2 has increased by 5-fold or more; and 2) A cell aggregate in which the expression level of the RNA of OLIG1 and / or OLIG2 is increased by 10-fold or more can be obtained. The production method according to any one of [1] to [6], wherein the step (3) is continued until at least one of the following is satisfied, and then the step (4) is started. [8] The production method according to any one of [1] to [7], wherein the step (3) is performed for 4 to 11 days. [9] In the step (4), compared with the start of the step (4), the following conditions: 1) A cell aggregate in which the expression level of the RNA of at least one of NFIA, NFIB, SLC1A3, S100B, and FABP7 is increased by 10-fold or more can be obtained, and 2) A cell aggregate in which the expression level of the RNA of PAX6 is decreased to 5-fold or less can be obtained The production method according to any one of [1] to [8], wherein the step (4) is continued until at least one of the following is satisfied, and then the step (5) is started.

[10] The production method according to any one of [1] to [9], wherein the step (4) is performed for 4 days or more.

[11] In the step (4), after dispersing the cell aggregate obtained in the step (3) at the start of the step, the dispersed cells are cultured in suspension to form cell aggregates again. The production method according to any one of [1] to

[10] .

[12] The above method includes the step (5). In the step (5), after dispersing the cell aggregate obtained in the step (4) at the start of the step, the dispersed cells are cultured in suspension for 5 to 100 days to form cell aggregates again. The production method according to any one of [1] to

[11] .

[13] The production method according to any one of [1] to

[12] , wherein the above method includes the step (5), and in the step (5), the step (5) is continued until one or more markers selected from O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP, and PLP are expressed.

[14] The production method according to any one of [1] to

[12] , wherein the above method includes the step (5), and in the step (5), in the medium for culturing cell aggregates, the step (5) is continued until one or more proteins selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL are detected.

[15] The production method according to any one of [1] to

[14] , wherein the SMAD signal transduction inhibitor is two types, a TGFβ inhibitor and a BMP inhibitor.

[16] The production method according to

[15] , wherein the TGFβ inhibitor is one or more selected from the group consisting of SB431542, A83-01, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LY580276, Galunisertib (LY2157299), LY3200882, SB525334, GW788388, RepSox, and Lefty-1.

[17] The production method according to

[15] or

[16] , wherein the BMP inhibitor is one or more selected from the group consisting of Noggin, LDN-193189, LDN-212854, Dorsomorphin, K02288, Chordin, and Follistatin.

[18] The production method according to any one of [1] to

[17] , wherein the Wnt signal transduction activator is one or more selected from the group consisting of a GSK3β inhibitor, Wnt3a, a Wnt agonist, Dkk, and R-Spondin.

[19] The production method according to any one of [1] to

[18] , wherein the Wnt signaling activator is one or more selected from the group consisting of CHIR99021, BIO, Kenpaullone, SB216763, and L803-mts.

[20] The production method according to any one of [1] to

[19] , wherein the SHH signaling activator is one or more selected from the group consisting of Purmorphamine, SAG, SHH protein, and SHH fragment.

[21] The production method according to any one of [1] to

[20] , wherein the pluripotent stem cell is an induced pluripotent stem cell.

[22] The production method according to any one of [1] to

[20] , wherein the pluripotent stem cell is a human induced pluripotent stem cell.

[23] The cell aggregate containing glial progenitor cells has the following characteristics: (a) containing oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells, (b) expressing a spinal cord region marker, and (c) not containing feeder cells and components derived from feeder cells The production method according to any one of [1] to

[22] .

[24] A method for producing a cell population containing oligodendrocytes, astrocytes, and neurons, comprising a step of culturing a cell aggregate containing glial progenitor cells produced by the production method according to any one of [1] to

[23] in a maturation medium for 5 to 60 days.

[25] The cell population containing oligodendrocytes, astrocytes, and neurons is (i) cells expressing one or more markers selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2, (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3, and (iii) Cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2 The production method according to

[24] , comprising

[26] The production method according to

[24] or

[25] , wherein the maturation medium is a medium containing at least one of T3, NT-3, and LIF.

[27] The production method according to

[26] , wherein the maturation medium further contains CNTF.

[28] A cell aggregate containing glial progenitor cells obtained by the production method according to any one of [1] to

[23] .

[29] The following characteristics: (a) Containing oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells; (b) Containing cells expressing a spinal cord region marker; (c) Not containing feeder cells and components derived from feeder cells; and (d) Having the ability to differentiate into a cell population containing oligodendrocytes, astrocytes, and neurons A cell aggregate containing glial cell progenitor cells, having

[30] The cell aggregate according to

[29] , wherein the spinal cord region marker is one or more markers selected from the group consisting of HOXB3, HOXB4, HOXB6, and HOXD8.

[31] The cell aggregate according to

[29] or

[30] , further containing cells expressing one or more markers selected from the group consisting of NKX2.1, NKX2.2, NKX6.1, and NKX6.2.

[32] The following characteristics: (I) Containing cells expressing one or more markers selected from the group consisting of NFIA, NFIB, SOX9, HEY1, HEY2, ZBTB20, SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4; (II) comprising cells that express one or more markers selected from the group consisting of OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP, and PLP; (III) comprising cells that express one or more markers selected from the group consisting of DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3; (IV) comprising cells that express one or more markers selected from the group consisting of SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3, and ASCL1; and, (V) (i) cells that express one or more markers selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2, (ii) cells that express one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3, and (iii) cells that express one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2 having the ability to differentiate into a cell population comprising such cells The cell aggregate according to any one of

[29] to

[31] , having the above.

[33] Furthermore, the cell aggregate according to any one of

[29] to

[32] , comprising cells that express one or more markers selected from the group consisting of C1ORF61 and SERPINE2.

[34] Furthermore, the cell aggregate according to any one of

[29] to

[33] , comprising cells that express or secrete one or more markers selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL.

[35] A cell population comprising oligodendrocytes, astrocytes, and neurons, obtained by the production method according to any one of

[24] to

[27] .

[36] A pharmaceutical composition comprising, as an active ingredient, a cell aggregate according to any one of

[28] to

[34] or a cell population according to

[35] .

[37] A method for treating a demyelinating disease and a disease based on or accompanied by a glial cell disorder, comprising transplanting an effective amount of a cell aggregate according to any one of

[28] to

[34] or a cell population according to

[35] into a subject in need of transplantation.

[38] The treatment method according to

[37] , wherein the demyelinating disease and the disease based on or accompanied by a glial cell disorder are acute, subacute or chronic spinal cord injuries.

[39] A cell aggregate according to any one of

[28] to

[34] or a cell population according to

[35] for use in the treatment of a demyelinating disease and a disease based on or accompanied by a glial cell disorder.

[40] The cell aggregate or cell population according to

[39] , wherein the demyelinating disease and the disease based on or accompanied by a glial cell disorder are acute, subacute or chronic spinal cord injuries.

[41] A method for evaluating the toxicity or efficacy of a test substance, the method comprising contacting the test substance with a cell aggregate according to any one of

[28] to

[34] or a cell population according to

[35] , and detecting or quantifying the effect of the test substance on the cell aggregate or the cell population.

[42] A method for determining whether a cell aggregate containing glial progenitor cells is suitable for transplantation, using as an index whether one or more markers selected from the group consisting of C1ORF61 and SERPINE2 are expressed.

[43] A method for identifying glial progenitor cells or neural stem cells with high differentiation potential towards glia, comprising detecting one or more genes selected from the group consisting of C1ORF61 and SERPINE2, the protein encoded by the gene, and fragments thereof.

Effect of the Invention

[0014] According to the method for producing a cell aggregate containing glial progenitor cells of the present invention, a cell aggregate containing glial progenitor cells that does not contain feeder cells and heterogeneous cell-derived components derived from feeder cells can be produced. When this cell aggregate containing glial progenitor cells is further cultured, a cell population containing oligodendrocytes, astrocytes, and neurons can be produced. The cell aggregate or cell population obtained by the production method of the present invention is useful as a transplantation material for cell therapy.

Brief Description of the Drawings

[0015]

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Modes for Carrying Out the Invention

[0016] 〔Definition〕 In this specification, the term "stem cell" means an undifferentiated cell having the ability to differentiate and the ability to proliferate while maintaining the ability to differentiate (particularly self-renewal ability). Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells according to their differentiation ability.

[0017] A pluripotent stem cell refers to a stem cell that can be cultured in vitro and has the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissues. A multipotent stem cell means a stem cell that has the ability to differentiate into multiple types of tissues and cells, although not all types. A unipotent stem cell means a stem cell that has the ability to differentiate into a specific tissue or cell.

[0018] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), etc. Muse cells (multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSC) and GS cells prepared from germ cells (e.g., testis) are also included in pluripotent stem cells.

[0019] Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing the inner cell mass of the blastocyst stage on feeder cells or in a medium containing FGF2. Methods for producing embryonic stem cells are described, for example, in WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from a predetermined institution or purchased as a commercial product. For example, KhES-1, KhES-2, and KhES-3, which are human embryonic stem cells, are available from the Institute for Frontier Medical Sciences, Kyoto University. The Crx::Venus strain (derived from KhES-1), which is a human embryonic stem cell, is available from the National Institute of Advanced Industrial Science and Technology (AIST).

[0020] As used herein, the term "induced pluripotent stem cell" refers to a cell in which pluripotency has been induced by reprogramming somatic cells by known methods or the like.

[0021] Induced pluripotent stem cells were established from mouse cells by Yamanaka et al. in 2006 (Cell, 2006, 126(4), pp. 663-676). Induced pluripotent stem cells were also established from human fibroblasts in 2007 and have pluripotency and self-renewal ability similar to those of embryonic stem cells (Cell, 2007, 131(5), pp. 861-872; Science, 2007, 318(5858), pp. 1917-1920; Nat. Biotechnol., 2008, 26(1), pp. 101-106).

[0022] Artificial pluripotent stem cells specifically include cells in which differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells are reprogrammed by the expression of any combination of a plurality of genes selected from a group of reprogramming genes including OCT3 / 4, SOX2, KLF4, MYC (c-MYC, N-MYC, L-MYC), GLIS1, NANOG, SALL4, LIN28, ESRRB, etc., to induce pluripotency. Preferred combinations of reprogramming factors include: (1) OCT3 / 4, SOX2, KLF4, and MYC (c-MYC or L-MYC); (2) OCT3 / 4, SOX2, KLF4, LIN28, and L-MYC (Stem Cells, 2013;31:458-466).

[0023] In addition to the method of manufacturing by direct reprogramming by gene expression, artificial pluripotent stem cells can also be induced from somatic cells by the addition of compounds, etc. (Science, 2013, 341, pp.651-654).

[0024] It is also possible to obtain established artificial pluripotent stem cells. For example, human artificial pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells, etc., established at Kyoto University, are available from Kyoto University and iPS Academia Japan, Inc. As established artificial pluripotent stem cells, for example, QHJI01s04 cells established at Kyoto University are available from Kyoto University.

[0025] In this specification, the pluripotent stem cells are preferably embryonic stem cells or artificial pluripotent stem cells, more preferably artificial pluripotent stem cells.

[0026] In this specification, the pluripotent stem cells are mammalian pluripotent stem cells, preferably pluripotent stem cells of rodents (e.g., mouse, rat) or primates (e.g., human, monkey), more preferably human pluripotent stem cells, and still more preferably human artificial pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0027] Pluripotent stem cells such as human iPS cells can be subjected to maintenance culture and expansion culture by methods well known to those skilled in the art.

[0028] In this specification, "Glia" is also referred to as "Glial cell" or "neuroglial cell", and is a general term for cells that are not neurons constituting the nervous system. It is estimated that in the human brain, there are more than 10 times as many Glia cells as neurons in terms of cell number. Examples of Glia cells include Astrocyte and Oligodendrocyte, which exist around neurons in neural tissue and undertake various functions.

[0029] In this specification, "marker" means a substance present in a cell, and based on its presence or abundance, a substance that can identify or distinguish the type or properties of the cell. Specific examples of markers include mRNA, the protein and sugar chain encoded by the mRNA, and fragments thereof.

[0030] In this specification, "differentiation marker" means a marker for detecting the degree (level) of differentiation of cells at a specific differentiation stage.

[0031] In this specification, "glial progenitor cell" means a cell having the ability to differentiate into glial cells, and examples include astrocyte progenitor cells and oligodendrocyte progenitor cells.

[0032] In this specification, an "oligodendrocyte" is a type of glial cell present in the central nervous system. It exists so as to wrap around the axons of neurons (nerve cells) and is responsible for accelerating the signal conduction speed between nerve cells by forming myelin (myelin sheath). Oligodendrocytes can be identified by markers that are specifically expressed in oligodendrocytes. Examples of such markers include PLP, GalC, APC, MBP, MAG, MOG, GST-π, SOX10, O4 (O four) antigen, NG2 (CSPG4), and CNP, etc. By analyzing using, for example, gene amplification methods, in situ hybridization, immunohistological techniques, flow cytometry analysis, etc., it is possible to discriminate cells that express at least one of the above-mentioned mRNAs, sugar chains, or proteins as oligodendrocytes. Incidentally, when using NG2, which is also expressed in astrocytes, as an index among the above markers, it is desirable to identify it in combination with another marker.

[0033] In this specification, an "oligodendrocyte progenitor cell" means a cell having the ability to differentiate into an oligodendrocyte. The presence of oligodendrocyte progenitor cells can be identified by markers that are significantly expressed in oligodendrocyte progenitor cells. Examples of markers for oligodendrocyte progenitor cells include PDGFRα and OLIG2, etc. By analyzing using, for example, gene amplification methods, in situ hybridization, immunohistochemical staining methods, etc., it is possible to discriminate cells that express at least one of the above-mentioned mRNAs or proteins as oligodendrocyte progenitor cells. Note that immature oligodendrocytes that have not formed myelin are also included in oligodendrocyte progenitor cells.

[0034] In this specification, an "astrocyte" is a type of glial cell present in the central nervous system. It is thought to contribute to the activation of neurotransmission by structurally supporting neurons and regulating neurotransmitters, energy, and extracellular ion concentrations. It is also thought to supply substances that promote myelination to the above oligodendrocytes and is a cell that plays an important role together with neurons and oligodendrocytes in nervous tissue. It can be identified by a marker that is specifically expressed in astrocytes, and examples of such markers include GFAP, S100B, AQP4, NG2, and SLC1A3 (also referred to as GLAST or EAAT1). For example, by analyzing using immunohistological techniques, it is possible to discriminate cells that express at least one of the above mRNAs, sugar chains, or proteins as astrocytes. Incidentally, among the above markers, when expression is also observed in oligodendrocytes and NG2 is used as an indicator, it is desirable to identify in combination with another marker.

[0035] In this specification, an "astrocyte progenitor cell" means a cell having the ability to differentiate into an astrocyte. The presence of astrocyte progenitor cells can be identified by markers that are significantly expressed in astrocyte progenitor cells. Examples of markers for astrocyte progenitor cells include NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, and ZBTB20.

[0036] During the process of differentiating from pluripotent stem cells into oligodendrocytes, first, pluripotent stem cells differentiate into neural stem cells, and then further differentiate into oligodendrocyte progenitor cells and then oligodendrocytes in sequence. Neural stem cells have multipotency and can differentiate into any of neural progenitor cells, astrocyte progenitor cells, and oligodendrocyte progenitor cells. However, once differentiated into oligodendrocyte progenitor cells, they are fated to differentiate into oligodendrocytes. Also, when differentiated into neural progenitor cells, they are fated to differentiate into neurons, and when differentiated into astrocyte progenitor cells, they are fated to differentiate into astrocytes.

[0037] Whether being fated to differentiate into astrocytes here can be confirmed by whether at least one of the following one or more markers: NFIA, NFIB, SOX9, HEY1, HEY2, ZBTB20, SLC1A3, and S100B is expressed, preferably by whether at least one of SOX9, HEY1, HEY2, ZBTB20, SLC1A3, and S100B is expressed. Here, S100B is also referred to as S100β.

[0038] In this specification, a nerve cell refers to a nerve unit composed of a cell body, dendrites, and an axon, and is also called a neuron. Nerve cells can be identified by markers that are significantly expressed, and examples of such markers include βIII tubulin, MAP2, and ELAVL3, etc.

[0039] A nerve cell means a cell that has the function of transmitting stimuli from other nerve cells or stimulus-receiving cells to other nerve cells, muscles, or gland cells. Nerve cells are classified, such as serotonergic nerves, motor nerves, etc., according to the differences in neurotransmitters produced by nerve cells, but the types of neurotransmitters are not particularly limited in this specification.

[0040] As used herein, "neural progenitor cells" refer to cells that have the ability to differentiate into neurons and are destined to differentiate into neurons, and can be identified by neural cell adhesion molecule (NCAM), polysialylated NCAM, neural markers (DCX, βIII tubulin, etc.).

[0041] As used herein, "neural stem cells" refer to cells that have both the ability to differentiate into neural progenitor cells and the ability to differentiate into glial progenitor cells including astrocyte progenitor cells or oligodendrocyte progenitor cells, and can be identified by intermediate filament proteins (nestin, vimentin, etc.), markers of primitive neuroectoderm and neural stem cells such as transcription factors SOX1, PAX6, etc.

[0042] As used herein, "spinal cord region marker" means a marker that is expressed during the developmental stage along with the differentiation into the spinal cord, and specifically includes genes such as HOXB3, HOXB4, HOXB6 and HOXD8, or proteins encoded by such genes.

[0043] As used herein, "ventral region marker" means a marker that is expressed during the developmental stage along with the differentiation into the ventral side, and specifically includes genes such as NKX2.1, NKX2.2, NKX6.1 and NKX6.2, or proteins encoded by such genes.

[0044] As used herein, "cell aggregate" (Cell Aggregate) is not particularly limited as long as a plurality of cells adhere to each other to form a three-dimensional structure. For example, it refers to a mass formed by the aggregation of cells dispersed in a medium such as a culture medium, or a mass of cells formed through cell division. Cell aggregates include cases where a specific tissue is formed. Embryoid bodies are also included in cell aggregates. As used herein, "cell population" may be a cell aggregate (spherical cell population) or a layered cell population.

[0045] In the present specification, the "medium" may be any medium commonly used for culturing animal cells, and is not particularly limited as long as the animal cells can maintain their viability. Preferably, it provides an environment in which target cells can proliferate. The medium may be prepared by oneself or purchased and used as a commercially available medium. In the present specification, the "embryoid body formation medium", the "medium for nerve / glia proliferation", and the "maturation medium" are based on the above-mentioned medium, and can be prepared by adding factors suitable for each purpose.

[0046] From the viewpoint of being used for producing cell aggregates suitable for transplantation, the medium used in the present invention is preferably a serum-free medium. The "serum-free medium" in the present invention means a medium that does not contain unadjusted or unpurified serum. In the present specification, even a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors) is included in the serum-free medium as long as it does not contain unadjusted or unpurified serum. The serum-free medium may appropriately contain fatty acids or lipids, amino acids (for example, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like.

[0047] The medium used in the present invention may contain a serum substitute. Examples of the serum substitute include those appropriately containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thioglycerol, or their equivalents. Such a serum substitute can be prepared, for example, by the method described in WO98 / 30679. Commercially available products may be used as the serum substitute. Examples of such commercially available serum substitutes include Knockout Serum Replacement (manufactured by Thermo Fisher Scientific; hereinafter may be referred to as KSR), "StemSure (registered trademark) Serum Replacement (SSR)", Chemically-defined Lipid concentrated (manufactured by Thermo Fisher Scientific), B27 supplement (manufactured by Thermo Fisher Scientific), N2 supplement (manufactured by Thermo Fisher Scientific), and ITS supplement (manufactured by Thermo Fisher Scientific), and preferably the B27 supplement.

[0048] The medium used in the present invention is preferably a xenogene-free medium. Here, "xenogene-free" means a condition in which components derived from a species different from the species of the cells to be cultured (also referred to as xenogenic factors or heterologous-derived components) are excluded. A part of the serum-free medium can be a xenogene-free medium.

[0049] In this specification, feeder cells refer to cells other than the stem cells to be co-cultured when culturing stem cells such as pluripotent stem cells. Examples of the feeder cells include mouse fibroblasts (such as MEF), human fibroblasts, SNL cells, and STO cells. The feeder cells may be feeder cells that have been subjected to a growth inhibition treatment. Here, examples of the growth inhibition treatment include treatment with a growth inhibitor (such as mitomycin C) or treatment by gamma irradiation or UV irradiation.

[0050] In this specification, the component derived from feeder cells means a component that is not contained in the stem cells but is contained in the feeder cells or secreted by the feeder cells.

[0051] In this specification, "heterologous cells" means cells derived from a different biological species from the cells to be cultured, and is used synonymously with "heterologous animal cells". "Component derived from heterologous cells" means a component derived from the heterologous cells and not contained in the cells to be cultured.

[0052] The component derived from heterologous cells of feeder cells means a component derived from the feeder cells and not contained in the cells to be cultured when the feeder cells are derived from a different biological species from the cells to be cultured. Specific components include at least protein components with different base sequences or amino acid sequences, sugars, lipids, and the like.

[0053] In the present invention, "suspension culture" means allowing cells to survive in a suspended state in a medium. In this specification, cells are suspension-cultured in the form of single cells or aggregates (cell aggregates or cell populations) formed by a plurality of cells aggregated. The suspension-cultured cells proliferate and / or differentiate.

[0054] [Method for producing cell aggregates containing glial progenitor cells] As one aspect of the method for producing cell aggregates containing glial progenitor cells of the present invention, (1) In an embryoid body formation medium containing one or more SMAD signal transduction inhibitors and one or more Wnt signal transduction activators, pluripotent stem cells are suspension-cultured for 5 to 10 days in the absence of feeder cells to form cell aggregates. (2) The cell aggregates obtained in (1) are suspension-cultured in an embryoid body formation medium containing retinoic acid. (3) The cell aggregates obtained in (2) are suspension-cultured in an embryoid body formation medium containing retinoic acid and one or more SHH signal transduction activators or a medium for nerve / glial cell proliferation, and Step of culturing the cell aggregates obtained in (4) and (3) in suspension in a neural / glial cell growth medium that does not contain retinoic acid and contains one or more SHH signaling pathway activators including, and further comprising the following steps: Step of culturing the cell aggregates obtained in (5) and (4) in suspension in a neural / glial cell growth medium that does not contain both retinoic acid and SHH signaling pathway activators may be included, Examples of the production method include.

[0055] <Step (1)> In step (1), pluripotent stem cells are cultured to form embryoid bodies. "Embryoid Body (EB)" means a three-dimensional cell aggregate formed by culturing pluripotent stem cells in suspension. In order to efficiently differentiate pluripotent stem cells into target cells, it is preferable that the embryoid bodies formed during differentiation contain many cells having the ability to differentiate into the germ layer (ectoderm, mesoderm or endoderm) containing the target cells. For example, when it is desired to differentiate pluripotent stem cells into nerve cells or glial cells, it is preferable that the embryoid bodies formed during differentiation contain many cells having the ability to differentiate into the ectoderm. That is, in the present specification, the embryoid body is preferably an embryoid body having the ability to differentiate into the ectoderm. However, differentiation of pluripotent stem cells has already started during the process of embryoid body formation, and the embryoid body may contain cells classified as ectoderm.

[0056] In step (1), the pluripotent stem cells are cultured using an embryoid body formation medium containing one or more differentiation-inducing factors such as an effective concentration of one or more SMAD signaling pathway inhibitors and one or more Wnt signaling pathway activators. The medium used in step (1) can be prepared by mixing the above-mentioned predetermined differentiation-inducing factors in an effective concentration in the embryoid body formation medium.

[0057] As used herein, the SMAD signal transduction inhibitor is not particularly limited as long as it can inhibit the signal transduction mediated by SMAD, and one or more SMAD signal inhibitors may be used in appropriate combination. Examples of the SMAD signal transduction inhibitor include, for example, inhibitors of the TGFβ superfamily that can inhibit SMAD signal transduction by inhibiting the phosphorylation of SMAD upstream of the SMAD signal, specifically, TGFβ inhibitors and BMP inhibitors. It is preferable to use two types of TGFβ inhibitors and BMP inhibitors. For example, one or more TGFβ inhibitors, preferably one TGFβ inhibitor, and one or more BMP inhibitors, preferably one BMP inhibitor, may be used in appropriate combination.

[0058] As used herein, the TGFβ inhibitor is not particularly limited as long as it can inhibit the signal transduction mediated by TGFβ, and it may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of the TGFβ inhibitor include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that inhibit the expression of the gene encoding TGFβ (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ to the TGFβ receptor, and substances that inhibit the biological activity resulting from signal transduction by the TGFβ receptor (e.g., inhibitors of the TGFβ receptor, etc.). Examples of the TGFβ inhibitor include substances that inhibit the binding to the receptor ALK family or substances that inhibit the phosphorylation of SMAD by the ALK family.

[0059] In this specification, specific TGFβ inhibitors include, for example, SB431542, SB202190 (R.K. Lindemann et al., Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO2009146408), Galunisertib (LY2157299), LY3200882, SB525334, GW788388, RepSox, Lefty-1 (exemplified by mouse: NM_010094, human: NM_020997 as NCBI Accession No.), Lefty-2 (exemplified by mouse: NM_177099, human: NM_003240 and NM_001172425 as NCBI Accession No.), and derivatives thereof, etc. At least one TGFβ inhibitor can be used. As the TGFβ inhibitor, preferably, it is one or more selected from the group consisting of SB431542 and A83-01, which are known as inhibitors of TGFβ receptor (ALK5) and activin receptor (ALK4 / 7).

[0060] The concentration of the TGFβ inhibitor in the medium can be appropriately set within a range capable of suppressing the signal transduction mediated by TGFβ. For example, the concentration of SB431542 is not particularly limited as long as it can inhibit ALK5, but is, for example, 100 nM to 100 μM, preferably 500 nM to 30 μM, more preferably 1 μM to 20 μM. In the case of other TGFβ inhibitors, the concentration showing the ALK inhibitory activity or TGFβ inhibitory activity corresponding to the above-mentioned concentration of SB431542 can be appropriately set.

[0061] In this specification, the BMP inhibitor is not particularly limited as long as it is an inhibitor involved in the inhibition of BMP signaling via the binding of BMP (Bone Morphogenetic Protein) and BMP receptors (type I or type II), and it may be any of nucleic acids, proteins, and small organic compounds. Examples of BMP inhibitors include substances that act directly on BMP (such as proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (such as antisense oligonucleotides, siRNA, etc.), substances having a biological activity of inhibiting the above-mentioned BMP signaling by inhibiting the binding of BMP to BMP receptors, substances that inhibit the physiological activity resulting from signal transduction by BMP receptors (such as inhibitors of BMP receptors, etc.), and BMP type I receptor kinase inhibitors, etc.

[0062] Specific examples of those having a biological activity of inhibiting the above-mentioned BMP signaling by inhibiting the binding of BMP to BMP receptors include Dorsomorphin, Noggin, and their derivatives. Specific examples of BMP type I receptor kinase inhibitors include LDN-193189 (that is, 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline) and its derivatives (such as LDN-212854). Other BMP inhibitors include K02288, Chordin, and Follistatin.

[0063] Preferably, the BMP inhibitor is one or more selected from the group consisting of Noggin, LDN-193189, Dorsomorphin, and K02288 (PLoS One. 2013; 8(4): e62721.).

[0064] The concentration of the BMP inhibitor in the medium can be appropriately set, for example, within a range capable of suppressing BMP-mediated signal transduction. For example, the concentration of LDN-193189 is not particularly limited as long as it can inhibit BMP type I receptor kinase, and is, for example, 1 nM to 10 μM, preferably 10 nM to 5 μM, more preferably 50 nM to 3 μM, and even more preferably 50 nM to 1 μM. In the case of other BMP inhibitors, the concentration showing BMP type I receptor kinase inhibitory activity or BMP inhibitory activity corresponding to the above-mentioned concentration of LDN-193189 can be appropriately set.

[0065] In the present specification, the Wnt signal activator is not particularly limited as long as it can activate Wnt-mediated signal transduction. Examples of the Wnt signal activator include Wnt proteins (such as Wnt3a), Wnt agonists, Dkk (inhibitor of Wnt signal transduction inhibitor protein), GSK3β (Glycogen Synthase Kinase 3β) inhibitors, R-Spondin, and the like.

[0066] In the present specification, the GSK3β inhibitor is defined as a substance that inhibits the kinase activity of Glycogen Synthase Kinase (GSK) 3β protein. Specific examples thereof include indirubin derivatives such as BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin-3'-oxime), maleimide derivatives such as SB216763, α-bromomethyl ketone compounds such as GSK-3β inhibitor VII, cell membrane-permeable phosphorylated peptides such as CHIR99021 and L803-mts, and derivatives thereof.

[0067] In the present specification, the Wnt signal activator is preferably a GSK3β inhibitor, and more preferably one or more selected from the group consisting of CHIR99021, BIO, Kenpaullone, SB216763, and L803-mts, which are GSK3β inhibitors.

[0068] The concentration of the Wnt signal activator in the medium can be appropriately set within a range that can enhance signal transduction mediated by, for example, Wnt3a. For example, the concentration of CHIR99021 is not particularly limited as long as it can inhibit GSK3β, and is, for example, 100 nM to 100 μM, preferably 500 nM to 30 μM, and more preferably 1 μM to 10 μM. In the case of other Wnt signal activators, the concentration showing GSK3β inhibitory activity corresponding to CHIR99021 at the aforementioned concentration or enhancing signal transduction mediated by Wnt3a can be appropriately set.

[0069] (1) In the step, the pluripotent stem cells are cultured in the absence of feeder cells. The absence of feeder cells, also referred to as feeder-free, refers to a state where feeder cells are not present in the medium. Examples of the culture conditions in the absence of feeder cells include culture conditions without adding feeder cells such as fibroblasts, SNL cells, and STO cells.

[0070] As used herein, the "embryoid body formation medium" is not particularly limited as long as it can be used to produce embryoid bodies by culturing pluripotent stem cells in suspension. The embryoid body formation medium may be a feeder-free medium suitable for the formation of embryoid bodies and for the feeder-free culture of pluripotent stem cells. The embryoid body formation medium can be prepared, for example, by adding additive factors such as serum substitutes and nutrient sources to a basal medium as needed.

[0071] Examples of the basal medium include media that can be used for culturing animal cells, such as BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F-12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or a mixed medium thereof. These basal media contain carbon sources such as carbohydrates and amino acids, vitamins, inorganic salts, and the like.

[0072] As the embryoid body formation medium, for example, a medium obtained by removing some or all of the factors (undifferentiated maintenance factors) necessary for maintaining pluripotent stem cells in an undifferentiated state from a medium (undifferentiated maintenance medium) for maintaining pluripotent stem cells in an undifferentiated state, or a medium in which the concentration of some or all of the undifferentiated maintenance factors is reduced to an effective concentration or less as described below can be used. Such a medium can be prepared, for example, by formulating it to contain components equivalent to the medium. Alternatively, for example, a medium obtained by adding nutritional factors such as a serum substitute and hormones to a basal medium such as DMEM can also be used. Or, in some cases, the undifferentiated maintenance medium is sold as a kit so that a plurality of liquids can be appropriately mixed and used. In that case, it is also possible to prepare an embryoid body formation medium by formulating it without adding the liquid containing the undifferentiated maintenance factor. For example, in the case of Stem Fit AK03N (manufactured by Ajinomoto Health Supply Co., Ltd.), since it is divided into Solution A, Solution B, and Solution C, an embryoid body formation medium can be prepared and used only with Solution A and Solution B excluding Solution C containing FGF2.

[0073] When using a medium based on an undifferentiated maintenance medium as the embryoid body formation medium, there is an advantage that at the start of the step (1), the basic composition of the medium does not need to be significantly changed, and the desired additive factors can be appropriately changed to the desired concentrations. As a result, there is an advantage that the culture environment does not change significantly for the cells.

[0074] The embryoid body formation medium preferably contains components that inhibit or antagonize the action of differentiation-inducing factors such as SMAD signal transduction inhibitors and Wnt signal transduction activators at a concentration that does not affect the step (1), for example, a concentration at which they do not act, or does not contain them.

[0075] Specific examples of the medium used in the step (1) include a medium obtained by removing FGF2, called solution C in the kit, from the StemFit AK03N medium (manufactured by Ajinomoto Health Supply Co., Ltd.), which is an undifferentiated maintenance medium, and using it as an embryoid body formation medium, and adding LDN-193189 (SMAD signal transduction inhibitor) at 50 nM to 3 μM, preferably 100 nM, SB431542 (SMAD signal transduction inhibitor) at 1 μM to 20 μM, preferably 3 μM, and CHIR99021 (Wnt signal transduction activator) at 1 μM to 10 μM, preferably 3 μM (all manufactured by Stemgent).

[0076] In the step (1), the pluripotent stem cells are cultured in suspension in the embryoid body formation medium for 5 to 10 days. In one embodiment, the suspension culture is carried out in a state where the cells form cell aggregates (or embryoid bodies). As the suspension culture, a suspension culture method known as the so-called SFEBq method (Non-Patent Document 3) is preferred. The SFEBq method is a method of forming cell aggregates by culturing pluripotent stem cells in suspension using a culture vessel having a plurality of pores of uniform shape. In the SFEBq method, the cells are cultured in suspension in the culture vessel (pore) as single cells dispersed at the start of the step (1), and eventually, the cells floating in one pore adhere to each other to take a three-dimensional structure and form a cell aggregate in each pore one by one.

[0077] As a culture vessel having a plurality of uniformly shaped holes used in the SFEBq method, one culture vessel has a plurality of, for example, 6, 12, 24, 96 or 384, independent spaces (holes) for culturing cells, and these holes preferably have the same shape and size. Each hole of the culture vessel may be columnar, and the shape of the bottom surface is not particularly limited, and may be a perfect circle, square, rectangle, polygon, ellipse, etc., and it is preferable that it is a perfect circle (that is, the hole is columnar). Also, the bottom surface of the hole may be a flat bottom, or may be a round bottom (U-bottom) or V-bottom or M-bottom, preferably a U-bottom or V-bottom or M-bottom, more preferably a V-bottom or M-bottom, and most preferably a V-bottom. This is because when the shape of the bottom is pointed, the cells suspended (floating) in the hole tend to gather and are likely to form a sphere within a certain period of time. Also, it is preferable that a cell non-adhesive material or a cell low-adhesive material is used on the bottom surface of the hole.

[0078] The holes in the culture vessel may have a bottom area of 0.1 to 2.0 cm in terms of flat-bottom conversion 2 , preferably 0.5 to 1.2 cm 2 , more preferably about 0.35 cm 2 , the inner diameter may be 0.3 to 16.5 mm, preferably 3 to 11 mm, more preferably about 6 to 8 mm, and the height may be 0.1 to 20 mm, preferably 0.3 to 17 mm, more preferably 8 to 12 mm.

[0079] As a culture vessel, a culture vessel that is usually commercially available, for example, a 6-well plate (bottom area is about 9.5 cm in terms of flat-bottom conversion 2 , inner diameter about 35 mm), 12-well plate (bottom area is about 3.8 cm in terms of flat-bottom conversion 2 , inner diameter about 23 mm) 24-well plate (bottom area is about 1.88 cm in terms of flat-bottom conversion 2 , inner diameter about 16 mm), 48-well plate (bottom area is about 1.0 cm in terms of flat-bottom conversion 2 , inner diameter about 11 mm), 96-well plate (bottom area is about 0.35 cm in terms of flat-bottom conversion 2 , inner diameter about 6 to 8 mm), 384-well plate (bottom area is about 0.1 cm in terms of flat-bottom conversion 2Degree, inner diameter of about 3 to 4 mm). Preferably, it is a 96-well plate, more preferably a V-bottom 96-well plate.

[0080] As the 96-well plate, PrimeSurface plate 96V, 96M, 96U, 96-slit well plate (manufactured by Sumitomo Bakelite Co., Ltd.), Ultra-Low Attachment Surface 96-well round bottom (manufactured by Corning Inc.) can be used. In addition, as long as the culture vessel has a shape capable of independently forming cell aggregates of uniform size, it is not necessary for each well to be clearly independent like a 96-well plate. For example, a dish with unevenness or depressions on the bottom surface can also be used. Such a culture vessel includes multi-dimples, and those with a size of 60 mm to 150 mm are used, but it is not limited thereto.

[0081] When the cell suspension is dispensed into all the wells of a 96-well plate, in principle, it is possible to obtain 96 uniform cell aggregates. By increasing the number of plates or the number of wells per well like multi-dimples, it is possible to obtain a large number of cell aggregates at once. As commercially available multi-dimples, for example, Elplasia (manufactured by Kuraray Co., Ltd.), AggreWell (manufactured by STEMCELL Technologies Inc.), EZSPHERE (manufactured by Asahi Techno Glass Co., Ltd.) can be used, but it is not limited thereto.

[0082] In the SFEBq method, first, a cell suspension having a uniform cell density is dispensed in equal amounts into each well of a culture vessel. In the case of a 96-well plate, the cell density may be 3,000 cells / well to 20,000 cells / well, preferably 5,000 cells / well to 10,000 cells / well, and more preferably about 9,000 cells / well. Then, within a short time from the start of culture, for example, within 24 hours, preferably within 12 hours, and more preferably within 8 hours, cell aggregates having substantially the same size are formed one by one at the central part of each well. Further, by adjusting the amount of the dispensed cell suspension and the cell density, it is possible to control the diameter and the number of cells of the cell aggregates. Furthermore, by using the SFEBq method, the obtained plurality of cell aggregates become spherical, and cell aggregates can be formed with high reproducibility.

[0083] (1) The suspension culture in step (1) may be carried out for approximately 5 days to 10 days, preferably approximately 7 days to 10 days, and more preferably approximately 7 days. The culture period may be determined according to the desired degree of cell differentiation. The degree of cell differentiation can be determined by measuring differentiation markers. In step (1), for example, the culture period can be determined by an increase in the expression levels of SOX1, PAX6, HES4, and HES5, a decrease in the expression level of OCT3 / 4, and a decrease in the expression level of NANOG. Specifically, compared with the start of step (1), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one of SOX1, PAX6, HES4, and HES5 has increased by 100-fold or more at the RNA level, 2) Obtaining cell aggregates in which the expression level of OCT3 / 4 has decreased to 1 / 200 or less at the RNA level, and 3) Obtaining cell aggregates in which the expression level of NANOG has decreased to 1 / 400 or less at the RNA level The step (2) may be started after continuing step (1) until at least one of the above conditions is satisfied.

[0084] As used herein, "expressing X-fold more at the RNA level", "the expression level increased X-fold at the RNA level", or "the expression level of RNA increased X-fold" means that one or more floating cells or cells constituting cell aggregates are obtained at two time points during the process, and as a result of comparative analysis using a method capable of absolutely or relatively quantifying the RNA expression level in the cells, the RNA expression level at the other time point is X-fold higher (increased X-fold) than that at one time point. Further, "expressing X-fold less at the RNA level", "the expression level decreased X-fold at the RNA level", or "the expression level of RNA decreased X-fold" means that the RNA expression level at the other time point is X-fold lower (decreased X-fold) than that at one time point. The method used is not particularly limited as long as it can quantify RNA, but measurement methods using RNA sequencing, real-time PCR assay, microarray, Northern blot, and in situ hybridization, etc. can be used, and preferably, comparative measurement is performed using RNA sequencing, real-time PCR assay, and microarray.

[0085] (1) The culture conditions for suspension culture in the step are not particularly limited. For example, conditions such as a temperature of 35°C to 37°C, preferably 37°C, a humidity of 90% to 95%, preferably 95%, and a CO2 concentration of 3% to 5%, preferably 5% are acceptable, and it is more preferable to be under hypoxic culture conditions. "Hypoxic culture conditions" means conditions for culturing cells at an oxygen concentration lower than the oxygen concentration in the atmosphere (about 21%). Here, the oxygen concentration means the concentration of oxygen in the air in contact with the culture medium containing cells in the culture apparatus. The oxygen concentration is not particularly limited as long as it is 20% or less, but is preferably in the range of 3% to 10%, more preferably in the range of 4% to 6%, and most preferably 5%.

[0086] Before differentiation, that is, before the step (1), the pluripotent stem cells may be maintained in an undifferentiated maintenance medium. The undifferentiated maintenance medium contains factors necessary to maintain the undifferentiated state (undifferentiated maintenance factors) and is a medium capable of maintaining pluripotency. Examples of undifferentiated maintenance factors include fibroblast growth factors, transforming growth factor beta (TGFβ family) factors, activin A, and the like.

[0087] As used herein, "Fibroblast Growth Factors (FGF)" means a factor that acts on fibroblast growth factor receptors to activate FGF signals. Examples of FGF include FGF2 (also referred to as bFGF), FGF4, and FGF8.

[0088] The undifferentiated maintenance medium herein is a medium capable of maintaining pluripotent stem cells under feeder-free conditions, that is, a feeder-free medium. Various feeder-free media have been developed and are commercially available. For example, Essential 8 (manufactured by Thermo Fisher Scientific) can be mentioned. The Essential 8 medium contains L-ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenite (14 μg / 1), insulin (19.4 mg / l), NaHCO3 (543 mg / l), transferrin (10.7 mg / l), FGF2 (100 ng / ml), and TGFβ (TGFβ1 (2 ng / ml) or Nodal (100 ng / ml)) as additives in DMEM / F-12 medium (Nature Methods, 8, p424-429 (2011)).

[0089] Other commercially available undifferentiated cell maintenance media include S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Thermo Fisher Scientific), hESF9 (Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13409-14), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), Cellartis DEF-CS 500 Xeno-Free Culture Medium (manufactured by Takara Bio), or StemFit (manufactured by Ajinomoto Health Supply).

[0090] From the components of the above-mentioned undifferentiated cell maintenance medium, a medium with a reduced concentration of undifferentiated cell maintenance factors or a medium with undifferentiated cell maintenance factors removed can be prepared and used as the embryoid body formation medium in step (1). Therefore, the embryoid body formation medium used in step (1) is preferably a feeder-free medium and an undifferentiated cell maintenance medium with a reduced concentration of undifferentiated cell maintenance factors or with undifferentiated cell maintenance factors removed. Specifically, an undifferentiated cell maintenance medium for feeder-free culture that does not contain FGF such as FGF2 can be mentioned. Also, a medium prepared by mixing a medium for neural / glial cell proliferation described below or a medium with a similar composition and an undifferentiated cell maintenance medium at a ratio such as 1:1 can be used as the embryoid body formation medium.

[0091] <Step (2)> In step (2), the cell aggregates obtained in step (1) are cultured in suspension in an embryoid body formation medium containing retinoic acid. The embryoid body formation medium and suspension culture are as described above. The medium used in step (2) can be prepared by mixing retinoic acid into the embryoid body formation medium.

[0092] (2) The medium used in the step may be a medium having the same composition as the medium used in the step (1), except for two points: containing retinoic acid and not containing one or more SMAD signal transduction inhibitors and one or more Wnt signal transduction activators. Examples of "retinoic acid" include retinoids (e.g., retinoic acid). As a specific example of the medium used in the step (2), from StemFit AK03N medium (manufactured by Ajinomoto Health Supply Co., Ltd.), a medium (AK03N-C) obtained by removing FGF2 called solution C in the kit is used as an embryoid body formation medium, and a medium supplemented with 1 μM retinoic acid (manufactured by Sigma-Aldrich) can be mentioned.

[0093] The concentration of retinoids in the medium can be appropriately set within a range capable of enhancing the signal transduction mediated by retinoic acid. For example, the concentration of retinoic acid is not limited as long as it shows activity, i.e., the posteriorizing effect of region specificity. For example, it is 10 nM to 100 μM, preferably 100 nM to 10 μM, and more preferably 500 nM to 5 μM. In the case of other retinoids, the concentration showing an activity corresponding to the above-mentioned concentration of retinoic acid can be appropriately set.

[0094] At the end of the step (1), the medium used in the step (1) is removed from the culture vessel, and the step (2) can be started by replacing the medium with the medium used in the step (2). When using the same culture vessel as in (1) in the step (2), from the viewpoint of protecting cell aggregates, it is preferable to replace about half of the medium, but by repeating this operation a plurality of times, more than half of the medium can be replaced. Also, at this time, if necessary, after washing the cell aggregates with a medium other than the medium used in the step (2) or a phosphate buffer solution or the like, the culture in the medium used in the step (2) can also be started. Alternatively, at the end of the step (1), all the cell aggregates obtained in (1) are collected, and if necessary, after washing the cell aggregates, the step (2) can be started by suspending them in the medium used in the step (2). The culture vessel used in the step (2) is not particularly limited as long as it is a culture device for suspension culture, and dishes and plates commonly used by those skilled in the art can be used. For example, PrimeSurface plate 96V and Ultra-Low Attachment Culture Flasks (manufactured by Corning) etc. can be mentioned.

[0095] The suspension culture in the step (2) may be carried out for approximately 3 days to 21 days, preferably approximately 4 days to 11 days, more preferably approximately 7 days. The culture period may be determined according to the desired degree of cell differentiation. The degree of cell differentiation can be determined by measuring differentiation markers. For example, the amount of mRNA or protein of the differentiation marker expressed by collecting cells can be measured. Alternatively, the culture supernatant can be collected and the amount of protein of the differentiation marker secreted from the cells into the medium can be measured. In the step (2), for example, the culture period can be determined by the expression level of RNA or protein of one or more markers selected from the group consisting of ASCL1, DCX, SLIT1, HEY1, ZBTB20, βIII tubulin, ELAVL3, HOXB3, HOXA4, HOXB4, HOXB6 and HOXB8. Specifically, compared with the start of the step (2), 1) Obtaining cell aggregates in which the expression level of at least one of ASCL1, DCX, HEY1, ZBTB20, βIII tubulin, ELAVL3, and SLIT1 is increased by 5 times or more at the RNA level, and 2) Obtaining cell aggregates in which the expression level of at least one of HOXB3, HOXA4, HOXB4, HOXB6, and HOXB8 is increased by 5 times or more at the RNA level The step (2) may be continued until at least one of the above is satisfied, and then the step (3) may be started.

[0096] The culture conditions for suspension culture in the step (2) may be the same as those in the step (1), and preferably, they are hypoxic culture conditions. The oxygen concentration under hypoxic culture conditions is not particularly limited as long as it is 20% or less, but preferably it is in the range of 3% to 10%, more preferably in the range of 4% to 6%, and most preferably 5%.

[0097] <(Step (3))> In the step (3), the cell aggregates obtained in (2) are subjected to suspension culture in an embryoid body formation medium or a medium for neural and glial cell growth containing retinoic acid and one or more SHH signaling activators. The embryoid body formation medium is as described above. The medium used in the step (3) can be prepared by mixing retinoic acid and one or more SHH signaling activators into the embryoid body formation medium or the medium for neural and glial cell growth. When using the embryoid body formation medium, it can also be prepared by further mixing one or more SHH signaling activators into the medium used in the step (2).

[0098] As used herein, the "culture medium for neural and glial cell growth" refers to a culture medium suitable for the growth and culture of glial progenitor cells, glial cells, neural progenitor cells, and neurons, and there is no particular limitation as long as it can maintain the survival of these cells and allow them to differentiate and proliferate. The culture medium for neural and glial cell growth can be prepared by appropriately adding nutrient factors suitable for the survival of glial progenitor cells, glial cells, neural progenitor cells, and neurons to the above-mentioned basal medium. However, commercially available media that are particularly suitable for the maintenance culture or differentiation induction of nervous system cells can also be appropriately combined or used after partially modifying the composition. For example, after selecting a commercially available basal medium such as Neurobasal or commercially available DMEM / F-12, the above-mentioned nutrient factors can be appropriately added thereto for use.

[0099] The aforementioned "nutrient factors suitable for survival" can be appropriately combined and used from those well-known to those skilled in the art as factors added when culturing nervous system cells. Specifically, examples include insulin, sodium selenite, progesterone, transferrin, putrescine, growth factors, vitamins, cAMP activators, hormones, neurotrophic factors, etc., and one or more factors can be selected from these for use.

[0100] The culture medium for neural and glial cell growth in this specification preferably contains one or more growth factors to appropriately differentiate and proliferate all of glial progenitor cells, glial cells, neural progenitor cells, and neurons, and preferably contains platelet-derived growth factor (PDGF). As the platelet-derived growth factor, PDGF-AA can be used. Other growth factors included in the culture medium for neural and glial cell growth in this specification also include FGF (for example, FGF2), EGF, IGF-1, or HGF, etc. In a preferred embodiment, the culture medium for neural and glial cell growth contains PDGF-AA, EGF, IGF-1, and FGF (preferably FGF2).

[0101] Examples of neurotrophic factors include human NT-3 (recombinant protein human neurotrophin 3), BDNF, NGF, GDNF, or CNTF. Examples of vitamins include biotin or ascorbic acid. Examples of cAMP activators include cAMP, dibutyryl-cAMP, or forskolin. Examples of hormones include progesterone or T3. In a preferred embodiment, the medium for nerve / glia growth contains thyroid hormones such as T3 and neurotrophic factors such as NT-3 in addition to the aforementioned growth factors.

[0102] Mixtures appropriately formulated with the above-mentioned nutritional factors, such as N1 supplement (hereinafter sometimes referred to as N1), N2 supplement (hereinafter sometimes referred to as N2), B27 supplement (including those with vitamin A removed; hereinafter sometimes referred to as B27), etc., are commercially available, and thus can be conveniently used and added to the basal medium. For example, N2 contains insulin, transferrin, progesterone, sodium selenite, and putrescine. When using N2, it is preferably further contained human NT-3 and / or B27 (preferably excluding vitamin A).

[0103] Examples of media for promoting neural and glial cell growth include, for example, DMEM / F-12 medium supplemented with N2, B27 (it is also possible to use the one excluding vitamin A), Glutamax (registered trademark), FGF2, EGF, T3, NT-3, PDGF-AA, and IGF-1. Further examples include DMEM / F-12 medium supplemented with glucose, L-glutamine, sodium bicarbonate, HEPES, insulin, transferrin, progesterone, sodium selenite, putrescine, B27, MEM NEAA, FGF2, EGF, T3, NT-3, PDGF-AA, and IGF-1. Additionally, examples include DMEM / F-12 medium supplemented with Glutamax, β-mercaptoethanol, B27, N2, MEM NEAA, FGF2, EGF, T3, NT-3, PDGF-AA, and IGF-1. Furthermore, examples include DMEM / F-12 medium supplemented with N1, B27, T3, biotin, dibutyryl cAMP, PDGF-AA, IGF-1, and NT-3.

[0104] From the perspective that the medium for promoting neural and glial cell growth contains one or more of the above-described trophic factors suitable for the survival of neural cells, it is preferable that the medium does not contain a substance that inhibits or suppresses the biological activity of the component, or contains it at a concentration less than the effective concentration.

[0105] As a specific example of a medium for nerve and glial cell proliferation, in a DMEM / F-12 medium (manufactured by Thermo Fisher Scientific), 1% N2 (manufactured by Thermo Fisher Scientific), B27 (manufactured by Thermo Fisher Scientific) with a vitamin A concentration of 5 μg / ml or less, preferably without vitamin A, 10 ng / ml to 100 ng / ml, preferably 60 ng / ml of T3 (manufactured by Sigma-Aldrich), 5 ng / ml to 100 ng / ml, preferably 10 ng / ml of PDGF-AA (manufactured by PeproTech), 10 ng / ml to 100 ng / ml, preferably 20 ng / ml of FGF2, 5 ng / ml to 100 ng / ml, preferably 10 ng / ml of EGF (manufactured by PeproTech), 10 ng / ml of insulin-like growth factor 1 (manufactured by R&D Systems), and 5 ng / ml to 100 ng / ml, preferably 10 ng / ml of NT-3 (Neurotrophin-3) (manufactured by R&D Systems) are added.

[0106] As used herein, an SHH signaling activator is defined as a substance that causes the de-repression of Smoothened (Smo) and subsequent activation of Gli2, which are caused by the binding of SHH (Sonic Hedgehog) to its receptor Patched (Ptch1). Examples of SHH signaling activators include proteins belonging to the Hedgehog family, specifically SHH or IHH (Indian Hedgehog), or partial peptides of SHH or IHH such as Sonic Hedgehog N-Terminus (Shh-N), recombinant Human Sonic Hedgehog (C24II) N-Terminus (SHH-C24II), and recombinant Mouse Sonic Hedgehog (C25II) N-Terminus (SHH-C25II), SHH receptors, SHH receptor agonists, Hh-Ag1.5 (Li, X. et al., Nature Biotechnology, 23, 215-221 (2005)), Smoothened Agonist (SAG), [N-Methyl-N’-(3-pyridinylbenzyl)-N’-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane], 20a-hydroxycholesterol, Purmorphamine (PMA; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), and derivatives thereof (Stanton BZ et al., Mol Biosyst. 6:44-54, 2010).

[0107] In the step (3), the SHH signal transduction activator may be one or more selected from the group consisting of Purmorphamine, SAG, SHH protein, and SHH fragment (for example, SHH C25II or SHH C24II), and preferably, it is one or more selected from the group consisting of SHH protein, SHH fragment, Purmorphamine, and SAG.

[0108] The concentration of the SHH signal transduction activator in the medium can be appropriately set within a range that can enhance the signal transduction mediated by SHH. For example, the concentration of Purmorphamine is not limited as long as it shows a region-specific ventralizing effect. For example, it is 10 nM to 10 μM, preferably 100 nM to 10 μM, and more preferably 500 nM to 5 μM. In the case of other SHH signal transduction activators, the concentration that causes the de-repression effect of Smoothened (Smo) and the subsequent activation effect of Gli2, or shows a region-specific ventralizing effect, corresponding to the above-mentioned concentration of Purmorphamine, can be appropriately set.

[0109] Specific examples of the medium used in the step (3) include a medium obtained by adding 1 μM retinoic acid (manufactured by Sigma-Aldrich) and 1 μM Purmorphamine (manufactured by Millipore) to an embryoid body formation medium or a medium for nerve and glial cell proliferation.

[0110] At the end of the step (2), all cell aggregates obtained in (2) are collected, and if necessary, the cell aggregates collected with the medium used in the step (3), other media (e.g., DMEM / F-12, but not limited thereto) or phosphate buffer solution, etc. are washed, and then suspended in the medium used in the step (3), whereby the step (3) can be started. Alternatively, from the perspective of protecting cell aggregates, it is also possible to change about half of the medium to avoid a sudden change in the culture environment, and by repeating this operation multiple times, it is also possible to change more than half of the medium. The culture vessel used in the step (3) is not particularly limited as long as it is a culture device for suspension culture, and flasks and plates commonly used by those skilled in the art can be appropriately used. For example, PrimeSurface plate 96V, 96-slit well plate, and Ultra-Low Attachment Culture Flasks (manufactured by Corning) etc. can be mentioned.

[0111] The suspension culture in the step (3) may be carried out for approximately 4 days to 11 days, preferably approximately 4 days to 7 days, more preferably approximately 7 days. The culture period may be determined according to the desired degree of cell differentiation. During the culture period, the whole or part of the medium may be changed appropriately once every several days (e.g., once every 3 days or 4 days).

[0112] The degree of cell differentiation can be determined by measuring differentiation markers. For example, the mRNA amount or protein amount of the differentiation markers expressed by collecting cells can be measured. Alternatively, the culture supernatant can be collected, and the protein amount of the differentiation markers secreted from the cells into the medium can be measured. In the step (3), for example, the culture period can be determined by the expression amount of RNA or protein of one or more markers selected from the group consisting of HEY2, NKX6.2, NKX2.2, OLIG1, and OLIG2. Specifically, compared with the start of the step (3), 1) Cell aggregates in which the expression amount of at least one of HEY2, NKX6.2, and NKX2.2 has increased by 5 times or more at the RNA level can be obtained, and 2) Cell aggregates with a more than 10-fold increase in the expression level of OLIG1 and / or OLIG2 at the RNA level can be obtained. The step (3) may be continued until at least one of them is satisfied, and then the step (4) may be started. The method for evaluating the expression level of the RNA is as described in the step (1).

[0113] The culture conditions for suspension culture in the step (3) are not particularly limited. For example, the temperature can be 35°C to 37°C, preferably 37°C, the humidity can be 90% to 95%, preferably 95%, the CO2 concentration can be 3% to 5%, preferably 5%, and the O2 concentration can be 10% to 20%, preferably 20%.

[0114] <(Step (4))> In the step (4), the cell aggregates obtained in (3) are suspension-cultured in a medium for nerve and glial cell growth that does not contain retinoic acid and contains one or more SHH signaling activators. The medium for nerve and glial cell growth and the SHH signaling activator are as described above.

[0115] In the step (4), at the end of the step (3), the cell aggregates obtained in the step (3) may be subcultured in the medium of the step (4) while remaining as cell aggregates, but cells that cannot survive and proliferate in the medium used in the step (4) are removed as unwanted cells, and in order to improve the content of the target oligodendrocyte progenitor cells, astrocyte progenitor cells, and nerve progenitor cells, the cell aggregates obtained in the step (3) may be dispersed and then the dispersed cells may be suspension-cultured to form cell aggregates again.

[0116] "Dispersing cell aggregates" means dissociating the cells forming the cell aggregates into single cells (single cell units), or collapsing the shape of the original cell aggregates while maintaining the state where multiple cells remain joined together. Examples of methods for dispersing include physical stimulation methods and methods using compounds, and by using either or both of these methods in combination, the cell aggregates can be collapsed. That is, the original cell aggregates contain approximately 1,000 to 100,000 cells, preferably approximately 3,000 to 50,000 cells, more preferably approximately 1,000 to 50,000 cells, and even more preferably approximately 3,000 to 30,000 cells. However, after dispersing the cell aggregates, even if multiple cells are joined together to form small cell masses, the number of cells contained in the mass is about 100 or less. Preferably, it is more preferable to disperse until 90% or more of the cells are in a single state compared to the cell aggregates before dissociation.

[0117] Specifically, trypsin-like enzymes (e.g., trypsin, TrypLE (registered trademark)), Accutase (registered trademark), Accumax (registered trademark), or metal chelating agents (EDTA, EGTA, etc.) can be used to dissociate cell-cell adhesion, and by further applying physical stimulation using a pipetteman, the cell aggregates can be effectively dispersed. Furthermore, as a method for dispersing the cells to single cells, a technique of passing them through a cell strainer can be utilized. For the implementation of the present invention, to aggregate the dispersed cell aggregates and form cell aggregates again, it can be achieved by culturing the cells for a certain period under suspension culture conditions. However, to obtain cell aggregates of a more uniform size, the aforementioned SFEBq method can be used.

[0118] The suspension culture in step (4) may be carried out for approximately 4 days or more, preferably approximately 10 days or more, more preferably approximately 14 days or more, and particularly more preferably approximately 14 days to 21 days. The culture period may be determined according to the desired degree of cell differentiation.

[0119] The degree of cell differentiation can be determined by measuring differentiation markers. For example, cells can be collected and the amount of mRNA or protein of the differentiation marker expressed can be measured. Alternatively, the culture supernatant can be collected and the amount of protein of the differentiation marker secreted from the cells into the medium can be measured. In the step (4), for example, the culture period can be determined by the expression level of RNA or protein of one or more markers selected from the group consisting of NFIA, NFIB, SLC1A3, S100B, FABP7, and PAX6. Specifically, compared with the start of the step (4), 1) cell aggregates in which the expression level of at least one of NFIA, NFIB, SLC1A3, S100B, and FABP7 has increased by 10-fold or more at the RNA level are obtained, and 2) cell aggregates in which the expression level of PAX6 has decreased to 5-fold or less at the RNA level are obtained The step (5) may be started after continuing the step (4) for a period until at least one of the above is satisfied. The method for evaluating the expression level of the RNA is as described in the step (1).

[0120] Examples include PrimeSurface plate 96V, 96-slit well plate, and Ultra-Low Attachment Culture Flasks (manufactured by Corning), etc.

[0121] The culture vessel used in the step (4) is not particularly limited as long as it is a culture device for suspension culture for cell culture, and flasks, plates, and dishes commonly used by those skilled in the art can be appropriately used. For example, Nunc (trademark) EasYFlask (trademark) Cell Culture Flasks (manufactured by Thermo Fisher Scientific), Ultra-Low Attachment Culture Flasks (manufactured by Corning), culture vessels having a plurality of uniformly shaped holes used in the SFEBq method, etc. can be mentioned.

[0122] The culture conditions for suspension culture in step (4) are not particularly limited. For example, the temperature can be 35°C to 37°C, preferably 37°C; the humidity can be 90% to 95%, preferably 95%; the CO2 concentration can be 2% to 5%, preferably 5%; and the O2 concentration can be 10% to 20%, preferably 20%.

[0123] During the culture period, the medium can be replaced approximately once every 2 to 7 days, or approximately once every 2 to 5 days, or approximately once every 3 to 4 days. The medium replacement can involve replacing the entire volume of the medium, or to avoid a large change in the cell environment, for example, about 1 / 3 or 1 / 2 of the medium can be replaced. At times other than medium replacement, components such as growth factors or neurotrophic factors contained in the medium can be appropriately supplemented. For example, in step (4), it is preferable to add PDGF-AA approximately once every 2 to 3 days so that the concentration of PDGF-AA (for example, 10 ng / ml) is maintained.

[0124] When using a medium for nerve and glial cell growth in step (4), if a medium for nerve and glial cell growth is used in step (3), the same composition of the medium can also be used as the medium for nerve and glial cell growth in step (4), or the components can be appropriately changed.

[0125] The medium for nerve and glial cell growth used in step (4) preferably contains 5 ng / ml to 100 ng / ml, more preferably approximately 10 ng / ml of PDGF-AA.

[0126] <Step (5)> In step (5), the cell aggregates obtained in (4) are subjected to suspension culture in a medium for nerve and glial cell growth that does not contain both retinoic acid and an SHH signaling activator. The medium for nerve and glial cell growth is as described above.

[0127] In step (5), at the end of step (4), the cell aggregates obtained in step (4) may be subcultured in suspension as cell aggregates by exchanging the medium with the medium of step (5). However, cells that cannot survive and proliferate in the medium used in step (5) are removed as unwanted cells, and in order to improve the content of the target oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells, the cell aggregates obtained in step (4) are dispersed and then the dispersed cells are subcultured in suspension to form cell aggregates again. The specific method is the same as in step (4).

[0128] The suspension culture in step (5) is not particularly limited in its period as long as subculture and medium exchange are appropriately performed under conditions where differentiation does not progress too far and the cell aggregates do not become too large, and the culture can be continued. The culture in step (5) may be carried out for approximately 5 days to 100 days, preferably approximately 5 days to 20 days or 7 days to 50 days, more preferably approximately 7 days to 35 days, still more preferably 7 days to 28 days, and most preferably 14 days to 28 days. In step (5), during the culture period, according to the number of cells and the amount of medium, medium exchange may be carried out once every few days, for example, once within 7 days, approximately once every 2 to 7 days, or approximately once every 2 to 5 days, or approximately once every 3 to 4 days. For this medium exchange, the total amount of the medium may be exchanged, or in order to avoid a large change in the cell environment, for example, about 1 / 3 or 1 / 2 of the medium may be exchanged. The number of subcultures is not limited, and for example, subculture may be carried out 0 to 5 times. For example, step (5) may be started, cultured for 14 days, and the process may be terminated at that time. Furthermore, subculture may be carried out once, and then, after continuing the culture for 8 days or 14 days, the process may be terminated.

[0129] The degree of cell differentiation can be determined by measuring differentiation markers. For example, cells can be collected and the amount of differentiation markers expressed or present can be measured. Examples of differentiation markers include at least mRNA or protein, sugar, lipid, etc. Alternatively, the culture supernatant can be collected and the amount of differentiation markers secreted from the cells into the medium can be measured.

[0130] In the step (5), for example, the culture period can be determined by the expression level or abundance of one or more markers selected from the group consisting of O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 (Epitope A2B5) antigen, CNP, and PLP.

[0131] As the medium for nerve / glia proliferation used in the step (5), a medium having the same composition as that in the step (4) may be used, or the components may be appropriately changed, as long as neither retinoic acid nor an SHH signaling activator is included.

[0132] The culture vessel used in the step (5) is not particularly limited as long as it is a device for cell culture, and flasks and plates commonly used by those skilled in the art can be appropriately used. For example, Nunc (trademark) EasYFlask (trademark) Cell Culture Flasks (manufactured by Thermo Fisher Scientific), Ultra-Low Attachment Culture Flasks (manufactured by Corning), 96-well plates, 96-well slit well plates, etc. can be mentioned. The culture conditions for suspension culture in the step (5) are not particularly limited, but for example, conditions such as a temperature of 37°C, a humidity of 95%, a CO2 concentration of 5%, and an O2 concentration of 20% may be used. During the culture period, it is preferable to change the medium approximately once every 7 days and add PDGF-AA (for example, 10 ng / ml) approximately once every 3 to 4 days.

[0133] The cell aggregate containing glial progenitor cells obtained after the step (5) (the cell aggregate containing the glial progenitor cells of the present invention) has the following characteristics: (a) Containing oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells; (b) Expressing a spinal cord region marker; and (c) Not containing feeder cells and components derived from feeder cells.

[0134] In the step (5), for the purpose of confirming that cells having the characteristics of (a) above are obtained, the step (5) can be continued until it is confirmed that the cell aggregate expresses one or more, preferably two or more, more preferably five or more markers selected from O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP, and PLP.

[0135] Furthermore, in the step (5), for the purpose of confirming that cells having the characteristics of (b) above are obtained, the step (5) can be continued until it is confirmed that the cell aggregate expresses one or more, preferably two or more markers selected from the group consisting of HOXB3, HOXB4, HOXB6, and HOXD8.

[0136] Furthermore, in the step (5), the step (5) can be continued until one or more proteins selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL are detected in the medium for culturing the cell aggregate.

[0137] The cell aggregate containing the above glial progenitor cells is spherical with a diameter of about 100 μm to 800 μm, for example, about 100 μm to 300 μm, about 200 μm to 400 μm, or about 400 μm to 600 μm. The cell aggregate containing the glial progenitor cells can be differentiated into a cell aggregate containing oligodendrocytes, astrocytes, and neurons by continuing culturing suitable for differentiation. Also, when the cell aggregate containing the glial progenitor cells is transplanted into a living body, it can be differentiated into a cell population containing oligodendrocytes, astrocytes, and neurons in vivo.

[0138] [Cell aggregate containing glial progenitor cells] One aspect of the cell aggregate containing the glial progenitor cells of the present invention has the following characteristics: (a) Containing oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells, (b) Expressing a spinal cord region marker, (c) Not containing feeder cells and components derived from feeder cells, and (d) Having the ability to differentiate into a cell population containing oligodendrocytes, astrocytes, and neurons.

[0139] (a) The cell aggregate containing glial progenitor cells of one embodiment contains, as the cells constituting the cell aggregate, oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells.

[0140] Glial progenitor cells is a concept that includes both oligodendrocyte progenitor cells and astrocyte progenitor cells.

[0141] The oligodendrocyte progenitor cells in the cell aggregate containing glial progenitor cells of the present invention are characterized by expressing one or more, preferably two or more, more preferably five or more markers selected from O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP, and PLP. The oligodendrocyte progenitor cells preferably express at least A2B5 antigen, O4 antigen, OLIG2, and FAM181B.

[0142] The astrocyte progenitor cells in the cell aggregate containing glial progenitor cells of the present invention are characterized by expressing one or more, preferably two or more, more preferably five or more markers selected from NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, ZBTB20, SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4. The astrocyte progenitor cells preferably express at least NFIA, NFIB, SLC1A3, and SPARCL1.

[0143] Among the markers expressed in the above astrocyte progenitor cells, NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, and ZBTB20 are classified as glial progenitor cells and are markers expressed in astrocyte progenitor cells with a low degree of maturity that are fated to be induced to differentiate into astrocytes although they have not differentiated into complete astrocyte progenitor cells. That is, the cell aggregate containing the glial progenitor cells of the present invention is characterized by containing the astrocyte progenitor cells with a low degree of maturity.

[0144] Therefore, more preferably, in the cell aggregate containing the glial progenitor cells of the present invention, 1) immature astrocyte progenitor cells characterized by expressing one or more, preferably two or more, more preferably five or more markers selected from NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, and ZBTB20, and 2) astrocyte progenitor cells characterized by expressing one or more, preferably two or more, more preferably five or more markers selected from SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4 are both included.

[0145] The cell aggregate containing the glial progenitor cells of the present invention, together with neural progenitor cells, further contains relatively immature (low degree of differentiation) neural stem cells. The neural stem cells are characterized by expressing one or more, preferably two or more, more preferably four or more markers selected from SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3, and ASCL1. The neural stem cells preferably express at least SOX1, ASCL1, and NESTIN.

[0146] The neural progenitor cells in the cell aggregate containing the glial progenitor cells of the present invention express one or more, preferably two or more, more preferably five or more markers selected from the group consisting of DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3. The neural progenitor cells preferably express at least DCX, βIII tubulin, and ELAVL3.

[0147] The cell aggregate containing the glial progenitor cells in one embodiment contains 5% or more, preferably 10% to 60%, more preferably 20% to 50%, still more preferably 20% to 40% of oligodendrocyte progenitor cells with respect to all the cells constituting the cell aggregate. Further, it preferably contains 10% or more, preferably 10% to 80%, more preferably 10% to 50%, still more preferably 20% to 30% of astrocyte progenitor cells. Furthermore, it preferably contains 10% or more, preferably 20% to 60%, more preferably 30% to 50% of neural progenitor cells. Here, the total of oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells does not exceed 100%, and accounts for 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more of the total number of cells contained in the cell aggregate.

[0148] Here, the ratio of each cell type in the cell aggregate containing the glial progenitor cells is not particularly limited, but as one aspect, a cell aggregate in which the content of astrocyte progenitor cells or neural progenitor cells is larger than the content of oligodendrocyte progenitor cells can be mentioned.

[0149] The proportion of each cell in all the cells that make up the cell aggregate can be determined by the expression of each cell marker. For example, by using methods well-known to those skilled in the art, such as FACS, immunostaining, or single-cell RNA sequence analysis, the proportion of target cells among all the cells present within a certain range can be judged using the numerical value calculated by measuring (detecting or quantifying) the mRNA, protein, or fragments thereof of the marker. The total number of cells can be counted, for example, by nuclear staining, and the number of target cells present within the same range can be counted as the number of cells expressing the marker specifically expressed by the target cells.

[0150] (b) The cell aggregate containing glial progenitor cells of one embodiment expresses a spinal cord region marker, and for example, expresses at least one, preferably at least two, spinal cord region markers selected from HOXB3, HOXB4, HOXB6, and HOXD8 at the mRNA level.

[0151] The cell aggregate containing glial progenitor cells of one embodiment expresses a ventral region marker, and for example, expresses at least one, preferably at least two, ventral region markers selected from NKX2.1, NKX2.2, NKX6.1, and NKX6.2 at the mRNA level.

[0152] The cell aggregate containing glial progenitor cells of one embodiment is characterized by containing cells that express at least one, preferably at least two, spinal cord region markers selected from HOXB3, HOXB4, HOXB6, and HOXD8 at the mRNA level and express at least one, preferably at least two, ventral region markers selected from NKX2.1, NKX2.2, NKX6.1, and NKX6.2.

[0153] (c) The cell aggregate containing glial progenitor cells of one embodiment is characterized in that it does not contain feeder cells and components derived from feeder cells, and does not contain components derived from heterologous cells when the feeder cells are derived from a heterologous animal. Such a cell aggregate can be obtained by culturing pluripotent stem cells in the absence of feeder cells. Here, the component derived from a heterologous cell means a component derived from a biological species different from the biological species of the cells to be cultured. In the cell aggregate containing glial progenitor cells of the present invention, cells other than iPS cells or cells differentiated from iPS cells, or cell-specific components thereof are not included. Note that the cell aggregate containing glial progenitor cells of the present invention includes not only components derived from feeder cells, for example, components derived from heterologous feeder cells, but also other components derived from heterologous cells and xenogenic factors. Such a cell aggregate can be obtained by using a xenofree medium in addition to the above-described condition in the absence of feeder cells.

[0154] (d) The cell aggregate containing glial progenitor cells of one embodiment has the ability to differentiate into a cell population including oligodendrocytes, astrocytes, and neurons. The fact that the cell aggregate containing glial progenitor cells of the present invention has such an ability can be confirmed by culturing for 5 to 60 days, more specifically 10 days, in the maturation medium described below by the culture method described below, and then checking whether it has differentiated into a cell population including oligodendrocytes, astrocytes, and neurons. That is, the differentiation into a cell population including oligodendrocytes, astrocytes, and neurons can be identified by detecting the expression of markers described below.

[0155] The cell aggregate containing glial progenitor cells of one embodiment is characterized by including cells that express one or more, preferably two or more, more preferably five or more markers selected from NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, ZBTB20, SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4, preferably at the mRNA level.

[0156] Preferably, the cell aggregate containing the glial progenitor cells of the embodiment expresses at least one, preferably at least two, more preferably at least five markers selected from NFIA, NFIB, SOX9, HEY1, HEY2, and ZBTB20 at the mRNA level, and contains cells that express at least one, preferably at least two, more preferably at least five markers selected from SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4 at the mRNA level.

[0157] The cell aggregate containing the glial progenitor cells of one embodiment contains cells that preferably express at least one, preferably at least two, more preferably at least five markers selected from (II) OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP, and PLP, preferably at the mRNA level.

[0158] The cell aggregate containing the glial progenitor cells of one embodiment contains cells that express at least one marker selected from the group consisting of (III) DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3.

[0159] The cell aggregate containing the glial progenitor cells of one embodiment contains cells that express at least one marker selected from the group consisting of (IV) SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3, and ASCL1.

[0160] The cell aggregate containing the glial progenitor cells of one embodiment is (V) (i) cells that express at least one marker selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2; (ii) cells that express at least one marker selected from the group consisting of βIII tubulin, MAP2, and ELAVL3, and (iii) cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2 have the ability to differentiate into a cell population containing such cells.

[0161] A cell aggregate containing glial progenitor cells of one embodiment, after undergoing the above culture, is characterized by having the ability to differentiate into a cell population containing at least 5% or more, preferably about 5% to 20% of O4-positive cells.

[0162] A cell aggregate containing glial progenitor cells of one embodiment, after undergoing the above culture, is characterized by having the ability to differentiate into a cell population containing at least 10% or more, preferably about 10% to 50% of APC-positive cells.

[0163] A cell aggregate containing glial progenitor cells of one embodiment, after undergoing the above culture, is characterized by having the ability to differentiate into a cell population containing at least 10% or more, preferably about 10% to 40% of GFAP-positive cells.

[0164] A cell aggregate containing glial progenitor cells of one embodiment, after undergoing the above culture, is characterized by having the ability to differentiate into a cell population containing at least 15% or more, preferably about 15% to 50% of βIII tubulin-positive cells.

[0165] A cell aggregate containing glial progenitor cells of one embodiment, after undergoing the above culture, is characterized by having the ability to differentiate into a cell population containing at least 10% or more, preferably about 10% to 30% of Hu-positive cells.

[0166] A cell aggregate containing glial progenitor cells of one embodiment is characterized by expressing one or more markers selected from (VI) C1ORF61 and SERPINE2 at the mRNA level.

[0167] A cell aggregate containing glial progenitor cells of one embodiment further has at least one of the following characteristics (A) to (C): (A) 60% or more, preferably 70% or more, more preferably 70% to 90%, even more preferably 75% to 85% of all the cells constituting the cell aggregate express NFIA. (B) 10% or more, preferably 20% or more, more preferably 20% to 50%, even more preferably 20% to 40% of all the cells constituting the cell aggregate express OLIG2, and (C) 20% or more, preferably 30% or more, more preferably 30% to 60%, even more preferably 30% to 50% of all the cells constituting the cell aggregate express one or more markers selected from HEY2, C1ORF61, FAM181B, NFIA, NFIB, ITM2B, LHFPL3, and MLC1.

[0168] Here, the ratio of each marker-positive cell can be calculated by measuring the number of cells stained by immunostaining or by gene expression analysis at the single-cell level.

[0169] The cell aggregate containing glial progenitor cells of one embodiment does not include (VIII) pluripotent stem cells. The fact that pluripotent stem cells are not included can be confirmed using as an index that neither OCT3 / 4 nor NANOG is expressed at the mRNA level or protein level, or that the expression level is decreased. Specifically, for example, it can be indicated by the fact that the expression level of OCT3 / 4 is decreased to 1 / 200 or less compared to iPS cells, and / or the expression level of NANOG is decreased to 1 / 400 or less compared to iPS cells.

[0170] The cell aggregate containing glial progenitor cells of one embodiment is characterized in that it has differentiated to the extent of not expressing PAX6. Here, it can be confirmed by using as an index that PAX6 is not expressed at the mRNA level or protein level, or the expression level is decreased. Specifically, for example, the expression level of PAX6 is reduced to 1 / 10 or less at the timing of performing step (4) or (5) and recovering the cell aggregate for transplantation compared with the timing of starting step (3) (for example, day 14 of differentiation in the examples of the present application) (for example, day 49 of differentiation in the examples of the present application).

[0171] The cell aggregate containing glial progenitor cells of one embodiment may be a spherical cell aggregate having a diameter of about 100 μm to 800 μm, for example, about 100 μm to 300 μm, about 200 μm to 400 μm, or about 400 μm to 600 μm. The number of cells per cell aggregate may be about 1,000 to 100,000, preferably about 3,000 to 50,000, and more preferably about 3,000 to 30,000.

[0172] The cell aggregate containing glial progenitor cells of one embodiment has at least 2, at least 4, at least 6, at least 8 or all of the above characteristics (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X).

[0173] The cell aggregate containing glial progenitor cells of one embodiment includes cells that express or secrete one or more markers selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL, or cells in which the above markers are detected in the culture supernatant of the cell aggregate. The cell aggregate containing glial progenitor cells of one embodiment preferably includes cells that express or secrete one or more markers selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, and GRO-α, or cells in which the above markers are detected in the culture supernatant of the cell aggregate, and more preferably includes cells that express or secrete one or more markers selected from the group consisting of SPARCL1, MIF, MCP-1, SCF, M-CSF, and HGF, or cells in which the above markers are detected in the culture supernatant of the cell aggregate.

[0174] In one embodiment, when SPARCL1 is detected in the culture supernatant of the cell aggregate containing glial progenitor cells, for example, when cultured for 48 hours, 0.1 ng or more, preferably 1 ng or more, more preferably 10 ng or more is detected per 1000 cells. In one embodiment, 0.1 ng to 1000 ng, preferably 1 ng to 1000 ng, more preferably 10 ng to 60 ng is detected per 1000 cells.

[0175] In one actual embodiment, when MIF is detected in the culture supernatant of the cell aggregate containing glial progenitor cells, for example, when cultured for 48 hours, 0.1 pg or more, preferably 0.2 pg or more, more preferably 0.5 pg or more is detected per 1000 cells. In one embodiment, 0.1 pg to 100 pg, preferably 0.1 pg to 10 pg, more preferably 0.5 pg to 5 pg is detected per 1000 cells.

[0176] In one actual embodiment, when MCP-1 is detected in the culture supernatant of cell aggregates containing glial progenitor cells, for example, when cultured for 48 hours, it is detected at 0.1 pg or more, preferably 0.2 pg or more, more preferably 0.5 pg or more per 1000 cells. In one embodiment, it is detected at 0.1 pg to 100 pg, preferably 0.1 pg to 10 pg, more preferably 0.5 pg to 8 pg per 1000 cells.

[0177] In one actual embodiment, when SCF is detected in the culture supernatant of cell aggregates containing glial progenitor cells, for example, when cultured for 48 hours, it is detected at 0.001 pg or more, preferably 0.01 pg or more, more preferably 0.02 pg or more per 1000 cells. In one embodiment, it is detected at 0.001 pg to 10 pg, preferably 0.01 pg to 1 pg, more preferably 0.02 pg to 0.2 pg per 1000 cells.

[0178] In one actual embodiment, when M-CSF is detected in the culture supernatant of cell aggregates containing glial progenitor cells, for example, when cultured for 48 hours, it is detected at 0.001 pg or more, preferably 0.005 pg or more, more preferably 0.01 pg or more per 1000 cells. In one embodiment, it is detected at 0.0001 pg to 10 pg, preferably 0.001 pg to 1 pg, more preferably 0.005 pg to 0.1 pg per 1000 cells.

[0179] In one actual embodiment, when HGF is detected in the culture supernatant of cell aggregates containing glial progenitor cells, for example, when cultured for 48 hours, it is detected at 0.0001 pg or more, preferably 0.001 pg or more, more preferably 0.01 pg or more per 1000 cells. In one embodiment, it is detected at 0.0001 pg to 10 pg, preferably 0.001 pg to 1 pg, more preferably 0.005 pg to 0.5 pg per 1000 cells.

[0180] In one actual aspect, when LIF is detected in the culture supernatant of cell aggregates containing glial progenitor cells, for example, when cultured for 48 hours, it is detected at 0.0001 pg or more, preferably 0.001 pg or more, more preferably 0.01 pg or more per 1000 cells. In one embodiment, it is detected at 0.0001 pg to 10 pg, preferably 0.001 pg to 1 pg, more preferably 0.005 pg to 0.5 pg per 1000 cells.

[0181] [Method for producing a cell population containing oligodendrocytes, astrocytes and neurons] As one aspect of the method for producing a cell population containing oligodendrocytes, astrocytes and neurons of the present invention, it is carried out by culturing cell aggregates containing glial progenitor cells for 5 days to 60 days using a maturation medium.

[0182] The "maturation medium" refers to a medium for the maturation of glial cells and neurons, and is a medium containing factors necessary for the maturation of glial cells and neurons. The base medium (basic medium) is not particularly limited as long as it is a medium in which nervous system cells can survive, and may be common with the above-mentioned medium for nerve / glial cell proliferation. Examples of the basic medium include a medium obtained by adding supplements for culturing nervous system cells such as N2 and / or B27 to a basic medium such as DMEM / F-12. However, it is desirable that the growth factors mainly contributing to cell growth contained in the medium for nerve / glial cell proliferation, such as FGF (e.g., FGF2), EGF, and PDGF (e.g., PDGF-AA), are not contained.

[0183] On the other hand, the maturation medium contains neurotrophic factors, thyroid hormones and / or cytokines that contribute to the differentiation of glial cells. Examples of the neurotrophic factor include NT-3. Examples of the thyroid hormone include T3. Examples of the cytokine include LIF.

[0184] As a maturation medium, for example, a medium containing at least one, preferably two, more preferably all of T3, NT-3, and LIF can be used. Further, as a maturation medium used for differentiation into astrocytes, a medium containing CNTF can also be used. These factors may be added to the medium for use, or a medium formulated with these factors may be used. The concentration of T3 in the maturation medium may be 5 ng / ml or more, preferably 30 ng / ml to 100 ng / ml, more preferably 60 ng / ml to 100 ng / ml. Also, the concentration of NT-3 in the maturation medium may be 10 ng / ml or more, preferably 10 ng / ml to 100 ng / ml, more preferably 10 ng / ml to 50 ng / ml. Further, the concentration of LIF in the maturation medium may be 10 ng / ml or more, preferably 10 ng / ml to 100 ng / ml, more preferably 10 ng / ml to 50 ng / ml. Additionally, the concentration of CNTF in the maturation medium may be 5 ng / ml or more, preferably 10 ng / ml to 50 ng / ml, more preferably 25 ng / ml to 50 ng / ml.

[0185] The "cell population containing oligodendrocytes, astrocytes, and neurons" (the cell population containing oligodendrocytes, astrocytes, and neurons of the present invention) obtained by the method for producing a cell population containing oligodendrocytes, astrocytes, and neurons refers to a cell population in which the three types of cells, oligodendrocytes, astrocytes, and neurons, are contained at a detectable level in the cells constituting the cell population, and the content ratio of each cell type is not particularly limited. The above cell population may be a cell aggregate having a three-dimensional structure (spherical shape) or a cell population having a secondary layered structure. The layered cell population can be produced by adherent culturing a cell aggregate containing glial progenitor cells in the presence of a cell adhesion factor such as Matrigel.

[0186] To detect each cell, for example, immunohistological techniques and gene analysis (such as RT-qPCR and RNA sequencing) can be used. When using gene analysis, according to the method described in Example 2 of this specification, the mRNA contained in the cell aggregate and its expression level can be identified.

[0187] The cell population containing the oligodendrocytes, astrocytes and neurons of the present invention obtained may contain oligodendrocyte progenitor cells, astrocyte progenitor cells and neural progenitor cells which are the starting cells. Preferably, the cell population contains oligodendrocytes, astrocytes and neurons at 50% or more of the total cell number, more preferably 70% or more. The cell population contains 2% or more oligodendrocytes, preferably 5% - 50%, more preferably 10% - 40%. Also, it contains 10% or more astrocytes, preferably 10% - 70%, more preferably 10% - 50%. Further, it contains 10% or more neurons, preferably 20% - 60%, more preferably 30% - 50%. Here, the total of oligodendrocytes, astrocytes and neurons does not exceed 100% and occupies 50% or more, preferably 70% or more of the total cell number contained in the cell aggregate.

[0188] The cell population containing the oligodendrocytes, astrocytes and neurons of the present invention (i) cells expressing one or more markers selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα and NG2, preferably three or more markers, more preferably five or more markers, even more preferably all markers, (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2 and ELAVL3, preferably two or more markers, more preferably all markers, and (iii) It contains cells that express one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2, preferably two or more markers, more preferably three or more markers, and even more preferably all markers.

[0189] The culture of cell aggregates containing glial progenitor cells may be suspension culture. The culture conditions are not particularly limited. For example, conditions such as a temperature of 35°C to 37°C, preferably 37°C, a humidity of 90% to 95%, preferably 95%, a CO2 concentration of 3% to 5%, preferably 5%, and an O2 concentration of 10% to 20%, preferably 20% are acceptable. The culture vessel is not particularly limited as long as it is a device for cell culture, and flasks and dishes commonly used by those skilled in the art can be used. For example, Nunc (trademark) EasYFlask (trademark) Cell Culture Flasks (manufactured by Thermo Fisher Scientific), Ultra-Low Attachment Culture Flasks (manufactured by Corning), etc. can be mentioned.

[0190] [Pharmaceutical Composition] As one aspect of the present invention, there is provided a pharmaceutical composition comprising, as an active ingredient, a cell aggregate containing the glial progenitor cells of the present invention, or a cell population containing the oligodendrocytes, astrocytes, and neurons of the present invention, or a cell population for transplantation containing cells derived from the cell aggregate or cell population. The cell aggregate containing the glial progenitor cells of the present invention is produced by the method for producing a cell aggregate containing the glial progenitor cells of the present invention. The cell population containing the oligodendrocytes, astrocytes, and neurons of the present invention is produced by the method for producing a cell population containing the oligodendrocytes, astrocytes, and neurons of the present invention.

[0191] The effective amount of the active ingredient varies depending on the purpose of administration, the method of administration, and the condition of the administration subject (gender, age, weight, disease state, etc.). For example, in terms of the number of cells, it is 1×10 4 cells to 1×10 8 cells, 1×10 5 cells to 3×10 5 cells, 1×10 6 cells to 3×106 individuals, or 1×10 7 ~3×10 7 individuals can be used.

[0192] In addition to the effective amount of the above active ingredient, the pharmaceutical composition of one embodiment may contain a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, a physiological aqueous solvent (such as physiological saline, buffer solution, serum-free medium, etc.) can be used. The pharmaceutical composition may contain, as necessary, preservatives, stabilizers, reducing agents, isotonic agents, etc. that are usually used in pharmaceuticals containing tissues or cells to be transplanted in transplantation medicine.

[0193] The cell aggregate for transplantation or the cell population for transplantation can be manufactured as a cell suspension by suspending it in an appropriate physiological aqueous solvent. If necessary, a cryopreservative can be added to cryopreserve the above cell population for transplantation, thaw it at the time of use, wash it with a buffer solution, and use it in transplantation medicine.

[0194] The cell population for transplantation of the present invention may be a suspension in which cell aggregates are suspended, or may be a suspension in which the cells are dispersed from the cell aggregates, or a sheet agent.

[0195] Furthermore, the cell population for transplantation obtained by the production method of the present invention can be three-dimensionally molded and made into a cell tissue structure that is a three-dimensional tissue by performing culturing on a scaffold after carrying out the step (4) in the production method of cell aggregates containing glial progenitor cells.

[0196] As described below, since the pharmaceutical composition containing the cells of the present invention can be obtained without serum, it does not contain components that are problematic in treatment such as serum-derived components, and in the administration to spinal cord injury model animals (for example, mice), it brings about remarkable effects such as the repair of the damaged nervous system at the damaged site and the recovery of motor function. Therefore, the pharmaceutical composition of the present invention is useful as a therapeutic agent for demyelinating diseases and diseases based on or accompanied by glial cell disorders for which there has been no effective treatment means so far, and diseases based on or accompanied by glial cell disorders in the acute, subacute or chronic phases, such as acute, subacute or chronic spinal cord injury.

[0197] Moreover, the cell population for transplantation composed of cell aggregates containing glial progenitor cells described in this specification is produced from immortalized pluripotent stem cells, identified by markers, etc., and quality-controlled, so that a cell population for transplantation with stable quality can be mass-produced and used for transplantation. In addition, since the cell population for transplantation can be stored, the cell population for transplantation can be prepared according to the transplantation time of the patient.

[0198] The "demyelinating disease" is not particularly limited as long as it is a disorder including inflammation or atrophy of myelin that forms a myelin sheath around the axons of nerve cells. Examples of demyelinating diseases include, for example, spinal cord injury, multiple sclerosis, adrenoleukodystrophy, leukodystrophy, Pelizaeus-Merzbacher disease (broadly, congenital leukodystrophy) and leukodystrophy. Examples of the above spinal cord injury include acute spinal cord injury, subacute spinal cord injury or chronic spinal cord injury.

[0199] "Diseases associated with glial cell disorders" (also referred to as "diseases based on glial cells") are not particularly limited as long as they are diseases associated with disorders of one or more cell types selected from the group consisting of glial cells, namely astrocytes, oligodendrocytes, ependymal cells, and microglia. Specific examples of diseases associated with glial cell disorders include spinal cord injury, cerebral infarction, neuropsychiatric diseases such as schizophrenia, demyelinating diseases, and neurodegenerative diseases. More specifically, neuromyelitis optica, amyotrophic lateral sclerosis, Parkinson's disease, Alexander's disease, congenital leukodystrophy, Huntington's disease, Alzheimer's disease, schizophrenia, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), Wallerian degeneration, adrenoleukodystrophy, macular hole, spinocerebellar degeneration, multiple system atrophy, and many other nerve disorders and neurodegenerative conditions. Those diseases that have passed a certain period of time since onset are called chronic demyelinating diseases or diseases associated with chronic glial cell disorders. For example, in the case of spinal cord injury, it is often observed that very firm scar tissue forms at the injury site and the pathological condition becomes fixed when three to six months have passed after the injury, and this pathological condition is called chronic spinal cord injury.

[0200] In diseases associated with glial cell disorders, examples of the damaged state of the membrane tissue containing glial cells include a state in which glial cells such as oligodendrocytes or astrocytes have undergone degenerative death.

[0201] [Treatment method and therapeutic agent] As one aspect of the present invention, there is provided a method for treating a demyelinating disease and a disease based on or associated with a glial cell disorder, which includes transplanting an effective amount of a cell aggregate containing the glial progenitor cells of the present invention, or a cell population containing the oligodendrocytes, astrocytes and neurons of the present invention, or a transplant cell population containing cells derived from the cell aggregate or the cell population, into a subject in need of transplantation. Here, the demyelinating disease and the disease based on or associated with the glial cell disorder may be a disease based on or associated with a chronic demyelinating disease and a chronic glial cell disorder. It should be noted that a condition in which a certain period of time has elapsed since the onset of the disorder or disease, for example, in the case of chronic spinal cord injury, a condition in which 3 months or more have elapsed since the injury is determined to be the chronic phase.

[0202] The cell aggregate containing the glial progenitor cells of the present invention is useful for transplantation medicine for demyelinating diseases and diseases associated with glial cell disorders. Therefore, the present invention provides a therapeutic agent for a demyelinating disease and a disease associated with a glial cell disorder, which includes the cell aggregate of the present invention or a transplant cell population containing cells obtained from the cell aggregate.

[0203] In addition, the present invention also provides a treatment method including administering (transplanting) the therapeutic agent to a patient in the form of a cell aggregate or its suspension, a suspension of dispersed cells, a suspension with a biodegradable biomaterial such as fibrin or hydrogel, and a cell tissue structure cultured using them as a scaffold. As a therapeutic agent for a demyelinating disease and a disease associated with a glial cell disorder, or in a damaged state of a tissue containing the glial cells, the cell aggregate (cell population) of the present invention or a transplant cell population containing cells obtained from the cell aggregate (cell population) can be used to replenish the corresponding damaged site.

[0204] Transplanting the cell aggregate of the present invention or a transplant cell population containing cells obtained from the cell aggregate into a patient with a demyelinating disease and a disease associated with a glial cell disorder, or a damaged state of a tissue containing glial cells, or replenishing the tissue itself containing the damaged glial cells can treat the demyelinating disease and the disease associated with a glial cell disorder, or the damaged state of a tissue containing glial cells.

[0205] In transplantation medicine, rejection due to differences in histocompatibility antigens often becomes a problem, but this problem can be overcome by using pluripotent stem cells (e.g., induced pluripotent stem cells) established from the somatic cells of the transplant recipient. That is, in a preferred embodiment of the present invention, by using pluripotent stem cells (e.g., induced pluripotent stem cells) established from the somatic cells of the recipient, an immunologically autologous cell aggregate (cell population) is produced for the recipient, and a cell population for transplantation containing the cell aggregate (cell population) or cells obtained from the cell aggregate (cell population) is transplanted into the recipient.

[0206] Alternatively, an allogeneic cell aggregate (cell population) may be produced from pluripotent stem cells (e.g., induced pluripotent stem cells) established from the somatic cells of another person whose immunity is compatible with the recipient (e.g., HLA type or MHC type is compatible), and a cell population for transplantation containing the cell aggregate (cell population) or cells obtained from the cell aggregate (cell population) may be transplanted into the recipient.

[0207] In addition, by producing the cell aggregate (cell population) of the present invention using iPS cells in which the expression of histocompatibility antigens (e.g., antigen proteins constituting HLA Class I and HLA Class II) or factors necessary for the expression of the antigen is suppressed, rejection can be avoided even in allogeneic cell transplantation.

[0208] In the treatment method of the present invention, the aforementioned pharmaceutical composition can be used as a therapeutic agent to be administered or transplanted to a patient or recipient.

[0209] As one embodiment of the present invention, there is provided the use of a cell aggregate containing the glial progenitor cells of the present invention, or a cell population containing the oligodendrocytes, astrocytes, and neurons of the present invention, for use in the treatment of demyelinating diseases and diseases based on glial cell disorders and diseases accompanied by glial cell disorders.

[0210] 〔Toxicity and Pharmacodynamic Evaluation Method〕 As one aspect of the present invention, there is provided a method for evaluating the toxicity or efficacy of a test substance, which comprises contacting the test substance with a cell aggregate containing the glial progenitor cells of the present invention or a cell population containing the oligodendrocytes, astrocytes and neurons of the present invention, and detecting or quantifying the effect of the test substance on the cell aggregate or the cell population.

[0211] The cell aggregate containing the glial progenitor cells of the present invention and the cell population containing oligodendrocytes, astrocytes and neurons can be used as model cells for diseases in screening for therapeutic agents or prophylactic agents for demyelinating diseases, diseases based on glial cell disorders and diseases accompanied by glial cell disorders (collectively also referred to as glial diseases), and for evaluating the efficacy of such agents.

[0212] In addition, the cell aggregate containing the glial progenitor cells of the present invention and the cell population containing oligodendrocytes, astrocytes and neurons can be used as normal model cells in safety tests, stress tests, toxicity tests, side effect tests, infection or contamination tests for chemicals and the like. Further, since the cell aggregate or cell population of the present invention contains glial progenitor cells such as oligodendrocyte progenitor cells and astrocyte progenitor cells, neural progenitor cells, oligodendrocytes, astrocytes, etc., it can also be used for functional tests of nerve tissues (central nervous system, spinal cord, neuromuscular junction) containing these cells, specifically, for evaluating the functions of serotonergic nerves, motor nerves, etc., and for evaluating the proliferation ability and differentiation ability of glial and neural progenitor cells.

[0213] Examples of the evaluation methods include stimulation and toxicity tests such as apoptosis evaluation, and tests for evaluating the effect of a chemical substance on the normal differentiation of glial progenitor cells into glial cells (expression protein analysis by RT-PCR of various gene markers, ELISA of cytokines, etc., phagocytosis ability test), etc. For example, it can be used for searching for compounds that promote the differentiation ability into oligodendrocytes, and for searching for compounds, proteins, etc. that rescue disease-specific phenotypes in cells differentiated from patient-derived iPS cells with demyelinating diseases, glial diseases, etc.

[0214] In addition, as cell materials for these tests, for example, plates on which cells of the cell aggregates of the present invention are dispersed, seeded, and adhered, cell suspensions, their sheets, or molded bodies can be provided.

[0215] Cell aggregates containing glial progenitor cells of the present invention, cell populations containing oligodendrocytes, astrocytes, and neurons, or cells dispersed therefrom can be used as extrapolation tests for human and animal tests.

[0216] [New markers for evaluating and discriminating cell aggregates containing glial progenitor cells] As one aspect of the present invention, there is provided a method for discriminating whether a cell aggregate containing glial progenitor cells is suitable for transplantation using as an index whether one or more markers selected from the group consisting of C1ORF61 and SERPINE2 are expressed.

[0217] The discrimination method of the present invention can be carried out by detecting the presence or absence of the expression of C1ORF61 or SERPINE2 by detecting the proteins, mRNAs, or fragments thereof of these markers. That is, as one aspect, the discrimination method of the present invention includes the following steps: (1) A step of detecting a protein or a fragment thereof derived from one or more markers selected from the group consisting of C1ORF61 and SERPINE2 expressed in a cell aggregate sample; (2) A step of determining that the cell aggregate sample is a cell aggregate containing glial progenitor cells suitable for transplantation when the expression level of the protein or the fragment thereof derived from the marker is greater than a reference value.

[0218] In addition, as one aspect, the discrimination method of the present invention includes the following steps: (1) A step of detecting an mRNA or a fragment thereof derived from one or more markers selected from the group consisting of C1ORF61 and SERPINE2 expressed in a cell aggregate sample; (2) When the expression level of the mRNA derived from the marker or a fragment thereof is greater than a reference value, determining that the cell aggregate sample is a cell aggregate containing glial progenitor cells suitable for transplantation.

[0219] As one aspect of the present invention, there is provided a method for identifying glial progenitor cells or neural stem cells with high glial differentiation potential, which includes detecting one or more genes selected from the group consisting of C1ORF61 and SERPINE2, proteins encoded by the genes, and fragments thereof. Here, "high glial differentiation potential" refers to the property of neural stem cells having a high proportion (for example, 30% or more) of differentiating into glia when terminal differentiation is induced in a maturation medium.

[0220] In the identification method of the present invention, the presence or absence of the expression of C1ORF61 or SERPINE2 can be determined by detecting the proteins, mRNAs, or fragments thereof of these markers. That is, as one aspect, the identification method of the present invention includes the following steps: (1) A step of detecting a protein or a fragment thereof derived from one or more markers selected from the group consisting of C1ORF61 and SERPINE2 expressed in a cell population sample; (2) When the expression level of the protein or fragment derived from the marker is greater than a reference value, identifying that the cell population sample is a glial progenitor cell or a neural stem cell with high glial differentiation potential.

[0221] Also, as one aspect, the identification method of the present invention includes the following steps: (1) A step of detecting an mRNA or a fragment thereof derived from one or more markers selected from the group consisting of C1ORF61 and SERPINE2 expressed in a cell population sample; (2) When the expression level of the mRNA or the fragment thereof derived from the marker is greater than a reference value, determining that the cell population sample is a glial progenitor cell or a neural stem cell with high glial differentiation potential.

[0222] Table 1 below shows the genes described in this specification and their GenBank accession numbers.

Table 1

Examples

[0223] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0224] <Example 1 Production of cell aggregates> As the iPS cell line, the QHJI01s04 strain, a clinical-grade peripheral blood-derived feeder-free iPS cell line established at the Kyoto University iPS Cell Research Institute, was used. Before differentiation, it was maintained in StemFit® AK03N medium (manufactured by Ajinomoto Co., Inc.) supplemented with laminin 511E8 fragment (iMatrix-511E8; manufactured by Nippi Inc.) (0.5 mg / ml).

[0225] To induce the differentiation of iPS cells into neural progenitor cells, the serum-free aggregation suspension culture method (SFEBq method) for embryoid body formation was used. Specifically, the maintained QHJI01s04 strain was dissociated into single cells with 0.5× TrypLE Select (manufactured by Thermo Fisher Scientific) / 0.25 mM EDTA (ethylenediaminetetraacetic acid) / PBS (phosphate-buffered saline, manufactured by Thermo Fisher Scientific), and seeded on a 96-well low-adhesion plate (product name: PrimeSurface® plate 96V, manufactured by Sumitomo Bakelite Co., Ltd.) at 9000 cells / well. Induction was started at 37°C under 5% CO2 / 5% O2 with the seeding day defined as day 0 of differentiation. An embryoid body formation medium (AK03N-C medium) supplemented with each factor shown in Table 2 was used, and half of the medium was exchanged daily.

Table 2

[0226] On day 14 of differentiation, embryoid bodies were collected from 96-well plates and washed once with DMEM / F-12 (product name: D-MEM / Ham’s F-12; Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12, manufactured by Fujifilm Wako Pure Chemical Corporation). Thereafter, from day 14 to day 21 of differentiation, under the conditions 1, 2, 3, and 4 shown in Table 3, in a low-attachment flask (product name: Ultra-Low Attachment 75cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap, manufactured by Corning), at 37 °C under 5% CO2 / 20% O2, the medium was changed once every 3 days and cultured for 7 days.

Table 3

[0227] As the medium for nerve / glial cell growth in Table 3, a medium obtained by adding B27 supplement (manufactured by Thermo Fisher Scientific), N2 supplement (manufactured by Thermo Fisher Scientific), 60 ng / ml T3 (3,3’,5-triiodo-L-thyronine sodium salt, manufactured by Sigma-Aldrich), 10 ng / ml PDGF-AA (Platelet-Derived Growth Factor-AA, manufactured by PeproTech), 10 ng / ml IGF-1 (Insulin-like Growth Factor-1, manufactured by R&D Systems), 10 ng / ml NT-3 (Neurotrophin-3, manufactured by PeproTech), 10 ng / ml EGF (Epidermal Growth Factor, manufactured by PeproTech), and 20 ng / ml FGF2 (Fibroblast Growth Factor 2, manufactured by PeproTech) to DMEM / F-12 (containing 3.151 g / L glucose, 15 mM HEPES, 2.5 mM L-glutamine, and 0.5 mM sodium pyruvate, manufactured by Fujifilm Wako Pure Chemical Corporation) was used.

[0228] On the 21st day of differentiation, embryoid bodies were collected, dispersed into single cells using TrypLE Select, and then cultured in a tissue culture flask (manufactured by Thermo Fisher Scientific) with a medium for neural and glial cell growth containing 1 μM PM. The medium was changed once every 7 days, and suspension culture was performed (passage number 1). After 14 days, at the 35th day of differentiation, the cells were again dispersed into single cells and then cultured in suspension with a medium for neural and glial cell growth (passage number 2). Bright-field images at the 35th and 49th days of differentiation are shown in Fig. 1A.

[0229] In addition, to confirm the terminal differentiation ability of the cell aggregates obtained on the 49th day of differentiation, the cell aggregates were seeded in an 8-well chamber slide glass (manufactured by Iwaki) treated with 1% Matrigel Basement Membrane Matrix Growth Factor Reduced (manufactured by Corning) diluted with DMEM / F-12, adhered onto the Matrigel, and cultured for 14 days with a maturation medium for terminal differentiation. As the maturation medium, a medium obtained by adding B27 supplement, 1% non-essential amino acids (manufactured by Thermo Fisher Scientific), 60 ng / ml T3, 10 ng / ml NT-3, 25 ng / ml CNTF, and 10 ng / ml LIF to KBM Neural Stem Cell (manufactured by Cosmo Bio) was used.

[0230] Using the cells on the 14th day of terminal differentiation (the 63rd day of differentiation), fluorescence immunostaining was performed for each marker. For fluorescence immunostaining, six types of primary antibodies and their corresponding fluorescently labeled secondary antibodies were used. As shown in Table 4, three types of primary antibodies were mixed together and diluted with a blocking solution to prepare two types of primary antibody solutions (primary antibody solution 1 and primary antibody solution 2). Similarly, three types of secondary antibodies were mixed together as shown in Table 4, and Hoechst 33342 (dilution ratio 1:1000, manufactured by Dojindo Laboratories) for nuclear staining was also added, and then diluted with a blocking solution to prepare two types of secondary antibody solutions (secondary antibody solution 1 and secondary antibody solution 2).

Table 4

[0231] For fluorescence immunostaining, on the 14th day of terminal differentiation (63rd day of differentiation), each cell obtained under Conditions 1, 2, 3, and 4 was fixed with 4% paraformaldehyde (PFA, manufactured by Fujifilm Wako Pure Chemical Corporation) at room temperature for 25 minutes, washed three times with PBS, and then incubated with 10% goat serum (manufactured by Fujifilm Wako Pure Chemical Corporation) / PBS as a blocking solution at room temperature for 1 hour. Thereafter, the primary antibody solution (Primary Antibody Solution 1 or Primary Antibody Solution 2) shown in Table 4 above was added and incubated at 4°C overnight for co-staining. Thereafter, it was washed three times with PBS, and further, the secondary antibody solution (Secondary Antibody Solution 1 or Secondary Antibody Solution 2) corresponding to the primary antibody solution was added and incubated at room temperature for 1 hour. Thereafter, it was washed three times with PBS and stored at 4°C.

[0232] Microscopic observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by Keyence Corporation). The fluorescence immunostaining is shown in Figure 1B. Note that Merge is an image obtained by overlapping three types of fluorescence. Neurons, astrocytes, and oligodendrocytes were detected under any of Conditions 1, 2, 3, and 4. However, at the 14th day of terminal differentiation under Condition 1, the morphology of O4-positive cells was immature, and GalC-positive cells were not detected, indicating delayed maturation. Under Condition 2, a GFAP-negative cell population was detected. Thus, it became clear that Condition 3 or 4 is the best condition.

[0233] From the above investigations, a method for producing cell aggregates containing glial progenitor cells was established (Figure 2). In Figure 2, the embryoid body formation medium is AK03N-C medium, the nerve / glia growth medium is the above-mentioned nerve / glia growth medium, S of the compound represents SB431542, L represents LDN193189, C represents CHIR99021, Y represents Y-27632, RA represents retinoic acid, and PM represents palmoformin, respectively.

[0234] <Example 2 Characterization Evaluation 1 of Cell Aggregates (Change in Gene Expression during Differentiation Induction)> To examine gene expression changes during the differentiation induction process, gene expression analysis by RNA-sequencing was performed on the intermediate products of differentiation induction. Differentiation induction was initiated using the QHJI01s04 strain according to the method of Condition 3 in Example 1. Total RNA was extracted from cells before differentiation (iPS cell line), on the 7th, 14th, 21st, and 35th days of differentiation using the RNeasy Plus Mini Kit (Qiagen), libraries were prepared using the TruSeq Stranded mRNA Library Prep kit from Illumina, and 80-cycle sequencing was performed using the HiSeq 2500. Gene expression analysis was performed with data of more than 30 million reads per cell (Figures 3A - 3D).

[0235] As a result of comparing the gene expression profiles before differentiation induction (iPS cells) and on the 7th day of differentiation, the expression levels of the pluripotency markers NANOG and POU5F1 (OCT3 / 4) were significantly decreased at the 7th day of differentiation. Also, at the 7th day of differentiation, the expression levels of the neural stem cell markers SOX1, PAX6, HES4, and HES5 were significantly increased (Figure 3A). As a result of comparing the gene expression profiles on the 7th and 14th days of differentiation, genes whose expression increased on the 14th day of differentiation compared to the 7th day of differentiation included ASCL1, HEY1, DCX, βIII tubulin (TUBB3), ELAVL3, ZBTB20, SLIT1, HOXB3, HOXA4, HOXB4, HOXB6, HOXB8, etc. (Figure 3B). Also, as a result of comparing the gene expression profiles on the 14th and 21st days of differentiation, genes whose expression increased on the 21st day of differentiation compared to the 14th day of differentiation included HEY2, NKX6.2, NKX2.2, OLIG1, OLIG2, etc. (Figure 3C). As a result of comparing the gene expression profiles on the 21st and 35th days of differentiation, genes whose expression levels increased on the 35th day of differentiation compared to the 21st day of differentiation included NFIA, NFIB, SLC1A3, S100B, FABP7 (Figure 3D). Also, the expression level of PAX6 was significantly decreased (Figure 3A).

[0236] <Example 3 Characterization Evaluation of Cell Aggregates 2 (Fluorescent Immunostaining of Cell Aggregates)> As an iPS cell line, using the Ff-WJ14s01 strain, a feeder-free iPS cell line derived from umbilical cord blood established at the Kyoto University Institute for Integrated Cell-Material Sciences, cell aggregates containing glial progenitor cells were prepared by the method of Condition 3 in Example 1, and fluorescence immunostaining for each differentiation marker was performed. For fluorescence immunostaining, eight types of primary antibodies and their corresponding fluorescently labeled secondary antibodies were used. As shown in Table 5, two or three types of primary antibodies were mixed together, diluted with a blocking solution, and three types of primary antibody solutions (primary antibody solution 1, primary antibody solution 2, primary antibody solution 3) were prepared. Similarly, for the secondary antibodies, two or three types were mixed as shown in Table 5, and in addition, Hoechst 33342 (dilution ratio 1:1000, manufactured by Dojindo Laboratories) for nuclear staining was added, and three types of secondary antibody solutions (secondary antibody solution 1, secondary antibody solution 2, secondary antibody solution 3) were prepared by diluting with a blocking solution.

Table 5

[0237] At the 77th day of differentiation, the cell aggregates were fixed with 4% PFA at room temperature for 20 minutes and washed three times with PBS. The fixed cells were cryo-embedded on dry ice using O.C.T. compound (manufactured by Sakura Finetek Japan Co., Ltd.), and frozen sections with a thickness of 10 μm were prepared and stored at -80°C. After washing the frozen sections once with PBS, they were incubated at room temperature for 1 hour with 10% goat serum (manufactured by FUJIFILM Wako Pure Chemical Corporation) / PBS as a blocking solution. Thereafter, the primary antibody solution (primary antibody solution 1, primary antibody solution 2, or primary antibody solution 3) shown in Table 5 above was added, and incubation was carried out at 4°C overnight for co-staining. Thereafter, they were washed three times with PBS, and then the secondary antibody solution (secondary antibody solution 1, secondary antibody solution 2, or secondary antibody solution 3) corresponding to the primary antibody solution was added, and incubation was carried out at room temperature for 1 hour. After mounting with mounting agent PermaFlo (manufactured by Thermo Fisher Scientific), they were stored at 4°C.

[0238] Microscopic observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by Keyence Corporation). The fluorescence immunostaining image is shown in Fig. 4A. GFAP-positive cells, NESTIN-positive cells, Tuj1-positive cells, OLIG2-positive cells, ELAVL3-positive cells, MAP2-positive cells, and NFIA-positive cells were observed. Also, the ratios of OLIG2-positive cells and NFIA-positive cells per total number of cells constituting 1 spheroid (cell aggregate) were quantified using the analysis software (BZ-H3C) attached to the BZ-X710, and the average of 6 spheroids was calculated. As a result, the ratios of OLIG2-positive cells and NFIA-positive cells were 30.9 ± 6.6% and 87.6 ± 1.8%, respectively (Fig. 4B).

[0239] <Example 4 Characterization Evaluation 3 of Cell Aggregates (Single-Cell Gene Expression Analysis)> Genes characteristic of cell aggregates containing glial progenitor cells were searched. Single-cell RNA-sequencing (RamDA-seq) was performed on cell aggregates containing glial progenitor cells (Gliogenic NPC) prepared from the Ff-WJ14s01 strain by the method of Condition 3 in Example 1 and cell aggregates (Neurogenic NPC) containing many cells with high neurogenic differentiation potential prepared from the Ff-WJ14s01 strain by the method of Non-Patent Document 2 according to the method described in "Hayashi et al., Nature Communications, volume 9, Article number 619 (2018)". Using Illumina's HiSeq 2500, 50 cycles of sequencing were performed in Single-End mode. Gene expression analysis was performed on 150 Gliogenic NPCs and 91 Neurogenic NPCs (total 241) that passed the quality evaluation.

[0240] From the gene expression profiles, 241 cells were classified into three clusters (C1 - C3). 91.2% of the total number of analyzed Neurogenic NPCs were classified into C1, 8.8% into C3, and none into C2. On the other hand, 5.3% of the total number of analyzed Gliogenic NPCs were classified into C1, 58.0% into C2, and 36.7% into C3 (Figure 5). Thus, it was suggested that the gene expression profiles of Neurogenic NPCs and Gliogenic NPCs are significantly different.

[0241] Table 6 shows the genes (top 10) characteristically expressed in each cluster. Genes highly expressed in cells belonging to C2, which only contains Gliogenic NPCs, were found to be HEY2, C1ORF61, FAM181B, NFIB, ITM2B, NFIA, LHFPL3, and MLC1 (Table 6, Figure 6A, Figure 6B). Additionally, genes with higher expression in Gliogenic NPCs compared to Neurogenic NPCs were found to be ASCL1, MEIS1, MEIS2, DLL3, HEY1, ZBTB20, SOX9, SLC1A3, S100B, SLIT1, SPARCL1, TIMP3, TIMP4, SPON1, KLF9, GRIK3, SOX6, EPHA3, NTRK2, GRIA2, PTPRO, KCND3, and SERPINE2 (Figure 6A, Figure 6B). Some of the GenBank accession numbers of the genes listed in Table 6 are shown in Table 1. Also, LOC541471 is described in Oncol Lett. 2019 Feb; 17(2): 2457 - 2464.

Table 6

[0242] <Example 5 Characterization Evaluation of Cell Aggregates 4 (Terminal Differentiation Ability Evaluation)> Regarding cell aggregates containing glial progenitor cells, it was examined by fluorescence immunostaining whether they differentiated into three lineages of neurons, astrocytes, and oligodendrocytes after terminal differentiation. For cell aggregates (77 days after differentiation) containing glial progenitor cells derived from the Ff-WJ14s01 strain prepared by the method of Condition 3 in Example 1, terminal differentiation induction was performed using a maturation medium by the method described in Example 1. 31 days after terminal differentiation (108 days after differentiation), the cells were fixed with 4% PFA at room temperature for 25 minutes by the method described in Example 1, washed three times with PBS, and then fluorescence immunostaining was performed. Microscopic observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by Keyence Corporation) and a confocal fluorescence microscope LSM880 (manufactured by Zeiss). As a result, Tuj1- and MAP2-positive neurons, GFAP-positive astrocytes, O4-positive, GalC-positive, and MBP-positive oligodendrocytes were detected. Therefore, the ability to differentiate into three lineages was confirmed (Figure 7A). In addition, the positive cell rate of each was quantified using the analysis software Developer Toolbox (manufactured by GE Healthcare) of the In Cell Analyzer. As a result, the positive cell rate was approximately 10% for O4-positive cells, approximately 30% for GFAP-positive cells, and approximately 30% for Tuj1-positive cells (Figure 7B).

[0243] <Example 6 Characterization Evaluation of Cell Aggregates 5 (Gene Expression Changes Before and After Terminal Differentiation> For cell aggregates containing glial progenitor cells, the gene expression levels of each differentiation cell marker before and after terminal differentiation were analyzed. For cell aggregates containing glial progenitor cells (48 days after differentiation) prepared from the QHJI01s04 strain by the method of Condition 3 in Example 1, terminal differentiation was induced using a maturation medium by the method described in Example 1. Total RNA was extracted from cells before terminal differentiation (48 days after differentiation) and 34 days after terminal differentiation (82 days after differentiation) using an RNeasy Plus Mini Kit (manufactured by Qiagen). The extracted total RNA was reverse-transcribed into cDNA using a SuperScript III First-Strand Synthesis System for RT-PCR (manufactured by Thermo Fisher Scientific). Thereafter, the expression levels of GFAP, CSPG4 (NG2), OLIG2, PLP1 (PLP), PDGFRA (PDGFRα), SOX10, CNP, MBP, TUBB3 (βIII tubulin), and MAP2 were measured by quantitative RT-PCR using Fast SYBR (trademark) Green Master Mix (manufactured by Thermo Fisher Scientific) with a Step One Plus Realtime PCR System (manufactured by Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.

[0244] Table 7 shows the primer sets used for the amplification reaction of the target gene. The results are shown in Fig. 8. The expression levels after terminal differentiation are shown as relative expression levels relative to the expression level before terminal differentiation (set as 1). Since the expression levels of astrocyte markers and oligodendrocyte markers increased in the cells after terminal differentiation, it was suggested that the terminal differentiation was induced by the maturation medium and the cells differentiated into mature astrocytes and oligodendrocytes. The gene expression levels of the neural markers βIII tubulin and MAP2 hardly changed before and after terminal differentiation. However, from the results of the fluorescence immunostaining of the cell aggregates examined in Example 3, it was considered that the cell aggregates containing glial progenitor cells expressed βIII tubulin and MAP2 from the time of the cell aggregates before terminal differentiation and the expression was maintained even after terminal differentiation.

Table 7

[0245] <Example 7 Transplantation experiment of cell aggregates containing glial progenitor cells into subacute spinal cord injury model mice> NOD-SCID (NOD.CB17-Prkdcscid·J) mice (Charles River Laboratories) were handled in accordance with the animal experiment guidelines of Keio University and the NIH. The animal experiment plan was approved by the Animal Experiment Committee within Keio University. Female NOD-SCID mice at 8 weeks of age were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). According to the method described in "Scheff et al., Journal of neurotrauma 20, 179-193 (2003)", a contusion injury was applied at the 10th thoracic vertebra level with an IH impactor (60-70 kdyn, manufactured by Precision Systems and Instrumentation), and immediately after that, ampicillin (12.5 mg / kg) was intramuscularly injected. Seven days after the injury, the hindlimb motor function was evaluated using the Basso Mouse Scale (BMS) according to the method described in "Basso et al., Journal of Neurotrauma, 23: 635-59 (2006)". Individuals with spontaneous recovery of BMS to 2.5 or more at 7 days after injury were excluded because the cell transplantation effect could not be measured, and they were divided into two groups (12 in the control group and 14 in the cell transplantation group), and the groups were divided so that the average BMS of each group was the same.

[0246] On the 9th day after injury (subacute phase), using a stereotactic injection system (manufactured by Muromachi Kikai Co., Ltd.), a cell suspension was transplanted into the cell transplantation group and PBS was transplanted into the control group at 2 μl into the center of the injury using a 28G metal needle. The cells were cell aggregates containing glial progenitor cells derived from the Ff-WJ14s01 strain (5.0×10 5The cells were collected in 2 μl of PBS and used for transplantation. Various motor function evaluations were performed until 12 weeks after transplantation. Also, at 12 weeks after transplantation, under deep anesthesia, the spinal cord was fixed by perfusing 4% PFA into the heart and stored at 4°C. Then, the fixed spinal cord was stored at 4°C in 30% sucrose / PBS and then cryo-embedded using O.C.T. compound. Sagittal (14 μm thick) and transverse (16 μm thick) sections were prepared and stored at -80°C. For histological staining, hematoxylin-eosin (HE) staining was performed for tissue image observation, and Luxol fast blue (LFB) staining was performed for myelin sheath tissue staining.

[0247] Furthermore, fluorescence immunostaining was performed for more detailed tissue evaluation. After washing three times with PBS, it was incubated at room temperature for 1 hour with Blocking One (manufactured by Nacalai Tesque) containing 0.1% Triton X-100 as a blocking solution. Then, a primary antibody solution prepared by diluting the primary antibody with the blocking solution was incubated overnight at 4°C. The primary antibodies used in this example were human anti-Hu antibody (dilution ratio: 1:1000, provided by Dr. Robert Darnell of Rockefeller University), mouse anti-GFAP antibody (dilution ratio 1:5000, manufactured by Abcam), mouse anti-APC antibody (dilution ratio 1:300, manufactured by Abcam), rabbit anti-human specific NESTIN antibody (dilution ratio 1:200, manufactured by IBL), rabbit anti-Ki67 antibody (dilution ratio 1:1000, manufactured by Leica Biosystems), mouse anti-OCT3 / 4 antibody (dilution ratio 1:100, manufactured by Santa Cruz), mouse anti-human specific nuclear antibody (HNA) (dilution ratio 1:100, manufactured by Millipore), mouse anti-human specific cytoplasmic antibody (STEM121) (dilution ratio 1:100, manufactured by Millipore), mouse anti-human Tau antibody (dilution ratio 1:500, manufactured by Thermo Fisher Scientific), mouse anti-Bassoon antibody (dilution ratio 1:200, manufactured by GeneTex), mouse anti-human Synaptophysin antibody (dilution ratio 1:200, manufactured by Millipore). Then, after washing three times with PBS, a secondary antibody solution prepared by diluting the Alexa Fluor-labeled secondary antibody corresponding to the primary antibody and Hoechst 33342 (dilution ratio 1:1000, manufactured by Dojindo Laboratories) for nuclear staining with the blocking solution was incubated at room temperature for 1 hour. After washing three times with PBS, it was mounted using a mounting agent PermaFlo (manufactured by Thermo Fisher Scientific) and stored at 4°C. The samples were observed and imaged using a fluorescence microscope BZ-X710 (manufactured by Keyence) and a confocal fluorescence microscope LSM700 (manufactured by Zeiss).

[0248] <Example 8 Evaluation of the Efficacy of Cell Aggregates Containing Glial Progenitor Cells in a Mouse Model of Subacute Spinal Cord Injury> (BMS Scoring) In the experiment described in Example 7, in order to examine the effectiveness evaluation of transplanted cells, the hindlimb motor function was evaluated by BMS scoring once every 7 days until 12 weeks after transplantation. In the cell transplantation group, the BMS score was significantly increased (*: p < 0.05) compared to the control group from the 14th day after injury (5th day after transplantation) to the final evaluation day on the 91st day after injury (82nd day after transplantation) (Figure 9).

[0249] <Example 9 Evaluation of the effectiveness of cell aggregates containing glial progenitor cells in subacute spinal cord injury model mice (Rotarod test)> The coordinated movement evaluation (Rotarod test) was carried out at 12 weeks after transplantation. The time that the mice could walk on a rotarod (manufactured by Muromachi Kikai Co., Ltd.) rotating 20 times per minute was measured. In the cell transplantation group, the mice could walk on the rotarod for a significantly longer time (*: p < 0.05) compared to the control group (Figure 10).

[0250] <Example 10 Evaluation of the effectiveness of cell aggregates containing glial progenitor cells in subacute spinal cord injury model mice (Gait pattern evaluation)> The gait pattern evaluation was carried out at 12 weeks after transplantation using a DigiGait system (manufactured by Mouse Specific). The mice were made to walk on a treadmill (7 cm / sec), and the stride length and walking angle were measured. In the cell transplantation group, the stride length was significantly longer (**: p < 0.01) compared to the control group (Figure 11A). Also, in the cell transplantation group, the angle formed by the limbs with respect to the advancing direction was significantly smaller (*: p < 0.05) compared to the control group, suggesting that both limbs were grounded in parallel and the walking pattern was closer to normal (Figure 11B).

[0251] <Example 11 Evaluation of the effectiveness of cell aggregates containing glial progenitor cells in subacute spinal cord injury model mice (Kinematics analysis)> At 12 weeks after transplantation, in order to evaluate joint movement, analysis was performed using the three-dimensional motion analysis software KinemaTracer (manufactured by Kissei Comtec Co., Ltd.). Mice labeled with marker labels on the shoulder joint, hip joint, knee joint, ankle joint, and toes were made to walk on a treadmill, and video was taken with a high-speed digital camera (manufactured by GoPro) from four directions in the front and back of both sides, and the trajectories of each marker were visualized three-dimensionally. Figure 12(A) shows a representative example of the trajectory from when the hind limb leaves the ground until it touches the ground. In the cell transplantation group, the limbs were more separated from the ground compared to the control group, showing a smoother trajectory. Also, Figure 12(B) shows the joint angle changes of the hip joint, knee joint, ankle joint, and toes in one walking cycle. Analysis was performed in 5-step increments for 6 individuals. Each figure shows that the thin line represents the average joint angle change of 5 steps for one individual, and the thick line represents the average of 6 individuals. It was suggested that in the cell transplantation group, the variation in joint movement for each walking was smaller compared to the control group.

[0252] <Example 12 Tissue Evaluation of the Spinal Cord of Subacute Spinal Cord Injury Model Mice> At 12 weeks after transplantation, in order to detect the transplanted cells, fluorescence immunostaining was performed on sagittal sections using the STEM121 antibody (Figure 13(A)). The transplanted cells were detected over a wide range even in sites away from the center of the injury, confirming the engraftment of the transplanted cells. Also, HE staining was performed on adjacent sections, and as a result of observing the tissue images over a wide range from the cranial side to the caudal side, no tumor-like structures were observed (Figure 13(B)). In addition, fluorescence immunostaining was performed on transverse sections of the center of the injury (2), 4 mm cranial to the center of the injury (1), and 4 mm caudal to the center of the injury (3) using the anti-HNA antibody (Figure 13(C)). Since HNA-positive cells were detected in the sections of the center of the injury and 4 mm cranial and 4 mm caudal, which are the transplantation sites, it was suggested that the transplanted cells had migrated widely.

[0253] For more detailed analysis of the characteristics of transplanted cells, fluorescence immunostaining of human-specific markers and various markers was performed on sagittal sections. First, based on the report (Non-Patent Document 8) that iPS cell-derived neural progenitor cells become OCT3 / 4 positive when tumorigenic, fluorescence immunostaining was performed using anti-OCT3 / 4 antibody, anti-Ki67 (proliferative marker) antibody, and anti-HNA antibody. Also, for the analysis of the characteristics of transplanted cells, fluorescence immunostaining was performed using anti-NESTIN (neural stem cell marker) antibody, anti-Hu (neuronal marker) antibody, anti-GFAP (astrocyte marker) antibody, anti-APC (oligodendrocyte marker) antibody, and anti-HNA antibody, and positive cells were quantified.

[0254] As a result, since no HNA + / OCT3 / 4 + double-positive cells were detected, it was suggested that the transplanted cells were not tumorigenic (Figure 14A). HNA + / Ki67 + double-positive cells were 4.71 ± 0.20%, suggesting the presence of proliferative cells in the transplanted cells. Also, HNA + / NESTIN + double-positive neural progenitor cells were 17.02 ± 3.10%, HNA + / Hu + double-positive neurons were 18.12 ± 1.22%, HNA + / GFAP + double-positive astrocytes were 27.23 ± 1.92%, HNA + / APC + double-positive oligodendrocytes were 36.56 ± 2.82% (Figure 14B). Therefore, at 91 days after transplantation, although some immature neural progenitor cells and glial progenitor cells were present, it was suggested that the majority of the transplanted cells differentiated into three lineages: neurons, astrocytes, and oligodendrocytes.

[0255] Next, to examine whether neurons derived from transplanted cells form neural circuits with host mouse neurons, sagittal sections were subjected to fluorescence immunostaining using anti-Tuj1 antibody, anti-HNA antibody, anti-mouse-specific Bassoon (presynaptic marker), and anti-human Synaptophysin (synaptic vesicle protein) antibody. As a result, HNA + / Tuj1 + double-positive transplanted cell-derived neurons were co-localized with mouse-specific Bassoon (Fig. 15(A)). Also, HNA - / Tuj1 + host neurons were co-localized with human-specific Synaptophysin (Fig. 15(B)). These results suggest that host mouse neurons and transplanted cell-derived neurons form neural circuits.

[0256] Next, the remyelination effect by cell transplantation was examined. Fig. 16A shows LFB staining images of each of rostral 0.48 mm, the epi-center of the injury, and caudal 0.48 mm. Myelin tissue was observed by LFB staining for transverse sections from 0.96 mm rostral and caudal to the injury center, and the LFB-positive area was quantified. As a result, in the cell transplantation group, the LFB-positive area was significantly increased (*: p < 0.05; **: p < 0.01) compared to the control group in all sections observed (Fig. 16A, Fig. 16B). Also, fluorescence immunostaining of mature oligodendrocyte markers MBP and STEM121 was performed on transverse sections. As a result, a STEM121 + / MBP + double-positive area was detected (Fig. 17). Furthermore, for more detailed observation, immunoelectron microscopy using STEM121 antibody was performed on transverse sections by the method described in "Shibata et al., Frontiers in Neural Circuits,.2019;13:29.", and the STEM121 antibody reaction site was detected with a secondary antibody labeled with gold colloid particles. As a result of observing myelin under an electron microscope, gold colloid particles were detected in the myelin (arrow in Fig. 18). These results suggest that mature oligodendrocytes derived from transplanted cells remyelinate nerve axons.

[0257] <Example 13 Transplantation experiment of cell aggregates containing glial progenitor cells in a rat model of chronic spinal cord injury> Nude (F344 / NJcl-rnu / rnu) rats (CLEA Japan, Inc.) were handled in accordance with the animal experiment guidelines of Keio University and NIH. The animal experiment plan was approved by the Animal Experiment Committee at Keio University. Female Nude rats at 8 weeks of age were anesthetized by intraperitoneal administration of a mixture of three anesthetics (medetomidine: 0.375 mg / kg, midazolam: 2 mg / kg, butorphanol: 2.5 mg / kg). Using the method described in "Scheff et al., Journal of neurotrauma 20, 179-193 (2003)", a contusion injury was applied at the 10th thoracic vertebral level with an IH impactor (220 kdyn, manufactured by Precision Systems and Instrumentation), orbifloxacin (5 mg / kg) was subcutaneously injected, and then arousal was promoted by intraperitoneal administration of a medetomidine antagonist (atipamezole: 0.75 mg / kg). On the day after injury and every week after injury, the hindlimb motor function was evaluated using the Basso Beattie Bresnahan (BBB) according to the method described in "Basso et al., Journal of Neurotrauma, 12: 1-21 (1995)". Individuals with a natural recovery of BBB of 9 or more at 41 days after injury were excluded because the cell transplantation effect could not be measured, and they were divided into two groups (13 in the control group and 18 in the cell transplantation group), and the groups were divided so that the average BBB of each group was the same.

[0258] On the 42nd day after injury (chronic phase), using a stereotaxic injection system (manufactured by Muromachi Kikai Co., Ltd.), a cell suspension was transplanted into the cell transplantation group and PBS was transplanted into the control group, 2 μl each, at two sites, the rostral and caudal sides of the injury center, using a 27G metal needle. The cell suspension was a cell aggregate containing glial progenitor cells derived from the QHJI01s04 strain (5.0×10 5Cells (2 μl) were collected in PBS and used for transplantation. Hind limb motor function was evaluated until 12 weeks after transplantation. In addition, at 12 weeks after transplantation, gait pattern evaluation was performed, and then under deep anesthesia, the spinal cord was fixed and harvested by perfusing 4% PFA throughout the body from the heart, and stored at 4 °C under 4% PFA immersion. Thereafter, the fixed spinal cord was stored at 4 °C in 30% sucrose / PBS, and then cryo-embedded using OCT compound. Sagittal (14 μm thick) and transverse (20 μm thick) sections were prepared and stored at -80 °C. For histological staining, hematoxylin-eosin (HE) staining and fluorescence immunohistochemistry were performed for tissue image observation.

[0259] <Example 14 Evaluation of the Efficacy of Cell Aggregates Containing Glial Progenitor Cells in a Chronic Spinal Cord Injury Model Rat> (BBB Scoring) In the experiment described in Example 13, to examine the efficacy evaluation of transplanted cells, hind limb motor function was evaluated by BBB scoring once every 7 to 14 days until 12 weeks after transplantation. In the cell transplantation group, the BBB score increased significantly (*: p < 0.05, **: p < 0.01) compared to the control group from day 63 after injury (day 21 after transplantation) to the final evaluation day on day 126 after injury (day 84 after transplantation) (Figure 19).

[0260] <Example 15 Evaluation of the Efficacy of Cell Aggregates Containing Glial Progenitor Cells in a Chronic Spinal Cord Injury Model Rat> (Gait Pattern Evaluation) Gait pattern evaluation was performed at 12 weeks after transplantation using the DigiGait system (manufactured by Mouse Specific). The rats were made to walk on a treadmill (10 cm / sec), and the stride width and walking angle were measured. In the cell transplantation group, the stride width was significantly longer (**: p < 0.01) than that of the control group (Figure 20(A)). In addition, in the cell transplantation group, the angle formed by the limbs with respect to the direction of progression was significantly smaller (**: p < 0.01) than that of the control group, suggesting that both limbs were grounded in parallel and the walking pattern was closer to normal (Figure 20(B)).

[0261] <Example 16 Histological Evaluation of the Spinal Cord of a Chronic Spinal Cord Injury Model Rat> As a result of performing HE staining on the sagittal sections prepared by the method described in Example 13, no tumor-like structure was observed (Fig. 21(A)). Fluorescent immunostaining was also performed on adjacent sections using an anti-HNA antibody. As a result, engraftment of the transplanted cells was confirmed because HNA-positive cells were detected extensively (Fig. 21(B)).

[0262] For more detailed characterization of the transplanted cells, fluorescent immunostaining of human-specific markers and various markers was performed on sagittal sections by the method described in Example 12. To evaluate tumorigenicity, fluorescent immunostaining was performed using an anti-OCT3 / 4 antibody and an anti-HNA antibody. As a result, no HNA+ / OCT3 / 4+ co-positive cells were detected, suggesting that the transplanted cells were not tumorigenic (Fig. 22(A)). In addition, to examine the terminal differentiation ability of the transplanted cells, fluorescent immunostaining was performed using an anti-Hu (neuronal marker) antibody, an anti-GFAP (astrocyte marker) antibody, an anti-APC (oligodendrocyte marker) antibody, and an anti-HNA antibody. As a result, HNA+ / Hu+ co-positive cells (Fig. 22(B)), HNA+ / GFAP+ co-positive cells (Fig. 22(C)), and HNA+ / APC+ co-positive cells were detected (Fig. 22(D)). Therefore, it was suggested that the transplanted cells differentiated into neurons, astrocytes, and oligodendrocytes in the rat spinal cord.

[0263] <Example 17 Preparation of Uniform-Size Cell Aggregates> Cell aggregates containing glial progenitor cells prepared from the QHJI01s04 strain by the method of Condition 3 of Example 1 were dispersed into single cells using TrypLE Select at the 49th day of differentiation, and then seeded at 10,000 cells and 20,000 cells / well into a 96-well low-adhesion plate (product name: PrimeSurface® 96-slit well plate, manufactured by Sumitomo Bakelite Co., Ltd.), and cultured in a neural / glial growth medium at 37 °C under 5% CO2 / 20% O2. As a result, the formation of cell aggregates was confirmed from the day after seeding. The medium was replaced with half the volume once every three days, and the cells were cultured for 8 days and 14 days (Fig. 23). At the time points of 8 days and 14 days of culture (57th and 63rd days of differentiation), one cell aggregate was transferred to one well of a 96-well plate (manufactured by Corning Inc.) treated with 1% Matrigel basement membrane matrix growth factor reduced (manufactured by Corning Inc.) diluted with DMEM / F-12, and cultured in a maturation medium for 28 days to induce terminal differentiation.

[0264] At the time points of 28 days of terminal differentiation (85th and 91st days of differentiation), the cells were fixed with 4% PFA at room temperature for 25 minutes, washed three times with PBS, and then incubated with 10% goat serum (manufactured by Fujifilm Wako Pure Chemical Corporation) / PBS as a blocking solution at room temperature for 1 hour. Thereafter, the primary antibody solution 3 shown in Table 4 above was added, and the cells were incubated at 4 °C overnight for co-staining. Thereafter, the cells were washed three times with PBS, and the secondary antibody solution 3 corresponding to the primary antibody solution 3 was added, and the cells were incubated at room temperature for 1 hour. Thereafter, the cells were washed three times with PBS and stored at 4 °C.

[0265] Microscopic observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by Keyence Corporation). The fluorescence immunostaining is shown in Fig. 24. O4-positive oligodendrocytes, GFAP-positive astrocytes, and MAP2-positive neurons were detected under all the conditions examined (seeding cell numbers (10,000 cells and 20,000 cells) and culture days (8 days (A) and 14 days (B))).

[0266] <Example 18 Detection of Secretion Factors Derived from Cell Aggregates Containing Glial Progenitor Cells> Using the method of Condition 3 of Example 1, cell aggregates containing glial progenitor cells were prepared from the QHJI01s04 strain. At the 45th day of differentiation, the medium was changed to a neural-glial growth medium and cultured at 37°C under 5% CO2 / 20% O2. After 48 hours, the culture medium was collected, and the culture supernatant was recovered by centrifugation (1000 rpm, 5 minutes). The remaining cell pellet was dispersed into single cells using TrypLE Select, and the total cell number was counted. Also, to measure the cytokine concentration contained in the neural-glial growth medium, only the neural-glial growth medium was treated under the same conditions (5% CO2 / 20% O2 / 37°C) for 48 hours and then recovered.

[0267] Secreted factors in the culture supernatant were detected by two methods. As the first method, using the Bio-Plex Pro® Human Cytokine Screening 48-Plex Panel (manufactured by Bio-Rad), according to the manufacturer's instructions, the concentrations of 48 cytokines (FGF basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-1α, IL-2Rα, IL-3, IL-12(p40), IL-16, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, GRO-α, HGF, IFN-α2, LIF, MCP-3, IL-10, IL-12(p70), IL-13, IL-15, IL-17, IP-10, MCP-1(MCAF), MIG, β-NGF, SCF, SCGF-β, SDF-1α, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF, CTACK, MIF, TRAIL, IL-18, M-CSF, and TNF-β) were measured.

[0268] First, 50 μl of conjugated magnetic beads was added to each well of a 96-well plate and washed twice with the Wash Buffer provided in the kit. The washing process was carried out using an automated magnetic washing system (Bio-Plex ProII Wash Station, manufactured by Bio-Rad). Next, 50 μl each of an 8-step 4-fold dilution series (n = 2) prepared using the Standard provided in the kit and samples (culture supernatants and medium only, n = 3) were added to each well, and the reaction was carried out at room temperature for 30 minutes with shaking at 850 rpm in the dark. Thereafter, it was washed three times with Wash Buffer. Next, 25 μl each of the Detection Antibodies provided in the kit was added to each well, and the reaction was carried out at room temperature for 30 minutes with shaking at 850 rpm in the dark. Thereafter, it was washed three times with Wash Buffer. Next, 50 μl each of the Streptavidin-Phycoerythrin provided in the kit was added to each well, and the reaction was carried out at room temperature for 10 minutes with shaking at 850 rpm in the dark. Thereafter, it was washed three times with Wash Buffer. 125 μl each of the Assay Buffer provided in the kit was added to each well, and the fluorescence value was measured using a Bio-Plex 200 system (manufactured by Bio-Rad).

[0269] From the measurement data of the standard dilution series of each cytokine, a standard curve was created by 5-parameter logistic curve regression using Bio-Plex Manager software ver 6.1 (Bio-Rad), and the cytokine concentration in each sample was quantified. Thereafter, by subtracting the measurement data of the medium only from the measurement data of the culture supernatant as the background, the cytokine concentration secreted into the culture supernatant was calculated. The top 10 concentrations of cytokines detected in the culture supernatant and the secretion amount per cell are shown in Table 8.

Table 8

[0270] MIF, MCP-1, IL-8, and GRO-α have been reported as secretory factors derived from astrocytes (Non-Patent Document 9). In addition, SCF, HGF, and MIF promote the proliferation and differentiation of neural progenitor cells (Non-Patent Documents 10 to 12), and M-CSF plays an important role in the survival and differentiation of oligodendrocyte progenitor cells (Non-Patent Document 13). Therefore, it was found that cytokines contributing to neurogenesis are secreted from cell aggregates containing glial progenitor cells.

[0271] As a second method, the concentration of SPARCL1 in the culture supernatant was measured using Human SPARC-like 1 / SPARCL1 DuoSet ELISA (manufactured by R&D Systems) and DuoSet Ancillary Reagent Kit 2 (manufactured by R&D Systems).

[0272] First, according to the manufacturer's instructions, 100 μl of Human SPARC-like 1 Capture Antibody was added to each well of a 96-well plate and adsorbed overnight at room temperature. Then, it was washed three times with the Wash Buffer provided in the kit. Next, 300 μl of the Reagent Diluent provided in the kit was added to each well and reacted at room temperature for 80 minutes. Then, it was washed three times with Wash Buffer. Next, 100 μl each of an 11-step two-fold dilution series (n = 2) prepared using the Standard provided in the kit and samples (culture supernatant and medium only, n = 3) was added to each well and reacted at room temperature for 2 hours in the dark. Then, it was washed three times with Wash Buffer. Next, 100 μl of Human SPARC-like 1 Detection Antibody provided in the kit was added to each well and reacted at room temperature for 2 hours in the dark. Then, it was washed three times with Wash Buffer. Next, 100 μl of Streptavidin-Horseradish peroxidase provided in the kit was added to each well and reacted at room temperature for 20 minutes in the dark. Then, it was washed three times with Wash Buffer. Next, 100 μl of Substrate Solution (a 1:1 mixture of Color Reagent A and Color Reagent B provided in the kit) was added to each well and reacted at room temperature for 20 minutes in the dark. Subsequently, 50 μl of Stop Solution provided in the kit was added to each well to stop the reaction, and immediately the absorbance was measured using a plate reader Enspire (manufactured by PerkinElmer), and the value obtained by subtracting the absorbance at 540 nm from the absorbance at 450 nm was calculated.

[0273] From the measurement data of the Standard dilution series, a calibration curve was created from a plot on a log-log scale using Excel (manufactured by Microsoft), and the SPARCL1 concentration in each sample was quantified. From the measurement data of the culture supernatant, the SPARCL1 concentration secreted into the culture supernatant was calculated by subtracting the measurement data of the medium only as the background. Also, the amount of SPARCL1 secreted per cell was calculated (Table 9).

Table 9

[0274] As a result of the analysis, it was revealed that a high concentration of SPARCL1 was present in the culture supernatant of cell aggregates containing glial progenitor cells. Since SPARCL1 is a secretory factor derived from astrocytes and is known to control synapse formation (Non-Patent Document 14), it is considered to be valid as a marker detected in the culture supernatant of glial progenitor cells. Therefore, SPARCL1 is useful as a marker for confirming that the target cells have been obtained in the manufacturing process, either alone or in combination with other markers.

Claims

1. A method for producing a cell aggregate containing glial progenitor cells, comprising the following steps: (1) Using a culture vessel having a plurality of pores of uniform shape, in an embryoid body formation medium containing one or more SMAD signaling inhibitors and one or more Wnt signaling activators, in the absence of feeder cells, pluripotent stem cells are suspended and cultured for 5 to 10 days to form cell aggregates; (2) Suspending and culturing the cell aggregates obtained in (1) in an embryoid body formation medium containing retinoic acid for 4 to 11 days; (3) Suspending and culturing the cell aggregates obtained in (2) in an embryoid body formation medium containing retinoic acid and one or more SHH signaling activators or a nerve / glia proliferation medium; and (4) Suspending and culturing the cell aggregates obtained in (3) in a nerve / glia proliferation medium containing no retinoic acid and one or more SHH signaling activators and further comprising the following step: (5) Suspending and culturing the cell aggregates obtained in (4) in a nerve / glia proliferation medium containing neither retinoic acid nor an SHH signaling activator The production method may include.

2. In the step of (1), compared with the start of the step of (1), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA among SOX1, PAX6, HES4, and HES5 has increased by 100 times or more; 2) Obtaining cell aggregates in which the expression level of OCT3 / 4 RNA has decreased to 1 / 200 or less; and 3) Obtaining cell aggregates in which the expression level of NANOG RNA has decreased to 1 / 400 or less The production method according to claim 1, wherein the step of (2) is started after the step of (1) is continued until at least one of the above is satisfied.

3. In the step of (2), compared with the start of the step of (2), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA among ASCL1, DCX, HEY1, ZBTB20, βIII tubulin, ELAVL3, and SLIT1 has increased by 5 times or more; and 2) Obtaining cell aggregates in which the expression level of at least one RNA among HOXB3, HOXA4, HOXB4, HOXB6, and HOXB8 has increased by 5 times or more The production method according to claim 1 or 2, wherein the step of (3) is started after the step of (2) is continued until at least one of the above is satisfied.

4. The production method according to any one of claims 1 to 3, wherein in the steps (1) and (2), the oxygen concentration is 3% to 10%.

5. In the step (3), compared with the start of the step (3), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA among HEY2, NKX6.2, and NKX2.2 has increased by 5 times or more, and 2) Obtaining cell aggregates in which the expression level of the RNA of OLIG1 and / or OLIG2 has increased by 10 times or more The production method according to any one of claims 1 to 4, wherein the step (4) is started after the step (3) is continued until at least one of the above is satisfied.

6. The production method according to any one of claims 1 to 5, wherein the step (3) is carried out for 4 days to 11 days.

7. In the step (4), compared with the start of the step (4), the following conditions: 1) Obtaining cell aggregates in which the expression level of at least one RNA among NFIA, NFIB, SLC1A3, S100B, and FABP7 has increased by 10 times or more, and 2) Obtaining cell aggregates in which the expression level of the RNA of PAX6 has decreased to 5 times or less The production method according to any one of claims 1 to 6, wherein the step (5) is started after the step (4) is continued until at least one of the above is satisfied.

8. The production method according to any one of claims 1 to 7, wherein the step (4) is carried out for 4 days or more.

9. In the step (4), after dispersing the cell aggregates obtained in the step (3) at the start of the step, the dispersed cells are suspended and cultured to form cell aggregates again. The production method according to any one of claims 1 to 8.

10. The method includes the step (5), and in the step (5), after dispersing the cell aggregates obtained in the step (4) at the start of the step, the dispersed cells are suspended and cultured for 5 days to 100 days to form cell aggregates again. The production method according to any one of claims 1 to 9.

11. The method includes the step (5), and in the step (5), the step (5) is continued until one or more markers selected from O4 antigen, NG2, OLIG2, PDGFRα, SOX10, Spon1, FAM181B, TIMP4, SOX6, GRIK3, LHFP L3, KLF9, A2B5 antigen, CNP, and PLP are expressed. The production method according to any one of claims 1 to 10.

12. The method according to any one of claims 1 to 10, wherein the method includes the step (5), and in the step (5), the step (5) is continued until one or more proteins selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL are detected in the medium for culturing the cell aggregates.

13. The production method according to any one of claims 1 to 12, wherein the SMAD signal transduction inhibitor is a TGFβ inhibitor and a BMP inhibitor.

14. The production method according to claim 13, wherein the TGFβ inhibitor is one or more selected from the group consisting of SB431542, A83-01, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LY580276, Galunisertib (LY2157299), LY3200882, SB525334, GW788388, RepSox, and Left-1.

15. The production method according to claim 13 or 14, wherein the BMP inhibitor is one or more selected from the group consisting of Noggin, LDN-193189, LDN-212854, Dorsomorphin, K02288, Chordin, and Follistatin.

16. The production method according to any one of claims 1 to 15, wherein the Wnt signal transduction activator is one or more selected from the group consisting of a GSK3β inhibitor, Wnt3a, a Wnt agonist, Dkk, and R-Spondin.

17. The production method according to any one of claims 1 to 15, wherein the Wnt signal transduction activator is one or more selected from the group consisting of CHIR99021, BIO, Kenpaullone, SB216763, and L803-mts.

18. The production method according to any one of claims 1 to 17, wherein the SHH signal transduction activator is one or more selected from the group consisting of Purmorphamine, SAG, SHH protein, and SHH fragment.

19. The production method according to any one of claims 1 to 18, wherein the pluripotent stem cells are induced pluripotent stem cells.

20. The production method according to any one of claims 1 to 18, wherein the pluripotent stem cells are human induced pluripotent stem cells.

21. The cell aggregates containing glial progenitor cells have the following characteristics: (a) comprising oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells, (b) expressing a spinal cord region marker, and (c) not containing feeder cells and components derived from heterogeneous cells derived from feeder cells The production method according to any one of claims 1 to 20, which has.

22. A method for producing a cell population containing oligodendrocytes, astrocytes, and neurons, comprising culturing a cell aggregate containing glial progenitor cells produced by the production method according to any one of claims 1 to 21 in a maturation medium for 5 to 60 days. A manufacturing method including steps.

23. The cell population containing oligodendrocytes, astrocytes, and neurons is (i) cells expressing one or more markers selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2, (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3, and (iii) cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2 The production method according to claim 22, which comprises.

24. The production method according to claim 22 or 23, wherein the maturation medium is a medium containing at least one of T3, NT-3, and LIF.

25. The production method according to claim 24, wherein the maturation medium further contains CNTF.

26. The following features: (a) comprising oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells, (b) containing cells expressing a spinal cord region marker, (c) not containing feeder cells and components derived from heterogeneous cells derived from feeder cells, and (d) having the ability to differentiate into a cell population containing oligodendrocytes, astrocytes, and neurons A cell aggregate containing glial cell progenitor cells, which has, and further contains cells expressing one or more markers selected from the group consisting of NKX2.1, NKX2.2, NKX6.1, and NKX6.

2.

27. The cell aggregate according to claim 26, wherein the spinal cord region marker is one or more markers selected from the group consisting of HOXB3, HOXB4, HOXB6, and HOXD8.

28. The following features: comprising cells that express one or more markers selected from the group consisting of NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, ZBTB20, SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4; comprising cells that express one or more markers selected from the group consisting of OLIG2, PDGFRα, SOX10, SPO1N1, FAM181B, TIMP4, SOX6, GRIK3, LHFP13, KLF9, A2B5 antigen, CNP, and PLP; comprising cells that express one or more markers selected from the group consisting of DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3; comprising cells that express one or more markers selected from the group consisting of SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3, and ASCL1; and (V) (i) cells that express one or more markers selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2, (ii) cells that express one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3, and (iii) cells that express one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2 having the ability to differentiate into a cell population comprising The cell aggregate according to claim 26 or 27, having the above.

29. Furthermore, the cell aggregate according to any one of claims 26 to 28, comprising cells that express one or more markers selected from the group consisting of C1ORF61 and SERPINE2.

30. Furthermore, the cell aggregate according to any one of claims 26 to 29, comprising cells that express or secrete one or more markers selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL.

31. A pharmaceutical composition comprising the cell aggregate according to any one of claims 26 to 30 as an active ingredient.

32. The pharmaceutical composition according to claim 31, for the treatment of demyelinating diseases and diseases based on or associated with glial cell disorders.

33. The pharmaceutical composition according to claim 32, wherein the demyelinating disease and the disease based on or accompanied by glial cell disorder is spinal cord injury in the acute, subacute or chronic phase.

34. The cell aggregate according to any one of claims 26 to 30 for the treatment of a demyelinating disease and a disease based on or accompanied by glial cell disorder.

35. A method for evaluating the toxicity or efficacy of a test substance, comprising contacting the test substance with the cell aggregate according to any one of claims 26 to 30 and detecting or quantifying the effect of the test substance on the cell aggregate.

36. A method for determining whether a cell aggregate containing glial progenitor cells is suitable for transplantation, using whether C1ORF61 is expressed as an indicator.

37. A method for identifying glial progenitor cells or neural stem cells with high differentiation potential into glia, comprising detecting mRNA of C1ORF61, a protein encoded by the C1ORF61 gene, and fragments thereof.

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