Method for preparing cell aggregates containing glial precursor cells

Abstract

The present invention relates to a method for preparing cell aggregates containing glial precursor cells, comprising the following steps: step (1), suspending and culturing pluripotent stem cells for 5 to 10 days 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, thereby forming cell aggregates; step (2), suspending and culturing the cell aggregates obtained in step (1) in an embryoid body formation medium containing retinoic acid; step (3), suspending and culturing the cell aggregates obtained in step (2) in an embryoid body formation medium or a neuroglia proliferation medium containing retinoic acid and one or more SHH signaling activators; and step (4), suspending and culturing the cell aggregates obtained in step (3) in a neuroglia proliferation medium that does not contain retinoic acid and contains one or more SHH signaling activators.

Description

Technical Field

[0001] The present invention relates to a cell aggregate containing glial precursor cells derived from pluripotent stem cells, and a preparation method thereof. Background Art

[0002] Demyelinating diseases including spinal cord injury and diseases based on damaged glial cells rarely have spontaneous cures, and most cases deteriorate due to not only the shedding of glial cells that form myelin, but also axonal degeneration and cell body degeneration. In particular, for spinal cord injury, although recovery effects can be observed in some patients, there is no radical treatment that regenerates lost nerve axons and glial cells, and the unmet medical needs of the disease are high. Cell supplementation through transplantation is considered to be a promising treatment strategy based on nerve regeneration and myelin regeneration.

[0003] A large number of research results for realizing the treatment of transplanting neural progenitor cells have been reported, and in recent years, it is known that there are methods (such as non-patent literature 1 and 2) for inducing neural progenitor cells by differentiation of pluripotent stem cells. In addition, as a culture method for nervous system cells, it is known that there are methods (such as non-patent literature 3) for forming embryoid bodies in serum-free culture medium for suspension culture.

[0004] Myelin loss is prominent in spinal cord injury, so transplantation of cells containing a large number of oligodendrocytes among glial cells is considered important for its treatment (Non-Patent Documents 4, 6, and 7). However, it has not been clarified to date what type of cells should be present to show a therapeutic effect.

[0005] In addition, for the practicality of cell transplantation, undifferentiated pluripotent stem cells must not remain to prevent the cells after transplantation from evolving into tumors, and it is expected that no xenogeneic cell-derived components are contained to avoid mixing with infectious agents. However, the cell aggregates (non-patent literature 4,5 and patent documentation 1) comprising oligodendrocyte precursor cells reported so far are prepared in the presence of feeder cells such as mouse cells, so feeder cells or xenogeneic cell-derived components derived from feeder cells may be contained. Therefore, it is necessary to establish a preparation method that is more suitable for clinical application and may not contain xenogeneic cell-derived components.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application No. 2017-524340

[0009] Non-patent literature

[0010] Non-patent document 1: Fukusumi H et al., Stem Cells International, Volume 2016, Article ID 7235757 (2016)

[0011] Non-patent document 2: Sugai K et al., Molecular Brain, 9: 85 (2016)

[0012] Non-patent document 3: Eiraku M et al., Cell Stem Cell 3, 519-532 (2008)

[0013] Non-patent document 4: Douvaras P et al., Nature Protocols 10, 1143-1154 (2015)

[0014] Non-patent document 5: Numasawa-Kuroiwa Y et al., Stem Cell Reports, 2: 648-661 (2014)

[0015] Non-patent document 6: Kawabata S et al., Stem Cell Reports, 6: 1-8 (2016)

[0016] Non-patent document 7: Yasuda A et al., Stem Cells 29, 1983-1994. (2011)

[0017] Non-patent document 8: Nori et al., Stem Cell Reports, 4: 360-73. (2015)

[0018] Non-patent document 9: Choi SS et al., PLoS ONE 9(4)(2014):e92325

[0019] Non-patent literature 10: Kunlin Jin et al., J Clin Invest. 2002; 110(3): 311-319

[0020] Non-patent literature 11: Thorsten R Doeppner et al., J Cereb Blood Flow Metab 2011; 31: 1251-1262

[0021] Non-patent document 12: Shigeki Ohta et al., Journal of Cell Science 2012 125:3210-3220

[0022] Non-patent document 13: Vincent Pons et al., Front. Cell. Neurosci. 2018 12:499

[0023] Non-patent literature 14: Jeff A Stogsdill et al., Current Opinion in Neurobiology 2017, 42: 1-8 Summary of the invention

[0024] Problems to be solved by the invention

[0025] An object of the present invention is to provide a cell aggregate useful for regenerative medicine that does not contain xenogeneic cell-derived components and has a high content of glial progenitor cells, and a method for producing a cell aggregate containing glial progenitor cells that is unlikely to contain xenogeneic cell-derived components.

[0026] Means for solving problems

[0027] The inventors of the present application focused on reducing the myelin-neuron-astrocyte interaction in the lesion, and believed that it was important for the cells used for transplantation to include all of oligodendrocyte precursor cells, neural precursor cells and astrocyte precursor cells. They studied a differentiation induction method that could induce such cell aggregates from iPS cells and was more suitable for clinical applications.

[0028] The inventors of the present application first studied the method described in non-patent literature 5 as a previous differentiation induction method. In the method described in non-patent literature 5, iPS cells are maintained and cultured on mouse cells as feeder cells. That is, since the iPS cells are differentiated and induced using a culture medium containing xenogeneic cell-derived components, the resulting cell aggregates containing oligodendrocyte precursor cells may contain xenogeneic cell-derived components. On the other hand, in clinical applications, it is desirable to exclude xenogeneic cell-derived components as much as possible. Therefore, the inventors of the present application have conducted careful research in order to develop a method for inducing cell aggregates containing oligodendrocyte precursor cells by differentiation induction of iPS cells that are independent of feeder cells (i.e., without a feeder layer) using a culture medium that does not contain xenogeneic cell-derived components.

[0029] The inventors of the present application have attempted differentiation induction using feeder-free iPS cells cultured in an undifferentiated maintenance medium without xenogeneic cell-derived components and without feeder cells. As a result, depending on the cell line of the iPS cells, embryoid bodies (hereinafter sometimes abbreviated as "EB") may not be formed, and it was found that the probability of finally inducing cell aggregates that do not have the ability to differentiate into oligodendrocytes is high, suggesting that the differentiation induction method is unstable.

[0030] In this regard, the method of forming EBs was studied, and the results showed that by changing the culture method to a rapid aggregation serum-free suspension culture method (Serum-Free Embryonic Body quick; hereinafter sometimes abbreviated as "SFEBq method"), and using a combination of a specific culture medium and a differentiation inducer during differentiation induction, EBs can be stably formed, and cell aggregates (neurospheres) containing glial precursor cells with the ability to differentiate into oligodendrocytes can be obtained. It is also known that the cell aggregates show a therapeutic effect when transplanted into injury model animals. That is, the inventors of the present application have successfully established for the first time a method for preparing cell aggregates containing glial precursor cells that can be prepared in large quantities without xenogeneic cell-derived components.

[0031] That is, the present invention relates to the following contents. [1]

[0033] A preparation method, which is a preparation method for cell aggregates containing glial precursor cells, comprising the following steps:

[0034] Step (1), culturing the pluripotent stem cells in suspension for 5 to 10 days 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, thereby forming cell aggregates;

[0035] Step (2) suspending and culturing the cell aggregates obtained in step (1) in an embryoid body formation medium containing retinoic acid;

[0036] Step (3), suspending and culturing the cell aggregates obtained in step (2) in an embryoid body formation medium or a neuroglia proliferation medium containing retinoic acid and one or more SHH signaling activators; and

[0037] Step (4), suspending and culturing the cell aggregates obtained in step (3) in a neuroglia proliferation medium that does not contain retinoic acid and contains one or more SHH signaling activators,

[0038] The method may further include the following steps:

[0039] Step (5) is to culture the cell aggregates obtained in step (4) in suspension in a neuro-glial proliferation medium that does not contain both retinoic acid and an SHH signaling activator. [2]

[0041] The preparation method as described in [1], wherein in step (1), pluripotent stem cells are cultured using a culture container having a plurality of holes of uniform shape. [3]

[0043] The preparation method as described in [1] or [2], wherein in step (1), step (1) is continued until at least one of the following conditions is satisfied compared to the start of step (1), and then step (2) is started:

[0044] 1) obtaining cell aggregates in which the expression level of at least one RNA among SOX1, PAX6, HES4 and HES5 is increased by more than 100 times;

[0045] 2) obtaining cell aggregates in which the expression level of OCT3 / 4 RNA is reduced to less than 1 / 200; and

[0046] 3) Obtaining cell aggregates in which the expression level of NANOG RNA is reduced to less than 1 / 400.

[0047] [4] The preparation method according to any one of [1] to [3], wherein

[0048] In step (2), step (2) is continued until at least one of the following conditions is satisfied compared to when step (2) is started, and then step (3) is started:

[0049] 1) obtaining cell aggregates in which the expression level of at least one RNA selected from ASCL1, DCX, HEY1, ZBTB20, βIII tubulin, ELAVL3 and SLIT1 is increased by more than 5 times; and

[0050] 2) Obtaining cell aggregates in which the expression level of at least one RNA of HOXB3, HOXA4, HOXB4, HOXB6, and HOXB8 is increased by 5 times or more. [5]

[0052] The preparation method as described in any one of [1] to [4], wherein step (2) is performed for 4 to 11 days. [6]

[0054] The production method according to any one of [1] to [5], wherein in step (1) and step (2), the oxygen concentration is 3% to 10%. [7]

[0056] The preparation method as described in any one of [1] to [6], wherein in step (3), step (3) is continued until at least one of the following conditions is satisfied compared to when step (3) is started, and then step (4) is started:

[0057] 1) obtaining cell aggregates in which the expression level of at least one RNA among HEY2, NKX6.2 and NKX2.2 is increased by more than 5 times; and

[0058] 2) Obtaining cell aggregates in which the expression level of OLIG1 and / or OLIG2 RNA is increased by 10 times or more. [8]

[0060] The preparation method as described in any one of [1] to [7], wherein step (3) is performed for 4 to 11 days. [9]

[0062] The preparation method as described in any one of [1] to [8], wherein in step (4), step (4) is continued until at least one of the following conditions is satisfied compared to when step (4) is started, and then step (5) is started:

[0063] 1) obtaining cell aggregates in which the expression level of at least one RNA selected from NFIA, NFIB, SLC1A3, S100B and FABP7 is increased by more than 10 times; and

[0064] 2) Obtaining cell aggregates in which the expression level of PAX6 RNA is reduced to less than 5 times.

[10]

[0066] The preparation method according to any one of [1] to [9], wherein step (4) is performed for 4 days or more.

[11]

[0068] The production method as described in any one of [1] to

[10] , wherein in step (4), the cell aggregates obtained in step (3) are dispersed at the beginning of this step, and the dispersed cells are then suspended and cultured to form cell aggregates again.

[12]

[0070] A preparation method as described in any one of [1] to

[11] , wherein the method includes step (5), in which the cell aggregates obtained in step (4) are dispersed at the beginning of the step, and then the dispersed cells are suspended and cultured for 5 to 100 days, thereby forming cell aggregates again.

[13]

[0072] A preparation method as described in any one of [1] to

[12] , wherein the method comprises step (5), in which 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]

[0074] A preparation method as described in any one of [1] to

[12] , wherein the method includes step (5), in which 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 culture medium of the cultured cell aggregates.

[15]

[0076] The production method according to any one of [1] to

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

[16]

[0078] The preparation method as described in

[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]

[0080] The preparation method as described in

[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]

[0082] The production method according to any one of [1] to

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

[19]

[0084] 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, kemperolone, SB216763, and L803-mts.

[20]

[0086] 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 fragments. [twenty one]

[0088] The production method according to any one of [1] to

[20] , wherein the pluripotent stem cells are induced pluripotent stem cells. [twenty two]

[0090] The production method according to any one of [1] to

[20] , wherein the pluripotent stem cells are human induced pluripotent stem cells. [twenty three]

[0092] The preparation method according to any one of [1] to

[22] , wherein the cell aggregates comprising glial precursor cells have the following characteristics:

[0093] (a) comprising oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells;

[0094] (b) expressing spinal cord regional markers; and

[0095] (c) It does not contain feeder cells or feeder cell-derived components. [twenty four]

[0097] A preparation method for a cell population comprising oligodendrocytes, astrocytes and neurons, comprising the following steps: culturing a cell aggregate comprising glial precursor cells prepared by the preparation method described in any one of [1] to

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

[25]

[0099] The preparation method as described in

[24] , wherein the cell population comprising oligodendrocytes, astrocytes and neurons comprises:

[0100] (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;

[0101] (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3; and

[0102] (iii) Cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.

[26]

[0104] 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]

[0106] The preparation method as described in

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

[28]

[0108] A cell aggregate comprising glial precursor cells, obtained by the production method described in any one of [1] to

[23] .

[29]

[0110] Cell aggregates containing glial precursor cells with the following characteristics:

[0111] (a) comprising oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells;

[0112] (b) contains cells expressing spinal cord region markers;

[0113] (c) does not contain feeder cells and feeder cell-derived components; and

[0114] (d) having the ability to differentiate into a cell population including oligodendrocytes, astrocytes and neurons.

[30]

[0116] 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]

[0118] The cell aggregate according to

[29] or

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

[32]

[0120] The cell aggregate as described in any one of

[29] to

[31] has the following characteristics:

[0121] (I) comprising 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;

[0122] (II) comprising cells expressing 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;

[0123] (III) comprising cells expressing one or more markers selected from the group consisting of DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3;

[0124] (IV) comprising cells expressing one or more markers selected from the group consisting of SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3 and ASCL1;

[0125] as well as,

[0126] (V) having the ability to differentiate into a cell population comprising:

[0127] (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;

[0128] (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3; and

[0129] (iii) Cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.

[33]

[0131] The cell aggregate according to any one of

[29] to

[32] further has the following feature: (VI) it contains cells expressing one or more markers selected from the group consisting of C10RF61 and SERPINE2.

[34]

[0133] The cell aggregate as described in any one of

[29] to

[33] further comprises cells expressing or secreting 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]

[0135] A cell population comprising oligodendrocytes, astrocytes and neurons, obtained by the production method described in any one of

[24] to

[27] .

[36]

[0137] A pharmaceutical composition comprising the cell aggregate described in any one of

[28] to

[34] or the cell population described in

[35] as an active ingredient.

[37]

[0139] A method for treating demyelinating diseases and diseases based on or accompanied by glial cell damage, comprising transplanting an effective dose of the cell aggregate described in any one of

[28] to

[34] or the cell population described in

[35] into a subject in need of transplantation.

[38]

[0141] The treatment method as described in

[37] , wherein the demyelinating disease and the disease based on or accompanied by glial cell damage is an acute, subacute or chronic spinal cord injury.

[39]

[0143] The cell aggregate described in any one of

[28] to

[34] or the cell population described in

[35] , for use in the treatment of demyelinating diseases and diseases based on or accompanied by glial cell damage.

[40]

[0145] The cell aggregate or cell population as described in

[39] , wherein the demyelinating disease and the disease based on glial cell damage or the disease accompanied by glial cell damage is an acute, subacute or chronic spinal cord injury.

[41]

[0147] An evaluation method for evaluating the toxicity or efficacy of a test substance, comprising contacting the cell aggregate described in any one of

[28] to

[34] or the cell group described in

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

[42]

[0149] A method for determining whether a cell aggregate containing glial precursor cells is suitable for transplantation, using as an indicator the expression of one or more markers selected from the group consisting of C1ORF61 and SERPINE2.

[43]

[0151] A method for identifying glial precursor cells or neural stem cells with a high glial differentiation tropism, comprising detecting one or more genes selected from the group consisting of C1ORF61 and SERPINE2, proteins encoded by the genes, and fragments thereof.

[0152] Effects of the Invention

[0153] According to the preparation method of the cell aggregate comprising glial precursor cells of the present invention, it is possible to prepare a cell aggregate comprising glial precursor cells without feeder cells and heterologous cell-derived components derived from feeder cells. If the cell aggregate comprising glial precursor cells is further cultured, a cell group comprising oligodendrocytes, astrocytes and neural cells can be prepared. The cell aggregate or cell group obtained by the preparation method of the present invention is useful as a cell therapy transplantation material. BRIEF DESCRIPTION OF THE DRAWINGS

[0154] [ Figure 1A ] are bright field images showing cell aggregates containing glial precursor cells on the 35th and 49th days of differentiation in Example 1.

[0155] [ Figure 1B ] is a fluorescence microscope image showing the results of immunofluorescence staining of the cell aggregates containing glial precursor cells in Example 1 after 14 days of terminal differentiation.

[0156] [ Figure 2 ] is a diagram showing an example of an experimental protocol for a method of preparing cell aggregates containing glial precursor cells.

[0157] [ Figure 3A ] is a diagram showing the results of gene expression analysis during the differentiation induction process in Example 2.

[0158] [ Figure 3B ] is a diagram showing the results of gene expression analysis during the differentiation induction process in Example 2.

[0159] [ Figure 3C ] is a diagram showing the results of gene expression analysis during the differentiation induction process in Example 2.

[0160] [ Figure 3D ] is a diagram showing the results of gene expression analysis during the differentiation induction process in Example 2.

[0161] [ Figure 4A ] is a fluorescence microscope image showing the results of immunofluorescence staining of cell aggregates containing glial precursor cells in Example 3.

[0162] [ Figure 4B ] is a graph showing the analysis results of the ratio of OLIG2-positive cells and NFIA-positive cells relative to the total number of cells constituting the cell aggregates containing glial precursor cells in Example 3.

[0163] [ Figure 5] is a graph showing the ratio of the number of cells belonging to each cluster in Gliogenic NPC and Neurogenic NPC in Example 4.

[0164] [ Fig. 6A ] is a graph showing the distribution of the expression level of each gene per cell in Gliogenic NPC and Neurogenic NPC in Example 4.

[0165] [ Figure 6B ] is a graph showing the distribution of the expression level of each gene per cell in Gliogenic NPC and Neurogenic NPC in Example 4.

[0166] [ Fig. 7A ] is a fluorescence microscope image showing the results of immunofluorescence staining of the cell aggregates containing glial precursor cells in Example 5 after 31 days of terminal differentiation.

[0167] [ Figure 7B ] is a graph showing the proportions of O4-positive cells, GFAP-positive cells, and Tuj1-positive cells 31 days after terminal differentiation of the cell aggregates containing glial precursor cells in Example 5.

[0168] [ Figure 8 ] is a graph showing the relative values ​​of the expression levels of each gene before and after terminal differentiation of the cell aggregates containing glial precursor cells in Example 6.

[0169] [ Fig. 9 ] is a graph showing the results of hind limb motor function evaluation based on BMS score after transplanting cell aggregates containing glial precursor cells into subacute spinal cord injury model mice in Example 8.

[0170] [ Fig.10 ] is a graph showing the results of coordinated movement evaluation based on a rotarod test after transplanting cell aggregates containing glial precursor cells into subacute spinal cord injury model mice in Example 9.

[0171] [ Fig.11A ] is a graph showing the results of gait pattern evaluation based on stride length after transplanting cell aggregates containing glial precursor cells into subacute spinal cord injury model mice in Example 10.

[0172] [ Fig. 11B ] is a graph showing the results of gait pattern evaluation based on the angle of the limbs relative to the direction of travel after transplanting cell aggregates containing glial precursor cells into subacute spinal cord injury model mice in Example 10.

[0173] [ Fig.12] are diagrams showing the results of evaluating joint motion based on kinematic analysis after transplanting cell aggregates containing glial precursor cells into subacute spinal cord injury model mice in Example 11. (A) shows a representative example of the trajectory of the hind limbs from leaving the ground to touching the ground. (B) shows the changes in joint angles of the hip joint, knee joint, ankle joint, and toes during one walking cycle.

[0174] [ Fig.13 ] are fluorescence microscope images showing the observation results of tissue sections after transplanting cell aggregates containing glial precursor cells into subacute spinal cord injury model mice in Example 12. (A) is the result of immunofluorescence staining of sagittal sections using STEM121 antibody, (B) is the result of sagittal sections stained with HE, and (C) is the result of immunofluorescence staining using anti-HNA antibody for (1) cross-sectional sections 4 mm from the center of the injury to the head; (2) cross-sectional sections at the center of the injury; (3) cross-sectional sections 4 mm from the center of the injury to the tail.

[0175] [ Fig.14A ] is a fluorescence microscope image of a sagittal section in Example 12 in which fluorescent immunostaining was performed using human-specific markers and various markers.

[0176] [ Fig. 14B ] is a graph showing various positive cell rates among HNA-positive transplanted cells in sagittal sections in Example 12.

[0177] [ Fig.15 ] is a fluorescence microscope image showing the sagittal section immunofluorescence staining using Tuj1 antibody, anti-HNA antibody, anti-mouse specific Bassoon (Bsn) and anti-human synaptophysin (hSyn) antibodies in Example 12.

[0178] [ Fig.16A ] are the LFB staining images of 0.48 mm from the center of the injury to the rostral side (Rostral), the LFB staining image of the center of the injury (Epi-center), and the LFB staining image of 0.48 mm from the center of the injury to the caudal side (Caudal) in Example 12.

[0179] [ Fig. 16B ] is a diagram showing the results of quantification of the LFB-positive area of ​​the myelin tissue from the center of the injury to 0.96 mm on the cranial and caudal sides in the LFB-stained cross-sectional slice in Example 12.

[0180] [ Fig.17 ] is a fluorescence microscope image of a cross-sectional slice immunofluorescently stained with STEM121 antibody and anti-MBP antibody in Example 12.

[0181] [ Fig.18 ] is an electron microscope image of a cross-sectional slice of the STEM121 antibody reaction site detected by immunoelectron microscopy in Example 12. The arrows indicate the STEM121 antibody reaction site (black dots) detected by the secondary antibody labeled with colloidal gold particles. (A) and (B) are images taken in different fields of view.

[0182] [ Fig.19 ] is a graph showing the results of hind limb motor function evaluation based on the BBB score after transplanting cell aggregates containing glial precursor cells into chronic spinal cord injury model rats in Example 14.

[0183] [ Fig. 20 ] are graphs showing the results of gait pattern evaluation 12 weeks after transplantation of cell aggregates containing glial precursor cells into chronic spinal cord injury model rats in Example 15. (A) shows the stride length, and (B) shows the angle of the limb relative to the direction of travel.

[0184] [ Fig.21 ] shows the results of observation of tissue sections after transplantation of cell aggregates containing glial precursor cells into chronic spinal cord injury model rats in Example 16. (A) is the result of sagittal sections stained with HE; (B) is the result of sagittal sections stained with immunofluorescence using anti-HNA antibodies.

[0185] [ Fig. 22 ] shows the results of observation of tissue sections after transplanting cell aggregates containing glial precursor cells into chronic spinal cord injury model rats in Example 16. (A) is the result of immunofluorescence staining of sagittal sections using anti-HNA antibody and anti-OCT3 / 4 antibody; (B) is the result of immunofluorescence staining of sagittal sections using anti-HNA antibody and anti-Hu antibody; (C) is the result of immunofluorescence staining of sagittal sections using anti-HNA antibody and anti-GFAP antibody; (D) is the result of immunofluorescence staining of sagittal sections using anti-HNA antibody and anti-APC antibody.

[0186] [ Fig.23 ] are bright field images showing cell aggregates containing glial precursor cells on the 1st day, the 8th day, and the 14th day after cell inoculation in Example 17.

[0187] [ Fig.24 ] are fluorescent microscope images of the cell aggregates containing glial precursor cells in Example 17 after terminal differentiation for 28 days using O4 antibody, anti-GFAP antibody, and Tuj1 antibody for immunofluorescence staining. (A) is a cell aggregate that began to differentiate after 8 days of culture and never differentiated; (B) is a cell aggregate that began to differentiate after 14 days of culture and never differentiated. DETAILED DESCRIPTION

[0188] 〔definition〕

[0189] In this specification, "stem cell" refers to an undifferentiated cell that has the ability to differentiate and the ability to proliferate (especially the ability to self-replicate) while maintaining the differentiation ability. Stem cells include subgroups such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells according to their differentiation ability.

[0190] Pluripotent stem cells refer to stem cells that can be cultured in vitro and have the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissues (pluripotency). Pluripotent stem cells refer to stem cells that have the ability to differentiate into multiple but not all types of tissues and cells. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues and cells.

[0191] Pluripotent stem cells can be induced by fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, etc. As pluripotent stem cells, embryonic stem cells (ES cells: Embryonic Stem cell), EG cells (Embryonic Germ cell), induced pluripotent stem cells (iPS cells: induced Pluripotent Stem cell), etc. can be cited. Muse cells (Multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (Mesenchymal Stem Cell; MSC) and GS cells made from germ cells (e.g., testes) are also included in pluripotent stem cells.

[0192] Human embryonic stem cells were established in 1998 and are gradually used in regenerative medicine. Embryonic stem cells can be prepared by culturing the inner cell mass of the blastocyst stage on feeder cells or in a culture medium containing FGF2. For example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. describe methods for preparing embryonic stem cells. Embryonic stem cells can be obtained from a prescribed institution, or commercially available products can be purchased. For example, KhES-1, KhES-2, and KhES-3 as human embryonic stem cells can be obtained from the Institute of Regenerative Medicine, Kyoto University. Crx::Venus strain (derived from KhES-1) as human embryonic stem cells can be obtained from the National Institute of Physical and Chemical Research.

[0193] In the present specification, "induced pluripotent stem cells" are cells induced to have pluripotency by reprogramming somatic cells using known methods or the like.

[0194] Induced pluripotent stem cells were established by Yamanaka et al. using mouse cells in 2006 (Cell, 2006, 126 (4), pp. 663-676). In 2007, induced pluripotent stem cells were also established using human fibroblasts, which have the same pluripotency and self-replication ability as 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).

[0195] Specifically, induced pluripotent stem cells include cells that induce multipotent differentiation by reprogramming differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells through the expression of any combination of multiple genes selected from a reprogramming gene set including OCT3 / 4, SOX2, KLF4, MYC (c-MYC, N-MYC, L-MYC), GLIS1, NANOG, SALL4, LIN28, ESRRB, etc. 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).

[0196] In addition to methods of preparing induced pluripotent stem cells by direct reprogramming based on gene expression, induced pluripotent stem cells can also be obtained by induction from somatic cells by adding compounds or the like (Science, 2013, 341, pp. 651-654).

[0197] In addition, it is also possible to obtain a lineage of induced pluripotent stem cells, such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells, etc., which are established at Kyoto University. Human induced pluripotent cell lines can be obtained from Kyoto University and iPS Academia Japan, Inc. As a lineage of induced pluripotent stem cells, for example, QHJI01s04 cells established at Kyoto University can be obtained from Kyoto University.

[0198] In the present specification, the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0199] In the present specification, the pluripotent stem cells are pluripotent stem cells of mammals, preferably pluripotent stem cells of rodents (e.g., mice, rats) or primates (e.g., humans, monkeys), more preferably human pluripotent stem cells, and further preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0200] Pluripotent stem cells such as human iPS cells can be maintained and expanded in culture by methods known to those skilled in the art.

[0201] In this specification, "glial (Glia)" is also referred to as "glial cell (Glial cell)", "neuroglia cell", which is a general term for cells other than nerve cells constituting the nervous system, and it is estimated that there are more than 10 times of nerve cells in the human brain by cell number. As glial cells, astrocytes (Astrocyte), oligodendrocytes (Oligodendrocyte) etc. can be cited, which are present around nerve cells in nervous tissue and undertake various functions.

[0202] In this specification, "marker" refers to a substance present in a cell, and refers to a substance that can identify or distinguish the type or property of the cell based on its presence or amount. Specific examples of markers include mRNA, proteins and sugar chains encoded by the mRNA, and fragments thereof.

[0203] In the present specification, the term "differentiation marker" refers to a marker for detecting the degree (level) of differentiation of cells at a specific differentiation stage.

[0204] In the present specification, "glial precursor cells" refer to cells having the ability to differentiate into glial cells, and examples thereof include astrocyte precursor cells and oligodendrocyte precursor cells.

[0205] In this specification, "oligodendrocyte" is a kind of glial cell present in the central nervous system, by existing in a way to be wound around the axon of neuron (nerve cell), myelin (myelin sheath) is formed, and the effect of accelerating the signal conduction speed between nerve cells is assumed. Oligodendrocyte can be identified by the marker of specific expression in oligodendrocyte, as this marker, for example, PLP, GalC, APC, MBP, MAG, MOG, GST-π, SOX10, O4 (O-4) antigen, NG2 (CSPG4) and CNP etc. are known, for example, by using gene amplification method, in situ hybridization, immunohistological method, flow cytometry analysis etc. to analyze, it is possible to distinguish the cell expressing at least one of the above-mentioned mRNA, sugar chain or protein as oligodendrocyte. It should be noted that when the NG2 also expressed in astrocytes in the above-mentioned marker is used as an index, it is expected to be identified together with other markers.

[0206] In this specification, "oligodendrocyte precursor cell" refers to a cell with the ability to differentiate into an oligodendrocyte. The presence of oligodendrocyte precursor cells can be identified by a marker known to be significantly expressed in oligodendrocyte precursor cells. As markers of oligodendrocyte precursor cells, for example, PDGFRα and OLIG2 are known, for example, by using gene amplification methods, in situ hybridization, immunohistochemical staining methods, etc. for analysis, cells expressing at least one marker in the above-mentioned mRNA or protein can be identified as oligodendrocyte precursor cells. It should be noted that immature oligodendrocytes that have not formed myelin are also included in oligodendrocyte precursor cells.

[0207] In this specification, " astrocyte " is a kind of glial cell present in the central nervous system, and it is believed that it helps to activate nerve conduction by supporting the function of neurons in structure, regulating neurotransmitters, energy and extracellular ion concentration. In addition, it is believed that it supplies the above-mentioned oligodendrocytes with substances that promote the formation of myelin, and is a cell that bears important functions together with neurons and oligodendrocytes in nervous tissue. It can be identified by the markers specifically expressed in astrocytes, as the markers, for example, GFAP, S100B, AQP4, NG2 and SLC1A3 (also referred to as GLAST or EAAT1) etc. are known, for example, by using immunohistological methods to analyze, it is possible to distinguish the cells expressing at least one of the above-mentioned mRNA, sugar chain or protein as astrocytes. It should be noted that when the NG2 known to be also expressed in oligodendrocytes in the above-mentioned markers is used as an index, it is expected to be identified together with other markers.

[0208] In this specification, "astrocyte precursor cell" refers to a cell having the ability to differentiate into astrocytes. The presence of astrocyte precursor cells can be identified by markers known to be significantly expressed in astrocyte precursor cells. As markers of astrocyte precursor cells, for example, NFIA, NFIB, SOX9, HEY1, HEY2, FABP7 and ZBTB20 can be cited.

[0209] In the process of differentiation from pluripotent stem cells to oligodendrocytes, first, pluripotent stem cells differentiate into neural stem cells (Neural stem cells), and then differentiate into oligodendrocyte precursor cells and oligodendrocytes in sequence. Neural stem cells have multipotency and can differentiate into any one of neural precursor cells, astrocyte precursor cells, and oligodendrocyte precursor cells. When differentiating into oligodendrocyte precursor cells, they are destined to differentiate into oligodendrocytes. In addition, when differentiating into neural precursor cells, they are destined to differentiate into neurons, and when differentiating into astrocyte precursor cells, they are destined to differentiate into astrocytes.

[0210] Here, the determination of differentiation into astrocytes can be confirmed by whether or not one or more of the following markers are expressed: NFIA, NFIB, SOX9, HEY1, HEY2, ZBTB20, SLC1A3, and S100B; preferably, by whether or not at least one of SOX9, HEY1, HEY2, ZBTB20, SLC1A3, and S100B is expressed. Here, S100B is also referred to as S100β.

[0211] In the present specification, a nerve cell refers to a nerve unit composed of a cell body, dendrites, and axons, and is also called a neuron. A nerve cell can be identified by a marker that is significantly expressed, and examples of such a marker include βIII tubulin, MAP2, and ELAVL3.

[0212] A nerve cell is a cell that has the function of transmitting stimulation from other nerve cells or cells that sense stimulation to other nerve cells, muscles or gland cells. Nerve cells can be classified into serotonergic nerves, motor nerves, etc. according to the difference in neurotransmitters produced by the nerve cells, but in this specification, the type of neurotransmitter is not particularly limited.

[0213] In this specification, "neural precursor cells" refer to cells that have the ability to differentiate into neural cells and are destined to differentiate into neural cells, and can be identified by neural cell adhesion molecule (NCAM), polysialylated NCAM, neural markers (DCX, βIII tubulin, etc.), etc.

[0214] In this specification, "neural stem cells" refer to cells that have the ability to differentiate into both neural progenitor cells and glial progenitor cells including astrocytes or oligodendrocytes. They can be identified by markers of primitive neural ectoderm and neural stem cells such as intermediate filament proteins (nestin, vimentin, etc.), transcription factors SOX1, PAX6, etc.

[0215] In the present specification, the "spinal cord region marker" refers to a marker expressed along with differentiation into the spinal cord during the developmental stage, and specific examples thereof include genes such as HOXB3, HOXB4, HOXB6, and HOXD8, or proteins encoded by the genes.

[0216] In the present specification, the "ventral region marker" refers to a marker expressed along with differentiation into the ventral region during the developmental stage, and specific examples thereof include genes such as NKX2.1, NKX2.2, NKX6.1, and NKX6.2, or proteins encoded by the genes.

[0217] In this specification, "cell aggregate" (Cell Aggregate) is not particularly limited as long as it is a three-dimensional structure formed by the adhesion of multiple cells to each other, for example, a mass formed by the aggregation of cells dispersed in a medium such as a culture medium, or a mass of cells formed by cell division, etc. Cell aggregates also include the formation of specific tissues. In addition, embryoid bodies are also included in cell aggregates. In this specification, a "cell group" can be a cell aggregate (spherical cell group) or a layered cell group.

[0218] In this specification, "culture medium" is not particularly limited as long as it is a culture medium commonly used in the culture of animal cells, as long as it can maintain the life of animal cells, and preferably provides a culture medium that can proliferate in the target cell environment. The culture medium can be prepared by itself, or it can be purchased and used in a commercially available culture medium. In this specification, "embryoid formation culture medium", "nerve·glial proliferation culture medium" and "maturation culture medium" use the above-mentioned culture medium as a basal culture medium, and can be prepared by adding factors that meet the respective purposes, etc.

[0219] From the perspective of preparing cell aggregates suitable for transplantation, it is preferred that the culture medium used in the present invention is a serum-free culture medium. "Serum-free culture medium" in the present invention refers to a culture medium that does not contain unadjusted or unpurified serum. In this specification, even if a culture medium is mixed with purified blood-derived components or animal tissue-derived components (such as growth factors), as long as it does not contain unadjusted or unpurified serum, it is included in the serum-free culture medium. Serum-free culture medium may also appropriately contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, etc.

[0220] The culture medium used in the present invention may also contain a serum substitute. As a serum substitute, for example, a substance appropriately containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'thiolglycerol (3'thiolglycerol) or their equivalents can be cited. The serum substitute can be prepared, for example, by the method described in WO98 / 30679. As a serum substitute, a commercially available product can also be used. As the commercially available serum substitute, for example, Knockout Serum Replacement (manufactured by Thermo Fisher Scientific; sometimes referred to as KSR below), "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), ITS supplement (manufactured by Thermo Fisher Scientific), preferably B27 supplement.

[0221] The culture medium used in the present invention is preferably a Xeno-free culture medium. Here, "Xeno-free" refers to a condition in which components (xeno-derived components, also called xeno factors) derived from species different from the species of the cells to be cultured have been excluded. A portion of the serum-free culture medium may be a Xeno-free culture medium.

[0222] In this specification, feeder cells refer to cells other than the stem cells that are coexisted when culturing stem cells such as pluripotent stem cells. As feeder cells, for example, mouse fibroblasts (MEF, etc.), human fibroblasts, SNL cells, STO cells, etc. can be enumerated. Feeder cells can also be feeder cells treated with proliferation inhibition. Here, as a proliferation inhibition treatment, a proliferation inhibitor (such as mitomycin C) treatment or a treatment based on gamma ray irradiation or ultraviolet irradiation, etc. can be enumerated.

[0223] In the present specification, the component derived from the feeder cell refers to a component that is not contained in the stem cell but is contained in the feeder cell or is secreted by the feeder cell.

[0224] In this specification, "xenogeneic cells" refer to cells derived from a species different from the cells to be cultured, and are used synonymously with "xenogeneic animal cells." "Xenogeneic cell-derived components" are components derived from the xenogeneic cells and are components not contained in the cells to be cultured.

[0225] The xenogeneic cell-derived component derived from feeder cells refers to a component derived from the feeder cells and not contained in the cells to be cultured, when the feeder cells are derived from a species different from that of the cells to be cultured.

[0226] Specific components include at least protein components that differ in base sequence, amino acid sequence, etc., sugars, lipids, and the like.

[0227] In the present invention, "suspension culture" refers to keeping cells alive in a suspended state in a culture medium. In this specification, cells are suspended in a state of a single cell or a mass of multiple cells (cell aggregate or cell group). Cells cultured in suspension will proliferate and / or differentiate.

[0228] [Method for preparing cell aggregates containing glial precursor cells]

[0229] As one embodiment of the method for producing a cell aggregate containing glial precursor cells of the present invention, there can be mentioned a production method comprising the following steps:

[0230] Step (1), culturing pluripotent stem cells in suspension for 5 to 10 days 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, thereby forming cell aggregates;

[0231] Step (2), suspending and culturing the cell aggregates obtained in step (1) in an embryoid body formation medium containing retinoic acid;

[0232] Step (3), suspending and culturing the cell aggregates obtained in step (2) in an embryoid body formation medium or a neuroglia proliferation medium containing retinoic acid and one or more SHH signaling activators; and

[0233] Step (4), suspending and culturing the cell aggregates obtained in step (3) in a neuroglia proliferation medium that does not contain retinoic acid and contains one or more SHH signaling activators,

[0234] The preparation method may further include the following steps:

[0235] Step (5) is to culture the cell aggregates obtained in step (4) in suspension in a neuro-glial proliferation medium that does not contain both retinoic acid and an SHH signaling activator.

[0236] <Step (1)>

[0237] In step (1), pluripotent stem cells are cultured to form embryoid bodies. "Embryoid bodies (Embryoid Body; EB)" refers to a three-dimensional cell aggregate formed by suspending and culturing pluripotent stem cells. In order to make pluripotent stem cells differentiate into target cells efficiently, the embryoid bodies formed midway in the differentiation preferably contain a large number of cells with the ability to differentiate into the germ layers (ectoderm, mesoderm or endoderm) containing the target cells. For example, in the case where the pluripotent stem cells are intended to differentiate into neural cells and glial cells, the embryoid bodies formed midway in the differentiation preferably contain a large number of cells with the ability to differentiate into ectoderm. That is, in this specification, the embryoid bodies are preferably embryoid bodies with the ability to differentiate into ectoderm. Of course, the differentiation of pluripotent stem cells will also begin in the process of embryoid body formation, and the embryoid bodies may also include cells that are classified into ectoderm.

[0238] In step (1), pluripotent stem cells are cultured using an embryoid body formation medium containing differentiation inducing factors such as one or more SMAD signaling inhibitors and one or more Wnt signaling activators at effective concentrations. The medium used in step (1) can be prepared by mixing the above-mentioned differentiation inducing factors at effective concentrations in the embryoid body formation medium.

[0239] In this specification, the SMAD signaling inhibitor is not particularly limited as long as it can inhibit the signaling mediated by SMAD, and more than one SMAD signaling inhibitor can also be used in combination appropriately. As a SMAD signaling inhibitor, for example, an inhibitor of the TGFβ superfamily that inhibits SMAD signaling by inhibiting the phosphorylation of SMAD upstream of the SMAD signal can be cited, specifically, TGFβ inhibitors and BMP inhibitors can be cited. With regard to SMAD signaling inhibitors, it is preferred to use both TGFβ inhibitors and BMP inhibitors, for example, more than one TGFβ inhibitor (preferably one TGFβ inhibitor) and more than one BMP inhibitor (preferably one BMP inhibitor) can also be used in combination appropriately.

[0240] In this specification, TGFβ inhibitors are not particularly limited as long as they can inhibit signal transduction mediated by TGFβ, and can be any of nucleic acids, proteins, and low molecular organic compounds. As TGFβ inhibitors, for example, substances that directly act on TGFβ (such as proteins, antibodies, aptamers, etc.), substances that inhibit the expression of genes encoding TGFβ (such as antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ receptors to TGFβ, and substances that inhibit physiological activity caused by signal transduction based on TGFβ receptors (such as inhibitors of TGFβ receptors, etc.). As TGFβ inhibitors, substances that inhibit binding to the ALK family as a receptor, or substances that inhibit the phosphorylation of SMAD based on the ALK family can be cited.

[0241] In the present specification, specific examples of TGFβ inhibitors include SB431542, SB202190 (RK 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 (as NCBI accession number, examples include mouse: NM_010094, human: NM_020997), Lefty-2 (as NCBI accession number, examples include mouse: NM_177099, human: NM_003240 and NM_001172425), and their derivatives, etc., at least one TGFβ inhibitor can be used. As the TGFβ inhibitor, it is preferably one or more selected from the group consisting of SB431542 and A83-01, which are known inhibitors of TGFβ receptor (ALK5) and activin receptor (ALK4 / 7).

[0242] The concentration of the TGFβ inhibitor in the culture medium can be appropriately set within the range capable of inhibiting signal transduction mediated by TGFβ. For example, the concentration of SB431542 is not particularly limited as long as it is a concentration that can inhibit ALK5, for example, 100nM to 100μM, preferably 500nM to 30μM, and more preferably 1μM to 20μM. In the case of other TGFβ inhibitors, the concentration can be appropriately set to a concentration that can show ALK inhibitory activity or TGFβ inhibitory activity equivalent to the above-mentioned concentration of SB431542.

[0243] In this specification, BMP inhibitors are not particularly limited as long as they are inhibitors of the inhibition of BMP signaling (BMP signaling) involved in the combination of BMP (bone morphogenetic protein, BoneMorphogenetic Protein) and BMP receptors (type I or type II), and can be any of nucleic acids, proteins, and low molecular organic compounds. As BMP inhibitors, substances (such as proteins, antibodies, aptamers, etc.) that directly act on BMP, substances (such as antisense oligonucleotides, siRNA, etc.) that inhibit the expression of genes encoding BMP, substances with biological activity that inhibits the above-mentioned BMP signaling by inhibiting the combination of BMP and BMP receptors, substances that inhibit physiological activity caused by signaling based on BMP receptors (for example, inhibitors of BMP receptors, etc.), BMP type I receptor kinase inhibitors, etc.

[0244] Specific examples of substances having biological activity that inhibit the above-mentioned BMP signaling by inhibiting the binding of BMP and BMP receptors include Dorsomorphin, Noggin, and their derivatives. In addition, specific examples of BMP type I receptor kinase inhibitors include LDN-193189 (i.e., 4-(6-(4-(piperazine-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline) and its derivatives (e.g., LDN-212854). Other BMP inhibitors include K02288, chordogen, and follistatin.

[0245] The BMP inhibitor is preferably one or more selected from the group consisting of noggin, LDN-193189, Dorsomorphin, and K02288 (PLoS One. 2013; 8(4): e62721).

[0246] The concentration of the BMP inhibitor in the culture medium can be appropriately set within the range of being able to inhibit signal transduction mediated by BMP. For example, the concentration of LDN-193189 is not particularly limited as long as it is a concentration that can inhibit BMP type I receptor kinase, for example, 1nM to 10μM, preferably 10nM to 5μM, preferably 50nM to 3μM, and more preferably 50nM to 1μM. In the case of other BMP inhibitors, it can be appropriately set to a concentration that can show BMP type I receptor kinase inhibitory activity or BMP inhibitory activity equivalent to the above-mentioned concentration of LDN-193189.

[0247] 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 inhibitory protein), GSK3β (glycogen synthase kinase 3β) inhibitors, R-Spondin, and the like.

[0248] In the present specification, a GSK3β inhibitor is defined as a substance that inhibits the kinase activity of the 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-bromomethylketone compounds such as GSK-3β inhibitor VII, phosphorylated cell penetrating peptides such as CHIR99021 and L803-mts, and their derivatives.

[0249] In the present specification, the Wnt signal activator is preferably a GSK3β inhibitor, and is preferably one or more selected from the group consisting of CHIR99021, BIO, kemperolone, SB216763, and L803-mts, which are GSK3β inhibitors.

[0250] The concentration of the Wnt signal activator in the culture medium can be appropriately set within a range that can enhance the signal transduction mediated by Wnt3a. For example, the concentration of CHIR99021 is not particularly limited as long as it is a concentration that can inhibit GSK3β, and is, for example, 100nM to 100μM, preferably 500nM to 30μM, and more preferably 1μM to 10μM. In the case of other Wnt signal activators, it can be appropriately set to a concentration that can show GSK3β inhibitory activity equivalent to the above-mentioned concentration of CHIR99021 or the promotion of signal transduction mediated by Wnt3a.

[0251] In step (1), pluripotent stem cells are cultured in the absence of feeder cells. The absence of feeder cells is also referred to as a feeder-free layer, which refers to a state in which feeder cells are not present in the culture medium. Examples of culture conditions in the absence of feeder cells include culture conditions in which feeder cells such as fibroblasts, SNL cells, and STO cells are not added.

[0252] In this specification, "embryoid formation culture medium" is not particularly limited as long as it can be the culture medium used when preparing embryoid by suspension culture pluripotent stem cells. As embryoid formation culture medium, it can be suitable for forming embryoid and suitable for the feeder-free culture medium of the feeder-free culture of pluripotent stem cells. Embryoid formation culture medium can be prepared by adding intercalation factors such as serum substitutes and nutrient sources as required in basal medium.

[0253] As the basal medium, for example, there can be mentioned BME medium, BGJb medium, CMRL 1066 medium, GlasgowMEM (GMEM) medium, Improved MEM ZincOption medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F-12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, or mixed medium thereof, which can be used for culturing animal cells. These basal media contain carbon sources such as sugars and amino acids, vitamins, inorganic salts, and the like.

[0254] As embryoid body formation culture medium, for example, it is possible to use a culture medium obtained by removing a part or all of the factors (undifferentiated maintenance factors) required for maintaining undifferentiation from the culture medium (undifferentiated maintenance culture medium) for maintaining pluripotent stem cells in an undifferentiated state, or a culture medium obtained by reducing the concentration of a part or all of the undifferentiated maintenance factors described later to below the effective concentration. Such a culture medium, for example, can be prepared by coordinating in a manner comprising a component equal to the culture medium. Alternatively, for example, a culture medium in which nutrients such as serum substitutes and hormones are added in basal culture media such as DMEM can also be used. Alternatively, undifferentiated maintenance culture medium is sometimes sold in the form of a test kit that can be appropriately mixed with a variety of liquids, in which case, it is also possible to coordinate in a manner not to add a liquid containing undifferentiated maintenance factors, thus preparing embryoid body formation culture medium. For example, when it is Stem Fit AK03N (AJINOMOTO HEALTHY SUPPLY CO., INC. system), it is divided into A liquid, B liquid and C liquid, so the C liquid containing FGF2 can be removed, and only A liquid and B liquid are used to prepare embryoid body formation culture medium and use it.

[0255] When a medium based on an undifferentiation maintenance medium is used as an embryoid body formation medium, it has the advantage that the desired additive factor can be appropriately changed to the desired concentration at the beginning of step (1) without significantly changing the basic composition of the medium. As a result, there is an advantage that the cell culture environment does not change significantly.

[0256] Preferably, the embryoid body formation medium contains or does not contain components that inhibit or antagonize the action of differentiation-inducing factors such as SMAD signaling inhibitors and Wnt signaling activators at a concentration that does not affect step (1), for example, does not have any effect.

[0257] As a specific example of the culture medium used in step (1), there can be cited a culture medium (AK03N-C) obtained by removing FGF2, referred to as solution C in the kit, from StemFit AK03N culture medium (manufactured by AJINOMOTO HEALTHY SUPPLY CO., INC.), which is an undifferentiated maintenance medium, as an embryoid body formation medium, and supplemented with 50 nM to 3 μM, preferably 100 nM, of LDN-193189 (SMAD signaling inhibitor), 1 μM to 20 μM, preferably 3 μM, of SB431542 (SMAD signaling inhibitor), and 1 μM to 10 μM, preferably 3 μM, of CHIR99021 (Wnt signaling activator) (all manufactured by StemGen).

[0258] In step (1), pluripotent stem cells are suspended and cultured in an embryoid body formation medium for 5 to 10 days. In one embodiment, for suspension culture, suspension culture is performed in a state where the cells have formed cell aggregates (or embryoid bodies). As suspension culture, the suspension culture method known as the so-called SFEBq method (non-patent document 3) is preferred. The SFEBq method is a method for forming cell aggregates by suspending and culturing pluripotent stem cells in a culture container with a plurality of holes of uniform shape. In the SFEBq method, the cells are suspended and cultured in a culture container (hole) as dispersed single cells at the beginning of step (1), and the cells suspended in one hole eventually adhere to each other to form a three-dimensional structure, forming one cell aggregate in each hole.

[0259] As a culture container with multiple holes of uniform shape used in the SFEBq method, it is preferred that: a culture container has multiple, for example 6, 12, 24, 96 or 384 independent spaces (holes) for culturing cells, and these holes have the same shape and size. Each hole of the culture container can be columnar, and the shape of the bottom surface is not particularly limited, and can be a perfect circle, square, rectangle, polygon, ellipse, etc., preferably a perfect circle (that is, the hole is cylindrical). In addition, the bottom surface of the hole can be a flat bottom, or a round bottom (U bottom) or a V bottom or an M bottom, preferably a U bottom or a V bottom or an M bottom, more preferably a V bottom or an M bottom, and most preferably a V bottom. This is because by making the shape of the bottom pointed, the cells suspended (suspended) in the hole are easy to aggregate and easily form a sphere within a certain period of time. In addition, it is preferred that the bottom surface of the hole uses a culture container with a cell non-adsorbent material or a cell low-adsorbent material.

[0260] The bottom area of ​​the hole in the culture container is 0.1 to 2.0 cm in terms of flat bottom. 2 , preferably 0.5 to 1.2 cm 2 , more preferably 0.35 cm 2 The inner diameter is about 0.3 to 16.5 mm, preferably 3 to 11 mm, more preferably about 6 to 8 mm, and the height is 0.1 to 20 mm, preferably 0.3 to 17 mm, more preferably 8 to 12 mm.

[0261] Examples of the culture container include a commercially available culture container, such as a 6-well plate (bottom area 9.5 cm in terms of flat bottom). 2 About 35mm in inner diameter), 12-well plate (bottom area is 3.8cm when converted to a flat bottom 2 24-well plate (with an inner diameter of about 23 mm) (the bottom area is 1.88 cm when converted to a flat bottom 2 About 16 mm in inner diameter), 48-well plate (the bottom area is 1.0 cm when converted to a flat bottom 2 96-well plate (the bottom area is 0.35cm when converted to a flat bottom) 2 About 6 to 8 mm in inner diameter), 384-well plate (bottom area is 0.1 cm when converted to a flat bottom 2 The inner diameter is about 3 to 4 mm). A 96-well plate is preferred, and a 96-well plate with a V-bottom is more preferred.

[0262] As 96-well plates, PrimeSurface 96V plates, 96M plates, 96U plates, 96 slit-well plates (96 Slit-well Plates) (manufactured by Sumitomo Bakelite Co., Ltd.), and Ultra-Low Attachment Surface 96 round bottom plates (manufactured by CORNING) can be used. In addition, as long as the culture container has a shape that can independently form cell aggregates of uniform size, it is not necessary for each well to be clearly independent like a 96-well plate. For example, a culture container with a concave or convex bottom surface of a culture dish can also be used. As such a culture container, a Multi-Dimple can be cited, and a Multi-Dimple of 60 mm to 150 mm can be used, but it is not limited to this.

[0263] When the cell suspension is dispensed into all the wells of a 96-well plate, 96 uniform cell aggregates can be obtained in principle, and a large number of cell aggregates can be obtained at one time by increasing the number of culture plates or the number of wells per unit well (well) such as Multi-Dimple. As commercially available Multi-Dimple, for example, Elplasia (manufactured by KURARAY), AggreWell (manufactured by STEMCELL Technologies), and EZSPHERE (manufactured by Asahi Techno Glass) can be used, but it is not limited thereto.

[0264] In the SFEBq method, first, a cell suspension with a uniform cell density is dispensed in equal amounts into each well of a culture container. In the case of a 96-well plate, the cell density can be 3000 cells / well to 20000 cells / well, preferably 5000 cells / well to 10000 cells / well, and more preferably about 9000 cells / well. In addition, within a short period of time from the start of culture, for example, within 24 hours, preferably within 12 hours, and more preferably within 8 hours, the central portion of each well is formed with a cell aggregate of almost the same size. In addition, the diameter and cell number of the cell aggregate can be controlled by adjusting the amount of the cell suspension dispensed and the cell density. Furthermore, by using the SFEBq method, the multiple cell aggregates obtained become spherical, and cell aggregates can be formed with high reproducibility.

[0265] The suspension culture in step (1) can be carried out for about 5 to 10 days, preferably for about 7 to 10 days, and more preferably for about 7 days. The culture time can 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 time can be determined by the increase in the expression levels of SOX1, PAX6, HES4 and HES5, the decrease in the expression level of OCT3 / 4, and the decrease in the expression level of NANOG. Specifically, step (1) is continued until at least one of the following conditions is met compared to the start of step (1), and then step (2) is started:

[0266] 1) obtaining cell aggregates in which the expression level of at least one of SOX1, PAX6, HES4 and HES5 increased by more than 100 times based on RNA level;

[0267] 2) obtaining cell aggregates in which the expression level of OCT3 / 4 is reduced to less than 1 / 200 in terms of RNA level; and

[0268] 3) Obtaining cell aggregates in which the expression level of NANOG is reduced to less than 1 / 400 in terms of RNA level.

[0269] In this specification, "expression is X times more in terms of RNA level", "expression level increases X times in terms of RNA level" or "expression level of RNA increases X times" means obtaining one or more suspended cells or cells constituting cell aggregates at two time points in the step, using a method capable of absolute or relative quantification of RNA expression in the cells, and performing comparative analysis, and the result is that the RNA expression level at another time point is X times more (increased X times) than at one time point. In addition, "expression is X times less in terms of RNA level", "expression level decreases X times in terms of RNA level" or "expression level of RNA decreases X times" means that the RNA expression level at another time point is X times less (decreased X times) than at one time point. The method used is not particularly limited as long as RNA quantification can be performed, and measurement methods utilizing RNA sequences, real-time PCR assays, microarrays, Northern blotting, and in situ hybridization can be used, preferably using RNA sequences, real-time PCR assays, and microarrays for comparative determination.

[0270] The culture conditions for the suspension culture in step (1) are not particularly limited, and may be, for example, 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%. Hypoxic culture conditions are also preferred. "Hypoxic culture conditions" refer to conditions for culturing cells at an oxygen concentration lower than the oxygen concentration in the atmosphere (about 21%). Here, oxygen concentration refers to the oxygen concentration in the air in contact with the culture medium containing the cells in the culture device. The oxygen concentration is not particularly limited as long as it is 20% or less, and is preferably in the range of 3% to 10%, more preferably in the range of 4% to 6%, and most preferably 5%.

[0271] It should be noted that, before differentiation, that is, before step (1), the pluripotent stem cells can also be maintained and cultured using an undifferentiated maintenance medium. The undifferentiated maintenance medium contains only factors required to maintain the undifferentiated state (undifferentiated maintenance factors) and is a medium capable of maintaining pluripotency. Examples of undifferentiated maintenance factors include fibroblast growth factor, transforming growth factor-β (TGFβ family) factor, activin A, and the like.

[0272] The term "fibroblast growth factor (FGF)" as used herein refers to a factor that acts on a fibroblast growth factor receptor and activates FGF signaling. Examples of FGF include FGF2 (also referred to as bFGF), FGF4, and FGF8.

[0273] The undifferentiation maintenance medium in this specification is a medium that can maintain and culture pluripotent stem cells under feeder-free conditions, i.e., feeder-free medium. As a usable feeder-free medium, various culture media have been developed and commercially available, for example, Essential 8 (manufactured by Thermo Fisher Scientific). Essential 8 medium contains L-ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenide (14 μg / l), 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)).

[0274] Commercially available undifferentiation maintenance media include S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Thermo Fisher Scientific), hESF9 (Proc Natl Acad Sci USA. 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 Inc.), or StemFit (manufactured by AJINOMOTHEALTHY SUPPLY CO., INC.).

[0275] The concentration of the undifferentiation maintenance factor can be reduced from the components of the above-mentioned undifferentiation maintenance medium, and a medium with a composition in which the undifferentiation maintenance factor is 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 is an undifferentiation maintenance medium in which the concentration of the undifferentiation maintenance factor is reduced or the undifferentiation maintenance factor is removed. Specifically, an undifferentiation maintenance medium for feeder-free culture that does not contain FGF such as FGF2 can be cited. In addition, a medium prepared by mixing the neuroglia proliferation medium described later or a medium with a similar composition and the undifferentiation maintenance medium at a ratio of 1:1 or the like can also be used as an embryoid body formation medium.

[0276] <Step (2)>

[0277] In step (2), the cell aggregates obtained in step (1) are suspended in an embryoid body formation medium containing retinoic acid. The embryoid body formation medium and the suspension culture are as described above. The medium used in step (2) can be prepared by mixing retinoic acid in the embryoid body formation medium.

[0278] The culture medium used in step (2) may be a culture medium having the same composition as the culture medium used in step (1) except that it contains retinoic acid and does not contain one or more SMAD signaling inhibitors and one or more Wnt signaling activators. As "retinoic acid", retinoids (e.g., retinoic acid) can be cited. As a specific example of the culture medium used in step (2), a culture medium (AK03N-C) obtained by removing FGF2 referred to as C solution in the kit from StemFit AK03N culture medium (manufactured by AJINOMOTO HEALTHY SUPPLY CO., INC.) as an embryoid body formation culture medium and supplemented with 1 μM retinoic acid (manufactured by Sigma-Aldrich) can be cited.

[0279] The concentration of retinoids in the culture medium can be appropriately set within a range that can enhance the signal transduction mediated by retinoic acid. For example, the concentration of retinoic acid is not limited as long as it can show activity, that is, a region-specific post-localization effect, and is, for example, 10nM to 100μM, preferably 100nM to 10μM, and more preferably 500nM to 5μM. In the case of other retinoids, the concentration can be appropriately set to show activity equivalent to the above concentration of retinoic acid.

[0280] When step (1) is completed, the culture medium used in step (1) is removed from the culture container, and the culture medium is replaced with the culture medium used in step (2), so that step (2) can be started. In step (2), when the same culture container as step (1) is used, from the perspective of protecting cell aggregates, it is preferred to replace about half of the culture medium. By repeating this operation multiple times, more than half of the culture medium can be replaced. In addition, at this time, as needed, after washing the cell aggregates with a culture medium other than the culture medium used in step (2) or a phosphate buffer, culture can be started with the culture medium used in step (2). Alternatively, all cell aggregates obtained in (1) can be recovered when step (1) is completed, and after washing the cell aggregates as needed, they are suspended in the culture medium used in step (2), so as to start step (2). The culture container used in step (2) is not particularly limited as long as it is a culture device for suspension culture, and culture dishes and culture plates commonly used by those skilled in the art can be used. For example, PrimeSurface 96V plates and ultra-low adsorption culture bottles (manufactured by CORNING) can be cited.

[0281] The suspension culture in step (2) can be carried out for about 3 days to 21 days, preferably for about 4 days to 11 days, and more preferably for about 7 days. The culture time can be determined according to the desired degree of cell differentiation. 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 expressed differentiation markers 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 culture medium can be measured. In step (2), for example, the culture time 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, step (2) is continued until at least one of the following conditions is met compared to the start of step (2), and then step (3) is started:

[0282] 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 more than 5 times in terms of RNA level; and

[0283] 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.

[0284] The culture conditions of the suspension culture in step (2) may be the same as those in step (1), and are preferably hypoxic culture conditions. The oxygen concentration in the hypoxic culture conditions 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%.

[0285] <Step (3)>

[0286] In step (3), the cell aggregates obtained in (2) are suspended and cultured in an embryoid body formation medium or a neuroglia proliferation medium containing retinoic acid and one or more SHH signaling activators. The embryoid body formation medium is as described above. The culture medium used in step (3) can be prepared by mixing retinoic acid and one or more SHH signaling activators in an embryoid body formation medium or a neuroglia proliferation medium. When an embryoid body formation medium is used, it can also be prepared by further mixing one or more SHH signaling activators in the culture medium used in step (2).

[0287] In this specification, "nerve·glial proliferation medium" refers to a medium suitable for the proliferation and culture of glial precursor cells, glial cells, neural precursor cells and neural cells (Neuron; Neuron), as long as it can maintain the survival of the above-mentioned cells and can differentiate and proliferate the medium, there is no particular limitation. The nerve·glial proliferation medium can also be made by appropriately adding nutrients suitable for the survival of glial precursor cells, glial cells, neural precursor cells and neural cells in the above-mentioned basal medium, and in particular, it can also be appropriately combined as a medium suitable for the maintenance and culture of nervous system cells or differentiation induction and a commercially available medium or partially change its composition and use. For example, a commercially available Neurobasal or a commercially available DMEM / F-12 basal medium, etc., can also be selected, and the above-mentioned nutrients, etc. are appropriately added thereto and used.

[0288] The above-mentioned "nutrients suitable for survival" can be used in appropriate combination with nutrients known to those skilled in the art as factors added when culturing nervous system cells. Specifically, they 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 the above and used.

[0289] In the neuro·glial proliferation medium in this specification, it is particularly preferred that glial precursor cells, glial cells, neural precursor cells and neurons are all appropriately differentiated and proliferated and contain more than one growth factor, preferably containing platelet-derived growth factor (PDGF). As platelet-derived growth factor, PDGF-AA can be used. In the neuro·glial proliferation medium in this specification, as other growth factors, FGF (such as FGF2), EGF, IGF-1 or HGF etc. can be mentioned. As a preferred embodiment, PDGF-AA, EGF, IGF-1 and FGF (preferably FGF2) are contained in the neuro·glial proliferation medium.

[0290] As neurotrophic factors, human NT-3 (human recombinant neurotrophin 3), BDNF, NGF, GDNF or CNTF can be mentioned. As vitamins, biotin or ascorbic acid can be mentioned. As cAMP activators, cAMP, dibutyryl cAMP or forskolin can be mentioned. As hormones, progesterone or T3 can be mentioned. As a preferred embodiment, the culture medium for neuro-glial proliferation contains thyroid hormones such as T3 and neurotrophic factors such as NT-3 in addition to the above-mentioned growth factors.

[0291] As a mixture appropriately blended with the above nutrients, N1 supplement (hereinafter, sometimes referred to as N1), N2 supplement (hereinafter, sometimes referred to as N2), B27 supplement (including supplements from which vitamin A has been removed; hereinafter, sometimes referred to as B27) and the like are commercially available, and thus can be conveniently used and added to the basal medium. For example, N2 contains insulin, transferrin, progesterone, sodium selenate, and putrescine. When N2 is used, it is preferred that human NT-3 and / or B27 (preferably vitamin A has been removed) are also contained.

[0292] As a culture medium for neuroglia proliferation, for example, a culture medium in which N2, B27 (a supplement in which vitamin A is removed may be used), Glutamax (registered trademark), FGF2, EGF, T3, NT-3, PDGF-AA, and IGF-1 are added to a DMEM / F-12 culture medium. In addition, a culture medium in which 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 are added to a DMEM / F-12 culture medium can be cited. In addition, a culture medium in which Glutamax, β-mercaptoethanol, B27, N2, MEM NEAA, FGF2, EGF, T3, NT-3, PDGF-AA, and IGF-1 are added to a DMEM / F-12 culture medium can be cited. Another example is a medium obtained by adding N1, B27, T3, biotin, dibutyryl cAMP, PDGF-AA, IGF-1, and NT-3 to a DMEM / F-12 medium.

[0293] From the viewpoint of containing one or more nutrients suitable for the survival of nervous system cells, it is preferred that the medium for neuro-glial proliferation does not contain a substance that suppresses or inhibits the biological activity of the component or contains it at a concentration lower than the effective concentration.

[0294] Specific examples of the medium for neuroglia proliferation include a medium obtained by adding the following components to a DMEM / F-12 medium (manufactured by Thermo Fisher Scientific): 1% N2 (manufactured by Thermo Fisher Scientific); B27 (manufactured by Thermo Fisher Scientific) having a vitamin A concentration of 5 μg / ml or less, preferably vitamin A-free; T3 (manufactured by Sigma-Aldrich) at 10 ng / ml to 100 ng / ml, preferably 60 ng / ml; PDGF-AA (manufactured by PeproTech) at 5 ng / ml to 100 ng / ml, preferably 10 ng / ml; FGF2 at 10 ng / ml to 100 ng / ml, preferably 20 ng / ml; EGF (manufactured by PeproTech) at 5 ng / ml to 100 ng / ml, preferably 10 ng / ml; and insulin-like growth factor 1 (R&D Biotechnology, Inc.) at 10 ng / ml. SYSTEMS Corporation); and 5 ng / ml to 100 ng / ml, preferably 10 ng / ml of NT-3 (Neurotrophin-3) (R&D SYSTEMS Corporation).

[0295] In the present specification, the SHH signaling activator is defined as a substance that causes the de-inhibition of Smoothened (Smo) caused by the binding of SHH (Sonic Hedgehog) to Patched (Ptch1) as a receptor and the subsequent activation of Gli2. Examples of SHH signaling activators include proteins belonging to the Hedgehog family, and specifically, partial peptides of SHH or IHH such as SHH or IHH (Indian Hedgehog), or 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, and 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; N-methyl-N'-(3-piperidinylphenyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-dimethylaminocyclohexane], 20a-hydroxycholesterol, purmorphamine (PMA; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthyloxy)-9H-purin-6-amine) and their derivatives, etc. (Stanton BZ et al., Mol Biosyst. 6: 44-54, 2010).

[0296] The SHH signaling activator in step (3) may be one or more selected from the group consisting of purmorphamine, SAG, SHH protein and SHH fragment (e.g., SHH C25II or SHH C24II), preferably one or more selected from the group consisting of SHH protein, SHH fragment, purmorphamine and SAG.

[0297] The concentration of the SHH signaling activator in the culture medium can be appropriately set within a range that can enhance the signaling mediated by SHH. For example, the concentration of purmorphamine is not limited as long as it exhibits a region-specific ventralization effect, and is, for example, 10nM to 10μM, preferably 100nM to 10μM, and more preferably 500nM to 5μM. In the case of other SHH signaling activators, the concentration can be appropriately set to show a de-inhibition effect of Smoothened (Smo) and a subsequent activation of Gli2, or a region-specific ventralization effect, which is equivalent to the above-mentioned concentration of purmorphamine.

[0298] Specific examples of the medium used in step (3) include an embryoid body formation medium or a neuro-glial proliferation medium supplemented with 1 μM retinoic acid (manufactured by Sigma-Aldrich) and 1 μM purmorphamine (manufactured by Millipore).

[0299] When step (2) is completed, all the cell aggregates obtained in (2) are recovered. If necessary, the recovered cell aggregates can be washed with the culture medium used in step (3), other culture medium (for example, DMEM / F-12 can be mentioned, but it is not limited to this) or phosphate buffer, and then suspended in the culture medium used in step (3), thereby starting step (3). Alternatively, from the perspective of protecting the cell aggregates, it is also possible to replace about half of the culture medium to avoid drastic changes in the culture environment, and it is also possible to replace more than half of the culture medium by repeating this operation multiple times. The culture container used in step (3) is not particularly limited as long as it is a culture equipment for suspension culture, and culture bottles and culture plates commonly used by technicians in this field can be appropriately used. For example, PrimeSurface 96V plates, 96 slit well plates and ultra-low adsorption culture bottles (manufactured by CORNING) can be mentioned.

[0300] The suspension culture in step (3) can be carried out for about 4 to 11 days, preferably for about 4 to 7 days, and more preferably for about 7 days. The culture time can be determined according to the desired degree of cell differentiation. During the culture period, all or part of the culture medium can be appropriately replaced every few days (for example, once every 3 days or every 4 days).

[0301] 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 expressed differentiation markers 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 culture medium can be measured. In step (3), for example, the culture time can be determined by the expression level of RNA or protein of one or more markers selected from the group consisting of HEY2, NKX6.2, NKX2.2, OLIG1 and OLIG2. Specifically, step (3) is continued until at least one of the following conditions is met compared to the time when step (3) is started, and then step (4) is started:

[0302] 1) obtaining cell aggregates in which the expression level of at least one of HEY2, NKX6.2 and NKX2.2 is increased by 5 times or more at the RNA level; and

[0303] 2) Obtaining cell aggregates in which the expression level of OLIG1 and / or OLIG2 is increased by 10 times or more at the RNA level.

[0304] The method for evaluating the expression level of the RNA is the same as that described in step (1).

[0305] The culture conditions for the suspension culture in step (3) are not particularly limited, and may be, for example, 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%.

[0306] <Step (4)>

[0307] In step (4), the cell aggregate obtained in (3) is suspended and cultured in a neuron / glial proliferation medium that does not contain retinoic acid and contains one or more SHH signaling activators. The neuron / glial proliferation medium and the SHH signaling activator are as described above.

[0308] In step (4), upon completion of step (3), the culture medium can be replaced with the culture medium of step (4), and the cell aggregates obtained in step (3) can be suspended and cultured in the form of cell aggregates; or the cell aggregates obtained in step (3) can be dispersed in order to remove cells that cannot survive and proliferate in the culture medium used in step (4) as non-target cells and to increase the content of target oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells, and then the dispersed cells can be suspended and cultured, thereby forming cell aggregates again.

[0309] "Dispersing cell aggregates" means that the cells forming the cell aggregates are dissociated into a cell unit (single cell), or the shape of the original cell aggregates is destroyed while maintaining the state of multiple cells adhering to each other. As a method of dispersion, a physical stimulation method and a method using a compound can be cited. By using any one or a combination of two methods, the cell aggregates can be destroyed. That is, the original cell aggregates contain about 1000 to 100,000, preferably about 3000 to 50,000, more preferably about 1000 to 50,000, and more preferably about 3000 to 30,000 cells. After preferably dispersing the cell aggregates, even in the state where multiple cells adhere to each other to form small cell clumps, the number of cells contained in the clumps is about 100 or less, and it is also preferably dispersed to a state where more than 90% of the cells become a single state relative to the cell aggregates before dissociation.

[0310] Specifically, trypsin (e.g., trypsin, TrypLE (registered trademark)), Accutase (registered trademark), Accumax (registered trademark), or metal chelators (EDTA, EGTA, etc.) are used to dissociate the adhesion of cells to each other, and a micropipette is further used to give physical stimulation, thereby effectively dispersing cell aggregates. As a method for further dispersing cells into single cells, a technique of passing through a cell filter can be utilized. In order to implement the present invention, the dispersed cell aggregates can be polymerized by culturing the cells under suspension culture conditions for a certain period of time to form cell aggregates again. In order to obtain cell agglomerates of more uniform size, the above-mentioned SFEBq method can be used.

[0311] The suspension culture in step (4) can be carried out for about 4 days or more, preferably about 10 days or more, more preferably about 14 days or more, and even more preferably about 14 to 21 days. The culture time can be determined according to the desired degree of cell differentiation.

[0312] 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 expressed differentiation markers 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 culture medium can be measured. In step (4), the culture time can be determined, for example, 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, step (4) is continued until at least one of the following conditions is met compared to the time when step (4) is started, and then step (5) is started:

[0313] 1) obtaining cell aggregates in which the expression level of at least one of NFIA, NFIB, SLC1A3, S100B and FABP7 is increased by more than 10 times in terms of RNA level; and

[0314] 2) Obtaining cell aggregates in which the expression level of PAX6 is reduced to less than 5 times based on RNA level.

[0315] The method for evaluating the expression level of the RNA is the same as that described in step (1).

[0316] For example, PrimeSurface 96V plate, 96-slot well plate, and ultra-low attachment culture flask (manufactured by CORNING) can be cited.

[0317] The culture container used in step (4) is not particularly limited as long as it is a culture device for suspension culture for cell culture, and culture bottles, culture plates and culture dishes commonly used by those skilled in the art can be used appropriately. For example, Nunc (trademark) EasYFlask (trademark) cell culture flasks (manufactured by Thermo Fisher Scientific), ultra-low adsorption culture flasks (manufactured by CORNING), and culture containers with multiple uniformly shaped holes used in the SFEBq method can be cited.

[0318] The culture conditions for the suspension culture in step (4) are not particularly limited, and may be, for example, a temperature of 35°C to 37°C, preferably 37°C, a humidity of 90% to 95%, preferably 95%, a CO2 concentration of 2% to 5%, preferably 5%, and an O2 concentration of 10% to 20%, preferably 20%.

[0319] During the culture period, the culture medium may be replaced once every about 2 to 7 days, or once every about 2 to 5 days, or once every about 3 to 4 days. For the culture medium replacement, the entire amount of culture medium may be replaced, or, for example, about 1 / 3 or 1 / 2 of the culture medium may be replaced in order to avoid significant changes in the cell environment. It is also possible to appropriately supplement the growth factors or neurotrophic factors contained in the culture medium at a time other than the culture medium replacement. For example, in step (4), PDGF-AA is preferably added once every 2 to 3 days in a manner that maintains the concentration of PDGF-AA (e.g., 10 ng / ml).

[0320] Regarding the neuroglia proliferation medium used in step (4), when the neuroglia proliferation medium is used in step (3), a medium with the same composition as the neuroglia proliferation medium can be used in step (4), or the composition can be changed as appropriate.

[0321] The neuron / glial proliferation medium used in step (4) preferably contains 5 ng / ml to 100 ng / ml, and more preferably about 10 ng / ml of PDGF-AA.

[0322] <Step (5)>

[0323] In step (5), the cell aggregates obtained in (4) are suspended and cultured in a neuroglia proliferation medium that does not contain both retinoic acid and an SHH signaling activator. The neuroglia proliferation medium is as described above.

[0324] In step (5), upon completion of step (4), the culture medium may be replaced with the culture medium of step (5), and the cell aggregates obtained in step (4) may be suspended and cultured in the form of cell aggregates; or the cell aggregates obtained in step (4) may be dispersed in order to remove cells that cannot survive and proliferate in the culture medium used in step (5) as non-target cells and to increase the content of oligodendrocyte precursor cells, astrocyte precursor cells, and neural precursor cells as targets, and then the dispersed cells may be suspended and cultured to form cell aggregates again. The specific method is the same as step (4).

[0325] The suspension culture in step (5) can be continued to be cultured as long as the culture medium is appropriately replaced and subcultured without excessive differentiation and the cell aggregates become too large, and the time is not particularly limited. The culture in step (5) can be carried out for about 5 days to 100 days, preferably about 5 days to 20 days or 7 days to 50 days, more preferably about 7 days to 35 days, still preferably 7 days to 28 days, and most preferably 14 days to 28 days. In step (5), during the culture period, the culture medium can be appropriately replaced once every few days according to the number of cells and the amount of culture medium, for example, once within 7 days, once every about 2 to 7 days, or once every about 2 days to 5 days, or once every about 3 to 4 days. The culture medium replacement can replace the entire amount of culture medium, or it can replace, for example, about 1 / 3 or 1 / 2 of the culture medium in order to avoid significant changes in the environment of the cells. There is no limit to the number of subcultures, for example, 0 to 5 subcultures can also be carried out. For example, step (5) may be started, culture may be continued for 14 days, and the step may be terminated at this time point; or one more subculture may be performed, and then culture may be continued for 8 or 14 days before the step is terminated.

[0326] 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. As differentiation markers, at least mRNA or protein, sugar, lipid, etc. can be cited. Alternatively, the culture supernatant can be collected and the amount of differentiation markers secreted from the cells into the culture medium can be measured.

[0327] In step (5), for example, the culture time can be determined by the expression level or presence 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.

[0328] As the medium for neuronal and glial proliferation used in step (5), a medium having the same composition as that of step (4) may be used, or the composition may be appropriately changed, as long as it does not contain retinoic acid and SHH signaling activator.

[0329] The culture container used in step (5) is not particularly limited as long as it is an equipment for cell culture, and culture bottles and culture plates commonly used by those skilled in the art can be appropriately used. For example, Nunc (trademark) EasYFlask (trademark) cell culture bottles (manufactured by Thermo Fisher Scientific), ultra-low adsorption culture bottles (manufactured by CORNING), 96-well plates, 96-well slit well plates, etc. can be mentioned. The culture conditions of the suspension culture in step (5) are not particularly limited, for example, the conditions of a temperature of 37°C, a humidity of 95%, a CO2 concentration of 5%, and an O2 concentration of 20% can be used. During the culture period, it is preferred to replace the culture medium once every approximately 7 days and add PDGF-AA (e.g., 10 ng / ml) once every approximately 3 to 4 days.

[0330] The cell aggregates containing glial precursor cells obtained after step (5) (the cell aggregates containing glial precursor cells of the present invention) have the following characteristics:

[0331] (a) comprising oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells;

[0332] (b) expressing spinal cord regional markers; and

[0333] (c) It does not contain feeder cells or feeder cell-derived components.

[0334] In step (5), for the purpose of confirming whether cells having the above-mentioned characteristics (a) are obtained, step (5) can be continued until it is confirmed that the cell aggregates express one or more, preferably two or more, and preferably five or more markers selected from the group consisting of O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP and PLP.

[0335] Furthermore, in step (5), for the purpose of confirming whether cells having the above-mentioned characteristic (b) are obtained, step (5) can be continued until it is confirmed that the cell aggregates express one or more, preferably two or more markers selected from the group consisting of HOXB3, HOXB4, HOXB6 and HOXD8.

[0336] Furthermore, in step (5), 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 culture medium of the cell aggregates.

[0337] The diameter of the cell aggregates comprising glial precursor cells is 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 aggregates comprising glial precursor cells can be differentiated into cell aggregates comprising oligodendrocytes, astrocytes and neurons by continuing to culture suitable for differentiation. In addition, when the cell aggregates comprising glial precursor cells are transplanted into an organism, they can be differentiated into cell groups comprising oligodendrocytes, astrocytes and neurons in the organism.

[0338] 〔Cell aggregates containing glial precursor cells〕

[0339] One embodiment of the cell aggregate containing glial precursor cells of the present invention has the following characteristics:

[0340] (a) comprising oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells;

[0341] (b) expression of spinal cord regional markers;

[0342] (c) does not contain feeder cells and feeder cell-derived components; and

[0343] (d) having the ability to differentiate into a cell population including oligodendrocytes, astrocytes and neurons.

[0344] (a) A cell aggregate containing glial precursor cells according to one embodiment contains oligodendrocyte precursor cells, astrocyte precursor cells, and neural precursor cells as cells constituting the cell aggregate.

[0345] The concept of glial precursor cells includes both oligodendrocyte precursor cells and astrocyte precursor cells.

[0346] The oligodendrocyte precursor cells in the cell aggregates containing glial precursor cells of the present invention are characterized by expressing one or more markers selected from the group consisting of O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP and PLP, preferably two or more markers, and more preferably five or more markers. The oligodendrocyte precursor cells preferably express at least A2B5 antigen, O4 antigen, OLIG2 and FAM181B.

[0347] The astrocyte precursor cells in the cell aggregates containing glial precursor cells of the present invention are characterized by expressing 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, preferably two or more markers, and more preferably five or more markers. The astrocyte precursor cells preferably express at least NFIA, NFIB, SLC1A3 and SPARCL1.

[0348] Among the markers expressed in the above-mentioned astrocyte precursor cells, NFIA, NFIB, SOX9, HEY1, HEY2, FABP7 and ZBTB20 are markers expressed in the following cells: the cells are classified as glial precursor cells, and although they have not differentiated into complete astrocyte precursor cells, they are destined to be induced to differentiate into astrocytes, and are low-maturity astrocyte precursor cells. That is, the cell aggregate containing glial precursor cells of the present invention is characterized in that it contains the low-maturity astrocyte precursor cells.

[0349] Therefore, it is more preferred that the cell aggregates comprising glial precursor cells of the present invention simultaneously comprise:

[0350] 1) Astrocyte precursor cells with low maturity, characterized by expressing one or more markers selected from NFIA, NFIB, SOX9, HEY1, HEY2, FABP7 and ZBTB20, preferably two or more markers, and more preferably five or more markers; and

[0351] 2) Astrocyte precursor cells, characterized in that they express one or more markers selected from the group consisting of SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP and AQP4, preferably two or more markers, and more preferably five or more markers.

[0352] The cell aggregates containing glial progenitor cells of the present invention contain neural progenitor cells and relatively more immature (lowly differentiated) neural stem cells. The neural stem cells are characterized by expressing one or more, preferably two or more, and 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.

[0353] The neural progenitor cells in the cell aggregates containing glial progenitor cells of the present invention are characterized by expressing one or more markers selected from the group consisting of DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO and EPHA3, preferably two or more markers, and more preferably five or more markers. The neural progenitor cells preferably express at least DCX, βIII tubulin and ELAVL3.

[0354] In a cell aggregate containing glial precursor cells of one embodiment, it is preferred that: relative to all cells constituting the cell aggregate, it contains more than 5%, preferably 10% to 60%, more preferably 20% to 50%, and preferably 20% to 40% of oligodendrocyte precursor cells. In addition, it is preferred that: it contains more than 10%, preferably 10% to 80%, more preferably 10% to 50%, and preferably 20% to 30% of astrocyte precursor cells. Furthermore, it is preferred that: it contains more than 10%, preferably 20% to 60%, and more preferably 30% to 50% of neural precursor cells. Here, the total of oligodendrocyte precursor cells, astrocyte precursor cells, and neural precursor cells does not exceed 100%, accounting for more than 80% of the total number of cells contained in the cell aggregate, preferably more than 85%, more preferably more than 90%, and preferably more than 95%.

[0355] The ratio of each cell type in the cell aggregate containing glial precursor cells is not particularly limited. One embodiment thereof includes a cell aggregate in which the content of astrocyte precursor cells or neural precursor cells is greater than that of oligodendrocyte precursor cells.

[0356] The proportion of each cell relative to all cells constituting the cell aggregate can be determined by the expression of each cell marker. For example, the proportion of target cells present in a certain range relative to all cells can be determined by measuring (detecting or quantifying) the mRNA, protein or fragments thereof and the calculated values ​​by FACS, immunostaining, or single-cell RNA sequence analysis, which are known to those skilled in the art. The total number of cells can be counted, for example, by nuclear staining, and the number of target cells present in the same range can be counted by the number of cells expressing markers specifically expressed in the target cells.

[0357] (b) The cell aggregate containing glial precursor cells according to one embodiment is characterized by expressing spinal cord region markers, for example, one or more, preferably two or more spinal cord region markers selected from HOXB3, HOXB4, HOXB6 and HOXD8 at the mRNA level.

[0358] The cell aggregates containing glial precursor cells according to one embodiment are characterized by expressing ventral region markers, for example, expressing one or more, preferably two or more ventral region markers selected from NKX2.1, NKX2.2, NKX6.1 and NKX6.2 at the mRNA level.

[0359] One embodiment of a cell aggregate comprising glial precursor cells is characterized in that it comprises cells expressing at the mRNA level one or more, preferably two or more, spinal cord region markers selected from HOXB3, HOXB4, HOXB6 and HOXD8, and expressing one or more, preferably two or more, ventral region markers selected from NKX2.1, NKX2.2, NKX6.1 and NKX6.2.

[0360] (c) The cell aggregates containing glial precursor cells of one embodiment are characterized in that they do not contain feeder cells and components derived from feeder cells; when the feeder cells are derived from a xenogeneic animal, they do not contain xenogeneic cell-derived components. This cell aggregate can be obtained by culturing pluripotent stem cells in the absence of feeder cells. Here, xenogeneic cell-derived components refer to components derived from species different from the species of the cells of the culture object. The cell aggregates containing glial precursor cells of the present invention do not contain cells other than iPS cells or cells induced by differentiation of iPS cells, or their cell-specific components. It should be noted that the cell aggregates containing glial precursor cells of the present invention also include the following forms: they do not contain components derived from feeder cells, such as components derived from xenogeneic feeder cells, and do not contain components derived from other xenogeneic cells, xenogeneic components (Xenogenic Factor). This cell aggregate can be obtained by using a Xeno-free culture medium on the basis of the above-mentioned conditions in which feeder cells are not present.

[0361] (d) The cell aggregates comprising glial precursor cells of one embodiment have the ability to differentiate into cell groups comprising oligodendrocytes, astrocytes and neurons. The cell aggregates comprising glial precursor cells of the present invention can be confirmed to have this ability by the following method: cultured for 5 to 60 days, more specifically 10 days, in the maturation medium described later using the culture method described later, and then confirming whether it has differentiated into a cell group comprising oligodendrocytes, astrocytes and neurons. That is, differentiation into a cell group comprising oligodendrocytes, astrocytes and neurons can be identified by detecting the expression of markers described later.

[0362] The cell aggregates comprising glial precursor cells according to one embodiment are characterized in that (I) they contain cells that preferably express at the mRNA level one or more, preferably two or more, and even more preferably five 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.

[0363] Preferably, the cell aggregates containing glial precursor cells of the embodiment are characterized in that they contain the following cells, wherein the cells are characterized in that: one or more, preferably two or more, and preferably five or more markers selected from NFIA, NFIB, SOX9, HEY1, HEY2 and ZBTB20 are expressed at the mRNA level, and one or more, preferably two or more, and preferably five or more markers selected from SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP and AQP4 are expressed at the mRNA level.

[0364] The cell aggregates containing glial precursor cells in one embodiment are characterized in that (II) they contain cells that preferably express one or more, preferably two or more, and even more preferably five or more markers selected from the group consisting of OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP and PLP at the mRNA level.

[0365] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that (III) it contains cells expressing one or more markers selected from the group consisting of DCX, βIII-tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3.

[0366] The cell aggregate containing glial precursor cells according to one embodiment is characterized by (IV) containing cells expressing one or more markers selected from the group consisting of SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3, and ASCL1.

[0367] In one embodiment, the cell aggregate comprising glial precursor cells (V) has the ability to differentiate into a cell population comprising:

[0368] (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;

[0369] (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3; and

[0370] (iii) Cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.

[0371] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that it has the ability to differentiate into a cell population containing at least 5% or more, preferably about 5% to 20% of O4-positive cells after the above-mentioned culture.

[0372] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that it has the ability to differentiate into a cell population containing at least 10% or more, preferably about 10% to 50% APC-positive cells through the above-mentioned culture.

[0373] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that it has the ability to differentiate into a cell population containing at least 10% or more, preferably about 10% to 40% of GFAP-positive cells after the above-mentioned culture.

[0374] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that it has the ability to differentiate into a cell population containing at least 15% or more, preferably about 15% to 50% of βIII-tubulin-positive cells after the above-mentioned culture.

[0375] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that it has the ability to differentiate into a cell population containing at least 10% or more, preferably about 10% to 30% of Hu-positive cells after the above-mentioned culture.

[0376] The cell aggregate containing glial precursor cells according to one embodiment is characterized in that (VI) one or more markers selected from C10RF61 and SERPINE2 are expressed at the mRNA level.

[0377] In one embodiment, the cell aggregate containing glial precursor cells (VII) further has at least one of the following features (A) to (C):

[0378] (A) 60% or more, preferably 70% or more, further preferably 70% to 90%, and further preferably 75% to 85% of all cells constituting the cell aggregate express NFIA;

[0379] (B) 10% or more, preferably 20% or more, further preferably 20% to 50%, and further preferably 20% to 40% of all cells constituting the cell aggregate express OLIG2; and

[0380] (C) With respect to all cells constituting the cell aggregate, more than 20%, preferably more than 30%, further preferably 30% to 60%, and still more preferably 30% to 50% of the cells express one or more markers selected from HEY2, C1ORF61, FAM181B, NFIA, NFIB, ITM2B, LHFPL3 and MLC1.

[0381] Here, the ratio of cells positive for each marker can be calculated by counting the number of cells stained by immunostaining or analyzing gene expression at the single cell level.

[0382] For the cell aggregates comprising glial precursor cells of one embodiment, (VIII) does not include pluripotent stem cells. The absence of pluripotent stem cells can be confirmed by using OCT3 / 4 and NANOG not expressed at the mRNA level or protein level, or the expression level is reduced as an indicator. Specifically, for example, the expression level of OCT3 / 4 can be reduced to less than 1 / 200 compared with iPS cells, and / or the expression level of NANOG can be reduced to less than 1 / 400 compared with iPS cells as an indicator.

[0383] The cell aggregates comprising glial precursor cells of one embodiment are characterized in that (IX) they are differentiated to the extent that PAX6 is not expressed. Here, it can be confirmed by taking PAX6 not expressed at the mRNA level or protein level, or the expression level is reduced as an indicator. Specifically, for example, the following situation can be used as an indicator: compared with the timing of starting step (3) (e.g., differentiation day 14 in the embodiment of the present application), the expression level of PAX6 is reduced to less than 1 / 10 at the time of implementing step (4) or (5) to recover the cell aggregates for transplantation (e.g., differentiation day 49 in the embodiment of the present application).

[0384] The cell aggregates containing glial precursor cells in one embodiment may be spherical cell aggregates with a diameter (X) 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 unit 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.

[0385] In one embodiment, a cell aggregate comprising glial precursor cells has at least 2, at least 4, at least 6, at least 8 or all of the characteristics of (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X).

[0386] The cell aggregates containing glial precursor cells of one embodiment include: cells expressing or secreting 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 aggregates. The cell aggregates containing glial precursor cells of one embodiment preferably include: cells expressing or secreting 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 aggregates; more preferably, cells expressing or secreting 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 aggregates.

[0387] In one embodiment, when SPARCL1 is detected in the culture supernatant of cell aggregates containing glial precursor cells, for example, when cultured for 48 hours, 0.1 ng or more, preferably 1 ng or more, more preferably 10 ng or more of SPARCL1 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 of SPARCL1 is detected per 1000 cells.

[0388] In one embodiment, when MIF is detected in the culture supernatant of cell aggregates containing glial precursor 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 of MIF is detected in every 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 of MIF is detected in every 1000 cells.

[0389] In one embodiment, when MCP-1 is detected in the culture supernatant of cell aggregates containing glial precursor 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 of MCP-1 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 8 pg of MCP-1 is detected per 1000 cells.

[0390] In one embodiment, when SCF is detected in the culture supernatant of cell aggregates containing glial precursor cells, for example, when cultured for 48 hours, 0.001 pg or more, preferably 0.01 pg or more, more preferably 0.02 pg or more of SCF is detected per 1000 cells. In one embodiment, 0.001 pg to 10 pg, preferably 0.01 pg to 1 pg, more preferably 0.02 pg to 0.2 pg of SCF is detected per 1000 cells.

[0391] In one embodiment, when M-CSF is detected in the culture supernatant of cell aggregates containing glial precursor cells, for example, when cultured for 48 hours, 0.001 pg or more, preferably 0.005 pg or more, more preferably 0.01 pg or more of M-CSF is detected per 1000 cells. In one embodiment, 0.0001 pg to 10 pg, preferably 0.001 pg to 1 pg, more preferably 0.005 pg to 0.1 pg of M-CSF is detected per 1000 cells.

[0392] In one embodiment, when HGF is detected in the culture supernatant of cell aggregates containing glial precursor cells, for example, when cultured for 48 hours, 0.0001 pg or more, preferably 0.001 pg or more, more preferably 0.01 pg or more of HGF is detected per 1000 cells. In one embodiment, 0.0001 pg to 10 pg, preferably 0.001 pg to 1 pg, more preferably 0.005 pg to 0.5 pg of HGF is detected per 1000 cells.

[0393] In one embodiment, when LIF is detected in the culture supernatant of cell aggregates containing glial precursor cells, for example, when cultured for 48 hours, 0.0001 pg or more, preferably 0.001 pg or more, more preferably 0.01 pg or more of LIF is detected per 1000 cells. In one embodiment, 0.0001 pg to 10 pg, preferably 0.001 pg to 1 pg, more preferably 0.005 pg to 0.5 pg of LIF is detected per 1000 cells.

[0394] [Method for preparing a cell population comprising oligodendrocytes, astrocytes and neural cells]

[0395] One embodiment of the method for preparing a cell population including oligodendrocytes, astrocytes, and neurons of the present invention is performed by culturing a cell aggregate including glial precursor cells for 5 to 60 days using a maturation medium.

[0396] "Maturation medium" refers to a medium for maturing glial cells and neural cells, and is a medium containing factors required for the maturation of glial cells and neural cells. As for the medium (basal medium) as the basis, as long as it is a medium that can make nervous system cells survive, it is not particularly limited, and can be common with the above-mentioned nerve·glial proliferation medium. As a basal medium, for example, a medium in which a nervous system cell culture supplement such as N2 and / or B27 is added to a basal medium such as DMEM / F-12 can be cited. Among them, it is expected that the growth factors contained in the nerve·glial proliferation medium, which mainly contribute to cell proliferation, are not contained: FGF (such as FGF2), EGF and PDGF (such as PDGF-AA).

[0397] On the other hand, the maturation medium contains neurotrophic factors, thyroid hormones and / or cytokines that contribute to differentiation into glial cells. As neurotrophic factors, NT-3 can be cited. As thyroid hormones, T3 can be cited. As the cytokine, LIF can be cited.

[0398] As a maturation medium, for example, a medium containing at least one, preferably two, and preferably all of T3, NT-3, and LIF can be used. In addition, as a maturation medium for differentiation into astrocytes, a medium further containing CNTF can be used. The above factors can be added to the culture medium for use, or a culture medium with the above factors can be used. The concentration of T3 in the maturation medium is 5ng / ml or more, preferably 30ng / ml to 100ng / ml, and more preferably 60ng / ml to 100ng / ml. In addition, the concentration of NT-3 in the maturation medium is 10ng / ml or more, preferably 10ng / ml to 100ng / ml, and more preferably 10ng / ml to 50ng / ml. In addition, the concentration of LIF in the maturation medium is 10ng / ml or more, preferably 10ng / ml to 100ng / ml, and more preferably 10ng / ml to 50ng / ml. Furthermore, the concentration of CNTF in the maturation medium may be 5 ng / ml or more, preferably 10 ng / ml to 50 ng / ml, and more preferably 25 ng / ml to 50 ng / ml.

[0399] "Cell group comprising oligodendrocytes, astrocytes and neurons" (cell group comprising oligodendrocytes, astrocytes and neurons of the present invention) obtained by the preparation method of the cell group comprising oligodendrocytes, astrocytes and neurons, as long as it is a cell group comprising these 3 cells of oligodendrocytes, astrocytes and neurons at a detectable level in the cells constituting the cell group, then the containing ratio of each cell type is not particularly limited. The above-mentioned cell group can be a cell aggregate with a three-dimensional structure (spherical), or a cell group with a two-dimensional layered structure. The layered cell group can be prepared by carrying out adherent culture to the cell aggregate comprising glial precursor cells in the presence of cell adhesion factors such as Matrigel.

[0400] To detect individual cells, for example, immunohistological methods and gene analysis (RT-qPCR and RNA sequencing, etc.) can be used. When gene analysis is used, mRNA contained in cell aggregates and its expression level can be identified according to the method described in Example 2 of this specification.

[0401] The cell group of oligodendrocytes, astrocytes and neurons obtained by the present invention may also include oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells as starting cells. The cell group preferably contains oligodendrocytes, astrocytes and neurons with more than 50% of the total number of cells, and preferably more than 70%. The cell group contains more than 2%, preferably 5% to 50%, and more preferably 10% to 40% of oligodendrocytes. In addition, it contains more than 10%, preferably 10% to 70%, and more preferably 10% to 50% of astrocytes. Furthermore, it contains more than 10%, preferably 20% to 60%, and more preferably 30% to 50% of neurons. Here, the total of oligodendrocytes, astrocytes and neurons does not exceed 100%, accounting for more than 50% of the total number of cells contained in the cell aggregate, preferably more than 70%.

[0402] The cell population comprising oligodendrocytes, astrocytes and neurons of the present invention comprises:

[0403] (i) cells expressing one or more markers, preferably three or more markers, more preferably five or more markers, and more preferably all of the markers selected from the group consisting of O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2;

[0404] (ii) cells expressing one or more markers, preferably two or more markers, and preferably all of the markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3; and

[0405] (iii) cells expressing one or more markers, preferably two or more markers, more preferably three or more markers, and more preferably all of the markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.

[0406] The culture of cell aggregates containing glial precursor cells can be a suspension culture. The culture conditions are not particularly limited, for example, 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%. The culture container is not particularly limited as long as it is a cell culture device, and culture bottles and culture dishes commonly used by those skilled in the art can be used. For example, Nunc (trademark) EasYFlask (trademark) cell culture bottles (manufactured by Thermo Fisher Scientific), ultra-low adsorption culture bottles (manufactured by CORNING), etc. can be cited.

[0407] [Drug composition]

[0408] As one aspect of the present invention, a pharmaceutical composition comprising a cell aggregate comprising a glial precursor cell of the present invention or a cell group comprising oligodendrocytes, astrocytes and neurons of the present invention or a cell group comprising a transplantation cell group of cells derived from the cell aggregate or cell group as an active ingredient can be enumerated. The cell aggregate comprising a glial precursor cell of the present invention can be prepared by the preparation method of the cell aggregate comprising a glial precursor cell of the present invention. The cell group comprising oligodendrocytes, astrocytes and neurons of the present invention can be prepared by the preparation method of the cell group comprising oligodendrocytes, astrocytes and neurons of the present invention.

[0409] The effective dose of the active ingredient varies depending on the purpose of administration, the method of administration, and the condition of the subject (sex, age, weight, condition, etc.). For example, it can be set as 1×10 4 ~1×10 8 pcs, 1×10 5 ~3×10 5 pcs, 1×10 6 ~3×10 6 or 1×10 7 ~3×10 7 indivual.

[0410] In addition to the above-mentioned effective dose of the active ingredient, the pharmaceutical composition of one embodiment may also contain a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, a physiological aqueous medium (physiological saline, buffer, serum-free medium, etc.) can be used. The pharmaceutical composition may also contain preservatives, stabilizers, reducing agents, isotonic agents, etc. commonly used in medicines containing transplanted tissues or cells in transplant medicine as needed.

[0411] The cell suspension can be prepared by suspending the cell aggregate or cell group for transplantation in an appropriate physiological aqueous medium. If necessary, the cell group for transplantation can be cryopreserved by adding a cryopreservative, thawed when used, and washed with a buffer solution for transplantation medicine.

[0412] The cell population for transplantation of the present invention may be a suspension obtained by suspending a cell aggregate, or may be a suspension or tablet obtained by dispersing the cell aggregate into cells.

[0413] In addition, the cell population for transplantation obtained by the preparation method of the present invention can be formed into a three-dimensional shape by culturing on a scaffold after implementing step (4) in the preparation method of the cell aggregate containing glial precursor cells to prepare a cell tissue structure as a three-dimensional tissue.

[0414] As described below, the pharmaceutical composition comprising the cells of the present invention can be obtained in the absence of serum, and therefore does not contain components such as serum-derived components that may cause problems in treatment. When administered to a spinal cord injury model animal (e.g., mouse), it can play a significant effect of repairing the damaged nervous system in the injured part and restoring motor function. Therefore, the pharmaceutical composition of the present invention is useful as a therapeutic drug for demyelinating diseases and diseases based on or accompanied by damaged glial cells, such as acute, subacute, or chronic spinal cord injuries, which have not had effective treatment means so far, and diseases based on or accompanied by damaged glial cells in the acute, subacute, or chronic phases.

[0415] Moreover, the cell group for transplantation formed by the cell aggregates comprising glial precursor cells recorded in this specification is prepared by the pluripotent stem cells of the strain system, and is identified and quality controlled by markers, etc., so that a large number of cell groups for transplantation with stable quality can be prepared and used for transplantation. In addition, since the cell group for transplantation can be preserved, the cell group for transplantation can be prepared according to the transplantation time of the patient.

[0416] "Demyelinating disease" is not particularly limited as long as it is an inflammation or a disappearance of myelin that forms myelin around the axons of nerve cells. As demyelinating disease, for example, spinal cord injury, multiple sclerosis, adrenoleukodystrophy, leukoablative encephalopathy, Pelizaeus-Merzbacherdisease (broadly speaking, congenital leukodystrophy) and leukodystrophy can be cited. As the above-mentioned spinal cord injury, acute spinal cord injury, subacute spinal cord injury or chronic spinal cord injury can be cited.

[0417] "Diseases associated with damaged glial cells" (also referred to as "glial cell-based diseases") are not particularly limited as long as they are diseases associated with damaged glial cells, i.e., one or more cell types selected from the group consisting of astrocytes, oligodendrocytes, ependymal cells, and microglia. Diseases associated with damaged glial cells include, specifically, spinal cord injury, cerebral infarction, schizophrenia and other mental and neurological diseases, demyelinating diseases, and neurodegenerative diseases. More specifically, neuromyelitis optica, amyotrophic lateral sclerosis, Parkinson's disease, Alexander disease, congenital leukodystrophy, Huntington's disease, Alzheimer's disease and schizophrenia, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), Wallerian degeneration, adrenoleukodystrophy, macular hole, spinocerebellar degeneration, multiple system atrophy, and various nerve damage and neurodegenerative states can be cited. The situation that a certain time has passed since the onset of the above diseases is called a chronic demyelinating disease or a chronic disease with glial cell damage. For example, for spinal cord injury, many cases in which very hard scar tissue is formed at the injured part after 3 to 6 months after injury and the condition is fixed are observed, and the condition is called a chronic spinal cord injury.

[0418] In diseases involving damage to glial cells, examples of the damaged state of the membrane tissue containing glial cells include a state in which glial cells such as oligodendrocytes and astrocytes degenerate and die.

[0419] [Treatment methods and medications]

[0420] As one embodiment of the present invention, a method for treating demyelinating diseases and diseases based on or accompanied by damaged glial cells can be cited, the method comprising transplanting an effective dose of a cell aggregate comprising glial precursor cells of the present invention, or a cell group comprising oligodendrocytes, astrocytes and neurons of the present invention, or a cell group for transplantation comprising cells derived from the cell aggregate or the cell group to an object requiring transplantation. Here, demyelinating diseases and diseases based on or accompanied by damaged glial cells may also be demyelinating diseases in the chronic phase and diseases based on or accompanied by damaged glial cells in the chronic phase. It should be noted that the situation after a certain period of time from the onset of the damage or disease (for example, in the case of chronic spinal cord injury, the situation after more than 3 to 6 months after the injury) is judged as the chronic phase.

[0421] The cell aggregates containing glial precursor cells of the present invention are useful for transplantation medicine for demyelinating diseases and diseases accompanied by damaged glial cells. Therefore, the present invention provides a therapeutic drug for demyelinating diseases and diseases accompanied by damaged glial cells, which comprises the cell aggregates of the present invention, or a cell population for transplantation comprising cells obtained from the cell aggregates.

[0422] In addition, the present invention also provides a treatment method, which includes administering (transplanting) the therapeutic drug to a patient in the form of a cell aggregate or a suspension thereof, a suspension of dispersed cells, a suspension formed with biodegradable biomaterials such as fibrin and hydrogel, and a cell tissue structure cultured with them as a scaffold. As a therapeutic drug for demyelinating diseases and diseases accompanied by damaged glial cells, or in order to supplement the damaged site in the damaged state of the tissue containing glial cells, the cell aggregate (cell group) of the present invention or a cell group for transplantation containing cells obtained from the cell aggregate (cell group) can be used.

[0423] Demyelinating diseases and diseases accompanied by glial cell damage, or damaged states of glial cell-containing tissues that require transplantation can be treated by transplanting the cell aggregates of the present invention or a transplantation cell population containing cells obtained from the cell aggregates, or by supplementing the damaged glial cell-containing tissues themselves.

[0424] In transplant medicine, the rejection reaction caused by the difference of tissue compatibility antigens often becomes a problem, and this problem can be overcome by using the pluripotent stem cells (such as induced pluripotent stem cells) established by the somatic cells of the transplanted recipient. That is, in a preferred embodiment of the present invention, by using the pluripotent stem cells (such as induced pluripotent stem cells) established by the somatic cells of the recipient as pluripotent stem cells, the cell aggregates (cell groups) of the recipient itself in immunology are prepared, and the cell aggregates (cell groups) or the transplantation cell groups comprising the cells obtained by the cell aggregates (cell groups) are transplanted to the recipient.

[0425] In addition, allogeneic (allo) (xenogeneic) cell aggregates (cell populations) can be prepared from pluripotent stem cells (e.g., induced pluripotent stem cells) established from somatic cells of other people that are immunocompatible (e.g., HLA type or MHC type compatible) with the recipient, and the cell aggregates (cell populations) or cell populations for transplantation containing cells obtained from the cell aggregates (cell populations) can be transplanted into the recipient.

[0426] In addition, by preparing the cell aggregate (cell population) of the present invention using iPS cells in which expression of tissue compatibility antigens (e.g., antigen proteins constituting HLA class I and HLA class II) or factors required for the expression of the antigens is suppressed, rejection reactions can be avoided even in allogeneic cell transplantation.

[0427] In the therapeutic method of the present invention, the above-mentioned pharmaceutical composition can be used as a therapeutic drug to be administered or transplanted into a patient or a recipient.

[0428] As one embodiment of the present invention, the use of the cell aggregates containing glial precursor cells of the present invention, or the cell populations containing oligodendrocytes, astrocytes and neurons of the present invention, can be cited for use in the treatment of demyelinating diseases, diseases based on glial cell damage and diseases accompanied by glial cell damage.

[0429] 〔Toxicity and efficacy evaluation methods〕

[0430] As one embodiment of the present invention, there can be cited a method for evaluating the toxicity or efficacy of a test substance, which comprises contacting a cell aggregate comprising glial precursor cells of the present invention, or a cell group comprising oligodendrocytes, astrocytes and neurons of the present invention with a test substance, and detecting or quantifying the effect of the test substance on the cell aggregate or the cell group.

[0431] The cell aggregates comprising glial precursor cells and the cell populations comprising oligodendrocytes, astrocytes and neurons of the present invention can be used as disease model cells for the screening and efficacy evaluation of therapeutic drugs for demyelinating diseases and diseases based on glial cell damage and diseases accompanied by glial cell damage (collectively referred to as glial diseases), or their preventive drugs.

[0432] In addition, the cell aggregates comprising glial precursor cells of the present invention, and the cell groups comprising oligodendrocytes, astrocytes and nerve cells can be used as healthy model cells for safety tests, load tests, toxicity tests, side effect tests, infection or mixing tests of chemical substances, etc. In addition, the cell aggregates or cell groups of the present invention, because they include glial precursor cells such as oligodendrocyte precursor cells and astrocyte precursor cells, neural precursor cells, oligodendrocytes, astrocytes, etc., can also be used for functional tests of neural tissues (central nervous system, spinal cord, muscle nerve junctions) comprising the above-mentioned cells, specifically, can be used to evaluate the functions of 5-hydroxytryptamine nerves, motor nerves, etc., the proliferation ability and differentiation ability of glial and neural precursor cells.

[0433] As its evaluation method, in addition to stimulation and toxicity tests such as apoptosis evaluation, tests for evaluating the influence of chemical substances on the normal differentiation of glial precursor cells into glial cells (expression protein analysis based on RT-PCR of various gene markers, ELISA of cytokines, phagocytosis test, etc.) can be cited. For example, compounds that can be used to explore the ability to promote differentiation into oligodendrocytes, compounds and proteins that rescue disease-specific phenotypes in cells differentiated from iPS cells of patients with demyelinating diseases, glial diseases, etc.

[0434] In addition, as a cell material used in the above test, for example, a culture plate, a cell suspension, a sheet thereof, or a formed body thereof in which cells of the cell aggregate of the present invention are dispersed and inoculated and adsorbed can be provided.

[0435] The cell aggregates containing glial precursor cells and the cell groups containing oligodendrocytes, astrocytes and neurons of the present invention, or cells obtained by dispersing them, can be used for extrapolation tests of human and animal tests.

[0436] [New marker for evaluation and identification of cell aggregates containing glial precursor cells]

[0437] One embodiment of the present invention includes a method for determining whether a cell aggregate containing glial precursor cells is suitable for transplantation, using the expression of one or more markers selected from the group consisting of C10RF61 and SERPINE2 as an indicator.

[0438] Regarding the discrimination method of the present invention, whether C1ORF61 or SERPINE2 is expressed can be implemented by detecting the protein, mRNA or fragments of these markers. That is, as one embodiment, the discrimination method of the present invention includes the following steps:

[0439] Step (1), detecting proteins or fragments thereof derived from one or more markers selected from the group consisting of C1ORF61 and SERPINE2 expressed in a cell aggregate sample;

[0440] Step (2), when the expression level of the protein or fragment thereof derived from the marker is greater than the reference value, the cell aggregate sample is determined to be a cell aggregate containing glial precursor cells suitable for transplantation.

[0441] In addition, as one embodiment, the identification method of the present invention includes the following steps:

[0442] Step (1), detecting mRNA or fragments thereof derived from one or more markers selected from the group consisting of C10RF61 and SERPINE2 expressed in a cell aggregate sample;

[0443] Step (2), when the expression level of the mRNA or its fragment derived from the above marker is greater than the reference value, the cell aggregate sample is determined to be a cell aggregate containing glial precursor cells suitable for transplantation.

[0444] As one embodiment of the present invention, there is provided a method for identifying glial precursor cells or neural stem cells with high glial differentiation tropism, which comprises 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 tropism" refers to the property of neural stem cells that have a high ratio (e.g., 30% or more) of differentiation into glial cells when terminal differentiation is induced in a maturation medium.

[0445] Regarding the identification method of the present invention, whether C1ORF61 or SERPINE2 is expressed can be implemented by detecting the protein, mRNA or fragments of these markers. That is, as one embodiment, the identification method of the present invention comprises the following steps:

[0446] Step (1), detecting proteins or fragments thereof derived from one or more markers selected from the group consisting of C10RF61 and SERPINE2 expressed in a cell population sample;

[0447] In step (2), when the expression level of the protein or fragment derived from the marker is greater than the reference value, the cell population sample is identified as a glial precursor cell or a neural stem cell with a high glial differentiation tropism.

[0448] In addition, as one embodiment, the identification method of the present invention comprises the following steps:

[0449] Step (1), detecting mRNA or fragments thereof derived from one or more markers selected from the group consisting of C10RF61 and SERPINE2 expressed in a cell population sample;

[0450] In step (2), when the expression level of the mRNA or fragment thereof derived from the marker is greater than the reference value, the cell population sample is determined to be a glial precursor cell or a neural stem cell with a high glial differentiation tropism.

[0451] Table 1 below shows the genes described in this specification and their GenBank accession numbers.

[0452] [Table 1]

[0453]

[0454]

[0455]

[0456] Example

[0457] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0458] <Example 1 Preparation of cell aggregates>

[0459] As an iPS cell line, QHJI01s04, a clinical-grade peripheral blood-derived feeder-free iPS cell line established by the iPS Cell Research Institute of Kyoto University, was used. Before differentiation, the cells were maintained in StemFit (registered trademark) AK03N medium (manufactured by Ajinomoto Co., Inc.) supplemented with laminin 511E8 fragment (iMatrix-511E8; manufactured by Nippi. Inc.) (0.5 mg / ml).

[0460] In order to induce iPS cell differentiation into neural progenitor cells, a rapid aggregation serum-free suspension culture method (SFEBq method) was used to form embryoid bodies. Specifically, the QHJI01s04 strain maintained in culture was separated into single cells using 0.5×TrypLE Select (Thermo Fisher Scientific) / 0.25mM EDTA (ethylenediaminetetraacetic acid) / PBS (phosphate buffered saline, Thermo Fisher Scientific) and inoculated into a 96-well low-adsorption culture plate (trade name: PrimeSurface (registered trademark) 96V plate, Sumitomo Bakelite Co., Ltd.) at 9000 cells / well. The inoculation day was set as differentiation day 0, and induction was started at 37°C under 5% CO2 / 5% O2. The embryoid body formation medium (AK03N-C medium) supplemented with each factor shown in Table 2 was used, and half of the medium was replaced every day.

[0461] [Table 2]

[0462]

[0463] On the 14th day of differentiation, embryoid bodies were collected from the 96-well plate and washed once with DMEM / F-12 (trade name: D-MEM / Ham's F-12; Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, manufactured by FUJIFILM Wako Pure Chemical Corporation). Then, from the 14th day to the 21st day of differentiation, low-attachment culture bottles (trade name: Ultra-Low Attachment 75 cm) were used under conditions 1, 2, 3, and 4 shown in Table 3. 2Rectangular Canted Neck Cell Culture Flask with Vent Cap, manufactured by CORNING), and the culture medium was replaced every 3 days at 37°C under 5% CO2 / 20% O2 for 7 days.

[0464] [Table 3]

[0465] condition Culture medium factor 1 Neuronal and glial proliferation culture medium 1 μM retinoic acid (RA, Sigma-Aldrich) 2 Neuronal and glial proliferation culture medium 1 μM Purmorphamine (PM, manufactured by Millipore) 3 Neuronal and glial proliferation culture medium 1μM RA and 1μM PM 4 AK03N-C medium 1μM RA and 1μM PM

[0466] As the medium for neuroglia proliferation in Table 3, DMEM / F-12 (containing 3.15 lg / L glucose, 15 mM HEPES, 2.5 mM L-glutamine, and 0.5 mM sodium pyruvate, manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with 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 The culture medium contained EGF (Epidermal Growth Factor, manufactured by PeproTech) and 20 ng / ml FGF2 (Fibroblast Growth Factor 2, manufactured by PeproTech).

[0467] On the 21st day of differentiation, embryoid bodies were recovered, dispersed into single cells using TrypLE Select, and then suspended in a tissue culture flask (manufactured by Thermo Fisher Scientific) using a neuro-glial proliferation medium containing 1 μM PM, with the medium replaced once every 7 days (passage number 1). After 14 days, at the 35th day of differentiation, the embryoid bodies were dispersed into single cells again and suspended in a neuro-glial proliferation medium (passage number 2). Figure 1A Bright field images at days 35 and 49 of differentiation are shown.

[0468] In addition, in order to confirm the terminal differentiation ability of the cell aggregates obtained on the 49th day of differentiation, the cell aggregates were inoculated in an 8-well chamber slide (manufactured by IWAKI) treated with 1% Matrigel Basement Membrane Matrix Growth Factor Reduced (manufactured by CORNING) diluted with DMEM / F-12, adhered to Matrigel, and cultured in a maturation medium for 14 days to allow terminal differentiation. As a maturation medium, a medium in which B27 supplement, 1% non-essential amino acids (manufactured by ThermoFisher Scientific), 60ng / ml T3, 10ng / ml NT-3, 25ng / ml CNTF, and 10ng / ml LIF were added to KBM neural stem cells (manufactured by KOHJIN BIO) was used.

[0469] Using the cell of terminal differentiation the 14th day (differentiation the 63rd day), each marker was subjected to immunofluorescence staining.Immunofluorescence staining used 6 kinds of primary antibodies and corresponding fluorescently labeled secondary antibodies.Primary antibodies are as shown in Table 4, each mixed with 3 kinds, diluted with blocking buffer, and 2 kinds of primary antibody solutions (primary antibody solution 1 and primary antibody solution 2) were prepared.Secondary antibodies are also as shown in Table 4, each mixed with 3 kinds, further added with Hoechst33342 (dilution factor 1: 1000, Tongren Chemical Co., Ltd. system) for nuclear staining, diluted with blocking buffer, and 2 kinds of secondary antibody solutions (secondary antibody solution 1 and secondary antibody solution 2) were prepared.

[0470] [Table 4]

[0471]

[0472] * : Dilution factor 1:1000, manufactured by Thermo Fisher Scientific

[0473] For immunofluorescence staining, on the 14th day of terminal differentiation (differentiation day 63), each cell obtained in conditions 1, 2, 3 and 4 was fixed at room temperature for 25 minutes using 4% paraformaldehyde (PFA, manufactured by FUJIFILM Wako Pure Chemical Corporation), washed 3 times with PBS, and incubated at room temperature for 1 hour with 10% goat serum (manufactured by FUJIFILM Wako Pure Chemical Corporation) / PBS as a blocking solution. Then, the primary antibody solution (primary antibody solution 1 or primary antibody solution 2) shown in Table 4 above was added, and co-staining was performed by incubation at 4°C overnight. Then, the cells were washed 3 times with PBS, and the secondary antibody solution corresponding to the primary antibody solution (secondary antibody solution 1 or secondary antibody solution 2) was further added, and incubated at room temperature for 1 hour. Then, the cells were washed 3 times with PBS and stored at 4°C.

[0474] Microscope observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by KEYENCE). Figure 1B Immunofluorescence staining images are shown. It should be noted that Merge is an image with three types of fluorescence superimposed. Neurons, astrocytes, and oligodendrocytes were detected under any of conditions 1, 2, 3, and 4, but at the 14th day of terminal differentiation under condition 1, the morphology of O4-positive cells was immature, and no GalC-positive cells were detected, so maturation was slow; under condition 2, a GFAP-non-positive cell population was detected, which shows that condition 3 or condition 4 is the best condition.

[0475] Based on the above studies, a method for preparing cell aggregates containing glial precursor cells was established ( Figure 2 ). It should be noted that Figure 2 In the above, the embryoid body formation medium is AK03N-C medium, the neuroglia proliferation medium is the above-mentioned neuroglia proliferation medium, S of the compound represents SB431542, L represents LDN193189, C represents CHIR99021, Y represents Y-27632, RA represents retinoic acid, and PM represents purmorphamine.

[0476] <Example 2 Evaluation of cell aggregate characteristics 1 (Gene expression changes during differentiation induction)>

[0477] In order to study the changes in gene expression during differentiation induction, gene expression analysis based on RNA sequencing was performed on the intermediate products of differentiation induction. Differentiation induction was started using the QHJI01s04 strain according to the method of condition 3 of 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 (manufactured by QIAGEN). Libraries were prepared using Illumina's TruSeq Stranded mRNA Library Prepkit, and 80 cycles of sequencing were performed using HiSeq 2500. Gene expression analysis was performed based on data with more than 30 million reads per cell ( Figures 3A to 3D ).

[0478] The gene expression profiles before differentiation induction (iPS cells) and on day 7 of differentiation were compared. The results showed that the expression levels of NANOG and POU5F1 (OCT3 / 4), which are pluripotency markers, decreased significantly on day 7 of differentiation. In addition, the expression levels of SOX1, PAX6, HES4, and HES5, which are neural stem cell markers, increased significantly on day 7 of differentiation ( Figure 3A ). The gene expression profiles on the 7th day of differentiation and the 14th day of differentiation were compared. As a result, ASCL1, HEY1, DCX, βIII tubulin (TUBB3), ELAVL3, ZBTB20, SLIT1, HOXB3, HOXA4, HOXB4, HOXB6, HOXB8, etc. were listed as genes with increased gene expression on the 14th day of differentiation compared with the 7th day of differentiation ( Figure 3B ). In addition, the gene expression profiles on the 14th day of differentiation and the 21st day of differentiation were compared. As a result, genes whose gene expression increased on the 21st day of differentiation compared with the 14th day of differentiation included HEY2, NKX6.2, NKX2.2, OLIG1, OLIG2, etc. ( Figure 3C ). The gene expression profiles on the 21st day of differentiation and the 35th day of differentiation were compared. As a result, genes with increased expression levels on the 35th day of differentiation compared to the 21st day of differentiation included NFIA, NFIB, SLC1A3, S100B, and FABP7 ( Figure 3D In addition, the expression of PAX6 was significantly decreased ( Figure 3A ).

[0479] <Example 3 Evaluation of characteristics of cell aggregates 2 (Immunofluorescence staining of cell aggregates)>

[0480] As iPS cell strain, using the Ff-WJ14s01 strain of the umbilical cord blood-derived feeder-free iPS cell strain established by the iPS cell research institute of Kyoto University, the cell aggregates comprising glial precursor cells were prepared by the method of condition 3 of embodiment 1, and the immunofluorescence staining for each differentiation marker was implemented. For immunofluorescence staining, 8 kinds of primary antibodies and corresponding fluorescently labeled secondary antibodies were used. Primary antibodies are as shown in Table 5, each mixed with 2 or 3 kinds, diluted with blocking solution, and 3 kinds of primary antibody solutions (primary antibody solution 1, primary antibody solution 2, primary antibody solution 3) were prepared. For secondary antibodies, also as shown in Table 5, each mixed with 2 or 3 kinds, further added Hoechst33342 (dilution factor 1: 1000, Tongren Chemical Co., Ltd. system) for nuclear staining, diluted with blocking solution, and 3 kinds of secondary antibody solutions (secondary antibody solution 1, secondary antibody solution 2, secondary antibody solution 3) were prepared.

[0481] [Table 5]

[0482]

[0483] ＊ : Dilution factor 1:1000, manufactured by Thermo Fisher Scientific

[0484] At the time point of differentiation day 77, the cell aggregates were fixed at room temperature for 20 minutes using 4% PFA and washed 3 times with PBS. The fixed cells were cryo-embedded on dry ice using OCT embedding agent (manufactured by Sakura Finetek Japan Co., Ltd.), and 10 μm thick frozen sections were made and stored at -80 ° C. After the frozen sections were washed once with PBS, 10% goat serum (manufactured by FUJIFILM Wako Pure Chemical Corporation) / PBS was incubated at room temperature for 1 hour as a blocking solution. Then, the primary antibody solution (primary antibody solution 1, primary antibody solution 2 or primary antibody solution 3) shown in Table 5 above was added, incubated overnight at 4 ° C, and co-stained. Then, it was washed 3 times with PBS, and the secondary antibody solution corresponding to the primary antibody solution (secondary antibody solution 1 or secondary antibody solution 2 or secondary antibody solution 3) was further added, and incubated at room temperature for 1 hour. After sealing with the sealing agent PermaFluor (manufactured by ThermoFisher Scientific), it was stored at 4 ° C.

[0485] Microscope observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by KEYENCE). Figure 4AImmunofluorescence staining images are shown. GFAP-positive cells, NESTIN-positive cells, Tuj1-positive cells, OLIG2-positive cells, ELAVL3-positive cells, MAP2-positive cells, and NFIA-positive cells were observed. In addition, the analysis software (BZ-H3C) included with BZ-X710 was used to quantify the ratio of OLIG2-positive cells and NFIA-positive cells relative to the total number of cells constituting one sphere (cell aggregate), and the average value of the six spheres was calculated. As a result, OLIG2-positive cells were present at a ratio of 30.9±6.6%, and NFIA-positive cells were present at a ratio of 87.6±1.8% ( Figure 4B ).

[0486] <Example 4 Evaluation of cell aggregate characteristics 3 (single cell gene expression analysis)>

[0487] The characteristic genes of cell aggregates containing glial precursor cells were studied. For cell aggregates containing glial precursor cells (Gliogenic NPCs) prepared from the Ff-WJ14s01 strain by the method of condition 3 of Example 1, and cell aggregates containing a large number of cells with high neural differentiation orientation (Neurogenic NPCs) prepared from the Ff-WJ14s01 strain by the method of non-patent literature 2, single-cell RNA sequencing (RamDA-seq) was performed using the method described in "Hayashi et al., Nature Communications, volume 9, Article number 619 (2018)". Using Illumina's HiSeq2500, 50 cycles of sequencing were performed in Single-End mode. Gene expression analysis was performed on 150 Gliogenic NPCs and 91 Neurogenic NPCs (a total of 241) that passed the quality evaluation.

[0488] Based on the gene expression profile, 241 cells were classified into three clusters (C1 to C3). 91.2% of the total number of Neurogenic NPC cells analyzed were classified as C1, 8.8% were classified as C3, and none were classified as C2. On the other hand, 5.3% of the total number of Gliogenic NPC cells analyzed were classified as C1, 58.0% were classified as C2, and 36.7% were classified as C3. Figure 5 ). This suggests that the gene expression profiles of Neurogenic NPCs and Gliogenic NPCs are very different.

[0489] Table 6 shows the genes characteristically expressed in each cluster (ranked top 10). As genes highly expressed in cells belonging to C2, where only GliogenicNPCs were classified, HEY2, C1ORF61, FAM181B, NFIB, ITM2B, NFIA, LHFPL3, and MLC1 were found (Table 6, Fig. 6A , Figure 6B ). In addition, ASCL1, MEIS1, MEIS2, DLL3, HEY1, ZBTB20, SOX9, SLC1A3, S100B, SLIT1, SPARCL1, TIMP3, TIMP4, SPON1, KLF9, GRIK3, SOX6, EPHA3, NTRK2, GRIA2, PTPRO, KCND3, SERPINE2 ( Fig. 6A , Figure 6B ). It should be noted that the GenBank accession numbers of some of the genes listed in Table 6 are shown in Table 1. In addition, LOC541471 is described in Oncol Lett. 2019 Feb; 17(2): 2457-2464.

[0490] [Table 6]

[0491]

[0492] <Example 5 Evaluation of cell aggregate characteristics 4 (Evaluation of terminal differentiation ability)>

[0493] For cell aggregates containing glial precursor cells, whether they differentiate into three types of neurons, astrocytes, and oligodendrocytes after terminal differentiation was studied by immunofluorescence staining. For cell aggregates containing glial precursor cells derived from the Ff-WJ14s01 strain prepared by the method of condition 3 of Example 1 (differentiation day 77), terminal differentiation induction was performed using a maturation medium by the method described in Example 1. 31 days after terminal differentiation (differentiation day 108), the cells were fixed at room temperature for 25 minutes using 4% PFA by the method described in Example 1, washed three times with PBS, and then immunofluorescence staining was performed. Microscope observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by KEYENCE) and a confocal fluorescence microscope LSM880 (manufactured by ZEISS). As a result, Tuj1 and MAP2-positive neurons, GFAP-positive astrocytes, and O4-positive, GalC-positive, and MBP-positive oligodendrocytes were detected. It can be seen that the ability to differentiate into three types of cells was confirmed ( Fig. 7AIn addition, the analysis software Developer Toolbox (manufactured by GE Healthcare) of In Cell Analyzer was used to quantify the positive cell rate. As a result, the positive cell rate was about 10% for O4 positive cells, about 30% for GFAP positive cells, and about 30% for Tuj1 positive cells ( Figure 7B ).

[0494] <Example 6 Evaluation of cell aggregate characteristics 5 (Changes in gene expression before and after terminal differentiation>

[0495] For the cell aggregates comprising glial precursor cells, the gene expression levels of each differentiation cell marker before and after terminal differentiation are analyzed. For the cell aggregates comprising glial precursor cells prepared by QHJI01s04 strain using the method of condition 3 of embodiment 1 (differentiation the 48th day), using the method described in embodiment 1, maturation medium is used to induce terminal differentiation. Using RNeasy Plus Mini kit (QIAGEN company system), total RNA is extracted from cells before terminal differentiation (differentiation the 48th day) and after terminal differentiation 34 days (differentiation the 82nd day). Using SuperScript III First-StrandSynthesis System for RT-PCR (Thermo Fisher Scientific company system), the total RNA extracted is reverse transcribed into cDNA. Then, using Fast SYBR (trademark) Green Master Mix (manufactured by Thermo Fisher Scientific), 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 Step One Plus Realtime PCR System (manufactured by Thermo Fisher Scientific). It should be noted that 3-phosphoglyceraldehyde dehydrogenase (GAPDH) was used as an endogenous control.

[0496] Table 7 shows the primer sets used in the target gene amplification reaction. Figure 8Results are shown. The relative expression of the expression level after terminal differentiation relative to the expression level before terminal differentiation (set to 1) is expressed. In the cells after terminal differentiation, the expression levels of astrocyte markers and oligodendrocyte markers increased, suggesting that they were differentiated into mature astrocytes and oligodendrocytes by terminal differentiation induction based on maturation medium. The gene expression levels of βIII tubulin and MAP2, which are neural markers, hardly changed before and after terminal differentiation, and according to the results of immunofluorescence staining of the cell aggregates studied in Example 3, it can be considered that the cell aggregates containing glial precursor cells express βIII tubulin and MAP2 from the time point of the cell aggregates before terminal differentiation, and also maintain expression after terminal differentiation.

[0497] [Table 7]

[0498]

[0499] <Example 7 Transplantation experiment of cell aggregates containing glial precursor cells in subacute spinal cord injury model mice>

[0500] NOD-SCID (NOD.CB17-Prkdcscid·J) mice (Charles River Laboratories) were treated in accordance with the animal experiment operation guidelines of Keio University and NIH. The animal experiment plan was approved by the Animal Experiment Management Committee of Keio University. Anesthesia was performed by intraperitoneal administration of ketamine (100 mg / kg) and xylazine (10 mg / kg) to female NOD-SCID mice aged 8 weeks. A crush injury was applied to the level of the 10th thoracic vertebra using an IH impactor (60-70 kdyn, manufactured by Precision Systems and Instrumentation) according to the method described in "Scheff et al., Journal of neurotrauma 20, 179-193 (2003)", and then ampicillin (12.5 mg / kg) was immediately injected intramuscularly. Seven days after the injury, the motor function of the hind limbs was evaluated using the Basso Mouse Scale (BMS) according to the method described in "Basso et al., Journal of Neurotrauma, 23: 635-59 (2006)". At the time point of 7 days after the injury, individuals that naturally recovered to a BMS of 2.5 or more could not be measured for the effect of cell transplantation, so they were excluded and divided into two groups (12 in the control group and 14 in the cell transplantation group), and the grouping was performed in a manner that the average BMS of each group was the same.

[0501] On the 9th day after injury (subacute phase), 2 μl of the cell suspension was transplanted into the center of the injury in the cell transplantation group using a fixed injection system (manufactured by Muromachi Machinery Co., Ltd.) with a 28G metal needle, and 2 μl of PBS was transplanted into the center of the injury in the control group. The cells were cell aggregates (5.0×10 5 Cells) were aggregated in 2 μl of PBS for transplantation. Various motor function evaluations were performed until 12 weeks after transplantation. In addition, at 12 weeks after transplantation, the spinal cord was fixed by perfusing 4% PFA into the heart under deep anesthesia and stored at 4°C. Then, the fixed spinal cord was stored in 30% sucrose / PBS at 4°C, cryo-embedded using OCT embedding agent, and sagittal sections (14 μm thick) and cross-sectional sections (16 μm thick) were made and stored at -80°C. For tissue staining, hematoxylin-eosin (HE) staining for observing tissue images and myelin staining solution (Luxol Fast Blue) (LFB) staining for myelin tissue staining were performed.

[0502] In addition, for more detailed tissue evaluation, immunofluorescence staining was performed. After washing with PBS three times, Blocking One (manufactured by NACALAI TESQUE) containing 0.1% TritonX-100 was used as a blocking solution and incubated at room temperature for 1 hour. Then, a primary antibody solution was prepared by diluting the primary antibody with the blocking solution and incubated at 4°C overnight. The primary antibodies used in this example are human anti-Hu antibody (dilution factor: 1:1000, provided by Dr. Robert Darnell of Rockefeller University), mouse anti-GFAP antibody (dilution factor 1:5000, manufactured by Abcam), mouse anti-APC antibody (dilution factor 1:300, manufactured by Abcam), rabbit anti-human specific Nestin antibody (dilution factor 1:200, manufactured by IBL), rabbit anti-Ki67 antibody (dilution factor 1:1000, manufactured by Leica Biosystems), mouse anti-OCT3 / 4 antibody (dilution factor 1:100, manufactured by Santa Cruz), mouse anti-human specific nuclear antibody (HNA) (dilution factor 1:100, manufactured by Millipore), mouse anti-human specific cytoplasmic antibody (STEM121) (dilution factor 1:100, manufactured by Millipore), mouse anti-human Tau antibody (dilution factor 1:500, manufactured by ThermoFisher Scientific), mouse anti-Bassoon antibody (dilution factor 1:200, GeneTech), mouse anti-human synaptophysin antibody (dilution factor 1:200, Millipore). Then, after washing with PBS three times, further prepare a secondary antibody solution by diluting the Alexa Fluor labeled secondary antibody corresponding to the primary antibody and Hoechst33342 for nuclear staining (dilution factor 1:1000, Tongren Chemical Co., Ltd.) with blocking solution, and incubate at room temperature for 1 hour. After washing with PBS three times, use the sealing agent PermaFluor (Thermo Fisher Scientific) to seal and store at 4°C. Use fluorescence microscope BZ-X710 (KEYENCE) and confocal fluorescence microscope LSM700 (ZEISS) to observe the samples and collect images.

[0503] <Example 8 Evaluation of the effectiveness of cell aggregates containing glial precursor cells on subacute spinal cord injury model mice> (BMS score)

[0504] In order to evaluate the effectiveness of transplanted cells in the experiment described in Example 7, hind limb motor function evaluation based on BMS score was performed every 7 days until 12 weeks after transplantation. From the time point of 14 days after injury (5 days after transplantation) to the final day of evaluation on the 91st day after injury (82 days after transplantation), the BMS score of the cell transplantation group was significantly increased compared with the control group (*: p < 0.05) ( Fig. 9 ).

[0505] <Example 9 Evaluation of the effectiveness of cell aggregates containing glial precursor cells on subacute spinal cord injury model mice (rotarod test)>

[0506] At 12 weeks after transplantation, motor coordination was evaluated (rotarod test). The time that the rats could walk on a rotarod (Muromachi Machinery Co., Ltd.) that rotated 20 times per minute was measured. The rats in the cell transplantation group could walk on the rotarod for a significantly longer time than the control group (*: p < 0.05) ( Fig.10 ).

[0507] <Example 10 Evaluation of the effectiveness of cell aggregates containing glial precursor cells on subacute spinal cord injury model mice (gait pattern evaluation)>

[0508] At 12 weeks after transplantation, gait pattern evaluation was performed using the DigiGait system (Mouse Specific). Mice were allowed to walk on a treadmill (7 cm / sec), and stride length and walking angle were measured. The stride length of the cell transplantation group was significantly longer than that of the control group (**: p<0.01) ( Fig.11A In addition, the angle of the limbs of the cell transplant group relative to the walking direction was significantly smaller than that of the control group (*: p < 0.05), suggesting that the two limbs were parallel to the ground, which is closer to the normal walking pattern ( Fig. 11B ).

[0509] <Example 11 Evaluation of the effectiveness of cell aggregates containing glial precursor cells on subacute spinal cord injury model mice (kinematic analysis)>

[0510] At 12 weeks after transplantation, the three-dimensional motion analysis software KinemaTracer (manufactured by KISSEI COMTEC) was used to evaluate joint movement. Mice with labels on the shoulder joint, hip joint, knee joint, ankle joint, and toes were made to walk on a treadmill, and videos were taken from four directions, namely, two sides and front and back, using a high-speed digital camera (manufactured by GoPro) to visualize the trajectory of each marker in three dimensions. Fig.12(A) shows a representative example of the trajectory of the hind limbs from leaving the ground to touching the ground. Compared with the control group, the limbs of the cell transplantation group were farther from the ground and showed a smoother trajectory. Fig.12 (B) shows the changes in the joint angles of the hip joint, knee joint, ankle joint, and toe joints during one walking cycle. Six individuals were analyzed with 5 steps, and the thin lines in each figure show the average joint angle changes of 5 steps of one individual, and the thick lines show the average values ​​of 6 individuals. This suggests that the cell transplantation group has less variation in joint motion each time it walks than the control group.

[0511] <Example 12 Evaluation of Spinal Cord Tissue in Subacute Spinal Cord Injury Model Mice>

[0512] At 12 weeks after transplantation, sagittal sections were immunofluorescently stained with STEM121 antibody ( Fig.13 (A)) to detect transplanted cells. Transplanted cells were detected in a wide range including the area far from the center of the injury, confirming the engraftment of the transplanted cells. In addition, HE staining was performed on adjacent sections to observe a wide range of tissue images from the head to the tail, and no tumor-like structure was observed ( Fig.13 In addition, immunofluorescence staining was performed using anti-HNA antibodies on cross-sectional sections at the center of the injury (2), 4 mm headward from the center of the injury (1), and 4 mm caudal from the center of the injury (3). Fig.13 (C) HNA-positive cells were detected in the lesion center and in sections 4 mm cephalad and 4 mm caudal, which were the transplanted sites, suggesting that the transplanted cells migrated over a wide range.

[0513] In order to analyze the characteristics of transplanted cells in more detail, fluorescent immunostaining of human-specific markers and various markers was performed on sagittal sections. First, according to reports that iPS cell-derived neural progenitor cells are OCT3 / 4 positive when they evolve into tumors (non-patent literature 8), immunofluorescence staining was performed using anti-OCT3 / 4 antibodies and anti-Ki67 (proliferation marker) antibodies, and anti-HNA antibodies. In addition, in order to analyze the characteristics of transplanted cells, immunofluorescence staining was performed using anti-NESTIN (neural stem cell marker) antibodies, anti-Hu (neural marker) antibodies, anti-GFAP (astroglial marker) antibodies, anti-APC (oligodendrocyte marker) antibodies, and anti-HNA antibodies, and positive cells were quantified.

[0514] As a result, no HNA was detected + / OCT3 / 4 + co-positive cells, suggesting that the transplanted cells did not evolve into tumors ( Fig.14A ). + / Ki67 +The total positive cells were 4.71±0.20%, suggesting that there were proliferative cells in the transplanted cells. + / NESTIN + The total positive neural progenitor cells were 17.02±3.10%, HNA + / Hu + The total positive neurons were 18.12±1.22%, HNA + / GFAP + The total positive astrocytes were 27.23±1.92%, HNA + / APC + The total number of positive oligodendrocytes was 36.56±2.82% ( Fig. 14B ). This suggests that at 91 days after transplantation, although some immature neural progenitor cells and glial progenitor cells still exist, most of the transplanted cells differentiate into three types: neurons, astrocytes, and oligodendrocytes.

[0515] Next, to investigate whether neurons derived from transplanted cells form neural circuits with host mouse neurons, sagittal sections were immunofluorescently stained using Tuj1 antibody, anti-HNA antibody, and anti-mouse-specific Bassoon (presynaptic marker) and anti-human synaptophysin (synaptic vesicle protein) antibodies. + / Tuj1 + The co-positive transplanted cell-derived neurons co-localized with mouse-specific Bassoon ( Fig.15 (A)). In addition, HNA - / Tuj1 + The host neurons colocalize with human-specific synaptophysin ( Fig.15 (B)). These results suggest that the host mouse neurons and the transplanted cell-derived neurons form a neural circuit.

[0516] Next, we studied the myelin regeneration effect brought about by cell transplantation. Fig.16A LFB staining images of 0.48 mm on the rostral side, epicenter of injury, and 0.48 mm on the caudal side are shown. For the cross-sectional slices from the epicenter of injury to 0.96 mm on the rostral and caudal sides, myelin tissue was observed using LFB staining, and the LFB-positive area was quantified. As a result, the LFB-positive area in all slices observed in the cell transplantation group was significantly increased compared with the control group (*: p < 0.05; **: p < 0.01) ( Fig.16A , Fig. 16B In addition, the cross-sectional slices were subjected to immunofluorescence staining for mature oligodendrocyte markers MBP and STEM121.+ / MBP + The co-positive region ( Fig.17 ). Furthermore, for more detailed observation, immunoelectron microscopy using the STEM121 antibody was performed on the cross-sectional slices using the method described in "Shibata et al., Frontiers in Neural Circuits, 2019; 13: 29.", and the STEM121 antibody reaction site was detected using a secondary antibody labeled with colloidal gold particles. The myelin sheath was observed under an electron microscope, and colloidal gold particles were detected in the myelin sheath ( Fig.18 (arrow). The above results suggest that mature oligodendrocytes derived from transplanted cells remyelinate nerve axons.

[0517] <Example 13 Transplantation experiment of cell aggregates containing glial precursor cells in rats with chronic spinal cord injury model>

[0518] Nude (F344 / NJcl-rnu / rnu) rats (CLEA, Japan) were treated in accordance with the animal experiment operation guidelines of Keio University and NIH. The animal experiment plan was approved by the Animal Experiment Management Committee of Keio University. Anesthesia was performed by intraperitoneal administration of three mixed anesthesia (medetomidine: 0.375 mg / kg, midazolam: 2 mg / kg, butorphanol: 2.5 mg / kg) to female Nude rats aged 8 weeks. According to the method described in "Scheff et al., Journal of neurotrauma 20, 179-193 (2003)", a crush injury was applied to the level of the 10th thoracic vertebra using an IH impactor (220 kdyn, manufactured by Precision Systems and Instrumentation Co., Ltd.), and after subcutaneous injection of orbifloxacin (5 mg / kg), a medetomidine antagonist (atimepazole: 0.75 mg / kg) was administered intraperitoneally to promote awakening. The motor function of the hind limbs was evaluated using the Basso Beattie Bresnahan (BBB) ​​according to the method described in "Basso et al., Journal of Neurotrauma, 12: 1-21 (1995)" on the day after the injury and every week after the injury. At the time point of 41 days after the injury, individuals that naturally recovered to a BBB of 9 or more could not be measured for the effect of cell transplantation, so they were excluded and divided into 2 groups (13 in the control group and 18 in the cell transplantation group), and the grouping was performed so that the average BBB values ​​of each group were the same.

[0519] On the 42nd day after injury (chronic phase), 2 μl of cell suspension was transplanted into two locations on the head and tail sides of the injury center of the cell transplantation group using a fixed injection system (manufactured by Muromachi Machinery Co., Ltd.) with a 27G metal needle, and 2 μl of PBS was transplanted into two locations on the head and tail sides of the injury center of the control group. The cell suspension was composed of cell aggregates containing glial progenitor cells derived from the QHJI01s04 strain (5.0×10 5 The state in which the cells / 2μl) were aggregated in PBS was used for transplantation. The evaluation of hind limb motor function was performed until 12 weeks after transplantation. In addition, at 12 weeks after transplantation, gait pattern evaluation was performed, and then under deep anesthesia, 4% PFA was perfused from the heart to the whole body to fix and collect the spinal cord, and it was stored at 4°C under 4% PFA immersion. Then, the fixed spinal cord was stored in 30% sucrose / PBS at 4°C, and then cryo-embedded using OCT embedding agent, and sagittal sections (14μm thick) and cross-sectional sections (20μm thick) were made and stored at -80°C. For tissue staining, hematoxylin-eosin (HE) staining and immunofluorescence staining for observing tissue images were performed.

[0520] <Example 14 Evaluation of the effectiveness of cell aggregates containing glial precursor cells in rats with chronic spinal cord injury model> (BBB score)

[0521] In order to evaluate the effectiveness of transplanted cells in the experiment described in Example 13, hind limb motor function evaluation based on BBB score was performed every 7 to 14 days until 12 weeks after transplantation. From the time point of 63 days after injury (21 days after transplantation) to the final day of evaluation on the 126th day after injury (84 days after transplantation), the BBB score of the cell transplantation group was significantly increased compared with the control group (*: p < 0.05, **: p < 0.01) ( Fig.19 ).

[0522] <Example 15 Evaluation of the effectiveness of cell aggregates containing glial precursor cells on chronic spinal cord injury model rats (gait pattern evaluation)>

[0523] At 12 weeks after transplantation, gait pattern evaluation was performed using the DigiGait system (Mouse Specific). Mice were allowed to walk on a treadmill (10 cm / sec), and stride length and walking angle were measured. The stride length of the cell transplantation group was significantly longer than that of the control group (**: p<0.01) ( Fig. 20 In addition, the angle of the limbs of the cell transplant group relative to the walking direction was significantly smaller than that of the control group (**: p < 0.01), suggesting that the two limbs were parallel to the ground, which is closer to the normal walking pattern ( Fig. 20 (B)).

[0524] <Example 16 Evaluation of Spinal Cord Tissue in Chronic Spinal Cord Injury Model Rats>

[0525] HE staining was performed on the sagittal sections prepared by the method described in Example 13, and no tumor-like structure was observed ( Fig.21 In addition, immunofluorescence staining was performed on adjacent sections using anti-HNA antibodies. As a result, HNA-positive cells were detected over a wide range, confirming the engraftment of transplanted cells ( Fig.21 (B)).

[0526] In order to analyze the characteristics of transplanted cells in more detail, the sagittal sections were subjected to fluorescent immunostaining of human-specific markers and various markers by the method described in Example 12. In order to evaluate the tumorigenicity, immunofluorescence staining was performed using anti-OCT3 / 4 antibody and anti-HNA antibody. As a result, no HNA was detected. + / OCT3 / 4 + co-positive cells, suggesting that the transplanted cells did not evolve into tumors ( Fig. 22 In addition, in order to study the terminal differentiation ability of transplanted cells, immunofluorescence staining was performed using anti-Hu (neural marker), anti-GFAP (astroglial marker), anti-APC (oligodendrocyte marker), and anti-HNA antibodies. + / Hu + Co-positive cells ( Fig. 22 (B)), HNA + / GFAP + Co-positive cells ( Fig. 22 (C)), HNA + / APC + Co-positive cells ( Fig. 22 (D)). It can be seen that the transplanted cells differentiated into neurons, astrocytes, and oligodendrocytes in the rat spinal cord.

[0527] <Example 17 Preparation of cell aggregates of uniform size>

[0528] After the cell aggregates containing glial precursor 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 time point of differentiation for 49 days, 10,000 cells / well and 20,000 cells / well were inoculated in 96-well low-adsorption culture plates (trade name: PrimeSurface (registered trademark) 96 slot well plates, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured in a neuro-glial proliferation medium at 37°C under 5% CO2 / 20% O2. As a result, the formation of cell aggregates was confirmed from the day after inoculation. Half of the medium was replaced every 3 days, and the culture was continued for 8 and 14 days ( Fig.23 At 8 and 14 days of culture (57 and 63 days of differentiation), one cell aggregate was transferred to one well of a 96-well plate (CORNING) treated with 1% Matrigel Matrix Basement Membrane Growth Factor Reduced (CORNING) diluted in DMEM / F-12, and cultured in a maturation medium for 28 days to induce terminal differentiation.

[0529] At the time point of 28 days of terminal differentiation (differentiation 85 days and 91 days), fix with 4% PFA at room temperature for 25 minutes, wash with PBS 3 times, and incubate with 10% goat serum (manufactured by FUJIFILM Wako Pure Chemical Corporation) / PBS as blocking solution at room temperature for 1 hour. Then, add the primary antibody solution 3 shown in Table 4 above, incubate at 4°C overnight, and perform co-staining. Then, wash with PBS 3 times, further add the secondary antibody solution 3 corresponding to the primary antibody solution 3, and incubate at room temperature for 1 hour. Then, wash with PBS 3 times and store at 4°C.

[0530] Microscope observation and image acquisition were performed using a fluorescence microscope BZ-X710 (manufactured by KEYENCE). Fig.24 Immunofluorescence staining images are shown. Under all conditions studied (number of cells seeded (10,000 cells and 20,000 cells) and culture days (8 days (A) and 14 days (B)), O4-positive oligodendrocytes, GFAP-positive astrocytes, and MAP2-positive neurons were detected.

[0531] <Example 18 Detection of secreted factors derived from cell aggregates containing glial precursor cells>

[0532] Cell aggregates containing glial precursor cells were prepared from the QHJI01s04 strain using the method of condition 3 of Example 1. At 45 days of differentiation, the culture medium for the neuro-glial proliferation medium was replaced and cultured at 37°C under 5% CO2 / 20% O2. The culture fluid was recovered after 48 hours, and the culture supernatant was recovered by centrifugation (1000rpm, 5 minutes). The remaining cell clumps were dispersed into single cells using TrypLE Select, and the total cell number was determined. In addition, in order to determine the cytokine concentration contained in the neuro-glial proliferation medium, only the neuro-glial proliferation medium was recovered after 48 hours of treatment under the same conditions (5% CO2 / 20% O2 / 37°C).

[0533] Secreted factors in the culture supernatant were detected by two methods. In the first method, 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) concentrations.

[0534] First, 50 μl of conjugated magnetic beads were added to a 96-well plate and washed twice with the wash buffer provided with the kit. It should be noted that the washing step was performed using an automated magnetic cleaning system (Bio-Plex ProII Wash Station, manufactured by Bio-Rad). Next, 50 μl of 8 gradient 4-fold dilution series (n=2) and samples (only culture supernatant and culture medium, n=3) made using the standard provided with the kit were added to each well, and the plates were allowed to react at room temperature and in the dark for 30 minutes while being shaken at 850 rpm. Then, the plates were washed three times with a wash buffer. Next, 25 μl of the detection antibodies provided with the kit were added to each well, and the plates were allowed to react at room temperature and in the dark for 30 minutes while being shaken at 850 rpm. Then, the plates were washed three times with a wash buffer. Next, 50 μl of streptavidin-phycoerythrin included in the kit was added to each well, and the cells were allowed to react for 10 minutes at room temperature under light shielding while shaking at 850 rpm. The cells were then washed three times with a washing solution. 125 μl of the assay buffer included in the kit was added to each well, and the fluorescence value was measured using a Bio-Plex 200 system (manufactured by Bio-Rad).

[0535] According to the measurement data of the standard dilution series of each cytokine, the standard curve was prepared by using Bio-Plex Manager software ver6.1 (Bio-Rad) and 5-parameter logistic curve regression to quantify the cytokine concentration in each sample. Then, the measurement data of only the culture medium as the background value was subtracted from the measurement data of the culture supernatant to calculate the cytokine concentration secreted into the culture supernatant. Table 8 shows the concentration of the top 10 cytokines detected in the culture supernatant and the secretion amount per unit cell.

[0536] [Table 8]

[0537] pg / ml pg / 1000 cells MIF 433 1.50 MCP-1 250 0.867 IL-8 11.5 0.0398 SCF 8.83 0.0306 M-CSF 6.65 0.0230 HGF 5.48 0.0190 GRO-α 5.43 0.0188 LIF 3.29 0.0114 IFN-γ 3.07 0.0106 TRAIL 3.01 0.0104

[0538] MIF, MCP-1, IL-8, and GRO-α have been reported as secretory factors derived from astrocytes (Non-patent Document 9). In addition, based on the fact that SCF, HGF, and MIF promote the proliferation and differentiation of neural precursor cells (Non-patent Documents 10 to 12), and that M-CSF plays an important role in the survival and differentiation of oligodendrocyte precursor cells (Non-patent Document 13), it is known that cytokines that contribute to neurogenesis are secreted from cell aggregates containing glial precursor cells.

[0539] In addition, 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).

[0540] First, according to the manufacturer's instructions, 100 μl of Human SPARC-like 1 Capture Antibody was added to each 96-well plate and adsorbed overnight at room temperature. Then, the plate was washed three times with the washing solution provided with the kit. Then, 300 μl of the Reagent Diluent provided with the kit was added to each well and reacted at room temperature for 80 minutes. Then, the plate was washed three times with the washing solution. Then, 100 μl of 11 gradient 2-fold dilution series (n=2) prepared using the standard provided with the kit and samples (only culture supernatant and medium, n=3) were added to each well and reacted at room temperature in the dark for 2 hours. Then, the plate was washed three times with the washing solution. Then, 100 μl of the Human SPARC-like 1 Detection Antibody provided with the kit was added to each well and reacted at room temperature in the dark for 2 hours. Then, the plate was washed three times with the washing solution. Then, 100 μl of the streptavidin-horseradish peroxidase provided with the kit was added to each well and reacted at room temperature in the dark for 20 minutes. Then, wash three times with a washing solution. Next, add 100 μl of substrate solution (mixed with Color Reagent A and Color Reagent B provided with the kit at a ratio of 1:1) to each well, and react for 20 minutes at room temperature under light shielding. Next, add 50 μl of stop solution provided with the kit to each well to terminate the reaction, and immediately measure the absorbance using a microplate reader Enspire (manufactured by PerkinElmer), and calculate the value obtained by subtracting the absorbance at 540 nm from the absorbance at 450 nm.

[0541] Based on the measurement data of the standard dilution series, a calibration curve was prepared by plotting on a double logarithmic scale using Excel (manufactured by Microsoft Corporation), and the SPARCL1 concentration in each sample was quantified. The measurement data of the culture medium alone as a background value was subtracted from the measurement data of the culture supernatant to calculate the concentration of SPARCL1 secreted into the culture supernatant. In addition, the amount of SPARCL1 secreted per unit cell was calculated (Table 9).

[0542] [Table 9]

[0543] ng / ml ng / 1000 cells SPARCL1 6489.09 22.47

[0544] The results of the analysis showed that SPARCL1 was present at a high concentration in the culture supernatant of the cell aggregates containing glial precursor cells. SPARCL1 is known to be a secretory factor derived from astrocytes and controls synapse formation (Non-Patent Document 14), so it is considered appropriate that SPARCL1 is detected in the culture supernatant of glial precursor cells. Therefore, SPARCL1 can be used as a marker to confirm that target cells can be obtained in the preparation step by using it alone or in combination with other markers.

Claims

1. A method for preparing a cell aggregate comprising glial precursor cells, the method comprising the following steps: Step (1), culturing induced pluripotent stem cells in suspension for 5 to 10 days 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, thereby forming cell aggregates; Step (2), suspending and culturing the cell aggregates obtained in step (1) in an embryoid body formation medium containing retinoic acid; Step (3), suspending and culturing the cell aggregates obtained in step (2) in an embryoid body formation medium or a neuroglia proliferation medium containing retinoic acid and one or more SHH signaling activators; as well as Step (4), suspending and culturing the cell aggregates obtained in step (3) in a neuroglia proliferation medium that does not contain retinoic acid and contains one or more SHH signaling activators, In step (1), step (1) is continued until at least one of the following conditions is satisfied compared to when step (1) is started, and then step (2) is started: 1) obtaining cell aggregates in which the expression level of at least one RNA among SOX1, PAX6, HES4 and HES5 is increased by more than 100 times; 2) obtaining cell aggregates in which the expression level of OCT3 / 4 RNA is reduced to less than 1 / 200; and 3) Obtaining cell aggregates in which the expression level of NANOG RNA is reduced to less than 1 / 400, In step (2), step (2) is continued until at least one of the following conditions is satisfied compared to when step (2) is started, and then step (3) is started: 1) obtaining cell aggregates in which the expression level of at least one RNA selected from ASCL1, DCX, HEY1, ZBTB20, βIII tubulin, ELAVL3 and SLIT1 is increased by more than 5 times; and 2) obtaining cell aggregates in which the expression level of at least one RNA of HOXB3, HOXA4, HOXB4, HOXB6 and HOXB8 is increased by 5 times or more, In step (3), step (3) is continued until at least one of the following conditions is satisfied compared to when step (3) is started, and then step (4) is started: 1) obtaining cell aggregates in which the expression level of at least one RNA among HEY2, NKX6.2 and NKX2.2 is increased by more than 5 times; and 2) obtaining cell aggregates in which the expression level of OLIG1 and / or OLIG2 RNA increases by more than 10 times, In step (4), step (4) is continued until at least one of the following conditions is satisfied compared to when step (4) is started: 1) obtaining cell aggregates in which the expression level of at least one RNA selected from NFIA, NFIB, SLC1A3, S100B and FABP7 is increased by more than 10 times; and 2) Obtaining cell aggregates in which the expression level of PAX6 RNA is reduced to less than 5 times.

2. The preparation method according to claim 1, further comprising the following steps: Step (5), culturing the cell aggregates obtained in step (4) in suspension in a neuro-glial proliferation medium that does not contain both retinoic acid and SHH signaling activator, In step (4), step (4) is continued until at least one of the following conditions is satisfied compared to when step (4) is started, and then step (5) is started: 1) obtaining cell aggregates in which the expression level of at least one RNA selected from NFIA, NFIB, SLC1A3, S100B and FABP7 is increased by more than 10 times; and 2) Obtaining cell aggregates in which the expression level of PAX6 RNA is reduced to less than 5 times, In step (5), step (5) is continued until one or more markers selected from the group consisting of O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, A2B5 antigen, CNP and PLP are expressed, and 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 culture medium of the cell aggregates.

3. The preparation method according to claim 1 or 2, wherein: In step (1), induced pluripotent stem cells are cultured using a culture container having a plurality of wells of uniform shape.

4. The preparation method according to claim 1 or 2, wherein Step (2) is performed for 4 to 11 days.

5. The preparation method according to claim 1 or 2, wherein: In step (1) and step (2), the oxygen concentration is 3% to 10%.

6. The preparation method according to claim 1 or 2, wherein: Step (3) is performed for 4 to 11 days.

7. The preparation method according to claim 1 or 2, wherein: Step (4) is performed for more than 4 days.

8. The preparation method according to claim 1 or 2, wherein: In step (4), the cell aggregates obtained in step (3) are dispersed at the beginning of this step, and then the dispersed cells are suspended and cultured, thereby forming cell aggregates again.

9. The preparation method according to claim 2, wherein: In step (5), the cell aggregates obtained in step (4) are dispersed at the beginning of this step, and then the dispersed cells are suspended and cultured for 5 to 100 days, thereby forming cell aggregates again.

10. The preparation method according to claim 1 or 2, wherein: SMAD signaling inhibitors are TGFβ inhibitors and BMP inhibitors.

11. The preparation method according to claim 10, 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.

12. The preparation method according to claim 10, wherein: The BMP inhibitor is one or more selected from the group consisting of Noggin, LDN-193189, LDN-212854, Dorsomorphin, K02288, chordogen, and follistatin.

13. The preparation method according to claim 1 or 2, wherein: The Wnt signaling activator is one or more selected from the group consisting of a GSK3β inhibitor, Wnt3a, a Wnt agonist, Dkk, and R-Spondin.

14. The preparation method according to claim 1 or 2, wherein: The Wnt signaling activator is one or more selected from the group consisting of CHIR99021, BIO, kemperolone, SB216763, and L803-mts.

15. The preparation method according to claim 1 or 2, wherein: The SHH signaling activator is one or more selected from the group consisting of purmorphamine, SAG, SHH protein, and SHH fragment.

16. The preparation method according to claim 1 or 2, wherein: Induced pluripotent stem cells are human induced pluripotent stem cells.

17. The preparation method according to claim 1 or 2, wherein: Cell aggregates containing glial precursor cells have the following characteristics: (a) comprising oligodendrocyte precursor cells, astrocyte precursor cells and neural precursor cells; (b) expressing spinal cord regional markers; and (c) It does not contain feeder cells or xenogeneic cell-derived components derived from feeder cells.

18. A method for preparing a cell population comprising oligodendrocytes, astrocytes and neurons, comprising the following steps: culturing a cell aggregate comprising glial precursor cells prepared by the preparation method according to any one of claims 1 to 17 for 5 to 60 days using a maturation medium.

19. The preparation method according to claim 18, wherein: The cell population that includes oligodendrocytes, astrocytes, and neurons includes: (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; as well as (iii) Cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.

20. The preparation method according to claim 18 or 19, wherein: The maturation medium is a medium containing at least one of T3, NT-3 and LIF.

21. The preparation method according to claim 20, wherein: The maturation medium further contained CNTF.

22. A cell aggregate comprising glial precursor cells, obtained by the production method according to any one of claims 1 to 17. 23 . The cell aggregate comprising glial precursor cells according to claim 22 , which has the ability to differentiate into a cell population comprising oligodendrocytes, astrocytes and neural cells.

24. A cell aggregate comprising glial precursor cells, which is obtained by the preparation method according to claim 17, and has the ability to differentiate into a cell group comprising oligodendrocytes, astrocytes and neurons, wherein: The spinal cord region marker is one or more markers selected from the group consisting of HOXB3, HOXB4, HOXB6, and HOXD8. 25 . The cell aggregate comprising glial precursor cells according to claim 23 or 24 , further comprising cells expressing one or more markers selected from the group consisting of NKX2.1, NKX2.2, NKX6.1 and NKX6.

2.

26. The cell aggregate comprising glial precursor cells as claimed in claim 23 or 24, having the following characteristics: (I) comprising cells expressing 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; (II) comprising cells expressing 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 expressing one or more markers selected from the group consisting of DCX, βIII tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3; (IV) comprising cells expressing one or more markers selected from the group consisting of SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3 and ASCL1; as well as, (V) having the ability to differentiate into a cell population comprising: (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. 27 . The cell aggregate comprising glial precursor cells according to claim 23 or 24 , further having the following characteristics: (VI) comprising cells expressing one or more markers selected from the group consisting of C10RF61 and SERPINE2.

28. The cell aggregate comprising glial precursor cells according to claim 23 or 24, further comprising cells expressing or secreting 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.

29. A cell population comprising oligodendrocytes, astrocytes and neurons, obtained by the production method according to any one of claims 18 to 21.

30. A pharmaceutical composition comprising the cell aggregate comprising glial precursor cells according to any one of claims 22 to 28 or the cell population comprising oligodendrocytes, astrocytes and neurons according to claim 29 as an active ingredient.

31. Use of the cell aggregate comprising glial precursor cells according to any one of claims 22 to 28, or the cell population comprising oligodendrocytes, astrocytes and neurons according to claim 29, in the manufacture of therapeutic drugs for demyelinating diseases and diseases based on or accompanied by glial cell damage.

32. The use according to claim 31, wherein Demyelinating diseases and diseases based on or accompanied by glial cell damage are acute, subacute or chronic spinal cord injuries.

33. A method for evaluating the toxicity or efficacy of a test substance, comprising the following steps: contacting a cell aggregate comprising glial precursor cells as described in any one of claims 22 to 28 or a cell group comprising oligodendrocytes, astrocytes and neurons as described in claim 29 with the test substance, and detecting or quantifying the effect of the test substance on the cell aggregate or the cell group.

34. A method for determining whether a cell aggregate comprising glial precursor cells is suitable for transplantation, wherein the expression of C1ORF61 is used as an indicator, wherein: The cell aggregate is a cell aggregate containing glial precursor cells obtained by the production method according to any one of claims 1 to 17.

35. A method for identifying glial precursor cells or neural stem cells with a high glial differentiation tropism, comprising detecting the expression of C1ORF61 in a cell aggregate containing glial precursor cells obtained by the production method according to any one of claims 1 to 17.

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