Novel method for producing neural stem cells / neural precursor cells

Abstract

Although the efficiency of cell differentiation thereafter can be greatly improved by temporarily subjecting pluripotent stem cells to CTraS, it has been found that stable induction of pluripotent stem cells to CTraS lacks reproducibility, and as a result, it has been the problem that the efficiency of differentiation induction into targeted nervous system cells is poor. In addition, when a target nervous system cell is expected to be differentiated from a pluripotent stem cell in the future, the target nervous system cell needs to be differentiated into the nervous system cell more efficiently. Disclosed is a novel method for stably inducing pluripotent stem cells into a state in which it is easy to induce differentiation into nervous system cells in the future. Specifically, the present invention discloses a method for culturing SB431542, LDN193189, SU5402, and EC23, which can solve the above problem by adding four kinds of small molecules SB431542, LDN193189, SU5402, and EC23 and culturing for 5 days.

Description

Technical Field

[0001] The present invention relates to: providing a cell culture method for more stably and reliably producing neural stem cells / neural progenitor cells from pluripotent stem cells. Background Art

[0002] Pluripotent stem cells (e.g., ES cells, iPS cells) are pluripotent cells that can differentiate into various tissues and cells constituting the body during the differentiation process, and are considered to be widely applicable to the fields of regenerative medicine and research. For such applications, it is necessary to rapidly and efficiently induce differentiation into target tissues / cells.

[0003] When pluripotent stem cells are cultured in a suspended state, spherical cell masses are formed, and within these cell masses, various cells interact with each other to form embryoid bodies (EBs) containing cells of the three germ layers (ectoderm, mesoderm, endoderm) system. In clinical or research settings, most target cell types start from EBs, and the target cells are obtained by applying a culture method most suitable for each cell type.

[0004] However, primitive pluripotent stem cells have variations in each cell, and this variation frequently causes problems such as differentiation bias and failure to obtain target cells. To solve such problems, the following techniques have been disclosed: for undifferentiated pluripotent stem cells, three small molecules that regulate signals related to the undifferentiated maintenance mechanism, namely SB431542, Dorsomorphin, and CHIR99021, are added to the culture medium, so that pluripotent stem cells can enter a state "Chemically Transitional EB-like State" (chemically transformed EB-like state, CTraS) in which both the differentiation speed and efficiency into target cells are significantly increased through only 5 days of culture (Patent Documents 1 to 3, Non-Patent Document 1).

[0005] It has been disclosed that pluripotent stem cells in CTraS are a mixture of ectoderm-derived cells, mesoderm-derived cells, and endoderm-derived cells, and regardless of the type of pluripotent stem cell line used as the source, the differentiation speed and efficiency into target cells are significantly increased. For example, even for pluripotent stem cell lines that are difficult to differentiate into the nervous system, cells that have undergone CTraS efficiently differentiate into the target nervous system cells, and compared with the fact that it takes 66 days to form a neural cell mass, i.e., a neurosphere, using the conventional method (Non-Patent Document 2), by undergoing CTraS, a neural cell mass, i.e., a neurosphere, can be formed in only 20 days (Non-Patent Document 3), and the cell differentiation efficiency is significantly improved.

[0006] Prior Art Documents

[0007] Patent Document

[0008] Patent Document 1: WO2017 / 141900

[0009] Patent Document 2: WO2017 / 170328

[0010] Patent Document 3: WO2018 / 043476

[0011] Non - Patent Document

[0012] Non - Patent Document 1: Fujimori et al., Stem Cell Reports, 9(5), 1675 - 1691, 2017

[0013] Non - Patent Document 2: Nori et al., PNAS, 108(40)16825 - 16830, 2011

[0014] Non - Patent Document 3: Fujimori et al., Nat Med., 24(10), 1579 - 1589, 2018 Summary of the Invention

[0015] Problems to be Solved by the Invention

[0016] However, although the efficiency of subsequent cell differentiation can be significantly improved by temporarily subjecting pluripotent stem cells to CTraS, the stable induction of pluripotent stem cells into CTraS lacks reproducibility, and as a result, there is a problem of poor induction efficiency of differentiating into the target nerve system cells. In addition, when it is desired to differentiate pluripotent stem cells into the target nerve system cells in the future, more efficient differentiation into nerve system cells is required.

[0017] Solutions to the Problems

[0018] It is disclosed that: The inventors of the present invention have discovered a new method that can stably induce pluripotent stem cells into a state that is easily induced to differentiate into nerve system cells in the future, thereby solving the above - mentioned problems. Specifically disclosed is that the above - mentioned problems can be solved by adding 4 small molecules, namely SB431542, LDN193189, SU5402, and EC23 and culturing for 5 days.

[0019] More specifically, in order to solve the above - mentioned problems, the present application provides the following methods:

[0020] [1]: A method for producing neural stem cells / neural progenitor cells from pluripotent stem cells, comprising the following steps: culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, retinoic acid (RA) or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days;

[0021] [2]: The method according to [1], wherein the expression of PAX6, SOX1, and NESTIN in neural stem cells / neural progenitor cells is upregulated and the expression of OCT4 is suppressed;

[0022] [3]: The method according to [1], wherein the pluripotent stem cells are selected from the group consisting of ES cells and iPS cells;

[0023] [4]: The method according to [2], wherein the pluripotent stem cells are selected from the group consisting of ES cells and iPS cells;

[0024] [5]: The method according to any one of [1] to [4], wherein the SMAD signaling pathway inhibitors are SB431542 and LDN193189;

[0025] [6]: The method according to any one of [1] to [4], wherein the RA analog is EC23;

[0026] [7]: The method according to any one of [1] to [4], wherein the FGF signaling pathway inhibitor is SU5402;

[0027] [8]: The method according to any one of [1] to [4], wherein the culture medium is selected from the group consisting of a culture medium for primate ES / iPS cells, AK02N medium, ciKIC (registered trademark) iPS Medium, mTeSR medium, NutriSTEM medium, and Essential 8 medium;

[0028] [9]: A method for producing neurospheres from pluripotent stem cells, comprising the following steps:

[0029] Step (i): Culturing pluripotent stem cells in a culture medium containing an SMAD signaling pathway inhibitor, RA or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days to produce neural stem cells / neural progenitor cells from the pluripotent stem cells;

[0030] Step (ii): Producing neurospheres formed from neural stem cells / neural progenitor cells;

[0031]

[10] : The method according to [9], wherein step (ii) is carried out by culturing in the presence of B-27 supplement (registered trademark), Y27632, LIF, FGF, and RA or an RA analog for 13 days;

[0032]

[11] The method according to

[10] , wherein the RA analog is EC23;

[0033]

[12] : The method according to any one of [9] to

[11] , wherein step (ii) is carried out under hypoxic conditions;

[0034]

[13] : The method according to any one of [9] to

[11] , wherein a Notch signal inhibitor is further added 12 days after the start of the culture in step (ii);

[0035]

[14] : The method according to

[13] , wherein the Notch signal inhibitor is a γ-secretase inhibitor;

[0036]

[15] : The method according to any one of [9] to

[11] , wherein after step (ii), the formed neurospheres are further dispersed into single cells and then the step of producing neurospheres is carried out one or more times.

[0037]

[16] : A method for producing nervous system cells from pluripotent stem cells, comprising the following steps:

[0038] Step (i): Culturing pluripotent stem cells in a medium containing an SMAD signal transduction pathway inhibitor, RA or an RA analog at a concentration of 100 nM or less, and an FGF signal pathway inhibitor for 5 days to produce neural stem cells / neural progenitor cells from the pluripotent stem cells;

[0039] Step (ii): Producing neurospheres formed from neural stem cells / neural progenitor cells;

[0040] Step (iii): Producing nervous system cells from the neurospheres.

[0041] Effects of the Invention

[0042] By using the method for producing neural stem cells / neural progenitor cells of the present invention, through the step of culturing pluripotent stem cells in a medium containing an SMAD signal transduction pathway inhibitor, RA or an RA analog at a concentration of 100 nM or less (low concentration), and an FGF signal pathway inhibitor for 5 days, the pluripotent stem cells can be stably induced into neural stem cells / neural progenitor cells with high reproducibility, and the differentiation efficiency into neurospheres and the differentiation efficiency into nervous system cells thereafter can be significantly improved compared with the previous methods. Description of the Drawings

[0043] Figure 1 A graph showing the mRNA expression levels of four factors, PAX6, SOX1, NESTIN, and OCT4, as markers when culturing cells under various culture conditions.

[0044] Figure 2A graph showing the expression of proteins of four factors, PAX6, SOX1, NESTIN, and OCT4, as markers when culturing cells under various culture conditions.

[0045] Figure 3 A graph showing the mRNA expression levels of four factors, PAX6, SOX1, NESTIN, and OCT4, as markers when culturing cells under various culture conditions.

[0046] Figure 4 A graph showing the mRNA expression levels of four factors, PAX6, SOX1, NESTIN, and OCT4, as markers when culturing cells under various culture conditions.

[0047] Figure 5 A graph showing the mRNA expression levels of three factors, PAX6, SOX1, and NESTIN, as markers when culturing cells under various culture conditions.

[0048] Figure 6 A graph showing the results of fluorescence labeling of the expression of various marker proteins during in vitro differentiation induction starting from the PNS.

[0049] Figure 7 A graph showing the results of fluorescence labeling of the expression of various marker proteins during in vitro differentiation induction starting from the SNS.

[0050] Figure 8 A graph showing the results of evaluating the walking state of mice transplanted with neurospheres 9 weeks after spinal cord injury at different time points after spinal cord injury on the 9th day by BMS score.

[0051] Figure 9 A graph showing the results of fluorescence labeling of the expression of various marker proteins of transplanted cells in the body of mice transplanted with neurospheres. Detailed implementation mode

[0052] Prior to the present invention, the following technology was disclosed: Undifferentiated pluripotent stem cells were cultured in a medium supplemented with three small molecules, SB431542, Dorsomorphin, and CHIR99021, which regulate signals related to the undifferentiated maintenance mechanism, so that pluripotent stem cells could enter a state of "Chemically Transitional EB-like State" (chemically transformed EB-like state, CTraS) with significantly improved differentiation speed and efficiency towards target cells through only 5 days of culture (Non-Patent Document 1).

[0053] It is disclosed that the pluripotent stem cells entering the CTraS are a mixture of ectodermal cells, mesodermal cells, and endodermal cells. Regardless of the type of pluripotent stem cell line used as the source, the differentiation rate and efficiency into target cells are significantly improved. However, it is known that as the cell characteristics of the pluripotent stem cells entering the CTraS, the OCT4 expression decreases, and the PAX6 expression is strongly positive, the SOX1 expression is strongly positive, and the NESTIN expression is strongly positive. The stable induction of pluripotent stem cells into CTraS lacks reproducibility, and as a result, there is a problem of poor differentiation induction efficiency into the target neural system cells. It is considered that a cell culture method for solving this problem can be provided, and research and development have been carried out.

[0054] Method for manufacturing neural stem cells / neural progenitor cells of the present invention

[0055] As a result of in-depth research, the inventors of the present invention found that by culturing pluripotent stem cells in a medium containing a SMAD signaling pathway inhibitor, retinoic acid (RA) or an RA analog at a concentration of 100 nM or less (low concentration), and an FGF signaling pathway inhibitor for 5 days, the pluripotent stem cells can be efficiently induced into neural stem cells / neural progenitor cells, thereby being able to solve the above problems.

[0056] Therefore, in one aspect, the present invention provides a method for producing neural stem cells / neural progenitor cells from pluripotent stem cells, which comprises the following steps: culturing pluripotent stem cells in a medium containing a SMAD signaling pathway inhibitor, low-concentration retinoic acid (RA) or an RA analog, and an FGF signaling pathway inhibitor for 5 days.

[0057] The method is characterized in that the SMAD signaling pathway inhibitor, retinoic acid (RA) or an RA analog at a concentration of 100 nM or less (low concentration compared to the concentration (2 μM) used in conventional neural differentiation induction), and the FGF signaling pathway inhibitor contained in the medium are included in the medium from the start of pluripotent stem cell culture (i.e., day 0), and the cells are cultured in the medium having this composition for 5 days.

[0058] The culture medium for pluripotent stem cells that can be used in the method for manufacturing neural stem cells / neural progenitor cells of the present invention can be any medium that is commonly used in this technical field. For example, a culture medium for primate ES / iPS cells (Primate ES Cell Medium), AK02N medium (Ajinomoto), ciKIC (registered trademark) iPS Medium culture medium (Kanto Chemical), mTeSR medium (StemCell Technologies), NutriSTEM medium (Sartorius), Essential 8 medium (Thermo Fisher), etc. can be used. In addition, since the method for manufacturing neural stem cells / neural progenitor cells of the present invention aims to culture cells transplanted into humans, it is not desirable to add serum other than human serum to the culture medium. Therefore, a serum-free culture medium is preferably used.

[0059] When using these culture media, in order to improve cell culture efficiency, coating of the culture plate can be combined. As the coating, coating using iMatrix, coating using Matrigel, and coating using vitronectin can be used. As combinations of these culture media and coatings, for example, a combination of AK02N medium and coating using iMatrix, a combination of ciKIC (registered trademark) iPS Medium culture medium and coating using iMatrix, a combination of StemFit medium and coating using iMatrix, a combination of NurtiSTEM medium and coating using Matrigel, a combination of mTeSR medium and coating using Matrigel, a combination of Essential 8 medium and coating using vitronectin, etc. can be used.

[0060] In the present invention, the culture period of 5 days means: culturing for 5 days starting from changing to a culture medium containing an SMAD signaling pathway inhibitor, low-concentration retinoic acid (RA) or an RA analog, and an FGF signaling pathway inhibitor. This culture period can also be changed while observing the cell condition. The culture period does not have to be strictly 5 days (i.e., 120 hours) and can fluctuate up and down by about 6 hours.

[0061] Characteristics of raw material cells

[0062] The pluripotent stem cells used as starting cells in the method for producing neural stem cells / neural progenitor cells of the present invention refer to stem cells that can differentiate into almost all cells constituting the body, and examples thereof include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), somatic stem cells, and EG cells. Regarding ES cells, cell lines established and supplied by the RIKEN BioResource Center, the Institute for Frontier Life and Medical Sciences of Kyoto University, etc. can be used in Japan. In addition, for iPS cells, cell lines established and supplied by the Center for iPS Cell Research and Application of Kyoto University, etc. can be used. In the present invention, from the viewpoints of ethics and cell supply stability, iPS cells are preferably used.

[0063] Human pluripotent stem cells, particularly iPS cells, have the properties that the differentiation efficiency varies among cell lines and the differentiation speed is relatively slow. Therefore, it is necessary to pre-screen pluripotent stem cell lines that are easily differentiated into target cells / tissues. Even when using the screened cell lines, there is a problem that a great deal of effort and time are required to achieve highly efficient differentiation induction. In contrast, according to the method for producing neural stem cells / neural progenitor cells from pluripotent stem cells of the present invention, neural stem cells / neural progenitor cells with high homogeneity can be produced, and subsequent differentiation induction of nervous system cells can also be carried out with high efficiency.

[0064] For example, it is known that when iPS cells are used for differentiation induction into neural stem cells, in the case of the conventional neural sphere production method, the induction into neural stem cells is carried out more efficiently when the source cells of the iPS cells are skin cells, while the induction into neural stem cells is difficult when the source cells of the iPS cells are blood cells. In contrast, with the method for producing neural stem cells / neural progenitor cells of the present invention, regardless of the type of source cells of the pluripotent stem cells, the induction from pluripotent stem cells into neural stem cells / neural progenitor cells is shown to proceed efficiently. Therefore, in the present invention, when using iPS cells as starting cells, the source of the iPS cells can be any cell, and iPS cells derived from blood cells, iPS cells derived from skin cells, etc. can be used.

[0065] Characteristics of neural stem cells / neural progenitor cells produced by using the present invention

[0066] The neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention are characterized in that, compared with the cells produced by the above-mentioned CTraS induction method, they are neural stem cells / neural progenitor cells that are already destined to become nervous system cells. The neural stem cells / neural progenitor cells can be characterized as follows: compared with pluripotent stem cells as raw material cells, as cell markers, the expression of OCT4 decreases, the expression of PAX6 is strongly positive, the expression of SOX1 is strongly positive, and the expression of NESTIN is strongly positive. Here, when referring to the decrease in OCT4 expression, it means that the expression of OCT4 is 50% or less, preferably 20% or less, and more preferably 10% or less compared with the expression of OCT4 in the iPS cells used as the raw material. When referring to the strong positive expression of PAX6, it means that it is 2 times or more, preferably 5 times or more, and more preferably 10 times or more compared with the expression of PAX6 in the iPS cells used as the raw material. When referring to the strong positive expression of SOX1, it means that it is 2 times or more, preferably 5 times or more, and more preferably 10 times or more compared with the expression of SOX1 in the iPS cells used as the raw material. When referring to the strong positive expression of NESTIN, it means that it is 2 times or more, preferably 5 times or more, and more preferably 10 times or more compared with the expression of NESTIN in the iPS cells used as the raw material.

[0067] Regardless of whether the neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention maintain an undifferentiated state, they have thereafter been reliably directed to differentiate into nervous system cells, and the differentiation maturity / speed has been greatly improved. Using this feature, various nervous system cells can be efficiently differentiated according to the methods for inducing differentiation into various nervous system cells established in this technical field.

[0068] The neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention can be defined as cells that have both the self-renewal ability to proliferate while maintaining an undifferentiated state and the multi-differentiation ability to differentiate into cells of three systems, namely nerve cells, astrocytes, and oligodendrocytes, that make up the central nervous system. The neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention, as described in this definition, have the self-proliferation ability, and by subsequently performing differentiation culture into nervous system cells, they can form cells of three systems, namely nerve cells, astrocytes, and oligodendrocytes, via a neural progenitor cell population and according to the differentiation conditions for various nervous system cells.

[0069] The neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention also have the following characteristics: as the characteristics of marker molecules expressed by cells, the expression of PAX6, SOX1, and NESTIN is hyperactive and the expression of OCT4 is inhibited.

[0070] OCT4 is a molecule known as a marker of pluripotent stem cells and is known to be highly expressed in pluripotent stem cells as a raw material. In the neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention, the expression of OCT4 decreases, indicating that they no longer have the pluripotency of pluripotent stem cells.

[0071] On the other hand, neural stem cells / neural progenitor cells are characterized by the expression of SOX family genes such as SOX1 and SOX2, and the expression of PAX6 gene. Usually, the neural induction efficiency in vitro can be monitored by analyzing the expression of NESTIN, PAX6, and SOX family genes, which are characteristic markers of neural stem cells / neural progenitor cells (Stem Cells 2005; 23: 1234-1241). Consistent with this view, the neural stem cells / neural progenitor cells produced by the method for producing neural stem cells / neural progenitor cells of the present invention also show enhanced expression of PAX6, SOX1, and NESTIN.

[0072] Additives in the culture medium

[0073] (1) Inhibitor of the SMAD signaling pathway

[0074] The method for producing neural stem cells / neural progenitor cells of the present invention is characterized in that an inhibitor of the SMAD signaling pathway is added to the cell culture medium. It is characterized in that SB431542 and LDN193189 are used as the inhibitor of the SMAD signaling pathway. Regarding the inhibition achieved by SB431542 and LDN193189 (this inhibition treatment is referred to as the LSB treatment), it is determined to use the dual inhibition (LSB) of SB431542 and LDN193189 according to the literature information capable of efficiently inducing PAX6-positive neural progenitor cells (Qi et al., Nat Biotechnol. 35(2): 154-163, 2017). The research in this specification also confirms that through the LSB treatment, compared with pluripotent stem cells as raw material cells, the expression of OCT4 decreases, and conversely, the expression of PAX6, SOX1, and NESTIN is enhanced.

[0075] For the culture medium of pluripotent stem cells, when using SB431542 as the inhibitor of the SMAD signaling pathway, it is preferably added at 1 to 10 μM, preferably 2 to 5 μM, and more preferably 3 μM. In addition, when using LDN193189 as the inhibitor of the SMAD signaling pathway, it is preferably added at 50 to 250 nM, preferably 100 to 200 nM, and more preferably 150 nM.

[0076] In the present invention, when referring to an inhibitor of the SMAD signaling pathway, it means a group of molecules characterized by inhibiting signal transduction from TGFβ intracellularly. It is known that in addition to the above-mentioned SB431542 and LDN193189, it also includes compounds such as noggin and dorsomorphin. In the present invention, as long as the expression of OCT4 is decreased compared to pluripotent stem cells as the starting cells, and conversely, the expression of PAX6, SOX1, and NESTIN is increased, any one of these can also be used in addition to or in place of SB431542 or LDN193189.

[0077] (2) Wnt agonist

[0078] In the conventional CTraS induction method (Non-Patent Document 1), the Wnt agonist CHIR99021 was added to the culture medium for cell culture. However, it was found that in this method, the expression of SOX10, which is often observed in differentiated neural crest cells, was increased. In order to maintain neural stem cells / neural progenitor cells in an undifferentiated state, it was considered that the addition of CHIR99021 was not preferable. Therefore, it was decided not to add the Wnt agonist CHIR99021 in the cell culture of the method for producing neural stem cells / neural progenitor cells of the present invention.

[0079] (3) Retinoic acid (RA) or RA analog

[0080] Regarding retinoic acid (RA) or RA analog, it was not used in the conventional CTraS induction method. Previous reports (Okada Y. et al, Stem Cells, 2008) disclosed that RA at a concentration of 100 nM or less (a relatively low concentration compared to the concentration (2 μM) used in conventional neural differentiation induction) was used to induce PAX6-positive and NESTIN-positive neural progenitor cells. Based on this insight, in the present invention, the use of RA or RA analog was studied. As a result, it was confirmed that when RA was added at a concentration of 100 nM or less, the expression levels of PAX6 and NESTIN were increased, and when the RA analog EC23 (Maltman et al., Mol. BioSyst., 5, 458 - 471, 2009) was added at a concentration of 100 nM or less, the expression levels of PAX6 and NESTIN were increased by almost 2 times compared to RA. Based on this result, it was decided to add either RA at a concentration of 100 nM or less or an RA analog at a concentration of 100 nM or less to the cell culture medium. In the manufacturing process of the neural stem cells / neural progenitor cells of the present invention, as a preferred mode, EC23, which is an RA analog, is used.

[0081] When using RA in the medium for pluripotent stem cells, it is preferably added in the range of 1 nM to 100 nM, more preferably 5 nM to 100 nM, and still more preferably 10 nM to 100 nM. Additionally, when using EC23, which is an RA analog, it is also preferably added in the range of 1 nM to 100 nM, more preferably 5 nM to 100 nM, and still more preferably 10 nM to 100 nM.

[0082] In the present invention, when referring to RA or an RA analog, it means retinoic acid or its natural or artificial analogs, which are a group of molecules that act by binding to retinoic acid receptors (RARs). In addition to the above-mentioned RA and EC23, compounds such as AM580, TTNPB, and Adapalene are also known. In the present invention, as long as the expression of OCT4 is decreased and, conversely, the expressions of PAX6, SOX1, and NESTIN are increased compared to pluripotent stem cells as the starting cells, any of these can be used on the basis of or in place of retinoic acid (RA) or EC23.

[0083] (4) FGF signaling pathway inhibitor

[0084] Regarding the FGF signaling pathway inhibitor, it has not been used in the conventional CTraS induction method. Since the FGF signal has the function of maintaining the stemness of pluripotent stem cells, it is considered that when inducing pluripotent stem cells into neural stem cells, by reversely using the FGF signal inhibitor, neural stem cells can be easily induced. Therefore, in the present invention, the use of the FGF signaling pathway inhibitor was studied, and as a result, it was confirmed that the expression levels of PAX6 and NESTIN increased when SU5402 was added.

[0085] When using SU5402 as the FGF signaling pathway inhibitor in the medium for pluripotent stem cells, it is preferably added in the range of 200 nM to 50 μM, more preferably 500 nM to 20 μM, and still more preferably 1 μM to 10 μM. As a particularly preferred embodiment, 5 μM of SU5402 can be used as the FGF signaling pathway inhibitor.

[0086] When referring to FGF signaling pathway inhibitors in the present invention, in addition to SU5402, FGF signaling pathway inhibitors that act through the same mechanism as SU5402 can also be used. As such FGF signaling pathway inhibitors, compounds such as Ponatinib (AP24534), Infigratinib (BGJ398), PD173074, Dovitinib (TKI-258), AZD4547, Danusertib (PHA-739358), Lenvatinib (E7080), Lucitanib (E3810), Sulfatinib, Pemigatinib (INCB054828), ODM-203, Futibatinib (TAS-120), ASP5878, Derazantinib (ARQ-087), Lenvatinib mesylate (E7080), PRN1371, PD-166866 (PD166866), S49076, ON123300, FIIN-2, Dovitinib lactate (TKI258), Zoligratinib (Debio-1347), LY2874455 can be used.

[0087] Regarding the research results of (1) to (4) of the additives in the above culture medium, it was found in the present invention that by culturing pluripotent stem cells in a culture medium containing (1) a SMAD signaling pathway inhibitor, (3) RA or an RA analog at a concentration of 100 nM or less, and (4) an FGF signaling pathway inhibitor, the expression of OCT4 in the cells can be decreased, and conversely, the expression of PAX6, SOX1, and NESTIN can be enhanced. Compared with the conventional method, the CTraS induction method, neural stem cells / neural progenitor cells derived from pluripotent stem cells can be maintained in an undifferentiated state (CTraS) reproducibly, and when neural cells are to be differentiated from pluripotent stem cells in the future, they can be differentiated into neural cells more efficiently.

[0088] Other culture conditions

[0089] In the present invention, when culturing pluripotent stem cells to produce neural stem cells / neural progenitor cells, the pluripotent stem cells can be cultured without using feeder cells. When culturing pluripotent stem cells, a method of culturing feeder cells in a sheet on a cell culture substrate and culturing pluripotent stem cells thereon is well known. However, since feeder cells are usually cells derived from animals other than humans such as mice, in consideration of the use of products such as regenerative medicine produced from pluripotent stem cells for humans, it is preferable not to use feeder cells in order to avoid risks such as immune responses. In the present invention, by using the above-mentioned culture medium and additives, a method for producing neural stem cells / neural progenitor cells from pluripotent stem cells without using feeder cells can be provided.

[0090] Method for manufacturing neurospheres from pluripotent stem cells

[0091] In the present invention, after producing neural stem cells / neural progenitor cells according to the above-mentioned method for producing neural stem cells / neural progenitor cells from pluripotent stem cells (this step is also referred to as step (i)), the following-described step (ii) of culturing is carried out, whereby neurospheres can be produced.

[0092] In the present invention, when producing neurospheres from pluripotent stem cells, specifically, neurospheres can be produced by carrying out the following steps:

[0093] Step (i): Culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, retinoic acid (RA) or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days to produce neural stem cells / neural progenitor cells from the pluripotent stem cells; then,

[0094] Step (ii): Producing neurospheres from the obtained neural stem cells / neural progenitor cells.

[0095] In the above step (ii), a method for producing neurospheres from neural stem cells / neural progenitor cells can be a method well known in the art.

[0096] The process of manufacturing neurospheres from the obtained neural stem cells / neural progenitor cells (process (ii)) can be carried out using culture methods known in the art. As such a method, for example, the method described in the paper by Okada et al. (Okada Y. et al, Stem Cells, 2008, 0293), or the method described in the paper by Matsumoto et al. (Matsumoto et al., Stem Cell Reports, 6, 422 - 435, 2016) can be used. Specifically, for example, as described in known literature (Matsumoto et al., Stem Cell Reports, 6, 422 - 435, 2016, etc.), it can be carried out starting from undifferentiated pluripotent stem cells, through CTraS, and then culturing under hypoxic conditions to promote differentiation into the ectoderm, or it can be carried out by adding B-27 supplement (registered trademark), bFGF, ROCK inhibitor (such as Y27632), hLIF, high-concentration RA or RA analog (such as EC23) to the spheroid medium (Hormone mix or KBM medium) when culturing neural stem cells / neural progenitor cells and culturing for 13 days.

[0097] Here, the B-27 supplement (registered trademark) added to the medium is a serum-free supplement for culturing nerve cells, and is composed of 20 factors including insulin (the components are not publicly disclosed). The B-27 supplement (registered trademark) is added for long-term culturing of nervous system cells in the process of manufacturing neurospheres. When using the B-27 supplement (registered trademark) for the medium in the process of manufacturing neurospheres, it can be used at a concentration recommended by the provider of the product relative to the amount of the medium. For example, it is preferably added at 2% relative to the amount of the medium.

[0098] The ROCK (Rho-associated protein kinase) inhibitor added to the medium is a substance that can inhibit the apoptosis phenomenon that occurs when cells are dispersed during cell manipulation. Examples of the ROCK inhibitor that can be used in the present invention include Y27632, Fasudil, H-1152, Wf-536, etc., but are not limited to these. In the process of manufacturing neurospheres, the ROCK inhibitor is added to induce the differentiation of neural stem cells / neural progenitor cells into nerve cells.

[0099] As an example, Y27632, which can be used as a ROCK inhibitor, is a specific and potent ROCK inhibitor with ATP antagonistic activity. It is known to act as a strong inhibitor of Ca2+ receptor agonists in myosin phosphorylation and smooth muscle contraction, inhibit cell spreading, and inhibit the formation of stress fibers in hepatic stellate cells induced by Rho-A. When using Y27632 in the culture medium during the process of manufacturing neurospheres, it is preferably added in the range of 500 nM to 50 μM, more preferably 1 μM to 30 μM, and still more preferably 5 μM to 15 μM. As a particularly preferred embodiment, 10 μM of Y27632 can be used as the ROCK inhibitor.

[0100] Regarding hLIF added to the culture medium, hLIF refers to human LIF (Leukemia Inhibitory Factor). LIF is a cytokine that was discovered as a factor that inhibits the proliferation of leukemia cells and induces their differentiation into macrophages. It belongs to the IL-6 family and activates JAK1 and JAK2 through the LIFR / gp130 receptor. LIF is known to have various functions and is also expressed in nerve cells, and it is known to have the function of making nerve cells extend. In the process of manufacturing neurospheres, hLIF is added to induce the differentiation of neural stem cells / neural progenitor cells into nerve cells. When using hLIF in the culture medium during the process of manufacturing neurospheres, it is preferably added in the range of 500 pg / mL to 50 ng / mL, more preferably 1 ng / mL to 30 ng / mL, and still more preferably 5 ng / mL to 15 ng / mL. As a particularly preferred embodiment, 10 ng / mL of human LIF can be used.

[0101] In previous reports (Okada Y. et al, Stem Cells, 2008) on adding RA or RA analogs during the process of manufacturing neurospheres, it was disclosed that a relatively high concentration of about 10 μM of RA induced more postmitotic nerve cells positive for βIII-tubulin. Based on this insight, in the process of manufacturing neurospheres, RA or RA analogs are added to induce the differentiation of neural stem cells / neural progenitor cells into nerve cells. In this process, as a preferred embodiment, EC23, which is an RA analog, is used.

[0102] During the process of manufacturing neurospheres, RA or RA analogs are used. For example, when using EC23, which is an RA analog, it is preferably added in the range of 1 μM to 50 μM, more preferably 2 μM to 30 μM, and still more preferably 2 μM to 10 μM.

[0103] In the present invention, step (ii) can be carried out entirely under hypoxic conditions during the 13-day culture period. By culturing step (ii) under hypoxic conditions, the production of neurospheres can be efficiently carried out.

[0104] In step (ii) of the present invention, a Notch signal inhibitor can be added 12 days after the 13-day culture period, that is, on the last day (24 hours before the neurospheres are recovered / frozen). The Notch signaling pathway is a gene regulation (signal transduction) pathway related to the processes of differentiating into various tissues such as nerves, hematopoiesis, blood vessels, and somites. In the information transfer between cells, it plays a very important role in the maintenance of development and homeostasis and the control of cell differentiation-related genes. In the transplantation of cells induced from pluripotent stem cells, after transplantation, the highly undifferentiated cells remaining in the transplanted cells may become cancerous. Therefore, in order to prevent this carcinogenesis, it is considered important to ensure safety. By treating pluripotent stem cell-derived neural stem cells / neural progenitor cells with an agent (such as a γ-secretase inhibitor) that can block the Notch signal closely related to their multi-differentiation ability and self-renewal ability in neural stem cells, the highly undifferentiated cells are removed or further differentiated, and transplantation in such a state can inhibit carcinogenesis. A Notch signal blocker refers to a group of agents that have the effect of blocking any pathway in the Notch signaling pathway.

[0105] In particular, it is known that a part of the γ-secretase complex is cleaved in the Notch signaling pathway, and its information migrates into the nucleus to cause the transcription / expression of target genes. The γ-secretase inhibitor acts as an inhibitor of the Notch signaling pathway by knowing this cleavage and promotes differentiation into various tissues represented by nerves. Therefore, as a Notch signal inhibitor, a γ-secretase inhibitor can be used. Examples of γ-secretase inhibitors that can be used in the method of the present invention include DAPT, Compound 34, etc., and DAPT is preferably used.

[0106] When using the γ-secretase inhibitor DAPT as a Notch signal inhibitor in the medium for pluripotent stem cells, it is preferably added in the range of 1 μM to 20 μM, preferably 5 μM to 15 μM, more preferably 7 μM to 13 μM. For example, it can be used at 10 μM.

[0107] In the present invention, when obtaining neurospheres in step (ii) as described above, the neurospheres can be directly used for subsequent applications, or the neurospheres can be further dissociated into single cells and then the same steps as in step (ii) above can be repeated one or more times, and then the obtained neurospheres can be used for subsequent applications. When the additional step of manufacturing neurospheres is carried out once, the obtained neurospheres are sometimes referred to as secondary neurospheres, and when the additional step of manufacturing neurospheres is carried out twice, the obtained neurospheres are sometimes referred to as tertiary neurospheres. Thus, depending on the number of times the additional step is carried out, the names of the neurospheres are also different.

[0108] The neurospheres produced by this method can be used for transplantation for the purpose of neural tissue regeneration, such as transplantation to the damaged site of spinal cord injury, transplantation to the damaged site of cerebral infarction, etc. As the cells of the transplanted neurospheres proliferate in the transplanted tissue, due to the action of the position of the transplanted tissue, they differentiate into appropriate nervous system cells at the transplanted position.

[0109] Method for manufacturing nervous system cells from pluripotent stem cells

[0110] The present invention further provides a method for manufacturing nervous system cells from pluripotent stem cells, which undergoes the above-mentioned method for manufacturing neurospheres from pluripotent stem cells via neural stem cells / neural progenitor cells.

[0111] Here, when referring to nervous system cells, it means any cells in the three systems of nerve cells, astrocytes, and oligodendrocytes. By starting with neural stem cells / neural progenitor cells produced by using the method of the present invention and culturing them under various differentiation induction conditions, they can differentiate into any cells.

[0112] In the present invention, when manufacturing nervous system cells from pluripotent stem cells, specifically, nervous system cells can be manufactured through the following steps:

[0113] Step (i): Culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, retinoic acid (RA) or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days to produce neural stem cells / neural progenitor cells from pluripotent stem cells;

[0114] Step (ii): A step of manufacturing neurospheres formed from the obtained neural stem cells / neural progenitor cells;

[0115] Step (iii): Manufacturing nervous system cells from neurospheres.

[0116] For example, after manufacturing neural stem cells / neural progenitor cells using the method of the present invention (step (i)), the neural stem cells / neural progenitor cells are used to manufacture neurospheres using the method described in Non-Patent Document 1 (step (ii)). Then, 5×10 3 cells derived from the neurospheres obtained in the previous step are inoculated into a 96-well multi-well plate (Falcon), 2% B-27 supplement (registered trademark) is added to the spheroid medium (also referred to as Hormone mix or KBM medium), and in addition, the cells are cultured for 6 weeks in a medium supplemented with EC23 instead of RA, whereby the maturation of the differentiation from neurospheres to neural cells can be efficiently induced (step (iii)).

[0117] The step of manufacturing nervous system cells from neurospheres (step (iii)) can also be carried out using a culture method known in the art. Specifically, in the culture of the neurospheres manufactured by the above step (ii), it can be carried out by adding 2% B-27 supplement (registered trademark) to the medium MHM (Media Hormone Mix) for neural stem cells and culturing for 5 to 40 days.

[0118] In the present invention, when obtaining neurospheres in step (ii) as described above, the neurospheres can be directly used in step (iii), or the neurospheres manufactured in step (ii) can be further dispersed into single cells and then the step of manufacturing neurospheres can be carried out one or more times through the same steps as step (ii) above, and then the obtained neurospheres can be used in the subsequent step (iii).

[0119] The differentiation into neural cells can be confirmed based on the increased expression levels of neural cell markers such as TUJ1 and MAP2 and the decreased expression levels of neural stem cell markers such as SOX1 and PAX6.

[0120] When induced and differentiated from neurospheres according to step (iii), it will differentiate into nervous system cells of three systems: neural cells, astrocytes, and oligodendrocytes.

[0121] Examples are listed below to specifically illustrate the present invention. In the following examples, no method is intended to limit the present invention.

[0122]

Examples

[0123] Example 1: Study on culture conditions when manufacturing neural stem cells / neural progenitor cells from pluripotent stem cells (1)

[0124] This example is carried out for the purpose of studying the culture conditions when manufacturing neural stem cells / neural progenitor cells from pluripotent stem cells.

[0125] That is, additives that can be used to more efficiently produce neural stem cells / neural progenitor cells from pluripotent stem cells were studied. Specifically, based on the "SMAD signaling pathway inhibitors" SB431542 (Tocris Bioscience), Dorsomorphin, and the "Wnt agonist" CHIR99021 (ReproCELL), which have been conventionally used to produce neural stem cells / neural progenitor cells from pluripotent stem cells (Non-Patent Document 1), conditions for more efficiently producing neural stem cells / neural progenitor cells were explored by changing these components.

[0126] First, as an additive used in the conventional method in the culture medium, Dorsomorphin was considered to be toxic, so it was decided not to use it.

[0127] On the other hand, regarding the "SMAD signaling pathway inhibitor", as reported previously (Chambers S.M. et al, Nat Biotechnol., 30(7), 715 - 720 (2012)), in the case of human ES cells, by inhibiting two types of SMAD signaling pathways, namely the BMP signaling pathway and the TGFβ signaling pathway, more than 80% of the cells differentiated into neural cells under adherent culture conditions. Therefore, it was decided to study a protocol using LDN193189 (StemRD) in addition to SB431542 (Tocris Bioscience). At this time, 3 μM of SB431542 (Tocris Bioscience) and 150 nM of LDN193189 (StemRD) were added to the culture medium. The addition amounts of these were determined values based on the descriptions in known literature (Chambers, S.M., et al., 2012 Nat. Biotechnol., 30(7), 715 - 720).

[0128] In addition, regarding the "Wnt agonist", it was decided to use CHIR99021 (ReproCELL) used in the conventional method (Non-Patent Document 1). At this time, 3 μM of CHIR99021 (ReproCELL) was added to the culture medium.

[0129] Previous reports (Okada Y. et al, Stem Cells, 2008) have disclosed that relatively low concentrations of retinoic acid (RA) around 10 nM can induce neural progenitor cells that are positive for PAX6 and NESTIN, which are indicators of neural stem cells / neural progenitor cells. Based on this insight, in this example, it was decided to adopt a protocol (LSBE treatment) in which "retinoic acid (RA) or an RA analog" is added on the basis of the LSB treatment and EC23 (Cayman) is used as the "RA analog". At this time, EC23 (Cayman) was added to the culture medium at 10 nM.

[0130] Based on the above research, the culture was carried out as follows in this example. As the starting cells, iPS cells (ReproCELL, RCRP002N), which are pluripotent stem cells, were used. These cells were seeded at a cell density of 1.5×10 4 cells per well on a cell culture substrate (6-well plate) coated with iMatrix-511silk (Matrixome) (2.4 μg / ml) and cultured in AK02N medium (Ajinomoto) for 1 week. In principle, the culture medium was changed daily and passage culture was performed. The iPS cells at 70 - 80% confluence were treated with 0.5×TrypLE select (Gibco) and seeded at a cell density of 3×10 4 cells per well on a 6-well plate coated with iMatrix-511silk (Matrixome) as before. 10 μM of a Rock inhibitor (Y27632, CultureSURE) was added to the AK02N medium (Ajinomoto). After seeding, the 6-well plate was incubated overnight at 37°C and 5% CO 2 . This time point was set as day 0.

[0131] On day 1, after observing whether the cells adhered well to the plate, the culture medium of the cells was changed to 3 ml of a culture medium (AK02N-based medium containing 3 μM of SB431542 (Tocris Bioscience) and 150 nM of LDN193189 (StemRD)), and then incubated overnight at 37°C and 5% CO 2 .

[0132] On days 2 and 5, the culture medium of the cells was changed to 3 ml of the following culture medium containing additives:

[0133] · "LSB": Culturing was carried out in AK02N-based medium containing two "SMAD signaling pathway inhibitors", namely 150 nM of LDN193189 (StemRD) and 3 μM of SB431542 (Tocris Bioscience) (hereinafter also referred to as LSB treatment);

[0134] · "LSBC": AK02N-based medium containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and also containing 3 μM of CHIR99021 (ReproCELL) as a "Wnt agonist" (hereinafter, culturing under this condition is also referred to as LSBC treatment);

[0135] · "LSBE": AK02N-based medium containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and also containing 10 nM of EC23 (Cayman) as a "retinoic acid (RA) or RA analog" (hereinafter, culturing under this condition is also referred to as LSBE treatment);

[0136] · "iPSC" is AK02N-based medium without additives;

[0137] And cultured until day 6, and an attempt was made to produce neural stem cells / neural progenitor cells from the starting cells.

[0138] In this example, as an index for neural stem cells / neural progenitor cells, the decrease in the expression of OCT4 compared to pluripotent stem cells (iPS cells) as the starting cells, and conversely, the increase in the expression of PAX6, SOX1, and NESTIN was used to investigate the ability to produce neural stem cells / neural progenitor cells.

[0139] In this example, regarding the expression of the four factors PAX6, SOX1, NESTIN, and OCT4, the expression at the nucleic acid level and the protein level were investigated respectively.

[0140] Regarding the expression at the nucleic acid level, for the genes of each factor, mRNA was extracted, cDNA was prepared, and quantitative PCR was performed using known primers for detecting the sequence, thereby quantifying the mRNA expression level of each gene for confirmation.

[0141] mRNA extraction was carried out using the RNeasy plus micro kit (Qiagen). 1×10 5Samples with 0 or fewer cells were dissolved in 350 μl of Buffer RLT plus + 3.5 μl of β-ME, and then mRNA extraction was performed according to the product manual. Thereafter, cDNA synthesis was carried out. To synthesize cDNA, 8 μl of the mRNA sample was added to a PCR tube together with 2 μl of SuperScript VILO (Thermo Fischer) and mixed well. Thereafter, it was placed in a PCR apparatus at 25 °C for 10 minutes, reacted at 42 °C for 1 hour, incubated at 85 °C for 5 minutes, and then stored at 4 °C. After cDNA synthesis, the concentration of each cDNA sample was diluted to 5 ng / μl.

[0142] For quantitative PCR, 5 μl of Thunderbird Sybr mix (TOYOBO), 0.3 μl of 10 μM forward primer, 0.3 μl of 10 μM reverse primer, 0.2 μl of 50X ROX, 3.2 μl of water, and 1 μl of 5 ng / μl cDNA template were mixed as each reaction mixture. Each sample was set to 3 replicates and then added to a 384-well plate. For analyzing each sample, it was subjected to the real-time PCR system ViAA7. The primer sets for each factor are as shown above:

[0143] PAX6: 5’-accacaccggtttcctccttcaca-3’ (SEQ ID NO: 1)

[0144] 5’-ttgccatggtgaagctgggcat-3’ (SEQ ID NO: 2)

[0145] SOX1: 5’-gatcagcaagcgcctggggg-3’ (SEQ ID NO: 3)

[0146] 5’-agcagcgtcttggtcttgcgg-3’ (SEQ ID NO: 4)

[0147] NESTIN: 5’-ttccctcagctttcaggaccccaa-3’ (SEQ ID NO: 5)

[0148] 5’-aaggctggcacaggtgtctcaa-3’ (SEQ ID NO: 6)

[0149] OCT4: 5’-ttgggctcgagaaggatgtggt-3’ (SEQ ID NO: 7)

[0150] 5’-tgcatagtcgctgcttgatcgc-3’ (SEQ ID NO: 8).

[0151] In addition, regarding the protein expression level, the protein expression levels of each factor were visualized by fluorescence labeling using an antibody against the protein of each factor, and thus confirmed.

[0152] Each cell was washed 3 times with PBS, and each sample was fixed with 70 μl of 4% PFA for 15 minutes. Thereafter, the cells were washed 3 times with PBS, and the cells were permeabilized with 0.1% triton-x for 15 minutes. The cells were washed 3 times with PBS and blocked overnight at 4°C with 3% BSA / PBS. The next day, the cells were washed 3 times with PBS, and primary antibodies (anti-OCT4 antibody (Millipore), anti-PAX6 antibody (Abcam), anti-SOX1 antibody (Abcam), anti-NESTIN antibody (Millipore), anti-TUJ1 (Beta-Tubulin III) antibody (Sigma), anti-MAP2 antibody (Sigma), anti-OLIG2 antibody (R&D), anti-GFAP antibody (Synaptic system)) were added to 1% BSA / PBS, and then stored overnight at 4°C. Here, OCT4 is an iPS cell marker; PAX6, SOX1, and NESTIN are neural stem cell markers; TUJ1 and MAP2 are neural cell markers; OLIG2 is an oligodendrocyte marker; GFAP is an astrocyte marker. On the third day, the cells were washed 3 times with PBS, and secondary antibodies (donkey anti-rabbit IgG (Alexa Flour 555), donkey anti-mouse IgG (Alex Flour 488), donkey anti-goat IgG (Alex Flour555), goat anti-rat IgG (Alex Flour 4888), goat anti-guinea pig IgG (Alex Flour 555), Hoechst33342 (all from Thermo Fisher)) and DAPI were added to 1% BSA / PBS, and the reaction was carried out in the dark at room temperature for one hour. After washing 3 times with PBS, observation was performed using a fluorescence microscope (Keyence).

[0153] The results of the mRNA expression levels are shown in Figure 1 . In this figure,

[0154] · "LSB": represents the mRNA expression level of each factor when LSB treatment is performed on iPS cells;

[0155] · "LSBC": represents the mRNA expression level of each factor when LSBC treatment is performed on iPS cells;

[0156] · "LSBE": represents the mRNA expression level of each factor when LSBE treatment is performed on iPS cells;

[0157] · "iPSC" represents the mRNA expression levels of each factor when culturing iPS cells without additives.

[0158] In this figure, for each factor, culture conditions were studied that could meet the criterion of a decrease in OCT4 expression and an increase in the expression of PAX6, SOX1, and NESTIN compared to pluripotent stem cells (iPS cells) as the starting cells. The results showed that:

[0159] · Decrease in OCT4 expression: Compared to "iPSC", any one of LSB treatment, LSBC treatment, and LSBE treatment is preferred, and LSBC treatment and LSBE treatment are more preferred;

[0160] · Increase in PAX6 expression: Compared to "iPSC", any one of LSB treatment and LSBE treatment is preferred, and LSBE treatment is more preferred;

[0161] · Increase in SOX1 expression: Compared to "iPSC", any one of LSB treatment and LSBE treatment is preferred, and LSBE treatment is more preferred;

[0162] · Increase in NESTIN expression: Compared to "iPSC", any one of LSB treatment and LSBE treatment is preferred, and LSBE treatment is more preferred.

[0163] This result indicates that in the present invention, the method of culturing using a medium supplemented with CHIR99021 (ReproCELL) as a Wnt agonist (LSBC treatment) is preferred for reducing the expression of OCT4, but is not preferred for increasing the expression of PAX6, SOX1, and NESTIN.

[0164] On the other hand, it was shown that the method of culturing using a medium supplemented with LDN193189 (StemRD) and SB43154 (Tocris Bioscience) 2 as SMAD signaling pathway inhibitors (LSB treatment) and the method of culturing using a medium further supplemented with EC23 (Cayman) as an RA analog to the LSB treatment (LSBE treatment) are both preferred methods for reducing the expression of OCT4 and increasing the expression of PAX6, SOX1, and NESTIN, and the LSBE treatment is a more preferred method. This result indicates that through 5 days of culture, cells with a preferred neural stem cell / neural progenitor cell marker phenotype were obtained.

[0165] The protein expression results of each factor are shown in Figure 2 . This protein expression result indicates that it is related to the mRNA expression level results of each factor described above. Figure 2 ​

[0166] Example 2: Study on culture conditions when manufacturing neural stem cells / neural progenitor cells from pluripotent stem cells (2)

[0167] This example was conducted to further study the culture conditions for manufacturing neural stem cells / neural progenitor cells from pluripotent stem cells.

[0168] In this example, the concentration of the RA analog EC23 (Cayman), which was the result of Example 1, was determined by comparing it with the relatively low concentration of retinoic acid (RA) of about 10 nM reported in a previous report (Okada Y. et al, Stem Cells, 2008). In this example, retinoic acid (RA) (Sigma-Aldrich) was added to the culture medium at 10 nM, and EC23 (Cayman) was added at 10 nM. This addition amount was determined based on prior studies of concentrations from 10 nM to 100 nM and confirmation that the effect of 10 nM was sufficient.

[0169] Based on the above research, in this example, iPS cells (ReproCELL, RCRP002N), which are pluripotent stem cells, were used as the starting cells in the same way as in Example 1, and cultured under the same conditions as in Example 1 until day 1.

[0170] For the cells, the culture medium was changed on days 2 and 5 to 3 ml of the following culture medium containing additives:

[0171] · "LSBC": A medium based on AK02N containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and also containing 3 μM of CHIR99021 (ReproCELL) as a "Wnt agonist" (hereinafter, culturing under this culture condition is also referred to as LSBC treatment);

[0172] · "LSBE": A medium based on AK02N containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and also containing 10 nM of EC23 (Cayman) as a "retinoic acid (RA) or RA analog" (hereinafter, culturing under this culture condition is also referred to as LSBE treatment);

[0173] · "LSBRA": A medium based on AK02N containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and 10 nM of retinoic acid (RA) as a "retinoic acid (RA) or RA analog"; hereinafter, culturing under this culture condition is also referred to as LSBRA treatment.

[0174] · "iPSC" is a medium based on AK02N without additives,

[0175] and cultured until day 6, and an attempt was made to produce neural stem cells / neural progenitor cells from the source cells.

[0176] In this example, as in Example 1, the expression of OCT4 decreased compared to pluripotent stem cells (iPS cells) as the source cells, and conversely, the expression of PAX6, SOX1, and NESTIN was upregulated, which are characteristics of neural stem cells / neural progenitor cells, was used as an index to investigate whether neural stem cells / neural progenitor cells could be produced.

[0177] In Example 1, it has been confirmed that for the expression of the four factors PAX6, SOX1, NESTIN, and OCT4, the expression at the nucleic acid level is related to the expression at the protein level. Therefore, in this example, the expression at the nucleic acid level of the four factors PAX6, SOX1, NESTIN, and OCT4 was investigated. Regarding the expression at the nucleic acid level, it was confirmed by the same method as in Example 1.

[0178] The results of the mRNA expression levels are shown in Figure 3 . In this figure,

[0179] · "LSBC" represents the expression levels of each factor when iPS cells are treated with LSBC;

[0180] · "LSBE" represents the expression levels of each factor when iPS cells are treated with LSBE;

[0181] · "LSBRA" represents the expression levels of each factor when iPS cells are treated with LSBRA;

[0182] · "iPS" represents the expression levels of each factor when iPS cells are cultured without additives.

[0183] The culture conditions that could meet the criterion of a decrease in the expression of OCT4 and an upregulation in the expression of PAX6, SOX1, and NESTIN compared to pluripotent stem cells (iPS cells) as the source cells, which are characteristics of neural stem cells / neural progenitor cells, were investigated. The results showed that:

[0184] ·To reduce the expression of OCT4, any one of LSBC treatment, LSBE treatment, and LSBRA treatment is preferred;

[0185] ·To enhance the expression of PAX6, any one of LSBE treatment and LSBRA treatment is preferred, and LSBE treatment is more preferred;

[0186] ·To enhance the expression of SOX1, any one of LSBC treatment, LSBE treatment, and LSBRA treatment is preferred, and LSBE treatment and LSBRA treatment are more preferred;

[0187] ·To enhance the expression of NESTIN, any one of LSBE treatment and LSBRA treatment is preferred, and LSBE treatment is more preferred.

[0188] The results show that when "retinoic acid (RA) or RA analog" is added in the present invention, it is more preferred to use the RA analog EC23 (Cayman) (LSBE treatment). The results show that through 5-day culture, cells with a more preferred marker phenotype of neural stem cells / neural progenitor cells are obtained.

[0189] Example 3: Study on culture conditions when manufacturing neural stem cells / neural progenitor cells from pluripotent stem cells (3)

[0190] This example is carried out to further study the culture conditions for manufacturing neural stem cells / neural progenitor cells from pluripotent stem cells.

[0191] In this example, based on the insight reported in a previous report (Qi Y. et al, Nat Biotechnol., 2017) that adding SU5402 (Cayman), an inhibitor of the FGF signaling pathway, can promote differentiation into nerve cells, the effect of adding SU5402 to the culture medium is confirmed. In this example, SU5402 (Cayman) is added to the culture medium at 5 μM.

[0192] Based on the above research, in this example, iPS cells (ReproCELL, RCRP002N), which are pluripotent stem cells, are used as raw material cells in the same way as in Example 1, and cultured under the same conditions as in Example 1 until the 1st day.

[0193] For the cells, the culture medium is changed on the 2nd day and the 5th day to the following 3 ml of culture medium containing additives:

[0194] · "LSBC": A medium based on AK02N containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and 3 μM of CHIR99021 (ReproCELL) as a "Wnt agonist" (hereinafter, culturing under this culture condition is also referred to as LSBC treatment);

[0195] · "LSBE": A medium based on AK02N containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), and 10 nM of EC23 (Cayman) as a "retinoic acid (RA) or RA analog" (hereinafter, culturing under this culture condition is also referred to as LSBE treatment);

[0196] · "LSBES": A medium based on AK02N containing 150 nM of LDN193189 (StemRD), 3 μM of SB431542 (Tocris Bioscience), 10 nM of EC23 (Cayman), and 5 μM of SU5402 (Cayman) as an "FGF signaling pathway inhibitor" (hereinafter, culturing under this culture condition is also referred to as LSBES treatment);

[0197] · "iPSC" is a medium based on AK02N without additives;

[0198] And cultured until the 6th day, attempting to produce neural stem cells / neural progenitor cells from the source cells.

[0199] In this example, similar to Example 1, using as an index the decrease in the expression of OCT4 compared to pluripotent stem cells (iPS cells) as the source cells, and conversely the increase in the expression of PAX6, SOX1, and NESTIN, which are characteristics of neural stem cells / neural progenitor cells, to investigate whether neural stem cells / neural progenitor cells can be produced.

[0200] In addition, in this example, similar to Example 2, the expression of the four factors PAX6, SOX1, NESTIN, and OCT4 was confirmed by the same method as in Example 1.

[0201] The results of the mRNA expression levels are shown in Figure 4 . In this figure,

[0202] · "LSBC" represents the expression levels of each factor when iPS cells are subjected to LSBC treatment;

[0203] · "LSBE" represents the expression levels of each factor when iPS cells are subjected to LSBE treatment;

[0204] · "LSBES" represents the expression levels of each factor when iPS cells are subjected to LSBES treatment;

[0205] · "iPSC" represents the expression levels of each factor when iPS cells are cultured without additives.

[0206] The culture conditions that meet the criteria of a decrease in the expression of OCT4 and an increase in the expression of PAX6, SOX1, and NESTIN compared to pluripotent stem cells (iPS cells) as the starting cells, which are characteristic of neural stem cells / neural progenitor cells, were investigated. The results showed that:

[0207] · To decrease the expression of OCT4, any one of LSBC treatment, LSBE treatment, and LSBES treatment is preferred;

[0208] · To increase the expression of PAX6, any one of LSBE treatment and LSBES treatment is preferred, and LSBES treatment is more preferred;

[0209] · To increase the expression of SOX1, any one of LSBC treatment, LSBE treatment, and LSBES treatment is preferred, LSBE treatment and LSBES treatment are more preferred, and LSBES treatment is further preferred;

[0210] · To increase the expression of NESTIN, any one of LSBC treatment, LSBE treatment, and LSBES treatment is preferred, and LSBE treatment and LSBES treatment are more preferred.

[0211] From the results of Examples 1 to 3, for producing neural stem cells / neural progenitor cells using iPS cells as the starting material, any one of LSB treatment, LSBE treatment, LSBRA treatment, or LSBES treatment is preferred, LSBE treatment or LSBES treatment is more preferred, and LSBES treatment is further preferred. This result indicates that cells with a more preferred marker phenotype of neural stem cells / neural progenitor cells were obtained through 5 days of culture.

[0212] Example 4: Formation of neurospheres (1)

[0213] This example was conducted to study the process of producing neurospheres using the neural stem cells / neural progenitor cells prepared based on the results of Examples 1 to 3.

[0214] In this example, as the raw material cells for manufacturing neural spheres, neural stem cells / neural progenitor cells produced by performing the LSBES treatment, which was confirmed as the most preferred method in Example 3, were used.

[0215] In a PrimeSurface low-adhesion 96-well multi-well plate (Sumitomo Bakelite), the neural stem cells / neural progenitor cells were inoculated at a cell density of 1.6×10 2 cells per well. As the culture medium, PNS medium (KBM medium containing 2% B-27 supplement (registered trademark), 20 ng / mL bFGF (Peprotech), 10 ng / mL hLIF (NACALAI TESQUE), 10 μM Y27632 (CultureSURE), 3 μM CHIR99021 (ReproCELL), 2 μM SB431542 (Tocris Bioscience), and 2 μM EC23 (Cayman)) was used, and half of the medium was changed every other day. When changing the medium, the cells that did not form cell clusters were removed.

[0216] After 5 days of cell culture, the culture (primary neural spheres, PNS) was passaged and inoculated into a 96-well multi-well plate (falcon) at a cell density of 4.2×10 2 cells per well. As the culture medium, SNS medium (KBM medium containing 2% B-27 supplement, 20 ng / mL bFGF (Peprotech), 10 ng / mL hLIF (NACALAI TESQUE), 10 μM Y27632 (CultureSURE), 2 μM SB431542 (Tocris Bioscience), and 2 μM EC23 (Cayman)) was used, and half of the medium was changed every other day. Thereafter, the cells were further cultured for 7 days (SNS), and the total culture time was 12 days.

[0217] Example 5: Formation of neurospheres (2)

[0218] This example was conducted to further study the process of manufacturing neural spheres studied in Example 4.

[0219] In this example, similar to Example 4, neural stem cells / neural progenitor cells produced by performing the LSBES treatment, which was confirmed as the most preferred method in Example 3, were used as the raw material cells for manufacturing neural spheres.

[0220] In Example 3, the LSBES-treated iPS cells that had reached an almost 90% confluent state on the 6th day of culture were washed twice with 2 ml of PBS. Thereafter, 1 ml of 0.5× TrypLE select (Gibco) was added, and the cells were incubated at 37 °C for 5 minutes to detach the cells. After 5 minutes, the cells were detached using a P1000 Pipetman and transferred to a 15-ml tube containing 9 ml of AK02N medium (Ajinomoto) (supplemented with Y27632 (CultureSURE)). The cells were centrifuged at 800 rpm for 5 minutes, and the supernatant was aspirated. Thereafter, 10 ml of AK02N medium (Ajinomoto) + Y27632 (CultureSURE) was further added to wash away TrypLE select (Gibco) (ES group).

[0221] After aspirating the supernatant, the pellet was resuspended in 1 ml of AK02N medium (Ajinomoto) (supplemented with Y27632 (CultureSURE)), and cell counting was performed.

[0222] Thereafter, the cells were seeded in a PrimeSurface low-adhesion 96-well multi-well plate (Sumitomo Bakelite) at a cell density of 1.6×10 2 cells per well. As the medium, PNS medium (KBM medium containing 2% of B-27 supplement (registered trademark), 20 ng / mL of bFGF (Peprotech), 10 ng / mL of hLIF (NACALAI TESQUE), 10 μM of Y27632 (CultureSURE), 3 μM of CHIR99021 (ReproCELL), 2 μM of SB431542 (Tocris Bioscience), and 2 μM of EC23 (Cayman)) was used, and half of the medium was replaced every other day, and the cells were cultured under hypoxic conditions for 6 days (until the 12th day). (In this example, this time point was the 6th day). As a comparison group, the cells of the LSBES group were cultured using PNS medium without adding EC23 (Cayman) (designated as LSBES PNS-E group).

[0223] This culture was different from Example 4 in that all the cells including the cells removed during medium replacement in Example 4 were combined and cultured to attempt to increase the expression of markers for neural stem cells / neural progenitor cells in all the cells.

[0224] In this example, by the same method as in Example 1, for the expression of PAX6, SOX1, and NESTIN, whether their mRNA expression levels were elevated was confirmed.

[0225] The mRNA expression results are shown in Figure 5 .Should Figure 5 middle,

[0226] "LSBES PNS" indicates the expression level of each factor in primary neurospheres (PNS) formed by culturing neural stem cells / neural precursor cells treated with LSBES using PNS medium;

[0227] "LSBES PNS-E" indicates the expression level of each factor in primary neurospheres (PNS) formed by culturing neural stem cells / neural precursor cells treated with LSBES using a PNS medium to which EC23 (Cayman) was not added.

[0228] The expression of PAX6, SOX1, and NESTIN, which are neural stem cell / neural progenitor cell markers, in the cells that make up the neurospheres was studied. The results showed that:

[0229] · The expression level of PAX6 was increased by adding EC23 (Cayman) to the culture medium;

[0230] By adding EC23 (Cayman) to the culture medium, the expression of SOX1 was slightly reduced, but there was almost no change;

[0231] · The expression level of NESTIN was increased by adding EC23 (Cayman) to the culture medium.

[0232] This result indicates that the addition of EC23 (Cayman) to the culture medium during neurosphere formation can also promote induction into neural stem cells.

[0233] Example 6: Formation of secondary neurospheres

[0234] This example was conducted to show that the PNS prepared in Example 4 or 5 has the same properties as the SNS intended for actual transplantation in the present invention.

[0235] On the 12th day of the neurosphere preparation step of Example 5, the PNS was collected and washed twice with PBS, and then 1 to 3 ml of 0.5×TrypLE select (Gibco) was added and treated at 37°C for 5 minutes to dissolve the spheres. After dissolution, the spheres were washed twice with TrypLE select (Gibco) using KBM medium supplemented with Y27632 (CultureSURE). Thereafter, the dissociated PNS cells were counted and 4.3×10 cells were added per well. 2Cells were seeded at a density of [number of cells] per well in a PrimeSurface low-adhesion 96-well multiwell plate (Sumitomo Bakelite). As the culture medium, 100 μl of SNS medium (KBM medium containing 2% B-27 supplement (registered trademark), 20 ng / mL bFGF (Peprotech), 10 ng / mL hLIF (NACALAI TESQUE), 10 μM Y27632 (CultureSURE), 2 μM SB431542 (Tocris Bioscience), and 2 μM EC23 (Cayman)) was used. Half of the medium was replaced every other day, and the cells were cultured for 7 days. On the 7th day (day 19) after seeding the cells in the plate, the SNS was used for further experiments.

[0236] Example 7: Manufacturing of nervous system cells in vitro

[0237] This example was conducted to produce nervous system cells using the primary neurospheres (PNS) formed in Example 4 or 5 and the secondary neurospheres (SNS) formed in Example 6.

[0238] That is, in this example, the PNS produced by performing the LSBES PNS treatment, which was confirmed to be the most preferred method in Example 5, the SNS produced using this PNS in Example 6, and the cells (ivN) differentiated and induced in vitro from this SNS by the following method were used.

[0239] Specifically, the SNS was recovered and washed twice with PBS. Thereafter, 1 - 3 ml of 0.5× TrypLE select (Gibco) was added, and the spheroids were dissociated by treating at 37°C for 5 minutes. After dissociation, TrypLE select (Gibco) was removed by washing twice with KBM medium supplemented with Y27632 (CultureSURE). Thereafter, the obtained single cells were counted, and seeded at a cell density of 5×10 3 ~7.5×10 3 per well in a 96-well multiwell plate (falcon) (in this example, this time point was set as day 0). As the culture medium, 150 μl of KBM medium containing 10 μM Y27632 (CultureSURE) was used. On day 1, the medium was replaced with KBM medium containing 2% B-27 supplement (registered trademark) and without vitamin A, and then the medium was replaced every other day, and the cells were cultured until day 33.

[0240] In this example, by the same method as in Example 1, in addition to OCT4 as an iPS cell marker, PAX6, SOX1, and NESTIN as neural stem cell / neural progenitor cell markers, it was also confirmed whether there was overexpression / reduction in the expression of SOX17 as an endoderm cell marker, T as a mesoderm marker, TUJ1 and MAP2 as neural cell markers, OLIG2 and NG2 as oligodendrocyte markers, and CD44 as an astrocyte marker. It should be noted that the primer sets for SOX17, T, TUJ1, MAP2, OLIG2, NG2, and CD44 are as follows:

[0241] SOX17: 5’-acgctttcatggtgtgggctaag-3’ (SEQ ID NO: 9)

[0242] 5’-gtcagcgccttccacgacttg-3’ (SEQ ID NO: 10)

[0243] T: 5’-ccttcagcaaagtcaagctcacc-3’ (SEQ ID NO: 11)

[0244] 5’-tgaactgggtctcagggaagca-3’ (SEQ ID NO: 12)

[0245] TUJ1: 5’-atttcatctttggtcagagtggggc-3’ (SEQ ID NO: 13)

[0246] 5’-tgcaggcagtcgcagttttcac-3’ (SEQ ID NO: 14)

[0247] MAP2: 5’-ggatcaacggagagctgac-3’ (SEQ ID NO: 15)

[0248] 5’-tcaggactgctacagcctca-3’ (SEQ ID NO: 16)

[0249] OLIG2: 5’-ggcgcgcaactacatcct-3’ (SEQ ID NO: 17)

[0250] 5’-cgctcaccagtcgcttcat-3’ (SEQ ID NO: 18)

[0251] NG2: 5’-ttgtcctgatggctaatgcct-3’ (SEQ ID NO: 19)

[0252] 5’-tgggctgctcgatggtgta-3’ (SEQ ID NO: 20)

[0253] CD44: 5’-tggcacccgctatgtcgag-3’ (SEQ ID NO: 21)

[0254] 5’-gtagcagggattctgtctg-3’ (SEQ ID NO: 22)

[0255] In the steps of this example, for the iPS cells (iPSC), primary neural spheres (PNS), and cells obtained by in vitro differentiation induction of secondary neural spheres (SNS) (ivN) used as raw materials,

[0256] · OCT4, an iPS cell marker,

[0257] · PAX6, SOX1, NESTIN, neural stem cell markers,

[0258] · SOX17, an endoderm marker,

[0259] · T, a mesoderm marker,

[0260] · TUJ1 and MAP2, neural cell markers,

[0261] · OLIG2, NG2, oligodendrocyte markers,

[0262] · The results of the expression levels of each mRNA of CD44, an astrocyte marker, are shown in Table 1.

[0263] The results of manufacturing neural spheres using the differentiation induction method of the present invention show that, compared with iPS cells, in the cases of PNS and SNS, the expression level of the iPS cell marker OCT4 is significantly reduced, and the expression levels of the neural stem cell markers PAX6, SOX1, and NESTIN are increased. On the other hand, it also shows that the expression levels of the endoderm marker SOX17 and the mesoderm marker T do not increase at all. These results indicate that specific differentiation into neural stem cells has been induced.

[0264] Next, the results of dispersing the SNS and performing in vitro differentiation induction show that (ivN) is that, compared with SNS, in the case of ivN, the expression levels of PAX6 and SOX1 among the neural stem cell markers decrease, the expression levels of the neural cell markers TUJ1 and MAP2 increase, and the expression levels of OLIG2 and NG2, oligodendrocyte markers, and CD44, an astrocyte marker, also increase.

[0265] These results indicate that by performing in vitro differentiation induction on neural spheres derived from neural stem cells, they are differentiated into cells of three systems: neural cells, glial cells, and oligodendrocytes.

[0266] [Table 1]

[0267] Next, the results of immunocytochemistry during differentiation induction starting from the PNS in vitro are shown in Figure 6 , and the results of immunocytochemistry during differentiation induction starting from the SNS are shown in Figure 7 . These results indicate that:

[0268] · When differentiating and inducing cells derived from the PNS in vitro, no OCT4-positive cells were found ( Figure 6 upper left), PAX6, NESTIN, and SOX1-positive cells were confirmed ( Figure 6 upper left, upper right), OLIG2-positive cells were confirmed ( Figure 6 lower left), TUJ1-positive cells were confirmed ( Figure 6 lower right), and GFAP-positive cells were confirmed ( Figure 6 lower right). These indicate that the cells constituting the PNS do not contain undifferentiated iPS cells, some maintain the state of neural stem cells / neural progenitor cells, but have the ability to differentiate into cells of three systems: neurons, astrocytes, and oligodendrocytes.

[0269] · When differentiating and inducing cells derived from the SNS in vitro, no OCT4-positive cells were found ( Figure 7 upper left), PAX6, NESTIN, and SOX1-positive cells were confirmed ( Figure 7 upper left, upper right), OLIG2-positive cells were confirmed ( Figure 7 lower left), TUJ1-positive cells were confirmed ( Figure 7 lower right), and GFAP-positive cells were confirmed ( Figure 7 lower right). These indicate that the cells constituting the SNS do not contain undifferentiated iPS cells, some maintain the state of neural stem cells / neural progenitor cells, but have the ability to differentiate into cells of three systems: neurons, astrocytes, and oligodendrocytes. The above results indicate that the PNS and SNS have the same differentiation ability.

[0270] Example 8: Induction of differentiation in vivo

[0271] This example was conducted to study the therapeutic effect of the secondary neurospheres prepared in Example 6 on spinal cord injury in vivo.

[0272] In this example, 8-week-old NOD-SCID mice were used. The spinal cord injury of these mice was made as follows: The vertebral arch of the 10th thoracic vertebra was resected, and a pressure of 70 kdyn was applied to the spinal cord using a special impactor with a pressure sensor, thus making the injury. On the 9th day after spinal cord injury, the SNS prepared in Example 6 was transplanted. That is, at the center of the spinal cord injury of mice with motor dysfunction confirmed 9 days after the injury surgery, 2 μl of a liquid containing 2.5×10 5 / μl of SNS (SNS; transplantation group) was injected over 2 minutes. As a control, mice injected with only 2 μl of PBS (PBS; non-transplantation group) were used.

[0273] The Basso Mouse Scale (BMS) was used to evaluate the movement of the hind limbs during walking for each group. BMS is an evaluation criterion for scoring the movement of the hind limbs during walking of a mouse spinal cord injury model with a full score of 9 points. The specific evaluation is as follows. For the transplantation group; SNS (n = 10) and the non-transplantation group; PBS (n = 11), the mean ± standard error of the mean (SEM) of these scores was calculated, and statistical analysis was performed by Repeated measure ANOVA. A p < 0.05 was judged as having a significant difference.

[0274] 0 points: The ankle joint cannot move at all

[0275] 1 point: The ankle joint can move slightly

[0276] 2 points: The ankle joint moves well

[0277] 3 points: The sole of the foot touches the ground or walks on the dorsum of the foot

[0278] 4 points: Occasionally walks on the sole of the foot

[0279] 5 points: Frequently walks on the sole of the foot, but does not move in coordination with the forelimbs

[0280] 6 points: Frequently walks on the sole of the foot, and occasionally moves in coordination with the forelimbs

[0281] 7 points: Frequently walks on the sole of the foot, and almost moves in coordination with the forelimbs. There is severe trunk instability

[0282] 8 points: Frequently walks on the sole of the foot, and almost moves in coordination with the forelimbs. There is slight trunk instability

[0283] 9 points: Normal

[0284] The results of evaluating the walking state of mice over time by BMS score up to 9 weeks after spinal cord injury are shown in Figure 8At the time of the downward arrow in the figure (9 days after spinal cord injury), SNS cells were transplanted or PBS was injected. Here, the SNS cells used for transplantation were prepared by the same method as the SNS cell preparation method of Example 7, except that they were cultured in a medium supplemented with the γ-secretase inhibitor DAPT (10 μM) (Selleck), which is a Notch signal inhibitor, only for the last 24 hours of the last 7 days of SNS production in the SNS cell preparation method of Example 7. From around 2 weeks after SNS transplantation, a tendency for improved walking was observed. The BMS scores 9 weeks later were 4.6 ± 0.3 for the SNS transplantation group and 3.5 ± 0.2 for the PBS injection group (p < 0.01, Repeated measure ANOVA), and significant motor function recovery was confirmed.

[0285] Then, for the differentiated cell types of the transplanted cells in vivo, antibodies against the marker proteins of each cell type were used, and the expression of each marker protein was visualized by fluorescence labeling for determination.

[0286] The frozen spinal cord sections of individuals whose walking status was evaluated by BMS score over time 9 weeks after spinal cord injury were air-dried and washed 3 times with PBS filled in the staining pool. Thereafter, they were blocked with Blocking One (NACALAI TESQUE) at room temperature for 20 minutes. Then, the primary antibodies diluted in Blocking One (anti-HNA antibody (Millipore), anti-OCT4 antibody (Millipore), anti-PAX6 antibody (Abcam), anti-HuC / D antibody (Invitrogen), anti-OLIG2 antibody (R&D), anti-GFAP antibody (Synaptic system)) were added, and then stored at 4 °C overnight. Here, HNA is a human nuclear marker; OCT4 is an iPS cell marker; PAX6 is a neural stem cell marker; HuC / D is a neural cell marker; OLIG2 is an oligodendrocyte marker; GFAP is an astrocyte marker.

[0287] On the next day, it was washed three times with PBS, and a secondary antibody diluted in Blocking One (donkey anti-rabbit IgG (AlexaFlour 555), donkey anti-mouse IgG (Alex Flour 488, Alex Flour 555), donkey anti-goat IgG (Alex Flour555), goat anti-rat IgG (Alex Flour 488), goat anti-guinea pig IgG (Alex Flour 555), Hoechst33342 (all from Thermo Fisher)) was added, and the reaction was carried out at room temperature for 1 hour. It was washed three times with PBS, Fluoromount (Diagnostic BioSystems) was added dropwise, a cover glass was covered, and it was left at room temperature for 30 minutes to dry the Fluoromount. The stained image was observed with a fluorescence microscope (Keyence).

[0288] The results of the fluorescence staining are shown in Figure 9 . Positive for HNA as a human nuclear marker indicates transplanted cells. Most of the HNA-positive cells expressed HuC / D as a neuronal marker ( Figure 9 (a) of), and a part expressed GFAP as an astrocyte marker and OLIG2 as an oligodendrocyte marker ( Figure 9 (b) and (c) of). In addition, HNA-positive cells expressing PAX6 as a neural stem cell marker were also confirmed ( Figure 9 (d) of). As shown in this figure, it can be seen that the SNS transplanted into the spinal cord injury site was mainly differentiated into neurons, and a part was differentiated into astrocytes and oligodendrocytes, and there were cells that maintained the neural stem cell state. On the other hand, there was no residual iPS cell positive for OCT4 ( Figure 9 (e) of).

[0289] Industrial applicability

[0290] By using the method of the present invention, pluripotent stem cells can be stably and highly reproducibly induced into neural stem cells / neural progenitor cells by culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, a low concentration of RA or an RA analog, and an FGF signaling pathway inhibitor for 5 days. Compared with the conventional method, the differentiation efficiency into nervous system cells can be greatly improved thereafter.

Claims

1. A method for producing neural stem cells / neural progenitor cells from pluripotent stem cells, comprising the following steps: culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, retinoic acid (RA) or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days.

2. The method according to claim 1, wherein, the expression of PAX6, SOX1, and NESTIN in the neural stem cells / neural progenitor cells is enhanced and the expression of OCT4 is inhibited.

3. The method according to claim 1, wherein, the pluripotent stem cells are selected from the group consisting of ES cells and iPS cells.

4. The method according to claim 2, wherein, the pluripotent stem cells are selected from the group consisting of ES cells and iPS cells.

5. The method according to any one of claims 1 to 4, wherein, the SMAD signaling pathway inhibitor is SB431542 and LDN193189.

6. The method according to any one of claims 1 to 4, wherein, the RA analog is EC23.

7. The method according to any one of claims 1 to 4, wherein, the FGF signaling pathway inhibitor is SU5402.

8. The method according to any one of claims 1 to 4, wherein, the medium is selected from the group consisting of a medium for primate ES / iPS cells, AK02N medium, ciKIC (registered trademark) iPS Medium, mTeSR medium, NutriSTEM medium, and Essential 8 medium.

9. A method for producing neural spheres from pluripotent stem cells, comprising the following steps: Step (i): culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, RA or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days to produce neural stem cells / neural progenitor cells from the pluripotent stem cells; Step (ii): producing neural spheres formed by the neural stem cells / neural progenitor cells.

10. The method according to claim 9, wherein, Step (ii) is carried out by culturing in the presence of B-27 supplement (registered trademark), Y27632, LIF, FGF, and RA or an RA analog for 13 days.

11. The method according to claim 10, wherein, the RA analog is EC23.

12. The method according to any one of claims 9 to 11, wherein, Step (ii) is carried out under hypoxic conditions.

13. The method according to any one of claims 9 to 11, wherein, 12 days after the start of the culture in Step (ii), a Notch signal inhibitor is further added.

14. The method according to claim 13, wherein, the Notch signal inhibitor is a γ-secretase inhibitor.

15. The method according to any one of claims 9 to 11, wherein, after the step of (ii) is carried out, the formed neural spheres are further dispersed into single cells and then the step of producing neural spheres is carried out one or more times.

16. A method for manufacturing nervous system cells from pluripotent stem cells, comprising the following steps: Step (i): Culturing pluripotent stem cells in a medium containing an SMAD signaling pathway inhibitor, RA or an RA analog at a concentration of 100 nM or less, and an FGF signaling pathway inhibitor for 5 days to produce neural stem cells / neural progenitor cells from the pluripotent stem cells; Step (ii): Producing neurospheres formed from neural stem cells / neural progenitor cells; Step (iii): Producing nervous system cells from the neurospheres.

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