Manufacturing method and expansion culture method for hindbrain neural stem cell, culture medium, cell, and frozen stock

Abstract

The purpose of the present invention is to provide a method whereby hindbrain neural stem cells can be cultured over a long period of time while maintaining multipotency. The present invention relates to a method for manufacturing hindbrain neural stem cells, the method involving: a step for inducing differentiation of hindbrain neural stem cells from pluripotent stem cells; and a step for culturing the hindbrain neural stem cells by using a culture medium that contains a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor and does not contain a basic fibroblast growth factor (bFGF).

Description

Method for producing hindbrain neural stem cells, method for expanding culture, culture medium, cells, and frozen stock

[0001] The present invention relates to a method for producing hindbrain neural stem cells, a method for expanding culture, a culture medium, cells, and a frozen stock. More specifically, the present invention relates to a method for producing hindbrain neural stem cells and a method for expanding culture, a culture medium used therefor, hindbrain neural stem cells, and a frozen stock containing hindbrain neural stem cells.

[0002] During the developmental period, the central nervous system is regionalized into the forebrain, midbrain, hindbrain, and spinal cord along the anterior-posterior axis. Each region is further regionalized until adulthood, and each region has its own specific function. Among them, the hindbrain is an important region that forms tissues responsible for basic life support and activities in humans, such as the medulla oblongata (control of life support functions), pons (transmission of movement and sensation), and cerebellum (regulation of movement). Therefore, the development of a method for culturing neural stem cells (hindbrain neural stem cells) present in this hindbrain can greatly contribute to regenerative medicine, elucidation of pathological conditions, and drug discovery research using these cells.

[0003] As one approach, a method has been reported for isolating hindbrain neural stem cells from the hindbrain region of the fetal period and maintaining them in culture as neural stem cells (Non-Patent Document 1).

[0004] As other approaches for obtaining neural stem cells, methods for inducing differentiation of neural stem cells from pluripotent stem cells such as ES cells and iPS cells (Non-Patent Documents 2 to 9), or methods for inducing neural stem cells from other cells such as fibroblasts by direct conversion or the like (Non-Patent Document 10) have been reported, and methods for maintaining these cells in culture as stem cells have also been reported.

[0005] Tailor J et al., J Neurosci. 2013 Jul 24; 33(30): 12407-12422.Chambers SM et al., Nat Biotechnol. 2009 Mar;27(3):275-80Kim DS et al., Stem Cell Rev Rep. 2010 Jun;6(2):270-81.Lu J et al., Nat Biotechnol. 2016 Jan;34(1):89-94.Zhang M et al., Cell Stem Cell. 2016 May 5;18(5):653-67.Rifes P et al., Nat Biotechnol. 2020 Nov;38(11):1265-1273.Togo K et al., Mol Brain. 2021 Oct 11;14(1):149.Valiulahi P et al., Stem Cell Reports. 2021 Aug 10;16(8):1938-1952.Nishimura K et al., Stem Cell Reports. 2023 Jan 10;18(1):337-353.Li W et al., Proc Natl Acad Sci US A. 2011 May 17;108(20):8299-304.

[0006] However, previous reports starting with pluripotent stem cells have been limited to induction studies and have not performed long-term culture, and no method has been reported for culturing hindbrain neural stem cells over the long term while maintaining their pluripotency. In particular, hindbrain neural stem cells inherently possess multipotency, the ability to differentiate into various cell types such as neurons and glial cells, but it is known that under culture conditions, multipotency gradually decreases, and the number of cell types that can differentiate decreases. Furthermore, previous reports on the maintenance culture of hindbrain neural stem cells have included bFGF in the culture medium. For example, the method in Non-Patent Document 1 has ethical issues, such as using fetuses as a cell source, and also has the issue of using expensive bFGF.

[0007] Therefore, the present invention aims to provide a method for culturing postbrain neural stem cells over a long period of time while maintaining their multipotency.

[0008] Therefore, after diligent research, the inventors unexpectedly discovered that hindbrain neural stem cells can be cultured for a long period of time while maintaining multipotency by culturing them in a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF), leading to the present invention.

[0009] Therefore, embodiments of the present invention can be expressed as follows.

[0010] [1] A method for producing hindbrain neural stem cells, comprising the steps of: inducing differentiation of pluripotent stem cells into hindbrain neural stem cells; and culturing the hindbrain neural stem cells using a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF). [2] The method according to [1], wherein the differentiation induction step is carried out using the medium. [3] The method according to any one of [1] to [2], wherein the culturing step is carried out following the differentiation induction step. [4] The method according to any one of [1] to [3], wherein the hindbrain neural stem cells are GBX2 positive and positive for at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. [6] The method according to any one of [1] to [5], wherein the medium does not contain any animal-derived components. [7] The method according to any one of [1] to [6], wherein in the culture step, the hindbrain neural stem cells are proliferated while maintaining multipotency. [8] The method according to any one of [1] to [7], wherein the culture step is carried out for 8 days or more. [9] The method according to any one of [1] to [8], wherein the culture step is carried out for 14 days or more.

[10] A culture medium for culturing hindbrain neural stem cells, comprising a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF).

[11] The culture medium according to any one of [1] to

[10] , wherein the culture medium does not contain animal-derived components.

[12] A method for expanding the culture of hindbrain neural stem cells having multipotency, comprising the step of culturing the hindbrain neural stem cells using a culture medium comprising a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF).

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

[12] , wherein the culture medium does not contain any animal-derived components.

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

[13] , wherein the culturing step is carried out for 8 days or more.

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

[14] , wherein the culturing step is carried out for 14 days or more.

[16] Hindbrain neural stem cells obtained by any one of the methods of [1] to

[15] .

[17] Frozen stock containing hindbrain neural stem cells obtained by any one of the methods of [1] to

[16] . This specification includes the disclosures of Japanese Patent Application No. 2024-197945, which forms the basis of the priority of this application.

[0011] The present invention provides a method for culturing postbrain neural stem cells over a long period of time while maintaining their multipotency.

[0012] This is a schematic diagram of the induction method used to induce human iPS cells into hindbrain neural stem cells (Hb-iNSCs). From left to right, the images show phase-contrast images of iPS cell colonies, Hb-iNSC colonies, and neurons differentiated from Hb-iNSCs (scale bar: 100 μm). These are phase-contrast images of Hb-iNSCs induced from three different iPS cell lines on day 7. This graph shows the changes in gene expression during the induction period to Hb-iNSCs, examined by qPCR. This graph shows the gene expression of E-cadherin (CDH1), N-cadherin (CDH2), SOX1, SOX2, PAX6, and POU5F1 during the induction period (days 1-7) relative to the gene expression of iPS cells (day 0), which is set to 1 (mean (n=3), error bars: standard deviation). These are fluorescence images showing the results of immunohistochemical staining for SOX1, SOX2, NESTIN, PAX6, NANOG, and POU5F1 in cells 7 days after induction from human iPS cells (scale bar: 50 μm). Gene expression on day 7 was confirmed by protein analysis. A heatmap comparing gene expression on day 0 (iPS cells) and day 7 (Hb-iNSCs) by bulk RNA sequencing (RNAseq) is shown. Three samples of 1231A3 iPS cells and three samples of PN0 day 7 1231A3 Hb-iNSCs were used for this analysis. The results for pluripotency markers in the heatmap of Figure 4A are shown as bar graphs. The results for neural stem cell markers in the heatmap of Figure 4A are shown as bar graphs. The results for hindbrain markers in the heatmap of Figure 4A are shown as bar graphs. This is a volcano plot comparing the expression levels of Hb-iNSC samples (3 samples) on day 7 and iPS cell controls (3 samples) on day 0. The y-axis represents the p-value, and the x-axis represents the log fold change value. Red dots represent genes with significant differences in expression (significant difference genes, SigDEGs). The vertical dashed line indicates the threshold of 1.5 log fold change, and the horizontal dashed line indicates the threshold of significance (p = 0.05).This figure shows the results of enrichment analysis using a dataset of genes whose expression was significantly increased (log2 fold change ≥ 1.5) in iPS cells (p < 0.05). The size of the dots indicates the number of genes that overlapped with the dataset, the intensity of the dot color indicates significance (top 10 terms sorted based on p-value), and the x-axis shows the score calculated by Enrichr. Neurological terms are highlighted in red. These are phase-contrast images of iPS cells after induction for one week using iPS cells, or A-83-01, CHIR99021, or LDN193189 individually or in combination. These are graphs showing the results of qPCR analysis in iPS cells obtained under each condition. This is a schematic diagram showing the timing of subculturing, RNA sample collection, and cell cryopreservation. Subsequent figures show sample collection according to this schematic diagram. Cell subculturing was performed weekly, and RNA sample collection and cell cryopreservation were performed every 5 subculturings. Based on passage number, samples were classified into early (PN5-20), mid-stage (PN25-40), and late-stage (PN45-60) groups. The image shows the karyotype analysis results for Hb-iNSCs at passage 53 (week 53) derived from 1231A3 human iPS cells. The fluorescence image (scale bar: 50 μm) shows the results of immunohistochemical staining for TUBB3, SOX1, NESTIN, PAX6, NANOG, and SOX2 in Hb-iNSCs at passage 60 (week 60) derived from iPS cells. The heatmap compares gene expression in iPS cells, Hb-iNSCs at passages 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60, and neurons differentiated from passages 5 and 60 (_diff), as determined by bulk RNA sequencing. As markers, we used pluripotent stem cell markers, neural stem cell markers, neural differentiation markers, and representative markers from different brain regions. Samples PN5_1 to PN5_3 and PN60_1 to PN60_3 were derived from the 1231A3 iPS cell line, while samples PN5_4 and PN5_5 were derived from the HLAKO and SgT5 iPS cell lines, respectively. The bar graph shows the gene expression of hindbrain markers in PN0 to PN60.This graph shows the gene expression of hindbrain markers in PN0–PN60 cells as a bar graph. It also shows the results of principal component analysis using bulk RNA-seq data. Red represents iPS cells (control), green represents early Hb-iNSCs and post-neuronal differentiation, purple represents mid-stage Hb-iNSCs, and blue represents late-stage Hb-iNSCs and post-neuronal differentiation. The graph shows the results of enrichment analysis for genes whose expression was significantly increased (log2 fold change ≥ 1.5) in late-stage Hb-iNSCs compared to iPS cells (control). The size of the dots indicates the number of overlapping genes in the dataset, and the intensity of the dot color indicates significance (top 10 terms sorted based on p-value). The x-axis represents the score calculated by Enrichr, and neurological terms are highlighted in red. Finally, there is a heatmap showing the correlation between samples. The intensity of the color indicates the degree of correlation: red = 1 indicates a positive correlation, white = 0 indicates no correlation, and green = -1 indicates a negative correlation. This is a bar graph of log fold change for genes with differing expression levels detected by bulk RNA sequencing. This is a comparison of late Hb-iNSCs and iPS cells (control). The green bars on the left show a list of genes whose expression was elevated in early Hb-iNSCs (upper x-axis), and the red bars on the right show a list of genes whose expression was decreased in early Hb-iNSCs (lower x-axis). This is a bar graph plot of RTqPCR data for GBX2, HOXB4, SIX3, and HOXB9 in PN5 and PN60 Hb-iNSC samples (n=3). Human RNA samples from the cerebellum, pons, cerebral cortex, and spinal cord (n=1) were used as a control group. Statistical significance is indicated by ns≧0.05, *p≦0.05, and **p≦0.01. This graph shows the results of enrichment analysis for 427 genes whose expression was significantly increased (log2 hold change > 1.5) in early Hb-iNSCs compared to iPS cells (control) (P < 0.05). The size of the dots indicates the number of overlapping genes in the dataset, and the intensity of the dot color indicates significance (top 10 terms sorted by p-value).The x-axis represents the score calculated by Enrichr, with neurological terms highlighted in red. This is a schematic diagram of the neurosphere preparation method in Example 2. This schematic diagram shows attachment to a dish (flat surface), attachment to a microelectrode array (MEA), and transplantation into a mouse brain in vivo. These are phase-contrast images of neurospheres 1 week and 8 weeks after the start of preparation (scale bar: 100 μm). This is a bar graph of the cross-sectional area of ​​the neurospheres 1 week and 8 weeks after the start of preparation. This is cellular immunostaining of neurospheres attached to a dish (scale bar: 100 μm). Nuclear staining was performed with TUBB3, OLIG2, GFAP, and DAPI. This graph shows the results of gene expression analysis by RT-qPCR of ventral hindbrain markers (NKX 2.2 and NKX 6.1) in ventralized Hb-iNSCs derived from Hb-iNSCs (relative values ​​between ventralized and non-ventralized Hb-iNSCs) (error bars: standard deviation, n=3). This graph shows the expression of serotonergic neuronal markers (FEV, TPH2, SLC6A4) in ventralized Hb-iNSCs (error bars: standard deviation, n=3). This is a histogram of electrode counts of spike amplitude detected by MEA. Dark colors indicate electrode counts of samples before tetrodotoxin (TTX) addition, and pink colors indicate electrode counts of samples after TTX addition (PN5 left plot and PN61 right plot). This is a raster plot of action potentials detected by MEA. Each dot represents an action potential from a sample derived from PN5 (upper plot) and PN61 (lower plot). Figures 21A and 21B are raster plots showing action potentials recorded after TTX addition for the same sample. This is a schematic diagram showing the transplantation site of neurospheres into the mouse brain. The area indicated by the orange square corresponds to the location in the enlarged view of Figure 22C. These are sagittal section images of the mouse brain 8 weeks after neurosphere transplantation. This shows the tracking results of hNCAM-positive cells detected with human-specific NCAM antibody along the spinocerebellar tract (1, 1'), cerebellum (2, 2'), corticospinal tract (3, 3'), and midbrain (4, 4'). Numbers without a single quote indicate cells derived from early Hb-iNSCs, and numbers with a single quote indicate cells derived from late Hb-iNSCs.These images show brain sections after transplantation of early-stage Hb-iNSC neurospheres and late-stage Hb-iNSC neurospheres, stained with human-specific NCAM antibody, TUBB3 antibody, GFAP antibody, and OLIG2 antibody.

[0013] In this specification, "pluripotency" refers to the ability to differentiate into tissues and cells with various different forms and functions, and to differentiate into cells of any of the three germ layers. "Pluripotency" is distinguished from "totipotency," which is the ability to differentiate into all tissues of a living organism, including the blastodisc, in that "pluripotency" cannot differentiate into the blastodisc and therefore does not have the ability to form an individual.

[0014] In this specification, "multipotency" means the ability to differentiate into a limited number of cell lineages. Preferably, "multipotency" refers to cells that can differentiate into multiple (preferably three or more) cell types from cells derived from all three germ layers, but not all of them. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0015] "Hidbrain neural stem cells" refer to cells differentiated from stem cells that possess the characteristics of neural stem cells in the hindbrain region, have self-renewal ability, and have the ability to differentiate into neurons or glial cells in the hindbrain region. Hindbrain neural stem cells can be identified by confirming the expression of GBX2 and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Hindbrain neural stem cells express GBX2 and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. In addition, hindbrain neural stem cells may express GBX2 and at least two (or at least three, or at least four) selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Furthermore, hindbrain neural stem cells may express GBX2 and at least one (or at least two, at least three, or at least four) selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN, in addition to at least one (or at least two, or at least three) selected from the group consisting of HOXA2, HOXB2, and HOXB4. In one embodiment, hindbrain neural stem cells are GBX2 positive and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Alternatively, hindbrain neural stem cells are GBX2 positive and at least two (or at least three, or at least four) selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Furthermore, in addition to being positive for GBX2 and at least one (or at least two, at least three, or at least four) selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN, the posterior neural stem cells may also be positive for at least one (or at least two, or at least three) selected from the group consisting of HOXA2, HOXB2, and HOXB4.

[0016] Furthermore, it is preferable that the hindbrain neural stem cells do not express at least one selected from the group consisting of POU5F1 and NANOG, and more preferably, they do not express at least two selected from the group consisting of POU5F1 and NANOG. Moreover, in addition to not expressing at least one (or at least two) selected from the group consisting of POU5F1 and NANOG, the hindbrain neural stem cells may also not express at least one (or at least two) selected from the group consisting of SIX3 and HOXB9. In one embodiment, it is preferable that the hindbrain neural stem cells are negative for at least one selected from the group consisting of POU5F1 and NANOG, and more preferably, they are negative for at least two selected from the group consisting of POU5F1 and NANOG. Moreover, in addition to being negative for at least one (or at least two) selected from the group consisting of POU5F1 and NANOG, the hindbrain neural stem cells may also be negative for at least one (or at least two) selected from the group consisting of SIX3 and HOXB9.

[0017] Hindbrain neural stem cells have the ability to differentiate into nerve cells or glial cells. In this specification, "culturing hindbrain neural stem cells with multipotency" means culturing the hindbrain neural stem cells while maintaining their multipotency.

[0018] In this specification, the SOX1 protein is a protein encoded by the SOX1 (SRY-Related HMG-Box 1) gene and is a transcription factor of the Sox protein family. SOX1 is involved in the differentiation and maintenance of neural stem cells and neural progenitor cells. The term SOX1 encompasses its gene, transcript, or polypeptide, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting SOX1.

[0019] In this specification, the SOX2 protein is a protein encoded by the SOX2 (SRY-Related HMG-Box 2) gene and is a transcription factor of the Sox protein family. SOX2 plays a crucial role in self-renewal and maintaining the undifferentiated state. The term SOX2 encompasses its gene, transcript, or polypeptide, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting SOX2.

[0020] In this specification, the PAX6 protein is the protein encoded by the PAX6 (Paired Box 6) gene. PAX6 is a marker of brain structures during embryonic development, including neural progenitor cells. The term PAX6 encompasses its gene, transcript, or polypeptide, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting PAX6.

[0021] In this specification, the NESTIN protein is a neuroepithelial stem cell protein encoded by the NES gene. The NESTIN protein is a type VI intermediate filament protein expressed in neural stem cells and neural progenitor cells. The term NESTIN encompasses its gene, transcript, or polypeptide, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting NESTIN.

[0022] In this specification, the GBX2 protein is the protein encoded by the GBX2 (Gastrulation Brain Homeobox 2) gene. GBX2 is a transcription factor involved in development and differentiation of neural stem cells in the hindbrain region. The term GBX2 encompasses its gene, transcript, or protein, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting GBX2.

[0023] In this specification, the HOXA2 protein, HOXB2 protein, or HOXB4 protein are each homeobox proteins encoded by the HOXA2 gene, HOXB2 gene, or HOXB4 gene, and are involved in hindbrain formation. The term HOXA2 encompasses its gene, transcript, or protein, and may also include its fragments or variants. The term HOXB2 encompasses its gene, transcript, or protein, and may also include its fragments or variants. The term HOXB4 encompasses its gene, transcript, or protein, and may also include its fragments or variants. Those skilled in the art will know how to detect HOXA2, HOXB2, and HOXB4.

[0024] In this specification, the POU5F1 protein is a protein encoded by the "POU class 5 homeobox 1" gene and is one of the markers of pluripotent cells. The term POU5F1 encompasses the gene, transcript, or protein, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting POU5F1.

[0025] In this specification, the NANOG protein is a protein encoded by the "Nanog homeobox" gene and is one of the markers of pluripotent cells. The term NANOG encompasses the gene, transcript, or protein, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting NANOG.

[0026] In this specification, the SIX3 protein is one of the SIX homeobox proteins, encoded by the SIX homeobox 3 gene. The term SIX3 encompasses the gene, transcript, or protein, and may also include their fragments or variants. Those skilled in the art are familiar with methods for detecting SIX3.

[0027] In this specification, the HOXB9 protein is one of the Antp homeobox proteins and is encoded by the HOXB9 gene. The term HOXB9 encompasses its gene, transcript, or protein, and may also include its fragments or variants. Those skilled in the art are familiar with methods for detecting HOXB9.

[0028] In this specification, “culture” means maintaining, growing, or proliferating cells in an in vitro environment. “To culture” means maintaining, growing, or proliferating cells outside of tissue or in vitro, for example, in a cell culture dish or flask. The number of cells may or may not increase during culture.

[0029] "Adherent culture" refers to culturing cells while they are attached to a container, for example, in the presence of a suitable culture medium, in a sterile plastic (or coated plastic) cell culture dish or flask. "Suspension culture," on the other hand, refers to culturing cells without attaching them to a container, by dispersing them in a suitable culture medium as a cell sphere consisting of a single cell or two or more cells.

[0030] "Expanded culture" refers to culturing cells while increasing their proliferation.

[0031] A "GSK3β inhibitor" is a substance that has inhibitory activity against GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase and is involved in many signaling pathways related to glycogen production, apoptosis, and stem cell maintenance. There are two isoforms of GSK3: α and β. The "GSK3β inhibitor" used in this invention is not particularly limited as long as it has GSK3β inhibitory activity, and may also be a substance that has both GSK3β inhibitory activity and GSK3α inhibitory activity.

[0032] In this specification, "TGFβ inhibitor" refers to a substance that inhibits the signal transduction from the binding of TGFβ to its receptor to SMAD, and is not particularly limited as long as it inhibits the resulting signal transduction pathway; it may be a nucleic acid, protein, or small organic compound. Examples of such substances include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGFβ (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ to the TGFβ receptor, and substances that inhibit the physiological activity resulting from signal transduction by the TGFβ receptor (e.g., TGFβ receptor inhibitors, SMAD inhibitors, etc.). Examples include substances that inhibit binding to the ALK family of receptors, or substances that inhibit the phosphorylation of SMAD by the ALK family.

[0033] In this specification, "BMP inhibitor" refers to a substance that inhibits signal transduction caused by BMPs, and may be a nucleic acid, protein, or small organic compound. Examples of BMPs include BMP2, BMP4, BMP7, or GDF7. Examples of BMP inhibitors include substances that act directly on BMPs (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMPs (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMPs, and substances that inhibit the physiological activity caused by signal transduction by BMP receptors. Examples of BMPRs include ALK2 or ALK3.

[0034] In this specification, "FGF" refers to fibroblast growth factor, that is, a member of the fibroblast growth factor family of signaling molecules. FGF may be natural or synthetic. For example, 22 types of FGF are known in humans. FGF is involved in cell proliferation and angiogenesis. In this specification, "bFGF" means basic fibroblast growth factor, and refers to FGF2, which belongs to the FGF family. bFGF is a signaling factor involved in cell proliferation, differentiation, angiogenesis, wound healing, neurogenesis, or the maintenance of pluripotent stem cells and neural stem cells. In this specification, "-free" means a state in which a specified component is substantially not added to the culture medium. Specifically, in this specification, "basic fibroblast growth factor (bFGF)-free" means a state in which bFGF is substantially not added to the culture medium. Here, "substantially not added" means that the specified component is not intentionally added in the preparation of the culture medium. For example, even if the specified component is present in trace amounts as an impurity in the culture medium components or is mixed in trace amounts via complex components such as serum, it is considered "not included" if it does not substantially affect the production of postbrain neural stem cells. Specifically, "substantially not added bFGF" means that bFGF is not intentionally added in the preparation of the culture medium. For example, even if bFGF is present in trace amounts as an impurity in the culture medium components or is mixed in trace amounts via complex components such as serum, it is considered "not included" if it does not induce bFGF-dependent proliferative activity of postbrain neural stem cells. In this specification, "culture medium that does not contain ~" means a culture medium in which the concentration of the specified component is less than 0.01 μM (more preferably 0.005 μM or less, even more preferably 0.001 μM or less, and particularly preferably 0 μM (below the detection limit)). In this specification, "bFGF-free culture medium" means a culture medium having a bFGF concentration that does not activate the bFGF-dependent signaling pathway in the culture of postbrain neural stem cells, preferably a culture medium in which the bFGF concentration is less than 0.01 μM (more preferably 0.005 μM or less, even more preferably 0.001 μM or less, and particularly preferably 0 μM (below the detection limit)).

[0035] In this specification, "marker" means a "marker protein" or "marker gene" that is specifically expressed on the cell surface, in the cytoplasm, and / or in the nucleus, etc., in a given cell type. The marker is preferably a "cell surface marker." The marker may be a positive selection marker or a negative selection marker. Preferably, the marker is a cell surface marker, and in particular, a cell surface positive selection marker enables the enrichment, isolation, and / or detection of viable cells. A "cell surface marker" refers to a protein expressed on the cell surface that can be labeled (stained) with a fluorescent substance, allowing for the easy detection, enrichment, and isolation of cells expressing the cell surface marker. The cell surface marker refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a given cell type, specifically a substance that is produced (positive marker) or not produced (negative marker) as mRNA by transcription of the gene in the genome, or as a protein by translation of that mRNA.

[0036] The detection of marker proteins can be performed using immunological assays with antibodies specific to the marker protein, such as ELISA, immunostaining, and flow cytometry. Antibodies specific to marker proteins can be antibodies that bind to a specific amino acid sequence in the marker protein or to specific glycans attached to the marker protein. Furthermore, in the case of marker proteins expressed intracellularly but not appearing on the cell surface (e.g., transcription factors or their subunits), the target marker protein can be detected by expressing a reporter protein together with the marker protein and detecting the reporter protein. This method is preferably used when no suitable cell surface marker is found. The detection of marker genes can be performed using nucleic acid amplification and / or nucleic acid detection methods known in the field, such as RT-PCR, microarrays, biochips, and RNA-seq.

[0037] In this specification, "expressed" means that the gene product (RNA or protein) can be identified by methods known to those skilled in the art, such as RT-PCR, in-situ hybridization, immunoassay, and chromatography.

[0038] In this specification, "not expressed" means that a gene product (RNA or protein) is not detected by methods known to those skilled in the art, such as RT-PCR, in-situ hybridization, immunoassay, or chromatography, or even if detected, it is expressed to such an extent that it cannot exert its function. If a specified marker is not expressed, it cannot be characterized by the marker because the expression level of the marker is absent or low. Regarding the term "not detected," even if a signal is detected using an extremely sensitive detection method such as RT-PCR, if there is a significant difference in signal intensity, or if the signal is below the detection level in a detection method with sufficient sensitivity from a practical standpoint, such as immunoassay, these are included in the definition of "not detected" as used in this specification.

[0039] In this specification, a marker being "positive" means that the expression level of the marker protein or gene (the measured value or signal reflecting it) exceeds (or is greater than) the detection limit or predetermined reference value using the methods known in the field as described above. In this specification, a marker being "negative" means that the expression level of the marker protein or gene is below (or less than) the detectable amount or predetermined reference value using all or any of the methods known in the field as described above. Some markers may be expressed at a certain level regardless of the cell type (or even in cells other than hindbrain neural stem cells). In such cases, if the expression level exceeds a predetermined reference value obtained by the measurement method known in the field, it may be determined as "positive," and if it falls below that, it may be determined as "negative." For example, POU5F1, NANOG, SIX3, and HOXB9 can be negative markers for hindbrain neural stem cells, but they may also be expressed at low levels in hindbrain neural stem cells. Therefore, it is preferable to determine POU5F1, NANOG, SIX3, and HOXB9 based on the above definition of "positive / negative." The detection limits and reference values ​​for protein or gene expression levels may vary depending on the method used and the purpose of the analysis. For example, in the case of the expression level (secretion level) of a marker protein, if the fluorescence signal in flow cytometry (typically fluorescence-activated cell sorting: FACS), which stains cells with a fluorescently labeled antibody, is higher than (greater than) a predetermined standard set based on the fluorescence signal of unstained cells, it can be determined as "positive," and if it is lower than (less than) a predetermined standard, it can be determined as "negative." A "+" symbol may be used to indicate positive expression, and a "-" symbol may be used to indicate negative expression. A person skilled in the art can appropriately select cells for comparison, but for example, pluripotent stem cells (e.g., iPS cells) can be used as starting cells for the production of hindbrain neural stem cells, and the expression level of pluripotent stem cells can be used as a predetermined standard. Furthermore, cells for comparison may be appropriately selected for each marker. For example, for POU5F1, NANOG, and SIX3, pluripotent stem cells (e.g., iPS cells) can be used as starting cells for the production of hindbrain neural stem cells.For HOXB9, for example, nervous system cells derived from the spinal cord (for example, spinal cord neural stem cells or spinal cord glial cells) can be cited as comparison targets.

[0040] As used herein, "comprise(s) or comprising" means indicating the inclusion of the elements following that phrase, but not limited thereto. Therefore, it suggests the inclusion of the elements following that phrase, but does not suggest the exclusion of any other elements.

[0041] As used herein, "about" or "approximately" indicates a value that varies by plus or minus 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% respectively from the reference value. Preferably, the term "about" or "approximately" indicates a range of plus or minus 15%, 10%, 5%, or 1% respectively from the reference value.

[0042] Hereinafter, preferred embodiments for carrying out the present invention will be described. The embodiments described below show an example of a representative embodiment of the present invention, and the scope of the present invention is not construed narrowly thereby.

[0043] One aspect of the present invention is a method for producing hindbrain neural stem cells, comprising a step of inducing differentiation of pluripotent stem cells into hindbrain neural stem cells, and a step of culturing the hindbrain neural stem cells using a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor and not containing basic fibroblast growth factor (bFGF).

[0044] The present invention provides a method for culturing hindbrain neural stem cells over a long period while maintaining multipotency. The inventors, through diligent research, unexpectedly discovered that hindbrain neural stem cells can be cultured over a long period while maintaining multipotency by culturing them in a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF), leading to this embodiment. Conventionally, in the culture of neuroectoderm, bFGF was added to the culture medium to inhibit terminal differentiation in the presence of BMP and maintain the pluripotency of neural stem cells. However, unexpectedly, it was found that bFGF is unnecessary for the culture of hindbrain neural stem cells. By avoiding the use of bFGF, low-cost, sustainable, and reproducible cell culture can be achieved. Conventionally, it was widely believed that bFGF-mediated FGFR signaling was essential for maintaining self-renewal and multipotency at the level of neural stem cells or neural progenitor cells, not just hindbrain neural stem cells. Therefore, in conventional reports on the maintenance culture of hindbrain neural stem cells, it was common for the culture medium to contain bFGF. In fact, some literature suggests that in groups without FGFR agonists, including bFGF, the survival rate of neural stem cells / neural progenitor cells decreases, and it becomes difficult to maintain self-renewal capacity (Reference 1). Against this background, it was conventionally thought that adding bFGF to the culture medium was essential for the maintenance culture of hindbrain neural stem cells. Unexpectedly, in the present invention, it was confirmed that even under conditions without bFGF, hindbrain neural stem cells can be cultured for a long period of time while maintaining their survival and self-renewal capacity by culturing them in a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor. According to the present invention, it is possible to maintain the culture of hindbrain neural stem cells without the addition of bFGF, and this has the remarkable effect of allowing hindbrain neural stem cells to be obtained stably and continuously while maintaining the multipotency of hindbrain neural stem cells, even without the bFGF addition step that was conventionally required. In other words, the present invention provides a novel culture method that enables a stable supply of postbrain neural stem cells.Reference 1: Liao et al., "Development of synthetic modulator enabling long-term propagation and neurogenesis of human embryonic stem cell-derived neural progenitor cells," Biological Research, 56:59 (2023).。

[0045] <Method for producing hindbrain neural stem cells> One aspect of the present invention is a method for producing hindbrain neural stem cells, comprising a step of inducing differentiation of pluripotent stem cells into hindbrain neural stem cells, and a step of culturing the hindbrain neural stem cells using a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor and not containing basic fibroblast growth factor (bFGF).

[0046] <Step of inducing differentiation of hindbrain neural stem cells> The present invention includes a step of inducing differentiation of pluripotent stem cells into hindbrain neural stem cells.

[0047] Examples of "pluripotent stem cells" usable in the present invention include pluripotent stem cells or "multipotent stem cells." In the present invention, "pluripotent stem cells" refer to stem cells that can differentiate into various tissues and cells with different forms and functions in the living body, and that have the ability to differentiate into any of the three germ layers (endoderm, mesoderm, and ectoderm). These include, but are not limited to, embryonic stem cells (ESCs), embryonic stem cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells, embryonic germ cells, and induced pluripotent stem cells (sometimes referred to as "iPSCs" or "iPS cells" in this specification). Furthermore, in the present invention, "multipotent stem cells" refer to stem cells that have the ability to differentiate into a limited number of cell lineages. Examples of "multipotent stem cells" usable in the present invention include dental pulp stem cells, oral mucosa-derived stem cells, hair follicle stem cells, cultured fibroblasts, and somatic stem cells derived from bone marrow stem cells. Preferred pluripotent stem cells are ESCs and iPSCs. In this specification, when simply referred to as "pluripotent stem cells," both "pluripotent stem cells" and "multipotent stem cells" are included.

[0048] For "ESCs," various mouse ESC strains established by inGenious Targeting Laboratory, RIKEN (Institute of Physical and Chemical Research), etc., are available, while various human ESC strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, etc., are available. For example, human ESC strains such as CHB-1 to CHB-12, RUES1, RUES2, HUES1 to HUES28, etc., distributed by ESI Bio, H1, H9, etc., distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, SSES3, etc., distributed by RIKEN can be used.

[0049] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells." These include iPSCs established by Yamanaka et al. by introducing four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting cells based on Nanog expression after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs produced using methods that do not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature). Biotechnology, (2008) 26, 101-106), and iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors—OCT3 / 4, SOX2, NANOG, and LIN28—created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (Japanese Patent Publication No. 2008-307007) can also be used.

[0050] In addition, all published papers (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, Any induced pluripotent stem cells known in the art described in 795-797) or patents (e.g., JP 2008-307007, JP 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0051] Various iPS cell lines established by institutions such as the NIH, RIKEN, and Kyoto University can be used as induced pluripotent stem cell lines. The following are examples of usable induced pluripotent stem cell lines:HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, Nips-B2 (all from RIKEN) 201B7, 201B7-Ff, 253G1, 253G4, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3 , 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 836B3, FF-I14s03, FF-I01s04, MH 09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs09, QHJI14s03 , QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all from iPS Academia Japan or Kyoto University iPS Research Foundation), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), Toe (JCRB1338 strain), and Lollipop (JCRB1336 strain) (National Center for Child Health and Development, National Institute of Biomedical Innovation, Department of Rare Diseases and Disease Resources, JCRB Cell Bank), UTA-1 strain and UTA-1-SF-2-2 strain (both from the University of Tokyo), 21526, 21528, 21530, 21531, 31536, 31538 strains (all from Fujifilm Cellular Dynamics), ATCC-DYP0730, ATCC-DYP0250, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYS0100, ATCC-HYS0103, ATCC-CYS0105, KYOU-DXR0109B, ATCC-BYS0110, ATCC-BYS0111, ATCC-BYS0112, ATCC-BYS0113, ATCC-BXS0114, ATCC-BXS0115, ATCC-BXS0116, ATCC-BXS0117 (all from the non-profit organization American Type Culture Collection), RPCiPS771-2 Stemgent (SgT5-2).Preferred induced pluripotent stem cell lines include strain 1231A3, strain RPCiPS771-2, and strain Ff-XT28s05-Abo_To.

[0052] The method for differentiating hyperbrain neural stem cells from pluripotent stem cells such as ES cells and iPS cells is not particularly limited, and may be carried out, for example, according to the methods described in Non-Patent Documents 2 to 9.

[0053] In one embodiment of the present invention, it is preferable to use a culture medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF), in order to induce differentiation of hindbrain neural stem cells from pluripotent stem cells. In the present invention, it is preferable to use the same culture medium in the hindbrain neural stem cell differentiation induction step and the subsequent culture step. This allows the same culture medium to be used in both the induction step to hindbrain neural stem cells and the subsequent culture step, which is advantageous in terms of cost reduction, ease of management, and operability in the manufacturing process. The description of the culture medium will be detailed in the section on [Steps for culturing hindbrain neural stem cells] below.

[0054] The culture period for the differentiation induction step is not particularly limited as long as the target cells are obtained, and is, for example, 2 to 10 days, preferably 3 to 8 days.

[0055] Hindbrain neural stem cells are cells that express GBX2 and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. In one embodiment, hindbrain neural stem cells are cells that are GBX2 positive and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. As mentioned above, negative markers for hindbrain neural stem cells include POU5F1, NANOG, SIX3, or HOXB9. By confirming the expression of these genes, it is possible to determine whether or not hindbrain neural stem cells have been produced. Detection of these genes can be performed using immunological assays with antibodies specific to the marker proteins, such as ELISA, immunostaining, or flow cytometry. Furthermore, detection of marker genes can be performed using nucleic acid amplification methods and / or nucleic acid detection methods known in the field, such as RT-PCR, microarrays, or biochips.

[0056] [Step for culturing hindbrain neural stem cells] The present invention includes a step of culturing the hindbrain neural stem cells using a medium that contains a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but does not contain basic fibroblast growth factor (bFGF).

[0057] The culture of hindbrain neural stem cells is carried out using a culture medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF). The culture medium used is not particularly limited as long as it contains a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF). For example, a basal culture medium to which a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor has been added is preferably used. The culture medium used in this invention does not contain bFGF, but it may also be composed of a medium that does not contain basic fibroblast growth factor (bFGF) or fibroblast growth factor (FGF). Furthermore, the culture medium used in this invention may also be composed of a medium that does not contain epidermal growth factor (EGF).

[0058] The basal medium used in the present invention is not particularly limited, but it must not contain basic fibroblast growth factor (bFGF). For example, StemFit medium (e.g., StemFit AK03N) and Chemically Defined Medium (CDM) medium are preferably used. In addition, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM (IMEM) medium, Improved MDM (IMDM) medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (High glucose, Low glucose), DMEM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof can also be used. The CDM medium is not particularly limited, but for example, a medium prepared from Iscove's modified Dulbecco's medium (manufactured by GE Healthcare) can be used. These basal culture media can be modified by adding a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor to prepare the culture medium used in the present invention.

[0059] In preferred embodiments of the present invention, the basal culture medium preferably does not contain animal-derived components. Here, animal-derived components refer to components and extracts collected or extracted from animals, such as serum (e.g., fetal bovine serum (FBS)), plasma, tissue extracts, bovine amniotic fluid, bovine fetal extract (BEE), chicken fetal extract (CEE), and other biological solutions such as ascites, and Tryptose Phosphate Broth (e.g., manufactured by DIFCO). By not using animal-derived components in the culture medium, safety can be improved and variability in the culture medium components can be suppressed. In particular, the culture medium used in the present invention preferably does not contain animal-derived components (e.g., bovine serum such as fetal bovine serum, human serum). Serum contains large amounts of protein, exhibits performance variations between batches, and carries risks such as infection with mad cow disease. By using a culture medium that does not contain such potentially harmful components, cells produced by the manufacturing method or expansion culture method according to this embodiment can be used in clinical trials and other applications.

[0060] Examples of GSK3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), CHIR98014 (2-[[2-[(5-nitro-6-aminopyridine-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazole-1-yl)pyrimidine), CP21R7 (3-(3-amino-phenyl)-4-( 1-methyl-1H-indole-3-yl)pyrrole-2,5-dione), LY2090314 (3-[9-Fluoro-1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1h-pyrrole-2,5-dione), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolid SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yloxy]phenol), Kenpaullone, 1-Azakenpaullone, SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrate Examples include (1H-pyrrole-2,5-dione) and AR-AO144-18 (1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea), CT99021, CT20026, BIO ((2'Z,3'E)-6-bromoindilbine-3'-oxime), BIO-acetoxime, pyridocarbazole-cyclopentadienylruthenium complex, OTDZT, alpha-4-dibromoacetophenone, lithium, etc. GSK3β inhibitors may be used individually or in combination of two or more.

[0061] GSK3β inhibitors are not limited to these; antisense oligonucleotides or siRNAs against GSK3β mRNA, antibodies that bind to GSK3β, dominant-negative GSK3β variants, etc., can also be used as GSK3β inhibitors, and these are commercially available or can be synthesized according to known methods.

[0062] The concentration of the GSK3β inhibitor in the culture medium during this process is adjusted as appropriate depending on the type of GSK3β inhibitor added, but is, for example, 0.01 to 20 μM, preferably 0.1 to 10 μM.

[0063] Examples of TGFβ inhibitors include A-83-01 (3-(6-methylpyridine-2-yl)-1-phenylthiocarbamoyl-4-quinoline-4-ylpyrazole), SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazole-2-yl]-benzamide), Wnt3a / BIO (Wnt Family Member 3A / (2'Z,3'E)-6-bromoindilbine-3'-oxime), BMP4 (Bone Morphogenetic protein 4), GW788388 (4-[4-[3-(pyridin-2-yl)-1H-pyrazole-4-yl]-pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16 (4-[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide), IN-1130 (3-[[5-(6- Examples include Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide, GW6604 (2-Phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine), and SB505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazole-4-yl)-6-methylpyridine). TGFβ inhibitors may be used individually or in combination of two or more.

[0064] The concentration of the TGFβ inhibitor in the culture medium is adjusted as appropriate depending on the type of TGFβ inhibitor added, but is, for example, 0.1 to 40 μM, preferably 0.5 to 20 μM, and preferably 1 to 10 μM.

[0065] Examples of BMP inhibitors include LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), Chordin, Noggin, Follistatin, Gremlin, DMH1, DMH2, ML347, Dorsomorphin (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), or their derivatives (PBYuetal.(2007), Circulation, 116: II_60; PB Yuetal. (2008), Nat. Chem. Biol., 4:33-41; J.Haoetal. (2008), PLoSONE, 3(8): e2904). LDN193189 is a well-known BMPR (ALK2 / 3) inhibitor (hereinafter referred to as a BMPR inhibitor) and is commercially available, for example, in the form of its hydrochloride salt. Dorsomorphin and LDN193189 are available from Sigma-Aldrich and Stemgent, respectively. The BMP inhibitor used in the present invention is preferably LDN193189. The BMP inhibitor may be used alone or in combination of two or more.

[0066] The concentration of the BMP inhibitor in the culture medium is adjusted as appropriate depending on the type of BMP inhibitor added, but is, for example, 0.01 to 20 μM, preferably 0.02 to 10 μM, and preferably 0.03 to 5 μM.

[0067] The cell culture method may be either adherent culture or suspension culture.

[0068] For adherent culture, culture vessels such as dishes, flasks, microplates, and cell culture sheets such as OptiCell (product name) (Nunc) are used. It is preferable that the culture vessels are surface-treated to improve cell adhesion (hydrophilicity) and coated with cell adhesion substrates such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel (e.g., BD Matrigel (Becton Deckinson Japan)), or vitronectin. "Matrigel" is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is rich in extracellular matrix proteins, and culture vessels coated with Matrigel are commercially available. The main components of Matrigel are laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen 1,2. In addition to these main components, Matrigel contains TGFβ, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator 3,4, and other growth factors that are naturally produced in EHS tumors.

[0069] In suspension culture, after preparing the cells, neural stem cells are detached from the culture vessel and dispersed in the culture medium. Cell aggregates can then be formed by stirring or shaking, while homogenizing the culture medium components and oxygen concentration. The appropriate stirring speed is set according to the cell density and the size of the culture vessel; however, excessive stirring or shaking can cause physical stress to the cells and inhibit cell aggregate formation. Therefore, the stirring or shaking speed should be controlled to homogenize the culture medium components and oxygen concentration without inhibiting cell aggregate formation. Suspension culture can also be performed by simply letting the cells stand without stirring or shaking.

[0070] The culture temperature is not particularly limited, but is typically 30-40°C (e.g., 37°C). The carbon dioxide concentration in the culture vessel is approximately 5%.

[0071] The culture period in this process can be any period required to obtain the desired number of cells. This process makes it possible to culture and proliferate hindbrain neural stem cells that maintain multipotency over a long period of time. The proportion of hindbrain neural stem cells that maintain multipotency in the cultured cell population can be maintained at, for example, 20% or more, preferably 30% or more, preferably 40% or more, preferably 50% or more, preferably 60% or more, preferably 70% or more, preferably 80% or more, preferably 90% or more, and preferably 95% or more. The method for measuring the proportion of hindbrain neural stem cells in the cultured cell population is not particularly limited, but can be done, for example, by identifying hindbrain neural stem cells by antibody staining and obtaining the number of hindbrain neural stem cells relative to the number of cells in the cultured cell population.

[0072] During this period, cell subculturing is performed as appropriate. Subculturing is performed, for example, every 2 to 21 days after sowing. The subculturing interval is preferably a period sufficient for cell expansion, and shorter than the period during which the cells are thought to proliferate too much and become deficient in oxygen and nutrients. Subculturing is preferably performed every 2 to 15 days, and more preferably every 3 to 8 days.

[0073] This process allows for the long-term culture of postbrain neural stem cells that maintain multipotency. While this "long-term" period is not particularly limited, it can be, for example, 7 days or more, preferably 8 days or more, preferably 14 days or more, preferably 21 days or more, preferably 28 days or more, preferably 35 days or more, preferably 42 days or more, preferably 49 days or more, preferably 56 days or more, preferably 63 days or more, preferably 70 days or more, preferably 77 days or more, preferably 84 days or more, preferably 91 days or more, preferably 98 days or more, preferably 105 days or more, preferably 112 days or more, preferably 119 days or more, preferably 126 days or more, preferably 133 days or more, preferably 140 days or more, preferably 147 days or more, preferably 154 days or more, preferably 161 days or more, preferably 168 days or more, preferably 175 days or more, preferably 182 days or more, preferably 189 days or more, preferably 196 days or more, preferably 203 days or more, and preferably 210 days. Preferably, the duration is 217 days or more, preferably 224 days or more, preferably 231 days or more, preferably 238 days or more, preferably 245 days or more, preferably 252 days or more, preferably 259 days or more, preferably 266 days or more, preferably 273 days or more, preferably 280 days or more, preferably 287 days or more, preferably 294 days or more, preferably 301 days or more, preferably 308 days or more, preferably 315 days or more, preferably 322 days or more, preferably 329 days or more, preferably 336 days or more, preferably 343 days or more, preferably 350 days or more, preferably 357 days or more, preferably 364 days or more, preferably 371 days or more, preferably 378 days or more, preferably 385 days or more, preferably 392 days or more, preferably 399 days or more, preferably 406 days or more, preferably 413 days or more, and preferably 420 days or more.

[0074] The culture temperature is not particularly limited, but it is typically 30-40°C (e.g., 37°C). The carbon dioxide concentration in the culture vessel is approximately 5%.

[0075] In one embodiment of the present invention, "postbrain neural stem cells that maintain multipotency" have the ability to differentiate into nerve cells or glial cells.

[0076] The ability of hindbrain neural stem cells to maintain multipotency can be evaluated by several methods. These methods are not particularly limited, but examples include inducing differentiation of the hindbrain neural stem cells to be evaluated into neurons or glial cells, respectively. If the hindbrain neural stem cells to be evaluated can actually differentiate into neurons and glial cells, they can be determined to be hindbrain neural stem cells that maintain multipotency. Another method involves measuring the expression of marker proteins or genes. In this invention, hindbrain neural stem cells are cells that express GBX2 and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. In one embodiment, hindbrain neural stem cells are cells that are GBX2 positive and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Detection of these genes can be performed using immunological assays with antibodies specific to the marker proteins, such as ELISA, immunostaining, or flow cytometry. The detection of marker genes can be performed using nucleic acid amplification and / or nucleic acid detection methods known in the field, such as RT-PCR, microarrays, and biochips. Furthermore, if the cells have a base sequence encoding a reporter protein (e.g., Nano-Lantern (Saito K. et al., "Luminescent proteins for high-speed single-cell and whole-body imaging." Nat. Commun., 2012; 3: 1262.)) inserted downstream of these marker genes, and express a fusion protein of the marker gene and the reporter protein under the control of the 0 promoter, then a method for detecting the reporter protein (e.g., measuring fluorescence intensity) can also be used.

[0077] The manufacturing method according to the present invention preferably does not include a step in which hindbrain neural stem cells are exposed to bFGF. Furthermore, the manufacturing method according to the present invention preferably does not include a step in which pluripotent stem cells are exposed to bFGF. In one embodiment, hindbrain neural stem cells are manufactured without exposure to bFGF. In other words, hindbrain neural stem cells are not exposed to bFGF in the process from pluripotent stem cells as starting cells to their manufacture. The manufacturing method according to the present invention preferably does not include a step in which hindbrain neural stem cells are exposed to FGF. Furthermore, the manufacturing method according to the present invention preferably does not include a step in which pluripotent stem cells are exposed to FGF. In one embodiment, hindbrain neural stem cells are manufactured without exposure to FGF. In other words, hindbrain neural stem cells are not exposed to FGF in the process from pluripotent stem cells as starting cells to their manufacture. The manufacturing method according to the present invention preferably does not include a step in which hindbrain neural stem cells are exposed to animal-derived components. Furthermore, the manufacturing method according to the present invention preferably does not include a step in which pluripotent stem cells are exposed to animal-derived components. In one embodiment, hindbrain neural stem cells are manufactured without exposure to animal-derived components. In other words, hindbrain neural stem cells are not exposed to animal-derived components during the process from the pluripotent stem cells used as starting cells to their production. Note that these descriptions do not specify the cells prepared as pluripotent stem cells used for differentiation induction into hindbrain neural stem cells in the production method according to the present invention.

[0078] As described above, the manufacturing method according to the present invention preferably uses a culture medium that contains a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but does not contain basic fibroblast growth factor (bFGF), for the step in inducing differentiation of pluripotent stem cells into hindbrain neural stem cells. This allows the same culture medium to be used for both the induction step into hindbrain neural stem cells and the subsequent culture step, which is advantageous in terms of cost reduction, ease of management, and operability in the manufacturing process. It should be noted that the culture medium used in the differentiation induction step and the culture step do not need to have exactly the same components; each can independently contain a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but does not contain basic fibroblast growth factor (bFGF).

[0079] <Method for Expanding the Culture of Hindbrain Neural Stem Cells> One aspect of the present invention is a method for expanding the culture of hindbrain neural stem cells having multipotency, comprising the step of culturing hindbrain neural stem cells using a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF). The method for expanding the culture is as described in the section [Steps for Culturing Hindbrain Neural Stem Cells] above.

[0080] Hindbrain neural stem cells may be prepared by a process of differentiating pluripotent stem cells into hindbrain neural stem cells, as explained in the section above on [Process of Differentiating Hindbrain Neural Stem Cells]. Alternatively, hindbrain neural stem cells may be prepared by purchasing commercially available hindbrain neural stem cells or by collecting naturally occurring hindbrain neural stem cells.

[0081] <Culture Medium> One aspect of the present invention is a culture medium for culturing hindbrain neural stem cells, comprising a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF). Another aspect of the present invention is a culture medium for long-term culture of hindbrain neural stem cells, comprising a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF). Another aspect of the present invention is a culture medium for large-scale culture of hindbrain neural stem cells, comprising a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF). The composition of the culture medium is as described above, and the contents described in the section [Steps for culturing hindbrain neural stem cells] apply.

[0082] Furthermore, one aspect of the present invention can also be expressed as the use of a culture medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF), for culturing hindbrain neural stem cells with multipotency. Furthermore, one aspect of the present invention can also be expressed as the use of a culture medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF), for long-term culture of hindbrain neural stem cells with multipotency. Furthermore, one aspect of the present invention can also be expressed as the use of a culture medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but without basic fibroblast growth factor (bFGF), for large-scale culture of hindbrain neural stem cells with multipotency. The composition of the culture medium is as described above, and the contents described in the section on "[Steps for culturing hindbrain neural stem cells]" apply.

[0083] <Cells> One aspect of the present invention is a hindbrain neural stem cell obtained by the production method or expansion culture method according to the present invention described above.

[0084] The hindbrain neural stem cells express GBX2 and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. It is also preferable that the hindbrain neural stem cells express GBX2 and at least two selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Furthermore, it is preferable that the hindbrain neural stem cells express GBX2 and at least three selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. It is also preferable that the hindbrain neural stem cells express GBX2, SOX1, SOX2, PAX6, and NESTIN. In one embodiment, the hindbrain neural stem cells are GBX2-positive and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Furthermore, it is preferable that the hindbrain neural stem cells are GBX2-positive and at least two selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Furthermore, it is preferable that the hindbrain neural stem cells are positive for GBX2 and also positive for at least three selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN.

[0085] Hindbrain neural stem cells can be used, for example, as a starting material for differentiating them to obtain neurons or glial cells. Furthermore, hindbrain neural stem cells can be used to create tissue models that are composed of hindbrain neural stem cells.

[0086] <Frozen Stock> One aspect of the present invention also provides a frozen stock containing hindbrain neural stem cells obtained by the method for producing hindbrain neural stem cells or the expansion culture method according to the present invention described above. These hindbrain neural stem cells express GBX2 and at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN. Preferably, the hindbrain neural stem cells express GBX2, SOX1, SOX2, PAX6, and NESTIN.

[0087] Frozen stock can be produced by separating hindbrain neural stem cells obtained by the method for producing hindbrain neural stem cells or the expansion culture method according to the present invention from the culture medium, suspending them in a cryopreservation solution, and freezing them. The separation of hindbrain neural stem cells from the culture medium is not particularly limited and can be done, for example, by a cell strainer or centrifugation. The separated cells may be washed as needed. The frozen stock may contain other cell populations in addition to hindbrain neural stem cells, but preferably contains purified hindbrain neural stem cells. Purification of hindbrain neural stem cells can be performed, for example, by separating them from other cell populations by cell sorting using the expression of the above-mentioned markers as an indicator. For the cell preservation solution, reagents conventionally used for cryopreservation of cells may be used. For example, Cryostem Freezing Medium and CELLBANKER® are commercially available.

[0088] Frozen stocks can be used as starting material for differentiating hindbrain neural stem cells to obtain neurons or glial cells, etc. Furthermore, frozen stocks can be used to create tissue models that use hindbrain neural stem cells as a component.

[0089] <Method for Induction into Various Cell Types> According to the method for producing and expanding culture hindbrain neural stem cells of the present invention, hindbrain neural stem cells that maintain multipotency, the ability to differentiate into nerve cells or glial cells, can be obtained.

[0090] The method for inducing differentiation from postbrain neural stem cells into neurons or glial cells is not particularly limited and can be carried out, for example, according to known methods.

[0091] The resulting nerve cells and glial cells can be used as cell therapies for regenerative medicine, respectively.

[0092] Hindbrain neural stem cells are cells that possess multipotency, the ability to differentiate into many types of cells such as nerve cells and glial cells, and also have the ability to self-replicate. Based on these capabilities observed in hindbrain neural stem cells, their application to cell therapies for regenerative medicine is expected. If hindbrain neural stem cells that maintain multipotency can be efficiently maintained or proliferated, their mass production will be possible. Furthermore, if a stock of hindbrain neural stem cells that maintain multipotency can be prepared, it will be useful as a raw material for cell therapies. For example, using cells that are located at an intermediate stage in the differentiation of stem cells to target cells (e.g., nerve cells) (e.g., hindbrain neural stem cells) as a starting material is being investigated in the manufacture of cell therapies with the aim of simplifying the manufacturing method, shortening the manufacturing period, and reducing manufacturing costs, and the present invention has the potential to provide a useful technology for this purpose. Furthermore, this can be considered similarly not only for the manufacture of cell therapies, but also for the construction of screening systems using various cells.

[0093] The present invention will be described below with reference to examples. However, the present invention is not limited to the following examples, and the configuration can be modified as appropriate without impairing the spirit of the invention.

[0094] <Experimental Methods and Evaluation Methods> [Culture of iPS cells (induced Pluripotent Stem Cells)] iPS cell line 1231A3, RPCiPS771-2 Stemgent (SgT5-2), and Ff-XT28s05-Abo_To (HLAKO) were cultured in 6-well plastic plates coated with iMatrix-511 silk (Nippi Corporation, 892021) using StemFit AK03N (Ajinomoto) under xeno-free conditions. The cells were dissociated with Accutase (registered trademark, Sigma Aldrich, A6964) and subcultured weekly.

[0095] [Induction and Culture of Hindbrain Neural Stem Cells (Hb-iNSCs) from iPS Cells] After dissociating iPS cells into a suspension, 20,000 iPS cells were seeded into a 12-well plastic plate coated with iMatrix-511. The culture medium used was the same medium used for culturing iPS cells (StemFit AK03N) to which A-83-01 (Tocris, 2939, TGFβ inhibitor, concentration in medium: 5 μM), CHIR99021 (Axon, 1386, GSK3β inhibitor, concentration in medium: 5 μM), and LDN193189 (MedChem Express, HY-12071, BMP inhibitor, concentration in medium: 0.1 μM) were added, and Y-27632 (WAKO, 253-00513, concentration in medium: 1 μM) was further added. bFGF was not added to the medium. The following day, the culture medium was changed to one that did not contain Y-27632. Specifically, the medium was changed to StemFit AK03N with A-83-01 (concentration in medium: 5 μM), CHIR99021 (concentration in medium: 5 μM), and LDN193189 (concentration in medium: 0.1 μM) added. Figure 1A shows a schematic diagram of the induction method used to induce human iPS cells into hindbrain neural stem cells (Hb-iNSCs). Hereinafter, A-83-01, CHIR99021, and LDN193189 will be collectively referred to as "ACL compounds." In addition, A-83-01 may be referred to as compound A, CHIR99021 as compound C, and LDN193189 as compound L.

[0096] Subculturing was performed weekly, and cells were dissociated with Accutase during subculturing. Y-27632 was always added to the culture medium on the first day after seeding, and the medium was changed to a medium without Y-27632 the following day. Throughout the entire cell culture period, Hb-iNSCs were cultured in AK03N medium (without bFGF) supplemented with ACL compounds to maintain multipotency.

[0097] [Formation of neurospheres and differentiation of Hb-iNSCs] Hb-iNSCs were harvested as dissociated cells and Neurobasal TMApproximately 10,000 cells were seeded in each well of a 96-well U-shaped low-adhesion plate in a neural differentiation medium prepared by mixing Thermofisher (21103049) and DMEM / F12 (GIBCO, 11320-033) in a 1:1 ratio. This medium was supplemented with 1% B27 (Thermofisher, 17504044), 1% N-2 (Thermofisher, 17502048), and 1% GlutaMAX. TM (Gibco, 35050), 200 μM ascorbic acid (nacalai, 03420-52), insulin 7 μg / ml (WAKO, 097-06474), BDNF 10 ng / ml (BioLegend, 788902), and GDNF 10 ng / ml (BioLegend, 760402) were added (hereinafter referred to as NDM). Cells were arranged to form spherical aggregates (neurospheres), and the medium was changed every other day. Neurospheres were removed from the U-shaped bottom wells and attached to the flat surface of culture plastic slides (ibidi, IB80606) for differentiation experiments. Neurospheres were used for MEA recording and in vivo experiments.

[0098] [Real-time quantitative PCR analysis] As previously explained (22) QuantStudio TM 3 and QuantStudio TM 7 Flex Real-Time PCR System (Applied Biosystems) with THUNDERBIRD TM The following analysis was briefly described using Next SYBR (registered trademark, Toyobo, QPX-201) and specially designed primers. Data from three biological replicas were analyzed to determine the relative ploidy level (2-ΔΔCT). Graphs were created using GraphPad Prism 9 software.

[0099]

[0100] [Immunocytochemical Staining] Hb-iNSCs and / or attached neurospheres were fixed with 4% PFA and stained with SOX1 (Cell signaling, 4194S), SOX2 (R&D systems, MAB2018), NESTIN (R&D systems, MAB1259), NANOG (R&D systems, AF1997D systems, AF1997), POU5F1 (Santa Cruz Biotechnology, sc-5279), PAX6 (abcam, EPR15858), TUBB3 (GeneTex, GTX85469), OLIG2 (GeneTex, GTX132732), and GFAP (Santa Cruz Biotechnology, sc-33673). The nuclei were stained with DAPI. Visualization was performed using Keyence microscopy systems (BZ-X710 and BZ-X810) and the Olympus 3000 confocal microscopy system.

[0101] [RNA Sequencing] In bulk RNA sequencing, cell lysis, RNA extraction, and sequencing were performed according to the previously described method (Non-Patent Literature 11). [Non-Patent Literature 11] Kamiya D et al., NPJ Regen Med. 2022 Sep 15;7(1):47.

[0102] For the analysis of count data, the DESeq2 method was used for normalization and differential gene expression analysis. Standard RStudio packages such as "EnhancedVolcano," "ComplexHeatmap," "ggVennDiagram," and "ggplot2" were used to create plots. Enrichr was used for enrichment analysis (24). For scRNA sequencing, 5,000 cells were processed from each sample, and the sequencing library was prepared using 10xGenomics' Chromium Next GEM Single Cell 3' Reagent Kits v3.1, following the manufacturer's standard procedure (Dual Index). After cDNA synthesis, sizing (TapeStation) and concentration measurement (Qubit) were performed. After library preparation, sizing (TapeStation) and concentration measurement (qPCR) were also performed. Data processing, filtering, analysis, and plot creation were performed using Scanpy.

[0103] [Multi-electrode array recording] To detect neuronal firing and network activity in neurospheres, a high-density multi-electrode array (HD-MEA) from MaxWell Biosystems was used. After one week, the neurospheres, formed in suspension, were plated to directly adhere to electrodes coated with iMatrix-511. After six weeks, recordings were performed using the MaxOne HD-MEA system. The same electrodes were recorded again one hour after treatment with 1 μM tetrodotoxin (TTX). The data were then analyzed using MaxLab Live software.

[0104] [In vivo neuronal cell transplantation] Mice approximately 4 weeks old were used for neurosphere transplantation one week prior. Using a stereotactic device and a Hamilton syringe, approximately 5 neurospheres were collected, and holes were made in the mouse skull according to the coordinates of the mouse brain atlas, and the neurospheres were delivered to the hindbrain region. Eight weeks after transplantation, the mice were sacrificed according to ethical guidelines and perfused and fixed. Whole brain samples were collected, fixed with 4% PFA, and then stored in 30% sucrose for 24 hours. Subsequently, the samples were embedded in a cryotherapy agent and cryopreserved. 30 μm sections were prepared in the sagittal plane of the transplanted hemisphere.

[0105] [Statistical Analysis] Parametric Welch's t-test without pairs was performed using GraphPad Prism 9 software. Significance levels were defined as ns > 0.05, *p ≤ 0.05, ***p ≤ 0.01, ***p ≤ 0.001, and ***p ≤ 0.0001.

[0106] <Example 1: Differentiation Induction and Expansion Culture of Hindbrain-like Induced Neural Stem Cells (Hb-iNSCs)> Following the method described in the section [Induction and Culture of Hindbrain Neural Stem Cells (Hb-iNSCs) from iPS Cells] above, iPS cells were induced into Hb-iNSCs and expanded culture was performed. Specifically, the formation of Hb-iNSCs from hiPS cells was achieved by inhibiting the TGFb / Activin / Nodal pathway, activating the Wnt signaling pathway, and inhibiting the BMP signaling pathway using a medium containing ACL compounds (A-83-1, CHIR99021, and LDN193189) (bFGF not included), thereby inhibiting the mesoderm and endoderm pathways. iPS cells were seeded in each coated culture well with the medium containing the ACL compounds. On the seeding day (first day after seeding), Y-27632 was added to the medium.

[0107] (Observation and evaluation in PN0) During the first week, several independent, dome-shaped colonies formed, distinct from the typical confluent, somewhat flat iPS cell colonies. These dome-shaped colonies were able to spontaneously change their morphology by forming neurite-like projections when the ACL compound was removed from the culture medium.

[0108] Figure 1B shows, from left to right, phase-contrast images of iPS cell colonies, Hb-iNSC colonies, and neurons differentiated from Hb-iNSCs. The ability to induce neurons from Hb-iNSCs demonstrates that Hb-iNSCs indeed possess neural stem cell properties. This colony morphology was maintained after cell passage and was similar across the three different iPS cell lines tested.

[0109] Figure 1C shows phase-contrast images of Hb-iNSCs induced from three iPS cell lines under the presence of ACL compounds, taken on day 7. It can be seen that all cells induced have similar morphologies.

[0110] To analyze gene expression, cell lysates were collected from each day during the induction period, and mRNA was extracted. Figure 2 is a graph showing the changes in gene expression during the induction period to Hb-iNSCs, as examined by qPCR. The graph shows the gene expression of E-cadherin (CDH1), N-cadherin (CDH2), SOX1, SOX2, PAX6, and POU5F1 during the induction period (days 1-7) relative to the gene expression of iPS cells (day 0), which is set to 1. As shown in Figure 2, the expression of pluripotent stem cell markers (CDH1, POU5F1) decreased, while the expression of neural stem cell markers (CDH2, SOX1, SOX2, PAX6) increased, indicating a gradual transition to neural stem cells. Specifically, it was confirmed that within just 3 days, the cells significantly decreased the expression of E-cadherin (CDH1), which is characteristic of epithelial cells, while increasing the expression of N-cadherin (CDH2), which is expressed in neural stem cells. Furthermore, the cells gradually upregulated the expression of neural stem cell markers such as SOX1, SOX2, and PAX6, while downregulating the expression of the pluripotency marker POU5F1.

[0111] Gene expression in cells 7 days after induction from human iPS cells was confirmed by immunocytochemistry. Figure 3 is a fluorescence image showing immunocytochemistry for SOX1, SOX2, NESTIN, PAX6, NANOG, and POU5F1 in cells 7 days after induction from human iPS cells. From the results of immunocytochemistry, the expression of neural stem cell markers was consistently observed in iNSC colonies 7 days after induction by immunocytochemistry, while the expression of the pluripotency marker (POU5F1) was not observed.

[0112] To further investigate the effects of induction by ACL compounds, gene expression on day 0 (iPS cells) and day 7 (Hb-iNSCs) was confirmed by bulk RNA sequencing (RNAseq). Figure 4A shows a heatmap comparing gene expression on day 0 (iPS cells) and day 7 (Hb-iNSCs) as determined by RNAseq. Figure 4B is a bar graph showing the results for pluripotency markers in the heatmap of Figure 4A. Figure 4C is a bar graph showing the results for neural stem cell markers in the heatmap of Figure 4A. Figure 4D is a bar graph showing the results for hindbrain markers in the heatmap of Figure 4A. The RNAseq results confirmed that pluripotency stem cell markers decreased and neural stem cell markers increased after 7 days of induction. Furthermore, since the cells on day 7 expressed representative markers of the hindbrain region (especially GBX2), it was found that they possessed hindbrain characteristics among neural stem cells. In iPSCs, pluripotency markers are expressed, but hindbrain markers are not. In contrast, in PN0 cells, pluripotency markers have already disappeared, and neural stem cell markers and hindbrain markers are confirmed to be expressed. Three samples of 1231A3 iPS cells and three samples of 1231A3 Hb-iNSCs at PN0 day 7 were used for this analysis.

[0113] Figure 5 is a volcano plot comparing the expression levels of Hb-iNSC samples (3 samples) on day 7 and iPS cell controls (3 samples) on day 0. The y-axis represents the p-value, and the x-axis represents the log fold change value. Red dots represent genes with significant differences in expression (significant difference genes, SigDEGs). The vertical dashed line indicates the threshold of 1.5 log fold change, and the horizontal dashed line indicates the threshold of significance (p = 0.05).

[0114] Figure 6 shows the results of enrichment analysis using a dataset of genes whose expression was significantly increased (log2 fold change ≥ 1.5) in iPS cells (p < 0.05). The size of the dots indicates the number of genes that overlapped with the dataset, the intensity of the dot color indicates significance (top 10 terms sorted based on p-value), and the x-axis shows the score calculated by Enrichr. Neurological terms are highlighted in red. In silico analysis results showed that the cells on day 7 possessed characteristics of hindbrain neural stem cells.

[0115] Figure 7A shows phase-contrast images of iPS cells after induction for one week using iPS cells, or A-83-01, CHIR99021, or LDN193189 individually or in combination.

[0116] Figure 7B is a graph showing the results of qPCR analysis in iPS cells obtained under each condition. The expression of pluripotent stem cell markers (POU5F1), endoderm marker (FOXA2), mesoderm marker (SMA), and ectoderm markers (SOX1, SOX2, PAX6) was analyzed. Under conditions in the absence of compound A (conditions containing only compound C, conditions containing only compound C and compound L, and conditions containing only compound L), the endoderm marker (FOXA2) increased, while under conditions containing only compound A, conditions containing only compound A and compound C, and conditions containing only compound A and compound L, the mesoderm marker (SMA) increased. On the other hand, it was confirmed that only when ACL was added, the expression of endoderm and mesoderm markers was suppressed, and the expression of ectoderm markers increased.

[0117] (Observation and evaluation from PN1 onwards, long-term maintenance of postbrain neural stem cells) We attempted to maintain cell culture for a long period (up to 60 weeks) by passage and proliferation of induced Hb-iNSCs every week. Figure 8 is a schematic diagram showing the timing of passage culture, RNA sample collection, and cell cryopreservation. In subsequent experiments, samples were collected according to this schematic diagram. Cell passage was performed every week, and RNA samples were collected and cells were cryopreserved every 5 passages, and this process was repeated until week 60. Based on the passage number, the samples were classified into early (PN5-20), mid-stage (PN25-40), and late-stage (PN45-60) groups. This allowed us to obtain an expandable cell stock that can be thawed and used for further experiments, as well as RNA samples from different points in time during the long-term maintenance period.

[0118] To confirm that chromosomal integrity was not compromised after long-term culture, karyotype analysis was performed. Figure 9 shows the results of karyotype analysis at passage 53 (week 53) of Hb-iNSCs induced from 1231A3 human iPS cells. It was confirmed that no karyotype abnormalities occurred after long-term culture. In addition, a benign inversion of chromosome 9, which was pre-existing in the 1231A3 iPS cell line, was confirmed.

[0119] Figure 10 is a fluorescence image showing the results of immunohistochemical staining for TUBB3, SOX1, NESTIN, PAX6, NANOG, and SOX2 in Hb-iNSCs induced from iPS cells at passage 60 (week 60) (scale bar is 50 μm). The results showed uniform expression of SOX1, SOX2, PAX6, and NESTIN, indicating that the cells maintained their multipotency.

[0120] Figure 11A is a heatmap comparing gene expression in iPS cells, Hb-iNSCs at passages 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60, and neurons differentiated from passages 5 and 60 (diff), as determined by bulk RNA sequencing. Pluripotency stem cell markers, neural stem cell markers, neural differentiation markers, and representative markers from different brain regions were used as markers. Samples PN5_1 to PN5_3 and PN60_1 to PN60_3 were derived from the 1231A3 iPS cell line, while samples PN5_4 and PN5_5 were derived from the HLAKO and SgT5 iPS cell lines, respectively. Figures 11B and 11C show bar graphs illustrating the gene expression of hindbrain markers in PN0 to PN60. The results of the heatmap shown in Figure 11A also confirmed that the cells possessed characteristics of hindbrain neural stem cells. Furthermore, Figures 11B and 11B confirm that postbrain neural stem cell markers are expressed from PN0 onward.

[0121] Figure 12 is a graph showing the results of principal component analysis using bulk RNA-seq data. Red represents iPS cells (control), green represents early Hb-iNSCs and post-neuronal differentiation, purple represents mid-stage Hb-iNSCs, and blue represents late-stage Hb-iNSCs and post-neuronal differentiation. It was confirmed that early, mid, and late-stage Hb-iNSCs all show similar expression patterns.

[0122] Figure 13 is a graph showing the results of enrichment analysis for genes whose expression was significantly increased (log2 fold change ≥ 1.5) in late-stage Hb-iNSCs compared to iPS cells (control) (p < 0.05). The size of the dots indicates the number of overlapping genes in the dataset, and the intensity of the dot color indicates significance (top 10 terms sorted based on p-value). The x-axis represents the score calculated by Enrichr, and neurological terms are highlighted in red. This analysis also shows that the cells possess characteristics of postbrain neural stem cells.

[0123] Figure 14 is a heatmap showing the correlation between Hb-iNSCs samples from passage 5 to passage 60. The intensity of the color indicates the degree of correlation: red = 1 indicates a positive correlation, white = 0 indicates no correlation, and green = -1 indicates a negative correlation. Hb-iNSCs from passage 5 to passage 60 show a strong correlation in gene expression patterns.

[0124] Figure 15 is a bar graph showing log fold change of genes with differing expression levels detected by bulk RNA sequencing. This is a comparison between late-stage Hb-iNSCs and iPS cells (control). The green bars on the left show a list of genes whose expression was elevated in late-stage Hb-iNSCs (upper x-axis), and the red bars on the right show a list of genes whose expression was decreased in late-stage Hb-iNSCs (lower x-axis). Figure 15 confirms the detection of hindbrain marker genes.

[0125] Figure 16 shows bar graph plots of RTqPCR data for GBX2, HOXB4, SIX3, and HOXB9 in PN5 and PN60 Hb-iNSC samples (n=3). Human RNA samples from the cerebellum, pons, cerebral cortex, and spinal cord (n=1) were used as a control group. Statistical significance is indicated by ns≧0.05, *p≦0.05, and **p≦0.01. Similar expression levels were observed in both early Hb-iNSCs (PN5) and late Hb-iNSCs (PN60).

[0126] Figure 17 is a graph showing the results of enrichment analysis for 427 genes whose expression was significantly increased (log2 hold change > 1.5) in early Hb-iNSCs compared to iPS cells (control) (P < 0.05). The size of the dots indicates the number of overlapping genes in the dataset, and the intensity of the dot color indicates significance (top 10 terms sorted based on p-value). The x-axis represents the score calculated by Enrichr, with neurological terms highlighted in red. This analysis also demonstrates that the cells possess characteristics of hindbrain neural stem cells.

[0127] <Example 2: Differentiation induction of hindbrain neural stem cells (Hb-iNSCs) into nerve cells and functional network activity> Neurospheres are commonly used to evaluate primary or induced nerve cells. Therefore, cultured Hb-iNSCs were differentiated using the neurosphere method, and then adhesion and migration, electrophysiological recording, and in vivo transplantation were performed. Neurospheres are aggregates of cells cultured in three dimensions (3D) and suspended on a round-bottom plate with low adhesion. In neurosphere formation, dissociated hindbrain neural stem cells could be seeded without the need for scaffolding, and after seeding, the cells formed spherical aggregates. Neuron differentiation medium (NDM) without ACL compounds was used as the culture medium. Figure 18A is a schematic diagram of the method for preparing neurospheres in Example 2. Figure 18B is a schematic diagram showing adhesion to a dish (flat surface), adhesion to a microelectrode array (MEA), and transplantation into an in vivo transplant (mouse brain).

[0128] Figure 19A shows phase-contrast images of neurospheres one week and eight weeks after the start of preparation (scale bar: 100 μm). Figure 19B is a bar graph of the cross-sectional area of ​​the neurospheres one week and eight weeks after the start of preparation. These neurospheres were relatively similar in size at the time of initial seeding and grew significantly after several weeks.

[0129] • Immunocellular staining of neurospheres Figure 20 shows immunocellular staining of neurospheres attached to a dish (scale bar: 100 μm). Nuclear staining was performed with TUBB3, OLIG2, GFAP, and DAPI. When the prepared neurospheres were attached to a flat surface coated with laminin, in addition to cells migrating to the outside of the sphere's edge, TUBB3-positive neurite-like structures were observed. Glial cells such as astrocytes and oligodendrocytes were also observed, being GFAP and OLIG2 positive, respectively. Neurospheres prepared from early (PN7) and late (PN58) HbL-iNSCs all similarly showed differentiation potential into nerves (TUBB3), oligodendrocytes (OLIG2), and astrocytes (GFAP). These observations indicate that Hb-iNSCs cultured over a long period maintain multipotency into nervous and glial cells.

[0130] • Induction of Hb-iNSCs to the ventral region: It has been shown that treating cells with sonic hedgehog (SHH) recombinant protein and FGF4 can further induce iNSCs to the ventral hindbrain region. This treatment enables differentiation into serotonergic neurons expressing serotonergic neuron transcription factor (FEV), tryptophan hydroxylase 2 (TPH2), and serotonin transporter (SLC6A4). Furthermore, it has been reported that this leads to the formation of a population of serotonergic progenitor cells expressing NXK2.2 and NKX6.1 (Non-Patent Literature 12). [Non-Patent Literature 12] Lu JF, Zhong XF, Liu HS, Hao L, Huang CTL, Sherafat MA, et al. Generation of serotonin neurons from human pluripotent stem cells. Nature Biotechnology. 2016;34(1):89-94.

[0131] Therefore, in this experiment, cultured Hb-iNSCs were treated with SAG (smoothed agonist) along with FGF4 to differentiate them into ventral Hb-iNSCs expressing NXK2.2 and NKX6.1.

[0132] Figure 21A is a graph showing the results of gene expression analysis of ventral hindbrain markers (NKX 2.2 and NKX 6.1) by RT-qPCR in ventralized Hb-iNSCs induced from Hb-iNSCs (relative values ​​between ventralized and non-ventralized Hb-iNSCs) (error bars represent standard deviation, n=3). Expression of ventral hindbrain markers (NKX2.2 and NKX6.1) was detected in ventralized Hb-iNSCs.

[0133] Figure 21B is a graph showing the expression of serotonergic neuronal markers (FEV, TPH2, SLC6A4) in ventralized Hb-iNSCs (error bars represent standard deviation, n=3). Elevated expression of serotonergic neuronal markers (FEV, TPH2, SLC6A4) was observed in neurons induced from ventralized Hb-iNSCs.

[0134] This finding demonstrates the importance of Hb-iNSCs, showing that cultured Hb-iNSCs can be ventralized and differentiate into a limited, highly specialized type of neuron present in that region of the brain.

[0135] • Evaluation of the electrophysiological activity of neurospheres To evaluate the electrophysiological activity of neurospheres derived from Hb-iNSCs, a microelectrode array (MEA) was used. After neurosphere formation in one week, maturation was carried out for 5-6 weeks. When adhered neurospheres migrated onto electrodes, bursts of action potentials (spikes) with amplitudes exceeding 50 μV were detected across multiple electrodes (Figure 22A). Figure 22A is a histogram of electrode counts of spike amplitudes detected by the MEA. Dark colors indicate electrode counts of samples before tetrodotoxin (TTX) addition, and pink colors indicate electrode counts of samples after TTX addition (PN5 left plot and PN61 right plot). Neurospheres were prepared from Hb-iNSCs, seeded on a microelectrode array (MEA), cultured for 5-6 weeks, and then analyzed. Action potentials were detected. Furthermore, the action potentials were suppressed by the addition of tetrodotoxin (TTX), a neurotoxin. Tetrodotoxin (TTX) is a neurotoxin that blocks sodium channels and inhibits the conduction of action potentials in neurons. When TTX was added to the culture medium of neurospheres, the spike amplitude decreased significantly, and the number of active electrodes also decreased compared to before TTX addition.

[0136] Figure 22B is a raster plot of action potentials detected by MEA. Each dot represents the action potential of samples derived from PN5 (upper plot) and PN61 (lower plot). Figure 22C is a raster plot of the same samples as in Figures 22A and 22B, recorded after the addition of TTX.

[0137] These findings indicate that long-term maintenance does not affect multipotency, and that Hb-iNSCs can still generate functional neurons.

[0138] • In vivo transplantation of neurospheres To elucidate the integration and migration of cultured Hb-iNSCs in vivo, neurospheres (1 week old) prepared from early and late Hb-iNSCs were transplanted into the anterior cerebellar and pontine regions of mouse brains. Figure 23A is a schematic diagram showing the transplantation sites of neurospheres in the mouse brain. The areas indicated by orange squares correspond to the locations shown in the enlarged view of Figure 23C. Figure 23B is a sagittal section image of the mouse brain 8 weeks after neurosphere transplantation. Figure 23C shows the tracking results of hNCAM-positive cells detected with human-specific NCAM antibodies along the spinocerebellar tract (1, 1'), cerebellum (2, 2'), corticospinal tract (3, 3'), and midbrain (4, 4'). Numbers without a single quote indicate cells derived from early Hb-iNSCs, and numbers with a single quote indicate cells derived from late Hb-iNSCs. As shown in Figure 23C, hNCAM-positive cells were detected along the spinocerebellar tract (1, 1'), cerebellum (2, 2'), corticospinal tract (3, 3'), and midbrain (4, 4').

[0139] Figure 23D shows photographs of brain sections after transplantation of early and late Hb-iNSC neurospheres, stained with human-specific NCAM antibody, TUBB3 antibody, GFAP antibody, and OLIG2 antibody. In both the early and late stages, TUBB3, GFAP, and OLIG2-positive cells co-stained with human-specific NCAM antibody were detected, confirming that the transplanted cells differentiated into neurons, oligodendrocytes, and astrocytes.

[0140] These studies demonstrated that neurons derived from Hb-iNSCs can differentiate and elongate alongside host neurons that simulate normal physiological development. This property suggests they could be a useful tool for disease modeling and potential cell therapies.

[0141] <Discussion> In this experiment, iPS cells were successfully induced into Hb-iNSCs, which primarily share identity with postbrain neural stem cells, by using a minimal approach involving three small molecule inhibitors without requiring bFGF. Furthermore, these Hb-iNSCs could be maintained for up to 60 passages while preserving their characteristics and differentiation potential. Bulk RNA sequencing was performed on samples from different time points, and a comparison was made between early and long-term cultures. The results showed that long-term culture and maintenance in the above medium did not affect maturity, and genes related to immature / fetal nerve cells were similarly abundantly expressed in both early and late-stage samples.

[0142] One application of this research is the modeling of DIPG, a deadly tumor affecting children whose cells originate in the ventral limbic system. Another application is the modeling of disorders related to the serotonergic system. Serotonergic neurons are neurons that utilize the neurotransmitter serotonin, and dysfunction of these neurons is associated with neuropsychiatric disorders such as depressive disorders, autism spectrum disorder, and Rett syndrome. This application involves establishing Hb-iNSCs from patient-derived iPS cells and combining these Hb-iNSCs with MEA (multi-electrode arrays) to analyze the spike activity (action potentials) of neurons and measure the activity, learning rate, and prediction accuracy of neural networks. Specifically, spike (action potential) data can be analyzed using systems such as MEA-NAP, DishBrain, and BiaemuS.

[0143] The upper and / or lower limits of the numerical ranges described herein can be arbitrarily combined to define a preferred range. For example, the upper and lower limits of the numerical ranges can be arbitrarily combined to define a preferred range, the upper limits of the numerical ranges can be arbitrarily combined to define a preferred range, and the lower limits of the numerical ranges can be arbitrarily combined to define a preferred range.

[0144] The claims following this disclosure are expressly incorporated herein into this disclosure, and each claim stands independently as a separate embodiment. This disclosure includes all instances in which an independent claim is replaced by its dependent claim. Furthermore, any additional embodiments derived from the independent claims and subsequent dependent claims are also expressly incorporated herein into this specification.

[0145] Although this embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and any design changes that do not depart from the gist of this disclosure are also included in this disclosure.

Claims

1. A method for producing hindbrain neural stem cells, comprising the steps of: differentiating hindbrain neural stem cells from pluripotent stem cells; and culturing the hindbrain neural stem cells in a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF).

2. The method according to claim 1, wherein the differentiation induction step is performed using the culture medium.

3. The method according to claim 1, wherein the differentiation induction step is followed by the culture step.

4. The method according to claim 1, wherein the postbrain neural stem cells are not exposed to bFGF.

5. The method according to claim 1, wherein the postbrain neural stem cells are positive for GBX2 and positive for at least one selected from the group consisting of SOX1, SOX2, PAX6, and NESTIN.

6. The method according to claim 1, wherein the culture medium does not contain any animal-derived components.

7. The method according to claim 1, wherein, in the culturing step, the postbrain neural stem cells are proliferated while maintaining their multipotency.

8. The method according to claim 1, wherein the culturing step is carried out over a period of 8 days or more.

9. The method according to claim 8, wherein the culturing step is carried out for 14 days or more.

10. A culture medium for culturing posterior neural stem cells, comprising a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but free from basic fibroblast growth factor (bFGF).

11. The culture medium according to claim 10, wherein the culture medium does not contain any animal-derived components.

12. A method for expanding the culture of hindbrain neural stem cells having multipotency, comprising the step of culturing hindbrain neural stem cells using a medium containing a GSK3β inhibitor, a TGFβ inhibitor, and a BMP inhibitor, but not containing basic fibroblast growth factor (bFGF).

13. The method according to claim 12, wherein the culture medium does not contain any animal-derived components.

14. The method according to claim 12, wherein the culturing step is carried out over a period of 8 days or more.

15. The method according to claim 14, wherein the culturing step is carried out for 14 days or more.

16. Postbrain neural stem cells obtained by the method of claim 1 or 12.

17. A frozen stock comprising postbrain neural stem cells obtained by the method of claim 1 or 12.

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