In-vitro cerebral cortex intermediate neuron precursor cell population and preparation method and application thereof

Abstract

The invention provides an in-vitro cerebral cortex intermediate neuron precursor cell population as well as a preparation method and application of the in-vitro cerebral cortex intermediate neuron precursor cell population. According to the cell population, the iNSC is taken as an initial cell to directionally differentiate to the neuroectoderm cell, so that neuroendoderm and neuromesoderm impurity cells generated in the differentiation process can be reduced, the cell differentiation direction is more accurate, and the differentiated cell purity is higher. The cell population can be used for the prevention, diagnosis and treatment of nervous system diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of neural stem cell biology, in particular to a population of cerebral cortex intermediate neuron precursor cells induced to differentiate from neural stem cells, and a preparation method and use thereof. BACKGROUND

[0002] The medial ganglion eminence (MGE) is differentiated from embryonic stem cells. During the development of mice, the MGE begins to appear around the 11th day of embryonic development, and then the MGE cells differentiate into different types of intermediate neurons and gradually migrate to the cortex to form the characteristic laminar structure of the mature cerebral cortex, and these intermediate cells can form neural circuits with excitatory neurons derived from the cortex. Early studies have shown that MGE precursor cells isolated from mouse embryonic tissues can produce inhibitory intermediate neurons after in vitro expansion, and when transplanted into the hippocampus of newborn mice, these neurons can migrate and differentiate into mature inhibitory intermediate neurons in the host brain, and form appropriate synaptic connections with native pyramidal neurons. Using this property, MGE-derived intermediate neuron inhibition therapy can have therapeutic effects on the pathophysiological defects of the nervous system.

[0003] The clinical application of embryonic stem cells has ethical problems and xenotransplantation rejection, and induced pluripotent stem cells (iPSC) are stem cells induced by reprogramming of somatic cells, which have similar developmental multipotency as embryonic stem cells, avoiding immune rejection and moral and ethical controversy. Human iPSCs can be differentiated into specialized cell types and have the potential to provide patient-specific, immune-matched cells for regenerative medicine, and the differentiation of human iPSCs into MGE-derived intermediate neurons provides a new therapeutic approach for pathophysiological defects of the human nervous system.

[0004] However, the differentiation cycle of iPSCs is generally more than one month (30 days or more), and during the differentiation process to specialized cell types, neural endoderm and neural mesoderm impurity cells are produced, which is not conducive to the purity, yield and clinical safety of the differentiated cells.

[0005] Therefore, it is of great significance to explore and develop cerebral cortex intermediate neuron precursor cells from different starting cell sources. SUMMARY

[0006] The present application provides a cerebral cortex intermediate neuron precursor cell population derived from human induced neural stem cells (iNSC), which is differentiated from iNSC to neuroectoderm cells to MGE type GABAergic intermediate neurons, the cell differentiation direction is more accurate, the neural endoderm and neural mesoderm impurity cells generated during the differentiation of iPSC can be reduced or avoided, and the purity of the differentiated cells is higher. The cell population can be used for preventing, diagnosing and treating nervous system diseases.

[0007] In a first aspect, the present application provides an ex vivo cerebral cortex intermediate neuron precursor cell population, which expresses markers including NKX2.1, FoxG1, GABA and SST, wherein the proportion of cells expressing NKX2.1 in the cell population is more than 40%, the proportion of cells expressing GABA in the cell population is more than 20%, and the proportion of cells expressing SST in the cell population is more than 30%.

[0008] In some embodiments, in the ex vivo cerebral cortex intermediate neuron precursor cell population, the proportion of cells expressing FoxG1 in the cell population is more than 35%.

[0009] In some embodiments, the ex vivo cerebral cortex intermediate neuron precursor cell population is derived from iNSC.

[0010] In a second aspect, the present application provides a preparation method of an ex vivo cerebral cortex intermediate neuron precursor cell population, which comprises: culturing iNSC under effective conditions to transform them into medial ganglionic eminence (MGE) like progenitor cells; and inducing the MGE like progenitor cells to form a cerebral cortex intermediate neuron precursor cell population.

[0011] In a third aspect, the present application provides a pharmaceutical composition comprising the aforementioned ex vivo cerebral cortex intermediate neuron precursor cell population, or the ex vivo cerebral cortex intermediate neuron precursor cell population prepared by the aforementioned method, and at least one pharmaceutically acceptable excipient.

[0012] In a fourth aspect, the present application provides a kit comprising the aforementioned ex vivo cerebral cortex intermediate neuron precursor cell population, or the ex vivo cerebral cortex intermediate neuron precursor cell population prepared by the aforementioned method.

[0013] In a fifth aspect, the present application provides use of the aforementioned ex vivo cerebral cortex intermediate neuron precursor cell population, or the ex vivo cerebral cortex intermediate neuron precursor cell population prepared by the aforementioned method, the pharmaceutical composition or the kit in preparation of a diagnostic agent for preventing, diagnosing and treating nervous system diseases.

[0014] The cerebral cortex intermediate neuron precursor cell population provided by the application, especially the cerebral cortex intermediate neuron precursor cell population derived from iNSCs, can avoid ethical issues, and is beneficial to improve the availability of drugs for treating nervous system diseases; the cell population has a relatively short preparation time and high purity, which is beneficial to improve the production quality of cell drugs, reduce the production cost, and improve the safety of cell drugs in preventing, diagnosing, and treating nervous system diseases. BRIEF DESCRIPTION OF DRAWINGS

[0015] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are intended to illustrate preferred embodiments of the application, and should not be considered limiting of the application.

[0016] Figure 1 A schematic diagram showing the strategy of in vitro directional differentiation of iNSCs to GABAergic intermediate neurons.

[0017] Figure 2 A fluorescence staining image of the cerebral cortex intermediate neuron precursor cell population of Example 1 is shown, and the staining markers are shown above the corresponding staining images. Merge represents the merged staining images.

[0018] Figure 3 A fluorescence staining identification quantitative result of the cerebral cortex intermediate neuron precursor cell population of Example 1 is shown.

[0019] Figure 4 A fluorescence staining image of the cerebral cortex intermediate neuron precursor cell population of Example 2 is shown, and the staining markers are shown above the corresponding staining images. Merge represents the merged staining images.

[0020] Figure 5 A fluorescence staining identification quantitative result of the cerebral cortex intermediate neuron precursor cell population of Example 2 is shown.

[0021] Figure 6 A fluorescence staining image of the cerebral cortex intermediate neuron precursor cell population of Comparative Example 1 is shown, and the staining markers are shown above the corresponding staining images. Merge represents the merged staining images.

[0022] Figure 7 A fluorescence staining identification quantitative result of the cerebral cortex intermediate neuron precursor cell population of Comparative Example 1 is shown.

[0023] Figure 8 A fluorescence staining image of the cerebral cortex intermediate neuron precursor cell population of Example 3 is shown, and the staining markers are shown above the corresponding staining images. Merge represents the merged staining images.

[0024] Figure 9 FIG. 8 shows the immunofluorescent staining identification quantitative results of the cerebral cortex intermediate neuron precursor cell population of Example 3, with and without the addition of 50 ng / mL FGF8 on the 8th to 12th day of differentiation (control).

[0025] Figure 10 FIG. 9 shows the migration results of the cerebral cortex intermediate neuron precursor cell population of Example 4 in two dimensions and three dimensions.

[0026] Figure 11 FIG. 10 shows the standard curve of the GABA (gamma-aminobutyric acid) content detection of the iNSC-derived cerebral cortex intermediate neuron organoids of Example 5 on the 60th day of in vitro induction.

[0027] Figure 12 FIG. 11 shows the neurotransmitter content detection results of the iNSC-derived cerebral cortex intermediate neuron organoids of Example 5 on the 60th day of in vitro induction.

[0028] Figure 13 FIG. 12 shows the hippocampal tissue immunofluorescent staining images of the C-NKG mice transplanted with the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 6, with the staining markers shown on the left side of the corresponding staining images; from left to right, the staining results of the cells on the 14th and 18th day of differentiation.

[0029] Figure 14 FIG. 13 shows the electroencephalogram and behavioral detection results of the chronic epilepsy mice after 6 weeks of transplantation with the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 7; A. the time domain graph and time-frequency graph of the cortical electroencephalogram of the cell transplantation group and the control group mice; B. the results statistics of the Barnes experiment, object location test (OLT), and total seizure duration.

[0030] Figure 15 FIG. 14 shows the hippocampal tissue immunofluorescent staining images of the chronic epilepsy mice after 12 weeks of transplantation with the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 8, with the staining markers shown on the left side of the corresponding staining images.

[0031] Figure 16 FIG. 15 shows the immunofluorescent staining identification quantitative results of the hippocampal tissue of the chronic epilepsy mice after 12 weeks of transplantation with the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 8.

[0032] Figure 17 FIG. 16 shows the brain slice electrophysiology results of the chronic epilepsy mice after 12 weeks of transplantation with the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 9, with the non-fluorescent group being the host pyramidal cells and the fluorescent group being the transplanted cells. DETAILED DESCRIPTION

[0033] The specific embodiments described herein should not be construed as limiting the scope of the application, which will be readily apparent to those skilled in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless otherwise specifically defined.

[0034] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratios, amounts, time, temperatures, thicknesses, and so forth, used herein are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and attached claims are approximations. It will be further understood that each of the values

[0035] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0036] Unless otherwise specified, all steps of the present application can be carried out in sequence or randomly, preferably in sequence.

[0037] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meaning of similar expressions can be extended in a similar manner.

[0038] Ex vivo cerebral cortical intermediate neuron precursor cell population The present application provides a population of ex vivo cerebral cortical intermediate neuron precursor cells derived from iNSCs, said population of cells expressing markers including NK2 homeobox 1 (NKX2.1), forkhead box G1 (FoxG1), gamma-aminobutyric acid (GABA), and somatostatin (SST).

[0039] In some embodiments, the number of cells expressing NKX2.1 in the cell population is more than 40%. In some embodiments, the number of cells expressing FoxG1 in the cell population is more than 35%. In some embodiments, the number of cells expressing GABA in the cell population is more than 20%. In some embodiments, the number of cells expressing SST in the cell population is more than 30%.

[0040] The cell population can avoid ethical issues, and is beneficial to improve the availability of drugs for treating nervous system diseases; is beneficial to in vivo differentiation, improves the therapeutic effect of cell transplantation, and improves the safety of cell drugs in preventing, diagnosing and treating nervous system diseases.

[0041] As described herein, "induced neural stem cell" or "iNSC" refers to a neural stem cell obtained by reprogramming technology from a somatic cell as a starting cell, which retains the ability of self-renewal through mitotic cells, and can differentiate into different types of specialized cell types.

[0042] The term "reprogramming" refers to the phenomenon of dedifferentiation or transdifferentiation of cells into pluripotent and / or unipotent. The dedifferentiated cells can then be redifferentiated into different types of cells, so that the cells show new or different morphological and functional characteristics of specific cell lineages.

[0043] As used herein, the term "somatic cell" refers to any differentiated cell that forms an organism, except stem cells, progenitor cells and germ cells (i.e. oocytes and spermatogonia) and cells derived therefrom (e.g. oocytes, sperm). For example, internal organs, skin, bone, blood and connective tissue are all composed of somatic cells. In some embodiments, the somatic cells of the present application can be mononuclear cells isolated from the peripheral blood of adult humans. The somatic cells are obtained from animals, preferably human subjects, and are cultured according to standard cell culture protocols available to those of ordinary skill in the art.

[0044] A "neural stem cell" is a multipotent stem cell of ectodermal origin, which has the ability to form neural progenitor cells under physiological conditions or in vitro conditions conducive to differentiation into neurons and glia. As an example, a neural stem cell has the ability to form MGE progenitor cells under suitable in vitro conditions.

[0045] The term "neural progenitor cell" refers to an immature cell of the nervous system, which can differentiate into intermediate neuron precursor cells (e.g. GABAergic interneurons) under suitable physiological conditions or in vitro conditions, and continue to differentiate into different types of neurons (e.g. GABAergic neurons) or glial cells (oligodendrocytes and astrocytes) under suitable physiological conditions or in vitro conditions.

[0046] The method for preparing the induced neural stem cells can use any known method in the prior art. For example, the iNSC can be prepared according to the method disclosed in ZL201510628868.0 and / or ZL201810372724.7. In some embodiments, the iNSC is prepared using peripheral blood mononuclear cells as starting cells.

[0047] In some embodiments, the iNSC has the same biomarkers as the iNSC in ZL201510628868.0 or ZL201810372724.7.

[0048] The population of cerebral cortex intermediate neuron precursor cells derived from iNSC can avoid the ethical issues of embryonic stem cells and improve the accessibility of cell drugs for nervous system diseases.

[0049] In some embodiments, the population of cerebral cortex intermediate neuron precursor cells is MGE-type GABAergic intermediate neurons, i.e., GABAergic intermediate neurons derived from the medial ganglionic eminence (MGE).

[0050] In some embodiments, the proportion of cells expressing NKX2.1 in the population of cerebral cortex intermediate neuron precursor cells is 50%-100%. In some embodiments, the proportion of cells expressing NKX2.1 in the population of cerebral cortex intermediate neuron precursor cells is 60%-100%, 70%-100%, 75%-100%, or 80%-100%.

[0051] In some embodiments, the proportion of cells expressing NKX2.1 in the population of cerebral cortex intermediate neuron precursor cells is 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a value or range between any two of the foregoing.

[0052] In the population of cerebral cortex intermediate neuron precursor cells, the proportion of cells expressing NKX2.1 in the population is 50%-100%, which helps to improve the quality of cell drugs, improve the effect of cell transplantation, and improve the safety of cell therapy.

[0053] In some embodiments, the number of cells expressing FoxG1 in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 50-100% of the population. In some embodiments, the number of cells expressing FoxG1 in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 65-100%, 70-100%, 75-100%, or 80-100% of the population.

[0054] In some embodiments, the number of cells expressing FoxG1 in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a value or a range between any two of the foregoing, of the population.

[0055] The number of cells expressing FoxG1 in the population of intermediate neuron precursor cells in the cerebral cortex, which is 60-100% of the population, helps to improve the effect of cell transplantation and the safety of cell drugs.

[0056] In some embodiments, the number of cells expressing GABA in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 40-100% of the population. In some embodiments, the number of cells expressing GABA in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 45-100%, 50-100%, 60-100%, 70-100%, 75-100%, or 80-100% of the population.

[0057] In some embodiments, the number of cells expressing GABA in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a value or a range between any two of the foregoing, of the population.

[0058] The number of cells expressing GABA in the population of intermediate neuron precursor cells in the cerebral cortex, which is 45-100% of the population, helps to improve the quality of cell drugs, improve the safety and therapeutic effect of cell drugs.

[0059] In some embodiments, the number of SST-expressing cells in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 40-100% of the population.

[0060] In some embodiments, the number of SST-expressing cells in the population of intermediate neuron precursor cells in the ex vivo cerebral cortex is 40%, 41%, 42%, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a value or range between any two of the foregoing.

[0061] The number of SST-expressing cells in the population of intermediate neuron precursor cells in the cerebral cortex is 40-100% of the population, which helps to improve the quality of cell drugs, improve the safety and therapeutic effect of cell drugs.

[0062] The population of intermediate neuron precursor cells in the cerebral cortex has expression markers of GABAergic intermediate neurons, particularly MGE-type GABAergic intermediate neurons, which can continue to differentiate into mature intermediate neurons at the lesion site in vivo, thereby repairing the pathophysiological defects of the nervous system and improving the nervous system diseases.

[0063] The iNSC-derived cerebral cortex intermediate neurons can continue to survive and differentiate after being transplanted into the body, and further have the ability to secrete GABA neurotransmitters.

[0064] The population of iNSC-derived cerebral cortex intermediate neuron precursor cells can avoid the ethical issues and xenotransplantation rejection reactions of embryonic stem cells or iPSCs, which is conducive to improving the accessibility of cell therapy drugs.

[0065] In some embodiments, the cell population contains at least two of the following cells: NKX2.1-expressing cells, FoxG1-expressing cells, GABA-expressing cells, and SST-expressing cells.

[0066] In some embodiments, the cell population contains cells that express two or more of the following markers: NKX2.1, FoxG1, GABA, and SST.

[0067] In some embodiments, the cell population contains cells that express FoxG1, NKX2.1, and GABA markers simultaneously.

[0068] In some embodiments, the cell population contains cells that express NKX2.1, FoxG1, GABA, and SST markers simultaneously.

[0069] In some embodiments, the number of cells expressing marker parvalbumin (PV) in the cell population is less than 30% of the cell population.

[0070] In some embodiments, the number of cells expressing marker parvalbumin (PV) in the cell population is 0%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any value or range between any of the foregoing.

[0071] The number of cells expressing SST in the cell population is less than 30% of the cell population in the cerebral cortex interneuron precursor cell population helps to improve cell purity and cell drug quality, reduce non-target cells in the in vivo differentiation of cell transplantation, and improve cell drug safety.

[0072] The cerebral cortex interneuron precursor cell population provided in the present application has high purity, which helps to improve the quality of cell drugs and improve the safety of cell drugs in the prevention, diagnosis, and treatment of nervous system diseases.

[0073] In addition, the cerebral cortex interneuron precursor cell population provided in the present application is derived from iNSC, and the starting cell of iNSC is derived from a somatic cell, particularly peripheral blood mononuclear cells, so that the cerebral cortex interneuron precursor cell population carries the VDJ rearrangement characteristics in the development and differentiation of peripheral blood mononuclear cells. The iPSC and iNSC derived differentiated cells can be distinguished by methods known to those skilled in the art (for example, see WO2013128204A1).

[0074] Preparation method The present application provides a preparation method of an in vitro cerebral cortex interneuron precursor cell population, which comprises: culturing iNSC under effective conditions to transform it into medial ganglionic eminence (MGE) like progenitor cells; and inducing the MGE like progenitor cells to form a cerebral cortex interneuron precursor cell population, particularly an iNSC derived cerebral cortex interneuron precursor cell population.

[0075] In some embodiments, the preparation method comprises: preparing the population of intermediate cerebral cortical neuron precursor cells ex vivo using a combination of neural stem cell medium and differentiation medium.

[0076] In some embodiments, the preparation method comprises: (B) differentiating the iNSCs using a medial ganglionic eminence (MGE) differentiation medium to obtain MGE-like progenitor cells; optionally, the MGE differentiation medium comprises a basal medium, an amino acid component, a serum replacement supplement, a TGF-β receptor kinase inhibitor, a WNT inhibitor, a Bone Morphogenetic Protein 1 (BMP I) type receptor inhibitor, and / or a Sonic hedgehog (SHH) pathway activator; (C) differentiating the MGE-like progenitor cells using a GABA differentiation medium; optionally, the GABA differentiation medium comprises a basal medium, an amino acid component, a serum replacement supplement, a SHH pathway activator, and / or a mitogen-activated protein kinase kinase (MEK) inhibitor; (D) continuing to differentiate the MGE-like progenitor cells using a terminal differentiation medium to obtain a population of iNSC-derived intermediate cerebral cortical neuron precursor cells; optionally, the terminal differentiation medium comprises a basal medium, an amino acid component, a serum replacement supplement, a MEK inhibitor, a cyclin-dependent kinases 4 / 6 (CDK4 / 6) inhibitor, a γ-secretase inhibitor, and / or a neurotrophic factor.

[0077] For example, Figure 1 One of the differentiation strategies for preparing MGE-type GABAergic intermediate neurons using human iNSCs as starting cells is shown.

[0078] In some embodiments, the preparation method further comprises, before the step (B) of treatment, a step (A) of culturing the iNSCs using a neural stem cell medium; optionally, the neural stem cell medium comprises a basal medium, an amino acid component, a serum replacement supplement, a recombinant human leukemia inhibitory factor, a WNT (wingless-related integration site) activator, and / or a transforming growth factor-β (TGF-β) receptor kinase inhibitor.

[0079] The base medium contains the carbon source, nitrogen source, trace elements, and inorganic salts required for cell growth and is a basic component for formulating different types and functions of culture media. The base medium can be a culture medium commonly used in the art as a base medium.

[0080] In some embodiments, the base medium in the cell culture medium of steps (A) to (D) is each independently selected from one or a combination of Neural Basal-A and DMEM / F12, and optionally, the base medium in steps (A) to (D) comprises Neural Basal-A and DMEM / F12.

[0081] In some embodiments, the base medium in steps (A) to (D) each independently comprises Neural Basal-A and DMEM / F12 in a volume ratio of (0.5-2): 1, optionally (0.5-1.5): 1, for example 1: 1.

[0082] The amino acid component in the cell culture medium of steps (A) to (D) each independently comprises non-essential amino acids, which can include, by way of example, non-essential amino acid (NEAA) cell culture supplements and L-glutamine or its alternative supplements (e.g., GlutaMAX medium). In some embodiments, the amino acid component in the neural stem cell culture medium, the MGE differentiation medium, the GABA differentiation medium, and the terminal differentiation medium each independently comprises NEAA medium and GlutaMAX medium.

[0083] In some embodiments, the amino acid component in the neural stem cell culture medium, the MGE differentiation medium, the GABA differentiation medium, and the terminal differentiation medium each independently comprises NEAA medium at a volume ratio of 0.25%-2% of the medium, optionally 0.5%-1.5%, for example 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%.

[0084] In some embodiments, the amino acid component in the neural stem cell culture medium, the MGE differentiation medium, the GABA differentiation medium, and the terminal differentiation medium each independently comprises GlutaMAX medium at a volume ratio of 0.25%-2% of the medium, optionally 0.5%-1.5%, for example 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%.

[0085] The present application uses serum-free medium to culture induced neural stem cells, which can effectively inhibit the proliferation of non-neural cells, and at the same time, help to improve the quality of cell products and avoid the safety hidden dangers brought by blood. In some embodiments, the serum replacement additives used in the amino acid components of the neural stem cell culture medium, the MGE differentiation medium, the GABA differentiation medium and the terminal differentiation medium independently include one or a combination of N2 additives and B27 additives.

[0086] In some embodiments, the amino acid components of the neural stem cell culture medium, the MGE differentiation medium, the GABA differentiation medium and the terminal differentiation medium independently include N2 additives at 0.25%-2% of the volume of the medium, optionally 0.5%-1.5%, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%.

[0087] In some embodiments, the amino acid components of the neural stem cell culture medium, the MGE differentiation medium, the GABA differentiation medium and the terminal differentiation medium independently include B27 additives at 0.25%-2% of the volume of the medium, optionally 0.5%-1.5%, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, or a value or range between any two of the foregoing.

[0088] In some embodiments, the neural stem cell culture medium includes human leukemia inhibitory factor (hLIF), preferably using recombinant human leukemia inhibitory factor (e.g., rhLIF medium), which helps to promote the proliferation of iNSC in the early stage.

[0089] In some embodiments, the concentration of the recombinant human leukemia inhibitory factor in the neural stem cell culture medium is 1 ng / mL-20 ng / mL. In some embodiments, the concentration of the recombinant human leukemia inhibitory factor in the neural stem cell culture medium is 5 ng / mL-15 ng / mL, 5 ng / mL-20 ng / mL, 1 ng / mL-10 ng / mL, 3 ng / mL-17 ng / mL.

[0090] In some embodiments, the recombinant human leukemia inhibitory factor is at a concentration of 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, or a value or a range between any two of the foregoing, in the neural stem cell culture medium.

[0091] In some embodiments, the neural stem cell culture medium includes a WNT activator to enhance self-renewal of iNSCs. The WNT activator in the neural stem cell culture medium includes one or more of CHIR99021, SB-216763, IM-12, TWS119, KY19382, and DIF-3. In some embodiments, the WNT activator in the neural stem cell culture medium includes CHIR99021.

[0092] In some embodiments, the neural stem cell culture medium includes a WNT activator at a concentration of 1 μΜ-10 μΜ. In some embodiments, the WNT activator in the neural stem cell culture medium is at a concentration of 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, or a value or a range between any two of the foregoing.

[0093] In some embodiments, the neural stem cell culture medium includes a TGF-β receptor kinase inhibitor that can inhibit TGF-β induced transcription, gene expression, apoptosis, and growth inhibition, and promote self-renewal of iNSCs.

[0094] In some embodiments, the TGF-β receptor kinase inhibitor in the neural stem cell culture medium includes one or more of SB431542, LY2157299, RepSox, A 83-01, and Galunisertib; optionally, SB431542.

[0095] In some embodiments, the TGF-β receptor kinase inhibitor in the neural stem cell culture medium is at a concentration of 1 μΜ-10 μΜ. In some embodiments, the TGF-β receptor kinase inhibitor in the neural stem cell culture medium is at a concentration of 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, or a value or a range between any two of the foregoing.

[0096] In some embodiments, the neural stem cell medium comprises Neural Basal-A, DMEM / F12, N2 supplement, NEAA, B27, GlutaMAX, rhLIF, CHIR99021, and SB431542.

[0097] In some embodiments, the neural stem cell medium comprises Neural Basal-A and DMEM / F12 in a volume ratio of (0.5-2): 1, and N2 supplement at 0.25%-2% by volume of the medium, NEAA at 0.25%-2% by volume, B27 at 0.25%-2% by volume, GlutaMAX at 0.25%-2% by volume; optionally, the neural stem cell medium comprises Neural Basal-A and DMEM / F12 in a volume ratio of (0.5-1.5): 1, and N2 supplement at 0.5%-1.5% by volume of the medium, NEAA at 0.5%-1.5% by volume, B27 at 0.5%-1.5% by volume, GlutaMAX at 0.5%-1.5% by volume; further optionally, the neural stem cell medium comprises Neural Basal-A and DMEM / F12 in a volume ratio of 1: 1, N2 supplement at 1% by volume, NEAA at 1% by volume, B27 at 1% by volume, GlutaMAX at 1% by volume.

[0098] In some embodiments, the neural stem cell medium further comprises recombinant human leukemia inhibitory factor rhLIF at a concentration of 1 ng / mL-20 ng / mL, CHIR99021 at a concentration of 1 μΜ -10 μΜ, SB431542 at a concentration of 1 μΜ -10 μΜ; optionally, rhLIF at a concentration of 5 ng / mL-15 ng / mL, CHIR99021 at a concentration of 1 μΜ-5 μΜ, SB431542 at a concentration of 1 μΜ-5 μΜ; further optionally, 10 ng / mL rhLIF, 3 μΜ CHIR99021, 2 μΜ SB431542.

[0099] In some embodiments, the neural stem cell medium comprises Neural Basal-A and DMEM / F12 in a volume ratio of 1: 1, N2 supplement at 1% by volume, NEAA at 1% by volume, B27 at 1% by volume, GlutaMAX at 1% by volume, 10 ng / mL rhLIF, 3 μΜ CHIR99021, 2 μΜ SB431542.

[0100] The neural cell culture medium helps to expand the induced neural stem cells and maintain the undifferentiated state of the induced neural stem cells, reduce impurity cells, and improve the quality of subsequent cell differentiation.

[0101] In some embodiments, the step (A) comprises culturing the iNSC for 1-3 days, optionally 1-2 days, using the neural stem cell culture medium. In some embodiments, the step (A) comprises culturing the iNSC for 1 day, 2 days, 3 days, or a range between any of the aforementioned values, or any value between the aforementioned ranges, using the neural stem cell culture medium. The short culturing time in step (A) is advantageous for shortening the preparation period of the MGE-like progenitor cells.

[0102] The step (A) can be performed using the cell culture conditions commonly used in the art, or can be performed under conditions containing a suitable amount of carbon dioxide. In some embodiments, the cells in the step (A) can be cultured in a cell culture incubator at 37°C ± 2°C and a CO2 gas concentration of 5% ± 1%.

[0103] In some embodiments, in the step (B), the MGE differentiation medium comprises a TGF-β receptor kinase inhibitor to promote self-renewal of the cells and induce differentiation of the cells into neuroectoderm. In some embodiments, the TGF-β receptor kinase inhibitor in the MGE differentiation medium comprises one or more of SB431542, LY2157299, RepSox, A 83-01, and Galunisertib; optionally, the TGF-β receptor kinase inhibitor comprises SB431542.

[0104] In some embodiments, the concentration of the TGF-β receptor kinase inhibitor in the MGE differentiation medium is 1 μM-20 μM, optionally 5 μM-15 μM. In some embodiments, the concentration of the TGF-β receptor kinase inhibitor in the MGE differentiation medium is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, or a value or a range between any two of the aforementioned values.

[0105] In some embodiments, the MGE differentiation medium comprises a WNT (Tankyrase) inhibitor to promote differentiation of the cells into forebrain cells and confer forebrain cell identity. In some embodiments, the WNT inhibitor in the MGE differentiation medium comprises one or more of XAV939, Dickkopf 1, IWR-1, JW 55, K-756, MN-64, and WIKI4; optionally, the WNT inhibitor comprises XAV939.

[0106] In some embodiments, the concentration of the WNT inhibitor in the MGE differentiation medium is 1 μΜ-20 μΜ. In some embodiments, the concentration of the WNT inhibitor in the MGE differentiation medium is 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, or a value or a range between any two of the foregoing.

[0107] In some embodiments, the MGE differentiation medium includes a BMP type I receptor inhibitor to regulate cell differentiation and proliferation and to induce cell differentiation into neural ectoderm. In some embodiments, the BMP type I receptor inhibitor in the MGE differentiation medium includes one or more of LDN193189, Noggin, BMPR1A-Fc, K02288, and Dorsomorphin; alternatively, the BMP type I receptor inhibitor includes LDN193189.

[0108] In some embodiments, the concentration of the BMP type I receptor inhibitor in the MGE differentiation medium is 50 nM-500 nM, alternatively 150 nM-350 nM, 200 nM-300 nM, or 100 nM-400 nM.

[0109] In some embodiments, the concentration of the BMP type I receptor inhibitor in the MGE differentiation medium is 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or a range between any two of the foregoing, or any value between the ranges.

[0110] In some embodiments, the MGE differentiation medium includes a SHH pathway activator to activate the SHH signaling pathway and induce cell differentiation into ventral forebrain MGE-like progenitor cells. In some embodiments, the SHH pathway activator in the MGE differentiation medium includes one or more of SAG1, SHH, and Purmorphamine; alternatively, the SHH pathway activator includes SAG1.

[0111] In some embodiments, the concentration of the SHH pathway activator in the MGE differentiation medium is 50 nM-1000 nM, optionally 100 nM-1000 nM. In some embodiments, the concentration of the SHH pathway activator in the MGE differentiation medium is 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1000 nM, or a range between any of the foregoing, or any value between the ranges.

[0112] In some embodiments, the MGE differentiation medium comprises NeuralBasal-A, DMEM / F12, N2 supplement, NEAA, B27, GlutaMAX, SB431542, XAV939, LDN193189, and SAG1.

[0113] In some embodiments, the MGE differentiation medium comprises NeuralBasal-A and DMEM / F12 at a volume ratio of (0.5-2): 1, and N2 supplement at 0.25%-2% by volume of the medium, NEAA at 0.25%-2% by volume, B27 at 0.25%-2% by volume, GlutaMAX at 0.25%-2% by volume.

[0114] In some embodiments, the MGE differentiation medium comprises NeuralBasal-A and DMEM / F12 at a volume ratio of (0.5-1.5): 1, and N2 supplement at 0.5%-1.5% by volume of the medium, NEAA at 0.5%-1.5% by volume, B27 at 0.5%-1.5% by volume, GlutaMAX at 0.5%-1.5% by volume.

[0115] In some embodiments, the MGE differentiation medium comprises NeuralBasal-A and DMEM / F12 at a volume ratio of 1: 1, N2 supplement at 1% by volume, NEAA at 1% by volume, B27 at 1% by volume, GlutaMAX at 1% by volume.

[0116] In some embodiments, the MGE differentiation medium further comprises SB431542 at a concentration of 1 μΜ-20 μΜ, XAV939 at a concentration of 1 μΜ-20 μΜ, LDN193189 at a concentration of 50 nM-500 nM, and SAG1 at a concentration of 50 nM-1000 nM. Optionally, SB431542 at a concentration of 5 μΜ-15 μΜ, XAV939 at a concentration of 5 μΜ-15 μΜ, LDN193189 at a concentration of 150 nM-350 nM, and SAG1 at a concentration of 100 nM-1000 nM are included.

[0117] In some embodiments, the MGE differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, 10 μΜ SB431542, 10 μΜ XAV939, 250 nM LDN193189, and 100 nM-1000 nM SAG1.

[0118] In some embodiments, the MGE differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, 10 μΜ SB431542, 10 μΜ XAV939, 250 nM LDN193189, and 100 nM SAG1.

[0119] The MGE differentiation medium helps induce differentiation of neural stem cells into MGE-like progenitor cells. In addition, since the starting cells are iNSCs, differentiation into neural endoderm and neural mesoderm can be reduced or avoided, allowing the cells to differentiate mainly or entirely into neural ectoderm, reducing the generation of impurity cells, and improving the yield and purity of MGE-like progenitor cells. Furthermore, the use of serum-containing medium in the preparation of MGE-like progenitor cells is avoided, which also reduces the safety risks associated with blood sources, further improving the safety of the cell product.

[0120] As described herein, "differentiation" refers to the process by which an unspecialized cell becomes a more specialized type with altered structural and / or functional properties. Mature cells typically have altered cellular structure and tissue-specific proteins.

[0121] During development, an embryo can generate three main embryonic germ layers, namely mesoderm, endoderm and ectoderm. "Endoderm" means the germinal layer that forms during the development of an animal embryo to produce the gastrointestinal tract, respiratory tract, endocrine glands and organs, certain structures of the auditory system, and certain structures of the urinary system. "Mesoderm" means the germinal layer that forms during the development of an animal embryo to produce muscle, cartilage, bone, dermis, reproductive system, adipose tissue, connective tissue of the intestine, peritoneum, certain structures of the urinary system, mesothelium, notochord, and spleen. "Ectoderm" means the germinal layer that forms during the development of an animal embryo to produce the nervous system, enamel of teeth, epidermis, hair, nails, and lining of mucosal tissue.

[0122] The culture medium for culturing induced neural stem cells and the culture medium for differentiating neural cells can also use the culture medium known in the art, as long as it has similar components and amounts as the culture medium of the present application. In addition, the culture medium can be moderately modified to achieve the predetermined goal. For example, the carbon source, nitrogen source, trace elements, growth factors, etc. known in the art can be adjusted and used afterwards to improve the differentiation efficiency of the cells.

[0123] In some embodiments, the step (B) comprises culturing for 5-9 days using the MGE differentiation medium. In some embodiments, the step (B) comprises culturing for 6-8 days using the MGE differentiation medium.

[0124] In some embodiments, the step (B) comprises culturing for 5 days, 6 days, 7 days, 8 days, 9 days, or a range between any of the aforementioned values, or any value between the aforementioned ranges.

[0125] In some embodiments, the step (B) comprises culturing for 5 days, 6 days, 7 days, 8 days, 9 days, or a range between any of the aforementioned values, or any value between the aforementioned ranges.

[0126] In some embodiments, the cells of the step (B) can be cultured in a cell culture incubator at 37℃±2℃, with a CO2 gas concentration of 5%±1%.

[0127] The differentiation period of the step (B) is short, which helps to reduce the preparation period of the MGE-like progenitor cells.

[0128] Through the culture and differentiation of steps (A) and (B), a population of MGE-like progenitor cells expressing NKX2.1 and / or FOXG1 markers can be prepared.

[0129] In some embodiments, the number of cells expressing NKX2.1 in the MGE-like progenitor cell population is 50-100% of the MGE-like progenitor cell population. In some embodiments, the number of cells expressing NKX2.1 in the MGE-like progenitor cell population is 60-100%, 70-100%, 80-100%, or 90-100% of the MGE-like progenitor cell population. In some embodiments, the number of cells expressing NKX2.1 in the MGE-like progenitor cell population is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a value or range between any two of the foregoing, of the MGE-like progenitor cell population.

[0130] In some embodiments, the number of cells expressing FOXG1 in the MGE-like progenitor cell population is 50-100% of the MGE-like progenitor cell population. In some embodiments, the number of cells expressing FOXG1 in the MGE-like progenitor cell population is 70-100%, 80-100%, or 85-100% of the MGE-like progenitor cell population. In some embodiments, the number of cells expressing FOXG1 in the MGE-like progenitor cell population is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a value or range between any two of the foregoing, of the MGE-like progenitor cell population.

[0131] The MGE-like progenitor cell population prepared in step (B) is further differentiated in a GABA differentiation medium to inhibit differentiation of the cells into non-target cells and development of the cells into cancer cells, and to increase the yield of the cerebral cortical intermediate neuron precursor cell population, particularly the GABAergic intermediate neuron cell population.

[0132] In step (C), the GABA differentiation medium includes a MEK inhibitor to increase the efficiency and specificity of the differentiation of the cells into the cerebral cortical intermediate neuron precursor cell population, particularly the GABAergic intermediate neuron cell population.

[0133] In some embodiments, the MEK inhibitor in the GABA differentiation medium comprises one or more of PD0325901, MEK162, RO4987655, AZD8330, AZD6244, BAY 869766, AZD6244, SL327, U0126, GSK1120212, Cobimetinib hemifumarate, TCS PIM-1 1, CI-1040, GDC-0973, Trametinib, Pimasertib, GDC-0623, PD98059, U0126-EtOH, TAK-733, and BIX02189; optionally, PD0325901.

[0134] In some embodiments, the concentration of the MEK inhibitor in the GABA differentiation medium is 1 μΜ-10 μΜ, optionally 1 μΜ-5 μΜ. In some embodiments, the concentration of the MEK inhibitor in the GABA differentiation medium is 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, or a value or range between any two of the foregoing.

[0135] In some embodiments, the GABA differentiation medium comprises a SHH pathway activator to activate the SHH signaling pathway and induce differentiation of the cells into GABAergic interneurons.

[0136] In some embodiments, the SHH pathway activator in the GABA differentiation medium comprises one or more of SAG1, SHH, and Purmorphamine, optionally SAG1.

[0137] In some embodiments, the concentration of the SHH pathway activator in the GABA differentiation medium is 50 nM-1000 nM, optionally 50 nM-500 nM, 100 nM-1000 nM.

[0138] In some embodiments, the concentration of the SHH pathway activator in the GABA differentiation medium is 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1000 nM, or a range between any two of the foregoing, or any value between the ranges.

[0139] In some embodiments, the GABA differentiation medium comprises Neural Basal-A, DMEM / F12, N2 supplement, NEAA, B27, GlutaMAX, SAG1 and PD0325901.

[0140] In some embodiments, the GABA differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of (0.5-1.5):1, and N2 supplement at a volume ratio of 0.5%-1.5%, NEAA at a volume ratio of 0.5%-1.5%, B27 at a volume ratio of 0.5%-1.5%, GlutaMAX at a volume ratio of 0.5%-1.5%; optionally, the GABA differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of (0.5-1.5):1, N2 supplement at a volume ratio of 0.5%-1.5%, NEAA at a volume ratio of 0.5%-1.5%, B27 at a volume ratio of 0.5%-1.5%, GlutaMAX at a volume ratio of 0.5%-1.5%; further optionally, the GABA differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%.

[0141] In some embodiments, the GABA differentiation medium further comprises SAG1 at a concentration of 10 nM-1000 nM, PD0325901 at a concentration of 1 μM-10 μM, optionally SAG1 at a concentration of 100 nM-1000 nM, PD0325901 at a concentration of 1 μM-5 μM.

[0142] In some embodiments, the GABA differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, 100 nM-1000 nM SAG1 and 2 μM PD0325901.

[0143] The GABA differentiation medium helps the differentiation of MGE-like progenitor cell population into GABAergic interneurons, with high differentiation efficiency and cell purity, which is conducive to improving the yield of cell preparation.

[0144] In some embodiments, the step (C) comprises rocking the MGE progenitor cells in the GABA differentiation medium. The cells can be rocked using a rocker, which is not limited to the model or type of instrument, nor is the rocking frequency of the rocker limited, as long as the needs of the cells for normal growth are met. As an example, an orbital shaker can be used to rock the cells at a speed of 80 rotations per hour.

[0145] In some embodiments, the step (C) comprises culturing the cells in half volume of fresh GABA differentiation medium every other day, which helps the cells to grow well.

[0146] In some embodiments, the step (C) comprises culturing the cells for 5-9 days, preferably 6-8 days. In some embodiments, the step (C) comprises culturing the cells for 5 days, 6 days, 7 days, 8 days, 9 days, or a range between any two of the foregoing values, or any value between the ranges. In some embodiments, the step (C) can be performed in a cell culture incubator at 37°C ± 2°C, with a CO2 gas concentration of 5% ± 1%.

[0147] In the step (D), the terminal differentiation medium comprises a MEK inhibitor, which further improves the efficiency and specificity of cell differentiation. In some embodiments, the MEK inhibitor in the terminal differentiation medium comprises one or more of PD0325901, MEK162, RO4987655, AZD8330, AZD6244, BAY 869766, AZD6244, SL327, U0126, GSK1120212, Cobimetinib hemifumarate, TCS PIM-1 1, CI-1040, GDC-0973, Trametinib, Pimasertib, GDC-0623, PD98059, U0126-EtOH, TAK-733, and BIX02189; and optionally, PD0325901.

[0148] In some embodiments, the concentration of the MEK inhibitor in the terminal differentiation medium is 1 μM-10 μM, and optionally 1 μM-5 μM. In some embodiments, the concentration of the MEK inhibitor in the terminal differentiation medium is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or a value or a range between any two of the foregoing values.

[0149] In some embodiments, the terminal differentiation medium comprises a CDK4 / 6 inhibitor, which helps the cells to exit the mitotic cycle, improves the homogeneity of the intermediate neuronal precursor cell population, reduces or eliminates the pluripotency of residual cells (or impurity cells), and avoids the formation of tumors after in vivo transplantation.

[0150] In some embodiments, the one or more of CDK4 / 6 inhibitor PD0332991, Lerociclib dihydrochloride, Abemaciclib, Lerociclib, Abemaciclib methanesulfonate, AT7519 Hydrochloride, AT7519, and Narazaciclib in the terminal differentiation medium; optionally, the CDK4 / 6 inhibitor comprises PD0332991.

[0151] In some embodiments, the terminal differentiation medium comprises 1 μΜ - 10 μΜ, optionally 1 μΜ - 5 μΜ, 1 μΜ - 8 μΜ, 1 μΜ - 4 μΜ of the CDK4 / 6 inhibitor. In some embodiments, the concentration of the CDK4 / 6 inhibitor in the terminal differentiation medium is 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, or a value or a range between any two of the foregoing.

[0152] In some embodiments, the terminal differentiation medium comprises a gamma secretase inhibitor for inhibiting Notch 1 signaling and inducing cell differentiation, inhibiting abnormal proliferation while promoting cell differentiation.

[0153] In some embodiments, the gamma secretase inhibitor in the terminal differentiation medium comprises one or more of DAPT, Compound E, Z-IL-CHO, Crenigacestat, L-685458, BMS-906024, Nirogacestat, LY-411575, Semagacestat, Avagacestat, and RO4929097; optionally, the gamma secretase inhibitor comprises DAPT.

[0154] In some embodiments, the terminal differentiation medium comprises a gamma secretase inhibitor at a concentration of 0.5 μΜ - 20 μΜ, optionally 1 μΜ - 20 μΜ, 1 μΜ - 15 μΜ, 5 μΜ - 15 μΜ, or 8 μΜ - 12 μΜ. In some embodiments, the concentration of the gamma secretase inhibitor in the terminal differentiation medium is 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, or a value or a range between any two of the foregoing.

[0155] In some embodiments, the terminal differentiation medium includes neurotrophic factors that promote normal proliferation and differentiation of the cells. The neurotrophic factors include one or more of cyclic adenosine monophosphate (cAMP), Forskolin, Rolipram, BDNF, NGF, IGF-I, and GDNF; alternatively, the neurotrophic factors include cAMP, BDNF, and GDNF.

[0156] In some embodiments, the terminal differentiation medium includes neurotrophic factors that promote normal proliferation and differentiation of the cells. The neurotrophic factors include one or more of cyclic adenosine monophosphate (cAMP), Forskolin, Rolipram, BDNF, NGF, IGF-I, and GDNF; alternatively, the neurotrophic factors include cAMP, BDNF, and GDNF.

[0157] In some embodiments, the brain-derived neurotrophic factor includes cyclic adenosine monophosphate (cAMP) to activate cAMP-dependent protein kinase, promote differentiation of the neural stem cells, enhance survival of the neurons, and promote growth.

[0158] In some embodiments, the concentration of cAMP in the terminal differentiation medium is 0.1 μΜ to 100 μΜ, alternatively 0.1 μΜ to 80 μΜ, 0.1 μΜ to 50 μΜ, 0.1 μΜ to 10 μΜ, 0.1 μΜ to 5 μΜ, or 0.1 μΜ to 3 μΜ. In some embodiments, the concentration of cAMP in the terminal differentiation medium is 0.1 μΜ, 0.5 μΜ, 1 μΜ, 3 μΜ, 5 μΜ, 10 μΜ, 20 μΜ, 30 μΜ, 40 μΜ, 50 μΜ, 60 μΜ, 70 μΜ, 80 μΜ, 90 μΜ, 100 μΜ, or a value or a range between any two of the foregoing.

[0159] In some embodiments, the brain-derived neurotrophic factor includes BDNF (brain-derived neurotrophic factor) to promote proliferation and differentiation of the neural stem cells to help maintain normal cognitive and emotional functions of the brain after transplantation in vivo.

[0160] In some embodiments, the concentration of BDNF in the terminal differentiation medium is 1 ng / ml to 50 ng / ml, alternatively 5 ng / ml to 50 ng / ml, 5 ng / ml to 30 ng / ml, or 5 ng / ml to 15 ng / ml. In some embodiments, the concentration of BDNF in the terminal differentiation medium is 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 45 ng / ml, 50 ng / ml, or a value or a range between any two of the foregoing.

[0161] In some embodiments, the glial cell line-derived neurotrophic factor (GDNF) promotes neural stem cell proliferation and differentiation to help maintain normal cognitive and emotional brain function after transplantation in vivo.

[0162] In some embodiments, the GDNF is at a concentration of 1 ng / ml-50 ng / ml, optionally 5 ng / ml-50 ng / ml, 5 ng / ml-30 ng / ml, 5 ng / ml-15 ng / ml in the terminal differentiation medium. In some embodiments, the GDNF is at a concentration of 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 45 ng / ml, 50 ng / ml, or a value or range between any two of the foregoing, in the terminal differentiation medium.

[0163] The neurotrophic factors can nourish cells, improve cell survival and promote cell differentiation and development, and improve the quality and yield of GABAergic interneuron precursor cell population in the cerebral cortex derived from iNSCs.

[0164] In some embodiments, the terminal differentiation medium comprises Neural Basal-A, DMEM / F12, N2 supplement, NEAA, B27, GlutaMAX, PD0325901, PD0332991, DAPT, cAMP, BDNF and GDNF.

[0165] In some embodiments, the terminal differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of (0.5-1.5): 1, and N2 supplement at 0.5%-1.5% of the volume of the medium, NEAA at 0.5%-1.5% of the volume, B27 at 0.5%-1.5% of the volume, and GlutaMAX at 0.5%-1.5% of the volume.

[0166] In some embodiments, the terminal differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of (0.5-1.5): 1, N2 supplement at 0.5%-1.5% of the medium volume, NEAA at 0.5%-1.5% of the volume, B27 at 0.5%-1.5% of the volume, GlutaMAX at 0.5%-1.5% of the volume. Alternatively, the terminal differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of 1: 1, N2 supplement at 1% of the volume, NEAA at 1% of the volume, B27 at 1% of the volume, GlutaMAX at 1% of the volume.

[0167] The terminal differentiation medium further comprises PD0325901 at a concentration of 1 μM-10 μM, PD0332991 at a concentration of 1 μM-10 μM, DAPT at a concentration of 0.5 μM-20 μM, cAMP at a concentration of 0.1 μM-100 μM, BDNF at a concentration of 1 ng / ml-50 ng / ml, GDNF at a concentration of 1 ng / ml-50 ng / ml. Alternatively, further comprises PD0325901 at a concentration of 1 μM-5 μM, PD0332991 at a concentration of 1 μM-5 μM, DAPT at a concentration of 5 μM-15 μM, cAMP at a concentration of 0.1 μM-3 μM, BDNF at a concentration of 5 ng / ml-15 ng / ml, GDNF at a concentration of 5 ng / ml-15 ng / ml.

[0168] In some embodiments, the terminal differentiation medium comprises Neural Basal-A and DMEM / F12 at a volume ratio of 1: 1, N2 supplement at 1% of the volume, NEAA at 1% of the volume, B27 at 1% of the volume, GlutaMAX at 1% of the volume, 2 μM PD0325901, 2 μM PD0332991, 10 μM DAPT, 1 μM cAMP, 10 ng / ml BDNF, 10 ng / ml GDNF.

[0169] In some embodiments, the step (D) comprises culturing with the terminal differentiation medium for 5-9 days, optionally 6-8 days. In some embodiments, the step (D) comprises culturing with the terminal differentiation medium for 5 days, 6 days, 7 days, 8 days, 9 days, or a range between any of the aforesaid values, or any value between the aforesaid ranges.

[0170] In some embodiments, the step (D) comprises culturing in fresh terminal differentiation medium with half the amount every other day. In some embodiments, the cells in the step (D) can be cultured in a cell incubator at 37°C ± 2°C, with a CO2 gas concentration of 5% ± 1%.

[0171] The method for preparing the cerebral cortex intermediate neuron precursor cell population, particularly the MGE-type GABAergic intermediate neuron, has a short preparation period, can obtain target cells with high purity, and significantly improves the yield and clinical safety.

[0172] The present application provides a cerebral cortex intermediate neuron precursor cell population prepared from human iNSCs, which expresses NKX2.1, FOXG1, GABA, and SST markers, has the characteristics of MGE-type GABAergic intermediate neurons, and can continue to differentiate at the site of neuron injury or the site of abnormal GABA expression level. The cell population has strong migration ability and GABA neurotransmitter secretion ability, and can significantly improve diseases related to abnormal GABA expression. The preparation method provided by the present application can induce iNSCs to differentiate into neuroectoderm in vitro in a relatively short time, the cell differentiation direction is more accurate, the generation of neural ectoderm and neural mesoderm impurity cells during the differentiation of iPSCs can be reduced or avoided, the differentiated cells have higher purity, which is helpful for efficiently and quickly preparing an ex vivo cerebral cortex intermediate neuron precursor cell population, particularly an MGE-type GABAergic intermediate neuron.

[0173] Pharmaceutical composition The present application provides a pharmaceutical composition comprising an ex vivo cerebral cortex intermediate neuron precursor cell population prepared by the aforementioned method as an active ingredient, and at least one pharmaceutically acceptable excipient.

[0174] In this document, unless otherwise specified, "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient, and / or salt / ester / hydrate formed by the same is generally chemically or physically compatible with other ingredients constituting a certain pharmaceutical dosage form, and is physiologically compatible with the subject.

[0175] The pharmaceutically acceptable excipient can be a carrier, excipient, diluent, preservative, etc., but is not limited thereto. For example, the carrier, excipient, and diluent are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, distilled water, physiological saline, glycerol, ethanol, human serum albumin (HSA), etc. For example, the preservative can be benzoic acid, sodium benzoate, sorbic acid, p-hydroxybenzoic acid, chlorobutanol, etc.

[0176] When the pharmaceutical composition is formulated, a filler, an extender, a binder, a wetting agent, and the like can be further included.

[0177] The pharmaceutical composition can be formulated for subcutaneous, intravenous, parenteral, nasal, oral, pulmonary, brain, topical, vaginal, or rectal administration.

[0178] The pharmaceutical composition can be prepared in a solid form, a liquid form, a capsule form, a semi-solid form, and the like. When the pharmaceutical composition is formulated for parenteral use, the pharmaceutical composition can be prepared as an injection, an aerosol, and the like. Preferably, it can be formulated as an injection.

[0179] Kit The present application provides a kit comprising the foregoing population of MGE-like progenitor cells or population of cerebral cortical intermediate neuron precursor cells ex vivo, or the foregoing population of MGE-like progenitor cells or population of cerebral cortical intermediate neuron precursor cells ex vivo prepared by the foregoing method.

[0180] The kit can further include instructions for use, for example, in diagnosing, treating a nervous system disease. The kit can further include containers, suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single or dual-chamber syringes), and test tubes. Containers can be formed from a variety of materials such as glass or plastic, and the containers hold the formulation. The container holding the formulation can be a single-use vial or a multi-use vial, allowing for repeated administration of the reconstituted formulation.

[0181] The kit can further include a label or package insert containing instructions for reconstituting and / or using the formulation. The label or package insert can further indicate that the formulation is used or intended to be used for subcutaneous, intravenous (e.g., intravenous infusion), or other modes of administration for treating a nervous system disease (e.g., mesial temporal lobe epilepsy) in a subject. The kit can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0182] Therapeutic methods or uses The present application provides the foregoing population of MGE-like progenitor cells ex vivo, population of cerebral cortical intermediate neuron precursor cells, pharmaceutical composition, or kit for use in the preparation of a medicament for preventing, diagnosing, and treating a nervous system disease.

[0183] In the present application, the nervous system disease can be a disease caused by or to neurons, or a disease caused by abnormal GABA expression levels, for example, a mental disease.

[0184] In some embodiments, the subject suffering from the nervous system disease has abnormal GABA expression level in the body, particularly in the brain lesion site, which is higher or lower than the normal expression level.

[0185] The iNSC-derived cerebral cortex interneuron precursor cells have excellent migration ability after being transplanted into the body, can continue to survive and differentiate into SST-positive interneurons, secrete GABA neurotransmitters and receive synaptic signals of host neurons, and are integrated into the neural circuit in function, which helps the treatment and improvement of diseases caused by abnormal GABA expression level and the repair of injury.

[0186] The nervous system disease can be one or more selected from the group consisting of stroke, spinal cord injury, apoplexy, Huntington's disease, amyotrophic lateral sclerosis, motor neuron injury, traumatic peripheral nerve injury, ischemic brain injury, neonatal hypoxic-ischemic brain injury, cerebral palsy, peripheral paralysis, central paralysis, quadriplegia, diplegia, epilepsy, neuronal developmental disorder, neuralgia, intractable epilepsy, Alzheimer's disease, congenital metabolic nervous system disease, traumatic brain injury, motor neuron injury, diseases caused by motor neuron injury, autoimmune encephalitis, and mental illness, but is not limited thereto.

[0187] In some embodiments, the nervous system disease includes epilepsy (e.g., mesial temporal lobe epilepsy), stroke, Alzheimer's disease, traumatic brain injury, autoimmune encephalitis, and mental illness, and the mental illness includes depression.

[0188] In some embodiments, the cell population can improve epilepsy, further, can reduce the frequency and total duration of seizures, and further, can improve the impairment of learning and memory ability caused by epilepsy.

[0189] The prophylactic, diagnostic, and therapeutic doses of the nervous system disease vary depending on the specific condition, the severity of the condition, individual patient parameters including age, physical condition, size, gender, and weight, the duration of the treatment, the properties of concomitant therapies, if any, the specific route of administration, and the knowledge of the skilled practitioner, and the like.

[0190] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by the combination are all considered part of the disclosure of the present application.

[0191] The technical solutions of the present application will be described more clearly and explicitly below by way of examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of protection of the present application. The scope of protection of the present application is only defined by the claims.

[0192] Examples The following describes the examples of the present application. The examples described below are exemplary and are for the purpose of explanation of the present application and cannot be understood as a limitation of the present application. In the examples, the specific techniques or conditions not noted are performed according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not noted for the manufacturer are all conventional products that can be obtained commercially.

[0193] Reagents and sources

[0194] Example 1: Preparation of cerebral cortical intermediate neuron precursor cell population Provided human induced neural stem cells; human induced neural stem cells (prepared according to the method of ZL201810372724.7) were inoculated on a low-adhesion 96-well culture plate at a density of 8x10 3 The neural stem cell culture medium consisted of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 ng / mL rhLIF, 3 μM CHIR99021 and 2 μM SB431542.

[0195] On day 2, the MGE differentiation medium was used for differentiation culture, and the composition of the MGE differentiation medium included Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1% of the culture medium, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 μM SB431542, 10 μM XAV939, 250 nM LDN193189, and 100 nM SAG1.

[0196] The process of the differentiation culture included: half the amount of fresh MGE differentiation medium was changed every other day, the culture device was tilted, one-half of the culture medium was aspirated, fresh MGE differentiation medium was added again, and the cells were cultured for 7 days to form cell clusters; the MGE-like progenitor cell population was obtained.

[0197] On day 8, the MGE was transferred to a low-adhesion 6-well plate, and the GABA differentiation medium was used for differentiation culture, and the composition of the GABA differentiation medium included Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1% of the culture medium, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 100 nM SAG1 and 2 μM PD0325901.

[0198] The process of the differentiation culture includes: placing on an orbital shaker and shaking at a speed of 80 rounds per minute. Fresh GABA differentiation medium is replaced at half volume every other day, the culture device is tilted, half of the medium is sucked out, fresh GABA differentiation medium is added again, and the culture is carried out for 7 days to obtain GABAergic interneurons.

[0199] On day 15, terminal differentiation culture is carried out using a terminal differentiation medium, and the composition of the terminal differentiation medium includes: Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at 1% of the volume of the medium, NEAA at 1% of the volume, B27 at 1% of the volume, GlutaMAX at 1% of the volume, and 2 μM PD0325901, 2 μM PD0332991, 10 μM DAPT, 1 μM cAMP, 10 ng / ml BDNF, and 10 ng / ml GDNF.

[0200] The process of the differentiation culture includes: replacing fresh terminal differentiation medium at half volume every other day, tilting the culture device, sucking out half of the medium, adding fresh terminal differentiation medium again, and culturing for 7 days to obtain a population of iNSC-derived cerebral cortex interneuron precursor cells, i.e., MGE-type GABAergic interneurons.

[0201] The iNSC-derived cerebral cortex interneuron precursor cell population prepared in Example 1 is subjected to immunofluorescence staining, including the following steps: (1) Rewarming: rewarming the frozen section at 30-37°C; (2) Membrane breaking: using 0.3% Triton X-100 at room temperature (25°C±5°C) for 15-20 min, and washing the section with PBS once; (3) Blocking: using PBST blocking solution containing 3% donkey serum, blocking at room temperature for 2 h; (4) Primary antibody incubation: preparing PBST diluent containing 1% donkey serum by mass, adding the primary antibody, incubating overnight at 4°C, and then washing with PBS solution 3 times; (5) Secondary antibody incubation: preparing PBST diluent containing 1% donkey serum by mass, adding the secondary antibody, incubating at room temperature for 2 h in the dark, and washing the section with PBS 3 times; (6) DAPI staining: using DAPI to fluorescently stain the cell nucleus, and washing with PBS 3 times after the staining is completed; (7) Mounting: adding mounting medium for mounting.

[0202] (8) Image fluorescence data statistics: the positive rate of fluorescent cells was statistically analyzed using image J.

[0203] Among them, the primary antibodies used are: anti-NKX2.1 antibody (Proteintech, 66034-1-lg), anti-FOXG1 antibody (Abcam, ab196868), anti-GABA antibody (Sigma, A2052), anti-SST antibody (Proteintech, 17512-1-AP) and Parvalbumin polyclonal antibody (Proteintech, 29312-1-AP).

[0204] The secondary antibody used is: donkey anti-mouse Cy5 secondary antibody (Jackson) and donkey anti-rabbit Cy3 secondary antibody (Jackson).

[0205] The fluorescence staining results are shown in Figure 2 , which show that NKX2.1, FoxG1, GABA and SST present positive results with positive signals. The fluorescence staining identification quantitative results are shown in Figure 3 , which show that the differentiation proportion of cells expressing NKX2.1 is 70%, the differentiation proportion of cells expressing FOXG1 is 65%, the differentiation proportion of cells expressing GABA is 45%, the differentiation proportion of cells expressing SST is 45%, and the differentiation proportion of cells expressing PV is 20%.

[0206] Example 2: Preparation of cerebral cortical intermediate neuron precursor cell population The human induced neural stem cells are provided; the human induced neural stem cells are inoculated on a low adhesion 96-well culture plate at a density of 8×10 3 cells per well, and are cultured using a neural stem cell culture medium, the composition of the neural stem cell culture medium comprising: Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement accounting for 1% of the volume of the culture medium, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 ng / mL rhLIF, 3 μM CHIR99021 and 2 μM SB431542.

[0207] On the 2nd day, the MGE differentiation culture medium is used for differentiation culture, the composition of the MGE differentiation culture medium comprising: Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement accounting for 1% of the volume of the culture medium, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 μM SB431542, 10 μM XAV939, 250 nM LDN193189, 1 μM SAG1.

[0208] The process of the differentiation culture comprises: replacing half of the fresh MGE differentiation medium every other day, tilting the culture device, sucking out half of the medium, adding fresh MGE differentiation medium again, and culturing for 7 days to form cell clusters.

[0209] On day 8, the MGEs are transferred to a low-adhesion 6-well plate and subjected to differentiation culture with GABA differentiation medium, which comprises: Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at 1% of the volume of the medium, NEAA at 1% of the volume, B27 at 1% of the volume, GlutaMAX at 1% of the volume, and 1 μM SAG1 and 2 μM PD0325901.

[0210] The process of the differentiation culture comprises: shaking at 80 rounds per minute on an orbital shaker. Half of the fresh GABA differentiation medium is replaced every other day, the culture device is tilted, half of the medium is sucked out, fresh GABA differentiation medium is added again, and the culture is continued for 7 days to obtain GABAergic interneurons.

[0211] On day 15, the iNSC-derived cerebral cortex interneuron precursor cell population is subjected to differentiation culture with terminal differentiation medium, which comprises: Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at 1% of the volume of the medium, NEAA at 1% of the volume, B27 at 1% of the volume, GlutaMAX at 1% of the volume, and 2 μM PD0325901, 2 μM PD0332991, 10 μM DAPT, 1 μM cAMP, 10 ng / ml BDNF, and 10 ng / ml GDNF.

[0212] The process of the differentiation culture comprises: replacing half of the fresh terminal differentiation medium every other day, tilting the culture device, sucking out half of the medium, adding fresh terminal differentiation medium again, and culturing for 7 days to obtain the iNSC-derived cerebral cortex interneuron precursor cell population, i.e., the MGE-type GABAergic interneuron precursor cell population.

[0213] The iNSC-derived cerebral cortex interneuron precursor cell population prepared in Example 2 is subjected to immunofluorescence staining, and the staining method and reagents are the same as those in Example 1.

[0214] The fluorescence staining results are shown in Figure 4 The fluorescence staining results are shown in Figure 5As shown, the differentiation ratio of cells expressing NKX2.1 was 90%, the differentiation ratio of cells expressing FOXG1 was 85%, the differentiation ratio of cells expressing GABA was 86%, the differentiation ratio of cells expressing SST was 80%, and the differentiation ratio of cells expressing PV was 8%.

[0215] Example 3: Preparation of cerebral cortical intermediate neuron precursor cell population The human induced neural stem cells were inoculated on a low-adhesion 96-well culture plate at a density of 8 x 10 3 The neural stem cell culture medium was composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 ng / mL rhLIF, 3 μM CHIR99021 and 2 μM SB431542.

[0216] On day 2, the MGE differentiation medium was used for differentiation culture, and the MGE differentiation medium was composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 μM SB431542, 10 μM XAV939, 250 nM LDN193189, and 100 nM SAG1.

[0217] The differentiation culture process included: fresh MGE differentiation medium was replaced at half the amount every other day, the culture device was tilted, one-half of the medium was aspirated, fresh MGE differentiation medium was added again, and the culture was performed for 7 days to form cell clusters; and the MGE-like progenitor cell population was obtained.

[0218] On day 8, the MGE was transferred to a low-adhesion 6-well plate, and the GABA differentiation medium was used for differentiation culture, and the GABA differentiation medium was composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 100 nM SAG1 and 2 μM PD0325901. On days 8 to 12, 50 ng / mL FGF8 was added or not added for comparative study.

[0219] The process of the differentiation culture includes: placing on an orbital shaker and shaking at a speed of 80 rounds per minute. Fresh GABA differentiation medium is replaced at half the amount every other day, the culture device is tilted, half of the medium is sucked out, fresh GABA differentiation medium is added again, and the culture is carried out for 7 days to obtain GABAergic interneurons.

[0220] On day 15, terminal differentiation culture is carried out using a terminal differentiation medium, and the composition of the terminal differentiation medium includes: Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at 1% of the volume of the medium, NEAA at 1% of the volume, B27 at 1% of the volume, GlutaMAX at 1% of the volume, and 2 μM PD0325901, 2 μM PD0332991, 10 μM DAPT, 1 μM cAMP, 10 ng / ml BDNF, and 10 ng / ml GDNF.

[0221] The process of the differentiation culture includes: replacing fresh terminal differentiation medium at half the amount every other day, tilting the culture device, sucking out half of the medium, adding fresh terminal differentiation medium again, and culturing for 7 days to obtain a group of cerebral cortex interneuron precursor cells derived from iNSC, i.e., MGE-type GABAergic interneurons.

[0222] The prepared group of cerebral cortex interneuron precursor cells derived from iNSC is subjected to immunofluorescence staining, including the following steps: (1) Rewarming: rewarming the frozen section at 30-37°C; (2) Membrane breaking: using 0.3% Triton X-100 at room temperature (25°C±5°C) for 15-20 min, and washing the section with PBS once; (3) Blocking: using PBST blocking solution containing 3% donkey serum for blocking treatment at room temperature for 2 h; (4) Primary antibody incubation: preparing PBST diluent containing 1% donkey serum by mass, adding the primary antibody, and washing with PBS solution 3 times after incubation at 4°C overnight; (5) Secondary antibody incubation: preparing PBST diluent containing 1% donkey serum by mass, adding the secondary antibody, incubating in the dark at room temperature for 2 h, and washing the section with PBS 3 times; (6) DAPI staining: using DAPI to perform fluorescent staining on the cell nucleus, and washing with PBS 3 times after the staining is completed; (7) Mounting: adding mounting medium for mounting.

[0223] (8) Image fluorescence data statistics: using image J to perform data statistics on the positive rate of fluorescent cells.

[0224] The primary antibodies used are: anti-NKX2.1 antibody (Proteintech, 66034-1-lg), anti-FOXG1 antibody (Abcam, ab196868), anti-GABA antibody (Sigma, A2052), anti-SST antibody (Proteintech, 17512-1-AP), and Parvalbumin polyclonal antibody (Proteintech, 29312-1-AP).

[0225] The secondary antibodies used are: donkey anti-mouse Cy5 secondary antibody (Jackson) and donkey anti-rabbit Cy3 secondary antibody (Jackson).

[0226] The results of the fluorescent staining are shown in Example 1, which shows that NKX2.1, FoxG1, GABA, and SST present positive results with positive signals. The results of the fluorescent staining and quantification of the cells with or without 50 ng / mL FGF8 added from day 8 to day 12 of differentiation are shown in FIGS. 8A-8D, respectively. Figure 8 (DAPI / NKX2.0 / GABA for the group without FGF8 added and DAPI / NKX2.0 / SST for the group with FGF8 added) and Figure 9 As shown, the proportion of cells expressing NKX2.1 is 76%, the proportion of cells expressing GABA is 44%, and the proportion of cells expressing SST is 44%. According to the test results of Example 1 and Figure 9 It can be seen that FGF8 has no obvious effect on the induced differentiation of the cells.

[0227] Comparative Example 1: Preparation of cerebral cortical intermediate neuron precursor cell population The human induced neural stem cells are inoculated on a low-adhesion 96-well culture plate at a density of 8 x 10 3 cells per well, and are cultured using a neural stem cell culture medium, which is composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, N2 supplement at a volume ratio of 1%, NEAA at a volume ratio of 1%, B27 at a volume ratio of 1%, GlutaMAX at a volume ratio of 1%, and 10 ng / mL rhLIF, 3 μM CHIR99021, and 2 μM SB431542.

[0228] On day 2, the cells were differentiated in MGE differentiation medium, which was composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, 1% N2 supplement, 1% NEAA, 1% B27, 1% GlutaMAX, and 10 μM SB431542, 10 μM XAV939, 250 nM LDN193189, and 10 nM SAG1.

[0229] The differentiation process included replacing half of the fresh MGE differentiation medium every other day, tilting the culture device, removing half of the medium, and adding fresh MGE differentiation medium again. The cells were cultured for 7 days to form cell clusters, i.e., a MGE-like progenitor cell population.

[0230] On day 8, the MGEs were transferred to a low-adhesion 6-well plate and differentiated in GABA differentiation medium, which was composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, 1% N2 supplement, 1% NEAA, 1% B27, 1% GlutaMAX, and 10 nM SAG1 and 2 μM PD0325901.

[0231] The differentiation process included shaking the cells at 80 rpm on an orbital shaker. Half of the fresh GABA differentiation medium was replaced every other day, the culture device was tilted, half of the medium was removed, and fresh GABA differentiation medium was added again. The cells were cultured for 7 days to obtain GABAergic interneurons.

[0232] On day 15, the cells were differentiated in terminal differentiation medium, which was composed of Neural Basal-A and DMEM / F12 at a volume ratio of 1:1, 1% N2 supplement, 1% NEAA, 1% B27, 1% GlutaMAX, and 2 μM PD0325901, 2 μM PD0332991, 10 μM DAPT, 1 μM cAMP, 10 ng / ml BDNF, and 10 ng / ml GDNF.

[0233] The differentiation process included replacing half of the fresh terminal differentiation medium every other day, tilting the culture device, removing half of the medium, and adding fresh terminal differentiation medium again. After 7 days of culture, a population of iNSC-derived cerebral cortex interneuron precursor cells, i.e., MGE-type GABAergic interneurons, was obtained.

[0234] The iNSC-derived cerebral cortex intermediate neuron precursor cell population prepared in Comparative Example 1 was subjected to immunofluorescence staining, and the staining method and reagents were the same as in Example 1.

[0235] The results of the fluorescence staining are shown in FIG. 6, and showed that NKX2.1, FoxG1, GABA, and SST were positively stained with positive signals. Figure 6 The quantitative results of the fluorescence staining are shown in FIG. 7, and showed that the differentiation ratio of cells expressing NKX2.1 was 14%, the differentiation ratio of cells expressing FOXG1 was 22%, the differentiation ratio of cells expressing GABA was 11%, the differentiation ratio of cells expressing SST was 14%, and the differentiation ratio of cells expressing PV was 0%. Figure 7

[0236] Example 4: Cerebral cortical intermediate neurons derived from iNSCs have strong migration ability For the Matrigel two-dimensional migration analysis, the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 1 was continuously differentiated into intermediate neuron organoids, and at day 21 of the differentiation, 10 organoids were aspirated using a 1 mL gun tip, placed in a 1.5 mL EP tube, and washed once with PBS to remove the culture medium. 200 μL of high potassium HBSS medium (60 mM KCL, 82 mM NaCl) containing γ-aminobutyric acid transaminase inhibitor Vigabatrin (3 mM) and Tiagabine (5 μM) was added to the EP tube, and incubated at 37°C for 45 minutes. After the incubation, the organoids were disrupted using ultrasonication, 180 μL of the homogenate was added to 20 μL of 1N perchloric acid (Merck), and stored at -80°C in the dark until analysis, or directly subjected to HPLC analysis. The results are shown in FIG. 8. Figure 10 As shown in FIG. 8, the iNSC-derived cerebral cortex intermediate neuron showed a strong migration ability in both two-dimensional and three-dimensional culture.

[0237] Example 5: Cerebral cortical intermediate neurons derived from iNSCs can have the ability to secrete GABA neurotransmitter For the Matrigel two-dimensional migration analysis, the iNSC-derived cerebral cortex intermediate neuron precursor cell population of Example 1 was continuously differentiated into intermediate neuron organoids, and at day 21 of the differentiation, 10 organoids were aspirated using a 1 mL gun tip, placed in a 1.5 mL EP tube, and washed once with PBS to remove the culture medium. 200 μL of high potassium HBSS medium (60 mM KCL, 82 mM NaCl) containing γ-aminobutyric acid transaminase inhibitor Vigabatrin (3 mM) and Tiagabine (5 μM) was added to the EP tube, and incubated at 37°C for 45 minutes. After the incubation, the organoids were disrupted using ultrasonication, 180 μL of the homogenate was added to 20 μL of 1N perchloric acid (Merck), and stored at -80°C in the dark until analysis, or directly subjected to HPLC analysis. The results are shown in FIG. 8. Figure 11 and Figure 12 ​As shown, the iNSC-derived cerebral cortex intermediate neuron organoids at day 60 of in vitro induction had the ability to secrete GABA neurotransmitters (5.707 μg / mL). Table 1 shows the specific results of neurotransmitter content detection.

[0238] Table 1 Neurotransmitter content detection

[0239] Example 6: Cerebral cortical intermediate neuron precursor cells can continue to survive and differentiate in vivo The iNSC-derived cerebral cortex intermediate neuron precursor cell population prepared in Example 1 was transplanted into a C-NKG immunodeficient mouse (purchased from China Sanyei Biotechnology) model to detect cell survival and differentiation. The specific steps include: The green fluorescent protein (GFP) labeled iNSC cell line was subjected to in vitro directional differentiation into MGE type GABAergic intermediate neurons according to the method of Example 1, and cells at 10, 14, and 18 days of differentiation were obtained. Cell transplantation surgery was performed in the hippocampus of 10 mice on one side. The cell concentration was 1E5 / µL, and the stereotactic injection site was AP (Y axis) 3.0 mm, ML (X value) 2.7 mm, and D (Z axis) 3.7 mm. After the transplantation surgery, the mice were normally fed. After 30 days of transplantation, the mice were perfused, and the mouse brain slices were obtained. The hippocampus of the mouse was taken and subjected to brain sectioning and immunofluorescence staining (referring to the staining method of Example 1).

[0240] The results showed that the cells at 10 days of differentiation had almost no survival after transplantation, and the cells at 14 and 18 days of differentiation survived well after transplantation. As shown in Figure 13 The results of detection after transplantation of cells at 14 days of differentiation on the left and 18 days of differentiation on the right showed that the transplanted cells survived and differentiated in vivo. Red labeling showed that the cells expressed FoxG1, NKX2.1, and GABA; and there was co-expression of STEM121 antibody (Takara, Y40410) and NKX2.1, FoxG1, GABA.

[0241] This indicates that the iNSC-derived cerebral cortex intermediate neuron precursor cell population obtained by in vitro differentiation can be further differentiated into ventral forebrain neurons positive for NKX2.1, FOXG1, and GABA when transplanted into a C-NKG immunodeficient mouse model, which helps to improve epilepsy and has significance for damage repair.

[0242] Example 7: Cerebral cortical intermediate neuron precursor cells can improve epilepsy and help restore memory learning ability Figure 14 Construction of epilepsy model: 18 C-NKG male severely immunodeficient mice (purchased from China Saiye Biological) aged 6 to 8 weeks and weighing 23-24 g were selected and divided into a control group, a high-dose treatment group (1E6 cells per mouse), and a low-dose treatment group (4E5 cells per mouse), with 6 mice in each group. Modeling surgery was performed by unilateral dorsal hippocampal injection of Kainic acid (KA), and a unilateral chronic temporal lobe epilepsy (MTLE) mouse model was constructed by electroencephalogram detection, behavioral assessment, and brain slice immunohistochemical staining evaluation. Cell transplantation surgery was performed on the modeling side of the mice 5 weeks after the KA modeling surgery. The iNSC-derived differentiated day 14-16 cerebral cortex intermediate neuron precursor cell population prepared in Example 1 was transplanted into the C-NKG immunodeficient mouse MTLE model, and the therapeutic effect on epilepsy and comorbidities was detected. After the transplantation surgery, the mice were normally fed. Six weeks after the cell transplantation surgery, the electroencephalogram and Barnes maze, object location recognition of the mice were collected. Electroencephalogram electrode implantation was performed 1 week before electroencephalogram detection. The mice were connected to a video electroencephalogram system, and the behavior and electroencephalogram activity occurring simultaneously in the free behavior mice were continuously monitored. Electroencephalogram recording lasted for one day, and the frequency of seizures and the total time of seizure activity were measured.

[0243] The Barnes maze was used to assess visual spatial learning and memory in mice. On day 1 of testing, animals were familiarized with the maze and escape chamber. Animals were returned to their home cage after a 1-minute stay in the escape chamber. Acquisition trials were conducted on days 1-3, with each animal having up to 3 minutes to find the hidden chamber, and if not found, the animal was gently guided to the hidden chamber. The location of the escape chamber remained constant during acquisition trials. The maze and hidden chamber were cleaned between each animal using Nolvasan® solution. A probe trial was conducted on day 5. In this trial, the escape chamber was removed, and each animal had up to 3 minutes to explore the maze. The latency to find the escape chamber was assessed.

[0244] Object location test (OLT) This test was used to assess the cognitive ability of rats to detect subtle changes in the environment. In trial 1, the animals were allowed to explore the open field for 5 minutes and then returned to their cage. In trial 2, the animals were again placed in the center of the open field, and two similar objects were placed on either side of the open field. The animals were allowed to freely explore the objects for 5 minutes and then returned to their cage. In trial 3, the animals were again placed in the center of the open field, and the left object was placed in its original position, while the right object was moved to the other corner. The animals were allowed to explore for 5 minutes, and the time spent exploring the new and old position objects was recorded.

[0245] The electroencephalogram results showed that there was no difference in the electroencephalogram results of the high-dose and low-dose groups. The electroencephalogram and behavioral statistics of the drug administration group and the control group of mice are as follows: Example 8: Cerebral cortical intermediate neuron precursor cells can improve epilepsy and have the significance of injury repairAs shown, the transplanted cells not only reduced the frequency and total duration of seizures in transplanted MTLE mice, but also improved the impairment of learning and memory ability caused by epilepsy.

[0246] Figure 15 Construction of epilepsy model: 18 C-NKG male severely immunodeficient mice (purchased from China Saiye Biological) aged 6 to 8 weeks and weighing 23-24 g were selected and divided into a control group, a high-dose treatment group (1E6 cells per mouse), and a low-dose treatment group (4E5 cells per mouse), with 6 mice in each group. The modeling operation was performed by unilateral dorsal hippocampal injection of Kainic acid (KA), and the mouse model of unilateral chronic temporal lobe epilepsy (MTLE) was constructed by electroencephalogram detection, behavioral assessment, and brain slice immunohistochemical staining evaluation. The modeling side cell transplantation operation was started at 5 weeks after the KA modeling operation. The iNSC-derived differentiated 14-16 day old cerebral cortex intermediate neuron precursor cell population prepared in Example 1 was transplanted into the C-NKG immunodeficient mouse MTLE model. After the transplantation operation, the mice were normally fed. At 12 weeks after the cell transplantation operation, the mice were perfused, the mouse brain slices were obtained, and the mouse hippocampal tissue was taken for brain sectioning and immunofluorescence staining to detect cell survival and differentiation. The specific steps include: (1) Rewarming: rewarming the frozen section at 30°C - 37°C; (2) Membrane breaking: using 0.3% Triton X-100 at room temperature (25°C ± 5°C) for 15 min-20 min, and washing the section with PBS once; (3) Blocking: using PBST blocking solution containing 3% donkey serum, blocking at room temperature for 2 h; (4) Primary antibody incubation: configuring PBST diluent containing 1% donkey serum by mass ratio, adding the primary antibody, and washing with PBS solution 3 times after incubation at 4°C overnight; (5) Secondary antibody incubation: configuring PBST diluent containing 1% donkey serum by mass ratio, adding the secondary antibody, and incubating at room temperature for 2 hours in the dark, and washing the section with PBS 3 times; (6) DAPI staining: using DAPI for fluorescent staining of cell nuclei, and washing with PBS 3 times after staining; (7) Mounting: adding mounting medium for mounting.

[0247] (8) Image fluorescence data statistics: using image J to statistically analyze the positive rate of fluorescent cells.

[0248] The primary antibodies used are: STEM121 antibody (Takara, Y40410), anti-GAD1 antibody (Sigma, G4913), anti-LHX6 antibody (Santa Cruz, sc-271433), anti-GABA antibody (Sigma, A2052), anti-SST antibody (Proteintech, 17512-1-AP), and Parvalbumin polyclonal antibody (Abeam, ab11427).

[0249] The secondary antibodies used are: donkey anti-mouse Cy5 secondary antibody (Jackson) and donkey anti-rabbit Cy3 secondary antibody (Jackson).

[0250] Figure 16 The fluorescence staining results are shown, and the markers of the staining figures are listed on the left side of each row. The second staining figure in each row is an enlarged view of the first staining figure in the box, and the fifth staining figure is a combined staining figure. The fluorescence staining results show that GAD1, GABA, and SST present positive results with positive signals, i.e., the transplanted cells survive and differentiate in vivo. The quantitative results of fluorescence staining identification are shown in Table 1. Example 9: Cerebral cortical intermediate neuron precursor cells have in vivo electrophysiological activity and form synaptic connections with host cells As shown in Table 1, the differentiation ratio of cells expressing GAD1 is 23%, the differentiation ratio of cells expressing GABA is 48%, and the differentiation ratio of cells expressing SST is 22%.

[0251] This indicates that the population of iNSC-derived cerebral cortex intermediate neuron precursor cells differentiated in vitro can be further differentiated into SST-positive and PV-positive inhibitory intermediate neurons after being transplanted into a C-NKG immunodeficient mouse model, which helps to improve epilepsy and has the significance of damage repair.

[0252] Figure 17 ​ Construction of epilepsy model: 18 C-NKG male severely immunodeficient mice (purchased from China Saiye Biological) aged 6 to 8 weeks and weighing 23-24 g were selected and divided into a control group, a high-dose treatment group (1E6 cells per mouse), and a low-dose treatment group (4E5 cells per mouse), with 6 mice in each group. Modeling surgery was performed by unilateral dorsal hippocampal injection of Kainic acid (KA), and a unilateral chronic temporal lobe epilepsy (MTLE) mouse model was constructed by electroencephalogram detection, behavioral assessment, and brain slice immunohistochemical staining evaluation. Cell transplantation surgery was performed on the 5th week after KA modeling surgery. The iNSC-derived differentiated 14-16 day-old cerebral cortex intermediate neuron precursor cell population prepared in Example 1 was transplanted into the C-NKG immunodeficient mouse MTLE model. After the transplantation surgery, the mice were normally fed. At 12 weeks after the cell transplantation surgery, the mice were perfused, brain slices were obtained, and the hippocampal tissue of the mice was taken for brain sectioning and immunofluorescence staining (referring to the staining method in Example 1) to detect cell survival and differentiation. The specific steps include: Brain slice preparation was completed within 10 minutes after decapitation, and the cut brain slices were incubated in artificial cerebrospinal fluid containing 95% oxygen concentration mixed gas at 32-33°C for half an hour, and then placed at room temperature for an hour before use. The recording instrument was an electrophysiological patch clamp, and the brain slices were placed in the brain slice recording tank, with the temperature controlled at 32°C. BIC was added to block the transmission of inhibitory synapses of neurons, potassium gluconate electrode internal solution was used, and the whole cell brain slice patch clamp recording method was used to clamp the voltage at -70 mV to record the changes in neuronal electrical activity. Data analysis and processing were performed using pClamp software. The measurement results are shown in ​ .

[0253] Baseline measurements showed that the transplanted cells had similar average resting membrane potential (RMP) and lower average membrane capacitance (Cm) compared to host pyramidal cells, indicating that the transplanted cells were not yet fully mature. In addition, the transplanted cells showed rapid inward and sustained outward currents when voltage pulses were applied, indicating the presence of voltage-gated Na+ and K+ channels. Importantly, 80% of human cerebral cortex intermediate neurons showed spontaneous excitatory postsynaptic currents (sEPSCs) when the voltage was maintained at -70 mV. Therefore, the transplanted population of human cerebral cortex intermediate neuron precursor cells (GABAPs) had synaptic connections with host neurons. These results indicate that the transplanted human cerebral cortex intermediate neurons can persist in the hippocampus of MTLE mice for a long time, receive synaptic signals from host neurons, and functionally integrate into neural circuits.

Claims

1. A population of cerebral cortical intermediate neuron precursor cells in vitro, characterized in that, The cell population expresses biomarkers including one or more of NKX2.1, FoxG1, GABA, and SST, wherein the number of cells expressing NKX2.1 accounts for more than 40% of the cell population, the number of cells expressing GABA accounts for more than 20% of the cell population, and the number of cells expressing SST accounts for more than 30% of the cell population; and the cerebral cortex intermediate neuron precursor cell population is derived from induced neural stem cells (iNSC). Optionally, in the ex vivo cerebral cortex intermediate neuron precursor cell population, the number of cells expressing FoxG1 accounts for more than 35% of the cell population.

2. The population of cells of claim 1, wherein the cells are derived from a human. In the cell population, the number of cells expressing NKX2.1 accounts for 50%-100%, optionally 60%-100% of the cell population. Alternatively, The number of cells expressing FoxG1 accounts for 50%-100%, optionally 60%-100% of the cell population. Alternatively, The number of cells expressing GABA accounts for 40%-100%, optionally 45%-100% of the cell population. Alternatively, The number of cells expressing SST accounts for 40%-100%, optionally 60%-100% of the cell population. The number of cells expressing the marker PV accounts for less than 40%, optionally 0%-30% of the cell population. Preferably, the cell population contains at least two of the cells expressing NKX2.1, FoxG1, GABA, and SST. Further preferably, the cell population contains cells expressing two or more of the markers NKX2.1, FoxG1, GABA, and SST.

3. The method for preparing a population of cerebral cortical intermediate neuron precursor cells ex vivo according to claim 1 or 2, characterized in that, The preparation method comprises: culturing iNSC under effective conditions to transform the iNSC into a medial ganglionic eminence (MGE)-like progenitor cell population; and inducing the MGE-like progenitor cell to form a cerebral cortex intermediate neuron precursor cell population.

4. The production method according to claim 3, characterized by, The method comprises: using a combination of neural stem cell medium and differentiation medium to prepare the ex vivo cerebral cortex intermediate neuron precursor cell population. The method comprises steps (B), (C), and (D), and optionally step (A), wherein, (A) culturing iNSC using neural stem cell medium; optionally, the neural stem cell medium comprises basal medium, amino acid component, serum replacement supplement, human leukemia inhibitory factor at a concentration of 1 ng / mL-20 ng / mL, WNT activator at a concentration of 1 μM (μmol / L)-10 μM (μmol / L), and / or TGF-β receptor kinase inhibitor at a concentration of 1 μM-10 μM. (B) differentiating the iNSCs in a medial ganglionic eminence (MGE) differentiation medium to obtain a MGE-like progenitor cell population; optionally, the MGE differentiation medium comprises a basal medium, an amino acid component, a serum replacement supplement, a TGF-beta receptor kinase inhibitor at a concentration of 1 µM-20 µM, a WNT inhibitor at a concentration of 1 µM-20 µM, a BMP type I receptor inhibitor at a concentration of 50 nM (nmol / L)-500 nM (nmol / L), and / or a SHH pathway activator at a concentration of 50 nM-1000 nM; (C) differentiating the MGE-like progenitor cell population in a GABA differentiation medium; optionally, the GABA differentiation medium comprises a basal medium, an amino acid component, a serum replacement supplement, a SHH pathway activator at a concentration of 50 nM-1000 nM, and / or a MEK inhibitor at a concentration of 1 µM-10 µM; (D) continuing to differentiate the MGE-like progenitor cell population in a terminal differentiation medium to obtain a population of iNSC-derived cerebral cortical intermediate neuron precursor cells; optionally, the terminal differentiation medium comprises a basal medium, an amino acid component, a serum replacement supplement, a neurotrophic factor, a MEK inhibitor at a concentration of 1 µM-10 µM, a CDK4 / 6 inhibitor at a concentration of 1 µM-10 µM, and / or a gamma secretase inhibitor at a concentration of 0.5 µM-20 µM.

5. The preparation method of claim 4, wherein the WNT activator comprises one or more of CHIR99021, SB-216763, IM-12, TWS119, KY19382, and DIF-3; optionally, the WNT activator comprises CHIR99021; the TGF-beta receptor kinase inhibitor, each occurrence independently, comprises one or more of SB431542, LY2157299, RepSox, A 83-01, and Galunisertib; optionally, in steps (A) and (B), the TGF-beta receptor kinase inhibitor, each occurrence independently, comprises SB431542; the WNT inhibitor comprises one or more of XAV939, Dickkopf 1, IWR-1, JW 55, K-756, MN-64, and WIKI4; optionally, the WNT inhibitor comprises XAV939; the BMP type I receptor inhibitor comprises one or more of LDN193189, Noggin, BMPR1A-Fc, K02288, and Dorsomorphin; optionally, the BMP type I receptor inhibitor comprises LDN193189; and the SHH pathway activator, each occurrence independently, comprises one or more of SAG1, SHH, and Purmorphamine; optionally, in steps (B) and (C), the SHH pathway activator, each occurrence independently, comprises SAG1. ​ ​ ​ ​ ​ each occurrence of the MEK inhibitor independently includes one or more of PD0325901, MEK162, RO4987655, AZD8330, AZD6244, BAY 869766, AZD6244, SL327, U0126, GSK1120212, Cobimetinib hemifumarate, TCS PIM-11, CI-1040, GDC-0973, Trametinib, Pimasertib, GDC-0623, PD98059, U0126-EtOH, TAK-733, and BIX02189; alternatively, in step (C) and step (D), the MEK inhibitor independently includes PD0325901; the CDK4 / 6 inhibitor includes one or more of PD0332991, Lerociclib dihydrochloride, Abemaciclib, Lerociclib, Abemaciclib methanesulfonate, AT7519 Hydrochloride, AT7519, and Narazaciclib; alternatively, the CDK4 / 6 inhibitor includes PD0332991; the gamma secretase inhibitor includes one or more of DAPT, Compound E, Z-IL-CHO, Crenigacestat, L-685458, BMS-906024, Nirogacestat, LY-411575, Semagacestat, Avagacestat, and RO4929097; alternatively, the gamma secretase inhibitor includes DAPT; and / or, the neurotrophic factor includes one or more of cAMP, Forskolin, Rolipram, BDNF, NGF, IGF-I, and GDNF; alternatively, the neurotrophic factor includes cAMP, BDNF, and GDNF.

6. The method of claim 4 or 5, wherein the step (C) and step (D) are performed simultaneously. The neural stem cell culture medium comprises recombinant human leukemia inhibitory factor at a concentration of 1 ng / mL-20 ng / mL, CHIR99021 at a concentration of 1 μM-10 μM, and SB431542 at a concentration of 1 μM-10 μM; optionally, the neural stem cell culture medium comprises recombinant human leukemia inhibitory factor at a concentration of 5 ng / mL-15 ng / mL, CHIR99021 at a concentration of 1 μM-5 μM, and SB431542 at a concentration of 1 μM-5 μM; further optionally, the neural stem cell culture medium comprises recombinant human leukemia inhibitory factor at a concentration of 10 ng / mL, CHIR99021 at a concentration of 3 μM, and SB431542 at a concentration of 2 μM; or, The MGE differentiation medium comprises SB431542 at a concentration of 1 μM-20 μM, XAV939 at a concentration of 1 μM-20 μM, LDN193189 at a concentration of 50 nM-500 nM, and SAG1 at a concentration of 50 nM-1000 nM; optionally, the MGE differentiation medium comprises SB431542 at a concentration of 5 μM-15 μM, XAV939 at a concentration of 5 μM-15 μM, LDN193189 at a concentration of 150 nM-350 nM, and SAG1 at a concentration of 100 nM-1000 nM; further optionally, the MGE differentiation medium comprises SB431542 at a concentration of 10 μM, XAV939 at a concentration of 10 μM, LDN193189 at a concentration of 250 nM, and SAG1 at a concentration of 100 nM-1000 nM; or, The GABA differentiation medium comprises SAG1 at a concentration of 50 nM-1000 nM and PD0325901 at a concentration of 1 μM-10 μM; optionally, the GABA differentiation medium comprises SAG1 at a concentration of 100 nM-1000 nM and PD0325901 at a concentration of 2 μM; or, The terminal differentiation medium comprises PD0325901 at a concentration of 1 μM-10 μM, PD0332991 at a concentration of 1 μM-10 μM, DAPT at a concentration of 0.5 μM-20 μM, cAMP at a concentration of 0.1 μM-100 μM, BDNF at a concentration of 1 ng / ml-50 ng / ml, and GDNF at a concentration of 1 ng / ml-50 ng / ml; optionally, the terminal differentiation medium comprises PD0325901 at a concentration of 1 μM-5 μM, PD0332991 at a concentration of 1 μM-5 μM, DAPT at a concentration of 5 μM-15 μM, cAMP at a concentration of 0.1 μM-3 μM, BDNF at a concentration of 5 ng / ml-15 ng / ml, and GDNF at a concentration of 5 ng / ml-15 ng / ml; further optionally, the terminal differentiation medium comprises PD0325901 at a concentration of 2 μM, PD0332991 at a concentration of 2 μM, DAPT at a concentration of 10 μM, cAMP at a concentration of 1 μM, BDNF at a concentration of 10 ng / ml, and GDNF at a concentration of 10 ng / ml.

7. The preparation method of any one of claims 3-6, wherein, the step (A) comprises culturing the neural stem cells under induction for 1-3 days, preferably 1-2 days, using the neural stem cell medium; or, the step (B) comprises culturing for 5-9 days, preferably 6-8 days, using the MGE differentiation medium; optionally, the MGE differentiation medium is replaced with fresh MGE differentiation medium at half the volume every other day; or, the step (C) comprises at least one of the following conditions: (1) culturing the MGE-like progenitor cells in the GABA differentiation medium with shaking; (2) culturing with fresh GABA differentiation medium at half the volume every other day; (3) culturing for 5-9 days, preferably 6-8 days; or, the step (D) comprises culturing for 5-9 days, preferably 6-8 days, using the terminal differentiation medium; optionally, the terminal differentiation medium is replaced with fresh terminal differentiation medium at half the volume every other day.

8. A pharmaceutical composition comprising the population of ex vivo cerebral cortex intermediate neuron precursor cells of any one of claims 1-2 or prepared by the method of any one of claims 3-7, and at least one pharmaceutically acceptable excipient.

9. A kit comprising the population of ex vivo cerebral cortex intermediate neuron precursor cells of any one of claims 1-2 or prepared by the method of any one of claims 3-7.

10. Use of the population of ex vivo cerebral cortex intermediate neuron precursor cells of any one of claims 1-2, prepared by the method of any one of claims 3-7, the pharmaceutical composition of claim 8, or the kit of claim 9, in the preparation of a medicament for the prevention, diagnosis, and treatment of nervous system diseases. Optionally, in the nervous system disease, the expression level of GABA is abnormal.

11. Use according to claim 10, characterized in that, The nervous system disease comprises one or more of the following diseases: stroke, spinal cord injury, apoplexy, Huntington's disease, amyotrophic lateral sclerosis, motor neuron injury, traumatic peripheral nerve injury, ischemic brain injury, neonatal hypoxic-ischemic brain injury, cerebral palsy, peripheral paralysis, central paralysis, quadriplegia, diplegia, epilepsy, neuronal development disorder, neuralgia, intractable epilepsy, Alzheimer's disease, congenital metabolic nervous system disease, traumatic brain injury, disease caused by motor neuron injury, autoimmune encephalitis, and mental illness; Optionally, the nervous system disease comprises epilepsy, stroke, Alzheimer's disease, traumatic brain injury, autoimmune encephalitis, and mental illness, and the mental illness comprises depression.

12. Use according to claim 10 or 11, characterized in that, The use comprises reducing the frequency, total duration of seizures, and / or improving the impairment of learning and memory ability caused by epilepsy.

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