Method for Differentiation of Neural Progenitor Cells from the Medial Ganglionic Eminence

Abstract

This application relates to a method for proliferating and differentiating pluripotent stem cells into medial ganglionic eminence (MGE) neural progenitor cells, comprising the following steps: (a) culturing the cells in a medium containing SAG for approximately 10 days; and (b) culturing the cells in a medium containing PD0325901 and DAPT for approximately 7 days. This application also provides a culture medium used in this method, a composition containing the culture medium, and applications thereof.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for differentiating neural precursor cells of the medial ganglionic eminence (MGE). Background Art

[0002] Normal brain function requires maintaining a balance between excitatory and inhibitory levels of neurotransmitters. Gamma-aminobutyric acid (GABA) interneurons are the primary inhibitory neurons of the central nervous system. They are believed to play a key role in shaping the dynamics of inhibitory networks in the cerebral cortex. Their dysfunction can contribute to numerous neurological disorders, such as epilepsy, major depression, anxiety, and autism.

[0003] The medial ganglionic eminence (MGE) is located on the lateral wall of the ventral ventricle of the brain and is part of the ganglionic eminence region. The MGE is an embryonic forebrain structure that is the primary source of inhibitory GABAergic interneurons. Transplantation of MGE precursor cells in the damaged brain can provide new GABA neurotransmitters and may participate in the reconstruction of neural circuits. Exogenous MGE precursor cell transplantation is considered a potential therapeutic strategy for treating neurological diseases related to GABAergic interneurons. Although various methods for differentiating MGE precursor cells from human pluripotent stem cells have been reported, obtaining a high proportion of MGE precursor cells remains challenging. Therefore, there is an urgent need to establish a time-saving, economical, and efficient non-xenogeneic differentiation system to generate MGE precursor cells in vitro. Summary of the Invention

[0004] The present application provides a method for generating MGE precursor cells in vitro, wherein the MGE precursor cells can differentiate into functional GABAergic interneurons, providing a potential treatment method for transplantation treatment of GABAergic interneuron-related neurological diseases. The MGE precursor cell differentiation method provided in the present application has one or more of the following advantages: short culture time, few additives used, low concentration, and the ability to obtain a large number of high-purity MGE precursor cells without genetic modification or stable and reproducible. On the other hand, after transplantation, MGE-derived GABAergic interneuron precursor cells can significantly reduce the number of epileptic seizures in epilepsy model mice, alleviate epileptic-like discharges in the brain organoid model of Angelman syndrome, and restore normal phenotypes, which has broad application prospects in the biopharmaceutical industry.

[0005] In one aspect, the present application provides a method for proliferating / differentiating pluripotent stem cells into medial ganglionic eminence (MGE) neural progenitor cells, comprising the following steps:

[0006] (a) culturing cells in a medium comprising SAG for about 10 days; and

[0007] (b) Cells were cultured in medium containing PD0325901 and DAPT for approximately 7 days.

[0008] The MGE neural precursor cells may be NKX2.1+ and LHX6+ double-positive cells.

[0009] In certain embodiments, the concentration of SAG is about 0.1-2 μM. In certain embodiments, the concentration of SAG is about 0.1-0.5 μM. In certain embodiments, the concentration of SAG is about 0.2 μM.

[0010] In certain embodiments, the concentration of PD0325901 is about 0.1-2 μM. In certain embodiments, the concentration of PD0325901 is about 0.5-2 μM. In certain embodiments, the concentration of PD0325901 is about 0.5 μM.

[0011] In certain embodiments, the concentration of DAPT is about 2.5-20 μM. In certain embodiments, the concentration of DAPT is about 5-20 μM. In certain embodiments, the concentration of DAPT is about 10 μM.

[0012] In certain embodiments, the method comprises the following steps:

[0013] (1) Cultivate cells in culture medium for approximately 9 days;

[0014] (2) culturing the cells in a medium containing SAG for about 10 days; and

[0015] (3) Cultivate in a medium containing PD0325901 and DAPT for approximately 7 days.

[0016] In certain embodiments, culturing the cells in a culture medium for about 9 days comprises culturing the cells in a culture medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for about 6 days, and culturing the cells in a culture medium containing Rock inhibitor for about 3 days.

[0017] In certain embodiments, the method comprises the following steps:

[0018] (1) Culture cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

[0019] (2) Cultivate cells in a medium containing Rock inhibitor for approximately 3 days;

[0020] (3) culturing the cells in a medium containing SAG for about 10 days; and

[0021] (4) Cultivate in a medium containing PD0325901 and DAPT for approximately 7 days.

[0022] In certain embodiments, the concentration of SB431542 is about 2-10 μM, the concentration of DMH-1 is about 2-10 μM, the concentration of IWR-1 is about 2-5 μM, and the concentration of Rock inhibitor is about 0.1-1 μM. In certain embodiments, the concentration of SB431542 is about 2 μM, the concentration of DMH-1 is about 2 μM, the concentration of IWR-1 is about 2.5 μM, and the concentration of Rock inhibitor is about 0.5 μM.

[0023] In certain embodiments, the culture medium comprises a basal medium.

[0024] In certain embodiments, the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, MEM.

[0025] In certain embodiments, the basal culture medium may be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, and Glutamax.

[0026] In certain embodiments, the basal medium comprises about 50% DMEM / F12, about 50% Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 0.5% Glutamax, and about 1% B27 supplement.

[0027] In some embodiments, the pluripotent stem cells may be induced pluripotent stem cells. In some embodiments, the pluripotent stem cells may be embryonic stem cells.

[0028] In certain embodiments, the method comprises the following steps:

[0029] (1) Culture cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

[0030] (2) Culture cells in a medium containing Rock inhibitor for approximately 3 days

[0031] (3) culturing the cells in a medium containing SAG for approximately 10 days;

[0032] (4) culturing the cells in a medium comprising PD0325901 and DAPT for about 7 days; and

[0033] (5) The cells were cultured in a medium containing GDNF, AA, BDNF, cAMP, IGF1, and Compound E for approximately 14 days to obtain GABAergic interneurons.

[0034] In certain embodiments, the concentration of GDNF is about 10-40 ng / ml, the concentration of AA is about 100-200 μM, the concentration of BDNF is about 10-40 ng / ml, the concentration of cAMP is about 0.2-1 μM, the concentration of IGF1 is about 10-40 ng / ml, and the concentration of Compound E is about 0.1-1 μM. In certain embodiments, the concentration of GDNF is about 20 ng / ml, the concentration of AA is about 200 μM, the concentration of BDNF is about 20 ng / ml, the concentration of cAMP is about 0.5 μM, the concentration of IGF1 is about 20 ng / ml, and the concentration of Compound E is about 0.1 μM.

[0035] In certain embodiments, the culture medium in step (5) comprises a basal culture medium, and the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, MEM.

[0036] In certain embodiments, the basal culture medium in step (5) may be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0037] In certain embodiments, the basal culture medium in step (5) comprises Neurobasal, about 1% MEMNEAA, about 1% N2 supplement, about 1% Glutamax, and about 1% B27 supplement.

[0038] In certain embodiments, the cells are cultured in suspension.

[0039] On the other hand, the present application provides a combined culture medium comprising:

[0040] (i) a first culture medium comprising SAG; and

[0041] (ii) A second culture medium comprising PD0325901 and DAPT.

[0042] In certain embodiments, the combination culture medium, wherein the concentration of SAG is about 0.1-2 μM. In certain embodiments, the combination culture medium, wherein the concentration of SAG is about 0.1-0.5 μM. In certain embodiments, the combination culture medium, wherein the concentration of SAG is about 0.2 μM.

[0043] In certain embodiments, the combination culture medium, wherein the concentration of PD0325901 is about 0.1-2 μM. In certain embodiments, the combination culture medium, wherein the concentration of PD0325901 is about 0.5-2 μM. In certain embodiments, the combination culture medium, wherein the concentration of PD0325901 is about 0.5 μM.

[0044] In certain embodiments, the combination culture medium wherein the concentration of DAPT is about 2.5-20 μM. In certain embodiments, the combination culture medium wherein the concentration of DAPT is about 5-20 μM. In certain embodiments, the combination culture medium wherein the concentration of DAPT is about 10 μM.

[0045] In certain embodiments, the combined culture medium, wherein the first culture medium and the second culture medium comprise a basal culture medium, and the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, MEM.

[0046] In certain embodiments, the combined culture medium, wherein the basal culture medium of the first culture medium and the second culture medium may be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0047] In certain embodiments, the combined culture medium, wherein the basal medium of the first culture medium and the second culture medium comprises about 50% DMEM / F12, about 50% Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 0.5% Glutamax, and about 1% B27 supplement.

[0048] In certain embodiments, the combined culture medium comprises a third culture medium comprising GDNF, AA, BDNF, cAMP, IGF1, and Compound E.

[0049] In certain embodiments, in the combined culture medium, the concentration of GDNF is about 10-40 ng / ml, the concentration of AA is about 100-200 μM, the concentration of BDNF is about 10-40 ng / ml, the concentration of cAMP is about 0.2-1 μM, the concentration of IGF1 is about 10-40 g / ml, and the concentration of Compound E is about 0.1-1 μM. In certain embodiments, in the combined culture medium, the concentration of GDNF is about 20 ng / ml, the concentration of AA is about 200 μM, the concentration of BDNF is about 20 ng / ml, the concentration of cAMP is about 0.5 μM, the concentration of IGF1 is about 20 ng / ml, and the concentration of Compound E is about 0.1 μM.

[0050] In certain embodiments, in the combined culture medium, the third culture medium comprises a basal culture medium, and the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, and MEM.

[0051] In certain embodiments, in the combined culture medium, the basal culture medium of the third culture medium may be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0052] In certain embodiments, in the combination culture medium, the basal medium of the third culture medium comprises Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 1% Glutamax, and about 1% B27 supplement.

[0053] On the other hand, the present application also provides a composition comprising cells and the culture medium, wherein the culture medium comprises SAG, DMEM / F12, Neurobasal, MEM NEAA, N2 supplement, Glutamax and B27 supplement.

[0054] In certain embodiments, in the composition, the cells are selected from one or more of the following groups: pluripotent stem cells, MGE neural precursor cells, or GABAergic interneurons.

[0055] In certain embodiments, the pluripotent stem cells in the composition may be induced pluripotent stem cells. In certain embodiments, the pluripotent stem cells in the composition may be embryonic stem cells.

[0056] In certain embodiments, the MGE neural precursor cells in the composition are NKX2.1+ and LHX6+ double positive cells. In certain embodiments, the GABAergic interneurons in the composition are LHX6+ positive cells.

[0057] On the other hand, the present application also provides a composition comprising cells and the culture medium, wherein the culture medium comprises PD0325901, DAPT, DMEM / F12, Neurobasal, MEM NEAA, N2 supplement, Glutamax and B27 supplement.

[0058] In certain embodiments, in the composition, the cells are selected from one or more of the following groups: pluripotent stem cells, MGE neural precursor cells, or GABAergic interneurons.

[0059] In certain embodiments, the pluripotent stem cells in the composition may be induced pluripotent stem cells. In certain embodiments, the pluripotent stem cells in the composition may be embryonic stem cells.

[0060] In certain embodiments, the MGE neural precursor cells in the composition are NKX2.1+ and LHX6+ double positive cells. In certain embodiments, the GABAergic interneurons in the composition are LHX6+ positive cells.

[0061] On the other hand, the present application also provides the use of the method, culture medium and / or composition described in the present application in preparing a medicament for preventing and / or treating a disease.

[0062] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. The detailed description below only illustrates and describes exemplary embodiments of the present application. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0064] Figure 1 Shown is a schematic diagram of the NKX2.1-mNeongreen / LHX6-tdTomato human pluripotent stem cell line described in this application ( Figure 1 A) and genotype identification diagram ( Figure 1 B).

[0065] Figure 2 The results shown are the flow cytometry and immunofluorescence staining results of MGE neural progenitor cells and GABAergic interneurons differentiated from the pluripotent stem cells described in this application. Figure 2 A shows a schematic diagram of the cell differentiation process described in this application. Figure 2 B-2C shows a typical flow cytometry result and related statistical graphs of the fluorescent cell ratio detected by flow cytometry at D28 as described in this application. Figure 2 D-2E shows typical images and related statistical graphs of immunofluorescence staining of cells fixed at D42 after mature culture as described in the present application.

[0066] Figure 3 Shown is the effect of adding different concentrations of SAG on the generation of MGE neural precursor cells and GABAergic interneurons as described in this application. Figure 3 A shows a schematic diagram of the SAG treatment conditions at different concentrations described in this application. Figure 3 B-3E shows the percentage of fluorescent cells detected by flow cytometry at D28 as described in this application ( Figure 3 B) and its statistical graph ( Figure 3 C-3E). Figure 3 F-3I shows a typical image (3F) of the immunofluorescence staining of GABA and LHX6-tdTomato positive cells and a statistical graph of the immunofluorescence staining results ( Figure 3 G-3I).

[0067] Figure 4 The results show the effects of adding SAG at different times on the generation of MGE neural precursor cells and GABAergic interneurons as described in this application. Figure 4 A shows a schematic diagram of the treatment of adding the SHH signaling pathway agonist SAG at D1, 4, 7, 10, and 13 as described in the present application. Figure 4 B-4E shows the percentage of fluorescent cells detected by flow cytometry at D28 as described in this application ( Figure 4 B) and its statistical graph ( Figure 4 C-4E). Figure 4 F-4I shows a typical image of GABA and LHX6-tdTomato positive cells characterized by immunofluorescence staining as described in this application ( Figure 4 F) and statistical graphs of immunofluorescence staining results ( Figure 4 G-4I).

[0068] Figure 5The results shown are the flow cytometry and immunofluorescence staining results of the cell flow cytometry assay and immunofluorescence staining assay for the differentiation of MGE neural progenitor cells and GABAergic interneurons induced by CB003 human pluripotent stem cells described in this application. Figure 5 A-5B shows a typical flow cytometry result and related statistical graphs of the fluorescent cell ratio detected by flow cytometry at D28 as described in this application. Figure 5 C-5F shows typical images of immunofluorescence staining of differentiated cells after mature culture at D42 as described in this application ( Figure 5 C, 5D) and related statistical graphs ( Figure 5 E, 5F).

[0069] Figure 6 It is shown that the mature GABAergic interneurons differentiated in vitro according to the present application can release the neurotransmitter GABA, and can release more GABA after being activated by KCL neurons, but does not release glutamate. DETAILED DESCRIPTION

[0070] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0071] Definition of terms

[0072] In this application, the term "comprising" generally refers to "containing" and "consisting of", for example, a composition "comprising" X may be completely composed of X, or may contain substances other than X, such as X+Y.

[0073] As used herein, the terms "culture medium" and "cell culture medium" generally refer to a nutrient source used to grow, maintain, or differentiate cells. As will be understood by those skilled in the art, a nutrient source can contain components required for cell growth, survival, and / or differentiation, or can contain components that aid in the growth, survival, and / or differentiation of cells. For example, the culture medium can be Minimum Essential Medium (MEM), Eagle's Medium, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F12), F10 Nutrient Mixture, Ham's F10 Nutrient Mixture, Ham's F12 Nutrient Mixture, Medium 199, RPMI, RPMI 1640, reduced serum culture medium, Basal Medium Emulsion (BME), Neurobasal medium, and the like, as well as combinations thereof. The culture medium or cell culture medium can be modified by adding one or more factors, such as supplements, differentiation factors, or anti-apoptotic agents.

[0074] In this application, the term "basal medium" generally refers to a medium that only meets the growth requirements of a culture. For example, the basal medium may be MEM, DMEM, DMEM / F12, Neurobasal medium, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, or Essential 6. Basal media can be supplemented with one or more factors to form a complete medium for cell growth, proliferation, or differentiation. These factors may include non-essential amino acids (NEAAs), N2 supplement, or B27 supplement.

[0075] In this application, the term "proliferation" generally refers to the generation of multiple individual cells by division of a starting cell. The multiple individual cells can be of the same type or of different types. The starting cells used for proliferation need not be the same as the cells produced by proliferation. For example, the proliferated cells can be generated from the growth and differentiation of a starting cell population.

[0076] As used herein, the terms "differentiation" and "differentiation process" generally refer to the process by which non-specific or less specific cells acquire specific cell characteristics. Differentiated or differentiation-induced cells are cells that occupy a more specific position in a cell lineage, typically exhibiting phenotypic characteristics of a specific cell lineage. For example, one or more steps in which pluripotent stem cells are converted into MGE neural precursor cells or GABAergic interneurons. In particular, by adding one or more differentiation agents to the cell culture medium, pluripotent stem cells can be induced to differentiate into MGE neural precursor cells or GABAergic interneurons.

[0077] In this application, the term "cell" generally refers to a living organism, a unit of tissue structure of a multicellular organism, surrounded by a membrane structure that separates it from the outside, and having genetic information and expression mechanisms. Further, a cell may have the ability to self-replicate. The term "cell" is used in its broadest sense in this article. A cell may be a naturally occurring cell or an artificially modified cell, such as a fused cell, a genetically modified cell, etc. A cell may be a single cell, a cell line, or a cell culture. A cell may be derived from any organism having one or more cells. A cell may include the offspring of a single cell.

[0078] In this application, the term "neural precursor cell" generally refers to a cell that is capable of migrating in vitro or in vivo and ultimately differentiating into an inhibitory interneuron. The precursor cell is preferably a migratory cell that has the ability to migrate from the transplant site to the desired treatment site. The neural precursor cell can be derived from the MGE, CGE (caudal ganglionic eminence), LGE (lateral ganglionic eminence) or another part of the mammalian brain. The neural precursor cell can be obtained by differentiation or reprogramming from other cell types. For example, the neural precursor cell can be an MGE neural precursor cell. For example, the inhibitory interneuron cell can be a GABAergic interneuron.

[0079] In this application, the terms "activator" and "agonist" generally refer to agents that stimulate or upregulate cell signaling pathways. Stimulation of cell signaling pathways can be initiated from outside the cell, for example, by using an agonist that activates a cell surface receptor involved in the signaling pathway. For example, an agonist can be a receptor ligand. Alternatively, stimulation of cell signaling pathway conduction can be initiated from within the cell, for example, by using a small molecule activator that interacts with components of the signaling pathway within the cell.

[0080] In this application, the terms "inhibitor" and "antagonist" generally refer to agents that inhibit or downregulate cell signaling pathways. Inhibition of cell signaling pathways can be initiated from outside the cell, for example, by using inhibitors that block cell surface receptors involved in the signaling pathway. Alternatively, inhibition of cell signaling pathway conduction can be initiated from within the cell, for example, by using small molecule inhibitors that interact with components of the signaling pathway within the cell.

[0081] In this application, the term "about" or "approximately" generally refers to a quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% compared to a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5% of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length.

[0082] In this application, the term "marker phenotype" generally refers to the identification of markers or antigens on cells to determine their phenotype (e.g., differentiation state and / or cell type). For example, immunophenotyping can be used, which uses antibodies to recognize antigens presented on cells. Antibodies can be monoclonal or polyclonal and are generally selected to have minimal cross-reactivity with other cell markers. These markers that determine the same cell type between species can be identified based on the same markers, which may differ in structure (e.g., amino acid sequence) between species. Cell markers can include cell differentiation markers, as well as gene expression markers. Gene expression markers can include expressed genes that can indicate cell type or differentiation state.

[0083] In this application, the term "positive" generally refers to the expression of a biomarker by the target cell, for example, while "negative" generally refers to the absence of a biomarker expression by the target cell. For example, the biomarker can be NKX2.1, LHX6, AS1, ATRNL1, CD200, CELSR3, CHRM4, CNTNAP4, CXCR4, CXCR7, DSCAML1, ELAVL2, ENSG00000260391, EPHA5, ERBB4, FAM5B, FAM65B, FNDC5, GAD1, GAD2, GNG2, GPD1, GRIA1, GRIA4, HMP19, INA, KALRN, KDM6B, KIF21B, L1CAM, LINC00340, LINC00599, MAF, MAFB, MAPT, MIAT, NCAM1, NMNAT2, NPAS1, NRCAM, NRXN3, NXPH1, PDZRN4, PIP5K1B, PLS3, PLXNA4, RAI2, ROBO1, ROBO2, RP11-384F7.2, RP4-7 91M13.3, RUNX1T1, SCG3, SCRT1, SCRT2, SIAH3, SLC32A1, SOX6, SRRM4, SST, ST8SIA5, STMN2, TAGLN3, TIAM1, TMEM2, TTC9B, or WI2-1896014.1.

[0084] In this application, the term "pluripotent stem cells" generally refers to cells that have the potential to proliferate and differentiate. Pluripotent stem cells can be functionally defined as cells that: (1) have the ability to differentiate into different cell types and, in some cases, only generate one specific cell type; (2) can undergo long-term self-renewal to produce one or more cells that are the same or different from the original cell type. The source and preparation method of pluripotent stem cells are not limited. For example, the pluripotent stem cells can be naturally obtained or artificially modified. For example, the pluripotent stem cells can include embryonic stem cells, induced pluripotent stem cells, etc. Therefore, the term "pluripotency" refers to a cell state with a degree of developmental potential that is less than or equal to totipotency.

[0085] In this application, the term "induced pluripotent stem cells" generally refers to a type of pluripotent stem cells artificially prepared from non-pluripotent cells, which can be abbreviated as "iPS cells" or "iPSCs." For example, the artificial method can be to introduce specific transcription factors to reprogram non-pluripotent cells. For example, the non-pluripotent cells can be adult somatic cells or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.

[0086] In this application, the term "embryonic stem cells," also known as "embryonic stem cells" and abbreviated as "ESC," generally refers to cells that possess the properties of unlimited proliferation, self-renewal, and multidirectional differentiation. Embryonic stem cells are derived from the undifferentiated inner cell mass of the blastocyst (early embryonic stage). Their source and preparation methods are not limited. Embryonic stem cells can be induced to differentiate into nearly any cell type in the body, both in vitro and in vivo. For example, these cell types may include hematopoietic stem cells, neural cells, and cardiomyocytes.

[0087] In this application, the term "GABAergic interneurons" generally refers to a type of neuron that activates GABA receptors on downstream neurons by releasing GABA, leading to an influx of chloride ions and thus achieving an inhibitory effect. Based on different molecular markers, the main subtypes of GABAergic interneurons include parvalbumin (PV) interneurons, somatostatin (SST) interneurons, and ionotropic 5-HT3aR interneurons, including vasoactive intestinal peptide (VIP) interneurons.

[0088] In this application, the term "composition" generally refers to a product comprising a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of the specified amounts of the specified ingredients. In this application, the composition may also include other inactive ingredients, such as carriers, excipients, adjuvants, stabilizers, etc.

[0089] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0090] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0091] In this application, the term "in vivo" generally refers to within a living organism. For example, in some cases, "in vivo" can refer to a specific location in a subject tissue or organ. Detailed Description of the Invention

[0093] method

[0094] In one aspect, the present application provides a method for proliferating / differentiating pluripotent stem cells into medial ganglionic eminence (MGE) neural progenitor cells, comprising the following steps:

[0095] (a) culturing cells in a medium comprising SAG for about 10 days; and

[0096] (b) Cells were cultured in medium containing PD0325901 and DAPT for approximately 7 days.

[0097] In the present application, non-limiting examples of biomarkers expressed by the MGE neural precursor cells include NKX2.1, LHX6, and DLX1. In certain embodiments, the biomarker is NKX2.1. In certain embodiments, the biomarker is NKX2.1 and LHX6.

[0098] In the present application, the method may include culturing pluripotent stem cells in a culture medium containing a specific activator and / or inhibitor of a cell signaling pathway.

[0099] The activators and inhibitors used in the methods disclosed herein can be those known in the art and commercially available. They are used in culture at concentrations effective to achieve the desired outcome, for example, to generate MGE neural precursor cells expressing NKX2.1 and LHX6 markers. Non-limiting examples of suitable activators and inhibitors and effective concentration ranges are further described below.

[0100] SHH pathway activators include agents, molecules, compounds, or substances that activate (upregulate) signaling in the SHH signaling pathway, which biologically involves SHH binding to the patch homolog-1 (PTCH1) receptor and transduction via the smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway activator can be selected from one or more of the following: SAG, purinemorphamine, and GSA10. In one embodiment, the SHH pathway activator can be SAG.

[0101] In the present application, the time for culturing cells using SAG in the method can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0102] For example, the culture time can be about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, about 11 days, about 11.5 days, about 12 days, about 12.5 days, about 13 days, about 13.5 days, about 14 days, about 14.5 days, or about 15 days.

[0103] MEK pathway inhibitors include agents, molecules, compounds, or substances that inhibit (downregulate) one or more components of the MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). In one embodiment, the MEK pathway inhibitor can be selected from one or more of the following: PD0325901, MEK162, XL518, selumetinib, GSK1120212, PD-184352, rifatinib, AZD-6244, PD98059, U0126, BI-847325, and RO5126766. In one embodiment, the MEK pathway inhibitor can be PD0325901.

[0104] In the present application, the time for culturing cells using PD0325901 can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0105] For example, the culture time can be about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, about 11 days, about 11.5 days, or about 12 days.

[0106] Notch pathway inhibitors include agents, molecules, compounds, or substances that can inhibit (downregulate) one or more components of the Notch pathway. In one embodiment, the Notch pathway inhibitor can be selected from one or more of the following groups: DAPT, Compound E, RO4929097, FLI-06, IMR-1, IMR-1A, GSI-XX, Semaside, Dibenzazepine, LY411575, Crenigacestat, Valproic acid, YO-01027, CB-103, Tangeretin, BMS-906024, Celecoxib, and Brucella D. In one embodiment, the Notch pathway inhibitor can be Compound E. In one embodiment, the Notch pathway inhibitor can be DAPT.

[0107] In the present application, the time for culturing cells using DAPT in the method can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0108] For example, the culture time can be about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, about 11 days, about 11.5 days, or about 12 days.

[0109] In the present application, the method may comprise the following steps:

[0110] (1) Cultivate cells in culture medium for approximately 9 days;

[0111] (2) culturing the cells in a medium containing SAG for about 10 days; and

[0112] (3) Cultivate in a medium containing PD0325901 and DAPT for approximately 7 days.

[0113] In the present application, the culturing time in step (1) can be about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 11 days, about 11.5 days, or about 12 days.

[0114] In the present application, culturing cells in a culture medium for about 9 days includes culturing cells in a culture medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for about 6 days, and culturing cells in a culture medium containing Rock inhibitor for about 3 days.

[0115] In the present application, the method may comprise the following steps:

[0116] (1) Culture cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

[0117] (2) Cultivate cells in a medium containing Rock inhibitor for approximately 3 days;

[0118] (3) culturing the cells in a medium containing SAG for about 10 days; and

[0119] (4) Cultivate in a medium containing PD0325901 and DAPT for approximately 7 days.

[0120] Inhibitors of the TGF-β (transforming growth factor β) pathway include agents, molecules, compounds, or substances that inhibit (downregulate) signaling through members of the TGF-β receptor family (a family of serine / threonine kinase receptors). In one embodiment, the TGF-β pathway inhibitor may be selected from one or more of the following: SB431542, A 83-01, GW788388, SB525334, TP0427736, RepSox, and SD-208. In one embodiment, the TGF-β pathway inhibitor may be SB431542.

[0121] In the present application, the time for culturing cells using SB431542 in the method can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0122] For example, the culture time can be about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, or about 11 days.

[0123] BMP (bone morphogenetic protein) pathway inhibitors include agents, molecules, compounds, or substances that can inhibit (downregulate) the BMP signaling pathway. This signaling pathway is biologically activated by the binding of BMPs to BMP receptors, such as activin receptor-like kinases (ALKs). For example, type I BMP receptors include, but are not limited to, ALK2 and ALK3. In one embodiment, the BMP pathway inhibitor can be selected from one or more of the following: DMH-1, DMH-2, LDN193189, Dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, and Noggin. In one embodiment, the BMP pathway inhibitor can be DMH-1.

[0124] In the present application, the time for culturing cells using DMH-1 in the method can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0125] For example, the culture time can be about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, or about 11 days.

[0126] WNT pathway inhibitors include agents, molecules, compounds, or substances that inhibit (downregulate) the Wnt / β-catenin signaling pathway. This pathway is biologically activated by the binding of Wnt protein ligands to Frizzled family receptors. In one embodiment, the WNT pathway inhibitor can be selected from one or more of the following: IWR-1, IWP-2, IWP-4, XAV939, ICG001, capmatinib, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797K, JW55, JW67, JW74, cardiomyocyte-1, NLS-StAx-h, TAK715, PNU74654, iCRT3, WIF-1, and DKK1. In one embodiment, the WNT pathway inhibitor can be IWR-1.

[0127] In the present application, the time for culturing cells using IWR-1 can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0128] For example, the culture time can be about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, or about 11 days.

[0129] ROCK pathway inhibitors include agents, molecules, compounds, or substances that can inhibit (downregulate) the ROCK signaling pathway. In one embodiment, the ROCK pathway inhibitor can be a Rho kinase inhibitor. In one embodiment, the ROCK pathway inhibitor can be selected from one or more of the following groups: Rock inhibitor (Y-27632), Y-30141, Y-33075, Y-39983, LX-7101, BA-210, AT-13148, AR-12286, AR-13324, GSK-429286A, WAY-624704, RKI-1447, TC-S 7001, H-1152, GSK-576371, GSK-269962A HCl, DJ4, ZINC00881524, and β-elemene. In one embodiment, the ROCK pathway inhibitor can be Rock inhibitor.

[0130] In the present application, the time for culturing cells using Rock inhibitor in the method can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0131] For example, the culture time can be about 0 day, about 0.5 day, about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, or about 11 days.

[0132] In the present application, the method comprises culturing pluripotent stem cells using a culture medium.

[0133] In the present application, the culture medium may comprise a basal medium. For example, the basal medium may comprise any culture medium known in the art. For example, the basal medium may comprise one or more of IMDM, MEM, Ham's F-12, mTeSR1, TeSR2, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, DMEM / F12, MEM / F12, APEL, StemSpan™ SFEM II, Nuwacell®ncTarget complete medium, and RPMI1640. For example, the basal medium may be DMEM / F12. For example, the basal medium may be DMEM / F12 mixed with other culture media in a specific ratio. For example, the basal medium may be Neurobasal. For example, the basal medium may be Neurobasal mixed with other culture media in a specific ratio. For example, the basal medium may be DMEM / F12 and Neurobasal. For example, the basal culture medium may be a mixed culture medium in which DMEM / F12 and Neurobasal are mixed in a ratio of 1:1.

[0134] In the present application, the culture medium may contain one or more nutrients, extracts, growth factors, hormones, cytokines and culture medium additives. The types and concentrations of the added components may be adaptively adjusted according to the culture conditions to achieve better culture effects.

[0135] For example, one or more of the following ingredients can be added: serum replacement, glutamine, NEAA (non-essential amino acids), AA (amino acids), N2 supplement, B27 supplement, ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), nerve growth factor (NGF), transforming growth factor β (TGF-β), cyclic adenosine monophosphate (cAMP), neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), 2-mercaptoethanol (2-mercaptoethanol), ROCK pathway inhibitor, SHH pathway activator, MEK pathway inhibitor, Notch pathway inhibitor, TGF-β pathway inhibitor, WNT pathway inhibitor. The sources of the additives are not limited and they can be obtained from commercial sources, or they can be natural or recombinant.

[0136] For example, one or more of the following ingredients can be added: Glutamax, NEAA, GDNF, AA, BDNF, cAMP, IGF-1, Compound E, N2 supplement, B27 supplement, SAG, PD0325901, DAPT, SB431542, DMH-1, IWR-1, and Rock inhibitor.

[0137] In the present application, the pluripotent stem cells that differentiate into MGE neural precursor cells or GABAergic interergic neurons can be of natural origin or modified. For example, the pluripotent stem cells can be artificially modified by physical methods, chemical methods and / or biological methods. For example, the expression of certain genes in the pluripotent stem cells can be adjusted. For example, the pluripotent stem cells can be selected from one or more of the following groups: induced pluripotent stem cells and embryonic stem cells.

[0138] In the present application, the source of the pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells is not limited and can be of mammalian or non-mammalian origin. For example, the pluripotent stem cells are human pluripotent stem cells. For example, the induced pluripotent stem cells are human induced pluripotent stem cells. For example, the embryonic stem cells are human embryonic stem cells.

[0139] In the present application, the method may comprise the following steps:

[0140] (1) Cells were cultured in a medium containing 2 μM SB431542, 2 μM DMH-1, 2.5 μM IWR-1, and 0.5 μM Rock inhibitor for approximately 6 days;

[0141] (2) Cultivate cells in a medium containing 0.5 μM Rock inhibitor for approximately 3 days;

[0142] (3) culturing the cells in a medium containing 0.2 μM SAG for approximately 10 days; and

[0143] (4) Culture in a medium containing 0.5 μM PD0325901 and 10 μM DAPT for approximately 7 days.

[0144] In the present application, the method may comprise the following steps:

[0145] (1) Culture cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

[0146] (2) Culture cells in a medium containing Rock inhibitor for approximately 3 days

[0147] (3) culturing the cells in a medium containing SAG for approximately 10 days;

[0148] (4) culturing the cells in a medium comprising PD0325901 and DAPT for about 7 days; and

[0149] (5) The cells were cultured in a medium containing GDNF, AA, BDNF, cAMP, IGF1, and Compound E for approximately 14 days to obtain GABAergic interneurons.

[0150] In the present application, the method may comprise the following steps:

[0151] (1) Cells were cultured in a medium containing 2 μM SB431542, 2 μM DMH-1, 2.5 μM IWR-1, and 0.5 μM Rock inhibitor for approximately 6 days;

[0152] (2) Cultivate cells in a medium containing 0.5 μM Rock inhibitor for approximately 3 days;

[0153] (3) culturing the cells in a medium containing 0.2 μM SAG for approximately 10 days; and

[0154] (4) culturing in a medium containing 0.5 μM PD0325901 and 10 μM DAPT for approximately 7 days; and

[0155] (5) GABAergic interneurons were obtained by culturing the cells in a medium containing 20 ng / ml GDNF, 200 μM AA, 20 ng / ml BDNF, 0.5 μM cAMP, 20 ng / ml IGF1, and 0.1 μM Compound E for approximately 14 days.

[0156] In the present application, the culture may be cultured at a temperature of about 35-39° C. For example, about 34.5° C., about 35° C., about 35.5° C., about 36° C., about 36.5° C., about 37° C., about 37.5° C., about 38° C., about 38.5° C., about 39° C., or about 39.5° C.

[0157] In the present application, the culture may be cultured under conditions of about 3-7% CO 2 . For example, about 3% CO 2 , about 3.5% CO 2 , about 4% CO 2 , about 4.5% CO 2 , about 5% CO 2 , about 5.5% CO 2 , about 6% CO 2 , about 6.5% CO 2 , about 7% CO 2 , about 7.5% CO 2 .

[0158] In the present application, the method can be culturing cells in an adherent or suspended manner. In certain embodiments, the method is culturing cells in a suspended manner.

[0159] In the present application, the method may comprise regularly or irregularly supplementing and / or replacing the culture medium. In certain embodiments, the method comprises regularly supplementing and / or replacing the culture medium. In certain embodiments, the method comprises supplementing and / or replacing the culture medium every 24 hours. In certain embodiments, the method comprises replacing the culture medium every 24 hours.

[0160] In the present application, the method can be performed under serum-containing culture conditions. In the present application, the culture method can be performed under serum-free culture conditions.

[0161] In the present application, the method may comprise digesting the cells into single cells after the cells have proliferated to a sufficient number.

[0162] In certain embodiments, the digestion in the method uses Accutase digestion solution. In certain embodiments, the digestion in the method uses Trypsin and EDTA. In certain embodiments, the digestion in the method uses TrypLE digestion enzyme.

[0163] In the present application, the method may comprise culturing cells on a matrix-coated surface.

[0164] In certain embodiments, the method comprises a method wherein the matrix is ​​laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel™. In certain embodiments, the method comprises a method wherein the matrix is ​​vitronectin or Matrigel™. In certain embodiments, the method comprises a method wherein the matrix is ​​Matrigel™.

[0165] In the present application, the method may be an in vitro method. In the present application, the method may be an ex vivo method.

[0166] In the present application, the method may be a method for the purpose of non-disease diagnosis and treatment.

[0167] culture medium

[0168] In another aspect, the present application provides a combination culture medium comprising a first culture medium comprising SAG and a second culture medium comprising PD0325901 and DAPT.

[0169] In the present application, the combined culture medium can be used as an MGE neural progenitor cell differentiation culture medium, and the method described in the present application can be applied to induce pluripotent stem cells to proliferate and / or differentiate into MGE neural progenitor cells to obtain MGE neural progenitor cells.

[0170] In the present application, the combined culture medium can be used as an MGE neural precursor cell differentiation medium, and the method described in the present application can be applied to induce pluripotent stem cells to proliferate and / or differentiate into MGE neural precursor cells, obtain MGE neural precursor cells, and then proliferate and / or differentiate the obtained MGE neural precursor cells into GABA intermediate neuronal cells.

[0171] In the present application, the combination culture medium may include specific activators and / or inhibitors of cell signaling pathways.

[0172] SHH pathway activators include agents, molecules, compounds, or substances that activate (upregulate) signaling in the SHH signaling pathway, which biologically involves SHH binding to the patch homolog-1 (PTCH1) receptor and transduction via the smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway activator can be selected from one or more of the following: SAG, purinemorphamine, and GSA10. In one embodiment, the SHH pathway activator can be SAG.

[0173] In the present application, the concentration of SAG can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0174] For example, the concentration can be about 0.1-2 μM, about 0.1-1.8 μM, about 0.1-1.6 μM, about 0.1-1.4 μM, about 0.1-1.2 μM, about 0.1-1 μM, about 0.1-0.8 μM, about 0.1-0.6 μM, about 0.1-0.5 μM, about 0.1-0.4 μM, about 0.1-0.2 μM, about 0.2-2 μM, about 0.4-2 μM, about 0.5-2 μM, about 0. .6-2μM, about 0.8-2μM, about 1-2μM, about 1.2-2μM, about 1.4-2μM, about 1.5-2μM, about 1.6-2μM, about 1.8-2μM, about 2μM, about 1.8μM, about 1.6μM, about 1.5μM, about 1.4μM, about 1.2μM, about 1μM, about 0.8μM, about 0.6μM, about 0.5μM, about 0.4μM, about 0.2μM, about 0.1μM.

[0175] MEK pathway inhibitors include agents, molecules, compounds, or substances that can inhibit (downregulate) one or more components of the MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). In one embodiment, the MEK pathway inhibitor can be selected from one or more of the following: PD0325901, MEK162, XL518, selumetinib, GSK1120212, PD-184352, rifatinib, AZD-6244, PD98059, U0126, BI-847325, and RO5126766. In one embodiment, the MEK pathway inhibitor can be PD0325901.

[0176] In this application, the concentration of PD0325901 can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0177] For example, the concentration can be about 0.1-2 μM, about 0.1-1.8 μM, about 0.1-1.6 μM, about 0.1-1.4 μM, about 0.1-1.2 μM, about 0.1-1 μM, about 0.1-0.8 μM, about 0.1-0.6 μM, about 0.1-0.5 μM, about 0.1-0.4 μM, about 0.1-0.2 μM, about 0.2-2 μM, about 0.4-2 μM, about 0.5-2 μM, about 0. .6-2μM, about 0.8-2μM, about 1-2μM, about 1.2-2μM, about 1.4-2μM, about 1.5-2μM, about 1.6-2μM, about 1.8-2μM, about 2μM, about 1.8μM, about 1.6μM, about 1.5μM, about 1.4μM, about 1.2μM, about 1μM, about 0.8μM, about 0.6μM, about 0.5μM, about 0.4μM, about 0.2μM, about 0.1μM.

[0178] Notch pathway inhibitors include agents, molecules, compounds, or substances that can inhibit (downregulate) one or more components of the Notch pathway. In one embodiment, the Notch pathway inhibitor can be selected from one or more of the following groups: DAPT, Compound E, RO4929097, FLI-06, IMR-1, IMR-1A, GSI-XX, Semaside, Dibenzazepine, LY411575, Crenigacestat, Valproic acid, YO-01027, CB-103, Tangeretin, BMS-906024, Celecoxib, and Brucella D. In one embodiment, the Notch pathway inhibitor can be Compound E. In one embodiment, the Notch pathway inhibitor can be DAPT.

[0179] In the present application, the concentration of DAPT can be adaptively adjusted according to different cells and culture conditions to achieve the best culture effect.

[0180] For example, the concentration can be about 2.5-20 μM, about 5-20 μM, about 7.5-20 μM, about 10-20 μM, about 12.5-20 μM, about 15-20 μM, about 17.5-20 μM, about 2.5-17.5 μM, about 2.5-15 μM, about 2.5-12.5 μM, about 2.5-10 μM, about 2.5-7.5 μM, about 2.5-5 μM, about 20 μM, about 15 μM, about 10 μM, about 5 μM, about 2.5 μM.

[0181] In the present application, the combination culture medium may include a first culture medium comprising SAG, a second culture medium comprising PD0325901 and DAPT, a third culture medium comprising GDNF, AA, BDNF, cAMP, IGF1 and Compound E, a fourth culture medium comprising SB431542, DMH-1, IWR-1 and Rock inhibitor, and a fifth culture medium comprising Rock inhibitor.

[0182] In actual use, one or more of the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium may be selected for use. For example, the first culture medium and the second culture medium may be used; the first culture medium, the second culture medium, and the third culture medium may be used; the first culture medium, the second culture medium, and the fourth culture medium may be used; the first culture medium, the second culture medium, and the fifth culture medium may be used; the first culture medium, the second culture medium, the third culture medium, and the fourth culture medium may be used; the first culture medium, the second culture medium, the third culture medium, and the fifth culture medium may be used; the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium may be used. For example, the order and timing of using the culture media may be adjusted.

[0183] In the present application, the culture medium may include a basal medium. For cell culture, the basal medium may be a single component or a combination of multiple culture media. For example, the basal medium may include any culture medium known in the art. For example, the basal medium may include one or more of IMDM, MEM, Ham's F-12, mTeSR1, TeSR2, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential6, DMEM, DMEM / F12, MEM / F12, APEL, StemSpan™ SFEM II, Nuwacell® ncTarget complete medium, and RPMI1640. For example, different basal media and / or combinations thereof may be used in different steps. For example, the basal medium may be DMEM / F12. For example, the basal medium may be DMEM / F12 mixed with other culture media in a specific ratio. For example, the basal medium may be Neurobasal. For example, the basal medium may be Neurobasal mixed with other culture media in a specific ratio. For example, the basal culture medium can be DMEM / F12 and Neurobasal. For example, the basal culture medium can be a mixed culture medium in which DMEM / F12 and Neurobasal are mixed in a ratio of 1:1.

[0184] In the present application, the culture medium may contain one or more nutrients, extracts, growth factors, hormones, cytokines and culture medium additives. The types and concentrations of the added components may be adaptively adjusted according to the culture conditions to achieve better culture effects.

[0185] For example, one or more of the following ingredients can be added: serum replacement, glutamine, NEAA (non-essential amino acids), AA (amino acids), N2 supplement, B27 supplement, ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), nerve growth factor (NGF), transforming growth factor β (TGF-β), cyclic adenosine monophosphate (cAMP), neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), 2-mercaptoethanol (2-mercaptoethanol), ROCK pathway inhibitor, SHH pathway activator, MEK pathway inhibitor, Notch pathway inhibitor, TGF-β pathway inhibitor, WNT pathway inhibitor. The sources of the additives are not limited and they can be obtained from commercial sources, or they can be natural or recombinant.

[0186] For example, one or more of the following ingredients can be added: Glutamax, NEAA, GDNF, AA, BDNF, cAMP, IGF-1, Compound E, N2 supplement, B27 supplement, SAG, PD0325901, DAPT, SB431542, DMH-1, IWR-1, and Rock inhibitor.

[0187] For example, the culture medium may contain GDNF, AA, BDNF, cAMP, IGF-1, and Compound E.

[0188] For example, in the culture, the concentration of GDNF is about 10-40 ng / ml, for example, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml.

[0189] For example, in the culture, the concentration of AA is about 100-200 μM, for example, about 100 μM, about 120 μM, about 140 μM, about 150 μM, about 160 μM, about 180 μM, or about 200 μM.

[0190] For example, in the culture, the concentration of BDNF is about 10-40 ng / ml, for example, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml.

[0191] For example, in the culture, the concentration of cAMP is about 0.2-1 μM, for example, about 0.2 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, or about 1 μM.

[0192] For example, in the culture, the concentration of IGF-1 is about 10-40 ng / ml, for example, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml.

[0193] For example, in the culture, the concentration of Compound E is about 0.1-1 μM, for example, about 0.1 μM, about 0.2 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, or about 1 μM.

[0194] For example, the culture medium may contain SB431542, DMH-1, IWR-1, and Rock inhibitor.

[0195] For example, in the culture, the concentration of SB431542 is about 2-10 μM, for example, about 2 μM, about 4 μM, about 6 μM, about 8 μM, or about 10 μM.

[0196] For example, in the culture, the concentration of DMH-1 is about 2-10 μM, for example, about 2 μM, about 4 μM, about 6 μM, about 8 μM, or about 10 μM.

[0197] For example, in the culture, the concentration of IWR-1 is about 2-5 μM, for example, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM.

[0198] For example, in the culture, the concentration of the Rock inhibitor is about 0.1-1 μM, for example, about 0.1 μM, about 0.2 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, or about 1 μM.

[0199] For example, the supplements are N2 supplement, B27 supplement, Glutamax and NEAA.

[0200] In the present application, the culture medium can be used alone or in combination with other culture media. For example, different differentiation culture media can be used for different differentiation steps.

[0201] Composition and use

[0202] In another aspect, the present application also provides a composition comprising cells and the culture medium, wherein the culture medium comprises SAG, DMEM / F12, Neurobasal, MEM NEAA, N2 supplement, Glutamax, and B27 supplement.

[0203] On the other hand, the present application also provides a composition comprising cells and the culture medium, wherein the culture medium comprises PD0325901, DAPT, DMEM / F12, Neurobasal, MEM NEAA, N2 supplement, Glutamax and B27 supplement.

[0204] In the present application, the cells are selected from one or more of the following groups: pluripotent stem cells, MGE neural precursor cells or GABAergic interneurons.

[0205] In the present application, the pluripotent stem cells may be one or more of induced pluripotent stem cells and embryonic stem cells.

[0206] In the present application, the cells and / or the state of the cells can be determined by cell markers. For example, the type of cells and / or the state of the cells can be determined by marker phenotype.

[0207] For example, the MGE neural precursor cells are NKX2.1+ and LHX6+ double positive cells. For example, the GABAergic interneurons are LHX6+ positive cells.

[0208] In certain embodiments, the cells described herein are isolated.

[0209] On the other hand, the present application also provides a method for preventing and / or treating a disease, which comprises administering MGE neural precursor cells and / or GABAergic interneurons to a subject in need thereof, wherein the MGE neural precursor cells and / or GABAergic interneurons are obtained by using the method, the culture medium, and the composition.

[0210] For example, the MGE neural precursor cells and / or GABAergic interneurons used can be prepared pharmaceutically according to any conventional method.For example, carrier, excipient or diluent can be used to mix or dilute the active ingredient. The example of suitable carrier, excipient or diluent is lactose, dextrose, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The preparation can additionally include for example filler, anti-agglomerant, lubricant, wetting agent, flavoring, emulsifier, preservative etc. Prepare the compositions of the present invention to provide quick, continuous or delayed release active ingredient after giving to the patient by using any method known in the art.

[0211] In this application, cells can be administered to an individual in need by any acceptable route, including but not limited to intravenous infusion, local injection, intrathecal injection, intraperitoneal injection, and implantation. In certain embodiments, the cells can be introduced into an individual in need by implantation. In certain embodiments, the cells can be transplanted into the brain of an individual in need.

[0212] In the present application, the administration dosage of the cells may be a single dose or multiple doses. For example, the actual administration amount of the cells may be determined based on a variety of relevant factors, such as the type of disease; the route of administration; the age, sex, and / or weight of the patient; and the severity of the patient's symptoms.

[0213] This application also provides the following implementation methods:

[0214] 1. A method for proliferating / differentiating pluripotent stem cells into medial ganglionic eminence (MGE) neural progenitor cells, comprising the following steps:

[0215] (a) culturing cells in a medium comprising SAG for about 10 days; and

[0216] (b) Cells were cultured in medium containing PD0325901 and DAPT for approximately 7 days.

[0217] 2. The method according to embodiment 1, wherein the MGE neural precursor cells are NKX2.1+ and LHX6+ double-positive cells.

[0218] 3. The method of any one of embodiments 1-2, wherein the concentration of SAG is about 0.1-2 μM.

[0219] 4. The method of any one of embodiments 1-3, wherein the concentration of SAG is about 0.1-0.5 μM.

[0220] 5. The method of any one of embodiments 1-4, wherein the concentration of SAG is about 0.2 μM.

[0221] 6. The method of any one of embodiments 1-5, wherein the concentration of PD0325901 is about 0.1-2 μM.

[0222] 7. The method of any one of embodiments 1-6, wherein the concentration of PD0325901 is about 0.5-2 μM.

[0223] 8. The method of any one of embodiments 1-7, wherein the concentration of PD0325901 is about 0.5 μM.

[0224] 9. The method of any one of embodiments 1-8, wherein the concentration of DAPT is about 2.5-20 μM.

[0225] 10. The method of any one of embodiments 1-9, wherein the concentration of DAPT is about 5-20 μM.

[0226] 11. The method of any one of embodiments 1-10, wherein the concentration of DAPT is about 10 μM.

[0227] 12. The method of any one of embodiments 1-11, wherein the method comprises the following steps:

[0228] (1) Cultivate cells in culture medium for approximately 9 days;

[0229] (2) culturing the cells in a medium containing SAG for about 10 days; and

[0230] (3) Cultivate in a medium containing PD0325901 and DAPT for approximately 7 days.

[0231] 13. The method according to embodiment 12, wherein the culturing the cells in the culture medium for about 9 days comprises culturing the cells in a culture medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for about 6 days, and culturing the cells in a culture medium containing Rock inhibitor for about 3 days.

[0232] 14. The method of any one of embodiments 12-13, wherein the method comprises the following steps:

[0233] (1) Culture cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

[0234] (2) Cultivate cells in a medium containing Rock inhibitor for approximately 3 days;

[0235] (3) culturing the cells in a medium containing SAG for about 10 days; and

[0236] (4) Cultivate in a medium containing PD0325901 and DAPT for approximately 7 days.

[0237] 15. The method of any one of embodiments 13-14, wherein the concentration of SB431542 is about 2-10 μM, the concentration of DMH-1 is about 2-10 μM, the concentration of IWR-1 is about 2-5 μM, and the concentration of Rock inhibitor is about 0.1-1 μM.

[0238] 16. The method of any one of embodiments 13-15, wherein the concentration of SB431542 is about 2 μM, the concentration of DMH-1 is about 2 μM, the concentration of IWR-1 is about 2.5 μM, and the concentration of Rock inhibitor is about 0.5 μM.

[0239] 17. The method of any one of embodiments 1-16, wherein the culture medium comprises a basal medium.

[0240] 18. The method of any one of embodiments 1-17, wherein the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, MEM.

[0241] 19. The method according to any one of embodiments 1-18, wherein the basal medium can be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0242] 20. The method of any one of embodiments 1-19, wherein the basal medium comprises about 50% DMEM / F12, about 50% Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 0.5% Glutamax, and about 1% B27 supplement.

[0243] 21. The method of any one of embodiments 1-20, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0244] 22. The method of any one of embodiments 1-20, wherein the pluripotent stem cells are embryonic stem cells.

[0245] 23. The method of any one of embodiments 1-22, comprising:

[0246] (1) Culture cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

[0247] (2) Culture cells in a medium containing Rock inhibitor for approximately 3 days

[0248] (3) culturing the cells in a medium containing SAG for approximately 10 days;

[0249] (4) culturing the cells in a medium comprising PD0325901 and DAPT for about 7 days; and

[0250] (5) The cells were cultured in a medium containing GDNF, AA, BDNF, cAMP, IGF1, and Compound E for approximately 14 days to obtain GABAergic interneurons.

[0251] 24. The method according to embodiment 23, wherein the concentration of GDNF is about 10-40 ng / ml, the concentration of AA is about 100-200 μM, the concentration of BDNF is about 10-40 ng / ml, the concentration of cAMP is about 0.2-1 μM, the concentration of IGF1 is about 10-40 ng / ml, and the concentration of Compound E is about 0.1-1 μM.

[0252] 25. The method of any one of embodiments 23-24, wherein the concentration of GDNF is about 20 ng / ml, the concentration of AA is about 200 μM, the concentration of BDNF is about 20 ng / ml, the concentration of cAMP is about 0.5 μM, the concentration of IGF1 is about 20 ng / ml, and the concentration of Compound E is about 0.1 μM.

[0253] 26. The method according to any one of embodiments 23-25, wherein the culture medium in step (5) comprises a basal culture medium, and the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, MEM.

[0254] 27. The method according to any one of embodiments 23-26, wherein the basal culture medium in step (5) can be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0255] 28. The method of any one of embodiments 23-27, wherein the basal medium in step (5) comprises Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 1% Glutamax, and about 1% B27 supplement.

[0256] 29. The method of any one of embodiments 1-28, wherein the cells are cultured in suspension.

[0257] 30. A combined culture medium comprising:

[0258] (i) a first culture medium comprising SAG; and

[0259] (ii) A second culture medium comprising PD0325901 and DAPT.

[0260] 31. The combined culture medium of embodiment 30, wherein the concentration of SAG is about 0.1-2 μM.

[0261] 32. The combined medium of any one of embodiments 30-31, wherein the concentration of SAG is about 0.1-0.5 μM.

[0262] 33. The combined medium of any one of embodiments 30-32, wherein the concentration of SAG is about 0.2 μM.

[0263] 34. The combined medium of any one of embodiments 30-33, wherein the concentration of PD0325901 is about 0.1-2 μM.

[0264] 35. The combined medium of any one of embodiments 30-34, wherein the concentration of PD0325901 is about 0.5-2 μM.

[0265] 36. The combined medium of any one of embodiments 30-35, wherein the concentration of PD0325901 is about 0.5 μM.

[0266] 37. The combined medium of any one of embodiments 30-36, wherein the concentration of DAPT is about 2.5-20 μM.

[0267] 38. The combined medium of any one of embodiments 30-37, wherein the concentration of DAPT is about 5-20 μM.

[0268] 39. The combined medium of any one of embodiments 30-38, wherein the concentration of DAPT is about 10 μM.

[0269] 40. A combination culture medium according to any one of embodiments 30-39, wherein the first culture medium and the second culture medium comprise a basal culture medium, and the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential6, DMEM, MEM.

[0270] 41. A combination culture medium according to any one of embodiments 30-40, wherein the basal culture medium of the first culture medium and the second culture medium can be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0271] 42. The combination culture medium of any one of embodiments 30-41, wherein the basal medium of the first culture medium and the second culture medium comprises about 50% DMEM / F12, about 50% Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 0.5% Glutamax, and about 1% B27 supplement.

[0272] 43. The combination culture medium of any one of embodiments 30-42, wherein the combination culture medium comprises a third culture medium comprising GDNF, AA, BDNF, cAMP, IGF1, and Compound E.

[0273] 44. The combined culture medium according to embodiment 43, wherein the concentration of GDNF is about 10-40 ng / ml, the concentration of AA is about 100-200 μM, the concentration of BDNF is about 10-40 ng / ml, the concentration of cAMP is about 0.2-1 μM, the concentration of IGF1 is about 10-40 g / ml, and the concentration of Compound E is about 0.1-1 μM.

[0274] 45. A combination culture medium according to any one of embodiments 43-44, wherein the concentration of GDNF is about 20 ng / ml, the concentration of AA is about 200 μM, the concentration of BDNF is about 20 ng / ml, the concentration of cAMP is about 0.5 μM, the concentration of IGF1 is about 20 ng / ml, and the concentration of Compound E is about 0.1 μM.

[0275] 46. ​​A combination culture medium according to any one of embodiments 43-45, wherein the third culture medium comprises a basal culture medium, and the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Neurobasal™, Essential 8™, TeSR™-E8™, Essential 6, DMEM, MEM.

[0276] 47. A combination culture medium according to any one of embodiments 43-46, wherein the base culture medium of the third culture medium can be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

[0277] 48. The combination medium of any one of embodiments 43-47, wherein the basal medium of the third culture medium comprises Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 1% Glutamax, and about 1% B27 supplement.

[0278] 49. A composition comprising cells and the culture medium of any one of embodiments 30-48, wherein the culture medium comprises SAG, DMEM / F12, Neurobasal, MEM NEAA, N2 supplement, Glutamax, and B27 supplement.

[0279] 50. The composition of embodiment 49, wherein the cells are selected from one or more of the following groups: pluripotent stem cells, MGE neural precursor cells, or GABAergic interneurons.

[0280] 51. The composition of any one of embodiments 49-50, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0281] 52. The composition of any one of embodiments 49-50, wherein the pluripotent stem cells are embryonic stem cells.

[0282] 53. A composition according to any one of embodiments 49-52, wherein the MGE neural precursor cells are NKX2.1+ and LHX6+ double positive cells.

[0283] 54. The composition of any one of embodiments 49-53, wherein the GABAergic interneurons are LHX6+ positive cells.

[0284] 55. A composition comprising cells and the culture medium of any one of embodiments 30-48, wherein the culture medium comprises PD0325901, DAPT, DMEM / F12, Neurobasal, MEM NEAA, N2 supplement, Glutamax, and B27 supplement.

[0285] 56. The composition of embodiment 55, wherein the cells are selected from one or more of the following groups: pluripotent stem cells, MGE neural precursor cells, or GABAergic interneurons.

[0286] 57. The composition of any one of embodiments 55-56, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0287] 58. A composition according to any one of embodiments 55-56, wherein the pluripotent stem cells are embryonic stem cells.

[0288] 59. A composition according to any one of embodiments 55-58, wherein the MGE neural precursor cells are NKX2.1+ and LHX6+ double positive cells.

[0289] 60. The composition of any one of embodiments 55-59, wherein the GABAergic interneurons are LHX6+ positive cells.

[0290] 61. Use of the method of any one of embodiments 1-29, the culture medium of any one of embodiments 30-48, and / or the composition of any one of embodiments 49-60 in the preparation of a medicament for preventing and / or treating a disease.

[0291] Without intending to be bound by any theory, the following examples are merely intended to illustrate the preparation method and application of the present application, and are not intended to limit the scope of the present invention.

[0292] Example

[0293] Example 1 Construction of fluorescence reporter system

[0294] Using CRISPR / Cas9 technology, we inserted the green fluorescent protein mNeongreen and the red fluorescent protein tdTomato into the loci of NKX2.1 and LHX6, markers of MGE neural progenitor cells, respectively, to construct the H9-NKX2.1-mNeongreen / LHX6-tdTomato human embryonic stem cell line for subsequent characterization of target cells using fluorescent proteins ( Figure 1 ).

[0295] Example 2: Generation of MGE neural precursor cells from induced pluripotent stem cells

[0296] 1) Grow human pluripotent stem cells in a 6-well plate to a density of approximately 70%-80%. Digest the cells into single cells and seed 2 million cells into a T25 cell culture flask. Culture in a cell culture incubator at 37°C and 5% CO2 for one day. The culture medium composition is shown in Table 1.

[0297] Table 1. Culture medium composition (pre-differentiation culture)

[0298]

[0299] 2) The second day of passage is designated as the first day of differentiation (D1). Replace the culture medium (components are shown in Table 2) and culture in a cell culture incubator at 37°C and 5% CO2 until D6, changing the medium every other day.

[0300] Table 2. Culture medium composition (D1-D6)

[0301]

[0302] 3) On days 7-9 of differentiation, replace the culture medium (medium composition is shown in Table 3) and culture in a cell culture incubator at 37°C and 5% CO2 until day 9, changing the medium every other day.

[0303] Table 3. Culture medium composition (D7-D9)

[0304]

[0305] 4) On differentiation days 10-20, replace the culture medium (medium composition is shown in Table 4) and culture in a cell culture incubator at 37°C and 5% CO2 until day 20, changing the medium every other day.

[0306] Table 4. Culture medium composition (D10-D20)

[0307]

[0308] 5) During differentiation days 21-28, replace the culture medium (medium composition is shown in Table 5) and culture in a cell culture incubator at 37°C and 5% CO2 until day 28, changing the medium every other day.

[0309] Table 5. Culture medium composition (D21-D28)

[0310]

[0311] 6) On day 28 of differentiation, cells were digested with TrplE at 37°C for 15-20 minutes. After digestion into single cells, cells were collected and fluorescent cells were detected by flow cytometry.

[0312] The above experiments show that:

[0313] like Figure 2 As shown in Figures AC, the vast majority of cells began to express LHX6-tdTomato, and the proportion of NKX2.1-mNeongreen and LHX6-tdTomato double-positive MGE neural progenitor cells reached 59.47±4.217%. The method listed in Example 2 can effectively induce pluripotent stem cells to generate MGE neural progenitor cells.

[0314] Example 3 Differentiation of MGE Neural Precursor Cells Generated from Pluripotent Stem Cells into GABA Interergic Neurons

[0315] The MGE neural progenitor cells obtained in Example 2 were attached to matrigel-coated glass slides for further maturation culture. After the cells were fully attached to the slides, culture medium was added, with 1 ml of culture medium added to each well of a 24-well plate. The cells were cultured in a 37°C incubator for two weeks or longer until they were fully mature. After fixation with 4% paraformaldehyde (PFA), immunofluorescence staining was performed. The culture medium composition is shown in Table 6.

[0316] Table 6. Medium composition (mature culture)

[0317]

[0318] The above experiments show that:

[0319] like Figure 2 As shown in Figures DE, 85.81±0.4268% of GABAergic interneuron-positive cells were obtained, and 58.05±1.773% of the cells were double-positive for GABA and LHX6-tdTomato, indicating that they were GABAergic interneurons derived from MGE. This result indicates that the MGE neural progenitor cells obtained by the method listed in Example 2 can be further differentiated to obtain a higher proportion of GABAergic interneurons.

[0320] Example 4 Effects of different concentrations of SAG on the proliferation and / or differentiation of MGE neural precursor cells and GABAergic interneurons

[0321] 1) Grow human pluripotent stem cells in a 6-well plate to a density of approximately 70%-80%. Digest the cells into single cells and seed 2 million cells into a T25 cell culture flask. Culture in a cell culture incubator at 37°C and 5% CO2 for one day. The culture medium composition is shown in Table 7.

[0322] Table 7. Medium Composition (Pre-differentiation Culture)

[0323]

[0324] 2) The second day of passage is designated as the first day of differentiation (D1). Culture the cells in a cell culture incubator at 37°C and 5% CO2 until D6. Change the medium every other day. The composition of the medium is shown in Table 8.

[0325] Table 8. Culture medium composition (D1-D6)

[0326]

[0327] 3) On differentiation days 7-9, replace the culture medium and culture the cells in a cell culture incubator at 37°C and 5% CO2 until day 9. Change the medium every other day. The composition of the culture medium is shown in Table 9.

[0328] Table 9. Culture medium composition (D7-D9)

[0329]

[0330] 4) During differentiation days 10-28, replace the culture medium and culture the cells in a cell culture incubator at 37°C and 5% CO2 until day 28. Change the medium every other day. The composition of the culture medium is shown in Table 10.

[0331] Table 10. Culture medium composition (D10-D28)

[0332]

[0333] On day 28 of differentiation, cells were digested with TrplE at 37°C for 15-20 minutes. After digestion into single cells, cells were collected and fluorescent cells were detected by flow cytometry or attached to matrigel-coated glass slides for further maturation culture.

[0334] 5) The cells were cultured for maturation according to the method in Example 3. After full maturation, the cells were fixed with 4% paraformaldehyde (PFA) and then immunofluorescence staining was performed.

[0335] The above experiments show that:

[0336] like Figure 3 As shown, under the action of 0.2 μM SAG, the ratios of NKX2.1-mNeongreen and LHX6-tdTomato reached the highest values, which were 77.88±0.3361% and 15.99±0.5460%, respectively, but there was no significant difference compared with the 0.1 μM and 0.5 μM treatment groups ( Figure 3 BC). In the group without SAG treatment, only 30.55±8.036% of cells were NKX2.1-mNeongreen positive cells, and almost no cells expressed LHX6-tdTomato ( Figure 3 BE). After mature culture, immunofluorescence staining results showed that there was no significant difference in the proportion of GABA-positive cells among the four groups. The group without SAG treatment almost did not express LHX6-tdTomato. In the other three groups treated with different concentrations of SAG, there was no significant difference in the proportion of GABA- and LHX6-tdTomato-double-positive cells. The proportion of double-positive cells was the highest at 0.2μM concentration, with a ratio of 54±7% ( Figure 3 FI).

[0337] Example 5 Effects of adding SAG at different times on the proliferation and / or differentiation of MGE neural precursor cells and GABAergic interneurons

[0338] According to the method in Example 4, 0.2 μM SAG was selected as an agonist of the SHH signaling pathway, and SAG treatment was performed on D1 (day 1), D4, D7, D10, D13 until the end of differentiation on D28. Figure 4 A) At the end of differentiation (D28), the proportion of fluorescent cells was determined by flow cytometry. Additionally, cells were cultured for maturation according to the method described in Example 3. After full maturation, cells were fixed with 4% paraformaldehyde (PFA) and then immunofluorescence stained.

[0339] The above experiments show that:

[0340] 1) The proportion of NKX2.1-mNeongreen positive cells in the group treated with SAG at D10 was the highest, with a positive cell rate of 79.56±1.989%. This was significantly different from the results of SAG treatment at D1, D4, and D7. The proportion of NKX2.1-mNeongreen positive cells in the group treated with SAG at D13 was 73.09±2.223% ( Figure 4BC). Since LHX6 is a marker for MGE neural progenitor cells to exit the cell cycle and enter the post-mitotic phase, a large number of cells have not yet entered the post-mitotic phase at D28, and the relative proportion of positive cells is relatively low. At D28, the highest proportion of NKX2.1-mNeongreen and LHX6-tdTomato double-positive cells was in the SAG-treated group at D10, with a double-positive cell proportion of 14.04±1.822% ( Figure 4 BE).

[0341] 2) The highest proportion of GABA-positive cells in the SAG-treated group on D10 reached 83.55±5.892%, which was significantly different from the SAG-treated groups on D1, D4, and D7. The proportion of GABA-positive cells in the SAG-treated group on D13 was 74.28±1.191%, which was slightly lower than that in the SAG-treated group on D10, but there was no significant difference ( Figure 4 After mature culture, cells exited the cell cycle, entered the post-mitotic stage, and began to express LHX6. Staining results showed that the proportion of GABA and LHX6-tdTomato double-positive cells reached the highest level in the SAG treatment group on D10, reaching 59.56±7.721%. This was significantly different from the SAG treatment groups on D1, D4, and D7, but not significantly different from the SAG treatment group on D13 ( Figure 4 FI).

[0342] Example 6 Testing experiments on different cell lines

[0343] Further testing was performed using the human embryonic stem cell line CB0003 according to the methods in Examples 1-3.

[0344] The above experiments show that:

[0345] Immunofluorescence staining of D28 cells showed that there were 78.2±2.107% NKX2.1-positive and 48.26±1.302% LHX6-positive MGE neural progenitor cells ( Figure 5 AB). After further maturation and culture of the differentiated post-mitotic neural progenitor cells, immunofluorescence staining revealed that more than 80% of GABA-positive neurons were obtained, with decreased NKX2.1 expression and increased LHX6 expression. 51.4±5.418% of the cells were NKX2.1 and GABA-positive cells, and 65.5±1.803% of the cells were GABA- and LHX6-positive GABAergic interneurons derived from the MGE ( Figure 5CF). The above results show that the differentiation method of the present invention is stable and reproducible, and different human embryonic stem cell lines can be used to differentiate into MGE neural progenitor cells and MGE-derived GABAergic interneurons.

[0346] Example 7 Functional Verification of Differentiated GABAergic Interneurons

[0347] 7.1 Detection of GABA Release in Differentiated GABAergic Interneurons

[0348] One month after the cells matured, the culture supernatant of the GABAergic interneurons obtained by the aforementioned differentiation method was subjected to high performance liquid chromatography (HPLC) to detect the amount of GABA released in the culture medium.

[0349] The above experiments show that:

[0350] like Figure 6 As shown, even without KCl stimulation, the differentiated cells released GABA, and after KCl activation, GABA release increased significantly. However, only a small amount of glutamate was detected in the supernatant, and after KCl stimulation, glutamate levels barely increased. This indicates that the majority of the differentiated cells are GABAergic neurons capable of releasing GABA.

[0351] 7.2 Differentiated GABAergic Interneurons Alleviate Seizures in Mouse Models of Epilepsy

[0352] The GABAergic interneuron precursor cells differentiated in this manner were transplanted into the hippocampus of a mouse model of epilepsy induced by kainic acid (KA). The maturation and development of the in vitro differentiated GABAergic interneuron precursor cells within the brain were examined. Furthermore, epilepsy model mice that were not transplanted with these cells but injected with artificial cerebrospinal fluid (ACSF) served as a control group. The number of epileptic seizures in these mice was compared 1-2 months and 6-7 months after transplantation.

[0353] The above experiments show that:

[0354] 1) After being transplanted into the hippocampus of epileptic mouse models, the differentiated GABAergic interneuron precursor cells can mature into various subtypes of GABAergic interneurons in the brain.

[0355] 2) Six to seven months after cell transplantation, the frequency of epileptic seizures in the transplanted mice was significantly reduced, while the frequency of epileptic seizures in the non-transplanted control mice did not change significantly.

[0356] 7.3 Differentiated GABAergic Interneurons Suppress Seizures in an Angelman Syndrome Organoid Model

[0357] A brain organoid model of Angelman syndrome was constructed through gene editing, the epileptic discharges of the brain organoid were observed, and the effects of differentiated GABAergic neurons on epileptic seizures in the brain organoid model were detected.

[0358] The above experiments show that:

[0359] Differentiated GABAergic interneuronal progenitor cells can suppress epileptic seizures in a brain organoid model of Angelman syndrome.

Claims

1. A method for proliferating / differentiating pluripotent stem cells into medial ganglionic eminence (MGE) neural progenitor cells, comprising the following steps: (a) culturing cells in a medium consisting of basal medium and 0.1-0.5 μM SAG for 10 days; and (b) Cells were cultured in a medium consisting of basal medium, 0.5-2 μM PD0325901, and 5-20 μM DAPT for 7 days; To obtain MGE neural progenitor cells expressing LHX6, NKX2.1+ and LHX6+ double positive. 2 . The method according to claim 1 , wherein the concentration of SAG is 0.2 μM, the concentration of PD0325901 is 0.5 μM, and the concentration of DAPT is 10 μM.

3. The method according to claim 1, wherein the method comprises the following steps: (1) Cultivate cells in culture medium for 9 days; (2) culturing the cells in a medium consisting of basal medium and 0.1-0.5 μM SAG for 10 days; and (3) Culture cells in a medium consisting of basal medium, 0.5-2 μM PD0325901, and 5-20 μM DAPT for 7 days.

4. The method according to claim 1, wherein the method comprises the following steps: (1) Cells were cultured in a medium consisting of basal medium, 2-10 μM SB431542, 2-10 μM DMH-1, 2-5 μM IWR-1, and 0.1-1 μM Y-27632 for 6 days; (2) Cultivate cells in a medium consisting of basal medium and 0.1-1 μM Y-27632 for 3 days; (3) culturing the cells in a medium consisting of basal medium and 0.1-0.5 μM SAG for 10 days; and (4) Culture the cells in a medium consisting of basal medium, 0.5-2 μM PD0325901, and 5-20 μM DAPT for 7 days. 5 . The method according to claim 4 , wherein the concentration of SB431542 is 2 μM, the concentration of DMH-1 is 2 μM, the concentration of IWR-1 is 2.5 μM, and the concentration of Y-27632 is 0.5 μM.

6. The method according to any one of claims 1 to 5, wherein the basal culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Essential 8™, TeSR™-E8™, Essential 6, DMEM, and MEM.

7. The method according to any one of claims 1 to 5, wherein the basal medium is supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement and Glutamax.

8. The method according to any one of claims 1 to 5, wherein the basal medium is DMEM / F12 and Neurobasal mixed in a 1:1 volume ratio and supplemented with 1% by volume of MEM NEAA, 1% by volume of N2 supplement, 0.5% by volume of Glutamax, and 1% by volume of B27 supplement, or the basal medium is DMEM / F12 and Neurobasal mixed in a 1:1 volume ratio.

9. The method according to claim 1, wherein the method comprises the following steps: (1) Cells were cultured in a medium consisting of basal medium, 2-10 μM SB431542, 2-10 μM DMH-1, 2-5 μM IWR-1, and 0.1-1 μM Y-27632 for 6 days; (2) Cultivate cells in a medium consisting of basal medium and 0.1-1 μM Y-27632 for 3 days; (3) Cultivate cells in a medium consisting of basal medium and 0.1-0.5 μM SAG for 10 days; (4) culturing the cells in a medium consisting of basal medium, 0.5-2 μM PD0325901, and 5-20 μM DAPT for 7 days; and (5) GABAergic interneurons were obtained by culturing the cells for 14 days in a medium consisting of basal medium, 10-40 ng / ml GDNF, 100-200 μM amino acids, 10-40 ng / ml BDNF, 0.2-1 μM cAMP, 10-40 ng / ml IGF1, and 0.1-1 μM Compound E.

10. The method according to claim 9, wherein the concentration of GDNF is 20 ng / ml, the concentration of the amino acid is 200 μM, the concentration of BDNF is 20 ng / ml, the concentration of cAMP is 0.5 μM, the concentration of IGF1 is 20 ng / ml, and the concentration of Compound E is 0.1 μM.

11. The method according to claim 9, wherein the basal culture medium in step (5) is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Essential 8™, TeSR™-E8™, Essential 6, DMEM, and MEM.

12. The method according to claim 11, wherein the basal culture medium in step (5) is supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement and Glutamax.

13. The method according to claim 9, wherein the basal culture medium in step (5) is Neurobasal, 1% by volume of MEM NEAA, 1% by volume of N2 supplement, 1% by volume of Glutamax, and 1% by volume of B27 supplement. The method according to claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

15. A combined culture medium comprising: (i) a first medium consisting of a basal medium and SAG, wherein the concentration of SAG is 0.1-0.5 μM; and (ii) A second culture medium consisting of a basal medium, PD0325901 and DAPT, wherein the concentration of PD0325901 is 0.5-2 μM, and the concentration of DAPT is 5-20 μM. The combined culture medium according to claim 15 , wherein the concentration of SAG is 0.2 μM, the concentration of PD0325901 is 0.5 μM, and the concentration of DAPT is 10 μM.

17. The combination culture medium according to claim 15, wherein the basal culture medium of the first culture medium and the second culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Essential 8™, TeSR™-E8™, Essential 6, DMEM and MEM.

18. The combined culture medium according to claim 17, wherein the basal culture medium of the first culture medium and the second culture medium is supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement and Glutamax.

19. The combination culture medium according to claim 15, wherein the basal culture medium of the first culture medium and the second culture medium is DMEM / F12 and Neurobasal mixed in a volume ratio of 1:1 and supplemented with 1% by volume of MEM NEAA, 1% by volume of N2 supplement, 0.5% by volume of Glutamax and 1% by volume of B27 supplement, or the basal culture medium of the first culture medium and the second culture medium is DMEM / F12 and Neurobasal mixed in a volume ratio of 1:

1.

20. The combination culture medium according to claim 15, wherein the combination culture medium comprises a third culture medium consisting of a basal culture medium, 10-40 ng / ml GDNF, 100-200 μM amino acids, 10-40 ng / ml BDNF, 0.2-1 μM cAMP, 10-40 g / ml IGF1 and 0.1-1 μM Compound E.

21. The combined culture medium according to claim 20, wherein the concentration of GDNF is 20 ng / ml, the concentration of amino acids is 200 μM, the concentration of BDNF is 20 ng / ml, the concentration of cAMP is 0.5 μM, the concentration of IGF1 is 20 ng / ml, and the concentration of Compound E is 0.1 μM.

22. The combination culture medium according to claim 20, wherein the basal culture medium of the third culture medium is selected from one or more of the following culture media: DMEM / F12, Neurobasal, Neurobasal™ Plus, Essential 8™, TeSR™-E8™, Essential 6, DMEM and MEM.

23. The combined culture medium according to claim 22, wherein the basal culture medium of the third culture medium is supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement and Glutamax.

24. The combination culture medium according to claim 20, wherein the basal culture medium of the third culture medium is Neurobasal, 1% by volume of MEM NEAA, 1% by volume of N2 supplement, 1% by volume of Glutamax, and 1% by volume of B27 supplement.

Images