Method for differentiating neural precursor cells of upheaval of medial ganglion

Abstract

The present application relates to a method of proliferating / differentiating pluripotent stem cells into medial ganglion elevation (MGE) neural precursor cells, comprising the steps of: (a) culturing the cells in a culture medium comprising SAG for about 10 days; and (b) culturing the cells in a culture medium comprising PD0325901 and DAPT for about 7 days. The invention further provides a culture medium used in the method, a composition containing the culture medium and application.

Description

Technical Field

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

[0002] Normal brain function requires maintaining a balance between excitatory and inhibitory levels of neurotransmitters. Gamma-aminobutyric acid (GABA) interneurons are the main inhibitory neurons of the central nervous system. They are thought to play a key role in shaping the dynamics of inhibitory networks in the cerebral cortex, and their dysfunction can induce many neurological diseases 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 and the main source of inhibitory GABAergic interneurons. Transplantation of MGE precursor cells in the damaged brain can provide new GABA neurotransmitters and may be involved 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 a variety of methods for differentiating MGE precursor cells from human pluripotent stem cells have been reported, it is still challenging to obtain a high proportion of MGE precursor cells. 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, and the MGE precursor cells can differentiate into functional GABAergic interneurons, providing a potential treatment method for transplantation treatment of GABAergic interneuron-related nervous system 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 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 about 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 some embodiments, the concentration of PD0325901 is about 0.1-2 μM. In some embodiments, the concentration of PD0325901 is about 0.5-2 μM. In some 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) Cultivating cells in culture medium for approximately 9 days;

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

[0015] (3) Cultivating 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) Cultivate cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

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

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

[0021] (4) Culturing 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 medium may be supplemented with one or more of the following substances: NEAA, N2supplement, B27 supplement, 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) Cultivate cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

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

[0031] (3) culturing the cells in a medium containing SAG for about 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 about 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 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 medium comprising PD0325901 and DAPT.

[0042] In some embodiments, the combined medium, wherein the concentration of SAG is about 0.1-2 μM. In some embodiments, the combined medium, wherein the concentration of SAG is about 0.1-0.5 μM. In some embodiments, the combined 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 medium, wherein the concentration of DAPT is about 2.5-20 μM. In certain embodiments, the combination medium, wherein the concentration of DAPT is about 5-20 μM. In certain embodiments, the combination 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 culture 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 combined culture medium, the basal culture 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 some embodiments, in the composition, the pluripotent stem cells may be induced pluripotent stem cells. In some embodiments, in the composition, the pluripotent stem cells may be embryonic stem cells.

[0056] In certain embodiments, in the composition, the MGE neural precursor cells are NKX2.1+ and LHX6+ double positive cells. In certain embodiments, in the composition, the GABAergic interneurons 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 some embodiments, in the composition, the pluripotent stem cells may be induced pluripotent stem cells. In some embodiments, in the composition, the pluripotent stem cells may be embryonic stem cells.

[0060] In certain embodiments, in the composition, the MGE neural precursor cells are NKX2.1+ and LHX6+ double positive cells. In certain embodiments, in the composition, the GABAergic interneurons 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 the preparation of a drug for preventing and / or treating a disease.

[0062] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The features and advantages of the inventions of the present 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 results of cell flow cytometry and immunofluorescence staining of MGE neural precursor cells and GABAergic interneurons differentiated from pluripotent stem cells described in the present application. Figure 2 A shows a schematic diagram of the cell differentiation process described in the present application. Figure 2 B-2C shows a typical graph and related statistical graphs of the flow cytometry results of detecting the proportion of fluorescent cells by flow cytometry at D28 as described in the present application. Figure 2 D-2E shows typical images of immunofluorescence staining of cells fixed at D42 after mature culture as described in the present application, as well as related statistical graphs.

[0066] Figure 3 The results show the effects 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 present application showing GABA and LHX6-tdTomato positive cells by immunofluorescence staining 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 described in the present application. Figure 4 A is a schematic diagram showing the treatment of adding 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 the present 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 results of cell flow cytometry and immunofluorescence staining of inducing CB003 human pluripotent stem cells to differentiate into MGE neural precursor cells and GABAergic interneurons as described in the present application. Figure 5 A-5B shows a typical graph and related statistical graphs of the flow cytometry results of detecting the proportion of fluorescent cells by flow cytometry at D28 as described in the present application. Figure 5 C-5F shows a typical picture of immunofluorescence staining of differentiated cells after maturation culture at D42 described in the present 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 described in the present application can release the neurotransmitter GABA, and can release more GABA after the activation of neuronal KCL, but not glutamate. DETAILED DESCRIPTION

[0070] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0071] Definition of terms

[0072] In the present application, the term “comprising” generally means “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] In this application, the terms "medium" and "cell culture medium" generally refer to a nutrient source for cell growth, maintenance or differentiation. As will be appreciated by those skilled in the art, a nutrient source may contain ingredients required for cell growth, survival and / or differentiation, or may contain ingredients that aid cell growth, survival and / or differentiation. For example, the culture medium may be a 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, HamF10 nutrient mixture, HamF12 nutrient mixture, medium 199, RPMI, RPMI1640, low serum medium, basal medium (BME), Neurobasal medium, etc., and combinations thereof. The culture medium or cell culture medium may be modified by adding one or more factors, for example, supplements, differentiation factors, anti-apoptotic agents.

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

[0075] In this application, the term "proliferation" generally refers to the generation of multiple individual cells by the division of a starting cell. The multiple individual cells can be cells of the same type or cells of different types. The starting cells used for proliferation do not need to be the same as the cells produced by proliferation. For example, the cells that proliferate 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 transformed 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 the present application, the term "cell" generally refers to a living body, which is a tissue structural unit of a multicellular organism, surrounded by a membrane structure that separates it from the outside, and has genetic information and a mechanism of expression. Further, a cell may be capable of self-replication. 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 fusion 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 the present application, the term "neural precursor cell" generally refers to a cell that can migrate in vitro or in vivo and eventually differentiate into an inhibitory interneuron. The precursor cell is preferably a migratory cell that has the ability to migrate from a transplant site to a desired treatment site. The neural precursor cell may be from MGE, CGE (caudal ganglionic eminence), LGE (lateral ganglionic eminence) or another part of the mammalian brain. The neural precursor cell may be obtained by differentiation or reprogramming from other cell types. For example, the neural precursor cell may be an MGE neural precursor cell. For example, the inhibitory interneuron cell may 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. In addition, 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. In addition, 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% around 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 usually selected to have less cross-reactivity with other cell markers. These markers that determine the same cell type between species can be identified based on the same markers, and there may be differences in the structure of these markers between species (e.g., amino acid sequence). Cell markers may include cell differentiation markers, as well as gene expression markers. Gene expression markers may include expressed genes that can indicate cell type or differentiation state.

[0083] In the present application, the term "positive" generally refers to the expression of a biomarker by the target cell, for example, while "negative" generally refers to the non-expression of a biomarker 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 with the potential for proliferation and differentiation. Pluripotent stem cells can be functionally defined as cells that: (1) have the ability to differentiate into different types of cells and, in some cases, only generate one specific cell type, and (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 omnipotence.

[0085] In the present application, the term "induced pluripotent stem cells" generally refers to a type of pluripotent stem cells prepared from non-pluripotent cells in an artificial manner, 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" is also called "embryonic stem cells", which can be abbreviated as "ESC", and generally refers to cells with the characteristics of unlimited proliferation, self-renewal and multidirectional differentiation. Embryonic stem cells are stem cells obtained from the undifferentiated internal cell mass of the blastocyst (early embryonic stage). There is no restriction on their source and preparation method. Embryonic stem cells can be induced to differentiate into almost all cell types of the body, whether in vitro or in vivo. For example, the cell types can be hematopoietic stem cells, neural cells, cardiomyocytes, etc.

[0087] In this application, the term "GABAergic interneurons" generally refers to a type of neuron that activates the γ-aminobutyric acid receptors of downstream neurons by releasing γ-aminobutyric acid, causing chloride ions to flow inward, thereby achieving an inhibitory effect. According to different molecular markers, the main subtypes of GABAergic interneurons are: 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 including a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of specified amounts of specified ingredients. In this application, the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0089] In this application, the term "ex vivo" generally refers to operations involving 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 by certain methods, or enter another organism.

[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" may 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 about 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 may be known in the art and commercially available. They are used in culture medium at concentrations effective to achieve the desired results, for example, to produce MGE neural precursor cells expressing NKX2.1 and LHX6 markers. Non-limiting examples of suitable activators, inhibitors, and effective concentration ranges are further described below.

[0100] SHH pathway activators include agents, molecules, compounds or substances that can activate (upregulate) signaling of the SHH signaling pathway, which biologically involves binding of SHH to the patch homolog-1 (PTCH1) receptor and transduction through the smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway activator can be selected from one or more of the following groups: SAG, purine morphamine 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 culturing 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 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 groups: PD0325901, MEK162, XL518, selumetinib, GSK1120212, PD-184352, refactinib, 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 in the method 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 Brucenin 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) Cultivating cells in culture medium for approximately 9 days;

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

[0112] (3) Cultivating 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 the cells in a culture medium for about 9 days includes 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.

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

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

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

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

[0119] (4) Culturing 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 can inhibit (downregulate) signaling through members of the TGF-β receptor family (serine / threonine kinase receptor family). In one embodiment, the TGF-β pathway inhibitor can be selected from one or more of the following groups: SB431542, A 83-01, GW788388, SB525334, TP0427736, RepSox and SD-208. In one embodiment, the TGF-β pathway inhibitor can 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] Inhibitors of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds or substances that can inhibit (downregulate) the BMP signaling pathway. The signaling pathway is biologically activated by the binding of BMP to a BMP receptor, which is an activin receptor-like kinase (ALK). For example, type I BMP receptors, including but not limited to ALK2 and ALK3. In one embodiment, the BMP pathway inhibitor can be selected from one or more of the following groups: 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 can inhibit (downregulate) the Wnt / β-catenin signaling pathway. The pathway is biologically activated by the binding of Wnt protein ligands to the Frizzled family receptors. In one embodiment, the WNT pathway inhibitor can be selected from one or more of the following groups: 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 in the method 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 include a basal medium. For example, the basal medium may include any culture medium known in the art. For example, the basal medium may include 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 culture medium and one or more of 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 of nutrients, extracts, growth factors, hormones, cytokines and culture medium additives, and 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 added substances are not limited to the source, can be obtained from commercial sources, or 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 differentiated into MGE neural precursor cells or GABAergic internergic 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 of 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 mammalian or non-mammalian. 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) Cultivate cells 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 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 about 10 days; and

[0143] (4) Cultivate 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) Cultivate cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days;

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

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

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

[0155] (5) The cells were cultured 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 to obtain GABAergic interneurons.

[0156] In the present application, the culture may be cultured under conditions 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., about 39.5° C.

[0157] In the present application, the culture can be carried out 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, 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 include regularly or irregularly supplementing and / or replacing the culture medium. In certain embodiments, the method is regularly supplementing and / or replacing the culture medium. In certain embodiments, the method is to supplement and / or replace the culture medium every 24 hours. In certain embodiments, the method is to replace 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 include digesting the cells into single cells after the cells have proliferated to a sufficient number.

[0162] In some embodiments, the digestion in the method uses Accutase digestion solution. In some embodiments, the digestion in the method uses Trypsin and EDTA. In some 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 laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel™. In certain embodiments, the method comprises laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel™. In certain embodiments, the method comprises 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] On the other hand, the present application provides a combined 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 a 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 to obtain MGE neural precursor 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 can activate (upregulate) signaling of the SHH signaling pathway, which biologically involves binding of SHH to the patch homolog-1 (PTCH1) receptor and transduction through the smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway activator can be selected from one or more of the following groups: SAG, purine morphamine 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 groups: PD0325901, MEK162, XL518, selumetinib, GSK1120212, PD-184352, refactinib, AZD-6244, PD98059, U0126, BI-847325 and RO5126766. In one embodiment, the MEK pathway inhibitor can be PD0325901.

[0176] In the present 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 Brucenin 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 combined 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 can be selected for use. For example, the first culture medium and the second culture medium can be used; the first culture medium, the second culture medium and the third culture medium can be used; the first culture medium, the second culture medium and the fourth culture medium can be used; the first culture medium, the second culture medium and the fifth culture medium can be used; the first culture medium, the second culture medium, the third culture medium and the fourth culture medium can be used; the first culture medium, the second culture medium, the third culture medium and the fifth culture medium can be used; the first culture medium, the second culture medium, the third culture medium, the fourth culture medium and the fifth culture medium can be used. For example, the order and time of using each culture medium can be adjusted.

[0183] In the present application, the culture medium may include a basal culture medium. For the culture of cells, the basal culture medium may be a single component or a combination of multiple culture media. For example, the basal culture medium may include any culture medium known in the art. For example, the basal culture 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 culture medium, and RPMI1640. For example, different basal culture media and / or combinations thereof may be used in different steps. For example, the basal culture medium may be DMEM / F12. For example, the basal culture medium may be DMEM / F12 mixed with other culture media in a specific ratio. For example, the basal culture medium may be Neurobasal. For example, the basal culture 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 medium may be a mixed 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 of nutrients, extracts, growth factors, hormones, cytokines and culture medium additives, and 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 added substances are not limited to the source, can be obtained from commercial sources, or 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, 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, 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, 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, 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, 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, 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, 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 in different differentiation steps.

[0201] Composition and use

[0202] 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.

[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 cell and / or the state of the cell can be determined by cell markers. For example, the type of cell and / or the state of the cell 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 fillers, anti-agglomerants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc. Prepare the composition of the present invention by using any known method in the art to provide fast, continuous or delayed release of active ingredients after giving to the patient.

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

[0212] In the present application, the administration dosage of the cells may also be a single dose or multiple doses. For example, the actual administration amount of the cells may be determined according to 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] The present application also provides the following implementation modes:

[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 about 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) Cultivating cells in culture medium for approximately 9 days;

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

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

[0231] 13. A method according to embodiment 12, wherein the culturing of cells in a culture medium for about 9 days comprises 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 Rockinhibitor for about 3 days.

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

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

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

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

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

[0237] 15. The method according to 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 according to 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 culture 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 according to 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 according to any one of embodiments 1-22, comprising wherein the method comprises the following steps:

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

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

[0248] (3) culturing the cells in a medium containing SAG for about 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 about 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. A 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 according to any one of embodiments 23-27, wherein the basal culture 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 medium comprising PD0325901 and DAPT.

[0260] 31. The combined culture medium according to 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 combined 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 combined 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. A combined culture medium according to any one of embodiments 30-41, wherein the basal culture 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. A combined culture medium according to any one of embodiments 30-42, wherein the combined culture medium comprises a third culture medium containing GDNF, AA, BDNF, cAMP, IGF1 and Compound E.

[0273] 44. A 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 combined 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 basal 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. A combination culture medium according to any one of embodiments 43-47, wherein the base culture 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. A composition according to any one of embodiments 49-50, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0281] 52. A composition according to 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. A composition according to 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. A composition according to 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. A composition according to 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. A composition according to any one of embodiments 55-59, wherein the GABAergic interneurons are LHX6+ positive cells.

[0290] 61. Use of the method described in any one of embodiments 1-29, the culture medium described in any one of embodiments 30-48 and / or the composition described in 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 use of the present application, and are not intended to limit the scope of the invention of the present application.

[0292] Example

[0293] Example 1 Construction of fluorescence reporter system

[0294] Using CRISPR / Cas9 technology, green fluorescent protein mNeongreen and red fluorescent protein tdTomato were inserted 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 by fluorescent protein ( 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 about 70%-80%, digest the cells into single cells, take 2 million cells and inoculate them into a T25 cell culture flask for culture at 37°C and 5% CO 2 The cells were cultured in a culture incubator for one day under the following conditions. The composition of the culture medium is shown in Table 1.

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

[0298]

[0299] 2) The second day of subculturing was set as the first day of differentiation, D1 (day 1). The culture medium (the composition of the culture medium is shown in Table 2) was replaced and incubated at 37°C and 5% CO 2 Under the same conditions, the cells were cultured in a cell culture incubator until D6, and the medium was changed every other day.

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

[0301]

[0302] 3) On differentiation days D7-9, replace the culture medium (the composition of the culture medium is shown in Table 3) and incubate at 37°C and 5% CO 2 Under the same conditions, the cells were cultured in a cell culture incubator until D9, and the medium was changed every other day.

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

[0304]

[0305] 4) On differentiation days D10-20, replace the culture medium (the composition of the culture medium is shown in Table 4) and incubate at 37°C and 5% CO 2 Under the same conditions, the cells were cultured in a cell culture incubator until D20, and the medium was changed every other day.

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

[0307]

[0308] 5) On differentiation days D21-28, replace the culture medium (the composition of the culture medium is shown in Table 5) and incubate at 37°C and 5% CO 2 Under the same conditions, the cells were cultured in a cell culture incubator until D28, and the medium was changed every other day.

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

[0310]

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

[0312] The above experiments show that:

[0313] like Figure 2 As shown in AC, most 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 this 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 precursor cells obtained in Example 2 were attached to a glass slide coated with matrigel for further maturation culture. After the cells were completely attached to the glass slide, culture medium was added, and 1 ml of culture medium was added to one well of each 24-well plate, and cultured in a 37°C incubator for two weeks or longer until the cells were fully mature. After fixation with 4% paraformaldehyde (PFA), immunofluorescence staining was performed. The composition of the culture medium is shown in Table 6.

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

[0317]

[0318] The above experiments show that:

[0319] like Figure 2DE shows that 85.81±0.4268% of GABAergic interneuron-positive cells can be obtained, and 58.05±1.773% of cells are GABA and LHX6-tdTomato double-positive, which are GABAergic interneurons derived from MGE. This result shows that the MGE neural precursor 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) Human pluripotent stem cells in a 6-well plate grow to a density of about 70%-80%, digest the cells into single cells, take 2 million cells and inoculate them into a T25 cell culture flask for culture at 37°C and 5% CO2 in a cell culture incubator 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 subculturing was set as the first day of differentiation, D1 (day 1). The cells were cultured in a cell culture incubator at 37°C and 5% CO2 until D6. The medium was changed every other day. The composition of the culture medium is shown in Table 8.

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

[0326]

[0327] 3) On differentiation days D7-9, change the culture medium and incubate at 37°C and 5% CO 2 Under the above conditions, the cells were cultured in a cell culture incubator until D9, and the medium was changed every other day. The composition of the culture medium is shown in Table 9.

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

[0329]

[0330] 4) On differentiation days D10-28, change the culture medium and incubate at 37°C and 5% CO 2 Under the above conditions, the cells were cultured in a cell culture incubator until D28, and the medium was changed every other day. The composition of the culture medium is shown in Table 10.

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

[0332]

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

[0334] 5) The cells were cultured for maturation according to the method in Example 3. After they were fully matured, they 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 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 in 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. Under the treatment of 0.2μM concentration, the proportion of double-positive cells was the highest, 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 D28 ( Figure 4 A) At the end of differentiation, D28, the ratio of fluorescent cells was detected by flow cytometry. In addition, the cells were cultured for maturation according to the method in Example 3. After full maturation, they were fixed with 4% paraformaldehyde (PFA) and then immunofluorescence staining was performed.

[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 4 BC). Since LHX6 is a marker for MGE neural progenitor cells to jump out of the cell cycle and enter the post-mitotic period, a large number of cells have not entered the post-mitotic period at D28, and the relative proportion of positive cells is low. At D28, the highest proportion of NKX2.1-mNeongreen and LHX6-tdTomato double-positive cells was in the SAG treatment group added 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 treatment group at D10 reached 83.55±5.892%, which was significantly different from the SAG treatment groups at D1, D4, and D7. The proportion of GABA-positive cells in the SAG treatment group at D13 was 74.28±1.191%, which was slightly lower than that in the SAG treatment group at D10, but there was no significant difference ( Figure 4 FG). After mature culture, the cells jumped out of the cell cycle, entered the post-mitotic stage, and began to express LHX6. The staining results showed that the proportion of GABA and LHX6-tdTomato double-positive cells reached the highest in the SAG treatment group at D10, with a proportion of 59.56±7.721%. At the same time, there was a significant difference with the SAG treatment group at D1, D4, and D7, but no significant difference with the SAG treatment group at D13 ( Figure 4 FI).

[0342] Example 6 Experiments on different cell lines

[0343] According to the method in Examples 1-3, further testing was performed using the human embryonic stem cell line CB0003.

[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 5AB). After further maturation and culture of the differentiated post-mitotic neural progenitor cells, more than 80% of GABA-positive neurons were obtained by immunofluorescence staining, and NKX2.1 expression was reduced, LHX6 expression was increased, 51.4±5.418% of the cells were NKX2.1 and GABA double-positive cells, and 65.5±1.803% of the cells were GABA and LHX6 double-positive GABAergic interneurons derived from MGE ( Figure 5 CF). The above results show that the differentiation method of the present invention is stable and repeatable, and different human embryonic stem cell lines can be used to differentiate into MGE neural precursor 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 above differentiation method was tested by 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 in the figure, when there is no KCL stimulation, the differentiated cells can release GABA, and after KCL activates the neurons, the amount of GABA released increases significantly; while only a small amount of glutamate can be detected in the supernatant, and after KCL stimulation, the glutamate content hardly increases. The vast majority of differentiated cells are GABAergic neurons that can release GABA.

[0351] 7.2 Differentiated GABAergic interneurons alleviate seizures in epilepsy mouse models

[0352] The GABAergic interneuron precursor cells differentiated above were transplanted into the hippocampus of the kainate (KA)-induced epilepsy mouse model to detect the maturation and development of the GABAergic interneuron precursor cells differentiated in vitro in the brain. In addition, epilepsy model mice that were not transplanted with cells but only injected with artificial cerebrospinal fluid (ACSF) were used as the control group to compare the number of epileptic seizures in the model mice 1-2 months and 6-7 months after transplantation.

[0353] The above experiments show that:

[0354] 1) After the differentiated GABAergic interneuron precursor cells were transplanted into the hippocampus of epilepsy mouse models, they were able to mature into various subtypes of GABAergic interneurons in the brain.

[0355] 2) The frequency of epileptic seizures in the transplanted mice was significantly reduced 6-7 months after cell transplantation, while the frequency of epileptic seizures in the control group mice that were not transplanted with cells did not change significantly.

[0356] 7.3 Differentiated GABAergic interneurons suppress seizures in the Angelman syndrome brain organoid model

[0357] A brain organoid model of Angelman syndrome was constructed through gene editing, the epileptic discharges of the brain organoids 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 interneuron progenitor cells can suppress epileptic seizures in the 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 comprising SAG for about 10 days; and (b) Cells were cultured in medium containing PD0325901 and DAPT for about 7 days.

2. The method according to claim 1, wherein the MGE neural precursor cells are NKX2.1+ and LHX6+ double positive cells. 3 . The method according to claim 1 , wherein the concentration of SAG is about 0.1-0.5 μM, the concentration of PD0325901 is about 0.5-2 μM, and the concentration of DAPT is about 5-20 μM. 4 . The method of claim 1 , wherein the concentration of SAG is about 0.2 μM, the concentration of PD0325901 is about 0.5 μM, and the concentration of DAPT is about 10 μM.

5. The method according to claim 1, wherein the method comprises the following steps: (1) Cultivating cells in culture medium for approximately 9 days; (2) culturing the cells in a medium comprising SAG for about 10 days; and (3) Cultivating in a medium containing PD0325901 and DAPT for approximately 7 days.

6. The method according to claim 1, wherein the method comprises the following steps: (1) Cultivate cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days; (2) Cultivate cells in medium containing Rock inhibitor for approximately 3 days; (3) culturing the cells in a medium comprising SAG for about 10 days; and (4) Culturing in a medium containing PD0325901 and DAPT for approximately 7 days. 7 . The method according to claim 6 , 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. 8 . The method of claim 6 , 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.

9. The method according to claim 1, wherein the 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. 10 . The method according to claim 9 , wherein the basal medium can be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

11. The method of claim 9, 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.

12. The method according to claim 1, wherein the method comprises the following steps: (1) Cultivate cells in a medium containing SB431542, DMH-1, IWR-1, and Rock inhibitor for approximately 6 days; (2) Cultivate cells in a medium containing Rock inhibitor for about 3 days (3) culturing the cells in a medium containing SAG for about 10 days; (4) culturing the cells in a medium comprising PD0325901 and DAPT for about 7 days; and (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.

13. The method according to claim 12, 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.

14. The method of claim 12, 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.

15. The method according to claim 12, 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.

16. The method according to claim 15, 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.

17. The method according to claim 15, wherein the basal culture medium in step (5) comprises Neurobasal, about 1% MEM NEAA, about 1% N2 supplement, about 1% Glutamax and about 1% B27 supplement.

18. A combined culture medium comprising: (i) a first culture medium comprising SAG, wherein the concentration of SAG is about 0.1-0.5 μM; and (ii) a second culture medium comprising PD0325901 and DAPT, wherein the concentration of PD0325901 is about 0.5-2 μM, and the concentration of DAPT is about 5-20 μM.

19. The combined culture medium according to claim 18, wherein the concentration of SAG is about 0.2 μM, the concentration of PD0325901 is about 0.5 μM, and the concentration of DAPT is about 10 μM.

20. The combined culture medium according to claim 18, 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.

21. The combined culture medium according to claim 20, 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.

22. The combined culture medium of claim 21, 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.

23. The combined culture medium according to claim 18, wherein the combined culture medium comprises a third culture medium containing GDNF, AA, BDNF, cAMP, IGF1 and Compound E.

24. The combined culture medium according to claim 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 g / ml, and the concentration of Compound E is about 0.1-1 μM.

25. The combined culture medium according to claim 23, 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.

26. The combined culture medium according to claim 23, 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.

27. The combined culture medium according to claim 26, wherein the basal culture medium of the third culture medium can be supplemented with one or more of the following substances: NEAA, N2 supplement, B27 supplement, Glutamax.

28. The combined culture medium of claim 27, 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.

Images