Method for inducing directional differentiation of human pluripotent stem cells into GABA neurons

Abstract

The invention belongs to the field of medicines, and particularly relates to a method, a product, a culture medium and a kit for directionally inducing human pluripotent stem cells to be differentiated into GABA neural precursor cells and GABA neuronal cells and application of the GABA neural precursor cells and the GABA neuronal cells.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a method, product, culture medium, kit and use thereof for directing inducing human pluripotent stem cells to differentiate into GABA neural precursor cells and GABA neuron cells. Background Art

[0002] GABA neurons originate from the fetal brain and are inhibitory neurons widely present in the human cerebral cortex. They are believed to be closely related to learning and memory, and are involved in the regulation of anxiety and depression. GABA neurons play an inhibitory role in the transmission pathway of pain, thereby reducing the pain sensation. Depression can be treated by screening drugs that enhance the function of GABA neurons, which has a large demand for GABA neuron cells. At present, there is an urgent need for a method for the in vitro directed differentiation of pluripotent stem cells into GABA neurons that can be stably and mass-produced. Summary of the invention

[0003] The present invention provides a method, product, culture medium, kit and use thereof for directed induction of differentiation of human pluripotent stem cells into GABA neural precursor cells and GABA neuron cells. The method for directed induction of differentiation of human pluripotent stem cells into GABA neural precursor cells and neuron cells has the advantages of being stable, rapid and efficient, with a shorter culture time and avoiding serious cell death during differentiation, thus ensuring a higher survival rate and fewer fiber-like impurity cells, thereby ensuring the purity of the finished product.

[0004] The first aspect of the present disclosure discloses a method for directed induction of differentiation of human pluripotent stem cells into GABA neural precursor cells, comprising: (1) culturing human pluripotent stem cells on matrigel, digesting the human pluripotent stem cells, preferably digesting them with EDTA, inoculating them in a complete medium and culturing them for 24-72 hours, replacing fresh complete medium every 24 hours, and preferably obtaining human pluripotent stem cells to be treated after 48 hours; (2) digesting the human pluripotent stem cells to be treated with digestive enzymes to obtain single cells, and aggregating the single cells to form EB spheres after treatment; (3) transferring the EB spheres into a neural stem cell induction differentiation medium and culturing them for 2-4 days, preferably 3 days later, transferring the EB spheres into a well plate for continued 3D culture for 5-7 days, preferably 6 days, replacing fresh medium every 2 days; (4) transferring the EB spheres into a GABA neural precursor cell differentiation medium and culturing them for 2-4 days, replacing fresh medium every 2 days, and preferably obtaining GABA neural precursor cells after 3 days.

[0005] The second aspect of the present disclosure discloses a method for inducing human pluripotent stem cells to differentiate into GABA neurons, comprising: (1) culturing human pluripotent stem cells on matrigel, digesting the pluripotent stem cells, preferably digesting them with EDTA, inoculating them in a complete medium and culturing them for 24-72 hours, replacing the complete medium with fresh medium every 24 hours, and preferably obtaining the human pluripotent stem cells to be processed after 48 hours; (2) digesting the human pluripotent stem cells to be processed with digestive enzymes to obtain single cells, and aggregating the single cells after treatment to form EB spheres; (3) transferring the EB spheres into a neural stem cell induction differentiation medium and culturing them for 2-4 days, preferably 3 days later, removing the EB spheres from the culture medium; and (4) removing the EB spheres from the culture medium. The B balls are transferred to a well plate and continue 3D culture for 5-7 days, preferably 6 days, and fresh culture medium is replaced every 2 days; (4) the EB balls are transferred to a GABA neural precursor cell differentiation culture medium and cultured for 2-4 days, and fresh culture medium is replaced every 2 days, and GABA neural precursor cells are obtained after 3 days; (5) the GABA neural precursor cells are transferred to a GABA neuron induction culture medium and cultured for 5-7 days, and fresh culture medium is replaced every 2 days, and initial GABA neuron cells are obtained after 6 days; (6) the initial GABA neuron cells are transferred to a GABA neuron maturation culture medium and cultured for 4-11 days, and GABA neuron cells are obtained after 5-10 days.

[0006] The third aspect of the present disclosure discloses GABA neural precursor cells prepared according to the method of the first aspect of the present disclosure.

[0007] The fourth aspect of the present disclosure discloses a GABA neuron cell prepared according to the method of the second aspect of the present disclosure.

[0008] The fifth aspect of the present disclosure discloses the use of GABA neural precursor cells and GABA neuronal cells prepared according to the methods of the first aspect and the second aspect of the present disclosure.

[0009] The sixth aspect of the present disclosure discloses a neural stem cell induction differentiation medium, which comprises a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG.

[0010] The seventh aspect of the present disclosure discloses a GABA neural precursor cell differentiation medium, which comprises a BMP inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the BMP inhibitor is preferably Noggin, the GSK-3 inhibitor is preferably CHIR99021, the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0011] The eighth aspect of the present disclosure discloses a GABA neuron induction medium, which comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0012] The ninth aspect of the present disclosure is a GABA neuron maturation medium, which comprises a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), the Hedgehog pathway antagonist is preferably Cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

[0013] According to a tenth aspect of the present disclosure, a kit comprises any one or more of the following reagent groups (a)-(d): (a) the reagent group comprises a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG; (b) the reagent group comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably The antagonist is preferably Cyclopamine; (c) the reagent group comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine; (d) the reagent group comprises a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The whole process of directed induction of differentiation of human pluripotent stem cells into GABA neurons in Example 1 is shown.

[0015] Figure 2 The microscope images of cells showing the differentiation of human pluripotent stem cells into mature GABA neurons on days 2, 3, 12, and 25 of directed induction in Example 2.

[0016] Figure 3 Figure 3 shows the use of immunofluorescence to detect specific indicators of GABA neurons obtained under different differentiation schemes in Example 3. + / Tublin + Mature GABA neurons are formed.

[0017] Figure 4 The figure shows the specific indicators of GABA neurons obtained under different differentiation schemes detected by immunofluorescence in Example 4. The expression of DAPI, SOX1, OLIG2, and PAX6 is shown.

[0018] Figure 5The figure shows the detection of specific markers of differentiated GABA neurons by flow cytometry in Example 5.

[0019] Figure 6 The figure shows the cell viability when the GABA neural precursor cells were revived after being frozen in Example 6.

[0020] Figure 7 The typical markers expressed by iPSCs in Example 7 are shown for identification. Immunofluorescence results and flow cytometry results show high expression of NANOG, OCT4, TR-1-81 and SSEA4; IPSCs have a dense morphology and a typical pluripotent stem cell morphology. DETAILED DESCRIPTION

[0021] The following is an explanation of the implementation of the present application by means of specific embodiments. Those familiar with the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification.

[0022] Unless otherwise indicated, all numbers used in this specification and claims to indicate content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, etc. should be understood as modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the attached claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through the present disclosure, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0023] Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximate, the numerical values ​​set forth in the specific embodiments are provided as accurately as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include each narrower numerical range that falls within the broader numerical range, just as if these narrower numerical ranges were all clearly written herein.

[0024] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0025] Method for preparing GABA neural precursor cells

[0026] The present disclosure provides a method for directed induction of differentiation of human pluripotent stem cells into GABA neural precursor cells, comprising: (1) culturing human pluripotent stem cells on matrigel, digesting the human pluripotent stem cells, preferably digesting them with EDTA, inoculating them in a complete medium and culturing them for 24-72 hours, preferably 48 hours later to obtain human pluripotent stem cells to be processed; (2) digesting the human pluripotent stem cells to be processed with a digestive enzyme to obtain single cells, and aggregating the single cells to form EB spheres after treatment; (3) transferring the EB spheres into a neural stem cell induction differentiation medium and culturing them for 2-4 days, preferably 3 days later, transferring the EB spheres into a well plate and continuing 3D culture for 5-7 days, preferably 6 days; (4) transferring the EB spheres into a GABA neural precursor cell differentiation medium and culturing them for 2-4 days, preferably 3 days later to obtain GABA neural precursor cells.

[0027] In some embodiments, the matrigel is selected from basement membrane matrix or vitronectin.

[0028] In some embodiments, the complete culture medium is selected from a growth medium or a maintenance medium, further preferably E8, E8plus, mTeSR1, mTesR plus or ncTarget, further preferably mTeSR1 or mTeSR Plus.

[0029] In some embodiments, the digestive enzyme is selected from proteases, lyases, lipases or ribonucleases, further selected from proteases, and further selected from proteolytic enzymes, preferably Accutase.

[0030] In some embodiments, the single cell treatment is to inoculate the single cell on the AggreWell plate, preferably inoculate the single cell on the AggreWell plate at a density of 5E5 / ml and then centrifuge the cell; the time for the centrifugation is selected from 3-5 minutes, preferably 3 minutes; the centrifugation unit for the centrifugation is selected from 100-300g, preferably 100g.

[0031] In some embodiments, digesting the human pluripotent stem cells is treating the human pluripotent stem cells at 25-40° C., preferably 37° C., for 3-15 minutes, preferably 8-10 minutes.

[0032] In some embodiments, the neural stem cell induction differentiation medium comprises: a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is selected from LDN193189, K02288, Dorsomorphin, DMH1 or Noggin, preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is selected from Asiaticoside, SB202190, Galunisertib, A83-01, LY2109761, SB-431542 or Hydrochlorothiazide, preferably SB431542, the GSK-3 inhibitor is selected from CHIR99021, AZD2858, SB216763, TWS119 or LY2090314, further, preferably CHIR99021, and the Smo receptor agonist is selected from SAG.

[0033] In some embodiments, the GABA neural precursor cell differentiation medium comprises: a BMP inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the BMP inhibitor is selected from LDN193189, K02288, Dorsomorphin, DMH1 or Noggin, preferably Noggin, the GSK-3 inhibitor is selected from CHIR99021, AZD2858, SB 216763, TWS119 or LY2090314, further, preferably CHIR99021, the RAR nuclear receptor activator is selected from retinoic acid or RA (retinoic acid), preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0034] In this article, the term "directed induction" refers to the process of guiding stem cells to differentiate into specific cell types by regulating the internal and external environment of the cell. In this process, specific factors and technical means can be used to control the differentiation direction of stem cells and transform them into target cell types. Directed induced differentiation is one of the key technologies in regenerative medicine. It is of great significance for stem cell therapy and the construction of tissue engineered tissues and organs. Through directed induced differentiation, cells with similar morphological structures and functions can be obtained, which is the basis and key to the successful realization of stem cell therapy and the construction of tissue engineered tissues and organs. The methods of directed induced differentiation can be divided into two categories: directed induced differentiation under the in vivo microenvironment and directed induced differentiation in vitro.

[0035] Herein, the term "pluripotent stem cells" refers to stem cells that have the ability to differentiate into a variety of cell types. These cells can self-renew and differentiate into any cell type of the three germ layers (endoderm, mesoderm and ectoderm), but they cannot form a complete individual. Pluripotent stem cells can be derived from a variety of different tissues and developmental stages, including embryonic stem cells and adult stem cells.

[0036] Herein, the term "GABA neural precursor cells" refers to those neural precursor cells that have the potential to differentiate into neurons that use gamma-aminobutyric acid (GABA) as the main neurotransmitter. These cells differentiate from pluripotent stem cells or neural stem cells during the development of the nervous system and have the potential to differentiate into specific neuronal and glial cell types, especially into GABAergic neurons.

[0037] In this article, the term "EDTA" refers to Ethylene Diamine Tetraacetic Acid, which is an organic compound with a chemical formula of C10H16N2O8 and is a white powder at room temperature and pressure. 2 + , Ca 2+ , Mn 2+ , Fe 2+ Chelating agents that bind to divalent metal ions. Most cell membrane surface proteins require calcium and magnesium ions to maintain stable bonds with the extracellular matrix. Without destroying the cell membrane surface proteins, EDTA can extract these ions, inactivate adhesion proteins, and reduce cell-cell and cell-substrate connections, making it easier for cells to detach from the culture substrate when external force is applied and promoting cell dispersion.

[0038] In this article, the term "EB" stands for Embryoid Body, which is a spherical structure formed by embryonic stem cells (ES) or induced pluripotent stem cells (iPS) under certain culture conditions in vitro. It has the structures of the three germ layers: endoderm, mesoderm and ectoderm. Its morphology is very similar to that of the early embryonic development stage of mammals.

[0039] In this article, the term "neural stem cells" refers to a special type of cells present in the nervous system, which have two core characteristics: self-renewal ability and multidirectional differentiation potential. Specifically, neural stem cells can differentiate into the three main cell types of the nervous system, namely neurons, astrocytes and oligodendrocytes. These cell populations can produce a large amount of brain cell tissue and are sufficient to meet the needs of brain tissue cells. Neural stem cells play an important role in the development, repair and regeneration of the nervous system. They can not only continuously produce new nerve cells to supplement and replace damaged or aging nerve cells, but also promote the repair of damaged cells by secreting a variety of neurotrophic factors.

[0040] In this article, the term "matrigel" is a soluble basement membrane matrix extracted from mouse tumors rich in extracellular matrix proteins. Its main components include laminin, collagen type IV, entactin / nidogen, and heparan sulfate proteoglycan (HSPG). In addition, matrix gel also contains a variety of growth factors, such as transforming growth factor β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), etc. Matrix gel can self-assemble into a supermolecular structure at 37°C, which is similar to the natural basement membrane in terms of physical properties, composition and functional characteristics. It provides a supportive and protective environment for cells, simulates the structure, composition, physical properties and function of the basement membrane of cells in vivo, and is conducive to the culture and differentiation of cells in vitro. Matrix gel plays an important role in various experimental or research fields such as cell culture, tissue engineering, disease model construction and regenerative medicine.

[0041] In this article, the term "basement membrane matrix" is a specialized structural form of the extracellular matrix, which exists in a variety of tissues. It is a thin film structure composed of type IV collagen, laminin, and heparan sulfate proteoglycans. The main functions of the basement membrane matrix include providing support and structural support for epithelial cells, endothelial cells, muscle cells and other tissues. It has multiple functions, such as providing mechanical support for cells, filtration, cell adhesion and migration, and signal transmission. The basement membrane matrix not only plays a vital role in maintaining the integrity of tissue structure, cell signal transmission and barrier function, but also abnormalities in its chemical or physical properties can induce a variety of diseases including cancer.

[0042] As used herein, the term “vitronectin”, also known as S-protein or serum spreading factor, is a multifunctional glycoprotein present in plasma and the extracellular matrix. It consists of two single-chain glycoproteins (65 kD and 75 kD). Vitronectin binds to specific cell surface receptors such as integrins αVβ3 and αVβ5 mediated by the Arg-Gly-Asp (RGD) sequence. It promotes endothelial cell attachment, regulates cell adhesion, and plays a key role in tissue remodeling; it promotes cell extension and proliferation, and has a regulatory effect on the differentiation of a variety of normal cells and cancer cells. In addition, vitronectin can bind to a variety of ligands such as glycosaminoglycans, collagen, plasminogen, and urokinase receptors, and stabilize the inhibitory conformation of plasminogen activator inhibitor-1. By localizing in the extracellular matrix and binding to plasminogen activator inhibitor-1, vitronectin may regulate protein degradation of the matrix. It also binds to complement, heparin, and the thrombin-antithrombin III complex, indicating that it is involved in the regulation of immune responses and clot formation.

[0043] Herein, the term "complete medium" refers to a medium that contains all the nutrients required for culturing cells in a basic medium and can meet the growth and reproduction requirements of a specific cell. It usually contains amino acids, vitamins, inorganic salts, etc. These components provide various growth factors required for cell proliferation and other substances that are beneficial to cell survival.

[0044] As used herein, the term "E8" refers to a xeno-free, feeder-free medium formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed by Guokai Chen et al. in the laboratory of James Thomson (published as "E8") and validated by Cellular Dynamics International, this medium has been extensively tested and demonstrated to maintain the pluripotency of multiple iPSC lines. In addition, this medium has been used to increase the yield of iPSCs, and there is evidence that it supports the growth of iPSCs for >50 generations without any signs of karyotypic abnormalities, while maintaining the ability of iPSCs to differentiate into all three germ lineages.

[0045] In this article, the term "E8plus" refers to a feeder-free, animal-free culture medium designed for the growth and expansion of human pluripotent stem cells (PSC). It is based on the E8 formula developed by James Thomson's laboratory and has been extensively tested to maintain the pluripotency of multiple induced pluripotent stem cell (iPSC) lines. The characteristic of E8Plus medium is that it contains only the basic components required to maintain PSC and does not contain any animal-derived components, further reducing the risk of potential immune reactions and batch-to-batch differences, thereby reducing uncertainty and variability in the culture medium and improving the consistency and reliability of cell culture. In addition, E8Plus medium is produced under cGMP conditions, ensuring high quality and consistency, suitable for preclinical research and applications.

[0046] In this article, the term "ncTARGET" refers to a serum-free, feeder-free culture medium designed for the in vitro culture of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This culture medium can maintain the three-germ layer differentiation potential and self-renewal ability of hPSCs for a long time.

[0047] As used herein, the term "mTeSR1" refers to a feeder-free cell culture medium specifically designed for human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). It is a complete, serum-free, defined medium that supports the maintenance and expansion of these cells in an undifferentiated state. mTeSR1 medium contains recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

[0048] In this article, the term "mTeSR Plus" refers to a serum-free, feeder-free maintenance medium designed for human embryonic stem cells (ES) and induced pluripotent stem cells (iPS). mTeSR Plus is optimized based on the mTeSR1 formula, and its key ingredients (such as FGF2) are stable and reliable, ensuring the quality and consistency of cell culture. mTeSR Plus medium can support the differentiation of stem cells into specific cell types. By adjusting the proportion of ingredients or adding specific induction factors, stem cells can be differentiated into various cell types such as neurons, cardiomyocytes, and hepatocytes. mTeSR Plus medium adopts a ready-to-use design and can be used directly without adding any additional ingredients, which simplifies the experimental process and improves experimental efficiency.

[0049] Herein, the term "digestive enzyme" refers to a class of enzymes that can break down various macromolecular nutrients (such as proteins, carbohydrates, and fats) in food into small molecules so that the body can absorb and use them. These enzymes work in the digestive tract to help break down food. Common digestive enzymes include amylase (breaks down carbohydrates), protease (breaks down proteins), and lipase (breaks down fats).

[0050] As used herein, the term "trypsin" refers to a specific protease that is produced by the pancreas and whose primary function is to break down proteins into small peptides and amino acids. It is particularly active in the small intestine, especially in an alkaline environment of about pH 8, which is the pH commonly found in the small intestine. Trypsin plays a vital role in the digestive process because it helps to further break down proteins that have already been partially digested by pepsin.

[0051] As used herein, the term "Accutase enzyme" refers to a commercial enzyme mixture used for gentle and efficient cell separation in cell culture. It typically contains enzymes that can break down the extracellular matrix (ECM) and intercellular connections, such as collagenase, hyaluronidase, and neutral protease. The formulation of Accutase is optimized to ensure that the activity and integrity of cells are maintained when separating cells, making it suitable for a variety of applications that require keeping cells alive and functional, such as cell culture, cell therapy, and research. The use of Accutase can reduce the reliance on mechanical force and reduce the risk of cell damage.

[0052] As used herein, the term "AggreWell Plate" refers to a laboratory tool used to generate large numbers of highly consistent 3D spheroid cultures. It is compatible with a variety of cell types and can be used in a variety of applications, including directing the differentiation of pluripotent stem cells (PSCs) using embryoid body (EB) protocols, as well as applications in cancer research, drug discovery research, and suspension culture. AggreWell Plates contain microwells that can be 400 microns (AggreWell TM 400) or 800 microns (AggreWell TM 800) size, providing flexibility to generate spheroids of desired size to meet research needs.

[0053] Herein, the term "BMP inhibitor" refers to a class of small molecule inhibitors / antagonists that can inhibit the bone morphogenetic protein (BMP) signaling pathway. The BMP signaling pathway plays an important role in embryonic patterning and a variety of disease processes, including anemia, bone formation, atherosclerosis, skin diseases and cancer.

[0054] In this article, the term "LDN193189" is a potent and selective BMP signaling pathway inhibitor that can inhibit the transcriptional activity of BMPI type receptors ALK2 and ALK3. Its molecular structure is:

[0055]

[0056] Herein, the term "K02288" is an effective bone morphogenetic protein (BMP) type I receptor inhibitor, with IC50s of 1.8, 1.1, and 6.4 nM for ALK1, ALK2, and ALK6, respectively. The inhibition of ALK3 and ALK6 is slightly weaker, with IC50s between 5-34 nM. Its molecular structure is:

[0057]

[0058] In this article, the term "Dorsomorphin" refers to a small molecule inhibitor that can inhibit BMP type I receptors and preferentially inhibit BMP signaling rather than other ligands such as TGF-β and Activin. Its molecular structure is:

[0059] In this article, the term "DMH1" is a selective BMP-1 receptor inhibitor, and its molecular structure is:

[0060] In this article, the term "Noggin" refers to a secreted homodimeric glycoprotein encoded by the NOG gene, which is widely expressed and functions in vertebrates, and plays an important role in early embryonic development, limb formation, and nervous system development. The Noggin protein consists of an acidic amino acid N-terminus and a cysteine-rich C-terminus. Its main mechanism of action is to specifically bind to and inhibit bone morphogenetic proteins (BMPs). By binding to BMPs, Noggin can prevent BMPs from binding to their receptors, thereby inhibiting the activation of the BMP signaling pathway. This inhibitory effect is essential for regulating processes such as cell survival, proliferation, and differentiation. Noggin also plays an important role in organoid culture. By inhibiting BMP signals, it can maintain the undifferentiated state of liver progenitor cells and promote their differentiation into mature hepatocytes and bile duct cells.

[0061] As used herein, the term "TGF-β1 / Smad signaling pathway inhibitors" refers to a class of compounds that are able to inhibit the transforming growth factor β (TGF-β) signaling pathway. TGF-β superfamily proteins regulate a variety of cellular processes, such as growth, development, and differentiation. Smad proteins, intracellular effector molecules of the TGF-β signaling pathway, are activated and translocate to the cell nucleus to regulate transcription.

[0062] In this article, the term "Asiaticoside" is a triterpenoid saponin compound isolated from Centella asiatica, which inhibits TGF-βRI and TGF-βRII and TGF-β / Smad signaling pathway in keloid fibroblasts by activating Smad7. Its molecular structure is:

[0063]

[0064] Herein, the term "SB202190" is a selective p38MAPK inhibitor with IC50 values ​​of 50nM and 100nM for p38α and p38β2, respectively. SB202190 binds to the ATP pocket of recombinant human active p38 kinase with a Kd value of 38nM. SB202190 has anticancer activity and can rescue memory impairment, and its molecular structure is:

[0065]

[0066] In this article, the term "Galunisertib" is a TGF-βRI kinase inhibitor that has shown safety in patients with a variety of solid tumors and reduced growth in lung cancer and breast cancer cell lines. Its molecular structure is:

[0067] Herein, the term "A83-01" is a small molecule TGF-βRI / ALK-5 kinase inhibitor with an IC50 value of 12nM and a molecular structure of:

[0068]

[0069] Herein, the term "LY2109761" is an orally active, selective TGF-βRI / II inhibitor with Ki values ​​of 38nM and 300nM, respectively. Its molecular structure is:

[0070]

[0071] As used herein, the term "SB-431542" refers to TGF-βR kinase inhibitors that block the phosphorylation of Smad2 / 3. Its molecular structure is:

[0072] In this article, the term "Hydrochlorothiazide" refers to hydrochlorothiazide, an orally effective thiazide diuretic that inhibits the transforming TGF-β / Smad signaling pathway. Its molecular structure is:

[0073]

[0074] As used herein, the term "GSK-3 inhibitor" refers to an inhibitor of glycogen synthase kinase 3 (GSK-3). GSK-3 is a multifunctional serine / threonine kinase present in all eukaryotic organisms, and it plays a central role in a variety of signaling pathways, including those activated by Wnt, PI3K, growth factors, and G protein-coupled receptors. GSK-3 is associated with the regulation of a variety of transcription factors, including β-catenin, NF-κB, c-Jun, CREB, etc. Therefore, changes in GSK-3 activity have a variety of effects on cytokine expression.

[0075] Herein, the term "CHIR99021" is an aminopyrimidine derivative, a highly effective and selective glycogen synthase kinase 3 (GSK-3) inhibitor, with IC50 values ​​of 6.7nM and 10nM for inhibition of GSK-3β and GSK-3α, respectively. GSK-3 is a serine / threonine kinase and a key inhibitor of the Wnt signaling pathway. Therefore, CHIR99021 activates the Wnt / β-catenin signaling pathway by inhibiting GSK-3β and GSK-3α. In addition, CHIR99021 also shows selectivity for other kinases, and its selectivity for GSK-3 is more than 500 times higher than that of CDC2, ERK2 and other protein kinases. CHIR99021 can induce cell autophagy and enhance the self-renewal ability of mouse and human embryonic stem cells. CHIR99021 plays an important role in various biological processes such as stem cell culture, maintenance and self-renewal, reprogramming and differentiation. Its molecular structure is:

[0076]

[0077] In this article, the term "AZD2858" is a potent, orally available GSK-3 inhibitor that can inhibit the activity of GSK-3α and GSK-3β with IC50 values ​​of 0.9 and 5nM, respectively, and can be used in the study of fracture healing. Its molecular structure is:

[0078]

[0079] Herein, the term "SB 216763" is a potent, selective and ATP-competitive GSK-3 inhibitor with an IC50 of 34.3 nM for inhibiting GSK-3α and GSK-3β, and its molecular structure is:

[0080]

[0081] In this article, the term "TWS119" is a GSK-3β inhibitor with an IC50 of 30nM in a cell-free assay; it can induce the differentiation of neural cells and is helpful for the study of stem cell biology. Its molecular structure is:

[0082]

[0083] Herein, the term "LY2090314" is a potent GSK-3 inhibitor that inhibits GSK-3α and GSK-3β with IC50 values ​​of 1.5 nM and 0.9 nM, respectively. Its molecular structure is:

[0084]

[0085] Herein, the term "Smo receptor agonist" refers to a class of small molecule compounds that can activate Smoothened (Smo) receptors. Smo is a transmembrane protein associated with the Hedgehog signaling pathway and can transmit Hedgehog signals. Smo receptor agonists activate the Hedgehog signaling pathway by directly binding to the Smo protein, thereby promoting signal transduction.

[0086] Herein, the term "SAG" is a potent Smo receptor agonist that activates the Hedgehog signaling pathway with a Kd value of 59nM. The Sonic hedgehog signaling pathway is important in developmental processes such as dorsal-ventral neural tube patterning, neural stem cell proliferation, and neuronal and glial cell survival. Shh is also involved in the regulation of adult hippocampal neurogenesis. Its molecular structure is:

[0087]

[0088] Herein, the term "RAR nuclear receptor activator" refers to a class of chemical substances that play a key role in regulating gene expression by interacting with nuclear receptors, retinoic acid receptors (RARs). These activators are very important for studying cell differentiation, development, and proliferation because they can regulate genes involved in various biological processes. RARs are a class of nuclear receptors that act as ligand-dependent transcription factors and can regulate the expression of specific genes after binding to ligands. RARs include three subtypes: α (NR1B1), β (NR1B2), and γ (NR1B3), which play an important role in cell growth, differentiation, immunity, metabolism, apoptosis, and autophagy. RAR nuclear receptor activators can promote or inhibit the transcription of genes containing retinoic acid response elements (RAREs) by binding to RARs, thereby playing an important role in the embryonic development, organ formation, and maintenance of normal physiological functions of the body.

[0089] In this article, the term "retinoic acid" is scientifically known as Tretinoin, also known as vitamin A acid, which is an important product of vitamin A metabolism in the body. Its main function is to affect bone growth and promote epithelial cell proliferation, differentiation, and keratin dissolution and other metabolic processes. Retinoic acid is a metabolite of vitamin A in the body. Current research shows that the biological activity of vitamin A is closely related to RA (retinoic acid).

[0090] In this article, the term "retinoic acid" is also called retinoic acid, which is a metabolite of vitamin A in the body. Current research shows that the biological activity of vitamin A is closely related to RA (retinoic acid). There are two active forms of RA (retinoic acid) that are of particular interest, namely all-trans retinoic acid (ATRA, RA) and 9-cis retinoic acid (9-cis retinoicacid).

[0091] In this article, the term "Hedgehog pathway antagonist" refers to a class of small molecule compounds or biological agents that can inhibit the Hedgehog signaling pathway. The Hedgehog signaling pathway plays a key role in cell proliferation, differentiation and tissue regeneration, and its abnormal activation is associated with the occurrence and development of various cancers. Hedgehog pathway antagonists play a role in anti-tumor therapy by directly inhibiting Smo receptors or other key components in the Hedgehog signaling pathway and blocking signal transduction.

[0092] In this article, the term "Cyclopamine" is an antagonist of the Hedgehog pathway with an IC50 of 46nM in cell experiments. It is also a selective Smo inhibitor with the molecular structure of:

[0093]

[0094] The method of directing the induction of pluripotent stem cells to differentiate into GABA neural precursor cells has the advantages of being stable, rapid and efficient, with a shorter culture time and avoiding severe cell death during the differentiation process, ensuring a higher survival rate and fewer fibrous cells to ensure the purity of the finished product. The GABA neural precursor cells prepared by this method can be frozen and can be passaged normally, saving the entire differentiation time and also providing a guarantee for stability between batches.

[0095] Method for preparing GABA neuron cells

[0096] The present disclosure provides a method for inducing human pluripotent stem cells to differentiate into GABA neurons, comprising: (1) culturing human pluripotent stem cells on matrigel, digesting the human pluripotent stem cells, preferably digesting them with EDTA, inoculating them in a complete culture medium and culturing them for 24-72 hours, replacing fresh culture medium every 24 hours, and preferably obtaining pluripotent stem cells to be processed after 48 hours; (2) digesting the pluripotent stem cells to be processed with digestive enzymes to obtain single cells, and aggregating the single cells to form EB spheres after treatment; (3) transferring the EB spheres into a neural stem cell induction differentiation culture medium and culturing them for 2-4 days, replacing fresh culture medium every 2 days, and preferably transferring the EB spheres into a neural stem cell induction differentiation culture medium after 3 days; Continue 3D culture in the well plate for 5-7 days, preferably 6 days, and replace fresh culture medium every 2 days; (4) transfer the EB ball into GABA neural precursor cell differentiation culture medium and culture for 2-4 days, replace fresh culture medium every 2 days, and preferably obtain GABA neural precursor cells after 3 days; (5) transfer the GABA neural precursor cells into GABA neuron induction culture medium and culture for 5-7 days, replace fresh culture medium every 2 days, and preferably obtain initial GABA neuron cells after 6 days; (6) transfer the initial GABA neuron cells into GABA neuron maturation culture medium and culture for 4-11 days, replace fresh culture medium every 2 days, and preferably obtain GABA neuron cells after 5-10 days.

[0097] In some embodiments, the matrigel is selected from basement membrane matrix or vitronectin.

[0098] In some embodiments, the complete culture medium is selected from a growth medium or a maintenance medium, further preferably E8, E8plus, mTeSR1, mTesR plus, ncTarget, further preferably mTeSR1 or mTeSR Plus.

[0099] In some embodiments, the digestive enzyme is selected from proteases, lyases, lipases or ribonucleases, further selected from proteases, further selected from proteolytic enzymes, preferably Accutase.

[0100] In some embodiments, the single cell treatment is to inoculate the single cell on the AggreWell plate, preferably inoculate the single cell on the AggreWell plate at a density of 5E5 / ml and then centrifuge the cell; the time for the centrifugation is selected from 3-5 minutes, preferably 3 minutes; the centrifugation unit for the centrifugation is selected from 100-300g, preferably 100g.

[0101] In some embodiments, digesting the human pluripotent stem cells is treating the human pluripotent stem cells at 25-40° C., preferably 37° C., for 3-15 minutes, preferably 8-10 minutes.

[0102] In some embodiments, the neural stem cell induction differentiation medium comprises: a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is selected from LDN193189, K02288, Dorsomorphin, DMH1 or Noggin, preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is selected from Asiaticoside, SB202190, Galunisertib, A83-01, LY2109761, SB-431542 or Hydrochlorothiazide, preferably SB431542, the GSK-3 inhibitor is selected from CHIR99021, AZD2858, SB216763, TWS119 or LY2090314, preferably CHIR99021, and the Smo receptor agonist is selected from SAG.

[0103] In some embodiments, the GABA neural precursor cell differentiation medium comprises: a BMP inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the BMP inhibitor is selected from LDN193189, K02288, Dorsomorphin, DMH1 or Noggin, preferably Noggin, the GSK-3 inhibitor is selected from CHIR99021, AZD2858, SB 216763, TWS119 or LY2090314, further, preferably CHIR99021, the RAR nuclear receptor activator is selected from retinoic acid or RA (retinoic acid), preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0104] In some embodiments, the GABA neuron induction medium comprises: a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is selected from retinoic acid or RA (retinoic acid), preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0105] In some embodiments, the GABA neuron maturation medium comprises: a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is selected from retinoic acid or RA (retinoic acid), preferably RA (retinoic acid), the Hedgehog pathway antagonist is preferably Cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

[0106] In this article, the term "insulin like IGF", also known as insulin-like growth factor (IGF), is a group of polypeptide substances with growth-promoting effects. Its secretory cells are widely distributed in human tissues such as liver, kidney, lung, heart, brain and intestine. There are two types of IGF family, IGF-Ⅰ and IGF-Ⅱ. The production of IGF-Ⅰ is more dependent on GH, and its growth-promoting effect is strong. It is an important growth factor in childhood. IGF-Ⅰ synthesized in various tissues mostly exerts its growth-promoting effect in an autocrine or paracrine manner, while IGFⅠ synthesized by the liver enters the blood circulation and acts on target cells in an endocrine manner. The level of IGF-Ⅰ in the body is regulated by GH, and IGF-Ⅰ also has a negative feedback regulatory effect on the secretion of GH. IGF-Ⅱ has a stronger insulin-like effect and plays an important role in fetal growth.

[0107] In this article, the term "BDNF" is also known as brain-derived neurotrophic factor (BDNF), which is a protein with neurotrophic effects first discovered in pig brain by Barde et al. in 1982. BDNF and its receptors are widely expressed in the nervous system. Structure, distribution and signal transduction of a small molecule dimer protein BDNF The BDNF molecule monomer is a secreted mature polypeptide composed of 119 amino acid residues. The protein isoelectric point is 9.99, the relative molecular mass is 3.5×103, and it is mainly composed of β-folding and random coil secondary structures. It contains 3 disulfide bonds and is a basic protein. BDNF is distributed in a wide range of areas such as the central nervous system, peripheral nervous system, endocrine system, bone and cartilage tissue, but is mainly expressed in the central nervous system, with the highest content in the hippocampus and cortex.

[0108] In this article, the term "GDNF" is also known as glial cell derived neurotrophie factor (GDNF), which is a neurotrophic factor isolated and purified from the conditioned medium of mouse glial cell line B49 by Lin et al. (1993), and is named after it. The amino terminal sequence of purified GDNF was used to make a probe, and the GDNF gene of rat and human was cloned. The human GDNF precursor protein is 211 amino acid residues (including 19 amino acids in the signal peptide). After processing, it forms a secretory mature protein with 134 amino acids. It is a glycosylated disulfide bond-linked homodimeric protein with a molecular weight of 32-34kD and is a basic protein.

[0109] The method of directing induction of pluripotent stem cells to differentiate into GABA neuron cells has the advantages of being stable, rapid and efficient, with a shorter culture time and avoiding severe cell death during the differentiation process, ensuring a higher survival rate and fewer fibrous cells, thereby ensuring the purity of the finished product.

[0110] GABA neural precursor cells and GABA neuron cells

[0111] The present disclosure provides GABA neural precursor cells and GABA neuron cells, which are prepared by the aforementioned methods for preparing GABA neural precursor cells and GABA neuron cells.

[0112] Uses of GABA neural precursor cells and GABA neuron cells

[0113] The present disclosure provides the use of GABA neural precursor cells and GABA neuron cells prepared by the aforementioned methods for preparing GABA neural precursor cells and GABA neuron cells in preparing drugs for treating neurological diseases related to GABA levels.

[0114] In some embodiments, the GABA neuron-related neurological disease is selected from depression, insomnia, fear, pain, epilepsy, convulsions, anxiety, schizophrenia, stroke, cerebral infarction, autoimmune encephalitis, Parkinson's disease, Lennox-Gastaut syndrome, narcolepsy, migraine, postherpetic neuralgia, autism or Alzheimer's disease, etc.

[0115] Culture medium

[0116] The present disclosure provides a neural stem cell induction differentiation medium, which comprises a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG.

[0117] The present disclosure provides a GABA neural precursor cell differentiation medium, which comprises a BMP inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the BMP inhibitor is preferably Noggin, the GSK-3 inhibitor is preferably CHIR99021, the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0118] The present disclosure provides a GABA neuron induction medium, which comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine.

[0119] The present disclosure provides a GABA neuron maturation culture medium, which comprises a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), the Hedgehog pathway antagonist is preferably Cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

[0120] Reagent test kit

[0121] The present disclosure provides a kit, which comprises any one or more of the following reagent groups (a)-(d): (a) the reagent group comprises a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG; (b) the reagent group comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Preferably, it is Cyclopamine; (c) the reagent group comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine; (d) the reagent group comprises a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably RA (retinoic acid), and the Hedgehog pathway antagonist is preferably Cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

[0122] Example

[0123] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0124] Example 1: Neural Progenitor Cell Culture

[0125] The differentiation reagents used in this example are shown in Table 1 below:

[0126] Table 1. Differentiation reagents used

[0127]

[0128] Methods for inducing the differentiation of human pluripotent stem cells into GABA neurons, such as Figure 1 As shown, it includes the following steps:

[0129] 1) Human pluripotent stem cells were cultured on Matrigel or Vitronectin. The culture medium could be mTeSR1 or mTeSR Plus. The cells were digested with ETDA at 37°C for 5-8 min. EDTA was discarded and complete culture medium was added for gentle blowing. The cells were then inoculated at a ratio of 1:4. After 48 h, the cells reached a density of more than 80%, which was considered a suitable result.

[0130] 2) Preparation of EBs: The prepared human pluripotent stem cells were digested with Accutase to obtain single cells, which were inoculated into aggrewell plate at a density of 5E5 / ML and centrifuged at 100g for 3 minutes to make the cells aggregate at the bottom of the aggrewell to form EBs. Different pluripotent stem cells have different growth rates and EB-forming abilities, so it is necessary to test a cell density suitable for the cells for inoculation. For example, some cells are inoculated at a density of 5E5 / ML, while others need to be inoculated at 6E5 / ML. Basically, the density is between 3-8E5 / ML.

[0131] 3) Add neural stem cell induction differentiation medium on the first day, change the medium every other day, and transfer EBs on the third day to a 6-well plate for further differentiation. The first differentiation medium used in this stage mainly includes BMP inhibitors, TGF-β1 / Smad signaling pathway inhibitors, GSK-3 inhibitors and Smo receptor agonists, Noggin, SB431542, CHIR99021 and SAG respectively;

[0132] 4) On the ninth day, the GABA neural precursor cell differentiation medium was replaced, and the medium was replaced every other day until the twelfth day for freezing or continued differentiation. At this stage, the second differentiation medium was used, which mainly included: BMP inhibitor, GSK-3 inhibitor, RAR nuclear receptor activator and Hedgehog (Hh) pathway antagonist, Noggin, CHIR99021, RA (retinoic acid) and Cyclopamine respectively.

[0133] Example 2: Neuronal cell culture

[0134] A method for directing induction of human pluripotent stem cells to differentiate into GABA neurons, comprising the following steps:

[0135] 1) During the two-day culture period, human pluripotent stem cells were cultured on Matrigel or Vitronectin. The culture medium could be mTeSR1 or mTeSR Plus. The cells were digested with ETDA at 37°C for 5-8 min, EDTA was discarded, complete culture medium was added and gently blown, and then the cells were inoculated at a ratio of 1:4. After 48 h, the cells reached a density of more than 80%, which was considered a suitable result.

[0136] 2) Preparation of EB: Wash the prepared human pluripotent stem cells with DPBS, then add Accutase enzyme to digest at 37 degrees for 10 minutes, blow gently to obtain single cells, transfer to a centrifuge tube and centrifuge at 300g for 5 minutes, resuspend in 1ml complete medium, pass through a 37um cell sieve to ensure that it is a single cell, take 10ul for live cell counting; inoculate in an aggrewell plate at a density of 5E5 / ML, add Y27632 with a final concentration of 10uM, centrifuge at 100g for 3min, let the cells aggregate at the bottom of the aggrewell to form EB balls. Different pluripotent stem cells have certain differences in growth rate and ability to form EB, so it is necessary to test a cell density suitable for the cell for inoculation, such as some are inoculated at a density of 5E5 / ML, and some need to be inoculated at 6E5 / ML, basically between 3-8E5 / ML;

[0137] 3) On the first day, discard the complete culture medium and add 1 ml of neural stem cell differentiation medium. Change the medium every other day.

[0138] 4) On the third day, EBs were transferred out and continued to differentiate in 6-well plates. The first differentiation medium used in this stage mainly included BMP inhibitors, TGF-β1 / Smad signaling pathway inhibitors, GSK-3 inhibitors and Smo receptor agonists, Noggin, SB431542, CHIR99021 and SAG, respectively; the medium was changed every other day;

[0139] 4) On the 9th day, the original culture medium was discarded and GABA neural precursor cell differentiation medium was added. The medium was changed every other day until day 12 for freezing or continued differentiation. At this stage, the second differentiation medium was used, which mainly included: BMP inhibitor, GSK-3 inhibitor, RAR nuclear receptor activator and Hedgehog (Hh) pathway antagonist, Noggin, CHIR99021, RA (retinoic acid) and Cyclopamine respectively;

[0140] 5) On the 12th day, discard the differentiation medium, wash with DPBS, then add Accutase to digest at 37 degrees for 10 minutes, collect the cells and centrifuge at 300g for 5 minutes, add the freezing solution to the cells for freezing, or add the GABA neuron induction medium, change the medium every other day, and start to change the third differentiation medium from day 12, which mainly includes: RAR nuclear receptor activator and Hedgehog (Hh) pathway antagonist, which are RA (retinoic acid) and Cyclopamine respectively;

[0141] 6) On day 18, add GABA neuron maturation medium, change the medium every other day, and obtain GABA neurons from day 23 to day 28. Day 18 is the best start. The fourth differentiation medium is also the GABA neuron maturation medium, which mainly includes: RAR nuclear receptor activator, Hedgehog (Hh) pathway antagonist and cytokines that promote neuronal growth, namely RA (retinoic acid), Cyclopamine, insulin-like IGF, BDNF and GDNF.

[0142] Figure 2 The culture process of neuronal cells is shown.

[0143] Example 3: Immunofluorescence verification of neuronal cell maps

[0144] Immunofluorescence was used to verify the cultured neuronal cells disclosed in the present invention, and the specific steps were as follows:

[0145] 1) Take the neuronal cells to be tested that have been induced to differentiate according to the disclosure, discard the cell supernatant, and wash twice with 1 ml / well of PBS.

[0146] 2) Fixation: Add an appropriate amount (1 ml / well for a 6-well plate) of 4% paraformaldehyde for fixation and let stand at room temperature for 30 min.

[0147] 3) After fixation, discard the 4% paraformaldehyde and wash twice with PBS.

[0148] 4) Blocking: Add appropriate amount of permeabilization solution for blocking and let stand at room temperature for 60 minutes.

[0149] 5) Primary antibody binding: Take the primary antibody required for the experiment, use permeabilization solution to prepare an appropriate amount of antibody solution, dilute according to the antibody concentration, and mix well.

[0150] 6) Discard the permeabilization solution, add appropriate antibody solution to completely cover the cells, and incubate at 4°C overnight.

[0151] 7) Secondary antibody binding: Use permeabilization solution to prepare an appropriate amount of corresponding fluorescent secondary antibody, dilute it according to the antibody concentration, add DAPI (2 drops / ml), and mix well.

[0152] 8) Discard the antibody solution, wash twice with PBS, add appropriate fluorescent secondary antibody to label the cells, and incubate at room temperature for 60 minutes (protected from light).

[0153] 9) Discard the fluorescent secondary antibody and wash twice with PBS.

[0154] 10) Add appropriate amount of PBS and observe and take pictures under a fluorescence microscope. The results are as follows Figure 3 shown. Figure 3It shows that 2D-1 cells die in large numbers, and 2D-2 produces a large number of fibroblast-like cells. The 2D differentiation method is a two-dimensional, adherent differentiation method, mainly referring to Zhang Suchun's 2013 paper: Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells and 2015 paper: Generation and expansion of highly-pure motor neuron progenitors from human pluripotent stem cells, that is, without EB balls, it is directly differentiated from iPSC single cells, and then induced to generate neural stem progenitor cells (NPC), and then further induced to differentiate into mature neurons. Among them, the 2D-1 method uses SB+LDN to induce NPC cells, and then further uses RA and CYC to induce GABA neurons. In order to solve the problem of cell survival, 2D-2 optimized the use of CYC and the basal culture medium. One of them is 2D-2-1, which uses neurobasal culture medium completely and increases the amount of B27; the other is 2D-2-2, which uses DMEM / F12:neurobasal culture medium, and adds 1.5% sodium pyruvate and NEAA. It is optimized from the aspects of factors and basal culture medium. Although a large number of neural cells are obtained, on the one hand, there are more fibroblasts, and on the other hand, although tublin is expressed, only a small number of cells express GABA. The method disclosed in the present disclosure obtains a large number of GABA neurons. The antibodies used in this embodiment are shown in Table 2.

[0155] Table 2. Antibodies used in neuronal cell validation experiments

[0156]

[0157]

[0158] Example 4: Neuronal Precursor Cell Validation

[0159] Use immunofluorescence to verify the differentiated neuronal precursor cells. The specific steps are as follows:

[0160] 1) Take the neuronal precursor cells induced and differentiated by the disclosed directed differentiation method to be tested, discard the cell supernatant, and wash twice with 1 ml / well of PBS.

[0161] 2) Fixation: Add an appropriate amount (1 ml / well for a 6-well plate) of 4% paraformaldehyde for fixation and let stand at room temperature for 30 min.

[0162] 3) After fixation, discard the 4% paraformaldehyde and wash twice with PBS.

[0163] 4) Blocking: Add appropriate amount of permeabilization solution for blocking and let stand at room temperature for 60 minutes.

[0164] 5) Primary antibody binding: Take the primary antibody required for the experiment, use permeabilization solution to prepare an appropriate amount of antibody solution, dilute according to the antibody concentration, and mix well.

[0165] 6) Discard the permeabilization solution, add appropriate antibody solution to completely cover the cells, and incubate at 4°C overnight.

[0166] 7) Secondary antibody binding: Use permeabilization solution to prepare an appropriate amount of corresponding fluorescent secondary antibody, dilute it according to the antibody concentration, add DAPI (2 drops / ml), and mix well.

[0167] 8) Discard the antibody solution, wash twice with PBS, add appropriate fluorescent secondary antibody to label the cells, and incubate at room temperature for 60 minutes (protected from light).

[0168] 9) Discard the fluorescent secondary antibody and wash twice with PBS.

[0169] 10) Add appropriate amount of PBS and observe and take pictures under a fluorescence microscope. The results are as follows Figure 4 shown. Figure 4 The results show that GABA neural precursor cells were successfully obtained. The cells expressed SOX1 and PAX6 as expected, and hardly expressed OLIG2. The antibodies used in this example are shown in Table 3.

[0170] Table 3. Antibodies used in the neuronal precursor cell validation assay

[0171]

[0172]

[0173] Example 5: Detection of differentiation efficiency of neuronal cells

[0174] The GABA markers of GABA neurons were detected by flow cytometry. The specific steps are as follows:

[0175] 1) Collect GABA neurons (2 x 10 5 -1x10 6 ) Wash once with DPBS;

[0176] 2) Add 150 μl of Cyto-Fast TM Mix the Fix / Perm solution and fix at room temperature for 20 minutes;

[0177] 3) Add 1 ml of 1X Cyto-Fast TM Wash once with Perm Wash solution and centrifuge at 300 g for 5 minutes;

[0178] 4) Add GABA primary antibody to 100ul cells and stain for 1 hour at room temperature;

[0179] 5) Add the corresponding secondary antibody for staining at room temperature for 30 minutes;

[0180] 6) Wash once with cell staining buffer, then resuspend in 200ul cell staining buffer for detection. Figure 5 shown. Figure 5 The method can differentiate more than 99% of neurons, of which more than 99% specifically express GABA markers unique to GABA neurons, and the efficiency of the disclosed method for differentiating GABA neurons is more than 99%. Specific material information is shown in Table 4 below.

[0181] Table 4. Materials used for neuronal cell differentiation efficiency testing

[0182]

[0183] Example 6: Detection of cryopreservation and resuscitation viability of neuronal cells

[0184] Specific experimental steps are as follows:

[0185] 1) Two batches of neuronal cells were treated by induced differentiation. The cell culture medium was removed, the cells were washed once with DPBS, and 1 ml of TrpLE was added for digestion at room temperature for 5 minutes.

[0186] 2) Collect the neural cells, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0187] 3) Add CS10 (stem cell) freezing solution to make the cell density 1-5E6 / mL, resuspend the cells, and dispense into cryopreservation tubes for cryopreservation.

[0188] 4) Place the cryo box in a -80 degree refrigerator overnight and then transfer it to a liquid nitrogen tank.

[0189] 5) After at least one week, remove the cells from the liquid nitrogen tank, quickly lyse at 37°C, and centrifuge at 300 g for 5 minutes.

[0190] 6) Add 1 ml of culture medium to resuspend, count the cells, and read the total cell count and cell viability.

[0191] 7) The viability of cells recovered after cryopreservation was counted in Excel. Figure 6Passaging and cryopreservation of neural cells are relatively difficult experimental techniques, mainly because the differentiated cells themselves are of poor quality and can hardly be cultured after passaging or cryopreservation. Most of them will die, and even the cells that survive are in poor condition. However, the survival rate of the cryopreserved cells disclosed in the present invention after revival is above 60%. Figure 6 The GABA neuron cells induced to differentiate by the disclosed scheme can be cryopreserved using CS10 cryopreservation solution, and the cells can be continuously cultured after cryopreservation, and the viability after recovery is above 60%.

[0192] Example 7: Immunofluorescence detection of IPSC markers

[0193] The specific experimental steps of immunofluorescence were the same as those in Example 4, and the flow cytometry was the same as that in Example 5. The reagent information is shown in Table 5. Figure 7 shown. Figure 7 It shows that iPSCs highly express NANOG, OCT4, TR-1-81 and SSEA4, and have a dense morphology with typical pluripotent stem cell morphology.

[0194] Table 5. Materials used for immunofluorescence detection of IPSC markers

[0195]

Claims

1. A method for inducing human pluripotent stem cells to differentiate into GABA neural precursor cells, comprising: (1) culturing human pluripotent stem cells on Matrigel, digesting the human pluripotent stem cells, preferably digesting them with EDTA, inoculating them in a complete medium and culturing them for 24-72 hours, replacing the complete medium with fresh medium every 24 hours, and obtaining the pluripotent stem cells to be processed preferably after 48 hours; (2) digesting the human pluripotent stem cells to be processed with digestive enzymes to obtain single cells, and aggregating the single cells to form EB spheres after processing; (3) transferring the EB spheres into a neural stem cell differentiation induction medium and culturing them for 2-4 days, preferably 3 days later, transferring the EB spheres into a well plate and continuing 3D culture for 5-7 days, preferably 6 days, and replacing the medium with fresh medium every 2 days; (4) The neural EB spheres are transferred into a GABA neural precursor cell differentiation medium and cultured for 2-4 days, with the medium replaced with fresh medium every 2 days. Preferably, GABA neural precursor cells are obtained after 3 days.

2. A method for inducing human pluripotent stem cells to differentiate into GABA neurons, comprising: (1) culturing human pluripotent stem cells on Matrigel, digesting the pluripotent stem cells, preferably digesting them with EDTA, inoculating them in a complete medium and culturing them for 24-72 hours, replacing the complete medium with fresh medium every 24 hours, and obtaining the pluripotent stem cells to be processed preferably after 48 hours; (2) digesting the human pluripotent stem cells to be processed with digestive enzymes to obtain single cells, and aggregating the single cells to form EB spheres after processing; (3) transferring the EB spheres into a neural stem cell differentiation induction medium and culturing them for 2-4 days, preferably 3 days later, transferring the EB spheres into a well plate and continuing 3D culture for 5-7 days, preferably 6 days, and replacing the medium with fresh medium every 2 days; (4) transferring the EB spheres into a GABA neural precursor cell differentiation medium and culturing them for 2-4 days, replacing the medium with fresh medium every 2 days, and obtaining GABA neural precursor cells preferably after 3 days; (5) transferring the GABA neural precursor cells into a GABA neuron induction medium and culturing them for 5-7 days, replacing the medium with fresh medium every 2 days, and preferably obtaining initial GABA neuron cells after 6 days; (6) The initial GABA neuron cells are transferred into a GABA neuron maturation medium and cultured for 4-11 days, preferably 5-10 days to obtain GABA neuron cells.

3. The method according to claim 1 or 2, wherein the matrix glue is selected from basement membrane matrix or vitronectin. The method according to claim 1 or 2, wherein the complete culture medium is selected from mTeSR1 or mTeSR Plus.

5. The method according to claim 1 or 2, wherein the digestive enzyme is selected from proteolytic enzymes, such as Accutase enzyme.

6. According to the method of claim 1 or 2, the treatment of the mono-pluripotent stem cells is performed by centrifuging the mono-pluripotent stem cells after they are seeded on an AggreWell plate, preferably after the mono-pluripotent stem cells are seeded on an AggreWell plate at a density of 5E5 / ml; the time for the centrifugation is selected from 3-5 minutes, preferably 3 minutes; the centrifugation unit for the centrifugation is selected from 100-300g, preferably 100g.

7. The method according to claim 1 or 2, wherein the digesting of the pluripotent stem cells is performed by treating the pluripotent stem cells at 25-40°C, preferably 37°C, for 3-15 minutes, preferably 8-10 minutes.

8. The method according to claim 1 or 2, wherein the neural stem cell induction differentiation medium comprises: a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG.

9. The method according to claim 1 or 2, wherein the GABA neural precursor cell differentiation medium comprises: a BMP inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the BMP inhibitor is preferably Noggin, the GSK-3 inhibitor is preferably CHIR99021, the RAR nuclear receptor activator is preferably retinoic acid, and the Hedgehog pathway antagonist is preferably cyclopamine.

10. The method according to claim 1 or 2, wherein the GABA neuron induction medium comprises: a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably retinoic acid, and the Hedgehog pathway antagonist is preferably cyclopamine.

11. The method according to claim 1 or 2, wherein the GABA neuron maturation medium comprises: a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably retinoic acid, the Hedgehog pathway antagonist is preferably cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

12. GABA neural precursor cells prepared by the method according to any one of claims 1 and 3 to 9.

13. A GABA neuronal cell prepared by the method according to any one of claims 2 to 11.

14. Use of the GABA neural precursor cell according to claim 12 or the GABA neuron cell according to claim 13 in the preparation of a medicament for treating GABA neuron-related neurological diseases.

15. The method of claim 14, wherein the GABA neuron-related neurological disease is selected from the group consisting of depression, insomnia, fear, pain, epilepsy, convulsions, anxiety, schizophrenia, stroke, cerebral infarction, autoimmune encephalitis, Parkinson's disease, Lennox-Gastaut syndrome, narcolepsy, migraine, postherpetic neuralgia, autism or Alzheimer's disease.

16. A neural stem cell induction differentiation medium, comprising a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG.

17. A GABA neural precursor cell differentiation medium, comprising a BMP inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the BMP inhibitor is preferably Noggin, the GSK-3 inhibitor is preferably CHIR99021, the RAR nuclear receptor activator is preferably retinoic acid, and the Hedgehog pathway antagonist is preferably cyclopamine.

18. A GABA neuron induction culture medium comprising a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably retinoic acid, and the Hedgehog pathway antagonist is preferably cyclopamine.

19. A GABA neuron maturation culture medium, comprising a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably retinoic acid, the Hedgehog pathway antagonist is preferably cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

20. A kit comprising any one or more of the following reagent groups (a)-(d): (a) the reagent group comprises a BMP inhibitor, a TGF-β1 / Smad signaling pathway inhibitor, a GSK-3 inhibitor and a Smo receptor agonist, wherein the BMP inhibitor is preferably Noggin, the TGF-β1 / Smad signaling pathway inhibitor is preferably SB431542, the GSK-3 inhibitor is preferably CHIR99021, and the Smo receptor agonist is preferably SAG; (b) the reagent set comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably retinoic acid, and the Hedgehog pathway antagonist is preferably cyclopamine; (c) the reagent set comprises a RAR nuclear receptor activator and a Hedgehog pathway antagonist, wherein the RAR nuclear receptor activator is preferably retinoic acid, and the Hedgehog pathway antagonist is preferably cyclopamine; (d) The reagent group comprises a RAR nuclear receptor activator, a Hedgehog pathway antagonist and a neuron growth factor, wherein the RAR nuclear receptor activator is preferably retinoic acid, the Hedgehog pathway antagonist is preferably cyclopamine, and preferably, the neuron growth factor is selected from one or more of the following: insulin-like IGF, BDNF and GDNF.

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