Expansion culture medium and culture method for neural cells

Abstract

To provide a culture medium and a culture method for neural cells that can efficiently obtain a large number of neural cells.SOLUTION: The present invention relates to a composition comprising an SMAD protein signaling pathway inhibitor, an SHH signaling pathway agonist, a Wnt signaling pathway agonist, and a Myosin II ATPase inhibitor, and optionally further comprising a ROCK inhibitor, or the composition composed of the SMAD protein signaling pathway inhibitor, the SHH signaling pathway agonist, the Wnt signaling pathway agonist, the Myosin II ATPase inhibitor, and optionally the ROCK inhibitor, as well as to a culture method using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority based on Chinese Patent Application No. 202010824749.3, filed on August 17, 2020. The disclosure of this Chinese patent application is incorporated herein by reference in its entirety.

[0002] This application relates to a neuronal cell culture technology, and in particular to a neuronal expansion culture medium and culture method. By using this culture medium and culture method, a large number of neurons can be efficiently obtained, and by obtaining neurons of stable quality during subsequent differentiation, cell therapy for diseases caused by neuronal loss (Parkinson's disease) can be realized. [Background technology]

[0003] Parkinson's disease is a degenerative disorder of the central nervous system that commonly affects middle-aged and elderly people. The primary etiology is the mass death of midbrain dopaminergic neurons in the substantia nigra, resulting in decreased synthesis of dopamine transmitters in the brain and thereby affecting behavior. Midbrain dopaminergic neurons in the substantia nigra differentiate from midbrain floor plate cells during embryonic development and project to the striatum, forming a loop. After maturation, midbrain dopaminergic neurons release dopamine transmitters, playing an important role in regulating individual movement.

[0004] Mesencephalic floor plate cells can be isolated from early embryos or obtained by in vitro differentiation from pluripotent stem cells. Existing differentiation methods simulate the signaling pathways of early embryonic development in vivo by dually inhibiting the SMAD signaling pathway using BMP and TGF inhibitors and activating Wnt and SHH signaling to induce the specialization of pluripotent stem cells into mesencephalic floor plate cells. Then, under treatment with FGF8, DAPT, BDNF, GDNF, etc., the mesencephalic floor plate cells further differentiate into dopaminergic neurons.

[0005] Midbrain dopaminergic neurons are a specific subpopulation of neurons. During the culture process, a small amount of Wnt signaling and a high dose of SHH are required during the precursor stage, followed by the addition of FGF8 to promote maturation. Compared to other cells, the culture conditions are quite unique. The current problems with the culture process are primarily due to: 1) the heterogeneity of the quality of midbrain floor plate cells differentiated in vitro from pluripotent stem cells; 2) poor batch stability; and 3) the lack of a stable bulk expansion process for midbrain dopaminergic neurons.

[0006] Furthermore, other neuronal or neuro-related cells, such as GABA progenitor cells, astrocytes and microglial cells, also play important roles in Parkinson's disease, but they also face the problem of unstable expansion. Summary of the Invention

[0007] After much creative research, the inventors have found that by adjusting the composition of the culture medium and adding the myosin II ATPase inhibitor blebbistatin, the expansion efficiency of neural or neural-related cells, such as midbrain dopaminergic precursors, GABA precursors, astrocytes, and microglial cells, can be significantly increased, and high-quality cells can be selected for batch expansion, thereby providing a stable method for obtaining high-quality neural or neural-related cells (e.g., dopaminergic neurons, GABA precursors, astrocytes, microglial cells, etc.), laying the foundation for cell therapy for Parkinson's disease and other diseases caused by a deficiency of dopaminergic cells.

[0008] In a first aspect, the present application provides a composition comprising one or more additives for maintaining and / or improving neuronal activity and / or function, and a myosin II ATPase inhibitor.

[0009] In some embodiments, the composition comprises a SMAD signaling pathway inhibitor, a SHH signaling pathway agonist, a Wnt signaling pathway agonist, and a myosin II ATPase inhibitor, and optionally further comprises a ROCK inhibitor; Alternatively, the composition consists of a SMAD signaling pathway inhibitor, an SHH signaling pathway agonist, a Wnt signaling pathway agonist, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor.

[0010] In embodiments of the present application, myosin II ATPase inhibitors refer to myosin II type ATPase inhibitors. In certain embodiments, myosin II ATPase inhibitors include blebbistatin and its derivatives, such as blebbistatin, i.e., [ka] (S)-(-)-blebbistatin having the structure: [ka] (S)-(-)-blebbistatin-O-benzoate having the structure:

[0011] In certain embodiments, the SMAD signaling pathway inhibitor is selected from a BMP inhibitor, a TGFβ / activin-nodal inhibitor, and combinations thereof.

[0012] In embodiments of the present application, the BMP (bone morphogenetic protein) inhibitor is an inhibitor of the BMP signaling pathway. In certain embodiments, the BMP inhibitor is selected from the group consisting of DMH-1, dorsomorphin, noggin, LDN193189, and any combination thereof. Among them, LDN193189 refers to the small molecule DM-3189, whose chemical formula is C 25 H 22N6, whose chemical name is 4-(6-(4-(piperidin-1-yl)phenyl)-pyrazolo[1,5-a]pyrimidin-3-yl)quinoline. LDN193189 acts as an inhibitor of the SMAD signaling pathway and is also a potent small molecule inhibitor of ALK2, ALK3, ALK6 and PTK. LDN193189 can inhibit the signaling pathway of TGFβ1 receptor ALK1 and ALK3 family members, thereby inhibiting multiple biological signaling pathways including BMP (e.g., BMP2, BMP4, BMP6, BMP7) and activin, and the subsequent phosphorylation of SMAD (e.g., Smad1, Smad5 and Smad8).

[0013] In an embodiment of the present application, the TGFβ / activin nodal inhibitor is a substance that inhibits the pathway in which TGFβ binds to its receptor and continuously transmits signals to SMAD, and can be selected from substances that inhibit binding to the ALK family as a receptor or substances that inhibit SMAD phosphorylation caused by the ALK family. In a specific embodiment, the TGFβ / activin nodal inhibitor is selected from the group consisting of SB431542, SB505124, A83-01, and any combination thereof. Among them, SB431542 has CAS number 301836-41-9 and molecular formula C 22 H 18 It refers to N4O3, and a small molecule with the chemical name 4-[4-(1,3-benzodioxan-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide, that can reduce or block transcription of the TGF / activin-nodal signaling pathway.

[0014] In embodiments of the present application, an SHH signaling pathway agonist refers to a substance that binds SHH to its receptor, Patched (Ptch1), resulting in disinhibition of Smoothened (Smo) and further activation of Gli2. In certain embodiments, the SHH signaling pathway agonist is selected from the group consisting of an SHH protein, a Smoothened agonist, and a combination thereof. In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHH and terminally modified SHH (e.g., SHH C25II). In certain embodiments, the Smoothened agonist is selected from the group consisting of SAG, Hh-Ag1.5, 20α-hydroxycholesterol, purmorphamine, and any combination thereof. SAG has the molecular formula C 28 H 28 ClN3OS, and has the CAS number 912545-86-9. Purmorphamine is a purine derivative with the CAS number 483367-10-8, which can activate the Hedgehog signaling pathway, including targeting smoothened.

[0015] In embodiments of the present application, the Wnt signaling pathway refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors. In certain embodiments, the Wnt signaling pathway agonist is selected from the group consisting of a GSK3β inhibitor, Wnt3A, Wnt1, and combinations thereof.

[0016] In embodiments of the present application, the GSK3β inhibitor refers to a compound that inhibits glycogen synthase kinase 3β. The GSK3β inhibitor of the present application can activate the Wnt signaling pathway. In certain embodiments, the GSK3β inhibitor is selected from the group consisting of CHIR99021, GSK3β inhibitor IX (6-bromoindirubin-3'-oxime, BIO), GSK3β inhibitor VII (4-dibromoacetophenone), indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azakempaullone, SB216763, or any combination thereof. Among them, CHIR99021 refers to 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidine-2-amino)ethylamino)nicotinonitrile, a small molecule inhibitor of GSK3β that can activate the Wnt signaling pathway.

[0017] In embodiments of the present application, the ROCK inhibitor is a substance that inhibits the function of Rho kinase (ROCK), such as Y-27632, HA100, or HA1152. In certain embodiments, the ROCK inhibitor is Y-27632.

[0018] In some embodiments, the composition comprises a BMP inhibitor, a TGFβ / activin-nodal inhibitor, an SHH protein, a smoothened agonist, a GSK3β inhibitor, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor; Alternatively, the composition consists of a BMP inhibitor, a TGFβ / activin-nodal inhibitor, an SHH protein, a smoothened agonist, a GSK3β inhibitor, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor.

[0019] In certain embodiments, the composition comprises LDN193189, SB431542, SHH, SAG, CHIR99021, blebbistatin, and optionally Y-27632; Alternatively, the composition consists of LDN193189, SB431542, SHH, SAG, CHIR99021, blebbistatin, and optionally Y-27632.

[0020] In certain embodiments, the composition comprises N2 supplement, B27 supplement, bFGF (recombinant basic fibroblast growth factor) and blebbistatin; Alternatively, the composition consists of N2 supplement, B27 supplement, bFGF and blebbistatin.

[0021] In a second aspect, the present application provides a culture medium comprising the aforementioned composition and a basal culture medium.

[0022] In certain embodiments, the basal culture medium is suitable for culturing neural cells.

[0023] In some embodiments, the basal culture medium is selected from the group consisting of N2 medium, DMEM medium, DMEM / F12 medium, Neurobasal culture medium, or / and Sciencell 1801 medium.

[0024] In certain embodiments, the basal culture medium is N2 medium.

[0025] In certain embodiments, the basal culture medium is a basal culture medium supplemented with one or more of the following substances: serum-free substitute, glutamine, or a stabilizing dipeptide of L-alanyl-L-glutamine.

[0026] In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following substances: N2 supplement, and a stabilizing dipeptide of L-alanyl-L-glutamine.

[0027] In certain embodiments, the basal culture medium is composed of the following: 49% CTS™ KnockOut™ DMEM / F-12 + 49% CTS™ Neurobasal + 1% CTS™ N2 Supplement + 1% CTS-GlutaMAX™-I.

[0028] In certain embodiments, the composition of N2 medium is as follows: 49% KODMEM + 49% Neurobasal + 1% N2 supplement + 1% GlutaMAX.

[0029] In some embodiments, the concentration of each component in the medium is an effective concentration to perform its respective biological function.

[0030] In some embodiments, the concentration of the BMP inhibitor may be, for example, but not limited to, 0.05 μM to 1 μM. In some specific embodiments, the concentration of the BMP inhibitor (e.g., LDN193189) may be 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM. A preferred concentration is 100 nM.

[0031] In certain embodiments, the concentration of the TGFβ / activin-nodal inhibitor can be, but is not limited to, 5 μM to 20 μM. In some specific embodiments, the concentration of the TGFβ / activin-nodal inhibitor (e.g., SB431542) can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM. A preferred concentration is 10 μM.

[0032] In certain embodiments, the concentration of the SHH protein can be, but is not limited to, 50 ng / mL to 200 ng / mL. In some specific embodiments, the concentration of the SHH protein (e.g., recombinant SHH) is, for example, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, or 200 ng / mL. A preferred concentration is 100 ng / mL.

[0033] In certain embodiments, the concentration of the smoothened agonist can be, but is not limited to, 0.5 μM to 3 μM. In some specific embodiments, the concentration of the smoothened agonist (e.g., SAG) is 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.2 μM, 1.4 μM, 1.6 μM, 1.8 μM, 2.0 μM, 2.5 μM, or 3.0 μM. A preferred concentration is 2.0 μM.

[0034] In certain embodiments, the concentration of the GSK3β inhibitor can be, but is not limited to, 0.5 μM to 1.0 μM. In some specific embodiments, the concentration of the GSK3β inhibitor (e.g., CHIR99021) is 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1.0 μM.

[0035] In certain embodiments, the concentration of the myosin II ATPase inhibitor can be, but is not limited to, 5 μM to 20 μM. In some specific embodiments, the concentration of the myosin II ATPase inhibitor (e.g., blebbistatin) is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM. A preferred concentration is 10 μM.

[0036] In certain embodiments, the concentration of the ROCK inhibitor can be, but is not limited to, 1 μM to 50 μM. In certain embodiments, the concentration of the ROCK inhibitor is 1 μM to 30 μM, for example, 5 μM to 30 μM. In some specific embodiments, the concentration of the ROCK inhibitor (e.g., Y-27632) is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM. A preferred concentration is 10 μM.

[0037] In some embodiments, the medium comprises N2 medium, 10 μM SB431542, 100 nM LDN193189, 100 ng / mL SHH, 2 μM SAG, 0.5 μM to 1.0 μM CHIR99021, and 10 μM blebbistatin.

[0038] In some embodiments, the culture medium is suitable for culturing, for example, to expand midbrain dopamine neuron precursors.

[0039] In certain embodiments, the basal culture medium is DMEM / F12 and Neurobasal.

[0040] In some embodiments, the medium contains 49.25% DMEM / F12, 49.25% Neurobasal, 0.5% N2 supplement, 1% B27 supplement, 10 ng / ml bFGF (recombinant basic fibroblast growth factor), and 5 μM to 20 μM blebbistatin.

[0041] In certain embodiments, the medium is suitable for culture, for example, to expand GABAergic neuron precursors.

[0042] In some embodiments, the basal culture medium is NIM (Neural Induction Media).

[0043] In some embodiments, the culture medium comprises DMEM / F12, Neurobasal, N2 supplement, B27 supplement, and Glutamax10, EGF, FGF2, TGFb1, and 5 μM to 20 μM blebbistatin.

[0044] In certain embodiments, the medium is suitable for culturing, for example, astrocytes for expansion.

[0045] In some embodiments, the basal culture medium is DMEM / F12, KOSR, 1× Glutamax, 1× NEAA, BMP4, VEGF, SCF, X-VIVO, IL-34, Y-27632, and 5 μM to 20 μM blebbistatin.

[0046] In certain embodiments, the culture medium is suitable for culturing, for example, to expand microglial cells.

[0047] In some embodiments, the medium contains 49.25% DMEM / F12, 49.25% Neurobasal, 0.5% N2 supplement, 1% B27 supplement, 10 ng / ml bFGF (recombinant basic fibroblast growth factor), and 5 μM to 20 μM blebbistatin.

[0048] In certain embodiments, the medium is suitable for culture, for example, to expand GABAergic neuron precursors.

[0049] In certain embodiments, the basal culture medium is Sciencell 1801.

[0050] In certain embodiments, the medium contains 5 μM to 20 μM blebbistatin.

[0051] In certain embodiments, the culture medium is suitable for culturing, for example, glial cells for expansion.

[0052] In certain embodiments, the medium is a basal culture medium supplemented with one or more of the following: one or more serum-free substitutes, glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following: N2 supplement, B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and blebbistatin. In certain embodiments, the medium is composed of: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 10 ng / ml EGF + 10 ng / ml FGF2 + 10 ng / ml TGFβ1 + 10 μM blebbistatin. In some embodiments, the medium is particularly suitable for the expansion of astrocytes.

[0053] In certain embodiments, the medium is a basal culture medium supplemented with one or more of the following substances: one or more serum-free substitutes, glutamine or L-alanyl-L-glutamine stabilizing dipeptide, an SHH signaling pathway inhibitor, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following substances: N2 supplement, B27 supplement, L-alanyl-L-glutamine stabilizing dipeptide, SHH, purmorphamine, and blebbistatin. In certain embodiments, the medium is composed of: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 100 ng / mL SHH + 1 μM purmorphamine + 10 μM blebbistatin. In certain embodiments, the medium is particularly suitable for expanding GABAergic neural progenitor cells.

[0054] In certain embodiments, the medium is a basal culture medium supplemented with the following substances: glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following substances: a stabilized dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof. In certain embodiments, the medium is composed of: X-VIVO15 + XGlutamax + 25 ng / ml IL-34 + 50 ng / ml M-CSF + 10 μM blebbistatin. In some embodiments, the medium is particularly suitable for the expansion of microglial cells.

[0055] In certain embodiments, the medium is a basal culture medium supplemented with one or more of the following: one or more serum-free substitutes, glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following: B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin, and blebbistatin. In certain embodiments, the medium is composed of: DMEM / F12 + 20 μg / mL EGF + 20 μg / mL bFGF + 5 μg / ml heparin + 2% B27 + 1×GlutaMAX + 10 μM blebbistatin. In some embodiments, the culture medium is particularly suitable for expanding neural stem cells.

[0056] In certain embodiments, the basal culture medium described in any of the above paragraphs is selected from the group consisting of DMEM / F12, Neurobasal, Neural Induction Media, and X-VIVO.

[0057] In another aspect, the present application provides a kit comprising the composition or culture medium described above, and optionally further comprising instructions for use.

[0058] In another aspect, the present application provides use of a myosin inhibitor (e.g., blebbistatin, or a derivative thereof (e.g., (S)-(-)-blebbistatin O-benzoate)), or a culture medium containing the myosin inhibitor, for maintaining or increasing the number of nerve cells or nerve-related cells in vitro.

[0059] In certain embodiments, the neuronal or neuron-related cells are selected from the group consisting of neural progenitor cells, motor neuron precursors, cortical neuron precursors, GABAergic neuron precursors, serotonin neuron precursors, midbrain dopaminergic neuron precursors, or neural stem cells, or glial cells (e.g., astrocytes or microglial cells).

[0060] In some embodiments, the medium is the medium described in the second aspect above, which is particularly useful for maintaining or expanding the number of midbrain dopaminergic neural progenitor cells in vitro.

[0061] In certain embodiments, the medium is a basal culture medium supplemented with one or more of the following: one or more serum-free substitutes, glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following: N2 supplement, B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and blebbistatin. In certain embodiments, the medium is composed of: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 10 ng / ml EGF + 10 ng / ml FGF2 + 10 ng / ml TGFβ1 + 10 μM blebbistatin. This is particularly useful for maintaining or expanding the number of midbrain dopaminergic neural progenitor cells in vitro. This is particularly useful for maintaining or increasing astrocyte numbers in vitro.

[0062] In certain embodiments, the medium is a basal culture medium supplemented with one or more of the following: a serum-free substitute, a stabilizing dipeptide of glutamine or L-alanyl-L-glutamine, an inhibitor of the SHH signaling pathway, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following: N2 supplement, B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, SHH, purmorphamine, and blebbistatin. In certain embodiments, the medium is composed of: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 100 ng / mL SHH + 1 μM purmorphamine + 10 μM blebbistatin. This is particularly useful for maintaining or expanding the number of GABAergic neural progenitor cells in vitro.

[0063] In certain embodiments, the medium is a basal culture medium supplemented with the following substances: glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following substances: a stabilized dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof. In certain embodiments, the medium is composed of: X-VIVO15 + XGlutamax + 25 ng / ml IL-34 + 50 ng / ml M-CSF + 10 μM blebbistatin, which is particularly useful for maintaining or increasing the number of microglial cells in vitro.

[0064] In certain embodiments, the medium is a basal culture medium supplemented with one or more of the following substances: one or more serum-free substitutes, glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin, and blebbistatin or a derivative thereof. In certain embodiments, the basal culture medium is a basal culture medium supplemented with the following substances: B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin, and blebbistatin. In certain embodiments, the medium is composed of: DMEM / F12 + 20 μg / mL EGF + 20 μg / mL bFGF + 5 μg / ml heparin + 2% B27 + 1×GlutaMAX + 10 μM blebbistatin, which is particularly suitable for maintaining or increasing the number of neural stem cells in vitro.

[0065] In certain embodiments, the basal culture medium described in any of the above is selected from the group consisting of DMEM / F12, Neurobasal, Neural Induction Media, and X-VIVO.

[0066] In another aspect, the present application provides a method for maintaining or increasing the number of cells in vitro, comprising culturing cells in the aforementioned medium, or use of the aforementioned medium for maintaining or increasing the number of cells in vitro.

[0067] In some embodiments, the method of culturing includes preparing the cells into a single cell suspension, and culturing the single cell suspension to a concentration of 2×10 4 / cm 2 ~6×10 4 / cm 2 (preferably 4×10 4 / cm 2 In certain embodiments, the cells are seeded in a culture medium at a density of 4 x 10, either adherent or suspension culture, and the cells are passaged once every 5 to 8 days, with the culture conditions for each passage remaining the same. 4 / cm 2 Inoculate at a density of .

[0068] In some embodiments, the conditions for adherent culture include preparing cells into a single cell suspension, and preparing the single cell suspension at a concentration of 2×10 4 / cm 2 ~6×10 4 / cm 2 In a specific embodiment, the cells are seeded in a culture medium at a density of 4×10 to 4×10, adherent culture is performed, and the cells are passaged once every 5 to 8 days, with the culture conditions for each passage being the same. 4 / cm 2 Inoculate at a density of .

[0069] In some embodiments, the conditions for suspension culture include preparing cells into a single cell suspension, and preparing the single cell suspension at a concentration of 2×10 5 / mL ~ 6 × 10 5 In certain embodiments, the cells are inoculated into a culture medium at a density of 4×10 / mL, cultured in suspension, and passaged once every 5 to 8 days, with the culture conditions for each passage being the same. 5 Inoculate at a density of 1000 / mL.

[0070] In certain embodiments, the cell is a motor neuron progenitor cell. In certain embodiments, the cell is a cortical neural progenitor. In certain embodiments, the cell is a GABAergic neuron progenitor. In certain embodiments, the cell is a serotonergic neural progenitor. In certain embodiments, the cell is an astrocyte. In certain embodiments, the cell is a neural stem cell. In certain embodiments, the cell is a microglial cell.

[0071] In certain embodiments, the expanded cells are genomically identical to the primary cells.

[0072] In another aspect, the present application provides a cell or cell population prepared or expanded by any of the methods described above. In another aspect, the present application provides a cell or cell population in which more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells express at least one or more of the following markers: PCDHGB1, SOX3, SEMA3D, VGF, NEFL, NTRK2, PCDHGA3, CNTN1, BDNF, STMN1, TNC, FAIM2, CHGB, GAP43, ARPP21, ALCAM, OTP, KCNF1, FOXP1 , RTN1, MAPT, IGFBP5, NNAT, CHRNA6, C1QL1, INA, TNR, PHLDA1, ELAVL3, TENM1, NRN1, CRMP1, SCG2, PMP22, and NSG1, preferably wherein the expression level of the marker is at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression level of the primary cell.

[0073] In certain embodiments, in a cell or population of cells, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells express at least one or more of the following markers: MTND4LP7, AL031777.3, HIST1H2AC, HIST1H1C, HIST1H4H, SYNPO2, LMO2, MGAT2, PDXP, DNAJC6, DNAJC22, ELN, MIR568, M IR1179, MIR6892, MIR7-3HG, ANGPTL1, HSPE1-MOB4, INO80B-WBP1, R3HDML, PMF1-BGLAP, PLP1, AP002748.4, MDFI, RCN3, FST, HSPH1, PCBP1, ASPN, TSPAN8, LINC01866, LEFTY2, GMNC, ATP5MF-PTCD1, CCDC96, ALG14, IL11, A2M, C4B, ITGB4, STC1, TMEM229B, MUC5AC, TAC1, CRABP1, CRABP2, H19, C22orf42, RCAN2, PCSK1, VAT1L, CXCL12, DCN, SSTR1, MAP7D2, PPP2R2C, L RFN5, DIRAS3, CA10, C4A, AP002373.1, AMIGO3, GDA, EDIL3, CFH, TGFBI, CLSTN2, FBLN5, HPCAL4, ADCYAP1R1, NNMT, CD44, S The cells specifically express MOC1, CLEC3B, DLX5, LYNX1, SYNC, TCAF1P1, CD9, COL3A1, CAVIN1, LMO4, TCF12, GDE1, GNG3, PEG10, TFPI2, CENPF, CAMK2N1, and MLLT11, and preferably the expression level of the markers is at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression level of the primary cells.

[0074] In certain embodiments, the method for preparing a cell or cell population comprises the steps defined in the method for maintaining or increasing cell numbers in vitro, as described in the above aspects.

[0075] In certain embodiments, the cell is a neuron or neuro-related cell.

[0076] In certain embodiments, the cells are selected from the group consisting of motor neuron precursors, cortical neuron precursors, GABAergic precursors, serotonin neuron precursors, midbrain dopaminergic precursors, astrocytes, neural stem cells, and microglial cells.

[0077] In certain embodiments, the cells are expanded from the primary cells by at least about 1.5x, 2.0x, 3x, 5x, 10x, 20x, 30x, 40x, 50x, 100x, 150x, 200x, 500x, 1000x, 10000x, or 100000x.

[0078] In another aspect, the cells or cell populations provided herein are midbrain dopaminergic progenitors. In certain embodiments, at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, e.g., 100%) of the cells express at least one marker of midbrain dopaminergic neuronal progenitors, such as FOXA2, LMX1A, and OTX2. In certain embodiments, the cells do not express TUJ1. In certain embodiments, the cells express TUJ1. In certain embodiments, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells in the midbrain dopamine neuron precursors express at least one or more of the following markers: PCDHGB1, SOX3, SEMA3D, VGF, NEFL, NTRK2, PCDHGA3, CNTN1, BDNF, STMN1, TNC, FAIM2, CHGB, GAP43, ARPP21, ALCAM. , OTP, KCNF1, FOXP1, RTN1, MAPT, IGFBP5, NNAT, CHRNA6, C1QL1, INA, TNR, PHLDA1, ELAVL3, TENM1, NRN1, CRMP1, SCG2, PMP22, and NSG1, and preferably the expression level of the markers is at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression level of the primary cells.

[0079] In certain embodiments, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells in the midbrain dopamine neuron precursors express at least one or more of the following markers: MTND4LP7, AL031777.3, HIST1H2AC, HIST1H1C, HIST1H4H, SYNPO2, LMO2, MGAT2, PDXP, DNAJC6, DNAJC22, ELN, MIR5. 68, MIR1179, MIR6892, MIR7-3HG, ANGPTL1, HSPE1-MOB4, INO80B-WBP1, R3HDML, PMF1-BGLAP, PLP1, AP002748.4, MDFI, RC N3, FST, HSPH1, PCBP1, ASPN, TSPAN8, LINC01866, LEFTY2, GMNC, ATP5MF-PTCD1, CCDC96, ALG14, IL11, A2M, C4B, ITGB4, ST C1, TMEM229B, MUC5AC, TAC1, CRABP1, CRABP2, H19, C22orf42, RCAN2, PCSK1, VAT1L, CXCL12, DCN, SSTR1, MAP7D2, PPP2R2C , LRFN5, DIRAS3, CA10, C4A, AP002373.1, AMIGO3, GDA, EDIL3, CFH, TGFBI, CLSTN2, FBLN5, HPCAL4, ADCYAP1R1, NNMT, CD44 , SMOC1, CLEC3B, DLX5, LYNX1, SYNC, TCAF1P1, CD9, COL3A1, CAVIN1, LMO4, TCF12, GDE1, GNG3, PEG10, TFPI2, CENPF, CAMK2N1, and MLLT11, and preferably the expression level of the markers is at least 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression level of the primary cells.

[0080] In another aspect, the present application provides a method comprising: (1) culturing the expanded midbrain dopamine neuron precursors in a first culture medium for 6 days; (2) culturing the cells obtained in step (1) in a second culture medium for 8 days to obtain a cell or a cell population; providing a cell or cell population prepared by the first culture medium is a basal culture medium supplemented with the following substances: BDNF, GDNF, TGF-β3, FGF8, AA, and Y-27632, preferably the first culture medium is a basal culture medium supplemented with the following substances: 10 ng / ml to 50 ng / ml BDNF, 10 ng / ml to 50 ng / ml GDNF, 0.5 ng / ml to 5 ng / ml TGF-β3, 50 ng / ml to 200 ng / ml FGF8, 0.1 mM to 1 mM AA, and 1 mM to 30 mM Y-27632, preferably the first culture medium is a basal culture medium supplemented with the following substances: 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, 100 ng / ml FGF8, 0.2 mM AA, and 10 mM Y-27632; The second culture medium is a basal culture medium supplemented with the following substances: BDNF, GDNF, TGF-β3, FGF8, AA, db-cAMP, and DAPT. Preferably, the second culture medium is a basal culture medium supplemented with the following substances: 10 ng / ml to 50 ng / ml BDNF, 10 ng / ml to 50 ng / ml GDNF, 0.5 ng / ml to 5 ng / ml TGF-β3, 500 to 200 FGF8, 0.1 mM to 1 mM AA, 200 μM to 1000 μM db-cAMP, and 5 μM to 20 μM DAPT. Preferably, the second culture medium is a basal culture medium supplemented with 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, 100 FGF8, 0.2 mM AA, 500 μM db-cAMP, and 10 μM DAPT.

[0081] In certain embodiments, the basal culture medium is B27.

[0082] In certain embodiments, the expanded midbrain dopamine neuron precursors obtained above are prepared into a single cell suspension, and 1×10 4 / cm 2 ~1×10 6 / cm 2 (e.g., 1×10 4 / cm 2 , 5×104 / cm 2 , 1×10 5 / cm 2 , 5×10 4 / cm 2 or 1 x 10 6 / cm 2 ) into the first culture medium at a density of 1000 x g.

[0083] In another aspect, the present application further provides a pharmaceutical composition comprising a cell or cell population described in any one of the above aspects.

[0084] In another aspect, the present application further provides use of a cell or cell population described in any one of the above aspects in the manufacture of a medicament for treating a nervous system disease (e.g., a disease caused by dopaminergic neuron damage (e.g., Parkinson's disease)).

[0085] Definition of Terms As used herein, the term "dopamine" refers to a chemical produced by dopaminergic neurons that send messages to neurons in the brain that control movement and coordination.

[0086] As used herein, the term "neural progenitor cells" refers to immature neural cells, including various types of neural progenitor cells, such as, for example, motor neuron precursors, cortical neuron precursors, GABAergic neuron precursors, and serotonin neuron precursors.

[0087] As used herein, the term "midbrain dopaminergic progenitors" has the same meaning as "midbrain floor plate cells" and refers to cells located in the midbrain that can develop into dopaminergic neurons, including midbrain dopaminergic neurons that are still in the embryonic developmental stage. In certain embodiments, midbrain dopaminergic progenitors express one or more of the following cell markers: EN1, FOXA2, LMX1A, OTX2, etc.

[0088] As used herein, the term "dopaminergic neurons" generally refers to cells that release dopamine. "Midbrain dopaminergic neurons" refer to cells in the forebrain that release dopamine. These cells express one or more of the following markers: FOXA2, LMX1A, TH, TUJ1, etc. As used herein, the term "signaling pathway" refers to a series of responses for downstream proteins to exert their effects, which are activated or otherwise influenced by ligand binding to a membrane protein or some other stimulus (e.g., the SMAD pathway, the Wnt pathway). Regulators of the Wnt pathway include β-catenin, GSK3β, etc. For many cell surface or intracellular receptor proteins, ligand-receptor interactions are not directly related to cellular responses. A receptor activated by a ligand must interact with other intracellular proteins before the ligand can exert a physiological effect on cellular behavior. Typically, the behavior of several cellular proteins that interact in a chain is altered by receptor activation or inhibition. The overall cellular changes induced by receptor activation are called cellular transduction mechanisms or signaling pathways.

[0089] As used herein, the terms "inhibit," "block," or "prevent" refer to a decrease in the activity of a particular signaling pathway in cells treated with a compound (i.e., an inhibitor). [Effects of the Invention]

[0090] Beneficial Effects of the Invention The present application has achieved at least one of the following technical effects by adjusting the components of the culture medium, adding the myosin II ATPase inhibitor blebbistatin, and expanding the culture to obtain various nerve cells or nerve-related cells, such as midbrain dopaminergic precursors.

[0091] 1. The expansion efficiency of neurons or neural-related cells, especially midbrain floor plate cells or midbrain dopaminergic precursors, is significantly improved, thereby enabling large-scale cell expansion and the establishment of cell lines useful for scientific research or commercialization.

[0092] 2. Based on the improvement of expansion efficiency, a technical procedure for expanding the intermediate products in the differentiation process of neural progenitor cells is designed, which makes it possible to obtain a large number of seed cells from the same batch and establish a seed cell bank.

[0093] 3. In some embodiments, the expanded midbrain dopaminergic precursors can still express floor plate cell markers FOXA2, LMX1A, OTX2, etc. and can still be used for subsequent cryopreservation.

[0094] 4. The manufacturing costs, especially the costs during the pluripotent stem cell culture phase, can be reduced, and as a result, the preparation process can be more easily controlled.

[0095] 5. Cells differentiated from expanded midbrain dopamine neuronal precursors specifically express various novel markers and show good therapeutic activity in a rat PD model.

[0096] BRIEF DESCRIPTION OF THE DRAWINGS The drawings described in this specification are used to provide a further understanding of the present invention and constitute a part of this application. The outline examples of the present invention and their description are used to explain the present invention and do not constitute undue limitations on the present invention. The drawings are as follows: [Brief explanation of the drawings]

[0097] [Figure 1] FIG. 1 shows the process of derivation and expansion of floor plate cells derived from human embryonic stem cells. [Figure 2] FIG. 1 shows cell growth curves for each passage of bottom plate expansion media 1 and 2. [Figure 3]FIG. 1 shows the expression of floor plate cell markers LMX1A and FOXA2 in floor plate cells obtained by subculture for 10 passages using floor plate cell expansion medium 1. [Figure 4] FIG. 1 shows the expression of dopamine neuron markers FOXA2, TH, and TUJ1 in cells obtained by differentiation of floor plate cells cultured for 10 passages using Floor Plate Cell Expansion Medium 1. [Figure 5] FIG. 1 shows the expression of cell markers FOXA2 and LMX1A after 10 passages of subculture using bottom plate cell expansion medium 2. [Figure 6] FIG. 1 shows the expression of dopaminergic neuron markers FOXA2, LMX1A, TH, and TUJ1 in cells obtained by differentiation of floor plate cells cultured for 12 passages using Floor Plate Cell Expansion Medium 2. [Figure 7] FIG. 1 shows the therapeutic effect of striatal injection of 2.5×10 5 cells in a rat PD model detected by rotation experiments. [Figure 8] Figure 1 shows the cell mass of astrocytes from p1 to p6 under the culture conditions of the experimental group and the control group: +SMI:NPM+10 ng / ml EGF+10 ng / ml FGF2+10 ng / ml TGFβ1+10 μm blebbistatin; −SMI:NPM+10 ng / ml EGF+10 ng / ml FGF2+10 ng / ml TGFβ1. [Figure 9] This figure shows the cell mass of GABA precursor cells from p4 to p5 under the culture conditions of the experimental group and the control group: +SMI:NPM+100 ng / mL SHH+1 μM purmorphamine+10 μM blebbistatin; −SMI:NPM+100 ng / mL SHH+1 μM purmorphamine. [Figure 10] Figure 1 shows the amount of suspended cells collected from EBs cultured on days 4-11 in microglial differentiation medium (collection 1): control: X-VIVO15 + 1xGlutamax + 25ng / ml IL-34 + 50ng / ml M-CSF; SMI: X-VIVO15 + 1xGlutamax + 25ng / ml IL-34 + 50ng / ml M-CSF + 10µM blebbistatin. [Figure 11]Figure 1 shows the amount of suspended cells collected from EBs cultured on days 11-18 in microglial differentiation medium (collection 2): control: X-VIVO15 + 1xGlutamax + 25ng / ml IL-34 + 50ng / ml M-CSF; SMI: X-VIVO15 + 1xGlutamax + 25ng / ml IL-34 + 50ng / ml M-CSF + 10µM blebbistatin. [Figure 12] Figure 1 shows the amount of suspended cells collected from EBs cultured on days 18-25 in microglial differentiation medium (collection 3): control: X-VIVO15 + 1xGlutamax + 25ng / ml IL-34 + 50ng / ml M-CSF; SMI: X-VIVO15 + 1xGlutamax + 25ng / ml IL-34 + 50ng / ml M-CSF + 10µM blebbistatin. [Figure 13] Figure 1 shows the total number of NSCs at various expansion passages: A: DMEM / F12 + 20 μg / mL EGF + 20 μg / mL bFGF + 5 μg / mL heparin + 2% B27 + 1×GlutaMAX; B: DMEM / F12 + 20 μg / mL EGF + 20 μg / mL bFGF + 5 μg / mL heparin + 2% B27 + 1×GlutaMAX + 10 μM blebbistatin. DETAILED DESCRIPTION OF THE INVENTION

[0098] Specific Modes for Carrying Out the Invention The technical solutions in the embodiments of the present invention are clearly and completely described with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely exemplary in nature and is not to be construed as any limitation of the present invention, its application, or use. Any other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the protection scope of the present invention. [Example]

[0099] Example 1. Floor plate cell expansion experiment 1. Reagents used in floor plate cell expansion experiments: (1) Preparation and working concentrations of each factor: 1) LDN193189 solution: The working concentration was 100 nM. This could be stored at 4°C for 2 weeks. 2) SB431542 solution: The working concentration was 10 μM. This could be stored at 4°C for 2 weeks. 3) SAG solution: The working concentration was 2 μM. This could be stored at 4°C for 2 weeks. 4) SHH (Sonic Hedgehog) solution: The working concentration was 100 ng / mL. This could be stored at 4°C for 2 weeks. 5) CHIR99021 solution: The working concentration was 0.5 μM to 1.0 μM. This could only be used once. 6) Blebbistatin solution: The working concentration was 10 μM. This solution could be stored at 4°C for 2 weeks.

[0100] (2) Preparation of basal culture medium N2 medium: 49%CTS(TM) KnockOut(TM) DMEM / F-12+49%CTS(TM) Neurobasal+1%CTS(TM) N2 Supplement+1%CTS-GlutaMAX(TM)-I

[0101] (3) Bottom Plate Cell Expansion Medium 1: N2 medium + LDN193189 (100 nM) + SB431542 (10 μM) + SHH (100 ng / mL) + SAG (2 μM) + CHIR99021 (0.5 μM–1.0 μM);

[0102] (4) Bottom Plate Cell Expansion Medium 2: N2 medium + LDN193189 (100 nM) + SB431542 (10 μM) + SHH (100 ng / mL) + SAG (2 μM) + CHIR99021 (0.5 μM–1.0 μM) + blebbistatin (10 μM);

[0103] 2. Reagents used in the differentiation experiment of dopamine neurons from expanded floor plate cells: (1) Preparation and working concentrations of each factor: 1) FGF8 solution: The working concentration was 100 ng / mL. This could be stored at 4°C for 2 weeks. 2) TGFβ3 solution: The working concentration was 1 ng / mL. This could be stored at 4°C for 2 weeks. 3) Ascorbic acid (AA) solution: The working concentration was 0.2 mM. This could be stored at 4°C for 2 weeks. 4) Dibutyryl cAMP (db-cAMP) solution: The working concentration was 0.5 mM. This could be stored at 4°C for 2 weeks. 5) BDNF solution: The working concentration was 20 ng / mL. This solution could be stored at 4°C for 2 weeks. 6) GDNF solution: The working concentration was 20 ng / mL. This could be stored at 4°C for 2 weeks.

[0104] (2) Preparation of basal culture medium B27 medium: 97%CTS(TM)Neurobasal+2%B27-CTS(TM)+1%CTS-GlutaMAX(TM)-I

[0105] (3) Preparation of dopamine neuron differentiation medium 1: B27 medium + BDNF (20 ng / mL) + GDNF (20 ng / mL) + TGF-β3 (1 ng / mL) + AA (0.2 mM) + FGF8 (100 ng / mL). After mixing each factor solution well, it was added little by little to obtain differentiation medium 1, which was then repeatedly pipetted.

[0106] (4) Preparation of dopamine neuron differentiation medium 2: B27 medium + BDNF (20 ng / mL) + GDNF (20 ng / mL) + TGF-β3 (1 ng / mL) + AA (0.2 mM) + db-cAMP (500 μM) + DAPT (10 μM) - After mixing each factor solution well, the solution was added little by little to obtain differentiation medium 2, which was then repeatedly pipetted.

[0107] 3. Method of floor plate cell expansion experiment: Referring to the method in Figure 1, human embryonic stem cells were subjected to induced differentiation to obtain floor plate cells, which were then used for subsequent expansion, cryopreservation, and differentiation experiments. Human embryonic stem cells were derived from the National Stem Cell Resource Bank, and commercially available human embryonic stem cell lines could also be used. For methods of deriving floor plate cells from human embryonic stem cells, please refer to the prior art, e.g., doi:10.1038 / nprot.2017.078.

[0108] (1) Method for obtaining floor plate cells: 1) Preparation of vitronectin matrix: At room temperature, 6 mL of DPBS was pipetted into a 15 mL centrifuge tube, and then 60 μL of vitronectin was pipetted into the DPBS. The mixture was mixed thoroughly by pipetting 10 times with a pipette gun to obtain a vitronectin matrix (prepared just before use). 1 mL of the prepared matrix was added to each well of a 6-well plate and incubated at room temperature for 1 hour.

[0109] 2) Human embryonic stem cells were digested into single cells with TrypLE or accutase, and 10 μL of the above suspension was added to 10 μL of trypan blue solution and mixed well. Counting was performed using a Countess cell counter. The vitronectin matrix was then discarded by pipetting, and the cells were digested with TrypLE or accutase to obtain single cells. These were then cultured at 2 × 10 in differentiation medium containing 10 μM Rock inhibitor (e.g., Y-27632). 4 / cm 2 The cells were seeded at a density of 1000 and cultured in a 5% CO2 incubator at 37°C. The differentiation medium contained BMP inhibitors, TGF inhibitors, Wnt activators, and SHH activators. After several days, mesencephalic base cells differentiated from human embryonic stem cells were obtained.

[0110] (2) Floor plate cell expansion experiment: 1) Preparation of vitronectin matrix: At room temperature, 6 mL of DPBS was pipetted into a 15 mL centrifuge tube, and then 60 μL of vitronectin was pipetted into the DPBS. The mixture was mixed thoroughly by pipetting 10 times with a pipette gun to obtain a vitronectin matrix (prepared just before use). 1 mL of the prepared matrix was added to each well of a 6-well plate and incubated at room temperature for 1 hour.

[0111] 2) The bottom plate cells were digested into single cells with TrypLE or accutase, and 10 μL of the above suspension was added to 10 μL of trypan blue solution, mixed well, and counted using a Countess cell counter. The vitronectin matrix was then discarded by pipetting, and the cells were cultured at 4 × 10 cells per well using the 2D method in Bottom Plate Cell Expansion Medium 1 and Bottom Plate Cell Expansion Medium 2 (both supplemented with 10 μM Rock inhibitor (e.g., Y-27632)). 4 / cm 2 The cells were re-inoculated at a density of 1000 and cultured in a 5% CO2 incubator at 37°C.

[0112] 3) The cells were passaged every 5 to 8 days, and the inoculation density, medium, and conditions for each passage were the same.

[0113] At each passage, the total number of cells was calculated using a countess cell counter and a cell growth curve was generated.

[0114] The proliferation efficiency of expansion performed using bottom plate cell expansion media 1 and 2 is shown in Figure 2. Under the treatment of Expansion Medium 1 and Expansion Medium 2, the cell numbers of Cell 1 and Cell 2 continued to increase with increasing cell passage number, while the proliferation rate gradually decreased. Cell 1 could be expanded for 10 passages, and Cell 2 could be expanded for 12 passages, and at the same time, the cell number of Cell 2 was 48 times that of Cell 1. The results demonstrated that Expansion Medium 2 could significantly increase the expansion capacity of cells.

[0115] In base plate cell expansion medium 1, the cell number gradually increased and the proliferation rate gradually decreased with increasing cell passage number. After 10 passages, the cells could be expanded 1000-fold, and immunofluorescence staining showed that the cells still stably expressed the base plate cell markers FOXA2 and LMX1A (Figure 3).

[0116] In base plate cell expansion medium 2, the cell number gradually increased with increasing passages, while the proliferation rate gradually decreased. After 10 passages, the cells could be expanded 50,000-fold, and immunofluorescence staining showed that the cells still stably expressed the base plate cell markers FOXA2 and LMX1A (Figure 5).

[0117] 4. Cryopreservation experiment of expanded floor plate cells: 1) The programmed cooling box and freezing solution were pre-cooled at 4°C for use.

[0118] 2) The condition of the cells was observed under an inverted microscope to confirm that the cells were in good condition and free of contamination.

[0119] 3) The culture medium was discarded in a biological safety cabinet, and 1 mL of DPBS was added to each well of the 6-well plate to wash the cells.

[0120] 4) Discard the DPBS, add 1 mL of Tryple to each well, and perform digestion for 3 to 4 minutes in a 37 °C incubator (the digestion time using Tryple could be adjusted depending on the degree of cell digestion).

[0121] 5) The trypsin was discarded, and 1 mL of basal culture medium N2 medium was added to each well to terminate the incubation. The cells were gently pipetted with a pipette gun to form small particles of uniform size, then collected into a centrifuge tube and centrifuged at 1200 rpm for 3 minutes at room temperature.

[0122] 6) The supernatant was discarded and pre-chilled freezing solution was added to resuspend the cells (one cryopreservation tube for every 1 / 6 well of the cell suspension, 1 mL / tube).

[0123] 7) The cell suspension was subpackaged into cryopreservation tubes (1 mL / tube). The cryopreservation tubes were then placed in a pre-cooled freezer box and stored in a -80°C refrigerator overnight. The next day, they were transferred to liquid nitrogen for long-term storage.

[0124] 5. Experiment to differentiate expanded floor plate cells into dopamine precursor cells: 1) Preparation of vitronectin matrix: At room temperature, 6 mL of DPBS was pipetted into a 15 mL centrifuge tube, and then 60 μL of vitronectin was pipetted into the DPBS and mixed well by pipetting 10 times with a pipette gun to obtain a vitronectin matrix (prepared just before use). 1 mL of the prepared matrix was added to each well of a 6-well plate and incubated at room temperature for 1 hour for later use.

[0125] 2) The bottom plate cells obtained by 10 passages using bottom plate cell expansion medium 1 and 2 were digested with TrypLE or accutase to form single cells, and then cultured at 5 × 10 cells each in dopamine neuron differentiation medium 1 (after mixing evenly) containing a Rock inhibitor (e.g., Y-27632) using the 2D method. 5 / cm 2 The cells were re-inoculated at a density of 1000 and cultured in a 5% CO2 incubator at 37°C.

[0126] 3) After culturing in Dopamine Neuron Differentiation Medium 1 for 6 days, the medium was replaced with Dopamine Neuron Differentiation Medium 2, and the cells were cultured for an additional 8 days.

[0127] 4) Differentiated dopamine neurons were identified by immunofluorescence, and the results are shown in Figures 4 and 6.

[0128] The results showed that immunofluorescence of markers identified dopamine neurons obtained by further differentiation of Cell 1 obtained after 10 passages of expansion in Floor Plate Cell Expansion Medium 1. The results also demonstrated that Cell 1 obtained after 10 passages of expansion still had the ability to differentiate into dopamine neurons and could stably express the dopamine neuron markers FOXA2, TH, and TUJ1.

[0129] Immunofluorescence of markers identified dopamine neurons obtained by further differentiation of cell 2 obtained after 12 passages of expansion in floor plate cell expansion medium 2. The results also demonstrated that cell 2 obtained after 12 passages of expansion still had the ability to differentiate into dopamine neurons and could stably express the dopamine neuron markers FOXA2, LMX1A, TH, and TUJ1.

[0130] Immunofluorescence Identification Method: a) Cell inoculation: A clean, sterile circular cover slip was placed at the bottom center of a 24-well cell culture dish and coated with vitronectin for 30 minutes before inoculation with cells.

[0131] b) Cell fixation: After the cells grew to an appropriate density, the culture medium was discarded, and the cells were washed once with PBS and fixed with 4% PFA at room temperature for 15–30 min, then washed three times with PBS.

[0132] c) Blocking: 320 μL of a mixed solution of 2% BSA and 0.3% Triton was added to each well, and blocking was carried out at room temperature for 2 hours.

[0133] d) Addition of primary antibody: The primary antibody was diluted with a mixture of 2% BSA and 0.3% Triton (see the instruction manual for the primary antibody for the ratio), mixed well by pipetting, discarded the blocking solution, and added 320 μL of the diluted primary antibody solution to each well. The wells were sealed with parafilm and left to stand overnight at 4°C.

[0134] e) Addition of secondary antibody: The primary antibody was discarded by pipetting, the cells were washed twice with PBS, and 320 μL of secondary antibody diluted in a mixture of 2% BSA and 0.3% Triton was added to each well (see the instruction manual for the primary antibody for the usage ratio), and the cells were left in the dark at room temperature for 2 hours.

[0135] f) Nuclear staining: The secondary antibody was discarded, the cells were washed twice with PBS, 320 μL of diluted Hoechst33342 solution (1 μl Hoechst33342 in 1 ml PBS) was added to each well, and the cells were incubated for 15 minutes.

[0136] g) Sealing: The diluted Hoechst33342 solution was discarded by pipetting, the cells were washed once with PBS, then 10 μL of antiquencher was added to each slide, the slide with the cells growing on it was carefully picked up using a syringe needle in conjunction with curved tweezers, and the side with the cells was placed on the slide with the antiquencher added, avoiding air bubbles, finally, colorless nail polish was lightly placed on the edge of the cover glass to fix the slide, then it was dried for 5 minutes and placed in a slide box.

[0137] 5) The differentiated cells were subjected to RNA extraction and expression profile sequencing. The specific method was based on the published literature of Kim et al., Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. 2021, Cell Stem Cell 28, 343-355.

[0138] The results showed that the expression levels of dopamine neurons obtained by differentiating floor plate cells cultured in expansion medium were more than 10-fold higher than those of non-expanded cells (Tables 1-2), and the markers are listed in the tables.

[0139] [Table 1]

[0140] [Table 2-1]

[0141] [Table 2-2]

[0142] [Table 2-3]

[0143] [Table 2-4]

[0144] 6. PD model behavior detection method (rotation experiment): Experimental equipment: rotary recorder, counter, weighing scale. Laboratory reagents: Apomorphine (sigma, A4393).

[0145] The rotation experiment steps are as follows: a) The animals were weighed. b) Animals were anesthetized by intraperitoneal injection of 5% chloral hydrate according to body weight. c) Preparation and injection of apomorphine: Powdered apomorphine was dissolved in saline to 1 mg / ml and injected intraperitoneally at a dose of 0.5 mg / kg. d) Counting began 5 to 10 minutes after the apomorphine injection or when the model rats began to circle around. Recording was continued for 30 minutes, and the average number of rotations per minute was calculated.

[0146] The cell injection procedure was as follows: Experimental equipment: stereotaxic apparatus, electric infusion pump, skull drill, surgical scissors, tweezers, needle forceps, 5-0 suture with needle, Hamilton 701 syringe, heating pad, ultra-clean workbench, etc. Laboratory reagents: povidone-iodine, saline, rubbing alcohol, Baytril, cyclosporine. a) Injection of immunosuppressant: 48 hours before transplantation, the immunosuppressant cyclosporine was intraperitoneally injected at a dose of 10 mg / kg, and injections were continued daily until the end of the experiment. b) Preparation of cell suspension: The culture supernatant of the cells to be transplanted was discarded, and the cells were washed once with PBS. Then, tryptophan was added and digested for 5 to 8 minutes. The cell suspension was collected in a centrifuge tube and centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and the cells were suspended in the cell injection solution at a concentration of 1.25 × 10 cells. 5 Resuspended at 1000µL / µL. c) Injection of cell suspension: 2 μL of the above cell suspension (2.5 × 10 5 Cells) were injected into the striatum of each animal on the model side, and the coordinates of the striatum were as follows: A, +1.0 mm; L, -3.0 mm; V, -6.0 mm, -5.0 mm; one needle, two depths, 1 μL injected at each depth, the injection rate was 1 μL / min, and the wound was sutured after injection. d) Postoperative management: After surgery, Baytril was administered subcutaneously at 0.5 mL / 200 g body weight daily for three consecutive days. Rotational tests were performed monthly after the injection, and the changes in rotational frequency over five months were plotted. Significant functional improvement was observed at three months (Figure 7).

[0147] Example 2. Astrocyte expansion experiment 1. Differentiation and culture methods: (1) hESCs were cultured and differentiated until they reached approximately 80% confluence. On day 0, the cells were dissociated with dispase (1 U / mL), and the bottom cell mass was collected and seeded onto an ultra-low-attachment 10-cm culture dish.

[0148] (2) Neural induction was initiated by replacing the medium with NIM supplemented with the neural induction molecules 100 nM LDN193189 and 10 μM SB431542 from day 1. The medium was changed daily until day 7.

[0149] (3) The cell spheres were spread onto Matrigel-coated 6-well plates on the 7th day.

[0150] (4) From day 9, the medium was replaced with NPM medium, and 20 ng / mL of FGF2 was added until day 13.

[0151] (5) On day 13, single cells were obtained by dissociation using Accutase, and 1.2 × 10 5 cells / cm 2 The cells were then re-spread onto Matrigel-coated 6-well plates at a density of 1000 μg / well.

[0152] (6) On day 14, the medium was replaced with astrocyte induction medium, and NPM medium was supplemented with 10 ng / ml EGF, 10 ng / ml FGF2, and 10 ng / ml TGFβ1, along with 10 μM blebbistatin (1:1000, not added to the control group). The medium was replaced every other day.

[0153] (7) After the astrocyte density reached 100% confluence, they were dissociated into single cells with Accutase, counted, and plated onto Matrigel-coated 6-well plates at 3.0 × 10 5 cells / cm 2 It was spread again at a density of .

[0154] (8) The cells were serially passaged five times, with the initial cell density of each passage being 3.0 × 10 5 cells / cm 2 and compared the proliferation of astrocytes when blebbistatin was added.

[0155] [Table 3]

[0156] [Table 4]

[0157] 2. Experimental results: From P2 to P6, the expansion capacity of astrocytes in the experimental group was significantly improved (Fig. 8).

[0158] Example 3. Proliferation of GABA precursor cells 1. Differentiation and culture methods: (1) hESCs were cultured and differentiated until they reached approximately 80% confluence. On day 0, the cells were dissociated with dispase (1 U / mL), and the bottom cell mass was collected and spread onto an ultra-low-attachment 10 cm culture dish.

[0159] (2) Neural induction was initiated using NIM medium supplemented with 100 nM LDN193189, 10 μM SB431542, and 2 μM XAV939 from day 1. The medium was changed daily until day 7.

[0160] (3) The cell spheres were spread onto Matrigel-coated 6-well plates on the 7th day.

[0161] (4) From day 9, the medium was replaced with NPM medium, and 20 ng / mL of FGF2 was added until day 13.

[0162] (5) On day 13, single cells were obtained by dissociation using Accutase and plated on Matrigel-coated 6-well plates at 3.0 × 10 5 cells / cm 2 It was spread again at a density of .

[0163] (6) On day 14, the medium was replaced with GABAergic cell induction medium, and 100 ng / mL SHH and 1 μM purmorphamine were added to the NPM medium, along with 10 μM blebbistatin (not added to the control group). The medium was changed every other day.

[0164] (7) After the density of GABA precursor cells reached 100% confluence, they were dissociated into single cells with Accutase, counted, and then plated on Matrigel-coated 6-well plates at 3.0 × 10 5 cells / cm 2It was spread again at a density of .

[0165] (8) The cells were passaged five times, with the initial cell density of each passage being 3.0 × 10 5 cells / cm 2 We compared the proliferation of GABA precursor cells in the presence or absence of blebbistatin.

[0166] 2. Experimental results: From P4 to P5, the expansion capacity of GABA precursor cells in the experimental group was significantly improved (Figure 9).

[0167] Example 4. Microglial precursor proliferation 1. Differentiation and culture methods: (1) On day 0, ESCs were digested with Accutase to single cells, counted, and seeded into V-bottom 96-well plates at a concentration of 10,000 / well for static culture.

[0168] (2) From day 0 to day 4, the culture medium was microglia induction medium: DMEM / F12, 20% KOSR, 1× Glutamax, 1× NEAA, 50 ng / ml BMP4, 50 ng / ml VEGF, 20 ng / ml SCF, 10 μM Y-27632.

[0169] (3) On day 4, the suspended EBs were cultured in 6-well plates for adherent culture. The culture medium was replaced on day 4, and static culture was continued until day 11. From day 4 to day 11, the culture medium was microglial differentiation medium: X-VIVO15, 1x Glutamax, 25 ng / ml IL-34, and 50 ng / ml M-CSF. At this stage, blebbistatin was added at a concentration of 10 μM.

[0170] (4) On day 11, single cells suspended in the medium were collected and counted. EBs were continued to be cultured in microglia differentiation medium, and the suspended cells in the supernatant were collected every 7 days for a total of three times.

[0171] (5) Microglial cell maturation: After centrifugation, the supernatant was discarded, and the cells were resuspended in microglial maturation medium and seeded onto Matrigel-coated culture plates. Microglial cells matured after 14 days of continuous culture. Microglial maturation medium: Advanced F12, 1x Glutamax, 50 ng / ml IL-34, 5 ng / ml M-CSF.

[0172] 2. Experimental results: On day 11 of differentiation, single cells suspended in the culture medium were collected and counted. EBs were then cultured in microglia differentiation medium, and the cells suspended in the supernatant were collected three times, every seven days. The cell counts at each collection were compared between the experimental and control groups (Figures 10-12). The cell counts at the first two collections in the experimental group were significantly higher, demonstrating that the small molecules promote the proliferation of microglial precursors and improve differentiation efficiency.

[0173] Example 5. Proliferation of neural stem cells 1. Differentiation and culture methods: The differentiation method was as described in Tchieu et al., A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages. 2017, Cell Stem Cell 21, 399-410.

[0174] After adhesion, NSCs were cultured in expansion medium (DMEM / F12 (1x, Gibco) supplemented with 20 μg / mL human EGF (R&D), 20 μg / mL bFGF (R&D), 5 μg / mL heparin (Sigma), 2% B27 (Gibco), 1x GlutaMAX, and 10 μM blebbistatin in the experimental group. Half of the culture medium was replaced daily.

[0175] 2. Experimental results: Small molecules significantly promoted the expansion of NSCs from P4 to P6 (see Figure 13).

[0176] Various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, journal articles, books, and any other publications, is hereby incorporated by reference in its entirety. This specification discloses the following inventions. [1] a SMAD signaling pathway inhibitor, a SHH signaling pathway agonist, a Wnt signaling pathway agonist, and a myosin II ATPase inhibitor, optionally further comprising a ROCK inhibitor (e.g., Y-27632); Alternatively, a composition consisting of a SMAD signaling pathway inhibitor, an SHH signaling pathway agonist, a Wnt signaling pathway agonist, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor (e.g., Y-27632). [2] The composition according to [1], wherein the myosin II ATPase inhibitor is selected from the group consisting of blebbistatin and its derivatives (e.g., (S)-(-)-blebbistatin O-benzoate). [3] the SMAD signaling pathway inhibitor is selected from the group consisting of a BMP inhibitor, a TGFβ / activin-nodal inhibitor, and a combination thereof; Preferably, the BMP inhibitor is selected from the group consisting of DMH-1, dorsomorphin, noggin, LDN193189, and any combination thereof; Preferably, the TGFβ / activin-nodal inhibitor is selected from the group consisting of SB431542, SB505124, A83-01, and any combination thereof. The composition according to [1] or [2]. [4] the SHH signaling pathway agonist is selected from the group consisting of an SHH protein, a Smoothend agonist, and a combination thereof; Preferably, the SHH protein is selected from the group consisting of recombinant SHH and terminally modified SHH (e.g., SHH C25II); Preferably, the Smoothened agonist is selected from the group consisting of SAG, Hh-Ag1.5, 20α-hydroxycholesterol, purmorphamine, and any combination thereof. The composition according to any one of [1] to [3]. [5] the Wnt signaling pathway agonist is selected from the group consisting of a GSK3β inhibitor, Wnt3A, Wnt1, and combinations thereof; Preferably, the GSK3β inhibitor is selected from the group consisting of CHIR99021, GSK3β inhibitor IX (6-bromoindirubin-3'-oxime, BIO), GSK3β inhibitor VII (4-dibromoacetophenone), indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azakempaullone, SB216763, or any combination thereof; The composition according to any one of [1] to [4]. [6] the composition comprises a BMP inhibitor, a TGFβ / activin-nodal inhibitor, a SHH protein, a smoothened agonist, a GSK3β inhibitor, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor, preferably the composition comprises LDN193189, SB431542, recombinant SHH, SAG, CHIR99021, blebbistatin, and optionally Y-27632; Alternatively, the composition consists of a BMP inhibitor, a TGFβ / activin-nodal inhibitor, an SHH protein, a smoothened agonist, a GSK3β inhibitor, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor, preferably, the composition consists of LDN193189, SB431542, recombinant SHH, SAG, CHIR99021, blebbistatin, and optionally Y-27632; The composition according to any one of [1] to [5]. [7] A culture medium comprising the composition according to any one of [1] to [6] and a basal culture medium, preferably a basal culture medium suitable for culturing nerve cells. [8] the basal culture medium is a basal culture medium supplemented with one or more of the following substances: a serum-free substitute, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine; Preferably, said basal culture medium is a basal culture medium supplemented with the following substances: N2 supplement and a stabilizing dipeptide of L-alanyl-L-glutamine, Preferably, the basal culture medium comprises the following components: 49% CTS™ KnockOut™ DMEM / F-12 + 49% CTS™ Neurobasal + 1% CTS™ N2 supplement + 1% CTS-GlutaMAX™-I; [7] culture medium. [9] The culture medium according to [7] or [8], wherein the contents of the components of the composition are as follows: 0.05 μM to 1 μM BMP inhibitor, 5 μM to 20 μM TGFβ / activin-nodal inhibitor, 50 ng / mL to 200 ng / mL SHH protein, 0.5 μM to 3 μM smoothon agonist, 0.5 μM to 1.0 μM GSK3β inhibitor, and 5 μM to 20 μM myosin II ATPase inhibitor; Preferably, 10 μM SB431542, 100 nM LDN193189, 100 ng / mL SHH, 2 μM SAG, 0.5 μM to 1.0 μM CHIR99021, and 10 μM blebbistatin.

[10] A culture medium comprising the composition according to any one of [1] to [6] and a basal culture medium, wherein the medium is selected from the following: (1) The culture medium according to any one of [7] to [9]; (2) a basal culture medium supplemented with one or more of the following substances: a serum-free substitute, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: N2 supplement, B27 supplement, stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1 and blebbistatin; Preferably, the culture medium contains the following components: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 10 ng / ml EGF + 10 ng / ml FGF2 + 10 ng / ml TGFβ1 + 10 μm blebbistatin; (3) a basal culture medium supplemented with one or more of the following substances: a serum-free substitute, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, an inhibitor of the SHH signaling pathway, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: N2 supplement, B27 supplement, stabilizing dipeptide of L-alanyl-L-glutamine, SHH, purmorphamine and blebbistatin; Preferably, the culture medium comprises the following components: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 100 ng / mL SHH + 1 μM purmorphamine + 10 μM blebbistatin; (4) a basal culture medium supplemented with the following substances: glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: a stabilizing dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof; Preferably, the culture medium comprises the following components: X-VIVO15 + xGlutamax + 25 ng / ml IL-34 + 50 ng / ml M-CSF + 10 μM blebbistatin; and (5) a basal culture medium supplemented with one or more of the following substances: serum-free substitute, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin and blebbistatin; Preferably, the culture medium comprises the following components: DMEM / F12 + 20 μg / mL EGF + 20 μg / mL bFGF + 5 μg / ml heparin + 2% B27 + 1×GlutaMAX + 10 μM blebbistatin; Preferably, the basal culture medium described in any of the above items is selected from the group consisting of DMEM / F12, Neurobasal, Neural Induction Media, or X-VIVO. Culture medium.

[11] A kit comprising the composition according to any one of [1] to [6] or the culture medium according to any one of [7] to

[10] , and optionally further comprising instructions for use.

[12] A method for maintaining or increasing the number of cells in vitro, comprising a step of culturing cells in the culture medium according to any one of [7] to

[10] , Preferably, the cells are selected from motor neuron progenitor cells, cortical neuron precursors, GABAergic precursors, serotonergic precursors, midbrain dopaminergic precursors, astrocytes, neural stem cells, or microglial cells; Preferably, the method of culturing comprises preparing the cells into a single cell suspension, and dissolving the single cell suspension in a solution of 2×10 4 / cm 2 ~6×10 4 / cm 2 inoculating the cells into the culture medium at a density of 0.1% by weight, performing adherent or suspension culture, and passage the cells once every 5 to 8 days, wherein the culture conditions for each passage are the same.

[13] A cell or cell population prepared by the method described in

[12] .

[14] The cell or cell population according to

[13] , wherein the cell is selected from a neuron or a neuron-related cell.

[15] The cell or cell population according to

[13] or

[14] , wherein the cell is selected from the group consisting of a motor neuron progenitor cell, a cortical neuron precursor, a GABAergic precursor, a serotonin neuron precursor, a midbrain dopaminergic neuron precursor, an astrocyte, a neural stem cell, or a microglial cell.

[16] More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells express at least one or more of the following markers: PCDHGB1, SOX3, SEMA3D, VGF, NEFL, NTRK2, PCDHGA3, CNTN1, BDNF, STMN1, TNC, FAIM2, CHGB, GAP43, ARPP21, ALCAM, OTP, KCNF1, FOXP1, RTN1, MAPT, IG The cell or cell population according to any one of

[13] to

[15] , which specifically expresses FBP5, NNAT, CHRNA6, C1QL1, INA, TNR, PHLDA1, ELAVL3, TENM1, NRN1, CRMP1, SCG2, PMP22, and NSG1, and preferably, the expression levels of the markers are at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression levels of primary cells.

[17] More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells express at least one or more of the following markers: MTND4LP7, AL031777.3, HIST1H2AC, HIST1H1C, HIST1H4H, SYNPO2, LMO2, MGAT2, PDXP, DNAJC6, DNAJC22, ELN, MIR568, MIR1179, MIR6892, MIR7-3HG, AN GPTL1, HSPE1-MOB4, INO80B-WBP1, R3HDML, PMF1-BGLAP, PLP1, AP002748.4, MDFI, RCN3, FST, HSPH1, PCBP1, ASPN, TSPAN 8, LINC01866, LEFTY2, GMNC, ATP5MF-PTCD1, CCDC96, ALG14, IL11, A2M, C4B, ITGB4, STC1, TMEM229B, MUC5AC, TAC1, CRABP 1, CRABP2, H19, C22orf42, RCAN2, PCSK1, VAT1L, CXCL12, DCN, SSTR1, MAP7D2, PPP2R2C, LRFN5, DIRAS3, CA10, C4A, AP002 373.1, AMIGO3, GDA, EDIL3, CFH, TGFBI, CLSTN2, FBLN5, HPCAL4, ADCYAP1R1, NNMT, CD44, SMOC1, CLEC3B, DLX5, LYNX1, SYN The cell or cell population according to any one of

[13] to

[16] , which specifically expresses C, TCAF1P1, CD9, COL3A1, CAVIN1, LMO4, TCF12, GDE1, GNG3, PEG10, TFPI2, CENPF, CAMK2N1, and MLLT11, and preferably, the expression levels of the markers are at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression levels of primary cells.

[18] The cell or cell population according to any one of

[13] to

[15] , which is expanded from the primary cell by at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 150-fold, 200-fold, 500-fold, 1000-fold, 10,000-fold, or 100,000-fold.

[19] A cell or cell population, the cells are midbrain dopamine neuron precursors, Preferably, at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, e.g., 100%) of the cells or cell population express at least one marker of the midbrain dopamine neuronal precursors, such as FOXA2, LMX1A, and OTX2.

[20] The cell or cell population described in

[19] , wherein the cells do not express TUJ1. [twenty one] The cell or cell population according to

[19] or

[20] , wherein the cell has one or more of the characteristics according to any one of

[13] to

[18] . [twenty two] The cell or cell population described in

[19] , wherein the cells express TUJ1. [twenty three] The cell or cell population according to

[22] , wherein the cell has one or more characteristics according to any one of

[13] to

[18] . [twenty four] A pharmaceutical composition comprising the cell or cell population according to any one of

[13] to

[23] . [twenty five] Use of the cell or cell population according to any one of

[13] to

[23] in the manufacture of a medicament for treating a nervous system disease (e.g., a disease caused by damage to dopamine neurons (e.g., Parkinson's disease)).

[26] A method for treating a nervous system disease (e.g., a disease caused by damage to dopamine neurons (e.g., Parkinson's disease)), comprising administering to a subject in need thereof an effective amount of the cell or cell population described in any one of

[13] to

[23] .

Claims

1. a SMAD signaling pathway inhibitor, a SHH signaling pathway agonist, a Wnt signaling pathway agonist, and a myosin II ATPase inhibitor, optionally further comprising a ROCK inhibitor (e.g., Y-27632); Alternatively, a composition consisting of an SMAD signaling pathway inhibitor, an SHH signaling pathway agonist, a Wnt signaling pathway agonist, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor (eg, Y-27632).

2. 2. The composition of claim 1, wherein the myosin II ATPase inhibitor is selected from the group consisting of blebbistatin and its derivatives (e.g., (S)-(-)-blebbistatin O-benzoate).

3. the SMAD signaling pathway inhibitor is selected from the group consisting of a BMP inhibitor, a TGFβ / activin-nodal inhibitor, and a combination thereof; Preferably, the BMP inhibitor is selected from the group consisting of DMH-1, dorsomorphin, noggin, LDN193189, and any combination thereof; Preferably, the TGFβ / activin-nodal inhibitor is selected from the group consisting of SB431542, SB505124, A83-01, and any combination thereof. The composition according to claim 1 or 2.

4. the SHH signaling pathway agonist is selected from the group consisting of an SHH protein, a Smoothened agonist, and combinations thereof; Preferably, the SHH protein is selected from the group consisting of recombinant SHH and terminally modified SHH (e.g. SHH C25II), Preferably, the Smoothend agonist is selected from the group consisting of SAG, Hh-Ag1.5, 20α-hydroxycholesterol, purmorphamine, and any combination thereof. The composition according to any one of claims 1 to 3.

5. the Wnt signaling pathway agonist is selected from the group consisting of a GSK3β inhibitor, Wnt3A, Wnt1, and combinations thereof; Preferably, the GSK3β inhibitor is selected from the group consisting of CHIR99021, GSK3β inhibitor IX (6-bromoindirubin-3'-oxime, BIO), GSK3β inhibitor VII (4-dibromoacetophenone), indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azakempaullone, SB216763, or any combination thereof; The composition according to any one of claims 1 to 4.

6. the composition comprises a BMP inhibitor, a TGFβ / activin-nodal inhibitor, an SHH protein, a smoothand agonist, a GSK3β inhibitor, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor, preferably the composition comprises LDN193189, SB431542, recombinant SHH, SAG, CHIR99021, blebbistatin, and optionally Y-27632; Alternatively, the composition consists of a BMP inhibitor, a TGFβ / activin-nodal inhibitor, an SHH protein, a smoothand agonist, a GSK3β inhibitor, a myosin II ATPase inhibitor, and optionally a ROCK inhibitor, preferably the composition consists of LDN193189, SB431542, recombinant SHH, SAG, CHIR99021, blebbistatin, and optionally Y-27632; The composition according to any one of claims 1 to 5.

7. A culture medium comprising the composition according to any one of claims 1 to 6 and a basal culture medium, preferably a basal culture medium suitable for culturing neuronal cells.

8. the basal culture medium is a basal culture medium supplemented with one or more of the following substances: a serum-free substitute, glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine; Preferably, said basal culture medium is a basal culture medium supplemented with the following substances: N2 supplement and a stabilizing dipeptide of L-alanyl-L-glutamine, Preferably, the basal culture medium comprises the following components: 49% CTS™ KnockOut™ DMEM / F-12 + 49% CTS™ Neurobasal + 1% CTS™ N2 supplement + 1% CTS-GlutaMAX™-I; The culture medium of claim 7.

9. The culture medium according to claim 7 or 8, wherein the content of each component of the composition in the culture medium is as follows: 0.05 μM to 1 μM BMP inhibitor, 5 μM to 20 μM TGFβ / activin-nodal inhibitor, 50 ng / mL to 200 ng / mL SHH protein, 0.5 μM to 3 μM smoothon agonist, 0.5 μM to 1.0 μM GSK3β inhibitor, and 5 μM to 20 μM myosin II ATPase inhibitor; Preferably, 10 μM SB431542, 100 nM LDN193189, 100 ng / mL SHH, 2 μM SAG, 0.5 μM to 1.0 μM CHIR99021, and 10 μM blebbistatin.

10. A culture medium comprising the composition of any one of claims 1 to 6 and a basal culture medium, wherein the medium is selected from: (1) The culture medium according to any one of claims 7 to 9; (2) a basal culture medium supplemented with one or more of the following substances: a serum-free substitute, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: N2 supplement, B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1 and blebbistatin; Preferably, the culture medium comprises the following components: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 10 ng / ml EGF + 10 ng / ml FGF2 + 10 ng / ml TGFβ1 + 10 μm blebbistatin; (3) a basal culture medium supplemented with one or more of the following substances: a serum-free substitute, a stabilizing dipeptide of glutamine or L-alanyl-L-glutamine, an inhibitor of the SHH signaling pathway, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: N2 supplement, B27 supplement, stabilizing dipeptide of L-alanyl-L-glutamine, SHH, purmorphamine and blebbistatin; Preferably, the culture medium comprises the following components: 48% DMEM / F-12 + 48% Neurobasal + 1% N2 + 2% B27 + 1% GlutaMAX + 100 ng / mL SHH + 1 μM purmorphamine + 10 μM blebbistatin; (4) a basal culture medium supplemented with the following substances: glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: a stabilizing dipeptide of L-alanyl-L-glutamine, IL-34, M-CSF, and blebbistatin or a derivative thereof; Preferably, the culture medium comprises the following components: X-VIVO15 + xGlutamax + 25 ng / ml IL-34 + 50 ng / ml M-CSF + 10 μM blebbistatin; and (5) a basal culture medium supplemented with one or more of the following substances: serum-free substitute, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin, and blebbistatin or a derivative thereof; Preferably, a basal culture medium supplemented with the following substances: B27 supplement, a stabilizing dipeptide of L-alanyl-L-glutamine, EGF, FGF2, heparin and blebbistatin; Preferably, the culture medium comprises the following components: DMEM / F12 + 20 μg / mL EGF + 20 μg / mL bFGF + 5 μg / ml heparin + 2% B27 + 1×GlutaMAX + 10 μM blebbistatin; Preferably, the basal culture medium described in any of the above items is selected from the group consisting of DMEM / F12, Neurobasal, Neural Induction Media, or X-VIVO. Culture medium.

11. A kit comprising the composition of any one of claims 1 to 6 or the culture medium of any one of claims 7 to 10, optionally further comprising instructions for use.

12. A method for maintaining or increasing the number of cells in vitro, comprising culturing cells in a culture medium according to any one of claims 7 to 10, Preferably, the cells are selected from motor neuron progenitor cells, cortical neuron precursors, GABAergic precursors, serotonergic precursors, midbrain dopaminergic precursors, astrocytes, neural stem cells, or microglial cells; Preferably, the method of culturing comprises preparing the cells into a single cell suspension, and dissolving the single cell suspension in a solution of 2 x 10 ４ / cm ２ ~6 x 10 ４ / cm ２ inoculating the cells into the culture medium at a density of 0.1% by weight, performing adherent or suspension culture, and passage the cells once every 5 to 8 days, wherein the culture conditions for each passage are the same.

13. A cell or cell population prepared by the method of claim 12.

14. 14. The cell or cell population of claim 13, wherein the cell is selected from a neuronal cell or a neuronal-related cell.

15. 15. The cell or cell population of claim 13 or 14, wherein the cell is selected from the group consisting of a motor neuron progenitor cell, a cortical neuron precursor, a GABAergic precursor, a serotonin neuron precursor, a midbrain dopaminergic neuron precursor, an astrocyte, a neural stem cell, or a microglial cell.

16. More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells express at least one or more of the following markers: PCDHGB1, SOX3, SEMA3D, VGF, NEFL, NTRK2, PCDHGA3, CNTN1, BDNF, STMN1, TNC, FAIM2, CHGB, GAP43, ARPP21, ALCAM, OTP, KCNF1, FOXP1, RTN1, MAPT, IGF 16. The cell or cell population of any one of claims 13 to 15, which specifically expresses BP5, NNAT, CHRNA6, C1QL1, INA, TNR, PHLDA1, ELAVL3, TENM1, NRN1, CRMP1, SCG2, PMP22, and NSG1, preferably wherein the expression level of said markers is at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than the expression level of primary cells.

17. More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells express at least one or more of the following markers: MTND4LP7, AL031777.3, HIST1H2AC, HIST1H1C, HIST1H4H, SYNPO2, LMO2, MGAT2, PDXP, DNAJC6, DNAJC22, ELN, MIR568, MIR1179, MIR6892, MIR7-3HG, AN GPTL1, HSPE1-MOB4, INO80B-WBP1, R3HDML, PMF1-BGLAP, PLP1, AP002748.4, MDFI, RCN3, FST, HSPH1, PCBP1, ASPN, TSPAN8 , LINC01866, LEFTY2, GMNC, ATP5MF-PTCD1, CCDC96, ALG14, IL11, A2M, C4B, ITGB4, STC1, TMEM229B, MUC5AC, TAC1, CRABP1 , CRABP2, H19, C22orf42, RCAN2, PCSK1, VAT1L, CXCL12, DCN, SSTR1, MAP7D2, PPP2R2C, LRFN5, DIRAS3, CA10, C4A, AP0023 73.1, AMIGO3, GDA, EDIL3, CFH, TGFBI, CLSTN2, FBLN5, HPCAL4, ADCYAP1R1, NNMT, CD44, SMOC1, CLEC3B, DLX5, LYNX1, SYNC , TCAF1P1, CD9, COL3A1, CAVIN1, LMO4, TCF12, GDE1, GNG3, PEG10, TFPI2, CENPF, CAMK2N1, MLLT11, and preferably the expression levels of said markers are at least about 1.5-fold, 2.0-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold higher than the expression levels of primary cells.

18. 16. The cell or cell population of any one of claims 13 to 15, which is expanded from said primary cell by at least about 1.5x, 2.0x, 3x, 5x, 10x, 20x, 30x, 40x, 50x, 100x, 150x, 200x, 500x, 1000x, 10000x or 100000x.

19. A cell or cell population, the cells are midbrain dopamine neuron precursors, Preferably, at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, e.g., 100%) of the cells or cell population express at least one marker of the midbrain dopamine neuronal precursors, such as FOXA2, LMX1A, and OTX2.

20. 20. The cell or cell population of claim 19, wherein the cells do not express TUJ1.

21. 21. The cell or cell population of claim 19 or 20, wherein the cells have any one or more of the characteristics of any one of claims 13 to 18.

22. 20. The cell or cell population of claim 19, wherein the cell expresses TUJ1.

23. 23. The cell or cell population of claim 22, wherein the cells have one or more of the characteristics of any one of claims 13 to 18.

24. A pharmaceutical composition comprising a cell or cell population according to any one of claims 13 to 23.

25. Use of a cell or cell population according to any one of claims 13 to 23 in the manufacture of a medicament for treating a nervous system disease (e.g., a disease caused by damage to dopamine neurons (e.g., Parkinson's disease)).

26. A method for treating a nervous system disease (e.g., a disease caused by dopamine neuron damage (e.g., Parkinson's disease)), comprising administering to a subject in need thereof an effective amount of a cell or cell population according to any one of claims 13 to 23.

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