EXPANSION CULTURE MEDIUM AND CULTURE METHOD FOR NEURAL CELLS

Abstract

A culture medium and a culture method for neural cells, particularly relating to a composition comprises a SMAD signaling pathway inhibitor, a SHH signaling pathway agonist, a Wnt signaling pathway agonist and a Myosin II ATPase inhibitor, optionally, it further comprises a ROCK inhibitor; or, the composition consists of the SMAD signaling pathway inhibitor, the SHH signaling pathway agonist, the Wnt signaling pathway agonist, the Myosin II ATPase inhibitor, and optionally the ROCK inhibitor. The same batch of neural cells, such as midbrain dopaminergic progenitors, can be efficiently obtained by using the culture method and culture medium designed on the basis of the composition.

Claims

1. A composition, which comprises a SMAD signaling pathway inhibitor, a SHE signaling pathway agonist, a Wnt signaling pathway agonist and a Myosin II ATPase inhibitor, optionally, which further comprises a ROCK inhibitor (e.g., Y-27632); alternatively, the composition consists of a SMAD signaling pathway inhibitor, a SHE 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 claim 1, which is characterized by one or more of the following items: a. the Myosin II ATPase inhibitor is selected from the group consisting of Blebbistatin and a derivative thereof (e.g., (S)-(−)-Blebbistatin O-Benzoate); b. the SMAD signaling pathway inhibitor is selected from the group consisting of a BMP inhibitor, a TGFIβ/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; c. the SHH signaling pathway agonist is selected from the group consisting of a SHH protein, a Smoothend agonist and a combination thereof; preferably, the SHH protein is selected from the group consisting of recombinant SHH and terminal-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; d. the Wnt signaling pathway agonist is selected from the group consisting of a GSK3β inhibitor, Wnt3A, Wnt1 and a combination 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-Azakenpaullone, SB216763 or any combination thereof.

3. (canceled)

4. (canceled)

5. (canceled)

6. The composition according to claim 1, which 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, it comprises LDN193189, SB431542, a recombinant SHH, SAG, CHIR99021, Blebbistatin, and optionally Y-27632; alternatively, the composition consists of 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 consists of LDN193189, SB431542, a recombinant SHH, SAG, CHIR99021, Blebbistatin and optionally Y-27632.

7. A culture medium, which comprises the composition according to claim 1, and a basal culture medium; preferably, the basal culture medium is suitable for the cultivation of a nerve cell; preferably, the basal culture medium is a basal culture medium supplemented with the following substances: one or more serum-free substitutes, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine; preferably, the basal culture medium is a basal culture medium supplemented with the following substances: a N2 supplement and a stabilized 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.

8. (canceled)

9. The culture medium according to claim 7, wherein, in the culture medium, the content of each component of the composition is as follows: 0.05-1 μM BMP inhibitor, 5-20 μM TGFβ/Activin-Nodal inhibitor, 50-200 ng/mL SHH protein, 0.5-3 μM Smoothened agonist, 0.5-1.0 μM GSK3β inhibitor, and 5-20 μM Myosin II ATPase inhibitor; preferably, 10 μM SB431542, 100 nM LDN193189, 100 ng/mL SHH, 2 μM SAG, 0.5-1.0 82 M CHIR99021, and 10 μM Blebbistatin.

10. A culture medium, which comprises the composition according to claim 1, and a basal culture medium which is selected from: (1) the culture medium comprising the composition according to claim 1, and a basal culture medium, preferably, the basal culture medium is suitable for the cultivation of a nerve cell; (2) a basal culture medium supplemented with the following substances: one or more serum-free substitutes, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, EGF, FGF2, TGFβ1, and Blebbistatin or a derivative thereof; preferably, a basal culture medium supplemented with the following substances: a N2 supplement, a B27 supplement, a stabilized 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 the following substances: one or more serum-free substitutes, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, a SHH signaling pathway inhibitor, and Blebbistatin or a derivative thereof; preferably, a basal culture medium supplemented with the following substances: a N2 supplement, a B27 supplement, a stabilized 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 stabilized 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+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF+10 μM Blebbistatin; and (5) a basal culture medium supplemented with the following substances: one or more serum-free substitutes, 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: a B27 supplement, a stabilized 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.

11. A kit, which comprises the composition according to claim 1, optionally, which further comprises an instruction for use.

12. A method for maintaining or increasing the number of cells in vitro, which comprises a step of culturing the cells in the culture medium according to claim 7; preferably, the cells are selected from motor neuron progenitor cells, cortical neuron progenitors, GABAergic progenitors, serotonergic progenitors, midbrain dopaminergic progenitors, astrocytes, neural stem cells, or microglial cells; preferably, the method to conduct the culturing is: preparing the cells into a single cell suspension, inoculating the single cell suspension into the culture medium at a density of 2×10.sup.4/cm.sup.2 to 6×10.sup.4/cm.sup.2, conducting adherent or suspension culture and passaging the cells once every 5-8 days, wherein the culture conditions of each passage are the same.

13. A cell or cell population, which is prepared by the method according to claim 12.

14. The cell or cell population according to claim 13, wherein the cell is selected from neural or nerve-related cells.

15. The cell or cell population according to claim 13, the cell is selected from the group consisting of motor neuron progenitor cell, cortical neuron progenitor, GABAergic progenitor, serotonin neuron progenitor, midbrain dopamine never cell progenitor, astrocyte, neural stem cell, or microglial cell.

16. A cell or cell population, or the cell or cell population according claim 13, wherein >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98% or >99% of the cells specifically 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, the expression level of the marker is at least about 1.5 times, 2.0 times, 3 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times or 100 times higher than that of primary cells.

17. The cell or cell population according to claim 13, wherein >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95% %, >98% or >99% of the cells specifically 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, 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, 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, preferably, the expression level of the marker is at least about 1.5 times, 2.0 times, 3 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times or 100 times higher than that of the primary cells.

18. The cell or cell population according to claim 13, which is expanded at least about 1.5 times, 2.0 times, 3 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 150 times, 200 times, 500 times, 1000 times, 10000 times or 100000 times from the primary cells.

19. A cell or cell population, wherein the cell is a midbrain dopamine never cell progenitor; 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%, such as 100%) of the cells express at least a marker of the midbrain dopamine never cell progenitor, for example FOXA2, LMX1A, and OTX2; preferably, the cell does not express TUJ1; further preferably, the cell has any one or more of the characteristics as defined in claim 13; preferably, the cell expresses TUJ1; further preferably, the cell has one or more of the characteristics as defined in claim 13.

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. A pharmaceutical composition, which comprises the cell or cell population according to claim 13.

25. (canceled)

26. (canceled)

27. The culture medium according to claim 10, wherein the basal culture medium in item (1) is a basal culture medium supplemented with the following substances: one or more serum-free substitutes, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine; preferably, the basal culture medium is a basal culture medium supplemented with the following substances: a N2 supplement and a stabilized 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; more preferably, in the culture medium, the content of each component of the composition is as follows: 0.05-1 μM BMP inhibitor, 5-20 μM TGFβ/Activin-Nodal inhibitor, 50-200 ng/mL SHH protein, 0.5-3 μM Smoothened agonist, 0.5-1.0 μM GSK3β inhibitor, and 5-20 μM Myosin II ATPase inhibitor; more preferably, in the culture medium, the content of each component of the composition is as follows: 10 μM SB431542, 100 nM LDN193189, 100 ng/mL SHH, 2 μM SAG, 0.5-1.0 μM CHIR99021, and 10 μM Blebbistatin.

28. A kit, which comprises the culture medium according to claim 7, optionally, which further comprises an instruction for use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The drawings described here are used to provide a further understanding of the present invention and constitute a part of the application. The schematic examples of the present invention and descriptions thereof are used to explain the present invention and do not constitute improper limitations to the present invention. In the drawings:

[0107] FIG. 1 shows the induction and expansion process of floor plate cells derived from human embryonic stem cells.

[0108] FIG. 2 shows the cell proliferation curve of each passage in the floor plate expansion media 1 and 2.

[0109] FIG. 3 shows the expression of floor plate cell markers LMX1A and FOXA2 of floor plate cells obtained by subculture for 10 passages using the floor plate cell expansion medium 1.

[0110] FIG. 4 shows the expression of dopamine neuron markers FOXA2, TH, and TUJ1 of cells obtained by differentiation of floor plate cells cultured for 10 passages using the floor plate cell expansion medium 1.

[0111] FIG. 5 shows the expression of cell markers FOXA2 and LMX1A after subculture for 10 passages using the floor plate cell expansion medium 2.

[0112] FIG. 6 shows the expression of dopaminergic neuron markers FOXA2, LMX1A, TH, and TUJ1 of cells obtained by differentiation of floor plate cells cultured for 12 passages using the floor plate cell expansion medium 2.

[0113] FIG. 7 shows the therapeutic effect of striatal injection of 2.5×10.sup.5 cells in rat PD models detected by the rotation experiment.

[0114] FIG. 8 shows the cell amount of the 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 g/ml FGF2+10 ng/ml TGFβ1.

[0115] FIG. 9 shows the cell amount of the GABA progenitor 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.

[0116] FIG. 10 shows the amounts of suspension cells collected from EBs cultured on days 4-11 in microglial differentiation medium (collection 1), CTRL: X-VIVO15+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF; SMI: X-VIVO15+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF+10 μM Blebbistatin.

[0117] FIG. 11 shows the amounts of suspension cells collected from EBs cultured on days 11-18 in microglial differentiation medium (collection 2), CTRL: X-VIVO15+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF; SMI: X-VIVO15+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF+10 μM M Blebbistatin.

[0118] FIG. 12 shows the amounts of suspension cells collected from EBs cultured on days 18-25 in microglial differentiation medium (collection 3), CTRL: X-VIVO15+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF; SMI: X-VIVO15+1× Glutamax+25 ng/ml IL-34+50 ng/ml M-CSF+10 μM M Blebbistatin.

[0119] FIG. 13 shows the total amount of NSCs at different expansion passages, wherein, 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.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0120] The technical solutions in the examples of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the examples of the present invention. Obviously, the described examples are only part, not all, examples of the present invention. The following description of at least one exemplary example is merely illustrative in nature and in no way taken as any limitation of the present invention, its application or uses. Based on the examples of the present invention, all other examples obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

EXAMPLE 1. Floor Plate Cell Expansion experiment

1. Reagents Used in the Floor Plate Cell Expansion Experiment:

[0121] (1) Preparation and working concentration of each factor: [0122] 1) LDN193189 solution: the working concentration was 100 nM. It could be stored at 4° C. for two weeks. [0123] 2) SB431542 solution: the working concentration was 10 μM. It could be stored at 4° C. for two weeks. [0124] 3) SAG solution: the working concentration was 2 μM. It could be stored at 4° C. for two weeks. [0125] 4) SHH (Sonic hedgehog) solution: the working concentration was 100 ng/mL. It could be stored at 4° C. for two weeks. [0126] 5) CHIR99021 solution: the working concentration was 0.5-1.0 μM. It could be used once. [0127] 6) Blebbistatin solution: the working concentration was 10 μM. It could be stored at 4° C. for two weeks.

[0128] (2) Preparation of basal culture medium N2 medium:

[0129] 49% CTS™ KnockOut™ DMEM/F-12+49% CTS™ Neurobasal+1% CTS™ N2 Supplement+1% CTS-GlutaMAX™-I

[0130] (3) Floor plate cell expansion medium 1:

[0131] N2 medium+LDN193189 (100 nM)+SB431542 (10 μM)+SHH (100 ng/mL)+SAG (2 μM)+CHIR99021 (0.5-1.0 μM);

[0132] (4) Floor plate cell expansion medium 2:

[0133] N2 medium+LDN193189 (100 nM)+SB431542 (10 μM)+SHH (100 ng/mL)+SAG (2 μM)+CHIR99021 (0.5-1.0 μM)+Blebbistatin (10 μM);

2. Reagents Used For the Differentiation Experiment of Dopamine Neurons From the Expanded Floor Plate cells:

[0134] (1) Preparation and working concentration of each factor: [0135] 1) FGF8 solution: the working concentration was 100 ng/mL. It could be stored at 4° C. for two weeks. [0136] 2) TGFβ33 solution: the working concentration was 1 ng/mL. It could be stored at 4° C. for two weeks. [0137] 3) Ascorbic acid (AA) solution: the working concentration was 0.2 mM. It could be stored at 4° C. for two weeks. [0138] 4) Dibutyryl cAMP (db-cAMP) solution: the working concentration was 0.5 mM. It could be stored at 4° C. for two weeks. [0139] 5) BDNF solution: the working concentration was 20 ng/mL. It could be stored at 4° C. for two weeks. [0140] 6) GDNF solution: the working concentration was 20 ng/mL. It could be stored at 4° C. for two weeks.

[0141] (2) Preparation of basal culture medium B27 medium:

[0142] 97% CTS™ Neurobasal+2% B27-CTS™+1% CTS-GlutaMAX™-I

[0143] (3) Preparation of dopamine neuron differentiation medium 1:

[0144] B27 medium+BDNF (20 ng/mL)+GDNF (20 ng/mL)+TGF-β3 (1 ng/mL)+AA (0.2 mM)+FGF8 (100 ng/mL), each of the factor solution was mixed well and then added in proportion, so as to obtain the differentiation medium 1, which was pipetted repeatedly;

[0145] (4) Preparation of dopamine neuron differentiation medium 2:

[0146] 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), each of the factor solution was mixed well and then added in proportion, so as to obtain the differentiation medium 2, which was pipetted repeatedly.

3. Method For Floor Plate Cell Expansion Experiment:

[0147] Referring to the method in FIG. 1, human embryonic stem cells were subjected to induced differentiation to obtain floor plate cells, which were used for subsequent expansion, cryopreservation and differentiation experiments. The 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 the method of inducing floor plate cells from human embryonic stem cells, refer to the prior art, for example doi:10.1038/nprot.2017.078.

[0148] (1) Method of obtaining the floor plate cells: [0149] 1) Preparation of Vitronectin matrix: At room temperature, 6 mL of DPBS was pipetted into a 15 mL centrifuge tube, then 60 μL of Vitronectin was pipetted into the above DPBS, mixed well by pipetting with a pipette gun for 10 times to obtain the Vitronectin matrix, which was prepared when being used. 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 use. [0150] 2) Human embryonic stem cells were digested into single cells with TrypLE or accutase, 10 μL of the above suspension was added to 10 μL of trypan blue solution and mixed well, and counting was performed with a countess cell counter; then the Vitronectin matrix was pipetted and discarded, and TrypLE or accutase was used for digestion to obtain single cells, which was inoculated at a density of 2×10.sup.4/cm.sup.2 in a differentiation medium containing 10 μM Rock inhibitor (e.g., Y-27632), and culture was conducted in a 37° C., 5% CO.sub.2 incubator. The differentiation medium contained BMP inhibitor and TGF inhibitor, Wnt and SHH activator, and the midbrain floor cells differentiated from the human embryonic stem cells could be obtained after several days.

[0151] (2) Floor plate cell expansion experiment: [0152] 1) Preparation of Vitronectin matrix: At room temperature, 6 mL of DPBS was pipetted into a 15 mL centrifuge tube, then 60 μL of Vitronectin was pipetted into the above DPBS, mixed well by pipetting with a pipette gun for 10 times to obtain the Vitronectin matrix, which was prepared when being used. 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 use. [0153] 2) The floor plate cells were digested into single cells with TrypLE or accutase, 10 μL of the above suspension was added to 10 μL of trypan blue solution and mixed well, and counting was performed with a countess cell counter; then the Vitronectin matrix was pipetted and discarded, the cells were re-inoculated at a density of 4×10.sup.4/cm.sup.2 by 2D method in the floor plate cell expansion medium 1 and the floor plate cell expansion medium 2 both of which supplemented with 10 μM Rock inhibitor (e.g., Y-27632), and cultured in a 37° C., 5% CO.sub.2 incubator. [0154] 3) The cells were passaged every 5-8 days, and the inoculation density, medium and other conditions of each passage were the same.

[0155] At each passage, the total number of cells was calculated using the countess cell counter, and the cell proliferation curve was made.

[0156] The proliferation efficiency of the expansion conducted in the floor plate cell expansion media 1 and 2 were shown in FIG. 2. Under the treatment of the expansion media 1 and 2, with the increase of cell passage number, the number of cells of Cell 1 and Cell 2 continued to increase, and the proliferation rate gradually decreased. Cell 1 could be expanded up to 10 passages, and Cell 2 could be expanded up to 12 passages; at the same time, the cell number of Cell 2 was 48 times that of Cell 1. The results proved that the expansion medium 2 could greatly increase the expansion ability of the cells.

[0157] In the floor plate cell expansion medium 1, with the increase of cell passage number, the number of cells gradually increased, and the proliferation rate gradually decreased. After 10 passages, the cells could be expanded for 1000 times, and the immunofluorescence staining showed that the cells still stably expressed the floor plate cell markers FOXA2 and LMX1A (FIG. 3).

[0158] In the floor plate cell expansion medium 2, with the increase of passages, the number of cells gradually increased, and the proliferation rate gradually decreased. After 10 passages, the cells could be expanded for 50,000 times, and the immunofluorescence staining showed that the cells still stably expressed the floor plate cell markers FOXA2 and LMX1A (FIG. 5).

4. Cryopreservation Experiment of Floor Plate Cells After Expansion:

[0159] 1) Programmed cooling boxes and freezing solution were pre-cooled at 4° C. for use. [0160] 2) The state of the cells was observed under an inverted microscope to ensure that the cells were in good condition and free from contamination. [0161] 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; [0162] 4) DPBS was discarded, 1 mL of Tryple was added to each well, and digestion was performed in a 37° C. incubator for 3-4 minutes (the time for digestion by using Tryple could be adjusted depending on the degree of digestion of the cells); [0163] 5) Tryple was discarded, 1 mL of basal culture medium N2 medium was added to each well for termination; the cells were gently pipetted into uniformly sized small pieces with a pipette gun, then collected into a centrifuge tube, and centrifuged at 1200 r/min for 3 min at room temperature; [0164] 6) The supernatant was discarded, and the pre-cooled freezing solution was added to resuspend the cells (every ⅙ well of cell suspension into one cryopreservation tube, 1 mL/tube); [0165] 7) The cell suspension was subpackaged into cryopreservation tubes, 1 mL/tube. Then the cryopreservation tubes were placed into a pre-cooled freezer box and stored in a −80° C. refrigerator overnight. It was transferred to liquid nitrogen for long-term storage on the next day.

5. Experiment of Differentiating the Expanded Floor Plate Cells Into Dopamine Progenitor Cells:

[0166] 1) Preparation of Vitronectin matrix: At room temperature, 6 mL of DPBS was pipetted into a 15 mL centrifuge tube, then 60 μL of Vitronectin was pipetted into the above DPBS, and mixed well by pipetting with a pipette gun for 10 times to obtain the Vitronectin matrix, which was prepared when being used. 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. [0167] 2) The floor plate cells obtained by culturing for 10 passages using the floor plate cell expansion media 1 and 2 were digested with TrypLE or accutase into single cells, which were re-inoculated at a density of 5×10.sup.5/cm.sup.2 by the 2D method in the dopamine neuron differentiation medium 1 containing Rock inhibitor (e.g., Y-27632), respectively, after being mixed evenly, they were cultured in a 37° C., 5% CO.sub.2 incubator. [0168] 3) After being cultured in the dopamine neuron differentiation medium 1 for 6 days, the cells were further cultured for 8 days by replacing the medium with the dopamine neuron differentiation medium 2. [0169] 4) The differentiated dopamine never cells were identified by immunofluorescence, and the results were shown in FIG. 4 and FIG. 6.

[0170] The results showed that the dopamine never cells obtained by further differentiation of Cell 1 obtained by 10 passages of expansion in the floor plate cell expansion medium 1 were identified by immunofluorescence of markers, and the results proved that Cell 1 obtained after 10 passages of expansion still had the ability to differentiate into dopamine nerve cells, and could stably express the markers FOXA2, TH, and TUJ1 of dopamine nerve cells.

[0171] The dopamine never cells obtained by further differentiation of Cell 2 obtained by 12 passages of expansion in the floor plate cell expansion medium 2 were identified by immunofluorescence of markers, and the results proved that Cell 2 obtained after 12 passages of expansion still had the ability to differentiate into dopamine never cells, and could stably express the markers FOXA2, LMX1A, TH, TUJ1 of dopamine nerve cells.

[0172] Immunofluorescence identification method: [0173] a) Cell inoculation: A clean and sterile circular coverslip was placed in the bottom center of a 24-well cell culture dish, and the cells were inoculated after being coated with vitronectin for 30 minutes; [0174] b) Cell fixation: After the cells grew to an appropriate density, the culture medium was discarded, the cells were washed once with PBS, and fixed with 4% PFA at room temperature for 15-30 min, and then washed 3 times with PBS. [0175] c) Blocking: 320 μL of a mixed solution of 2% BSA and 0.3% Triton was added to each well, and blocking was performed at room temperature for 2 h. [0176] d) Addition of primary antibody: The primary antibody was diluted with a mixed solution of 2% BSA and 0.3% Triton (referring to the instruction manual of the primary antibody for the usage ratio), pipetted and mixed well, the blocking solution was discarded, the diluted primary antibody solution was added, 320 μL for each well, and allowed to stand overnight at 4° C. after being sealed with parafilm. [0177] e) Addition of secondary antibody: The primary antibody was discarded by pipetting, the cells were washed twice with PBS; 320 μL of the secondary antibody diluted in a mixed solution of 2% BSA and 0.3% Triton was added to each well (referring to the instruction manual of the primary antibody for the usage ratio), and allowed to stand in the dark for 2 hours at room temperature. [0178] f) Staining nuclei: The secondary antibody was discarded, the cells were washed twice with PBS; 320 μL of a diluted Hoechst33342 solution (1 ul of Hoechst33342 in 1 ml of PBS) was added to each well, and the cells were incubated for 15 min. [0179] g) Sealing: The diluted Hoechst33342 solution was discarded by pipetting, the cells were washed once with PBS; then 10 μL of anti-quencher was added to each slide, the glass slide with cells growing thereon was carefully picked up using a syringe needle in cooperation with curved tweezers, and the side with the cells was put down on the glass slide with anti-quencher, avoiding air bubbles; finally, a colorless nail polish was lightly placed on the edge of the cover glass to fix the glass slide, which was then allowed to dry for 5 minutes and put into the slide box. [0180] 5) The differentiated cells were subjected to RNA extraction and expression profile sequencing, and the specific method referred to 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.

[0181] The results showed that the expression level of dopamine nerve cells obtained by differentiating the floor plate cells cultured with the expansion medium was more than 10 times that of the unexpanded cells (Tables 1 to 2), and the markers were shown in the table.

TABLE-US-00001 TABLE 1 Statistics of expression profile results of the expanded cells, part 1 Expression level Expression level of formula 1 of formula 2 Gene name (not expanded) (expanded) Relative PCDHGB1 3.400765 40.19527 mRNA SOX3 0.1409564 2.220847 expression SEMA3D 0.60748 10.76362 level VGF 14.195183 236.2871 NEFL 8.330079 98.3712 NTRK2 3.228189 33.12535 PCDHGA3 0.044837 6.693222 CNTN1 1.182307 20.33537 BDNF 0.480524 8.822195 STMN1 0.526314 14.26352 TNC 24.353844 277.0916 FAIM2 1.192175 12.36466 CHGB 1.089562 15.23698 GAP43 17.4865 361.2452 ARPP21 0.14378 5.665673 ALCAM 0.22283 7.826541 OTP 0 5.263871 KCNF1 2.366999 26.23453 FOXP1 1.23052 15.26389 RTN1 2.35628 74.21589 MAPT 11.732004 161.4916 IGFBP5 0.15872 21.33698 NNAT 0.36381 4.125873 CHRNA6 0 3.820327 C1QL1 0.25362 3.259631 INA 0.25741 4.218901 TNR 0.109791 4.099195 PHLDA1 0.695623 7.584162 ELAVL3 0.2513601 8.574123 TENM1 0.524152 10.55022 NRN1 4.235861 71.26374 CRMP1 5.217891 59.325743 SCG2 12.978907 279.7323 PMP22 0.41231201 8.251431 NSG1 12.559338 164.1721

TABLE-US-00002 TABLE 2 Statistics of expression profile results of the expanded cells, part 2 Expression level Expression level of formula 1 of formula 2 Gene name (not expanded) (expanded) Relative MTND4LP7 0 76.03553 mRNA AL031777.3 0.171489 4.468266 expression HIST1H2AC 2.476634 48.57276 level HIST1H1C 1.247798 18.05772 HIST1H4H 0.117923 2.009719 SYNPO2 0.244708 3.128491 LMO2 2.664059 29.57198 MGAT2 0 3.6155988 PDXP 1.274254 20.21273 DNAJC6 3.984809 44.89279 DNAJC22 0.01079 7.323679 ELN 25.384838 271.8633 MIR568 0 10.6673 MIR1179 0 2.305298 MIR6892 0 1.868145 MIR7-3HG 0.363221 17.53261 ANGPTL1 0.241169 5.236946 HSPE1-MOB4 0 22.99689 INO80B-WBP 0 15.52343 R3HDML 0 3.719844 PMF1-BGLAP 1.174822 12.88243 PLP1 2.91422 56.07297 AP002748.4 0 4.168712 MDFI 2.53376 69.14996 RCN3 1.022739 19.856729 FST 1.768944 27.64677 HSPH1 6.78339 71.87888 PCBP1 0 22.56321 ASPN 1.067857 17.3869 TSPAN8 0.425994 4.261259 LINC01866 0 2.114547 LEFTY2 1.675307 18.6166 GMNC 0.157274 4.895082 ATP5MF-PTCD1 0.258379 5.889129 CCDC96 0 2.845406 ALG14 0.50492 13.754033 IL11 0.638582 6.99416 A2M 0.639516 83.66215 C4B 0.326981 19.03333 ITGB4 0.164464 9.896965 STC1 1.107299 14.11464 TMEM229B 0.21548 6.56191 MUC5AC 1.282185 16.63354 TAC1 7.292313 247.3887 CRABP1 0 18.25214 CRABP2 0 10.23211 H19 0.335919 10.90753 C22orf42 0.693401 10.93448 RCAN2 2.200737 25.01389 PCSK1 9.467299 110.4257 VAT1L 7.56819 92.87996 CXCL12 3.896376 39.65608 DCN 3.248732 37.4826 SSTR1 0.839088 8.842989 MAP7D2 0.667324 7.378552 PPP2R2C 0.842985 15.11611 LRFN5 0.364771 8.302847 DIRAS3 3.174719 42.63766 CA10 0.123762 4.743845 C4A 0.913978 18.53574 AP002373.1 0 7.396976 AMIGO3 0.096202 6.43058 GDA 0.569416 8.455555 EDIL3 3.225003 46.04006 CFH 0.711477 29.47776 TGFBI 2.282342 31.66396 CLSTN2 4.044774 41.36876 FBLN5 3.726905 45.37458 HPCAL4 3.027599 39.77068 ADCYAP1R1 0.562132 7.302859 NNMT 3.632776 71.54955 CD44 2.824466 35.9178 SMOC1 10.995736 118.502 CLEC3B 0.532358 8.722571 DLX5 0.015 0.51 LYNX1 0 4.318749 SYNC 1.189385 13.70889 TCAF1P1 0 8.423484 CD9 1.433858 16.51166 COL3A1 61.333252 643.8076 CAVIN1 1.949901 28.39877 LMO4 0.836252 9.543621 TCF12 0.543213 7.642513 GDE1 0.668022 8.071253 GNG3 0.522036 19.23625 PEG10 0.985125 10.23561 TFPI2 0.549224 7.642502 CENPF 0.254056 38.31252 CAMK2N1 2.301012 53.263121 MLLT11 0.782102 16.230112

6. Behavior Detection Method of PD Model (Rotation Experiment):

[0182] Experimental equipment: rotation recorder, counter, weighing scale.

[0183] Experimental reagent: apomorphine (sigma, A4393).

[0184] The rotation experiment steps were as follows: [0185] a) The animals were weighed; [0186] b) The animals were anesthetized by intraperitoneal injection of 5% chloral hydrate according to body weight; [0187] c) Preparation and injection of apomorphine: The powdered apomorphine was dissolved in normal saline to 1 mg/ml, and injected intraperitoneally at a dose of 0.5 mg/kg; [0188] d) Counting was started 5-10 minutes after the injection of apomorphine or when the model rats started to spin in circles steadily, recorded for 30 minutes, and the average number of turns per minute was calculated.

[0189] The cell injection steps were as follows:

[0190] Experimental equipment: brain stereotaxic instrument, electric injection pump, cranial drill, surgical scissors, tweezers, needle-holding forceps, 5-0 suture with needle, Hamilton 701 syringe, heat preservation pad, ultra-clean workbench, etc.

[0191] Experimental reagents: povidone iodine, normal saline, disinfectant alcohol, Baytril, cyclosporine. [0192] a) Injection of immunosuppressant: 48 hours before transplantation, the immunosuppressant cyclosporine was intraperitoneally injected at a dose of 10 mg/kg, and the injection was performed every day until the end of the experiment; [0193] b) Preparation of cell suspension: The culture supernatant of the cells to be transplanted was discarded, the cells were washed once with PBS, and then added with tryple to digest for 5-8 minutes; the cell suspension was collected into a centrifuge tube, centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, the cells were resuspended to 1.25×10.sup.5/μL with cell injection solution; [0194] c) Injection of cell suspension: 2 μL (2.5×10.sup.5 cells) of the above cell suspension was injected into the striatum of each animal at the modeling side, and the coordinates of the striatum were: A, +1.0 mm; L, −3.0 mm; V, −6.0 mm, −5.0 mm; 1 needle track, 2 depths, 1 μL was injected for each depth, injection speed was 1 μL/min, and the wound was sutured after injection. [0195] d) Post-operative care: After the operation, Baytril was subcutaneously injected daily, 0.5 mL/200 g body weight, for 3 consecutive days. Rotation experiment was performed every month after injection, the change in the number of turns over 5 months was plotted, in which significant functional improvement was observed in the 3.sup.rd month (FIG. 7).

EXAMPLE 2. Expansion Experiment of Astrocytes

[0196] 1. Differentiation and Culture method:

[0197] (1) hESCs were cultured and differentiation was conducted until about 80% confluence. On the 0.sup.th day, the cells were dissociated with Dispase (1 U/mL), and the bottom cell mass was harvested and plated in an ultra-low adsorption 10 cm culture dish. [0198] (2) From the 1.sup.st day, the neural induction was started by replacing with NIM medium supplemented with neural induction molecules 100 nM LDN193189 and 10 μM SB431542. The medium was changed daily until the 7.sup.th day. [0199] (3) The cell spheres were spread on a Matrigel-coated 6-well plate on the 7.sup.th day. [0200] (4) From the 9.sup.th day, the medium was replaced with NPM medium, and 20 ng/mL FGF2 was added until the 13.sup.th day. [0201] (5) On the 13.sup.th day, Accutase was used for dissociation to obtain single cells, which were re-spread on a Matrigel-coated six-well plate at a density of 1.2×10.sup.5 cells/cm.sup.2. [0202] (6) On the 14.sup.th day, the medium was replaced with astrocyte induction medium, the NPM medium was added with 10 ng/ml EGF+10 ng/ml FGF2+10 ng/ml TGFβ1, and 10 μm Blebbistatin (1:1000, not added to the control group) was added with at the same time, and the medium was replaced every other day. [0203] (7) After the density confluence of astrocytes reached 100%, they were dissociated into single cells with Accutase and counted, and re-spread on a Matrigel-coated six-well plate at a density of 3.0×10.sup.5 cells/cm.sup.2. [0204] (8) The cells were passaged for 5 times in succession, the initial cell density of each passage was 3.0×10.sup.5 cells/cm.sup.2, and the proliferation of astrocytes under the addition of Blebbistatin was compared.

TABLE-US-00003 Neural induction medium (NIM) H9 hESC Volume DMEM/F12 243.75 ml Neurobasal 243.75 ml N2 (100X) 2.5 ml B27 (50X) 5 ml GlutaMAX (100X) 5 ml Total 500 ml

TABLE-US-00004 Neural proliferation medium (NPM): H9 hESC Volume DMEM/F12 240 ml Neurobasal 240 ml N2 (100X) 5 ml B27 (50X) 10 ml GlutaMAX (100X) 5 ml Total 500 ml

2. Experimental Results:

[0205] From P2 to P6, the expansion ability of astrocytes in the experimental group was significantly improved (FIG. 8).

EXAMPLE 3. Proliferation of GABA Progenitor Cells

1. Differentiation and Culture Method:

[0206] (1) hESCs were cultured and differentiation was conducted until about 80% confluence. On the 0.sup.th day, the cells were dissociated with Dispase (1 U/mL), and the bottom cell mass was harvested and spread on an ultra-low adsorption 10 cm culture dish.

[0207] (2) From the 1.sup.st day, the neural induction was started by using NIM medium supplemented with 100 nM LDN193189, 10 μM SB431542, and 2 μM XAV939. The medium was changed daily until the 7.sup.th day. [0208] (3) The cell spheres were spread on a Matrigel-coated 6-well plate on the 7.sup.th day. [0209] (4) From the 9.sup.th day, the medium was replaced with NPM medium, and 20 ng/mL FGF2 was added until the 13.sup.th day. [0210] (5) On the 13.sup.th day, Accutase was used for dissociation to obtain single cells, which were re-spread on a Matrigel-coated 6-well plate at a density of 3.0×10.sup.5 cells/cm.sup.2. [0211] (6) On the 14.sup.th day, the medium was replaced with GABA cell induction medium, the NPM medium was added with 100 ng/mL SHH and 1 μM purmorphamine, and 10 μm Blebbistatin was added at the same time (not added to the control group), and the medium was replaced every other day. [0212] (7) After the density confluence of GABA progenitor cells reached 100%, they were dissociated into single cells with Accutase and counted, and then re-spread on a Matrigel-coated six-well plate at a density of 3.0×10.sup.5 cells/cm.sup.2. [0213] (8) The cells were passaged for 5 times, the initial cell density of each passage was 3.0×10.sup.5 cells/cm.sup.2, and the proliferation of GABA progenitor cells with or without Blebbistatin was compared.

2. Experimental Results:

[0214] From P4 to P5, the expansion ability of GABA progenitor cells in the experimental group was significantly improved (FIG. 9).

EXAMPLE 4. Proliferation of Microglial Progenitors

1. Differentiation and Culture Method:

[0215] (1) On the 0.sup.th day, ESCs were digested with Accutase into single cells, counted and inoculated into a V-bottom 96-well plate at a concentration of 10,000/well, and cultured statically. [0216] (2) From the 0.sup.th to 4.sup.th day, the medium was microglial 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. [0217] (3) On the 4.sup.th day, the suspended EBs were subjected to adherent culture in a 6-well plate, the culture medium was changed on the 4.sup.th day and then the static culture was carried out until the 11.sup.th day. From the 4.sup.th to 11.sup.th day, the culture medium was microglial differentiation medium: X-VIVO15, 1× Glutamax, 25 ng/ml IL-34, 50 ng/ml M-CSF. At this stage, Blebbistatin was added at a concentration of 10 μM. [0218] (4) On the 11.sup.th day, the single cells suspended in the medium were collected and counted, and the EBs were continued to be cultured with the microglial differentiation medium, and the suspended cells in the supernatant were collected every 7 days for a total of 3 collections. [0219] (5) Maturation of microglial cells: The supernatant was discarded after centrifugation, the cells were resuspended in a microglial maturation medium and inoculated on Matrigel pre-coated culture plates. The microglial cells matured after being continuously cultured for 14 days. Microglial maturation medium: Advanced F12, 1× Glutamax, 50 ng/ml IL-34, 5 ng/ml M-CSF.

2. Experimental Results:

[0220] On the 11.sup.th day of differentiation, the single cells suspended in the culture medium were collected and counted, and the EBs were continued to be cultured in the microglial differentiation medium, and the suspended cells in the supernatant were collected every 7 days for a total of 3 collections. Number of cells in each time of collection between the experimental group and the control group was compared (FIGS. 10 to 12). The number of cells in the first two collections of the experimental group increased significantly, which proved that the small molecule could promote the proliferation of microglial progenitors and improve the differentiation efficiency.

EXAMPLE 5. Proliferation of Neural Stem Cells

1. Differentiation and Culture Method:

[0221] The differentiation method was referred to Tchieu et al., A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages. 2017, Cell Stem Cell 21, 399-410.

[0222] NSCs were cultured with expansion medium after adherence, DMEM/F12 (1×, Gibco) supplemented with 20 μg/mL human (EGF) (R&D), 20 μg/mL (bFGF) (R&D), 5 μg/ml Heparin (Sigma), 2% B27 (Gibco), 1× GlutaMAX, and 10 μM Blebbistatin was added to the experimental group. Half of the culture medium was changed every day.

2. Experimental Results:

[0223] The small molecule significantly promoted the expansion of NSCs from P4 to P6 (see: FIG. 13).

[0224] 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 the present application, including all patents, patent applications, journal articles, books, and any other publications, is hereby incorporated by reference in its entirety.