Kit for inducing stem cells to differentiate into motor nerve precursor cells and / or motor neurons and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a kit for inducing stem cells to be differentiated into motor nerve progenitor cells, motor nerve precursor cells and / or motor neurons and application of the kit. The kit adopts a culture medium with definite chemical components, small chemical molecules and cytokines, does not involve animal-derived components and other substances which easily bring unstable factors such as batch difference, can be clinically used, makes up for the deficiency of reagents adopted by the existing differentiation method, and has a good application prospect. The prepared motor nerve progenitor cells, motor nerve precursor cells and motor neurons have high purity, are suitable for cell transplantation and related disease treatment, and can be applied to clinical product development.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for inducing stem cells to differentiate into motor nerve progenitor cells, motor nerve precursor cells and / or motor neurons and an application thereof. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Its main pathological features are the lesions or death of motor neurons in the cortex, brainstem or spinal cord, and the loss of neural connections between the brain and muscle tissue, which leads to the body's loss of motor control. Its symptoms include progressive muscle atrophy, paralysis, and difficulty eating and breathing. ALS patients eventually die of respiratory failure within 3-5 years after the onset of the disease. The vast majority of ALS cases are sporadic cases with unknown causes, so there is currently no effective treatment. There are currently only two FDA-approved drugs for the treatment of ALS, but they can only extend the patient's survival time by 2-3 months and delay the progression of the disease, but they cannot reverse the disease or improve the patient's quality of life.

[0003] Cell replacement therapy is currently an important direction for the treatment of ALS. Many research results have shown that in animal models, transplanted neural stem cells or stem cell-derived motor nerve precursors can replace dead neurons, rebuild motor nerve circuits, and restore voluntary muscle control. Induced pluripotent stem cells (iPSCs) have unlimited proliferation capacity and multiple differentiation potentials. They can differentiate into motor nerve precursors in vitro and are expected to be used in the treatment of ALS. At present, many studies have reported on the differentiation methods of motor nerve precursor cells, but the existing differentiation methods still have defects such as long culture time, low differentiation efficiency, cumbersome operation, and the use of animal-derived components, which are not conducive to the expansion of production and clinical treatment applications. Therefore, there is an urgent need for an efficient, easy-to-operate, low-cost motor neuron induction differentiation program that can be expanded. Summary of the invention

[0004] The first aspect of the present invention aims to provide a kit for inducing stem cells to differentiate into motor neuron progenitor cells.

[0005] The second aspect of the present invention aims to provide a kit for inducing stem cells to differentiate into motor progenitor cells, motor precursor cells or motor neurons.

[0006] The purpose of the third aspect of the present invention is to provide an application of the kit of the first aspect or the second aspect.

[0007] The fourth aspect of the present invention aims to provide a method for inducing stem cells to differentiate into motor progenitor cells, motor precursor cells or motor neurons.

[0008] The fifth aspect of the present invention aims to provide a motor neuron progenitor cell.

[0009] The sixth aspect of the present invention aims to provide a motor nerve precursor cell.

[0010] The seventh aspect of the present invention aims to provide a motor neuron.

[0011] The eighth aspect of the present invention aims to provide a pharmaceutical composition.

[0012] The purpose of the ninth aspect of the present invention is to provide the use of the motor progenitor cells of the fifth aspect of the present invention, the motor progenitor cells of the sixth aspect of the present invention, the motor neurons of the seventh aspect of the present invention and / or the pharmaceutical composition of the eighth aspect.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, there is provided a kit for inducing stem cells to differentiate into motor neuron progenitor cells, comprising: a first culture medium and a second culture medium;

[0015] The first culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor and a GSK-3 inhibitor;

[0016] The second culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator, a SHH signaling pathway activator and a ROCK inhibitor.

[0017] In some embodiments of the present invention, the BMP inhibitor is independently selected from at least one of: DMH1, LDN-212854, dorsomorphin, noggin, LDN-193189, follistatin, chordin, and gremlin.

[0018] In some embodiments of the present invention, the Nodal inhibitor is independently selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0019] In some embodiments of the present invention, the GSK-3 inhibitor is at least one of GSK3β inhibitor IX (6-bromoindirubin 3'-oxime), SB216763, GSK3β inhibitor VII (4-dibromoacetophenone), L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, and CHIR99021.

[0020] In some embodiments of the present invention, the RAR nuclear receptor activator is independently selected from at least one of retinoic acid and tazarotene.

[0021] In some embodiments of the present invention, the SHH signaling pathway activator is independently selected from at least one of SHH, SHHC25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine.

[0022] In some embodiments of the present invention, the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632 and SNJ-1656.

[0023] In some embodiments of the invention, the BMP inhibitor of the first culture medium comprises DMH1.

[0024] In some embodiments of the present invention, the concentration of the BMP inhibitor in the first culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 3 μM; further 2 μM.

[0025] In some embodiments of the invention, the BMP inhibitor of the second culture medium comprises DMH1.

[0026] In some embodiments of the present invention, the concentration of the BMP inhibitor in the second culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 3 μM; further 2 μM.

[0027] In some embodiments of the invention, the Nodal inhibitor of the first culture medium comprises SB431542.

[0028] In some embodiments of the present invention, the concentration of the Nodal inhibitor in the first culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 10 μM; further 2 μM.

[0029] In some embodiments of the invention, the Nodal inhibitor of the second culture medium comprises SB431542.

[0030] In some embodiments of the present invention, the concentration of the Nodal inhibitor in the second culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 10 μM; further 2 μM.

[0031] In some embodiments of the present invention, the GSK-3 inhibitor of the first culture medium comprises CHIR99021.

[0032] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the first culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 4 μM; further 3 μM.

[0033] In some embodiments of the present invention, the GSK-3 inhibitor of the second culture medium comprises CHIR99021.

[0034] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the second culture medium is 0.2 μM to 100 μM; further 0.5 to 20 μM; further 1 to 3 μM; further 1 μM.

[0035] In some embodiments of the present invention, the RAR nuclear receptor activator of the second culture medium comprises retinoic acid.

[0036] In some embodiments of the present invention, the concentration of the RAR nuclear receptor activator in the second culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.1 μM.

[0037] In some embodiments of the present invention, the SHH signaling pathway activator of the second culture medium comprises SAG.

[0038] In some embodiments of the present invention, the concentration of the SHH signaling pathway activator in the second culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.5 μM.

[0039] In some embodiments of the invention, the ROCK inhibitor of the second culture medium comprises Y-27632.

[0040] In some embodiments of the present invention, the concentration of the ROCK inhibitor in the second culture medium is 1 to 100 μM; further 5 to 15 μM; further 10 μM.

[0041] In some embodiments of the present invention, the basal culture medium of the first culture medium and the second culture medium is independently selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, GMEM medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, and Neurobasal medium.

[0042] In some embodiments of the present invention, the basal medium of the first culture medium and the second culture medium is a basal medium containing non-essential amino acids, N2 additives and B-27 additives.

[0043] In some embodiments of the present invention, the concentration of the non-essential amino acids in the basal culture medium is 0.2-5% (v / v); further 0.5-2% (v / v); further 1% (v / v).

[0044] In some embodiments of the present invention, the concentration of the N2 additive in the basal culture medium is 0.2-5% (v / v); further 0.3-2% (v / v), and further 0.5% (v / v).

[0045] In some embodiments of the present invention, the concentration of the B-27 additive in the basal culture medium is 0.2-5% (v / v); further 0.5-2% (v / v), and further 1% (v / v).

[0046] In some embodiments of the present invention, the basal medium of the first culture medium comprises DMEM / F12.

[0047] In some embodiments of the present invention, the basal medium of the second culture medium comprises DMEM / F12.

[0048] In some embodiments of the present invention, the first culture medium is used to induce stem cells to differentiate into neuroepithelial cells.

[0049] In some embodiments of the present invention, the second culture medium is used to induce neuroepithelial cells to differentiate into motor neuron progenitor cells.

[0050] In some embodiments of the present invention, the stem cells are stem cells with multidirectional differentiation potential.

[0051] In some embodiments of the present invention, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; more preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0052] In some embodiments of the present invention, the stem cells are derived from mammals; further derived from primates, and further derived from humans.

[0053] In some embodiments of the present invention, the stem cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0054] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0055] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0056] The kit according to the first aspect of the present invention can be used to prepare a high-purity population of OLIG2+HOXB4+OCT4- motor progenitor cells.

[0057] A second aspect of the present invention provides a kit for inducing stem cells to differentiate into motor neuron progenitor cells and / or motor neuron precursor cells, comprising: a first culture medium, a second culture medium, a third culture medium and a fourth culture medium;

[0058] The first culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor and a GSK-3 inhibitor;

[0059] The second culture medium is a basal culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, an RAR nuclear receptor activator, an SHH signaling pathway activator, and a ROCK inhibitor;

[0060] The third culture medium is a basal culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, an RAR nuclear receptor activator, an SHH signaling pathway activator and a ROCK inhibitor;

[0061] The fourth culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator, an SHH signaling pathway activator and a gamma secretase inhibitor.

[0062] In some embodiments of the present invention, the BMP inhibitor is independently selected from at least one of DMH1, LDN-212854, doxorubicin, Nogin, LDN-193189, activin binding protein, tenascin, and gremlin.

[0063] In some embodiments of the present invention, the Nodal inhibitor is independently selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0064] In some embodiments of the present invention, the GSK-3 inhibitor is at least one of GSK3β inhibitor IX (6-bromoindirubin 3'-oxime), SB216763, GSK3β inhibitor VII (4-dibromoacetophenone), L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparolone, indirubin, and CHIR99021.

[0065] In some embodiments of the present invention, the RAR nuclear receptor activator is independently selected from at least one of retinoic acid and tazarotene.

[0066] In some embodiments of the present invention, the SHH signaling pathway activator is independently selected from at least one of SHH, SHHC25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine.

[0067] In some embodiments of the present invention, the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632 and SNJ-1656.

[0068] In some embodiments of the present invention, the gamma-secretase inhibitor is independently selected from at least one of DAPT, YO-01027, and Compound E.

[0069] In some embodiments of the invention, the BMP inhibitor of the first culture medium comprises DMH1.

[0070] In some embodiments of the present invention, the concentration of the BMP inhibitor in the first culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 3 μM; further 2 μM.

[0071] In some embodiments of the invention, the BMP inhibitor of the second culture medium comprises DMH1.

[0072] In some embodiments of the present invention, the concentration of the BMP inhibitor in the second culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 3 μM; further 2 μM.

[0073] In some embodiments of the present invention, the BMP inhibitor of the third culture medium comprises DMH1.

[0074] In some embodiments of the present invention, the concentration of the BMP inhibitor in the third culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 3 μM; further 2 μM.

[0075] In some embodiments of the present invention, the BMP inhibitor of the fourth culture medium comprises DMH1.

[0076] In some embodiments of the present invention, the concentration of the BMP inhibitor in the fourth culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 3 μM; further 2 μM.

[0077] In some embodiments of the invention, the Nodal inhibitor of the first culture medium comprises SB431542.

[0078] In some embodiments of the present invention, the concentration of the Nodal inhibitor in the first culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 10 μM; further 2 μM.

[0079] In some embodiments of the invention, the Nodal inhibitor of the second culture medium comprises SB431542.

[0080] In some embodiments of the present invention, the concentration of the Nodal inhibitor in the second culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 10 μM; further 2 μM.

[0081] In some embodiments of the present invention, the Nodal inhibitor of the third culture medium comprises SB431542.

[0082] In some embodiments of the present invention, the concentration of the Nodal inhibitor in the third culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 10 μM; further 2 μM.

[0083] In some embodiments of the present invention, the Nodal inhibitor of the fourth culture medium comprises SB431542.

[0084] In some embodiments of the present invention, the concentration of the Nodal inhibitor in the fourth culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 10 μM; further 2 μM.

[0085] In some embodiments of the present invention, the GSK-3 inhibitor of the first culture medium comprises CHIR99021.

[0086] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the first culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 4 μM; further 3 μM.

[0087] In some embodiments of the present invention, the GSK-3 inhibitor of the second culture medium comprises CHIR99021.

[0088] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the second culture medium is 0.2 μM to 100 μM; further 0.5 to 20 μM; further 1 to 3 μM; further 1 μM.

[0089] In some embodiments of the present invention, the GSK-3 inhibitor of the third culture medium comprises CHIR99021.

[0090] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the third culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 4 μM; further 3 μM.

[0091] In some embodiments of the present invention, the GSK-3 inhibitor of the fourth culture medium comprises CHIR99021.

[0092] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the fourth culture medium is 0.2 μM to 100 μM; further 1 to 100 μM; further 1 to 4 μM; further 3 μM.

[0093] In some embodiments of the present invention, the RAR nuclear receptor activator of the second culture medium comprises retinoic acid.

[0094] In some embodiments of the present invention, the concentration of the RAR nuclear receptor activator in the second culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.1 μM.

[0095] In some embodiments of the present invention, the RAR nuclear receptor activator of the third culture medium comprises retinoic acid.

[0096] In some embodiments of the present invention, the concentration of the RAR nuclear receptor activator in the third culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.1 μM.

[0097] In some embodiments of the present invention, the RAR nuclear receptor activator in the fourth culture medium is retinoic acid.

[0098] In some embodiments of the present invention, the concentration of the RAR nuclear receptor activator in the fourth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.1 μM.

[0099] In some embodiments of the present invention, the SHH signaling pathway activator of the second culture medium comprises SAG.

[0100] In some embodiments of the present invention, the concentration of the SHH signaling pathway activator in the second culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.5 μM.

[0101] In some embodiments of the present invention, the SHH signaling pathway activator of the third culture medium comprises SAG.

[0102] In some embodiments of the present invention, the concentration of the SHH signaling pathway activator in the third culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.5 μM.

[0103] In some embodiments of the present invention, the SHH signaling pathway activator of the fourth culture medium comprises SAG.

[0104] In some embodiments of the present invention, the concentration of the SHH signaling pathway activator in the fourth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.5 μM.

[0105] In some embodiments of the invention, the ROCK inhibitor of the second culture medium comprises Y-27632.

[0106] In some embodiments of the present invention, the concentration of the ROCK inhibitor in the second culture medium is 1 to 100 μM; further 5 to 15 μM; further 10 μM.

[0107] In some embodiments of the present invention, the ROCK inhibitor of the third culture medium comprises Y-27632.

[0108] In some embodiments of the present invention, the concentration of the ROCK inhibitor in the third culture medium is 1-100 μM; further 5-15 μM; further 10 μM.

[0109] In some embodiments of the present invention, the gamma-secretase inhibitor of the fourth culture medium comprises Compound E.

[0110] In some embodiments of the present invention, the concentration of the gamma secretase inhibitor in the fourth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.1 μM.

[0111] In some embodiments of the present invention, the basal culture medium of the first culture medium, the second culture medium, the third culture medium and the fourth culture medium is independently selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium) culture medium, Eagle's Basal Medium (BME) culture medium, GMEM culture medium, MEM culture medium, DMEM culture medium, Ham's F-12 culture medium, RPMI1640 culture medium and Neurobasal culture medium.

[0112] In some embodiments of the present invention, the basal medium of the first culture medium, the second culture medium, the third culture medium and the fourth culture medium is a basal medium containing non-essential amino acids, N2 additives and B-27 additives.

[0113] In some embodiments of the present invention, the concentration of the non-essential amino acids in the basal culture medium is 0.2-5% (v / v); further 0.5-2% (v / v); further 1% (v / v).

[0114] In some embodiments of the present invention, the concentration of the N2 additive in the basal culture medium is 0.2-5% (v / v); further 0.3-2% (v / v), and further 0.5% (v / v).

[0115] In some embodiments of the present invention, the concentration of the B-27 additive in the basal culture medium is 0.2-5% (v / v); further 0.5-2% (v / v), and further 1% (v / v).

[0116] In some embodiments of the present invention, the basal medium of the first culture medium comprises DMEM / F12.

[0117] In some embodiments of the present invention, the basal medium of the second culture medium comprises DMEM / F12.

[0118] In some embodiments of the present invention, the basal medium of the third culture medium comprises DMEM / F12.

[0119] In some embodiments of the present invention, the basal medium of the fourth culture medium comprises DMEM / F12.

[0120] In some embodiments of the present invention, the first culture medium is used to induce stem cells to differentiate into neuroepithelial cells.

[0121] In some embodiments of the present invention, the second culture medium is used to induce neuroepithelial cells to differentiate into motor neuron progenitor cells.

[0122] In some embodiments of the present invention, the third culture medium is used to induce motor neuron progenitor cells to differentiate into motor neuron precursor cells.

[0123] In some embodiments of the present invention, the fourth culture medium is used to induce motor neuron progenitor cells to differentiate into motor neuron precursor cells.

[0124] In some embodiments of the present invention, the stem cells are stem cells with multidirectional differentiation potential.

[0125] In some embodiments of the present invention, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; more preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0126] In some embodiments of the present invention, the stem cells are derived from mammals; further derived from primates, and further derived from humans.

[0127] In some embodiments of the present invention, the stem cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0128] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0129] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0130] The kit according to the second aspect of the present invention can be used to prepare a high-purity OLIG2+HOXB4+OCT4- motor progenitor cell population and / or a high-purity HB9+OLIG2- motor progenitor cell population.

[0131] The second aspect of the present invention also provides a kit for inducing stem cells to differentiate into motor neurons, comprising: the kit for inducing stem cells to differentiate into motor progenitor cells and / or motor precursor cells of the second aspect above, a fifth culture medium and a sixth culture medium; the fifth culture medium is a basic culture medium containing an RAR nuclear receptor activator, an SHH signaling pathway activator, a gamma secretase inhibitor and a ROCK inhibitor; the sixth culture medium is a basic culture medium containing brain-derived growth factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), an SHH signaling pathway activator, a RAR nuclear receptor activator and ascorbic acid (AA).

[0132] In some embodiments of the present invention, the RAR nuclear receptor activator of the fifth culture medium comprises retinoic acid.

[0133] In some embodiments of the present invention, the concentration of the RAR nuclear receptor activator in the fifth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.5 μM.

[0134] In some embodiments of the present invention, the SHH signaling pathway activator of the fifth culture medium comprises SAG. SAG can activate the SHH signaling pathway while reducing toxicity to cells, thereby improving differentiation efficiency.

[0135] In some embodiments of the present invention, the concentration of the SHH signaling pathway activator in the fifth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.2 μM.

[0136] In some embodiments of the present invention, the gamma secretase inhibitor of the fifth culture medium comprises compound E. Compound E can efficiently induce motor neuron progenitor cells to differentiate into motor neuron precursor cells.

[0137] In some embodiments of the present invention, the concentration of the gamma-secretase inhibitor in the fifth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.1 μμM.

[0138] In some embodiments of the present invention, the ROCK inhibitor of the fifth culture medium comprises Y-27632.

[0139] In some embodiments of the present invention, the concentration of the ROCK inhibitor in the fifth culture medium is 1 to 100 μM; further 5 to 15 μM; further 10 μM.

[0140] In some embodiments of the present invention, the basal culture medium of the fifth culture medium is independently selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, GMEM medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, and Neurobasal medium.

[0141] In some embodiments of the present invention, the basal medium of the fifth culture medium is a basal medium containing non-essential amino acids, N2 additives and B-27 additives.

[0142] In some embodiments of the present invention, the concentration of the non-essential amino acids in the basal culture medium is 0.2-5% (v / v); further 0.5-2% (v / v); further 1% (v / v).

[0143] In some embodiments of the present invention, the concentration of the N2 additive in the basal culture medium is 0.2-5% (v / v); further 0.3-2% (v / v), and further 0.5% (v / v).

[0144] In some embodiments of the present invention, the concentration of the B-27 additive in the basal culture medium is 0.2-5% (v / v); further 0.5-2% (v / v), and further 1% (v / v).

[0145] In some embodiments of the present invention, the basal medium of the fifth culture medium comprises DMEM / F12.

[0146] In some embodiments of the present invention, the fifth culture medium is used to induce motor neuron precursor cells to differentiate into mature motor neurons.

[0147] In some embodiments of the present invention, the concentration of the brain-derived growth factor (BDNF) in the sixth culture medium is 1 ng / mL to 100 ng / mL; further 5 to 15 ng / mL; further 10 ng / mL.

[0148] In some embodiments of the present invention, the concentration of the glial cell line-derived neurotrophic factor (GDNF) in the sixth culture medium is 1 ng / mL to 100 ng / mL; further 5 to 15 ng / mL; further 10 ng / mL.

[0149] In some embodiments of the present invention, the RAR nuclear receptor activator of the sixth culture medium comprises retinoic acid.

[0150] In some embodiments of the present invention, the concentration of the RAR nuclear receptor activator in the sixth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.5 μM.

[0151] In some embodiments of the present invention, the SHH signaling pathway activator of the sixth culture medium comprises SAG.

[0152] In some embodiments of the present invention, the concentration of the SHH signaling pathway activator in the sixth culture medium is 0.01 μM to 10 μM; further 0.05 to 1 μM; further 0.2 μM.

[0153] In some embodiments of the present invention, the concentration of ascorbic acid (AA) in the sixth culture medium is 10 μM to 1 mM; further 150 to 250 μM; further 200 μM.

[0154] In some embodiments of the present invention, the basal medium of the sixth culture medium is Neurobasal containing N2 additive and B-27 additive.

[0155] In some embodiments of the present invention, the concentration of the N2 additive in the basal culture medium is 0.2-5% (v / v); further 0.3-2% (v / v), and further 1% (v / v).

[0156] In some embodiments of the present invention, the concentration of the B-27 additive in the basal medium is 0.2-5% (v / v); further 0.5-3% (v / v), and further 2% (v / v).

[0157] In the present invention, the sixth culture medium is used to induce motor neuron precursor cells to differentiate into motor neurons.

[0158] The kit of the second aspect of the present invention can be used to prepare a high-purity OLIG2+HOXB4+OCT4- motor progenitor cell population, a high-purity HB9+OLIG2- motor progenitor cell population and / or a high-purity HB9+CHAT+ motor neurons.

[0159] The third aspect of the present invention provides the use of the kit of the first aspect or the kit of the second aspect of the present invention in any one of (1) to (6):

[0160] (1) Preparation of motor neuron progenitor cells

[0161] (2) preparing motor nerve precursor cells;

[0162] (3) Preparation of motor neurons;

[0163] (4) preparing products for inducing stem cells to differentiate into motor neuron progenitor cells;

[0164] (5) preparing products for inducing stem cells to differentiate into motor nerve precursor cells;

[0165] (6) Prepare products for inducing stem cells to differentiate into motor neurons.

[0166] In some embodiments of the present invention, the stem cells are stem cells with multidirectional differentiation potential.

[0167] In some embodiments of the present invention, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; more preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0168] In some embodiments of the present invention, the stem cells are derived from mammals; further derived from primates, and further derived from humans.

[0169] In some embodiments of the present invention, the stem cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0170] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0171] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0172] The fourth aspect of the present invention provides a method for inducing stem cells to differentiate into motor neuron progenitor cells, comprising the steps of using the kit for inducing stem cells to differentiate into motor neuron progenitor cells of the first aspect of the present invention or the kit for inducing stem cells to differentiate into motor neuron progenitor cells and / or motor neuron precursor cells of the second aspect of the present invention or the kit for inducing stem cells to differentiate into motor neurons.

[0173] In some embodiments of the present invention, the method comprises the following steps:

[0174] (1) culturing stem cells for the first time using the first culture medium in the kit of the first aspect of the present invention to obtain neuroepithelial cells;

[0175] (2) The neuroepithelial cells are resuspended in the second culture medium in the kit of the first aspect of the present invention, and cultured for a second time to obtain motor neuron progenitor cells.

[0176] In some embodiments of the present invention, the first culture period in step (1) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0177] In some embodiments of the present invention, the first culture in step (1) is adherent culture; further, it is adherent culture on a culture device coated with Laminin.

[0178] In some embodiments of the present invention, the second culture period in step (2) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0179] In some embodiments of the present invention, the second culture in step (2) further includes a digestion step.

[0180] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0181] In some embodiments of the present invention, the method comprises the following steps:

[0182] (1) culturing the stem cells for the first time using the first culture medium in the kit according to the second aspect of the present invention to obtain neuroepithelial cells;

[0183] (2) resuspending the neuroepithelial cells in the second culture medium of the kit according to the second aspect of the present invention, and performing a second culture to obtain motor neuron progenitor cells;

[0184] In some embodiments of the present invention, the first culture period in step (1) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0185] In some embodiments of the present invention, the first culture in step (1) is adherent culture; further, it is adherent culture on a culture device coated with Laminin.

[0186] In some embodiments of the present invention, the second culture period in step (2) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0187] In some embodiments of the present invention, the second culture in step (2) further includes a digestion step.

[0188] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0189] In some embodiments of the present invention, the above-mentioned culture conditions are 32-38°C, 4-6% CO 2 .

[0190] In some embodiments of the present invention, the stem cells further include a culturing step before differentiation, and the steps are as follows: when the stem cells are cultured to a polymerization degree of 70-80%, they are digested to obtain single cells, and resuspended in a culture medium containing a ROCK inhibitor to obtain a cell suspension; the cell suspension is inoculated in a culture device coated with Laminin and cultured for 20-28 hours.

[0191] In some embodiments of the present invention, the culture medium used for stem cell culture is StemFit culture medium.

[0192] In some embodiments of the present invention, the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632 and SNJ-1656.

[0193] In some embodiments of the present invention, the stem cells are stem cells with multidirectional differentiation potential.

[0194] In some embodiments of the present invention, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; more preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0195] In some embodiments of the present invention, the stem cells are derived from mammals; further derived from primates, and further derived from humans.

[0196] In some embodiments of the present invention, the stem cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0197] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0198] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0199] The method according to the fourth aspect of the present invention can be used to prepare a high-purity population of OLIG2+HOXB4+OCT4- motor neuron progenitor cells.

[0200] The fourth aspect of the present invention also provides a method for inducing stem cells to differentiate into motor nerve precursor cells, comprising the steps of using the kit for inducing stem cells to differentiate into neural progenitor cells and / or motor nerve precursor cells or the kit for inducing stem cells to differentiate into motor neurons according to the second aspect of the present invention.

[0201] In some embodiments of the present invention, the method comprises the following steps:

[0202] (1) culturing the stem cells for the first time using the first culture medium in the kit according to the second aspect of the present invention to obtain neuroepithelial cells;

[0203] (2) resuspending the neuroepithelial cells in the second culture medium of the kit according to the second aspect of the present invention, and performing a second culture to obtain motor neuron progenitor cells;

[0204] (3) The motor neuron progenitor cells are cultured for a third time using the third culture medium in the kit of the second aspect of the present invention, and then cultured for a fourth time using the fourth culture medium in the kit of the second aspect of the present invention to obtain motor neuron progenitor cells.

[0205] In some embodiments of the present invention, the first culture period in step (1) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0206] In some embodiments of the present invention, the first culture in step (1) is adherent culture; further, it is adherent culture on a culture device coated with Laminin.

[0207] In some embodiments of the present invention, the second culture period in step (2) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0208] In some embodiments of the present invention, the second culture in step (2) further includes a digestion step.

[0209] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0210] In some embodiments of the present invention, the duration of the third culture in step (3) is 2 to 7 days; further 3 to 5 days; further 4 days.

[0211] In some embodiments of the present invention, the third culture in step (3) further includes a digestion step.

[0212] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0213] In some embodiments of the present invention, the duration of the fourth culture in step (3) is 1 to 4 days; further 1 to 3 days; further 2 days.

[0214] In some embodiments of the present invention, the above-mentioned culture conditions are 32-38°C, 4-6% CO 2 .

[0215] In some embodiments of the present invention, the stem cells further include a culturing step before differentiation, and the steps are as follows: when the stem cells are cultured to a polymerization degree of 70-80%, they are digested to obtain single cells, and resuspended in a culture medium containing a ROCK inhibitor to obtain a cell suspension; the cell suspension is inoculated in a culture device coated with Laminin and cultured for 20-28 hours.

[0216] In some embodiments of the present invention, the culture medium used for stem cell culture is StemFit culture medium.

[0217] In some embodiments of the present invention, the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632 and SNJ-1656.

[0218] In some embodiments of the present invention, the stem cells are stem cells with multidirectional differentiation potential.

[0219] In some embodiments of the present invention, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; more preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0220] In some embodiments of the present invention, the stem cells are derived from mammals; further derived from primates, and further derived from humans.

[0221] In some embodiments of the present invention, the stem cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0222] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0223] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0224] The method of the fourth aspect of the present invention can be used to prepare a high-purity OLIG2+HOXB4+OCT4- motor progenitor cell population and / or a high-purity HB9+OLIG2- motor progenitor cell population.

[0225] The fourth aspect of the present invention also provides a method for inducing stem cells to differentiate into motor neurons, comprising the steps of using the kit for inducing stem cells to differentiate into motor neurons according to the second aspect of the present invention.

[0226] In some embodiments of the present invention, the method comprises the following steps:

[0227] (1) culturing the stem cells for the first time using the first culture medium in the kit according to the second aspect of the present invention to obtain neuroepithelial cells;

[0228] (2) resuspending the neuroepithelial cells in the second culture medium of the kit according to the second aspect of the present invention, and performing a second culture to obtain motor neuron progenitor cells;

[0229] (3) The motor neuron progenitor cells are cultured for a third time using the third culture medium in the kit of the second aspect of the present invention, and then cultured for a fourth time using the fourth culture medium in the kit of the second aspect of the present invention to obtain motor neuron progenitor cells.

[0230] (4) The motor neuron precursor cells are cultured for a fifth time using the fifth culture medium in the kit of the second aspect of the present invention, and then cultured for a sixth time using the sixth culture medium in the kit of the second aspect of the present invention to obtain motor neurons.

[0231] In some embodiments of the present invention, the first culture period in step (1) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0232] In some embodiments of the present invention, the first culture in step (1) is adherent culture; further, adherent culture on a culture device coated with Laminin. Laminin, as a differentiation matrix, can promote cell proliferation and differentiation and maturation, and improve the efficiency of MNP cell harvesting.

[0233] In some embodiments of the present invention, the second culture period in step (2) is 4 to 10 days; further 5 to 7 days; further 6 days.

[0234] In some embodiments of the present invention, the second culture in step (2) further includes a digestion step.

[0235] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0236] In some embodiments of the present invention, the second culture in step (2) is adherent culture; further, it is adherent culture on a culture device coated with Laminin.

[0237] In some embodiments of the present invention, the duration of the third culture in step (3) is 2 to 7 days; further 3 to 5 days; further 4 days.

[0238] In some embodiments of the present invention, the third culture in step (3) further includes a digestion step.

[0239] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0240] In some embodiments of the present invention, the third culture in step (3) is adherent culture; further, it is adherent culture on a culture device coated with Laminin.

[0241] In some embodiments of the present invention, the duration of the fourth culture in step (3) is 1 to 4 days; further 1 to 3 days; further 2 days.

[0242] In some embodiments of the present invention, the fifth culture in step (4) lasts for 1 to 5 days; further, 1 to 4 days; and further, 3 days.

[0243] In some embodiments of the present invention, the fifth culture in step (4) further includes a digestion step.

[0244] In some embodiments of the present invention, the digestion is performed using TrypLE digestion solution.

[0245] In some embodiments of the present invention, the fifth culture in step (4) is adherent culture; further, it is adherent culture on a culture device coated with Matrigel.

[0246] In some embodiments of the present invention, the sixth culture in step (4) lasts for 6 to 12 days or 7 to 12 days; further for 6 to 10 days or 8 to 10 days; further for 6 days or 9 days.

[0247] In some embodiments of the present invention, the above-mentioned culture conditions are 32-38°C, 4-6% CO 2 .

[0248] In some embodiments of the present invention, the stem cells further include a culturing step before differentiation, and the steps are as follows: when the stem cells are cultured to a polymerization degree of 70-80%, they are digested to obtain single cells, and resuspended in a culture medium containing a ROCK inhibitor to obtain a cell suspension; the cell suspension is inoculated in a culture device coated with Laminin and cultured for 20-28 hours.

[0249] In some embodiments of the present invention, the culture medium used for stem cell culture is StemFit culture medium.

[0250] In some embodiments of the present invention, the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632 and SNJ-1656.

[0251] In some embodiments of the present invention, the stem cells are stem cells with multidirectional differentiation potential.

[0252] In some embodiments of the present invention, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; more preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0253] In some embodiments of the present invention, the stem cells are derived from mammals; further derived from primates, and further derived from humans.

[0254] In some embodiments of the present invention, the stem cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0255] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0256] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0257] The method of the fourth aspect of the present invention can be used to prepare a high-purity OLIG2+HOXB4+OCT4- motor progenitor cell population, a high-purity HB9+OLIG2- motor progenitor cell population and / or a high-purity HB9+CHAT+ motor neurons.

[0258] The fifth aspect of the present invention provides a motor neuron progenitor cell, which is prepared by the kit for inducing stem cells to differentiate into motor neuron progenitor cells according to the first aspect of the present invention, the kit for inducing stem cells to differentiate into motor neuron progenitor cells and / or motor neuron precursor cells or the kit for inducing stem cells to differentiate into motor neurons according to the second aspect of the present invention, and / or the method for inducing stem cells to differentiate into motor neuron progenitor cells according to the fourth aspect of the present invention.

[0259] In some embodiments of the present invention, the content of OLIG2+HOXB4+OCT4- cells in the motor neuron progenitor cells is greater than 70%.

[0260] In some embodiments of the present invention, the content of OLIG2+HOXB4+OCT4- cells in the motor neuron progenitor cells is greater than 70%.

[0261] In some embodiments of the present invention, the content of OLIG2+HOXB4+OCT4- cells in the motor neuron progenitor cells is more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%; further is 70%; further is 90%.

[0262] In some embodiments of the invention, the motor neuron progenitor cells comprise a population of motor neuron progenitor cells.

[0263] The motor progenitor cells of the fifth aspect of the present invention can improve the limb muscle strength of the subject (e.g., ALS model mice or ALS patients); can improve the motor coordination ability of the subject (e.g., ALS model mice or ALS patients); can increase the survival rate of the subject (e.g., ALS model mice or ALS patients) and prolong their survival; can alleviate the reduction in the number of neurons in the subject (e.g., ALS model mice or ALS patients); and / or can differentiate into motor neurons in the subject (e.g., ALS model mice or ALS patients), but have almost no proliferation in the body and do not cause tumor formation.

[0264] The sixth aspect of the present invention provides a motor nerve precursor cell, which is prepared by the kit for inducing stem cells to differentiate into motor nerve progenitor cells and / or motor nerve precursor cells or the kit for inducing stem cells to differentiate into motor neurons according to the second aspect of the present invention, and / or the method for inducing stem cells to differentiate into motor nerve precursor cells according to the fourth aspect of the present invention.

[0265] In some embodiments of the present invention, the content of HB9+OLIG2- cells in the motor nerve precursor cells is greater than 70%.

[0266] In some embodiments of the present invention, the content of HB9+OLIG2- cells in the motor nerve precursor cells is greater than 70%.

[0267] In some embodiments of the present invention, the content of HB9+OLIG2- cells in the motor nerve precursor cells is greater than 72%, greater than 75%, greater than 80%, greater than 90%; further greater than 76%.

[0268] In some embodiments of the present invention, the content of cells expressing HB9 in the motor nerve precursor cells is greater than 80%.

[0269] In some embodiments of the present invention, the content of cells expressing HB9 in the motor nerve precursor cells is greater than 80%.

[0270] In some embodiments of the present invention, the content of cells expressing HB9 in the motor nerve precursor cells is 81% or more, 82% or more, 83% or more, 85% or more, 90% or more; further 84%.

[0271] In some embodiments of the invention, the motor nerve precursor cells comprise a population of motor nerve precursor cells.

[0272] The motor precursor cells of the sixth aspect of the present invention can improve the limb muscle strength of the subject (e.g., ALS model mouse or ALS patient); can improve the motor coordination ability of the subject (e.g., ALS model mouse or ALS patient); can increase the survival rate of the subject (e.g., ALS model mouse or ALS patient) and prolong its survival period; can alleviate the reduction in the number of neurons in the subject (e.g., ALS model mouse or ALS patient); and / or can differentiate into motor neurons in the subject (e.g., ALS model mouse or ALS patient), but have almost no proliferation in the body and do not cause tumor formation.

[0273] The seventh aspect of the present invention provides a motor neuron, which is prepared by the kit for inducing stem cells to differentiate into motor neurons according to the second aspect and / or the method for inducing stem cells to differentiate into motor neurons according to the fourth aspect of the present invention.

[0274] In some embodiments of the invention, the motor neurons comprise a population of motor neurons.

[0275] The motor neurons of the seventh aspect of the present invention can improve the limb muscle strength of the subject (e.g., ALS model mice or ALS patients); can improve the motor coordination ability of the subject (e.g., ALS model mice or ALS patients); can increase the survival rate and prolong the survival period of the subject (e.g., ALS model mice or ALS patients); can alleviate the reduction in the number of neurons in the subject (e.g., ALS model mice or ALS patients); and / or, have almost no proliferation in the body and do not cause tumor formation.

[0276] The eighth aspect of the present invention provides a pharmaceutical composition comprising: the motor neuron progenitor cells of the fifth aspect of the present invention, the motor neuron precursor cells of the sixth aspect of the present invention and / or the motor neurons of the seventh aspect of the present invention.

[0277] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0278] In some embodiments of the present invention, the pharmaceutical composition is a liquid preparation.

[0279] In some embodiments of the present invention, the pharmaceutically acceptable carrier or excipient includes but is not limited to: at least one of physiological saline, water, cell buffer, compound electrolyte injection, Ringer's solution and trehalose solution.

[0280] In some embodiments of the present invention, the water is ddH 2 O.

[0281] In some embodiments of the present invention, the cell buffer comprises: PBS, HBSS, EBSS, HEPES and other commonly used cell buffers.

[0282] In some embodiments of the present invention, the cell buffer comprises at least one of PBS and HBSS.

[0283] In some embodiments of the present invention, the pharmaceutically acceptable carrier or excipient is cerebrospinal fluid. In some embodiments of the present invention, the cerebrospinal fluid is artificial cerebrospinal fluid.

[0284] In some embodiments of the present invention, the pharmaceutical composition further comprises other drugs for preventing and / or treating nervous system diseases.

[0285] In some embodiments of the present invention, the nervous system disease includes at least one of a neurodegenerative disease and a neurodamage disease.

[0286] In some embodiments of the present invention, the nervous system disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's chorea, stroke, intracranial tumor, epilepsy, craniocerebral trauma, hereditary nervous system disease, cerebrovascular disease, paralysis, myasthenia gravis, multiple sclerosis, neuritis, cerebral palsy, and depression.

[0287] In some embodiments of the present invention, the nervous system disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis, stroke, craniocerebral trauma, and Alzheimer's disease.

[0288] In some embodiments of the present invention, the nervous system disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis, stroke, and craniocerebral trauma.

[0289] The pharmaceutical composition of the eighth aspect of the present invention can improve the limb muscle strength of the subject (e.g., ALS model mouse or ALS patient); can improve the motor coordination ability of the subject (e.g., ALS model mouse or ALS patient); can increase the survival rate of the subject (e.g., ALS model mouse or ALS patient) and prolong its survival period; can alleviate the reduction in the number of neurons in the subject (e.g., ALS model mouse or ALS patient); and / or can differentiate into motor neurons in the subject (e.g., ALS model mouse or ALS patient), but have almost no proliferation in the body and do not cause tumor formation.

[0290] The ninth aspect of the present invention provides the use of the motor progenitor cells of the fifth aspect of the present invention, the motor progenitor cells of the sixth aspect of the present invention, the motor neurons of the seventh aspect of the present invention and / or the pharmaceutical composition of the eighth aspect of the present invention.

[0291] The ninth aspect of the present invention provides the use of the motor neuron progenitor cells of the fifth aspect of the present invention, the motor neuron precursor cells of the sixth aspect of the present invention and / or the motor neurons of the seventh aspect of the present invention in constructing a cell bank.

[0292] The ninth aspect of the present invention also provides the use of the motor progenitor cells of the fifth aspect of the present invention, the motor progenitor cells of the sixth aspect of the present invention, the motor neurons of the seventh aspect of the present invention and / or the pharmaceutical composition of the eighth aspect of the present invention in the preparation of drugs for preventing and / or treating nervous system diseases.

[0293] The present invention also provides a method for treating nervous system diseases, comprising the following steps: administering to a subject the motor progenitor cells of the fifth aspect of the present invention, the motor precursor cells of the sixth aspect of the present invention, the motor neurons of the seventh aspect of the present invention and / or the pharmaceutical composition of the eighth aspect of the present invention.

[0294] In some embodiments of the present invention, the subject is a mammal, more preferably a primate, and even more preferably a human.

[0295] In some embodiments of the present invention, the nervous system disease includes at least one of a neurodegenerative disease and a neurodamage disease.

[0296] In some embodiments of the present invention, the nervous system disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's chorea, stroke, intracranial tumor, epilepsy, craniocerebral trauma, hereditary nervous system disease, cerebrovascular disease, paralysis, myasthenia gravis, multiple sclerosis, neuritis, cerebral palsy, and depression.

[0297] In some embodiments of the present invention, the nervous system disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis, stroke, craniocerebral trauma, and Alzheimer's disease.

[0298] In some embodiments of the present invention, the nervous system disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis, stroke, and craniocerebral trauma.

[0299] Implementation

[0300] Embodiment 1. A kit comprising a first culture medium, a second culture medium, a third culture medium and a fourth culture medium;

[0301] The first culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor and a GSK-3 inhibitor;

[0302] The second culture medium is a basal culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, an RAR nuclear receptor activator, an SHH signaling pathway activator, and a ROCK inhibitor;

[0303] The third culture medium is a basal culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, an RAR nuclear receptor activator, an SHH signaling pathway activator and a ROCK inhibitor;

[0304] The fourth culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator, an SHH signaling pathway activator and a gamma secretase inhibitor.

[0305] Embodiment 2. The kit according to embodiment 1 is characterized in that: the BMP inhibitor is independently selected from at least one of DMH1, LDN-212854, dorsomorphin, noggin, LDN-193189, follistatin, chordin, and gremlin; and / or the Nodal inhibitor is independently selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761; and / or the GSK-3 inhibitor is at least one of GSK3β inhibitor IX, SB216763, GSK3β inhibitor VII, L803-mts, 6-bromo-indirubin 3'-oxime, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, CHIR99021; and / or the RAR nuclear receptor activator is independently selected from: retinoic acid (Retinoic acid) and tazarotene; and / or the SHH signaling pathway activator is independently selected from: at least one of SHH, SHH C25II, SAG, SAG21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine; and / or the ROCK inhibitor is independently selected from: at least one of AR-12286, Y-27632 and SNJ-1656; and / or the gamma secretase inhibitor is independently selected from: at least one of DAPT, YO-01027, and Compound E.

[0306] Embodiment 3. The kit according to Embodiment 1 is characterized in that: the kit further comprises: a fifth culture medium and a sixth culture medium; the fifth culture medium is a basic culture medium containing RAR nuclear receptor activator, SHH signaling pathway activator, gamma secretase inhibitor, ROCK inhibitor and gamma secretase inhibitor; the sixth culture medium is a basic culture medium containing brain-derived growth factor, glial cell line-derived neurotrophic factor, SHH signaling pathway activator, RAR nuclear receptor activator and ascorbic acid.

[0307] Embodiment 4. Use of the kit described in Embodiment 3 in any one of (1) to (4):

[0308] (1) Preparing motor nerve precursor cells;

[0309] (2) preparing motor neurons;

[0310] (3) preparing products for inducing stem cells to differentiate into motor nerve precursor cells;

[0311] (4) Prepare products for inducing stem cells to differentiate into motor neurons.

[0312] Embodiment 5. Any one of the following methods a1) to a2):

[0313] a1) A method for inducing stem cells to differentiate into motor nerve precursor cells, comprising the steps of using the kit described in any one of embodiments 1 to 3;

[0314] a2) A method for inducing stem cells to differentiate into motor neurons, comprising the steps of using the kit described in embodiment 3.

[0315] Embodiment 6. The method according to embodiment 5, characterized in that:

[0316] The method of inducing stem cells to differentiate into motor nerve precursor cells in a1) comprises the following steps:

[0317] (1) culturing the stem cells for the first time using the first culture medium in the kit according to any one of embodiments 1 to 3 to obtain neuroepithelial cells;

[0318] (2) resuspending the neuroepithelial cells in the second culture medium described in any one of Embodiments 1 to 3, and culturing them for a second time to obtain motor neuron progenitor cells;

[0319] (3) culturing the motor neuron progenitor cells for a third time using the third culture medium in the kit described in any one of Embodiments 1 to 3, and then culturing them for a fourth time using the fourth culture medium described in any one of Embodiments 1 to 3, to obtain motor neuron progenitor cells;

[0320] Or the method for inducing stem cells to differentiate into motor neurons described in a2) comprises the following steps:

[0321] (1) culturing the stem cells for the first time using the first culture medium in the kit according to any one of embodiments 1 to 3 to obtain neuroepithelial cells;

[0322] (2) resuspending the neuroepithelial cells in the second culture medium in the kit according to any one of embodiments 1 to 3, and performing a second culture to obtain motor neuron progenitor cells;

[0323] (3) culturing the motor neuron progenitor cells for a third time using the third culture medium in the kit described in any one of Embodiments 1 to 3, and then culturing them for a fourth time using the fourth culture medium described in any one of Embodiments 1 to 3, to obtain motor neuron progenitor cells;

[0324] (4) culturing the motor neuron precursor cells for a fifth time using the fifth culture medium in the kit described in Embodiment 3, and then culturing them for a sixth time using the sixth culture medium in the kit described in Embodiment 3 to obtain motor neurons;

[0325] Preferably, the first culture time in step (1) of a1) or a2) is 4 to 10 days;

[0326] Preferably, the second culture time in step (2) of a1) or a2) is 4 to 10 days;

[0327] Preferably, the duration of the third culture in step (3) of a1) or a2) is 2 to 7 days;

[0328] Preferably, the duration of the fourth culture in step (3) of a1) or a2) is 1 to 4 days;

[0329] Preferably, the fifth culture in step (4) of a2) lasts for 1 to 5 days;

[0330] Preferably, the sixth culture in step (4) of a2) lasts for 7 to 12 days.

[0331] Embodiment 7. A motor nerve precursor cell, prepared by the kit described in any one of Embodiments 1 to 3 and / or the method for inducing stem cells to differentiate into motor nerve precursor cells described in any one of Embodiments 5 to 6;

[0332] Preferably, the content of HB9+OLIG2- cells in the motor nerve precursor cells is greater than 70%;

[0333] Preferably, the content of cells expressing HB9 in the motor nerve precursor cells is greater than 80%.

[0334] Embodiment 8. A motor neuron prepared by the kit described in any one of Embodiments 1 to 3 and / or the method for inducing stem cells to differentiate into motor neurons described in any one of Embodiments 5 to 6.

[0335] Embodiment 9. A pharmaceutical composition comprising: the motor nerve precursor cell of embodiment 7 and / or the motor neuron of embodiment 8;

[0336] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient;

[0337] Preferably, the pharmaceutical composition further comprises other drugs for preventing and / or treating nervous system diseases.

[0338] Embodiment 10. Any one of the following b1) to b2) is used:

[0339] b1) Use of the motor nerve precursor cells described in Embodiment 7 and / or the motor neurons described in Embodiment 8 in constructing a cell bank;

[0340] b2) Use of the motor nerve precursor cells described in Embodiment 7, the motor neurons described in Embodiment 8 and / or the pharmaceutical composition described in Embodiment 9 in the preparation of drugs for preventing and treating nervous system diseases.

[0341] The beneficial effects of the present invention are:

[0342] The present invention provides a kit, which uses a culture medium, chemical small molecules and cytokines with clear chemical compositions, and does not involve substances such as animal-derived components that are prone to bring about unstable factors such as batch differences. At the same time, the differentiation cycle is reduced and can be used clinically, making up for the shortcomings of reagents used in existing differentiation methods. The prepared motor progenitor cells, motor precursor cells and motor neurons have high purity, are suitable for cell transplantation and treatment of related diseases, and can be applied to clinical product development.

[0343] The kit or method for preparing motor progenitor cells, motor precursor cells and / or motor neurons of the present invention or the motor progenitor cells, motor precursor cells and motor neurons of the present invention can show excellent effects, such as but not limited to: (1) obtaining a high-purity OLIG2+HOXB4+OCT4- motor progenitor cell population; (2) obtaining a high-purity HB9+OLIG2- motor precursor cell population; (3) obtaining a high-purity HB9+CHAT+ motor neuron; (4) transplanting the cells of the present invention can improve the Limb muscle strength; (5) Transplantation of the cells of the present invention can improve the motor coordination ability of the subject (e.g., ALS model mice or ALS patients); (6) Transplantation of the cells of the present invention can increase the survival rate of the subject (e.g., ALS model mice or ALS patients) and prolong their survival; (7) Transplantation of the cells of the present invention can alleviate the reduction in the number of neurons in the subject (e.g., ALS model mice or ALS patients); (8) After transplantation, the cells of the present invention can differentiate into motor neurons in the subject (e.g., ALS model mice or ALS patients), but have almost no proliferation in the body and do not cause tumor formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0344] Figure 1 This is a graph showing the results of flow cytometry analysis of OLIG2+, OCT4+, and HOXB4+ of human pluripotent stem cells cultured for 12 days in the test example that differentiated into motor neuron progenitor cells.

[0345] Figure 2 This is a diagram showing the results of HB9 and OLIG2 staining of motor neuron precursor cells cultured to day 18 in the test example.

[0346] Figure 3 This is a statistical graph showing the results of HB9 and OLIG2 staining of motor neuron precursor cells cultured to day 18 in the test example.

[0347] Figure 4 This is a graph showing the detection results of the motor neurons in the test case.

[0348] Figure 5A and Figure 5B The results of the gripping strength test of SOD1 G93A mice in the vehicle group and the iPS-MNP cell treatment group are shown; the control group: 8 male mice and 9 female mice, the cell treatment group: 9 male mice and 9 female mice.

[0349] Figure 6 The rotarod test results of SOD1 G93A mice in the vehicle group and SOD1 G93A mice in the iPS-MNP cell treatment group are shown; the control group: 8 male mice and 9 female mice, the cell treatment group: 9 male mice and 9 female mice.

[0350] Figure 7 The survival statistics of SOD1 G93A mice in the vehicle group and the iPS-MNP cell treatment group are shown; the control group: 8 male mice and 9 female mice, the cell treatment group: 9 male mice and 9 female mice.

[0351] Figure 8 The results show the fluorescence distribution in NOG mice after intrathecal transplantation of luciferase-labeled iPS-MNP cells.

[0352] Fig. 9A and Fig. 9B The figure shows the survival, proliferation and differentiation of iPS-MNP cells in NOG mice after intrathecal transplantation of luciferase-labeled iPS-MNP cells. DETAILED DESCRIPTION

[0353] The present invention is further described in detail below through specific examples.

[0354] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0355] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0356] The human pluripotent stem cells used in this example are human induced pluripotent stem cells (iPSCs), which were obtained by CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat. No. A34546) was prepared (see the instruction manual for specific methods), and the initial cells were human peripheral blood cells.

[0357] Example 1 Kit for inducing human pluripotent stem cells to differentiate into motor nerve precursor cells

[0358] This embodiment prepares a kit for inducing stem cells to differentiate into motor nerve precursor cells, comprising: a first culture medium, a second culture medium, a third culture medium and a fourth culture medium;

[0359] The first culture medium is a neurobasal medium containing 2 μM SB431542 (purchased from selleck, catalog number S106707), 2 μM DMH-1 (purchased from selleck, catalog number S714602) and 3 μM CHIR99021 (purchased from Tocris, catalog number 442310);

[0360] The second culture medium was neurobasal medium containing 2 μM SB431542, 2 μM DMH-1, 1 μM CHIR99021, 0.1 μM retinoic acid (purchased from selleck, catalog number S106707), 10 μM Y-27632, and 0.5 μM SAG (purchased from Tocris, catalog number 4366);

[0361] The third medium was neurobasal medium containing 2 μM SB431542, 2 μM DMH-1, 3 μM CHIR99021, 0.1 μM retinoic acid, 10 μM Y-27632, and 0.5 μM SAG;

[0362] The fourth culture medium is a neurobasal medium containing 2 μM SB431542, 2 μM DMH-1, 3 μM CHIR99021, 0.1 μM retinoic acid, 0.5 μM SAG and 0.1 μM compound E (purchased from Tocris, catalog number 6476);

[0363] The neurobasal medium is DMEM / F12 (Gibco, catalog number 10565042) containing 0.5% (v / v) N2 supplement (N2 supplement, Gibco), 1% (v / v) non-essential amino acids (Non-essential amino acid, Gibco) and 1% (v / v) B-27.

[0364] Example 2 Kit for inducing human pluripotent stem cells to differentiate into motor neurons

[0365] A kit for inducing human pluripotent stem cells to differentiate into motor neurons, comprising: the kit for inducing human pluripotent stem cells to differentiate into motor nerve precursor cells of Example 1, a fifth culture medium and a sixth culture medium;

[0366] Among them, the fifth culture medium is a neural basal medium containing 0.1 μM compound E, 0.2 μM SAG, 0.5 μM retinoic acid, and 10 μM Y-27632; the neural basal medium is DMEM / F12 (Gibco, catalog number 10565042) containing 0.5% (v / v) N2 supplement (N2 supplement, Gibco), 1% (v / v) non-essential amino acids (Non-essential amino acid, Gibco) and 1% (v / v) B-27.

[0367] The sixth culture medium is a neural differentiation basal medium containing 10 ng / mL BDNF (purchased from peprotech, catalog number 450-02-1MG), 10 ng / mL GDNF (purchased from peprotech, catalog number 450-10-1MG), 0.2 μM SAG, 0.5 μM retinoic acid and 0.2 mM AA (L-Ascorbic acid, purchased from Sigma).

[0368] The neural differentiation basal medium is Neurobasal (Gibco) containing 1% (v / v) N2 supplement (N2 supplement, Gibco) and 2% (v / v) B-27 (purchased from thermo, product number A3653401).

[0369] Example 3 Method for inducing human pluripotent stem cells to differentiate into motor nerve precursor cells and motor neurons

[0370] The method for inducing human pluripotent stem cells to differentiate into motor nerve precursor cells and motor neurons comprises the steps of using the kit of Example 2, specifically as follows:

[0371] 1. Inducing human pluripotent stem cells to differentiate into motor nerve precursor cells:

[0372] (1) Culture of human pluripotent stem cells:

[0373] When iPSC cells were cultured to 70-80% polymerization degree using StemFit medium, TrypLE digestion solution was added and incubated in a 37°C incubator for 15 minutes (12-16 minutes is acceptable) to digest into complete single cells, resuspended in StemFit medium with 10 μM Y27632 (purchased from Sigma), counted and added with appropriate amount of medium (StemFit medium), and inoculated in a T75 cell culture flask pre-coated with Laminin at an inoculation density of 5000 / cm 2 Place the plate at 37°C, 5% CO 2 Culture in an incubator for 24 h.

[0374] (2) Differentiation of motor nerve precursor cells:

[0375] 1) Differentiation of pluripotent stem cells into neuroepithelial cells (D0-D6)

[0376] After the cells adhered to the wall for 24 h, the culture medium was replaced with the first culture medium in the kit of Example 1 (as day 0 of differentiation, D0) and incubated at 37°C and 5% CO 2 Culture in a carbon dioxide cell culture incubator for 6 days (change medium every two days);

[0377] 2) Differentiation of neuroepithelial cells into motor neuron progenitor cells (D6-D12)

[0378] On the 6th day of differentiation, the supernatant was discarded from the cell culture flask, washed once with 1×DPBS buffer, and then TrypLE digestion solution was added. The flask was placed in a 37°C incubator and incubated for 15 minutes (12-16 minutes is acceptable). The cells were gently shaken to completely detach from the bottom of the culture dish, and the neural basal medium was added for neutralization and digestion to obtain a cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, the supernatant was discarded, and the second culture medium in the kit of Example 1 was added. The cells were gently blown 1-2 times to disperse them into single cells as much as possible, and the concentration was 50000 / cm 2 The cells were seeded into Laminin-coated T75 cell culture flasks at a density of 1.54 μg / mL and incubated at 37°C with 5% CO. 2 Culture in an incubator, change the medium every 2 days, and continue culturing for 6 days to obtain motor neuron progenitor cells;

[0379] 3) Differentiation of motor progenitor cells into motor precursor cells (D12-D16)

[0380] On the 12th day of differentiation, the supernatant was discarded from the cell culture flask containing the motor neuron progenitor cells, washed once with 1×DPBS buffer, and then added with TrypLE digestion solution, and incubated in a 37°C incubator for 18 minutes (14-20 minutes are acceptable), and gently shaken to completely detach the cells from the bottom of the culture dish, and then added with neural basal medium for neutralization and digestion to obtain a cell suspension, which was transferred to a 50 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant was discarded, and the third culture medium in the kit of Example 1 was added, and the cells were cultured at 100,000 / cm 2 The cells were seeded into Laminin-coated T75 cell culture flasks at a seeding density of 1.54 μg / mL and incubated at 37°C with 5% CO. 2 The cells were cultured in an incubator with the medium changed every 2 days for 4 days.

[0381] 4) Differentiation of motor progenitor cells into motor precursor cells (D16-D18)

[0382] On the 16th day of differentiation, the culture medium was replaced with the fourth culture medium in the kit of Example 1 and the cells were incubated at 37°C with 5% CO 2 The motor nerve precursor cells can be obtained by culturing in an incubator for 2 days.

[0383] 2. Induce motor nerve precursor cells to differentiate into motor neurons (D18-D27):

[0384] (1) On the 18th day of differentiation, the motor progenitor cells obtained in step 1 were aspirated and the culture medium was discarded. The cells were washed once with 1×DPBS, added with 0.75×TrypLE digestion solution, and incubated in a 37°C incubator for 8 minutes (6-10 minutes). The cells were gently shaken to completely detach from the bottom of the culture dish, and the neural basal culture medium was added for neutralization and digestion. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 minutes, the supernatant was aspirated, and the fifth culture medium in the kit of Example 2 was added. The cells were counted and re-seeded into a 24-well cell culture plate coated with Matrigel at a seeding density of 100,000 / cm 2 Place the cell culture plate at 37°C, 5% CO 2 Culture in an incubator for 3 days.

[0385] (2) On the 21st day of differentiation, the culture medium was replaced with the sixth culture medium in the kit of Example 2 and the cells were incubated at 37°C with 5% CO 2 The cells were cultured in an incubator with half of the medium changed every three days. Mature motor neurons were obtained after 6 days of differentiation culture.

[0386] Comparative Example 1 Kit for inducing human pluripotent stem cells to differentiate into motor precursor cells

[0387] The kit for inducing human pluripotent stem cells to differentiate into motor progenitor cells is different from that of Example 1 in that the fourth culture medium does not contain compound E.

[0388] Comparative Example 2 Kit for inducing human pluripotent stem cells to differentiate into motor neurons

[0389] The kit for inducing human pluripotent stem cells to differentiate into motor neurons is different from Example 2 in that the kit for inducing human pluripotent stem cells to differentiate into motor neuron precursor cells in Example 1 is replaced by the kit for inducing human pluripotent stem cells to differentiate into motor neuron precursor cells prepared in Comparative Example 1.

[0390] Comparative Example 3 Method for inducing human pluripotent stem cells to differentiate into motor nerve precursor cells and motor neurons

[0391] The method for inducing human pluripotent stem cells to differentiate into motor nerve precursor cells and motor neurons is the same as that in Example 3, except that the kit of Comparative Example 2 is used.

[0392] Test Case

[0393] 1. Flow cytometry detection of motor neuron progenitor cells

[0394] The motor progenitor cells prepared by differentiation culture for 12 days in Example 3 were transferred to a 1.5 mL centrifuge tube, centrifuged and the supernatant was discarded, resuspended with a staining solution (PBS buffer containing 0.5% BSA), and evenly distributed into 4 new centrifuge tubes. Isotype control (Isotype Contro1), OLIG2, OCT4, and HOXB4 antibodies were added to the 4 new centrifuge tubes, respectively, mixed and incubated for 25 minutes (20 to 30 minutes), washed once with staining solution, centrifuged and the supernatant was discarded, and detected by flow cytometry.

[0395] Test results such as Figure 1 As shown in the figure, it can be seen that among the motor progenitor cells prepared by differentiation culture for 12 days, more than 90% of the cells expressed motor progenitor cell-related proteins OLIG2 and HOXB4, and almost no omnipotent stem cell marker OCT4.

[0396] 2. Immunofluorescence staining

[0397] The MNP cells obtained after induction culture to the 18th day in Example 3 were used as the experimental group, and the MNP cells obtained after induction culture to the 18th day in Comparative Example 3 and the motor neuron progenitor cells obtained after induction culture to the 12th day in Example 3 were used as the control group.

[0398] Equal amounts of cells in the experimental group and the control group were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS, fixed with paraformaldehyde at room temperature for 15 minutes, washed three times with PBS, permeabilized with PBST (0.2% Triton + PBS) for 10 minutes, washed once with PBS, added with blocking solution (PBST + 3% donkey serum) at room temperature for 30 minutes, and then added with HB9 primary antibody (1:500) and OLIG2 primary antibody (1:500) at 4°C overnight, washed three times with PBS, added with secondary antibody (1:500; Jackson Lab), incubated at 37°C in the dark for 1 hour, washed three times with PBS, added with DAPI to stain the nucleus for 5 minutes, and washed three times with PBS for observation under a fluorescence microscope and photography in the dark.

[0399] Test results such as Figure 2 and 3 As shown in the figure, it can be seen that the motor progenitor cells obtained by induction culture in Example 3 to the 12th day are further differentiated for 6 days (adding the fourth culture medium in Example 1, i.e., the MNP cells obtained after induction culture in Example 3 to the 18th day), and a high-purity HB9+OLIG2- motor progenitor cell population can be obtained (about 84% of the cells express HB9, of which HB9+OLIG2- cells account for about 76%). While the HB9 marker of the control cells is lowly expressed, the OLIG2 marker is highly expressed.

[0400] 3. Neuron Detection

[0401] The MN neurons obtained after induction culture for 27 days in Example 3 were discarded, washed twice with PBS, fixed with paraformaldehyde at room temperature for 15 minutes, washed three times with PBS, permeabilized with PBST (0.2% Triton + PBS) for 10 minutes, washed once with PBS, added with blocking solution (PBST + 3% donkey serum) at room temperature for 30 minutes, and then added with HB9 primary antibody (1: 500), CHAT primary antibody (1: 500) at 4°C overnight, washed three times with PBS, added with secondary antibody (1: 500; Jackson Lab), incubated at 37°C in the dark for 1 hour, washed three times with PBS, added with DAPI to stain the nucleus for 5 minutes, washed three times with PBS, observed under a fluorescence microscope and photographed in the dark.

[0402] Test results such as Figure 4 As shown in the figure, it can be seen that the cells were identified by HB9 and CHAT immunofluorescence staining on the 27th day of culture. The D18 motor progenitor cells were further differentiated for 9 days to obtain high-purity HB9+CHAT+ motor neurons. Among them, HB9 and CHAT are specific markers of motor neurons.

[0403] Example 4 Kit for inducing human pluripotent stem cells to differentiate into motor neuron progenitor cells

[0404] This embodiment prepares a kit for inducing stem cells to differentiate into motor neuron progenitor cells, comprising: a first culture medium and a second culture medium;

[0405] The first culture medium is a neurobasal medium containing 2 μM SB431542 (purchased from selleck, catalog number S106707), 2 μM DMH-1 (purchased from selleck, catalog number S714602) and 3 μM CHIR99021 (purchased from Tocris, catalog number 442310);

[0406] The second culture medium was a neurobasal medium containing 2 μM SB431542, 2 μM DMH-1, 1 μM CHIR99021, 0.1 μM retinoic acid (purchased from selleck, catalog number S106707), 10 μM Y-27632, and 0.5 μM SAG (purchased from Tocris, catalog number 4366);

[0407] The neurobasal medium is DMEM / F12 (Gibco, catalog number 10565042) containing 0.5% (v / v) N2 supplement (N2 supplement, Gibco), 1% (v / v) non-essential amino acids (Non-essential amino acid, Gibco) and 1% (v / v) B-27.

[0408] Example 5 Method for inducing human pluripotent stem cells to differentiate into motor neuron progenitor cells

[0409] The method for inducing human pluripotent stem cells to differentiate into motor neuron progenitor cells comprises the steps of using the kit of Example 4, specifically as follows:

[0410] (1) Culture of human pluripotent stem cells:

[0411] When iPSC cells were cultured to 70-80% polymerization degree using StemFit medium, TrypLE digestion solution was added and incubated in a 37°C incubator for 15 minutes (12-16 minutes is acceptable) to digest into complete single cells, resuspended in StemFit medium with 10 μM Y27632 (purchased from Sigma), counted and added with appropriate amount of medium (StemFit medium), and inoculated in a T75 cell culture flask pre-coated with Laminin at an inoculation density of 5000 / cm 2 Place the plate at 37°C, 5% CO 2 Culture in an incubator for 24 h.

[0412] (2) Differentiation of motor neuron progenitor cells:

[0413] 1) Differentiation of pluripotent stem cells into neuroepithelial cells (D0-D6)

[0414] After the cells adhered to the wall for 24 h, the culture medium was replaced with the first culture medium in the kit of Example 4 (as day 0 of differentiation, D0) and incubated at 37°C and 5% CO 2 The cells were cultured in a carbon dioxide cell culture incubator for 6 days (the medium was changed every two days).

[0415] 2) Differentiation of neuroepithelial cells into motor neuron progenitor cells (D6-D12)

[0416] On the 6th day of differentiation, the supernatant was discarded from the cell culture flask, washed once with 1×DPBS buffer, and then TrypLE digestion solution was added. The flask was placed in a 37°C incubator and incubated for 15 minutes (12-16 minutes is acceptable). The cells were gently shaken to completely detach from the bottom of the culture dish, and the neural basal medium was added for neutralization and digestion to obtain a cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, the supernatant was discarded, and the second culture medium in the kit of Example 4 was added. The cells were gently blown 1-2 times to disperse them into single cells as much as possible, and the concentration was 50000 / cm 2 The cells were seeded into Laminin-coated T75 cell culture flasks at a density of 1.54 μg / mL and incubated at 37°C with 5% CO. 2 The cells were cultured in an incubator with the medium changed every 2 days. Motor neuron progenitor cells were obtained after 6 days of continuous culture.

[0417] Test Case

[0418] 4. Evaluation of drug efficacy in ALS animal models

[0419] ALS-SOD1 gene mutation mice (from Jackson Laboratory, USA (B6SJL-Tg(SOD1*G93A)1Gur / J, #002726)) were used for pharmacodynamic evaluation. Specifically, SOD1 G93A gene positive mice (ALS model group) were selected for behavioral tests and survival statistics. Behavioral tests include grip strength test, rotarod test, etc.

[0420] The SOD1 G93A gene-positive mice were randomly and evenly divided into 2 groups (wherein the vehicle group had 8 male mice and 9 female mice, and the cell therapy group had 9 male mice and 9 female mice), and the motor progenitor cells (hereinafter referred to as iPS-MNP cells) prepared in Example 5 or the vehicle (artificial cerebrospinal fluid) were administered to each group of mice on the 40th, 60th, 80th, and 100th days after birth. Specifically, the mice were anesthetized and disinfected, and a 10 μL injection needle was inserted vertically to the dorsal tissue spinous process gap (i.e., L5 and L6) above the spine, and the needle tip entered the subarachnoid space, and a sample or vehicle with a volume of 10 μL was injected into the subarachnoid space. In addition, from three days before cell therapy (i.e., the 37th day), each mouse was intraperitoneally injected with an immunosuppressant 5 mg / kg rapamycin (selleckchem, S1039) + FK506 (selleckchem, S5003) once a day until the end of the experiment.

[0421] 4.1 Grip test

[0422] The gripping strength experiment was conducted on the 73rd day after the mice were born. The specific experimental method is as follows: Place the mouse's limbs on the grip strength meter (Jiangsu Saiangsi Biotechnology Co., Ltd., SA417) accessories, apply force to the mouse's tail, and drag it backwards. The mouse instinctively grasps the accessories with force, and the maximum grip strength value will be displayed on the grip strength meter display. Two researchers each performed the above operations and recorded the maximum grip strength value. The average of the two test data was counted by Graphpad, and the trend of the decrease in grip strength value (relative to the grip strength value 105 days ago) and the time progression of the grip strength value decreasing by 40% were counted.

[0423] According to the experimental results, the gripping strength of mice at a certain point in time or during a certain period of time during the onset of the disease is compared. If the average gripping strength value of mice in the cell therapy group is significantly higher than that of mice in the negative control group (solvent group), or the downward trend of the gripping strength value is slowed down or the time for the gripping strength value to decrease by 40% is prolonged, it means that cell therapy can effectively improve the gripping strength of mice.

[0424] The results of the gripping strength test showed that around 100-110 days after birth, the gripping strength of the limbs of female and male SOD1 G93A mice gradually decreased, and in the late stage (120-153 days), the hind limbs became stiff until the gripping strength test could no longer be performed. The researchers compared the gripping strength values ​​of mice after 105 days after birth with those of mice before 105 days to obtain the decrease in the gripping strength values ​​of mice at different time points. The results showed that compared with the vehicle group, the gripping strength values ​​of mice in the iPS-MNP cell treatment group decreased more slowly from 132 days to 139 days after birth, and there was a significant improvement (such as Figure 5A As shown, P < 0.05); and Figure 5BAs shown, the time progression data from a 40% reduction in grip strength values ​​(i.e., at different time points, the proportion of mice with a 40% reduction in grip strength values ​​compared to the grip strength values ​​of mice before day 105; 100% - Figure 5B The results of the ordinate value (%) are the proportion of mice whose gripping strength value has decreased by 40%). The disease progression was analyzed and it was found that iPS-MNP cell treatment can significantly delay the process of gripping strength decrease (there is a significant difference in the progress curve, P<0.05, log rank test). Compared with the median progression period of mice in the vehicle group (the age when the gripping strength value of 50% of mice has not decreased by 40%, that is, the age when the gripping strength value of 50% of mice has decreased by 40%) of 125 days, the median progression period of mice in the iPS-MNP cell treatment group was 133.5 days, which was delayed by 8.5 days and was significant (P<0.05). The above results show that transplantation of iPS-MNP cells can improve the limb muscle strength of SOD1 G93A mice (ALS model mice).

[0425] 4.2 Rotarod test

[0426] Therefore, this embodiment uses an accelerated rotarod instrument (Jiangsu Saiangsi Biotechnology Co., Ltd., SA102) to perform a rotarod experiment to test motor coordination. The specific experimental method is as follows: all mice are pre-trained for 3 days before the test, the rotarod speed is 15rpm, and the training is 300s. After 3 days of training, a formal test is carried out: the rotarod speed is 15rpm, and the test is 180s. If the time the mouse stays on the rotarod (hereinafter referred to as the time on the rod) reaches 180s, the time on the rod is recorded as 180s. If it does not reach 180s, the average value of the time on the rod of three tests is taken as the test result, and the above data is counted by Graphpad.

[0427] According to the experimental results, the time that mice spend on the rod at a certain time point or during a certain period of time during the onset of the disease is compared. If the time that mice in the cell therapy group spend on the rod is significantly longer than that of mice in the negative control group (solvent group), it means that cell therapy can effectively improve the movement and coordination ability of mice. Generally, the movement and coordination ability of ALS model mice decreases significantly around 110 days, and male mice generally decline earlier than female mice.

[0428] The results are as follows Figure 6As shown, the rod time of mice in the vehicle group began to decrease significantly on the 119th day after birth, while the rod time of mice in the iPS-MNP cell treatment group began to decrease at a later time point, on the 129th day after birth, and the rod time of mice in the iPS-MNP cell treatment group was significantly longer than that in the vehicle group from the 119th day after birth, especially on the 122nd day, the rod time of mice in the iPS-MNP cell treatment group was significantly prolonged (P < 0.05). The above results show that transplantation of iPS-MNP cells can improve the motor coordination ability of SOD1 G93A mice (ALS model mice).

[0429] 4.3 Survival Statistics

[0430] The mice were observed every day, and mice that died suddenly in the late stage of the disease or could not turn over within 30 seconds (i.e., the mice were turned over and could not return to their normal position within 30 seconds) were euthanized. The time of death was recorded and used as the experimental endpoint. The survival period of the mice at the experimental endpoint was counted. If the survival period of the mice in the cell therapy group was significantly longer than that of the mice in the negative control group (solvent group), it means that cell therapy can effectively prolong the survival period of mice and improve the survival rate.

[0431] During this process, it was found that as the disease progressed, SOD1 G93A mice gradually developed flexion of both lower limbs, weakness, muscle atrophy and paralysis, with a survival period of 120 to 150 days; among them, the median survival period of male ALS model mice was 127 days, and the median survival period of female SOD1G93A mice was 140 days.

[0432] The results are as follows Figure 7 As shown, compared with the median survival of 133.5 days in the vehicle group, the median survival of the mice in the iPS-MNP cell treatment group was 145 days, which was 11.5 days longer, and it was significant (there was a significant difference in the survival curve, P < 0.05). This result shows that iPS-MNP cells have the effect of improving the survival rate of SOD1 G93A mice (ALS model mice) and prolonging their survival.

[0433] 4.4 Tissue staining to study the in vivo distribution, proliferation and differentiation of iPS-MNP cells

[0434] The iPS-MNPs derived from iPS cells were transfected with a virus (Gene Gene, LVCON156) to make them luciferase-labeled. The iPS-MNPs labeled with luciferase were intrathecally transplanted (injected at L5 and L6) into 6-8 week old female NOG mice (Viton Lihua, Cat. No. 408), and then the survival of the transplanted cells in NOG mice was monitored in real time once a week using a small animal in vivo imager (Guangzhou Bolu Teng Instrument Co., Ltd., AniView100).

[0435] The researchers found that the fluorescence signal of iPS-MNP cells reached a stable state 3 weeks after transplantation. After continuous observation, it was found that iPS-MNP cells still survived stably in the body 40 days after transplantation, and were mainly distributed in the injection site and target sites (including the thoracic spinal cord and brain) (such as Figure 8 The researchers further examined the survival, proliferation and differentiation of transplanted cells by histology.

[0436] Two months after iPSC-derived motor neuron progenitor cells (iPS-MNPs) were intrathecally transplanted into 6-8 week old female NOG mice (Vitamin Liva, Cat. No. 408), the NOG mice were fixed by cardiac perfusion, and the whole spinal cord tissue of the mice was removed and collected, fixed in 4% paraformaldehyde, embedded with embedding agent, and frozen. The slice thickness was 20 mm, and 5 slices were collected every 100 mm. Tissue sections were analyzed by immunofluorescence staining. Specifically, spinal cord tissue sections of cell-transplanted mice were subjected to immunofluorescence staining with human nuclear antibody stem101 (Takara Bio Inc, Catalog No.: Y40400), human nuclear antibody HNA (Abcam, Catalog No.: ab191181), motor neuron marker HB9 (Abcam, Catalog No.: ab221884), astrocyte marker GFAP (Abcam, Catalog No.: ab68428) / Stem123 (Takara Bio Inc, Y40420) and cell proliferation marker Ki67 (Abcam, Catalog No.: ab15580).

[0437] The staining results showed that the transplanted cells were located in the subarachnoid space, attached to the dura mater and colonized in the spinal cord. Further observation of the motor neurons and human astrocytes in the mice revealed that some of the transplanted cells differentiated into motor neurons (expressed as HNA+HB9+ co-positive cells, results not shown) and astrocytes (expressed as Stem123+GFAP+ co-positive cells, results not shown) in vivo. Fig. 9A As shown in Figure 2, astrocytes can assist motor neurons in their function. The above results indicate that iPSC-derived motor neuron progenitor cells (iPS-MNPs) can survive after in vivo transplantation, colonize in the spinal cord, and differentiate into motor neurons and astrocytes. At the same time, the results of Ki67+ staining showed that Ki67+ was very rare in iPS-MNP cells (e.g. Fig. 9BAs shown, it is expressed as Stem101+Ki67+ co-positive cells, white arrows), which indicates that the cells have almost no proliferation in vivo. In addition, in the above-mentioned SOD1 G93A mouse efficacy evaluation experiment, when the mice with intrathecal iPS-MNP cells reached the end of life, no tumor formation was observed in the cell transplantation injection site, spinal cord and various organs, which further indicates that the cells have almost no proliferation in vivo.

[0438] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A kit comprising a first culture medium and a second culture medium; The first culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor and a GSK-3 inhibitor; The second culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator, a SHH signaling pathway activator and a ROCK inhibitor.

2. The kit according to claim 1, characterized in that: The BMP inhibitor is independently selected from at least one of DMH1, LDN-212854, doxorubicin, Nogin, LDN-193189, activin binding protein, tenascin, and gremlin; and / or the Nodal inhibitor is independently selected from SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, and SX-00 7. Antp-Sm2A, LY2109761; and / or the GSK-3 inhibitor is at least one of GSK3β inhibitor IX, SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparolone, indirubin, CHIR99021; and / or the RAR nuclear receptor activator is independently selected from: at least one of retinoic acid and tazarotene; and / or the SHH signaling pathway activator is independently selected from: SHH, SHH At least one of C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine; and / or the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632, and SNJ-1656.

3. A kit comprising a first culture medium, a second culture medium, a third culture medium and a fourth culture medium; The first culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor and a GSK-3 inhibitor; The second culture medium is a basal culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, an RAR nuclear receptor activator, an SHH signaling pathway activator, and a ROCK inhibitor; The third culture medium is a basal culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, an RAR nuclear receptor activator, an SHH signaling pathway activator and a ROCK inhibitor; The fourth culture medium is a basic culture medium containing a BMP inhibitor, a Nodal inhibitor, a GSK-3 inhibitor, a RAR nuclear receptor activator, an SHH signaling pathway activator and a gamma secretase inhibitor.

4. The kit according to claim 3, characterized in that: The BMP inhibitor is independently selected from at least one of DMH1, LDN-212854, doxorubicin, Nogin, LDN-193189, activin binding protein, tenascin, and gremlin; and / or the Nodal inhibitor is independently selected from SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, and SX-00 7. Antp-Sm2A, LY2109761; and / or the GSK-3 inhibitor is at least one of GSK3β inhibitor IX, SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparolone, indirubin, CHIR99021; and / or the RAR nuclear receptor activator is independently selected from: at least one of retinoic acid and tazarotene; and / or the SHH signaling pathway activator is independently selected from: SHH, SHH At least one of C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine; and / or the ROCK inhibitor is independently selected from at least one of AR-12286, Y-27632, and SNJ-1656; and / or the gamma-secretase inhibitor is independently selected from at least one of DAPT, YO-01027, and compound E.

5. The kit according to claim 3, characterized in that: The kit also contains: a fifth culture medium and a sixth culture medium; the fifth culture medium is a basic culture medium containing a RAR nuclear receptor activator, an SHH signaling pathway activator, a gamma secretase inhibitor and a ROCK inhibitor; the sixth culture medium is a basic culture medium containing brain-derived growth factor, glial cell-derived neurotrophic factor, an SHH signaling pathway activator, a RAR nuclear receptor activator and ascorbic acid.

6. Use of the kit according to any one of claims 1 to 5 in any one of (1) to (6): (1) preparing motor neuron progenitor cells; (2) preparing motor nerve precursor cells; (3) Preparation of motor neurons; (4) preparing products for inducing stem cells to differentiate into motor neuron progenitor cells; (5) preparing products for inducing stem cells to differentiate into motor nerve precursor cells; (6) Prepare products for inducing stem cells to differentiate into motor neurons.

7. Any of the following methods a1) to a3): a1) A method for inducing stem cells to differentiate into motor neuron progenitor cells, comprising the steps of using the kit according to any one of claims 1 to 5; a2) A method for inducing stem cells to differentiate into motor nerve precursor cells, comprising the steps of using the kit according to any one of claims 3 to 5; a3) A method for inducing stem cells to differentiate into motor neurons, comprising the step of using the kit according to claim 5.

8. The method according to claim 7, characterized in that: The method for inducing stem cells to differentiate into motor neuron progenitor cells in a1) comprises the following steps: (1) culturing stem cells for the first time using the first culture medium in the kit according to any one of claims 1 to 5 to obtain neuroepithelial cells; (2) resuspending the neuroepithelial cells in the second culture medium according to any one of claims 1 to 5, and performing a second culture to obtain motor neuron progenitor cells; and / or The method of inducing stem cells to differentiate into motor nerve precursor cells in a2) comprises the following steps: (1) culturing stem cells for the first time using the first culture medium in the kit according to any one of claims 3 to 5 to obtain neuroepithelial cells; (2) resuspending the neuroepithelial cells in the second culture medium according to any one of claims 3 to 5, and performing a second culture to obtain motor neuron progenitor cells; (3) culturing the motor neuron progenitor cells for a third time with the third culture medium in the kit according to any one of claims 3 to 5, and then culturing them for a fourth time with the fourth culture medium according to any one of claims 3 to 5, to obtain motor neuron progenitor cells; and / or The method for inducing stem cells to differentiate into motor neurons described in a3) comprises the following steps: (1) culturing stem cells for the first time using the first culture medium in the kit according to any one of claims 3 to 5 to obtain neuroepithelial cells; (2) resuspending the neuroepithelial cells in the second culture medium of the kit according to any one of claims 3 to 5, and performing a second culture to obtain motor neuron progenitor cells; (3) culturing the motor neuron progenitor cells for a third time with the third culture medium in the kit according to any one of claims 3 to 5, and then culturing them for a fourth time with the fourth culture medium according to any one of claims 3 to 5, to obtain motor neuron progenitor cells; (4) culturing the motor neuron precursor cells for a fifth time using the fifth culture medium in the kit according to claim 5, and then culturing them for a sixth time using the sixth culture medium in the kit according to claim 5 to obtain motor neurons; Preferably, the first culture time in step (1) of a1), a2) or a3) is 4 to 10 days; Preferably, the second culture time in step (2) of a1), a2) or a3) is 4 to 10 days; Preferably, the duration of the third culture in step (3) of a2) or a3) is 2 to 7 days; Preferably, the duration of the fourth culture in step (3) of a2) or a3) is 1 to 4 days; Preferably, the fifth culture in step (4) of a3) lasts for 1 to 5 days; Preferably, the sixth culture in step (4) of a3) lasts for 6 to 12 days or 7 to 12 days.

9. A motor neuron progenitor cell, prepared by the kit according to any one of claims 1 to 5 and / or the method for inducing stem cells to differentiate into motor neuron progenitor cells according to any one of claims 7 to 8; Preferably, the content of OLIG2+HOXB4+OCT4- cells in the motor neuron progenitor cells is greater than 70%.

10. A motor nerve precursor cell, prepared by the kit according to any one of claims 3 to 5 and / or the method for inducing stem cells to differentiate into motor nerve precursor cells according to any one of claims 7 to 8; Preferably, the content of HB9+OLIG2- cells in the motor nerve precursor cells is greater than 70%; Preferably, the content of cells expressing HB9 in the motor nerve precursor cells is greater than 80%.

11. A motor neuron prepared by the kit according to any one of claims 3 to 5 and / or the method for inducing stem cells to differentiate into motor neurons according to any one of claims 7 to 8.

12. A pharmaceutical composition comprising: the motor neuron progenitor cell according to claim 9, the motor neuron precursor cell according to claim 10 and / or the motor neuron according to claim 11; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; Preferably, the pharmaceutical composition further comprises other drugs for preventing and / or treating nervous system diseases.

13. Any application of b1) to b2) below: b1) Use of the motor neuron progenitor cell according to claim 9, the motor neuron precursor cell according to claim 10 and / or the motor neuron according to claim 11 in constructing a cell bank; b2) Use of the motor neuron progenitor cells according to claim 9, the motor neuron precursor cells according to claim 10, the motor neurons according to claim 11 and / or the pharmaceutical composition according to claim 12 in the preparation of drugs for preventing and / or treating nervous system diseases.

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