In vitro differentiation method of stem cells

Abstract

Provided are methods for in vitro induced differentiation of embryonic stem cells or pluripotent stem cells into dopaminergic neural progenitor cells, dopaminergic neural precursor cells and / or dopaminergic neurons. Also provided are media for use in the method, cell populations obtained using the method, and the use of the obtained cell populations in the prevention and / or treatment of diseases and / or dysfunctions associated with dopaminergic neuron degenerative disorders.

Description

Technical Field

[0001] This invention relates to the in vitro differentiation and culture of stem cells, and more particularly to a method for inducing embryonic stem cells or pluripotent stem cells to differentiate in vitro into dopaminergic neural progenitor cells, dopaminergic neural precursor cells, and / or dopaminergic neurons. It also relates to a culture medium for said method, a cell population obtained using said method, and the use of the obtained cell population in the prevention and / or treatment of diseases and / or functional impairments (e.g., Parkinson's disease) associated with dopaminergic neurodegenerative disorders. Background Technology

[0002] Parkinson's disease (PD) is a neurodegenerative disease primarily caused by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain and insufficient dopamine secretion from the striatum, widely affecting middle-aged and elderly people. Clinical symptoms of Parkinson's disease include typical motor symptoms such as resting tremor, rigidity, bradykinesia, and postural instability, as well as non-motor symptoms such as constipation, olfactory dysfunction, sleep disturbances, autonomic dysfunction, and mental and cognitive impairment. Traditional drug treatments not only fail to cure the disease but have also been observed to cause various serious side effects, including involuntary movements.

[0003] Based on the results of numerous global clinical trials, cell therapy holds promise as the most effective treatment for these neurodegenerative diseases. In vitro induced differentiation technology allows pluripotent stem cells (PSCs) to differentiate in a controlled manner along the dopaminergic lineage, becoming dopaminergic precursor cells (DAPs), which are ideal cells for treating Parkinson's disease. After intracranial transplantation, DAPs can continue to differentiate into mature dopaminergic (DA) neurons and integrate with the recipient's nervous system, restoring normal dopamine secretion and other functions. Several methods for in vitro differentiation of human pluripotent stem cells into cell populations containing DAPs have been reported. For example, Chambers et al. were the first to construct a method for inducing a single type of neural lineage cell from hPSCs using dual SMAD inhibitors (Chambers et al., Nat Biotechnol 27, (2009)), but the protein-based inducing factors they used were expensive and unstable. Building upon this, PCT international application publication number WO2022221765A1 and Chinese patent number CN115354028B, among others, have reported a series of improved induction methods to enhance the induction efficiency of dopaminergic cells. However, the cell populations produced by these methods exhibit excessive heterogeneity, often requiring the use of markers to assist in sorting and increase the proportion of dopaminergic lineage cells, thus rendering these methods unsuitable for practical industrial production. Furthermore, the excessively long induction differentiation cycle and low yield are also major factors restricting the research and industrialization of such cell therapy products.

[0004] Therefore, there is a need to provide a method that can efficiently prepare cell populations that can be directly used for in vivo transplantation by increasing the proportion of target dopaminergic neural progenitor cells and / or neurons without additional differentiation or specific killing. Summary of the Invention

[0005] Through in-depth research, the inventors of this application discovered that in the early stages of in vitro differentiation of pluripotent stem cells into dopaminergic neural progenitor cells or neurons, higher concentrations of dual SMAD inhibitors (especially higher concentrations of TGFβ inhibitors) reduce cell survival rate and inhibit cell growth and proliferation. Therefore, by using low concentrations of dual SMAD signaling inhibitors (including low concentrations of TGFβ inhibitors and BMP4 inhibitors) in the initial stage of differentiation induction, cell doubling time can be effectively shortened, cell growth and proliferation rates can be promoted, and cell survival rate can be improved, ultimately resulting in a significantly increased cell yield per unit time. Furthermore, even in the later stages of differentiation induction after the withdrawal of dual SMAD inhibitors, a significant increase in cell yield can be continuously observed. In other words, by using low concentrations of dual SMAD inhibitors (especially low concentrations of TGFβ inhibitors) in the initial stage, not only can a larger population of midbrain basal plate cells be obtained at the end of the initial stage, but also a larger population of dopaminergic neural progenitor cells and dopaminergic neural progenitor cells can be obtained in the middle and later stages of differentiation induction, respectively.

[0006] The inventors of this application have also discovered that, in the initial stage of induced pluripotent stem cell differentiation, it is not necessary to perform complex regulation of Wnt signal intensity in a manner similar to that described in US20210123018A1. Instead, by adding a BMP4 inhibitor within a specific window period after the removal of the reagent combination for inducing midbrain lamina, it is possible to efficiently promote the further differentiation of midbrain lamina cells into dopaminergic neural progenitor cells and dopaminergic neural precursor cells.

[0007] Therefore, the first objective of this invention is to provide a method for differentiating stem cells in vitro.

[0008] A second objective of this invention is to provide a kit for differentiating stem cells into dopaminergic neural progenitor cells or dopaminergic neurons in vitro.

[0009] A third object of the present invention is to provide a cell population obtained from stem cells by means of the in vitro differentiation method disclosed herein or by using the kits disclosed herein.

[0010] A fourth object of the present invention is to provide compositions comprising the cell populations disclosed herein, optionally pharmaceutical compositions.

[0011] A fifth object of the present invention is to provide the use of the cell populations or compositions disclosed herein in the preparation of cell banks.

[0012] A sixth object of the present invention is to provide the use of the cell populations or compositions disclosed herein in the preparation of medicaments for the prevention and / or treatment of diseases and / or dysfunctions associated with midbrain dopaminergic neuronal degeneration.

[0013] A seventh object of the present invention is to provide a method for preventing and / or treating diseases and / or dysfunctions associated with midbrain dopaminergic neuronal degeneration using the cell populations or compositions disclosed herein.

[0014] An eighth object of the present invention is to provide the cell populations or compositions disclosed herein for the prevention and / or treatment of diseases and / or functional impairments associated with midbrain dopaminergic neuronal degeneration.

[0015] To achieve the above objectives, in some embodiments, this document provides a method for in vitro differentiation of stem cells, the method comprising step A) contacting an initial cell population containing a plurality of stem cells with at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor, and at least one SHH agonist to transform the initial cell population into a first differentiated cell population containing midbrain basal plate cells, and step B) contacting the first differentiated cell population with at least one BMP4 inhibitor, at least one SHH agonist, and at least one fibroblast growth factor to transform the first differentiated cell population into a second differentiated cell population containing dopaminergic neural progenitor cells; wherein, the total concentration of at least one TGFβ inhibitor in step A is greater than 1.0 μM and less than 5.0 μM, preferably 1.1–4.0 μM, more preferably 1.5–3.0 μM. In some embodiments, the total concentration of at least one TGFβ inhibitor in step A is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 μM. In a preferred embodiment, the total concentration of at least one TGFβ inhibitor in step A is 2.0 μM.

[0016] In some embodiments, the total concentration of at least one BMP4 inhibitor in step A is 0.2–3 μM, preferably 0.5–2 μM, and more preferably 0.5–1.5 μM. In some embodiments, the total concentration of at least one BMP4 inhibitor in step A is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM. In a preferred embodiment, the total concentration of at least one BMP4 inhibitor in step A is 1.0 μM.

[0017] In some embodiments, the method disclosed herein further includes step C) contacting the second differentiated cell population with at least one SHH agonist and at least one fibroblast growth factor, and then contacting it with at least one SHH agonist, at least one fibroblast growth factor and at least one ROCK inhibitor, so that the second differentiated cell population is transformed into a third differentiated cell population containing dopaminergic neural progenitor cells.

[0018] In some embodiments, the method disclosed herein further includes step D) contacting the third differentiated cell population with a neuronal differentiation culture medium to transform it into a terminally differentiated cell population containing dopaminergic neurons.

[0019] In some embodiments, step A of the method disclosed herein further includes: after maintaining contact between the initial cell population and at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor and at least one SHH agonist for a first time window, further contacting the cell population with at least one ROCK inhibitor and maintaining that contact until the end of step A.

[0020] In some embodiments, the total time for step A is 7 to 13 days, preferably 8.5 to 11.5 days. In some embodiments, the total time for step A is 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or 13 days. In a preferred embodiment, the total time for step A is 11 days.

[0021] In some embodiments, the first time window in step A is 6 to 10 days, preferably 7 to 9 days. In some embodiments, the first time window in step A is 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 days. In a preferred embodiment, the first time window in step A is 9 days.

[0022] In some embodiments, the further contact time with at least one ROCK inhibitor in step A is 1 to 3 days, preferably 1.5 to 2.5 days. In some embodiments, the further contact time with at least one ROCK inhibitor in step A is 1, 1.5, 2, 2.5, or 3 days. In a preferred embodiment, the further contact time with at least one ROCK inhibitor in step A is 2 days.

[0023] In some embodiments, the cells in the first time window of step A are in adherent culture. In a preferred embodiment, the cells in the first time window of step A are in adherent culture on a culture device coated with Vitronectin. In some embodiments, the cells in step A that are further contacted with at least one ROCK inhibitor are in suspension culture. In some embodiments, step A further includes cell digestion before suspension culture.

[0024] In some embodiments, the total time for step B is 3 to 7 days, preferably 4 to 6 days. In some embodiments, the total time for step B is 3, 4, 5, 6, or 7 days. In a preferred embodiment, the total time for step B is 5 days.

[0025] In some implementations, the cells in step B are in suspension culture.

[0026] In some embodiments, the total time for step C is 5.5 to 10.5 days, preferably 7 to 9 days. In some embodiments, the total time for step C is 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or 10.5 days. In a preferred embodiment, the total time for step C is 8 days.

[0027] In some embodiments, the initial contact time with at least one SHH agonist and at least one fibroblast growth factor in step C is 5 to 9 days, preferably 6 to 8 days. In some embodiments, the initial contact time with at least one SHH agonist and at least one fibroblast growth factor in step C is 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9 days. In a preferred embodiment, the initial contact time with at least one SHH agonist and at least one fibroblast growth factor in step C is 7 days.

[0028] In some embodiments, the cells in step C that are first contacted with at least one SHH agonist and at least one fibroblast growth factor are cultured in an adherent culture. In a preferred embodiment, the cells are cultured in an adherent culture device coated with Vitronectin.

[0029] In some embodiments, the re-contact time with at least one SHH agonist, at least one fibroblast growth factor, and at least one ROCK inhibitor in step C is 0.5 to 1.5 days. In a preferred embodiment, the re-contact time with at least one SHH agonist, at least one fibroblast growth factor, and at least one ROCK inhibitor in step C is 1 day.

[0030] In some embodiments, the cells in step C that are further contacted with at least one SHH agonist, at least one fibroblast growth factor, and at least one ROCK inhibitor are in suspension culture.

[0031] In some embodiments, the TGFβ inhibitors in steps A through C may be independently selected from any 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, LY2109761, or any combination thereof. In a preferred embodiment, the TGFβ inhibitor in steps A through C is SB431542.

[0032] In some embodiments, the BMP4 inhibitors in steps A through C may be independently selected from any one of dorsomorphin, noggin, LDN-193189, follistatin, chordin, gremlin, DMH-1, or any combination thereof. In a preferred embodiment, the BMP4 inhibitor in steps A through C is DMH-1.

[0033] In some embodiments, the GSK3β inhibitors in steps A through C may be independently selected from GSK3β inhibitor IX (6-bromoindirubin 3'-oxime), SB216763, GSK3β inhibitor VII (4-dibromoacetophenone), L803-mts, 6-bromo-indirubin- The GSK3β inhibitor may be any one of the following: -oxime (BIO), TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, CHIR99021, or any combination thereof. In a preferred embodiment, the GSK3β inhibitor in steps A through C is CHIR99021.

[0034] In some embodiments, the SHH agonist in steps A to C may be independently selected from any one of SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20α-hydroxycholesterol, puromorphamine, or any combination thereof. In a preferred embodiment, the SHH agonist in steps A to C is SAG.

[0035] In some embodiments, the ROCK inhibitor in steps A and C may be independently selected from any one of Y-27632, HA100, HA1152, Blebbistatin, HA-1077, KD-025, Y-33075, Narciclasine, or any combination thereof. In a preferred embodiment, the ROCK inhibitor in steps A and C is Y-27632.

[0036] In some embodiments, the fibroblast growth factor in steps B and C may be independently selected from any one of FGF8b, FGF18, FGF8, FGF8a, FGF17, FGF2, and FGF4, or any combination thereof. In a preferred embodiment, the fibroblast growth factor in steps B and C is FGF8b.

[0037] In some embodiments, the total concentration of at least one GSK3β inhibitor in step A is 0.2–3.0 μM, preferably 0.2–1 μM, more preferably 0.4–0.6 μM. In some embodiments, the total concentration of at least one GSK3β inhibitor in step A is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM. In a preferred embodiment, the total concentration of at least one GSK3β inhibitor in step A is 0.4 μM.

[0038] In some embodiments, the total concentration of at least one SHH agonist in step A is 0.5–2.5 μM, preferably 1–1.75 μM, and more preferably 1 μM. In some embodiments, the total concentration of at least one SHH agonist in step A is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 μM. In a preferred embodiment, the total concentration of at least one SHH agonist in step A is 1.0 μM.

[0039] In some embodiments, the total concentration of at least one ROCK inhibitor in step A is 5–15 μM. In some embodiments, the total concentration of at least one ROCK inhibitor in step A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μM. In a preferred embodiment, the total concentration of at least one ROCK inhibitor in step A is 10 μM.

[0040] In some embodiments, the total concentration of at least one BMP4 inhibitor in step B is lower than the total concentration of at least one BMP4 inhibitor in step A. In a preferred embodiment, the total concentration of at least one BMP4 inhibitor in step B is 0.2–10 μM, preferably 0.8–1.2 μM. In some embodiments, the total concentration of at least one BMP4 inhibitor in step B is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In a more preferred embodiment, the total concentration of at least one BMP4 inhibitor in step B is 1.0 μM.

[0041] In some embodiments, the total concentration of at least one SHH agonist in step B is lower than the total concentration of at least one SHH agonist in step A. In a preferred embodiment, the total concentration of at least one SHH agonist in step B is 0.1–1.0 μM, preferably 0.1–0.5 μM. In some embodiments, the total concentration of at least one SHH agonist in step B is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μM. In a preferred embodiment, the total concentration of at least one SHH agonist in step B is 0.2 μM.

[0042] In some embodiments, the total concentration of at least one fibroblast growth factor in step B is 10–1000 ng / mL, preferably 80–120 ng / mL. In some embodiments, the total concentration of at least one fibroblast growth factor in step B is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / mL. In a preferred embodiment, the total concentration of at least one fibroblast growth factor in step B is 100 ng / mL.

[0043] In some embodiments, the total concentration of at least one SHH agonist and the total concentration of at least one fibroblast growth factor in step C are substantially the same as the corresponding concentrations in step B.

[0044] In some embodiments, the total concentration of at least one SHH agonist in step C is 0.1–1.0 μM, preferably 0.1–0.5 μM. In some embodiments, the total concentration of at least one SHH agonist in step C is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μM. In a preferred embodiment, the total concentration of at least one SHH agonist in step C is 0.2 μM.

[0045] In some embodiments, the total concentration of at least one fibroblast growth factor in step C is 10–1000 ng / mL, preferably 80–120 ng / mL. In some embodiments, the total concentration of at least one fibroblast growth factor in step C is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / mL. In a preferred embodiment, the total concentration of at least one fibroblast growth factor in step C is 100 ng / mL.

[0046] In some embodiments, the total concentration of at least one ROCK inhibitor in step C is 5–15 μM. In some embodiments, the total concentration of at least one ROCK inhibitor in step C is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μM. In a preferred embodiment, the total concentration of at least one ROCK inhibitor in step C is 10 μM.

[0047] In some implementations, the stem cells are pluripotent stem cells.

[0048] In some embodiments, the stem cells are selected from any one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells. In a preferred embodiment, the stem cells are embryonic stem cells or induced pluripotent stem cells.

[0049] In some embodiments, the stem cells are derived from mammals. In a preferred embodiment, the stem cells are derived from primates. In a more preferred embodiment, the stem cells are derived from humans.

[0050] In some implementations, the embryonic stem cells are commercially available human embryonic stem cell lines, such as the H1 or H9 embryonic stem cell lines.

[0051] In some implementations, human embryonic stem cells are stem cells isolated from or obtained from human ligands within 14 days of fertilization that have not undergone in vivo development.

[0052] In some implementations, induced pluripotent stem cells are human induced pluripotent stem cells, such as WC50 or IMR90.

[0053] In some embodiments, the neuronal differentiation culture medium in step D comprises brain-derived growth factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ascorbic acid, cyclic adenosine monophosphate (cAMP), and compound E. In some embodiments, the concentration of BDNF is 1–100 ng / mL, preferably 15–25 ng / mL, more preferably 20 ng / mL. In some embodiments, the concentration of GDNF is 1–100 ng / mL, preferably 15–25 ng / mL, more preferably 20 ng / mL. In some embodiments, the concentration of ascorbic acid is 10–1000 μM, preferably 150–250 μM, more preferably 200 μM. In some embodiments, the concentration of cAMP is 0.1–10 μM, preferably 0.5–1.5 μM, more preferably 1 μM. In some embodiments, the concentration of Compound E is 0.05–1 μM, preferably 0.05–0.15 μM, more preferably 0.1 μM. In some embodiments, the neuronal differentiation medium further comprises a basal medium selected from any one of IMDM medium, BME medium, GMEM medium, MEM medium, DMEM medium, Hams F-12 medium, RPMI 1640 medium, and Neurobasal medium, preferably Neurobasal medium. In some embodiments, the basal medium further comprises an N2 additive, non-essential amino acids, and a B27 additive. In some embodiments, the concentration of the N2 additive in the basal medium is 0.2–5% (v / v), preferably 0.5–2% (v / v), more preferably 0.5% (v / v). In some embodiments, the total concentration of non-essential amino acids in the basal medium is 0.2–5% (v / v), preferably 0.5–2% (v / v), more preferably 1% (v / v). In some embodiments, the concentration of the B27 additive in the basal medium is 0.2–5% (v / v), preferably 0.5–2% (v / v), more preferably 1% (v / v).

[0054] In some embodiments, the total time for step D is 18 to 24 days, preferably 20 to 22 days. In some embodiments, the total time for step D is 18, 19, 20, 21, 22, 23, or 24 days. In a preferred embodiment, the total time for step D is 21 days.

[0055] In some embodiments, the method disclosed herein further includes a pre-culture step of stem cells prior to step A. In some embodiments, the pre-culture is performed using a basal stem cell culture medium containing a ROCK inhibitor. In some embodiments, the basal stem cell culture medium is selected from any of the following: IMDM (Iscoves Modified Dulbeccos Medium), Eagle's Basal Medium (BME), MEM, DMEM, Hams F-12, RPMI 1640, Fischers, E8, mTESR, StemFit Basic 03, StemFit Basic 04, NutriStem hPSC XF, StemMACS iPS Brew, Stem-Partner ACF, TeSR-AOF, TeSR2, StemFit complete medium, or any combination thereof. In a preferred embodiment, the basal stem cell culture medium is StemFit complete medium. In some embodiments, the concentration of the ROCK inhibitor used in the pre-culture is 5–15 μM. In some embodiments, the concentration of the ROCK inhibitor used for pre-culturing is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μM. In a preferred embodiment, the total concentration of at least one ROCK inhibitor in step A is 10 μM. In some embodiments, the pre-culturing time is 0.5 to 1.5 days, preferably 1 day. In some embodiments, the pre-culturing conditions are 32 to 38°C and 4 to 6% CO2.

[0056] In some embodiments, the first differentiated cell population obtained in step A comprises approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% or more midbrain basal plate cells. In some embodiments, the markers for the midbrain basal plate cells may be selected from any one of FOXA2, OTX2, EN1, SHH, Corin, or any combination thereof.

[0057] In some embodiments, the second differentiated cell population obtained in step B comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more dopaminergic neural progenitor cells. In some embodiments, the markers for dopaminergic neural progenitor cells may be selected from FOXA2, FOXA1, LMX1A, EN1, and OTX2.

[0058] In some embodiments, the third differentiated cell population obtained in step C comprises approximately 80% or more dopaminergic neural progenitor cells. In some embodiments, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more dopaminergic neural progenitor cells. In some embodiments, the dopaminergic neural progenitor cells are selected from OTX2 and EN1 double-positive cells, FOXA2 and LMX1A double-positive cells, CD166 positive cells, Corin positive cells and / or CD166 and Corin double-positive cells, NURR1 positive cells, and LMX1B positive cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more OTX2 and EN1 double-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more FOXA2 and LMX1A double-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more CD166 positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% or more Corin-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% or more CD166 and Corin double-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% or more NURR1-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, or more than 96% LMX1B positive dopaminergic neural progenitor cells.In a more preferred embodiment, the third differentiated cell population comprises approximately 90.1% or more of OTX2 and EN1 double-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 87.5% or more of FOXA2 and LMX1A double-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 97.07% or more of CD166-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 87.74% or more of Corin-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 82.42% or more of CD166 and Corin double-positive dopaminergic neural progenitor cells.

[0059] In some embodiments, the terminally differentiated cell population obtained in step D comprises approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more dopaminergic neurons. In some embodiments, the markers for dopaminergic neurons may be selected from any one of MAP2, TH, GIRK2, PITX3, ALDH1A1, TUJ1, or any combination thereof.

[0060] In another aspect, the present invention also provides a kit for differentiating stem cells into dopaminergic neural progenitor cells, characterized in that the kit comprises a) a first differentiation composition comprising at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor and at least one SHH agonist, and b) a second differentiation composition comprising at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor, at least one SHH agonist and at least one ROCK inhibitor;

[0061] The total concentration of at least one TGFβ inhibitor in the first and second differentiation compositions is greater than 1.0 μM and less than 5.0 μM, preferably 1.1–4.0 μM, and more preferably 1.5–3.0 μM. In some embodiments, the total concentration of at least one TGFβ inhibitor in step A is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 μM. In a preferred embodiment, the total concentration of at least one TGFβ inhibitor in the first and second differentiation compositions is 2.0 μM.

[0062] In some embodiments, the total concentration of at least one BMP4 inhibitor in the first differentiation composition and the second differentiation composition is 0.2–3.0 μM, preferably 0.5–2.0 μM. In some embodiments, the total concentration of at least one BMP4 inhibitor in step A is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM. In a preferred embodiment, the total concentration of at least one BMP4 inhibitor in the first differentiation composition and the second differentiation composition is 1.0 μM.

[0063] In some embodiments, the total concentration of at least one GSK3β inhibitor in the first differentiation composition and the second differentiation composition is 0.2–3.0 μM, preferably 0.2–1.0 μM, more preferably 0.4–0.6 μM. In some embodiments, the total concentration of at least one GSK3β inhibitor in the first differentiation composition and the second differentiation composition is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM. In a preferred embodiment, the total concentration of at least one GSK3β inhibitor in the first differentiation composition and the second differentiation composition is 0.4 μM.

[0064] In some embodiments, the total concentration of at least one SHH agonist in the first and second differentiation compositions is 0.5–2.5 μM, preferably 1.0–1.75 μM. In some embodiments, the total concentration of at least one SHH agonist in the first and second differentiation compositions is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 μM. In a preferred embodiment, the total concentration of at least one SHH agonist in the first and second differentiation compositions is 1.0 μM.

[0065] In some embodiments, the total concentration of at least one ROCK inhibitor in the second differentiation composition is 5–15 μM. In some embodiments, the total concentration of at least one ROCK inhibitor in the second differentiation composition is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μM. In a preferred embodiment, the total concentration of at least one ROCK inhibitor in the second differentiation composition is 10 μM.

[0066] In some embodiments, the kits disclosed herein also include a third differentiation composition comprising at least one BMP4 inhibitor, at least one SHH agonist, and at least one fibroblast growth factor.

[0067] In some embodiments, the total concentration of at least one BMP4 inhibitor in the third differentiation composition is lower than the total concentration of at least one BMP4 inhibitor in the first and second differentiation compositions. In a preferred embodiment, the total concentration of at least one BMP4 inhibitor in the third differentiation composition is 0.2–10 μM, preferably 0.8–1.2 μM. In some embodiments, the total concentration of at least one BMP4 inhibitor in the third differentiation composition is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In a more preferred embodiment, the total concentration of at least one BMP4 inhibitor in the third differentiation composition is 1.0 μM.

[0068] In some embodiments, the total concentration of at least one SHH agonist in the third differentiation composition is lower than the total concentration of at least one SHH agonist in the first and second differentiation compositions. In a preferred embodiment, the total concentration of at least one SHH agonist in the third differentiation composition is 0.1–1.0 μM, preferably 0.1–0.5 μM. In some embodiments, the total concentration of at least one SHH agonist in the third differentiation composition is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μM. In a preferred embodiment, the total concentration of at least one SHH agonist in the third differentiation composition is 0.2 μM.

[0069] In some embodiments, the total concentration of at least one fibroblast growth factor in the third differentiation composition is 10–1000 ng / mL, preferably 80–120 ng / mL. In some embodiments, the total concentration of at least one fibroblast growth factor in the third differentiation composition is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / mL. In a preferred embodiment, the total concentration of at least one fibroblast growth factor in the third differentiation composition is 100 ng / mL.

[0070] In some embodiments, the kits disclosed herein also include d) a fourth differentiation composition comprising at least one SHH agonist and at least one fibroblast growth factor; and e) a fifth differentiation composition comprising at least one SHH agonist, at least one fibroblast growth factor, and at least one ROCK inhibitor.

[0071] In some embodiments, the total concentration of at least one SHH agonist and the total concentration of at least one fibroblast growth factor in the fourth and fifth differentiation compositions are substantially the same as the corresponding concentrations in the third differentiation composition.

[0072] In some embodiments, the total concentration of at least one SHH agonist in the fourth and fifth differentiation compositions is 0.1–1.0 μM, preferably 0.1–0.5 μM. In some embodiments, the total concentration of at least one SHH agonist in the fourth and fifth differentiation compositions is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μM. In a preferred embodiment, the total concentration of at least one SHH agonist in the fourth and fifth differentiation compositions is 0.2 μM.

[0073] In some embodiments, the total concentration of at least one fibroblast growth factor in the fourth and fifth differentiation compositions is 10–1000 ng / mL, preferably 80–120 ng / mL. In some embodiments, the total concentration of at least one fibroblast growth factor in the fourth and fifth differentiation compositions is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / mL. In a preferred embodiment, the total concentration of at least one fibroblast growth factor in the fourth and fifth differentiation compositions is 100 ng / mL.

[0074] In some embodiments, the total concentration of at least one ROCK inhibitor in the fifth differentiation composition is 5–15 μM. In some embodiments, the total concentration of at least one ROCK inhibitor in the fifth differentiation composition is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μM. In a preferred embodiment, the total concentration of at least one ROCK inhibitor in the fifth differentiation composition is 10 μM.

[0075] In some embodiments, the first, second, third, fourth, and fifth differentiation compositions may each independently comprise a basal medium selected from any one of IMDM (Iscoves Modified Dulbeccos Medium), Eagle's Basal Medium (BME), GMEM, MEM, DMEM, Hams F-12, RPMI 1640, Neurobasal, or any combination thereof. In a preferred embodiment, the basal medium is DMEM / F12. In some embodiments, the basal medium contained in the first, second, third, fourth, and fifth differentiation compositions is a basal medium containing an N2 additive and non-essential amino acids. In some embodiments, the concentration of the N2 additive in the basal medium is 0.2–5% (v / v), preferably 0.5–2% (v / v), more preferably 1% (v / v). In some embodiments, the total concentration of non-essential amino acids in the basal medium is 0.2–5% (v / v), preferably 0.5–2% (v / v), more preferably 1% (v / v).

[0076] In some embodiments, the kits disclosed herein also include f) a sixth differentiation composition comprising the neuronal differentiation culture medium disclosed herein.

[0077] In some aspects, the present invention provides cell populations obtained by differentiating stem cells using the in vitro differentiation methods disclosed herein, or using the kits disclosed herein.

[0078] In some embodiments, the cell population disclosed herein is a first differentiated cell population comprising more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of midbrain basal plate cells. In some embodiments, the markers for midbrain basal plate cells may be selected from any one of FOXA2, OTX2, EN1, SHH, Corin, or any combination thereof.

[0079] In some embodiments, the cell population disclosed herein is a secondary differentiated cell population comprising more than 80%, more than 85%, more than 90%, 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% dopaminergic neural progenitor cells. In some embodiments, the markers for dopaminergic neural progenitor cells may be selected from any one of FOXA2, FOXA1, LMX1A, EN1, OTX2, or any combination thereof.

[0080] In some embodiments, the cell population disclosed herein is a third-differentiated cell population comprising approximately 80% or more dopaminergic neural progenitor cells. In some embodiments, the third-differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more dopaminergic neural progenitor cells. In some embodiments, the dopaminergic neural progenitor cells are selected from any one of the following: OTX2 and EN1 double-positive cells, FOXA2 and LMX1A double-positive cells, CD166 positive cells, Corin positive cells, CD166 and Corin double-positive cells, NURR1 positive cells, LMX1B positive cells, or any combination thereof. In a preferred embodiment, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more OTX2 and EN1 double-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more FOXA2 and LMX1A double-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, or approximately 96% or more CD166 positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% or more Corin-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% or more CD166 and Corin double-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% or more NURR1-positive dopaminergic neural progenitor cells. In a preferred embodiment, the third differentiated cell population comprises more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, or more than 96% LMX1B positive dopaminergic neural progenitor cells.In a more preferred embodiment, the third differentiated cell population comprises approximately 90.1% or more of OTX2 and EN1 double-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 87.5% or more of FOXA2 and LMX1A double-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 97.07% or more of CD166-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 87.74% or more of Corin-positive dopaminergic neural progenitor cells. In a more preferred embodiment, the third differentiated cell population comprises approximately 82.42% or more of CD166 and Corin double-positive dopaminergic neural progenitor cells.

[0081] In some embodiments, the cell population disclosed herein is a terminally differentiated cell population comprising more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% dopaminergic neurons. In some embodiments, the markers for dopaminergic neurons may be selected from any one of MAP2, TH, GIRK2, PITX3, ALDH1A1, TUJ1, or any combination thereof.

[0082] In some aspects, the present invention provides compositions comprising the cell populations disclosed herein.

[0083] In some embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions disclosed herein are liquid formulations. In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, at least one of physiological saline, water, cell buffer, cerebrospinal fluid, compound electrolyte injection, Ringer's solution, and trehalose solution. In a preferred embodiment, the water is ddH2O. In a preferred embodiment, the cell buffer is a commonly used buffer such as PBS, HBSS, EBSS, or HEPES. In a more preferred embodiment, the cell buffer is PBS and / or HBSS. In a preferred embodiment, the cerebrospinal fluid is artificial cerebrospinal fluid.

[0084] In some embodiments, the compositions disclosed herein also include a specification. In some embodiments, the specification describes the dosage, frequency of administration, and / or route of administration of the cell populations and / or compositions disclosed herein. In some embodiments, the dosage of the cell populations and / or compositions disclosed herein is 1 x 102 5 Up to 4x10 8 Cells / dose. In a preferred embodiment, the dosage of the cell population and / or composition disclosed herein is 2 x 10-1. 5 Up to 2x108 Cells / dose. In some embodiments, the cell populations and / or compositions disclosed herein are administered 1 to 3 times.

[0085] In some embodiments, the compositions disclosed herein also comprise other medicaments for the prevention and / or treatment of neurological diseases. In some embodiments, the neurological disease is a disease and / or dysfunction associated with degenerative changes in midbrain dopaminergic neurons. In some embodiments, the neurological disease includes at least one of neurodegenerative diseases and neurodegenerative diseases. In some embodiments, the neurological disease includes at least one of Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, spinal cord injury, stroke, intracranial tumor, epilepsy, traumatic brain injury, hereditary neurological diseases, cerebrovascular diseases, paralysis, myasthenia gravis, multiple sclerosis, peripheral nervous system diseases, neuritis, cerebral palsy, and depression. In a preferred embodiment, the neurological disease includes at least one of Parkinson's disease, ALS, Alzheimer's disease, spinal cord injury, stroke, and traumatic brain injury. In a more preferred embodiment, the neurological disease includes at least one of Parkinson's disease, ALS, stroke, and traumatic brain injury.

[0086] In some aspects, the present invention provides the use of the cell populations or compositions disclosed herein in the preparation of medicaments for the prevention and / or treatment of nervous system diseases.

[0087] In some aspects, the present invention provides the use of the cell populations or compositions disclosed herein in the construction of cell banks.

[0088] In some aspects, the present invention provides methods for preventing and / or treating neurological diseases using the cell populations or compositions disclosed herein. In some embodiments, the method is applied to mammals. In some embodiments, the method is applied to primates. In a preferred embodiment, the method is applied to humans.

[0089] In some aspects, the present invention provides the cell populations or compositions disclosed herein for the prevention and / or treatment of nervous system diseases.

[0090] The cell populations (especially dopaminergic neural progenitor cells) prepared by the method disclosed herein exhibit excellent purity and differentiation specificity. After transplantation into vivo, most differentiate into midbrain A9 dopaminergic neurons, demonstrating excellent therapeutic effects on Parkinson's disease-like symptoms in animal models. Simultaneously, the cell populations prepared by this method also exhibit extremely high safety, including very low proliferative activity after transplantation, minimal iPSC residue, almost no differentiation into non-target cells that pose a risk of tic disorders, and no proliferation or metastasis in vivo. Therefore, this method holds promise as an effective and safe treatment for diseases related to A9 dopaminergic neurons. Attached Figure Description

[0091] Figure 1A The immunofluorescence staining results of the neurospheres differentiated in Example 1 are shown. Figure 1B The immunofluorescence staining results of the neurospheres differentiated by Comparative Example 1 are shown; the magnification of each small image is 20x, and the scale bar is 100μm.

[0092] Figure 2 The flow cytometry results of neurosphere cell populations obtained by the methods of Example 1 and Comparative Example 1 are shown.

[0093] Figure 3 The results of immunofluorescence staining of terminally differentiated cell populations obtained by the differentiation method exemplified in Example 2 with dopaminergic neuron markers MAP2 and TH are shown; in the figure, green fluorescence represents MAP2 and red fluorescence represents TH.

[0094] Figure 4 The results of immunofluorescence staining of frozen sections of cell populations obtained by the differentiation method exemplified in Example 1 after one month in transplanted mice are shown; in the figure, red fluorescence represents STEM101, green fluorescence represents KI67, and blue represents DAPI-stained cell nuclei.

[0095] Figure 5A The results of immunofluorescence staining of frozen sections of cell populations obtained by the differentiation method of Example 1 into mice 6 months after transplantation are shown. TH (red), STEM101 (green), and FOXA2 (white) are used to stain the sections. TH is a key enzyme in dopamine synthesis, STEM101 specifically marks human cell nuclei, and FOXA2 is expressed at multiple stages of midbrain dopaminergic neuron development until adulthood. Blue indicates DAPI-stained cell nuclei.

[0096] Figure 5B The results of immunohistochemical staining of brain slices based on hNCAM antibody are shown.

[0097] Figure 6The results of immunofluorescence staining of frozen sections of cell populations obtained by the differentiation method of Example 1 into mice 6 months after transplantation are shown. The blue section represents the cell nuclei stained with DAPI.

[0098] Figure 7 The results of immunofluorescence staining of frozen sections of cells obtained by the differentiation method of Example 1 into mice 6 months after transplantation are shown. The blue section represents the cell nuclei stained with DAPI.

[0099] Figure 8 The results of Stem101 (red) and 5-HT (green) immunofluorescence staining of frozen sections of cell populations obtained by the differentiation method of Example 1 six months after transplantation into mice are shown. Blue indicates DAPI-stained cell nuclei.

[0100] Figure 9 The results of immunofluorescence staining of frozen sections of the cell population obtained by the differentiation method of Example 1 six months after transplantation into mice are shown. Stem101 (red) and OCT4 (green) staining are shown. Blue indicates DAPI-stained cell nuclei.

[0101] Figure 10 The results of the apomorphine rotation test in a mouse model of Parkinson's disease (PD) after administration of DAP or solvent are shown; the vertical axis represents the ratio of the total number of rotations after apomorphine injection to the total number of rotations before injection. Detailed Implementation

[0102] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0103] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are the average values ​​of three replicate experiments.

[0104] definition

[0105] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0106] As used herein, the terms “comprising” or “including” mean that a composition and method comprises the described components, but does not exclude other components. “Mainly composed of”, when used to define a composition and method, should exclude any other components that are obviously essential to the composition. Therefore, compositions defined herein as mainly composed of these components will not exclude trace contamination from separation and purification methods and pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Composed of” should exclude trace components of other ingredients used in the application of the compositions of the invention and of substantial process. Examples defined by these provisional terms are within the scope of this invention.

[0107] Unless otherwise stated herein, the listing of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were listed separately herein. For example, in this specification, if the concentration range is expressed as 1 to 10 μM, it is intended to explicitly list values ​​such as 2 to 9 μM, 5 to 6 μM, or 1 to 5 μM. These are merely examples of specific intentions, and all possible combinations of values ​​between and including the listed minimum and maximum values ​​will be considered explicitly stated in this disclosure. The use of the word “about” to describe a particular listed quantity or range of quantities means indicating that a value very close to the listed quantity is included in that quantity, such as values ​​that can or naturally be considered due to manufacturing tolerances, instrumental and human errors in the formation of the measurement. For example, a value described herein as “about” means covering ±10% of the indicated value. In some cases, “about” may mean ±20%, or ±5%, or ±1%. A value or parameter described herein as “about” includes the value or parameter itself.

[0108] As used herein, the terms “first” and “second” are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Unless otherwise expressly stated, a feature defined as “first” or “second” may explicitly or implicitly include at least one of that feature.

[0109] As used herein, the terms “treatment” and “improvement” are used interchangeably to refer to a method of obtaining a beneficial or desired outcome, including but not limited to therapeutic benefits and / or preventive benefits. A therapeutic benefit means the eradication or reduction of an underlying disease being treated. A therapeutic benefit can be achieved by eradicating or reducing one or more physiological symptoms associated with an underlying disease, thereby resulting in an improvement observed in the patient. For the purposes of this invention, therapeutic benefits include, but are not limited to: symptom reduction, disease severity reduction, delay or slowing of disease progression, improvement or mitigation of a disease state, and remission (whether partial or complete), whether detectable or undetectable. For preventive benefits, the pharmaceutical compositions disclosed herein may also be administered to patients at risk of developing a specific disease or to patients reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed. As used herein, the term “prevention” means a reduction in the frequency of the disease or condition in a treated sample relative to an untreated control sample, or a delay in the onset and / or reduction in the severity of one or more symptoms of a disease or condition relative to an untreated control sample.

[0110] In some respects, the term "treatment" includes both preventative and therapeutic treatment. Generally, treatment is preventative (i.e., protecting the subject from developing the unwanted condition) if administered before the clinical manifestation of an unwanted condition (e.g., a hallmark behavior such as tremor, or the observation of typical pathological molecular markers); and therapeutic (i.e., aiming to reduce, improve, or stabilize the existing unwanted condition or its side effects) if administered after the manifestation of the unwanted condition.

[0111] As used herein, the term "pharmaceutical-acceptable carrier" refers to various compounds used in the preparation of pharmaceutical compositions that are generally safe and non-toxic and do not possess biologically or otherwise undesirable properties.

[0112] As used herein, the term "BMP inhibitor" refers to a class of SMAD inhibitors, including, non-limiting examples, those disclosed in WO2011 / 149762, Chambers et al., Nat Biotechnol. 2009 Mar, 27(3):275-80, Kriks et al., Nature. 2011 Nov 6, 480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1, 30(7):715-20, all of which are incorporated herein by reference in their entirety. In some embodiments, the BMP inhibitor may be selected from DMH-1 or its active derivatives, or pharmaceutically acceptable salts thereof. Derivatives of these small molecule inhibitors can be readily obtained by those skilled in the art through substituent modification, substitution, or addition to the core backbone of the inhibitor.

[0113] "DMH-1" refers to the molecule with CAS number 1206711-16-1-41-9 and name 4-[6-[4-(1-methoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline. In in vitro kinase assays, DMH-1 selectively inhibits the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2) receptor. Its selectivity for ALK-2 is 6 times that of ALK-1 and 19 times that of ALK-3, with no significant inhibitory effect on AMPK, ALK5, KDR (VEGFR-2), or PDGFRβ receptors. DMH-1 has been reported to block BMP4-induced phosphorylation of Smads 1, 5, and 8 in HEK293 cells (Neely et al., ACSChem. Neurosci., 2012; 3:482).

[0114] As used herein, the term "TGF-β1 inhibitor" can also be referred to as "TGFβ / Activin-Nodal inhibitor," which is another type of SMAD inhibitor. Therefore, the differentiation step using both BMP inhibitors and TGF-β1 inhibitors can also be termed "dual SMAD inhibition." In some embodiments, TGF-β1 inhibitors can inactivate receptors including TGFβ, Nodal, and / or Activin, and / or block signal transduction pathways by blocking these receptors and their downstream effectors. Non-limiting examples of TGF-β1 inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar, 27(3):275-80, Kriks et al., Nature. 2011 Nov 6, 480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1, 30(7):715-20, all of which are incorporated herein by reference in their entirety. In some embodiments, the TGF-β1 inhibitor may be selected from SB431542 or its active derivatives (such as A83-01), or pharmaceutically acceptable salts thereof. “SB431542” refers to a small molecule compound with the CAS number 301836-41-9, the name 4-[4-(l,3-benzodioxolane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide, and the molecular formula C22H18N4O3.

[0115] It has been reported that Wnt signaling activators can activate Wnt signaling by reducing the activity of glycogen synthase kinase 3β (GSK3β). Therefore, the term "Wnt activator" in this article can be used interchangeably with "GSK3β inhibitor". Non-limiting examples of Wnt activators include CHIR99021, WNT1, WNT5A, WNT3A, CHIR98014, AMBMP hydrochloride, LP922056, lithium, deoxycholic acid, BIO, or SB-216763, as well as those described in WO2011 / 149762, WO13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul 1, 30(7):715-20, Kriks et al., Nature. 2011 Nov 6, 480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug 19, 35(33):11462-81, all of which are incorporated herein by reference in their entirety.

[0116] In some embodiments, the Wnt activator may be selected from CHIR99021 or its active derivatives, or pharmaceutically acceptable salts thereof. “CHIR99021” refers to a small molecule inhibitor of the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, also known as “aminopyrimidine” or “3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene]-2-indolone”.

[0117] As used herein, the term “SHH” is used interchangeably with “Sonic hedgehog” and refers to a protein of one of at least three proteins in the mammalian signaling pathway family known as hedgehog factor. SHH regulates the expression of certain downstream genes by interacting with transmembrane molecules Patched (PTC) and Smoothened (SMO) (see Gilbert, 2000 Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers)). Non-limiting examples of SHH activators include those described in WO10 / 096496, WO13 / 067362, Chamberset al., Nat Biotechnol. 2009 Mar, 27(3):275-80, and Kriks et al., Nature. 2011 Nov 6, 480(7378):547-51. In some embodiments, the SHH activator may be selected from SAG (N-methyl-N'-(3-pyridylphenyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane) or its active derivatives, or pharmaceutically acceptable salts thereof.

[0118] As used herein, the term "ROCK inhibitor" is an abbreviation for "Rho-associated kinase inhibitor," referring to any substance that inhibits or reduces the function of Rho-associated kinases or their signaling pathways in cells. In some embodiments, the ROCK inhibitor may be selected from Y27632 or its active derivatives, or pharmaceutically acceptable salts thereof (e.g., dihydrochloride Y276322HCl).

[0119] As used herein, the term “substantially identical” means that two or more objects being compared have approximately the same quantity or level of the characteristics being compared, for example, when taking into account the error range of the measurement methods used.

[0120] The term "cell population" generally refers to a group of cells. A cell population can consist of cells with a common phenotype (e.g., pluripotent stem cells), or may contain at least a subset of cells with a common phenotype. Cells are considered to have a common phenotype when they are substantially similar or identical in one or more provable characteristics, including but not limited to morphological appearance, the presence, absence, or level of expression of specific cellular components or products (e.g., RNA, proteins, or other markers), activity of certain biochemical pathways, proliferative capacity and / or kinetics, differentiation potential and / or response to differentiation signals, or behavior during in vitro culture (e.g., adhesion, non-adhesion, monolayer growth, proliferation kinetics, etc.). Thus, such provable characteristics can define a cell population or its fractions.

[0121] The preferred embodiments for carrying out the present invention will now be described. It should be noted that the embodiments described below are examples illustrating representative embodiments of the present invention, but the present invention is not limited to these embodiments.

[0122] Two or more of the methods described below can be combined, and such combinations are also included in this invention.

[0123] Example

[0124] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0125] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0126] Experimental materials, reagents, instruments and experimental methods

[0127] Cell lines:

[0128] According to the manufacturer's instructions, use CTS. TM CytoTune TM -iPS 2.1 Sendai Virus Reprogramming Kit (CTS) TM CytoTune TM -iPS 2.1 Sendai Reprogramming Kit (Catalog No.: A34546) is used to prepare induced pluripotent stem cells by reprogramming human PBMC cells.

[0129] Human induced pluripotent stem cells were plated in clumps on a pre-coated laminin matrix at a density of approximately 100,000 per well. TeSR was used. TM Cells were cultured in E8 complete medium (purchased from STEMCELL Technologies) at 37°C and 5% CO2, and passaged by digestion every 4-5 days.

[0130] (S1 stage): One day before using cultured hPSCs for induced differentiation (D-1), human pluripotent stem cells with a confluence of approximately 70%–80% are digested into single cells using TrypLE enzyme at a density of approximately 4000 cells / cm³. 2The samples were seeded at a density of 10 μM into 6-well plates pre-coated with Vitronectin (Gibco) and cultured in E8 complete medium supplemented with 10 μM Y27632.

[0131] Neural basal medium: 98% (v / v) DMEM / F12 (Gibco), 1% (v / v) non-essential amino acids (Gibco) and 1% (v / v) N2 supplement (Gibco).

[0132] Neural differentiation culture medium:

[0133] 97.5% (v / v) Neurobasal (Gibco), 0.5% (v / v) N2 supplement (Gibco), 1% (v / v) B27 supplement (Gibco) and 1% (v / v) Non-essential amino acid (Gibco).

[0134] Antibody:

[0135]

[0136] Chemical reagents:

[0137] Reagent Name supplier Item number Cell cycle and apoptosis detection kit Beyotime C1052 CCK-8 reagent kit Beyotime C0039 Fixative Servicebio G1101 Permeable fluid Beyotime P0096 HumanFOXP3BufferSer Reagent Kit BD 560098

[0138] Example 1

[0139] This embodiment describes an exemplary method for inducing human pluripotent stem cells to differentiate into dopaminergic neural progenitor cells in vitro.

[0140] Inducing pluripotent stem cells to differentiate into midbrain basal plate cells:

[0141] Human pluripotent stem cells that had adhered to the culture medium for 24 hours were replaced with neural basal medium containing 2 μM SB431542, 1 μM MMH-1, 0.4 μM CHIR99021 and 1 μM SAG, and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 (D0); the medium was changed every two days and cultured for another 8 days (S2 stage). On day 9 of differentiation (D9), cells were digested with Accutase (purchased from Innovative), and then resuspended in neural basal medium containing 10 μM Y27632, 2 μM MSB431542, 1 μM DMH-1, 0.4 μM CHIR99021, and 1 μM SAG. Approximately 15 million cells per flask were seeded into T75 suspension culture flasks (purchased from Thermo Fisher Scientific) and cultured in suspension for 2 days in a carbon dioxide cell culture incubator at 37°C and 5% CO2 to obtain neurospheres containing midbrain basal plate cells (S3 stage).

[0142] Inducing differentiation of dopaminergic neural progenitor cells:

[0143] On day 11 (D11), the culture medium for neurospheres was replaced with neural basal medium containing 1 μM DMH-1, 0.2 μM SAG, and 100 ng / mL fibroblast growth factor 8b (FGF8b), and cultured for 5 days in a 37°C, 5% CO2 cell culture incubator. The medium was changed every 2.5 days during this period to obtain a cell population containing dopaminergic neural progenitor cells (S4 phase).

[0144] Inducing differentiation of dopaminergic neural progenitor cells:

[0145] On day 16 (D16), after treating neurosphere cells into single cells, approximately 5 million cells / flask were seeded into T75 culture flasks pre-coated with Vitronectin (purchased from Gibco) and cultured in an adherent cell culture incubator at 37°C and 5% CO2 for 7 days. The differentiation medium was then replaced with neural basal medium containing 0.2 μM SAG and 100 ng / mL FGF8b, with the medium changed every 2.5 days during this period (S5 phase). On day 23 (D23), adherent cells were digested into single cells using Accutase (purchased from Innovative) and then resuspended in neural basal medium containing 10 μM Y27632, 0.2 μM SAG and 100 ng / mLFGF8b. Approximately 15 million cells / flask were seeded into T75 suspension culture flasks and cultured in suspension at 37°C and 5% CO2 for 1 day to obtain neurospheres (S6 stage) containing dopaminergic neural progenitor (mDAP) cells.

[0146] Example 2

[0147] This embodiment describes an exemplary method for inducing dopaminergic neural progenitor cells to differentiate into dopaminergic neurons in vitro.

[0148] The neurospheres obtained by the method in Example 1 were digested into single cells using Accutase, at a density of approximately 2 × 10⁻⁶ cells. 5 cells / cm 2 Cells were seeded at a density of 100% onto pre-coated Matrigel glass slides (Thermo Fisher Scientific), and a neural differentiation medium containing 20 ng / mL brain-derived growth factor (BDNF), 20 ng / mL glial cell-derived neurotrophic factor (GDNF), 200 μM ascorbic acid (AA), 1 μM cyclic adenosine monophosphate (cAMP), and 0.1 μM compound E (Compound E) was added. The cells were cultured at 37°C in a 5% CO2 incubator for 3 weeks, with the medium changed every 3.5 days, to obtain a cell population containing dopamine neurons.

[0149] Comparative Example 1

[0150] The in vitro differentiation of pluripotent stem cells was induced using essentially the same method as in Example 1, except that the concentration of the TGFβ inhibitor SB431542 added to the culture medium during the induction of pluripotent stem cells into midbrain basal plate cells was 10 μM, the concentration of the BMP4 inhibitor DMH-1 was 2 μM, and the concentration of the Wnt agonist CHIR99012 was 0.6 μM.

[0151] Example 3

[0152] This embodiment compares the differentiation-inducing effects of the exemplary method of Embodiment 1 of the present invention with those of the method in Comparative Example 1.

[0153] Immunofluorescence staining

[0154] For neurospheres at stage S6 obtained in Example 1 and Comparative Example 1, the culture medium was removed from the 6-well plates, and 500 μL of fixative was added to each well for 15 min. After discarding the fixative, the cells were washed twice with PBS and then discarded. Next, 250 μL of immunostaining permeabilization buffer was added to each well, permeabilized for 10 min and then discarded, followed by washing twice with PBS. 250 μL of pre-prepared 5% BSA blocking buffer was added to each well, and the cells were blocked at room temperature for 1 h. After removing the blocking buffer, 250 μL each of primary antibody solutions diluted with 1% BSA (goat anti-human FOXA2 (1:200 dilution), rabbit anti-human EN1 (1:500 dilution), and rat anti-human Corin monoclonal antibody (1:200 dilution)) was added to each well, and the cells were incubated overnight at 2–8 °C. Finally, the cells were washed three times with PBS. Add 250 μL of a 1:500 diluted solution of fluorescently labeled secondary antibody (anti-goat, rabbit, and rat fluorescent antibodies) to each well, and incubate at room temperature in the dark for 1 hour. Remove the incubated antibody solution and wash three times with PBS. Finally, add 250 μL of DAPI staining solution to each well, incubate at room temperature in the dark for 10 minutes, then discard the solution and wash three times with PBS. Observe and photograph the immunofluorescence-stained neurospheres using a fluorescence inverted microscope (Nikon TS2FL model). The results are shown in [Figure 1]. Figure 1A and 1B .

[0155] Compare Figure 1A and 1B As can be seen, in the neurospheres obtained using the method exemplified in Example 1 of the present invention, the vast majority of cells highly express the markers Corin, FOXA2 or EN1, which are associated with midbrain dopaminergic neural progenitor cells, and no significant difference was observed between the proportion of positive cells for each marker and that in Comparative Example 1.

[0156] Flow cytometry

[0157] The neurospheres at stage S6 obtained from Example 1 and Comparative Example 1 were enzymatically digested into single cells, and approximately 5 x 10⁶ cells were taken from each. 6Each cell line was divided into two tubes, and 1 mL of flow cytometry staining solution was added to each tube. The cells were then centrifuged at 400 g for 5 min, and the supernatant was discarded. Following the manufacturer's instructions (Human FOXP3 Buffer Ser), 1 mL of 1×Buffer A was added to each tube to resuspend the cells. The cells were then incubated at room temperature in the dark for 10 min. The cells were then centrifuged at 500 g for 5 min, and the supernatant was discarded. 0.25 mL of Buffer C was added to each tube to resuspend the cells. The cells were then incubated at room temperature in the dark for 30 min. 1 mL of flow cytometry staining solution was added directly to each tube, and the cells were centrifuged at 500 g for 5 min. The supernatant was discarded, and the cells were resuspended in flow cytometry staining solution. The resuspended cell solutions were combined, and the cell solutions from Example 1 and Comparative Example 1 were divided into 10 tubes each. The flow cytometry antibody or isotype control antibody shown in Table 1 was added to each tube, and the cells were incubated at 2–8 °C in the dark for 40 min.

[0158] Table 1

[0159]

[0160] After washing with 1 mL / tube of flow cytometry staining buffer, centrifuge at 500g for 5 min, discard the supernatant, and repeat the washing process twice. Finally, resuspend the cells in flow cytometry staining buffer and analyze them using a flow cytometer (Agilent Novocyte 3000). The results are shown below. Figure 2 .

[0161] Depend on Figure 2 As can be seen, no cells expressing the pluripotency factor OCT4 and the early neural stem cell factor PAX6 were observed in the neurospheres obtained using the method exemplified in Example 1 of this invention. Furthermore, consistent with the results of the aforementioned immunostaining, Figure 2 The results showed that most of the cells in the neurosphere obtained by the method of the present invention were cells that highly expressed midbrain dopaminergic progenitor cell markers FOXA2, OTX2 or LMX1A, and the proportion of positive cells for each marker was not significantly different from that of the comparative method in Example 1.

[0162] The above results demonstrate that the method of this invention can effectively induce human pluripotent stem cells to differentiate into dopaminergic precursor cells in vitro. High-purity (e.g., over 80%) midbrain dopaminergic neural precursor cells can be directly obtained without additional purification of dopaminergic cells or specific killing of non-dopaminergic cells. This offers significant advantages for industrial applications.

[0163] Example 4

[0164] Immunofluorescence staining was performed on S7 stage differentiated cells prepared by the method illustrated in Example 2, using the same immunostaining method as in Example 3. The primary antibodies used were an antibody against the dopaminergic neuron marker TH (Pel-Freez, catalog number P40101) at a concentration of 1:1000; and an antibody against the neuron marker MAP2 (Millipore, catalog number MAB378) at a concentration of 1:1000. The staining results are shown in [image / image / description]. Figure 3 .

[0165] Depend on Figure 3 As can be seen, the terminally differentiated cell population obtained by the exemplary method of the present invention includes dopaminergic neurons at the terminal differentiation stage that highly express mature neuronal surface markers such as MAP2 and TH.

[0166] Example 5

[0167] This embodiment compares the cell proliferation effect using the exemplary method of the present invention with that of Comparative Example 1.

[0168] Measuring cell cycle distribution

[0169] The neurospheres from stage S6 in Example 1 and Comparative Example 1 were enzymatically digested into single cells. The single cells resuspended in PBS were then divided into approximately 5 x 10⁻⁶ cells. 5 Cells / tube: Add cell suspension to 1.5 mL centrifuge tubes, centrifuge at 1000 g for 5 min, and discard the supernatant. Add 1 mL of pre-chilled PBS, centrifuge at 1000 g for 5 min, and discard the supernatant. Then add 1 mL of pre-chilled 70% ethanol and fix at 2–8 °C for 2 h. Centrifuge at 1300 g for 5 min, and discard the supernatant. Then add 1 mL of pre-chilled PBS, centrifuge at 1300 g for 5 min, and discard the supernatant. Finally, add 0.2 mL of propidium iodide staining solution and incubate at 37 °C in the dark for 30 min. Flow cytometry was used to detect the percentage of cells in G1, S, and G2 phases. The results are shown in Table 2.

[0170] Measuring cell doubling time

[0171] The neurospheres at stage S6 were enzymatically digested into single cells. (Approximately 5 x 10⁻⁶ cells) 4Cells from Example 1 and Comparative Example 1 were seeded into 24-well culture plates and cultured at 37°C and 5% CO2 for 3 days. CCK-8 reagent was added at the 3rd hour of culture on the day of seeding (i.e., after cell adhesion) and on the 3rd day of culture, respectively. The absorbance (OD) at the dominant wavelength of 450 nm and the reference wavelength of 630 nm was measured using a microplate reader (TECAN). Three wells were measured in parallel for each group, with two cell-free blank wells used as blank controls. The average value of each well was calculated by subtracting the average value of the blank wells from the average value of the sample wells. The doubling time (ΔT) was calculated using the following formula: ΔT = t × [Lg2 / (LgNt-LgN0)] (N0: OD value measured a hours after seeding on day 0; Nt: OD value measured b hours after seeding; t: culture time (ba) hours). The results are also shown in Table 2.

[0172] Table 2:

[0173] Testing items Comparative Example 1 Example 1 G1 75.35％ 72.43％ S 19.62％ 21.46+％ G2 4.85％ 5.93％ S+G2 24.47％ 27.39％ Group doubling time 24.2h 18.7h

[0174] As shown in Table 2, compared with Comparative Example 1, the DAP cell population obtained by the method shown in Example 1 has a slightly higher proportion of cells in S phase and G2 phase, and a significantly shorter population cell doubling time. This indicates that the cell population obtained by the method of the present invention has higher proliferative capacity and can obtain significantly more target cells with the same differentiation culture time. The improved yield and production efficiency are of great significance for large-scale industrial production.

[0175] It should be noted that, based on the results of Examples 3 and 5, the improvement of the doubling time and cell proliferation capacity of the differentiated cell population by the method of the present invention did not lead to changes in the expression of pluripotency markers and midbrain dopaminergic differentiation markers, indicating that it has no adverse effect on the safety and efficacy of the obtained cells.

[0176] Example 6

[0177] This embodiment further compares the proliferative capacity of cell populations obtained at each differentiation stage using the exemplary method of Embodiment 1 of the present invention and the method in Comparative Example 1.

[0178] Cell populations at stages S2 to S6 were prepared using the same methods as in Example 1 and Comparative Example 1. Cell counts were performed on the different stages of the cell populations using a Rigel S2 cell counter (purchased from Countstar), and the results are shown in Table 3.

[0179] Table 3:

[0180]

[0181] As shown in Table 3, the cell population induced by the method of the present invention has a significantly higher proliferation capacity than the control method at each stage, resulting in a total number of harvested cells that is more than 10 times higher than the control.

[0182] Example 7

[0183] This embodiment describes the application of a cell population of dopaminergic neural progenitor cells (mDAP cells) prepared in Example 1 in the treatment of Parkinson's disease.

[0184] The cell population at the end of stage S6 (i.e., dopaminergic neural progenitor cells (mDAP cells)) obtained in Example 1 was prepared into a cell suspension suspended in artificial cerebrospinal fluid (ACSF, Maokang Biotechnology, MX0951), at a concentration of 1×10⁻⁶. 5 Stereotactic injections were administered to the brains of mice that had successfully developed 6-OHDA models (see, for example, Chinese Patent Application No. 202410187903.9) at a rate of 1 cell / μL. Each successful mouse was injected once into the left striatum of the brain, for a total of 2 μL.

[0185] Behavioral tests and evaluations were performed on mice that successfully developed the model and mice that received the transplant. Behavioral tests included the APO rotation and rotarod tests, and the specific methods are as follows:

[0186] APO-induced rotation: Mice were injected intraperitoneally with apomorphine (APO) 0.5 mg / kg. Five minutes later, the mice were placed in a cylinder with a diameter of 20 cm. The movement trajectory of the mice was recorded by video over 30 minutes, and the number of rotations in 30 minutes was recorded.

[0187] Rotary bar experiment: The mice were placed on the rotary bar. Day 1 training: 10s, the rotation speed was increased from 1 rpm to 10 rpm, 300s, repeated 3 times; Day 2 training: 15s, the rotation speed was increased from 1 rpm to 15 rpm, 300s, repeated 3 times; Day 3 formal experiment: 15s, the rotation speed was increased from 1 rpm to 15 rpm, and the time it took for the mice to fall was recorded.

[0188] Blood and brain tissue samples were collected from mice 1, 4, and 6 months after cell transplantation. The collected brain tissue was sectioned and stained with immunofluorescence. Specifically: ① Brain tissue obtained from PFA perfusion was immersed in 20% sucrose until it settled (2-3 days). ② Brain tissue was immersed in 30% sucrose until it settled (2-3 days). ③ The dehydrated brain tissue was removed, fixed to a sample holder with OCT glue, and rapidly frozen at -80°C. ④ The desired area was cut to a thickness of 40 μm. ⑤ After rinsing in a 6-well plate containing 1×PBS buffer, the sample was placed in a 96-well plate containing 150 μL of cryopreservation solution (1L cryopreservation solution: 300g sucrose + 400mL 1×PBS buffer, then brought to a final volume of 1L with ethylene glycol) and stored at -20°C. Remove the brain slices from the -20℃ freezer, wash three times with 1×PBS buffer, 5 min each time; add 5% donkey antiserum (product number SL050, manufacturer Solarbio) and block at room temperature for 1 h; add primary antibodies (Stem101 primary antibody, product number Y40400, manufacturer Takara; TH primary antibody, product number AB152, manufacturer Millipore; PSD95 primary antibody, product number ab12093, manufacturer Abcam; anti-serotonin rabbit anti-antibody, manufacturer Sigma, product number s5545-100 μl), put into a 96-well plate, and incubate overnight at 4℃. Remove the brain slices that have been incubated overnight at 4℃, recover the primary antibody (store at 4℃), wash 3 times with 1×PBS buffer, 5 min each time; add secondary antibodies (R(488) secondary antibody, catalog number A-21206, manufacturer Invitrogen; G(546) secondary antibody, catalog number A-11056, manufacturer Invitrogen; M(647) secondary antibody, catalog number A-31571, manufacturer Invitrogen), incubate at room temperature for 2 h; wash 3 times with 1×PBS buffer, 5 min each time; add DAPI (DAPI, catalog number F6057, manufacturer Sigma), mount the slices, and take pictures.

[0189] Figure 4 The results of fluorescence staining of mouse brain tissue sections one month after transplantation are shown. It can be seen that most of the cells at the transplantation site are transplanted cells expressing the human marker Stem101 (red), indicating that the cell population prepared by the method of the present invention can successfully survive and integrate into the host brain tissue after transplantation. Figure 4 Some cells in the study expressed the cell proliferation marker gene Ki67 (green), indicating that the cell population prepared by the method of this invention has the potential to continue to proliferate and increase the number of dopaminergic neurons one month after transplantation.

[0190] Example 8

[0191] This embodiment describes the single-cell transcriptome sequencing of the cell population prepared by the method of the present invention.

[0192] (1) Cell preparation

[0193] Using the same method as in Example 2, cell populations at stages S2, S3 (midbrain basal plate cell population), S4 (dopaminergic neural progenitor cell population), S5, S6 (dopaminergic neural precursor cell population), and S7 (dopaminergic neuron cell population) were prepared, respectively. Single-cell suspensions of each cell population were obtained through enzymatic digestion and washing. Cell suspensions used for transcriptome sequencing required a cell viability of over 80% and a cell concentration of 2.5–3.5 × 10⁻⁶ cells / mL. 5 Between cells / mL.

[0194] (2) Tagging

[0195] Inject the single-cell suspension The microfluidic chip (purchased from Novogene) was used to separate individual cells based on the principles of Poisson distribution and physical sedimentation, according to the chip manufacturer's instructions. Millions of magnetic beads (provided by Novogene, compatible with the microfluidic chip) carrying unique cell barcodes and molecular tags (UMIs) were then added to the chip's microwells. Cells were lysed within the microwells, and the mRNA released from cell lysis was captured by the binding of the unique cell barcodes and molecular tags (UMIs) carried by the magnetic beads to their poly(A) tails, thus achieving cell and mRNA labeling.

[0196] (3) Collect the magnetic beads in the chip, reverse transcribe the mRNA captured by the magnetic beads into cDNA and amplify it.

[0197] (4) Library construction and quantitative analysis

[0198] Next-generation sequencing libraries were constructed using the amplified cDNA. The libraries were then quantified through fragmentation, ligation of sequencing adapters, and sample index PCR to ensure their quality met sequencing standards.

[0199] (5) Sequencing

[0200] The constructed library was then subjected to next-generation sequencing using the Illumina HiSeq or NovaSeq platform to obtain 10× genomic data. The PE150 sequencing mode, typically with a sequencing throughput of 50 kreads / cell or higher, was generally used.

[0201] (6) Sequencing results analysis

[0202] For the single-cell sequencing data obtained in Section (5) above, the CellRanger single-cell toolkit provided by 10× Genomics was used, and the Seurat R package (v4.4.0) was used for downstream analysis. Cell types were identified through data quality control, cell clustering and visualization, and differentially expressed genes specific to cell populations were determined. Heatmaps, featureplots, vlnplots and dotplots were then used to display the data.

[0203] Example 9

[0204] This embodiment describes how the DAP cells of this disclosure can survive long-term in the brain (striatum) of PD model mice and correctly colonize the transplantation area.

[0205] Neurospheres were prepared using the same method as described in Example 1. The test sample was prepared and transplanted into the striatum of 6- to 8-week-old NOG mice (AP: +0.6 mm, ML: +1.8 mm, DV: -3.2 mm) using the same method as described in Example 7. At week 24 post-transplantation, mice were sacrificed, and intact brain tissue was extracted within 24 hours and immersed in 4% PFA. After fixation, the tissue was dehydrated at 4°C using a 20% and 30% sucrose gradient, and then OCT-embedded sections (40 μm thickness). Immunofluorescence staining was then performed using anti-TH antibody (Millipore, AB152), human HNF-3beta / FOXA2 antibody (R&D, AF2400), and Stem101 antibody (Takara, Y40400). The results were shown in... Figure 5A .Depend on Figure 5A As can be seen, in the brain slices of transplanted mice, most TH+, FOXA2+, and STEM101+ cells were co-localized within the transplanted area. This suggests that the dopaminergic neurons in this region are all human midbrain dopaminergic neurons differentiated from the transplanted test sample.

[0206] hNCAM is a marker specific to human nerve cells, and its antibody can be used to label human cells in animal tissues. Immunohistochemical staining of brain slices using an hNCAM-based antibody (Santa Cruz, sc-106) yielded the following results: Figure 5B It can be seen that hNCAM+ cells can be detected in the striatum of mice 24 weeks after transplantation, and the hNCAM+ cells are aggregated in well-defined areas of the striatum.

[0207] Example 10

[0208] This embodiment describes how the DAP cells of this disclosure can differentiate into A9 type dopaminergic neurons in the substantia nigra of the midbrain after transplantation.

[0209] Sox6 in the ventral substantia nigra pars compacta + The cell population contains one Aldh1a1 + This subpopulation of cells is more prone to degeneration and loss in PD, exhibiting characteristics of midbrain A9 dopaminergic neurons. Immunofluorescence staining of brain slices prepared according to the method described in Example 9 was performed using anti-TH antibody, ALDH1A1 antibody (R&D, AF5869), and human SOX6 antibody (R&D, MAB7759). The results are shown in... Figure 6 .

[0210] Depend on Figure 6 It was observed that at week 24 post-transplantation, a subset of TH+ cells differentiated from human cells in the transplantation area co-localized with ALDH1A1+ cells and also with SOX6+ cells. This indicates that the cells disclosed herein successfully differentiated into substantia nigra A9 dopaminergic neurons at the transplantation site after transplantation into the striatum.

[0211] Example 11

[0212] This embodiment describes the proliferation of DAP cells of this disclosure in the mouse striatum.

[0213] Ki-67 is a nuclear antibody associated with cell proliferation. Immunofluorescence staining was performed on brain slices prepared according to the method described in Example 9 using anti-Ki-67 antibody (Proteintech, 27309-1-AP) and Stem101 antibody. The results are shown in... Figure 7 .

[0214] Depend on Figure 7 As can be seen, at week 24 post-transplantation, the proportion of STEM101 and Ki-67 double-positive cells among STEM101 positive cells was relatively small. This indicates that the DAP cells disclosed in this study exhibit low proliferative activity after transplantation, with almost no observed proliferating cells generated by them, resulting in low proliferation-related risks, including teratogenicity, and high safety.

[0215] Example 12

[0216] This embodiment uses 5-hydroxytryptamine labeling to explore whether the DAP cells of this disclosure differentiate into serotonergic neurons in vivo.

[0217] Serotonin is associated with diseases such as Tourette syndrome (Wei Xijun et al., A Study on Changes in Serum Monoamine Neurotransmitters and Sleep Structural Parameters in Children with Tourette Syndrome and Their Relationship with Overall Severity, *Chinese Medical Innovation*, 2022, Vol. 30, pp. 44-48). Serotonin is a cytoplasmic antibody characterizing serotonergic neurons. Immunofluorescence staining of brain slices prepared according to the method described in Example 9 was performed using Stem101 antibody and Serotonin antibody (Merck, S5545). The results are shown in... Figure 8 .

[0218] Depend on Figure 8 As can be seen, no colocalization of STEM101 and Serotonin was observed in the host striatum at 24 weeks post-transplantation. This indicates that the DAP of this disclosure did not differentiate into serotonergic neurons after transplantation into the striatum.

[0219] Example 13

[0220] This embodiment measures the residual iPSCs of the DAP of this disclosure after transplantation and differentiation into target cells (A9).

[0221] OCT4 is a nuclear antibody characterizing iPS cells. Immunofluorescence staining was performed on brain slices prepared according to the method described in Example 9 using Stem101 antibody and OCT4 / POU5F1 antibody (Proteintech, 11263-1-AP). The results are shown in... Figure 9 .

[0222] Depend on Figure 9 As can be seen, no colocalization of STEM101 and OCT4 was observed in the striatum of the host at 24 weeks post-transplantation. This indicates that the pluripotency of the DAP disclosed in this study is lost during the differentiation process after transplantation into the striatum, and ultimately, almost no residual iPSCs are observed.

[0223] Example 14

[0224] This example describes the construction of a Parkinson's disease (PD) model and the results of behavioral tests conducted using the model.

[0225] SPF-grade Nod Scid mice (immunodeficient mice) (Scidbeige strain) of similar body weight were randomly divided into a model group and a control group, with 30 mice in each group. After anesthetizing the mice, 2 μL of a 3 mg / mL solution of 6-hydroxydopamine (6-OHDA, Sigma, H4381-100 mg) (L-ascorbic acid (VC) dissolved in physiological saline to a concentration of 1%) was slowly injected into the substantia nigra of the left brain of the model group mice using a stereotaxic instrument; 2 μL of L-ascorbic acid (Sigma-Aldrich, A92902-100G) was slowly injected into the same site of the control group mice. Rotation tests were performed at week 4 post-surgery. The number of rotations in the model group mice was significantly higher than that in the control group, indicating that the PD mouse model was successfully established.

[0226] Twenty-four successfully modeled PD mice were randomly divided into a control group (n=8) and a treatment group (DAP, n=10). Cell transplantation (drug administration) was performed one month after modeling. Mice in the treatment group were anesthetized with isoflurane, and 2 μL of the disclosed DAP cell preparation (2 × 10⁻⁶ cells / mL) was slowly injected into the left brain using a stereotaxic instrument. 5 (2 μL / cell, single-point injection). Mice were fed normally after recovery. Control group mice were treated with the same solvent as the treatment group. All mice were able to move normally, drink water, and eat during the treatment process.

[0227] Mice were assessed for behavioral changes using a rotation test at 2, 3, 4, 5, and 6 months post-administration. Specifically, mice were intraperitoneally injected with 0.5 mg / kg apomorphine. Five to ten minutes later, a camera recorded the total number of rotations (turns) within 30 minutes. The difference between the total number of rotations (turns) in the drug-treated group and the solvent-treated group within 30 minutes post-injection and the total number of rotations (turns) in the 30 minutes before cell transplantation was measured (rotations / 30 minutes). The results are shown below. Figure 10 .

[0228] Depend on Figure 10 As can be seen, throughout the observation period, the total number of rotations in the solvent group mice within 30 minutes after apomorphine injection was not statistically significantly different from the total number of rotations before injection, indicating that the solvent lacked efficacy in treating Parkinson's-like symptoms in mice. In contrast, starting from the second month after administration, the total number of rotations in the DAP-treated group mice within 30 minutes after apomorphine injection was significantly lower than the total number of rotations before injection (p<0.05), with a significantly greater reduction than that in the solvent group mice at the same time point. This indicates that the DAP disclosed in this paper can show significant improvement in Parkinson's-like biased gyratory behavioral symptoms starting from 3 months after transplantation.

[0229] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for in vitro differentiation of stem cells, the method comprising: A. An initial cell population containing multiple stem cells is contacted with at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor and at least one SHH agonist to transform the initial cell population into a first differentiated cell population containing midbrain basal plate cells. B. Contact the first differentiated cell population with at least one BMP4 inhibitor, at least one SHH agonist and at least one fibroblast growth factor to transform the first differentiated cell population into a second differentiated cell population containing dopaminergic neural progenitor cells. C. Optionally, the second differentiated cell population is first contacted with at least one SHH agonist and at least one fibroblast growth factor, and then contacted with at least one SHH agonist, at least one fibroblast growth factor and at least one ROCK inhibitor, so that the second differentiated cell population is transformed into a third differentiated cell population containing dopaminergic neural progenitor cells. D. Optionally, the third differentiated cell population is brought into contact with a neuronal differentiation culture medium to transform it into a terminally differentiated cell population containing dopaminergic neurons. in, In step A, the total concentration of the at least one TGFβ inhibitor is greater than 1.0 μM and less than 5.0 μM, preferably 1.1 to 4.0 μM, more preferably 1.5 to 3.0 μM, and most preferably 2.0 μM; Optionally, in step A, the total concentration of the at least one BMP4 inhibitor is 0.2–3.0 μM, preferably 0.5–2.0 μM, more preferably 0.5–1.5 μM, and most preferably 1.0 μM.

2. The method according to claim 1, wherein, Step A includes, after maintaining the contact for a first time window, further contacting the cell population with at least one ROCK inhibitor while continuing to maintain the contact until the end of Step A; Optionally, the total time for step A is 7 to 13 days, preferably 8.5 to 11.5 days, and more preferably 11 days; Optionally, the first time window in step A is 6 to 10 days, preferably 7 to 9 days, and more preferably 9 days; Optionally, the further contact time with at least one ROCK inhibitor in step A is 1 to 3 days, preferably 1.5 to 2.5 days, and more preferably 2 days.

3. The method according to claim 1 or 2, wherein, The total time for step B is 3 to 7 days, preferably 4 to 6 days, and more preferably 5 days.

4. The method according to any one of claims 1 to 3, wherein the total time for step C is 5.5 to 10.5 days, preferably 7 to 9 days, more preferably 8 days; Optionally, the contact time with at least one SHH agonist and at least one fibroblast growth factor in step C is 5 to 9 days, preferably 6 to 8 days, and more preferably 7 days; Optionally, the contact time with at least one SHH agonist, at least one fibroblast growth factor and at least one ROCK inhibitor in step C is 0.5 to 1.5 days, preferably 1 day.

5. The method according to any one of claims 1 to 4, wherein, The TGFβ inhibitors in steps A to C can be independently selected from any 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, LY2109761, or any combination thereof, with SB431542 being preferred; Optionally, the BMP4 inhibitor in steps A to C may be independently selected from any one of dorsomorphin, noggin, LDN-193189, follistatin, chordin, gremlin, DMH-1, or any combination thereof, preferably DMH-1; Optionally, the GSK3β inhibitor in steps A to C may be independently selected from GSK3β inhibitor IX (6-bromoindirubin 3'-oxime), SB216763, GSK3β inhibitor VII (4-dibromoacetophenone), L803-mts, 6-bromoindirubin- -oxime (BIO), TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, CHIR99021, or any combination thereof, preferably CHIR99021; Optionally, the SHH agonist in steps A to C may be independently selected from any one of SHH, SHH C25II, SAG, SAG21K, Hh-Ag1.5, 20α-hydroxycholesterol, puromorphamine, or any combination thereof, preferably SAG; Optionally, the ROCK inhibitor in steps A and C may be independently selected from any one of Y-27632, HA100, HA1152, Blebbistatin, HA-1077, KD-025, Y-33075, Narciclasine, or any combination thereof, with Y-27632 being preferred. Optionally, the fibroblast growth factor in steps B and C may be independently selected from any one of FGF8b, FGF18, FGF8, FGF8a, FGF17, FGF2, FGF4, or any combination thereof, with FGF8b being preferred.

6. The method according to any one of claims 1 to 5, wherein, The total concentration of the at least one GSK3β inhibitor in step A is 0.2–3.0 μM, preferably 0.2–1 μM, and more preferably 0.4–0.6 μM; Optionally, the total concentration of the at least one SHH agonist in step A is 0.5–2.5 μM, preferably 1–1.75 μM, and more preferably 1 μM; Optionally, the total concentration of the at least one ROCK inhibitor in step A is 5–15 μM, preferably 10 μM.

7. The method according to any one of claims 1 to 6, wherein, The total concentration of the at least one BMP4 inhibitor in step B is lower than the total concentration of the at least one BMP4 inhibitor in step A. Preferably, the total concentration of the at least one BMP4 inhibitor in step B is 0.2 to 10 μM, more preferably 0.8 to 1.2 μM, and more preferably 1 μM. Optionally, the total concentration of the at least one SHH agonist in step B is lower than the total concentration of the at least one SHH agonist in step A. Preferably, the total concentration of the at least one SHH agonist in step B is 0.1 to 1.0 μM, more preferably 0.1 to 0.5 μM, and more preferably 0.2 μM. Optionally, the total concentration of the at least one fibroblast growth factor in step B is 10 to 1000 ng / mL, preferably 80 to 120 ng / mL, and more preferably 100 ng / mL.

8. The method according to any one of claims 1 to 7, wherein, The total concentration of at least one SHH agonist and the total concentration of at least one fibroblast growth factor in step C are substantially the same as the corresponding concentrations in step B. Optionally, the total concentration of the at least one SHH agonist in step C is 0.1–1.0 μM, preferably 0.1–0.5 μM, more preferably 0.2 μM; Optionally, the total concentration of the at least one fibroblast growth factor in step C is 10 to 1000 ng / mL, preferably 80 to 120 ng / mL, and more preferably 100 ng / mL; Optionally, the total concentration of the at least one ROCK inhibitor in step C is 5–15 μM, preferably 10 μM.

9. The method according to any one of claims 1 to 8, wherein, The stem cells are pluripotent stem cells; Optionally, the stem cells are selected from any one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells, preferably embryonic stem cells or induced pluripotent stem cells; Optionally, the stem cells are derived from mammals, preferably from primates, and more preferably from humans; Optionally, the embryonic stem cells are commercially available human embryonic stem cell lines; Optionally, the human embryonic stem cells are stem cells isolated from or obtained from human ligands within 14 days of fertilization that have not undergone in vivo development.

10. The method according to any one of claims 1 to 9, wherein, The neuronal differentiation culture medium in step D contains brain-derived growth factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ascorbic acid, cyclic adenosine monophosphate (cAMP), and compound E. Optionally, the concentration of BDNF is 1 to 100 ng / mL, preferably 15 to 25 ng / mL, and more preferably 20 ng / mL; Optionally, the concentration of GDNF is 1 to 100 ng / mL, preferably 15 to 25 ng / mL, and more preferably 20 ng / mL; Optionally, the concentration of the ascorbic acid is 10-1000 μM, preferably 150-250 μM, and more preferably 200 μM; Optionally, the concentration of cAMP is 0.1–10 μM, preferably 0.5–1.5 μM, and more preferably 1 μM; Optionally, the concentration of Compound E is 0.05–1 μM, preferably 0.05–0.15 μM, and more preferably 0.1 μM; Optionally, the neuronal differentiation medium further comprises a basal medium selected from any one of IMDM medium, BME medium, GMEM medium, MEM medium, DMEM medium, Hams F-12 medium, RPMI1640 medium, and Neurobasal medium, preferably Neurobasal medium; Optionally, the basal culture medium further comprises N2 additive, non-essential amino acids, and B27 additive; Optionally, the concentration of the N2 additive in the basal culture medium is 0.2-5% (v / v), preferably 0.5-2% (v / v), and more preferably 0.5% (v / v); Optionally, the total concentration of the non-essential amino acids in the basal culture medium is 0.2-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v); Optionally, the concentration of the B27 additive in the basal culture medium is 0.2-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v); Optionally, the total time for step D is 18 to 24 days.

11. The method according to any one of claims 1 to 10, wherein, The first differentiated cell population contains approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% of the aforementioned midbrain basal plate cells; Optionally, the markers for the midbrain basal plate cells are selected from any one of FOXA2, OTX2, EN1, SHH, Corin, or any combination thereof; Optionally, the second differentiated cell population comprises more than 80%, more than 85%, more than 90%, 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% of the dopaminergic neural progenitor cells. Optionally, the markers for the dopaminergic neural progenitor cells are selected from any one of FOXA2, FOXA1, LMX1A, EN1, OTX2, or any combination thereof; Optionally, the third differentiated cell population comprises about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the dopaminergic neural progenitor cells; Optionally, the markers for the dopaminergic neural progenitor cells are selected from any one of OTX2, EN1, FOXA2, LMX1A, CD166, Corin, NURR1, LMX1B, or any combination thereof; Optionally, the terminally differentiated cell population comprises more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the dopaminergic neurons; Optionally, the markers of the dopaminergic neurons are selected from any one of MAP2, TH, GIRK2, PITX3, ALDH1A1, TUJ1, or any combination thereof.

12. A reagent kit, characterized in that, The kit contains a) A first differentiation composition comprising at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor, and at least one SHH agonist, and b) A second differentiation composition comprising at least one TGFβ inhibitor, at least one BMP4 inhibitor, at least one GSK3β inhibitor, at least one SHH agonist and at least one ROCK inhibitor; Wherein, the total concentration of the at least one TGFβ inhibitor in the first differentiation composition and the second differentiation composition is greater than 1.0 μM and less than 5.0 μM, preferably 1.1 to 4.0 μM, more preferably 1.5 to 3.0 μM, and most preferably 2.0 μM; Optionally, the total concentration of the at least one BMP4 inhibitor in the first differentiation composition and the second differentiation composition is 0.2 to 3.0 μM, preferably 0.5 to 2.0 μM, more preferably 1.0 μM; Optionally, the total concentration of the at least one GSK3β inhibitor in the first differentiation composition and the second differentiation composition is 0.2–3.0 μM, preferably 0.2–1.0 μM, more preferably 0.4–0.6 μM; Optionally, the total concentration of the at least one SHH agonist in the first differentiation composition and the second differentiation composition is 0.5 to 2.5 μM, preferably 1.0 to 1.75 μM, more preferably 1.0 μM; Optionally, the total concentration of the at least one ROCK inhibitor in the second differentiation composition is 5–15 μM, preferably 10 μM.

13. The kit according to claim 12, further comprising: c) A third differentiation composition comprising at least one BMP4 inhibitor, at least one SHH agonist and at least one fibroblast growth factor; in, The total concentration of the at least one BMP4 inhibitor is lower than the total concentration of the at least one BMP4 inhibitor in the first differentiation composition and the second differentiation composition. Preferably, the total concentration of the at least one BMP4 inhibitor is 0.2 to 10 μM, more preferably 0.8 to 1.2 μM, and more preferably 1.0 μM. Optionally, the total concentration of the at least one SHH agonist is lower than the total concentration of the at least one SHH agonist in the first differentiation composition and the second differentiation composition. Preferably, the total concentration of the at least one SHH agonist is 0.1 to 1.0 μM, more preferably 0.1 to 0.5 μM, and more preferably 0.2 μM. and Optionally, the total concentration of the at least one fibroblast growth factor is 10 to 1000 ng / mL, preferably 80 to 120 ng / mL, and more preferably 100 ng / mL.

14. The kit according to claim 13, further comprising: d) A fourth differentiation composition comprising at least one SHH agonist and at least one fibroblast growth factor; and e) The fifth differentiation composition, wherein the fifth differentiation composition comprises at least one SHH agonist, at least one fibroblast growth factor and at least one ROCK inhibitor; in, The total concentrations of at least one SHH agonist and at least one fibroblast growth factor in the fourth and fifth differentiation compositions are substantially the same as the corresponding concentrations in the third differentiation composition. Optionally, the total concentration of the at least one SHH agonist in the fourth differentiation composition and the fifth differentiation composition is 0.1 to 1.0 μM, preferably 0.1 to 0.5 μM, more preferably 0.2 μM; Optionally, the total concentration of the at least one fibroblast growth factor in the fourth differentiation composition and the fifth differentiation composition is 10 to 1000 ng / mL, preferably 80 to 120 ng / mL, and more preferably 100 ng / mL; Optionally, the total concentration of the at least one ROCK inhibitor in the fifth differentiation composition is 5–15 μM, preferably 10 μM.

15. The kit according to claim 14, further comprising: f) The sixth differentiation composition, wherein the sixth differentiation composition comprises a neuronal differentiation culture medium; in, The neuronal differentiation culture medium contains brain-derived growth factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ascorbic acid, cyclic adenosine monophosphate (cAMP), and compound E. Optionally, the concentration of BDNF is 1 to 100 ng / mL, preferably 15 to 25 ng / mL, and more preferably 20 ng / mL; Optionally, the concentration of GDNF is 1 to 100 ng / mL, preferably 15 to 25 ng / mL, and more preferably 20 ng / mL; Optionally, the concentration of the ascorbic acid is 10-1000 μM, preferably 150-250 μM, and more preferably 200 μM; Optionally, the concentration of cAMP is 0.1–10 μM, preferably 0.5–1.5 μM, and more preferably 1 μM; Optionally, the concentration of Compound E is 0.05–1 μM, preferably 0.05–0.15 μM, and more preferably 0.1 μM; Optionally, the neuronal differentiation medium further comprises a basal medium selected from any one of IMDM medium, BME medium, GMEM medium, MEM medium, DMEM medium, Hams F-12 medium, RPMI1640 medium, and Neurobasal medium, preferably Neurobasal medium; Optionally, the basal culture medium further comprises N2 additive, non-essential amino acids, and B27 additive; Optionally, the concentration of the N2 additive in the basal culture medium is 0.2-5% (v / v), preferably 0.5-2% (v / v), and more preferably 0.5% (v / v); Optionally, the total concentration of the non-essential amino acids in the basal culture medium is 0.2-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v); Optionally, the concentration of the B27 additive in the basal culture medium is 0.2-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v).

16. A cell population obtained by differentiating stem cells using the method of any one of claims 1 to 11 or using the kit of any one of claims 12 to 15.

17. A composition comprising the cell population according to claim 16; Optionally, it may also contain a pharmaceutically acceptable carrier.

18. The use of the cell population according to claim 16 or the composition according to claim 17 for any one of the following (i) to (iii): (i) constructing a cell bank; (ii) preparing a medicament for the prevention and / or treatment of nervous system diseases; (iii) preventing and / or treating nervous system diseases.

19. A method for preventing and / or treating nervous system diseases using the cell population according to claim 16 or the composition according to claim 17.

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