Method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells

Abstract

Disclosed is a method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural progenitor cells, comprising the following steps: seeding a neural progenitor cell culture medium containing pluripotent stem cells onto a cell culture plate, and performing directed differentiation culturing, to obtain dopaminergic neural progenitor cells, the neural progenitor cell culture medium containing a cell mechanotransduction pathway inhibitor; seeding a neural precursor cell culture medium containing the dopaminergic neural progenitor cells onto a cell culture plate, and performing directed differentiation culturing, to obtain dopaminergic neural precursor cells, the neural precursor cell culture medium containing a cell mechanotransduction pathway activator. The present invention combines the addition of biochemical factors with regulation of the cell mechanotransduction pathway. In the early stage of induced differentiation, a small molecule drug is used to inhibit the cell mechanotransduction signaling pathway; in the later stage of differentiation, a small molecule drug is used to activate this pathway. This allows for the rapid acquisition of high-purity and high-yield cells, and is suitable for the production of clinical-grade cells.

Description

A method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells Technical Field

[0001] The present invention belongs to the interdisciplinary field of stem cell biology and mechanics, and specifically relates to a highly efficient differentiation method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells. Background Art

[0002] Human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells, are a type of cell with self-renewal and extensive differentiation potential, capable of giving rise to nearly every cell type in the body. Their unique biological properties and broad differentiation potential make them of immense value in modern medicine, offering potentially revolutionary treatments and limitless possibilities. For example, in cell therapy, human pluripotent stem cells can be induced to differentiate into specific cell types, such as neurons, cardiomyocytes, or pancreatic islet cells, for the treatment of neurodegenerative diseases, heart disease, or diabetes.

[0003] Parkinson's disease (PD) is one of the most common progressive neurodegenerative disorders, affecting over 10 million people worldwide. Its pathological hallmark is the death or damage of A9 dopamine-producing (mDA) neurons in the substantia nigra pars compacta of the midbrain. This leads to a loss of the neurotransmitter dopamine in the striatum, which in turn causes the hallmark motor symptoms of PD, including tremor, muscle rigidity, bradykinesia, and postural and gait disturbances. While the exact cause of PD remains unknown, research suggests that genetic, environmental, and age-related factors may contribute to its onset. Existing treatments for PD primarily include medication, deep brain stimulation (DBS), and physical therapy. While these approaches can alleviate symptoms to some extent, they all have limitations. Drug treatments, such as dopamine prodrugs, can provide short-term symptom relief, but long-term use can lead to side effects, such as motor complications and drug tolerance. While DBS is effective in some patients, the risks and high cost of the procedure make it unsuitable for all. Furthermore, these treatments are not a definitive cure for Parkinson's disease, providing only temporary relief rather than halting disease progression. Therefore, finding more effective and safer treatments remains an important direction of PD research.

[0004] Stem cell therapy for Parkinson's disease aims to transform stem cells into mDA neural progenitor cells, which are then transplanted into the patient's brain. These cells can continue to mature in vivo and replace dead or damaged mDA neurons, restoring dopamine production and release. Therefore, unlike traditional treatments, stem cell therapy has great potential to restore the body's ability to produce its own dopamine, thereby delaying or even reversing Parkinson's disease progression. Early clinical trials have also shown that transplantation of mDA neurons from human fetuses can improve symptoms in Parkinson's patients to a certain extent. In recent years, researchers have successfully transformed human pluripotent stem cells into mDA neurons in the laboratory and transplanted them into the brains of Parkinson's patients. However, this therapy still faces challenges, such as obtaining high-purity mDA neural progenitor cells, ensuring the safety of transplanted cells, and improving cell survival and functional integration. Nevertheless, stem cell therapy offers a promising approach for the treatment of Parkinson's disease, and ongoing research is striving to overcome these challenges and make it an effective and reliable treatment.

[0005] In vivo, mDA neurons originate from the differentiation of midbrain neural plate cells. During early embryonic neural tube formation, bone morphogenetic proteins (BMPs) and sonic hedgehog (SHH) work together to determine cell differentiation along the ventral-dorsal axis. WNTs, retinoic acid, fibroblast growth factor 8, and BMPs collectively determine cell fate along the anterior-posterior axis. Mirroring the generation of mDA neurons in vivo, human pluripotent stem cells can also be induced to generate mDA neurons in experimental settings. Numerous studies have described how to induce human pluripotent stem cells into mDA neural progenitors in vitro. Currently, a relatively effective strategy involves initially using inhibitors of BMPs and transforming growth factor-β (TGF-β) in combination with SHH agonists to drive the differentiation of human pluripotent stem cells into neural plate cells. Subsequently, activation of WNT signaling further directs them into midbrain cells, ultimately leading to the formation of mDA neural progenitors. After obtaining mDA neural progenitor cells, they are further induced to develop into mDA neural precursor cells by combining neurotrophic factors (such as brain-derived and glial cell line-derived neurotrophic factors), WNT signaling activators, inducing factors TGF-β3, ascorbic acid, cyclic adenosine monophosphate and DAPT. Technical issues

[0006] The conversion efficiency of the above method is not ideal, especially after further differentiation in vivo, as the proportion of A9 mDA neurons still needs to be optimized. Therefore, further research is needed to address these issues and ensure the efficacy and safety of this method in clinical application. Technical Solutions

[0007] The present invention provides a novel induced differentiation technology for mDA neural precursor cells and its potential application in the treatment of Parkinson's disease. This method can rapidly produce large quantities of high-purity mDA neural precursor cells, significantly shortening the differentiation time and improving the purity and output of cell products. Unlike traditional stem cell induced differentiation methods, the present invention combines biochemical factor induction with regulation of cell mechanical conduction pathways. In the early stages of differentiation, small molecule drugs are used to inhibit cell mechanical conduction signals; and in the later stages of differentiation, small molecule drugs are used to enhance cell mechanical conduction signals. Therefore, since the method of the present invention has the characteristics of high-speed differentiation, high purity and large-scale output of mDA neural precursor cells, it is very suitable for the preparation of clinical-grade cell products and stem cell treatment of Parkinson's disease.

[0008] The present invention provides a method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells, comprising the following steps:

[0009] Step S1, plating a neural progenitor cell culture medium containing pluripotent stem cells on a cell culture plate, and culturing the culture medium to obtain dopaminergic neural progenitor cells through directed differentiation; the neural progenitor cell culture medium contains a cell mechanotransduction pathway inhibitor;

[0010] Step S2: Spreading a neural precursor cell culture medium containing dopaminergic neural progenitor cells on a cell culture plate, and culture the cells through directed differentiation to obtain dopaminergic neural precursor cells; the neural precursor cell culture medium contains a cell mechanotransduction pathway activator.

[0011] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the neural progenitor cell culture medium comprises: neural cell basal culture medium, N2 supplement, B27 supplement without vitamin A, L-glutamine, TGF-β signaling pathway inhibitor, BMP signaling pathway inhibitor, GSK-3β inhibitor, i.e. WNT signaling pathway activator, sonic hedgehog pathway activator, and cell mechanical transduction pathway inhibitor; wherein the concentration of L-glutamine is 1-5mM; the concentration of the TGF-β signaling pathway inhibitor is 1-15μM; the concentration of the BMP signaling pathway inhibitor is 100-500nM; the concentration of the GSK-3β inhibitor, i.e. WNT signaling pathway activator, is 0.5-9µM; and the concentration of the cell mechanical transduction signal pathway inhibitor is 5-15µM.

[0012] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the TGF-β inhibitor is SB431542 or RepSox; the BMP signaling pathway inhibitor is LDN193189 or DMH-1; the GSK-3β inhibitor is CHIR99021 or BIO; and the cell mechanical transduction signaling pathway inhibitor is the Rho kinase inhibitor Y27632 or the myosin II inhibitor Blebbistatin.

[0013] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the concentration of L-glutamine is 2mM; the concentration of the TGF-β inhibitor is 10μM; the concentration of the BMP signaling pathway inhibitor is 250nM; the concentration of the GSK-3β inhibitor is 0.7μM, which is then increased to 7.5µM; and the concentration of the cell mechanical transduction signaling pathway inhibitor is 10µM.

[0014] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the sonic hedgehog pathway activator is SAG at a concentration of 0.5-2 μM, SHH protein at a concentration of 100-600 ng / mL, or Purmorphamine at a concentration of 1-10 μM.

[0015] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the sonic hedgehog pathway activator is SAG at a concentration of 1 μM, SHH protein at a concentration of 500 ng / mL, or Purmorphamine at a concentration of 5 μM.

[0016] The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural progenitor cells as described above is characterized in that a neural progenitor cell culture medium containing pluripotent stem cells that have been blown into single cells is plated on a coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 35-39°C and 3-7% CO2 for 5-12 days, wherein the culture medium is replaced every 1-3 days.

[0017] The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural progenitor cells as described above is characterized in that a neural progenitor cell culture medium containing pluripotent stem cells that have been blown into single cells is plated on a coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 37°C and 5% CO2 for 10 days, wherein the culture medium is replaced every 2 days.

[0018] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural progenitor cells as described above is characterized in that the neural progenitor cell culture medium comprises: neural cell basal culture medium, B27 supplement, L-glutamine, nutrient factors required for the growth of dopaminergic neural progenitor cells, L-ascorbic acid, TGF-β3, GSK-3β inhibitor, i.e., WNT signaling pathway activator, activator cAMP, DAPT, cell mechanical transduction pathway activator, wherein the concentration of L-glutamine is 1-5 mM; The trophic factors required for the growth of dopaminergic neural progenitor cells include brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor; the concentration of the brain-derived neurotrophic factor is 10-50 ng / mL, and the concentration of the glial cell line-derived neurotrophic factor is 10-50 ng / mL; the concentration of the L-ascorbic acid is 0.1-1 mM; the concentration of the TGF-β3 is 0.5-5 ng / mL; the GSK-3β inhibitor, also known as the WNT signaling pathway activator, is CHIR99021 at a concentration of 2-4 µM; the concentration of the activator cAMP is 0.2-2 mM; the concentration of the DAPT is 1-15 µM; and the concentration of the cell mechanotransduction pathway activator is 1-20 µM.

[0019] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the GSK-3β inhibitor, i.e., the WNT signaling pathway activator, is CHIR99021 or BIO; and the cell mechanical transduction pathway activator is the Hippo-YAP / Taz signaling pathway activator PY-60 or the RhoA activator lysophosphatidic acid.

[0020] The method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells as described above is characterized in that the concentration of L-glutamine is 2 mM; the concentration of brain-derived neurotrophic factor is 20 ng / mL; the concentration of glial cell line-derived neurotrophic factor is 20 ng / mL; the concentration of L-ascorbic acid is 0.2 mM; the concentration of TGF-β3 is 1 ng / mL; the concentration of GSK-3β inhibitor, i.e., WNT signaling pathway activator, is 3 µM; the concentration of the activator cAMP is 0.5 mM; the concentration of DAPT is 10 µM; and the concentration of the cell mechanotransduction pathway activator is 10 µM.

[0021] The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural progenitor cells as described above is characterized in that a neural progenitor cell culture medium containing dopaminergic neural progenitor cells that have been blown into single cells is plated on a coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directing differentiation and culturing at 35-39°C and 3-7% CO2 for 3-8 days, wherein the culture medium is replaced every 1-3 days.

[0022] The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural progenitor cells as described above is characterized in that a neural progenitor cell culture medium containing dopaminergic neural progenitor cells that have been blown into single cells is plated on a coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 37°C and 5% CO2 for 5 days, wherein the culture medium is replaced once a day. Beneficial effects

[0023] Compared with the prior art, the beneficial effects of the present invention are: by using small molecule drugs to precisely regulate cell mechanical transduction signals at different stages of induced differentiation, pluripotent stem cells can be converted into high-purity mDA neural precursor cells in a relatively short period of time. These obtained mDA neural precursor cells can further develop into mature mDA neurons. These cells are identified, including detecting their specific protein expression (such as by immunofluorescence and flow cytometry), and can then be cryopreserved for subsequent medical research. This method is not only simple to operate, has rich output and high purity, but also the entire differentiation process takes only 15 days and does not contain any xenogeneic components. This provides an efficient and practical strategy for using the patient's own cells to mass-produce clinically suitable mDA neural precursor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 shows a flow chart of an embodiment of a method for efficiently inducing pluripotent stem cells to differentiate into mDA neural precursor cells disclosed in the present invention;

[0025] Figure 2 shows a microscopic photograph of human pluripotent stem cells;

[0026] FIG3 shows the identification of characteristic protein expression of dopaminergic neural precursor cells obtained by the present invention using cell immunofluorescence staining;

[0027] FIG4 is a quantitative comparison of the dopaminergic neural precursor cells obtained in Examples 1 and 3 and the cells in the control group using flow cytometry;

[0028] FIG5 shows a comparison of the yields of dopaminergic neural precursor cells obtained by Examples 1 and 3 of the present invention and a control group;

[0029] FIG6 shows the identification of characteristic protein expression in mature dopaminergic neurons terminally differentiated from dopaminergic neural precursor cells using cell immunofluorescence staining. Best Mode for Carrying Out the Invention

[0030] Example 1:

[0031] This example demonstrates a method for efficiently obtaining high-purity mDA neural progenitor cells, as shown in Figure 1. This method involves two key steps. In the first step, human pluripotent stem cells are cultured for 10 days in mDA neural progenitor cell induction medium to differentiate into midbrain neural floor plate cells, or mDA neural progenitor cells. This mDA neural progenitor cell induction medium consists primarily of the following components: Neurobasal Medium (NB), 1× N2 Supplement, 1× B27 Supplement without Vitamin A, 2mM L-glutamine, 10μM SB431542, 250nM LDN193189, CHIR99021 (0.7µM on Days 0-3; 7.5µM on Days 4-9), 1μM SAG, and 10μM Y27632. The specific components of the mDA neural progenitor cell induction medium should be adjusted according to the instructions in Figure 1 at different time points. The mDA neural progenitor cell induction medium contains the following: TGF-β signaling pathway inhibitor SB431542, BMP signaling pathway inhibitor LDN193189, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, SHH pathway activator SAG, and cellular mechanotransduction pathway inhibitor Y27632. In the second step, the mDA neural progenitor cells are cultured in the mDA neural progenitor cell induction medium for 5 days to promote their further differentiation into mDA neural progenitor cells. The mDA neural progenitor cell induction medium is mainly composed of NB, B27 supplement without vitamin A, 2mM L-glutamine, 20ng / ml BDNF, 20ng / ml GDNF, 0.2mM ascorbic acid, 1ng / ml TGF-β3, 0.5mM cAMP, 10μM DAPT, 3μM CHIR99021, and 10μM PY-60. At different time stages, the specific components of the mDA neural progenitor cell induction medium need to be adjusted as shown in Figure 1. The mDA neural progenitor cell induction medium is supplemented with: neurotrophic factors (BDNF and GDNF), induction factors ascorbic acid, cAMP, DAPT and TGF-β3, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, and cell mechanotransduction pathway activator PY-60.

[0032] The specific operation steps of this embodiment are as follows:

[0033] 1. Culture of human pluripotent stem cells:

[0034] Human pluripotent stem cells (HSCs) were cultured in Essential 8 medium on vitronectin- or laminin-coated plates in a 37°C, 5% CO2, humidified incubator. Every 4-5 days, when HSCs reach 70-80% confluency, they were passaged using EDTA or dispase, using a subculturing ratio of 1:4 to 1:6. HSCs used should have been rigorously validated for pluripotency (expression of various pluripotency markers and the ability to form teratomas encompassing the endoderm, mesodermal, and ectoderm in immunodeficient mice) and regularly tested for mycoplasma. Fresh Essential 8 medium was replaced daily, and cell growth was monitored. The normal morphology of HSCs is shown in Figure 2.

[0035] 2. Inducing human pluripotent stem cells to differentiate into mDA neural progenitor cells:

[0036] (1) On day 0, when human pluripotent stem cells grow to a confluence of 70-80%, confirm that the cells are in good condition and digest them with Accutase. After counting, the cells are plated on Vitronectin or Laminin coated well plates at a cell density of 400,000 cells / cm 2 Add fresh mDA neural progenitor cell induction medium.

[0037] (2) From day 1 to day 3, replace the mDA neural progenitor cell induction medium with fresh one every day.

[0038] (3) On the 4th day, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added, but without the cell mechanotransduction inhibitor Y27632, and the concentration of CHIR99021 in the culture medium was increased.

[0039] (4) On the 6th day, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added. The composition of the culture medium was the same as that used on the 4th day.

[0040] (5) On day 7, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added, but excluding SB, LDN and SAG.

[0041] (6) On day 9, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added. The composition of the culture medium was the same as that used on day 7.

[0042] 3. Induce mDA neural progenitor cells to differentiate into mDA neural precursor cells:

[0043] (1) On day 10, aspirate the culture medium added the day before and add fresh mDA neural progenitor cell induction medium.

[0044] (2) On day 11, cells were digested with Accutase for 30-40 minutes and seeded on a plate coated with 15 μg / ml poly-L-ornithine solution (PO) + 1 μg / ml Laminin + 2 μg / ml fibronectin at a seeding density of 800,000 cells / cm 2 , cultured using the same medium as on day 10.

[0045] (3) On day 12, replace the culture medium with fresh mDA neural progenitor cell induction medium, but without CHIR99021. In addition, add 10 μM DAPT to the culture medium.

[0046] (4) From day 13 to day 14, replace the neural progenitor cell induction medium with fresh one every day.

[0047] (5) Collection and cryopreservation of mDA neural progenitor cells: On day 15, cells were digested with Accutase for 20-40 minutes and then filtered through a 40 mm pore cell strainer to obtain single cells. Cells were counted, centrifuged, and resuspended in cryopreservation buffer at a density of 8 million cells / ml. The cells were then aliquoted into cryopreservation tubes. Finally, the collected cells were stored in a cryopreservation facility. Modes for Carrying Out the Invention

[0048] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments, and the present invention is not limited to the scope of the described implementation cases. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0049] In a method of inducing pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neural progenitor cells of the present invention, a culture medium for inducing pluripotent stem cells to differentiate into mDA neural progenitor cells, i.e., an mDA neural progenitor cell induction culture medium, comprises: neural cell basal medium (Neurobasal, NB, manufacturer: Life Technologies, catalog number: 21103-049), N2 supplement CTS (manufacturer: Thermo Fisher, catalog number: A1370701), B27 supplement minus vitamin A (manufacturer: Thermo Fisher, catalog number: 12587010), L-glutamine, a TGF-β signaling pathway inhibitor, a BMP signaling pathway inhibitor, a GSK-3β inhibitor, i.e., a WNT signaling pathway activator, a sonic hedgehog pathway activator, and a cell mechanotransduction pathway inhibitor. The TGF-β signaling pathway inhibitor is SB431542; the BMP signaling pathway inhibitor is LDN193189; the GSK-3β inhibitor, i.e., WNT signaling pathway activator, is CHIR99021; the sonic hedgehog pathway activator is SAG; and the cell mechanical transduction pathway inhibitor is Y27632.

[0050] In a preferred embodiment of the present invention, in the mDA neural progenitor cell induction culture medium, the N2 supplement is diluted 100 times, the B27 supplement is diluted 50 times (the purchased stock solutions of the N2 supplement and the B27 supplement are 100X and 50X respectively), the concentration of L-glutamine is 1-5mM, preferably 2mM; the concentration of the TGF-β signaling pathway inhibitor is 1-15µM, preferably 10µM; the concentration of the BMP signaling pathway inhibitor is 100-500nM, preferably 250nM; the GSK-3β inhibitor is WNT The concentration of the signal pathway activator is 0.5-9µM, preferably 0.7µM for differentiation D0-D3, and preferably 7.5µM for differentiation D4-D9; the concentration of the cell mechanical transduction signal pathway inhibitor is 5-15µM, preferably 10µM; the sonic hedgehog pathway activator can be SAG, with a concentration of 0.5-2µM, preferably 1µM; it can also be SHH protein, with a concentration of 100-600ng / mL, preferably 500ng / mL; it can also be Purmorphamine, with a concentration of 1-10µM, preferably 5µM.

[0051] The present invention discloses a culture medium for inducing the differentiation of mDA neural progenitor cells into mDA neural precursor cells, namely, an mDA neural precursor cell induction culture medium, comprising: Neurobasal (NB), B27 supplement minus vitamin A (manufacturer: Thermo Fisher, catalog number: 12587010), L-glutamine, nutrient factors required for the growth of mDA neural progenitor cells, L-ascorbic acid, TGF-β3, a GSK-3β inhibitor (i.e., a WNT signaling pathway activator), an activator of cAMP, DAPT, and a cell mechanotransduction pathway activator. The nutrient factors required for the growth of mDA neural progenitor cells include brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF); the GSK-3β inhibitor (i.e., a WNT signaling pathway activator) is CHIR99021; and the cell mechanotransduction pathway activator is PY-60.

[0052] In a preferred embodiment of the present invention, in the mDA neural precursor cell induction culture medium, the B27 supplement is diluted 50 times (the purchased B27 supplement stock solution is 50×), the concentration of L-glutamine is 1-5 mM, preferably 2 mM; the concentration of the brain-derived neurotrophic factor (BDNF) is 10-50 ng / mL, preferably 20 ng / mL; the concentration of the glial cell line-derived neurotrophic factor (GDNF) is 10-50 ng / mL, preferably 20 ng / mL; the concentration of the L-ascorbic acid is 10-50 ng / mL, preferably 20 ng / mL; The concentration of the acid is 0.1-1mM, preferably 0.2mM; the concentration of the TGF-β3 is 0.5-5ng / mL, preferably 1ng / mL; the GSK-3β inhibitor, i.e., the WNT signaling pathway activator, can be CHIR99021, with a concentration of 2-4µM, preferably 3µM; the concentration of the activator cAMP is 0.2-2mM, preferably 0.5mM; the concentration of the DAPT is 1-15µM, preferably 10µM; the concentration of the cell mechanical transduction pathway activator is 1-20µM, preferably 10µM.

[0053] The components of the mDA neural progenitor cell induction medium work together to convert pluripotent stem cells into mDA neural progenitor cells. The components of the mDA neural progenitor cell induction medium assist the mDA neural progenitor cells in further developing into mDA neural progenitor cells. Neurobasal medium is a serum-free basal medium that provides essential nutrients for neural stem cells and brain neurons. N2 and B27 supplements are serum-free, nutrient-rich supplements used to enhance the basal medium. L-glutamine is a cellular energy source and is involved in protein and nucleic acid metabolism. SB431542 (CAS No. 301836-41-9) is used as the TGF-β inhibitor. LDN193189 (CAS No. 1062368-24-4) is used as the BMP signaling pathway inhibitor. RepSox (CAS No. 446859-33-2) or DMH-1 (CAS No. 1206711-16-1) may also be used. The GSK-3β inhibitor, or WNT signaling pathway activator, can be CHIR99021 (CAS No. 252917-06-9), or BIO (CAS No. 667463-62-9). The cellular mechanotransduction signaling pathway inhibitor can be Y27632 (CAS No. 146986-50-7), or Blebbistatin (CAS No. 856925-71-8). The sonic hedgehog pathway activator can be SAG (CAS No. 912545-86-9), or Purmorphamine (CAS No. 483367-10-8) or SHH protein (manufacturer: R&D systems, Cat. No. 8908-SH). The cellular mechanotransduction signaling pathway activator can be PY-60 (CAS No. 2765218-56-0), or the RhoA activator lysophosphatidic acid (LPA, CAS No. 65528-98-5).

[0054] The method of inducing pluripotent stem cells to differentiate into mDA neural precursor cells of the present invention comprises the following steps:

[0055] Step S1, plating the mDA neural progenitor cell induction culture medium containing pluripotent stem cells on a cell culture plate, and performing directed differentiation culture to obtain mDA neural progenitor cells, wherein the neural progenitor cell induction culture medium contains a cell mechanotransduction signaling pathway inhibitor;

[0056] Step S2: The mDA neural progenitor cell induction culture medium containing mDA neural progenitor cells is plated on a cell culture plate, and directed differentiation culture is performed to obtain mDA neural progenitor cells. The neural progenitor cell induction culture medium contains a cell mechanotransduction signaling pathway activator.

[0057] Specifically, step S1 involves plating the aforementioned mDA neural progenitor cell induction culture medium containing pluripotent stem cells that have been processed into a single-cell state onto a coated cell culture plate. Subsequently, the cells are cultured at 35-39°C, preferably 37°C, and 3-7%, preferably 5%, CO2 for 5-12 days, preferably 10 days, to induce differentiation into mDA neural progenitor cells. During this process, the culture medium is replaced every 1-3 days, preferably every 2 days.

[0058] In step S2, the mDA neural progenitor cell induction culture medium containing the single-cell mDA neural progenitor cells is plated onto a coated cell culture plate. The cells are then cultured for 3 to 8 days, preferably 5 days, at 35-39°C, preferably 37°C, and 3 to 7% (preferably 5%) CO₂, to differentiate into mDA neural progenitor cells. During this stage, the culture medium is replaced every 1 to 3 days, preferably every day.

[0059] Example 1:

[0060] This example demonstrates a method for efficiently obtaining high-purity mDA neural progenitor cells, as shown in Figure 1. This method involves two key steps. In the first step, human pluripotent stem cells are cultured for 10 days in mDA neural progenitor cell induction medium to differentiate into midbrain neural floor plate cells, or mDA neural progenitor cells. This mDA neural progenitor cell induction medium consists primarily of the following components: Neurobasal Medium (NB), 1× N2 Supplement, 1× B27 Supplement without Vitamin A, 2mM L-glutamine, 10μM SB431542, 250nM LDN193189, CHIR99021 (0.7µM on Days 0-3; 7.5µM on Days 4-9), 1μM SAG, and 10μM Y27632. The specific components of the mDA neural progenitor cell induction medium should be adjusted according to the instructions in Figure 1 at different time points. The mDA neural progenitor cell induction medium contains the following: TGF-β signaling pathway inhibitor SB431542, BMP signaling pathway inhibitor LDN193189, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, SHH pathway activator SAG, and cellular mechanotransduction pathway inhibitor Y27632. In the second step, the mDA neural progenitor cells are cultured in the mDA neural progenitor cell induction medium for 5 days to promote their further differentiation into mDA neural progenitor cells. The mDA neural progenitor cell induction medium is mainly composed of NB, B27 supplement without vitamin A, 2mM L-glutamine, 20ng / ml BDNF, 20ng / ml GDNF, 0.2mM ascorbic acid, 1ng / ml TGF-β3, 0.5mM cAMP, 10μM DAPT, 3μM CHIR99021, and 10μM PY-60. At different time stages, the specific components of the mDA neural progenitor cell induction medium need to be adjusted as shown in Figure 1. The mDA neural progenitor cell induction medium is supplemented with: neurotrophic factors (BDNF and GDNF), induction factors ascorbic acid, cAMP, DAPT and TGF-β3, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, and cell mechanotransduction pathway activator PY-60.

[0061] The specific operation steps of this embodiment are as follows:

[0062] 1. Culture of human pluripotent stem cells:

[0063] Human pluripotent stem cells (HSCs) were cultured in Essential 8 medium on vitronectin- or laminin-coated plates in a 37°C, 5% CO2, humidified incubator. Every 4-5 days, when HSCs reach 70-80% confluency, they were passaged using EDTA or dispase, using a subculturing ratio of 1:4 to 1:6. HSCs used should have been rigorously validated for pluripotency (expression of various pluripotency markers and the ability to form teratomas encompassing the endoderm, mesodermal, and ectoderm in immunodeficient mice) and regularly tested for mycoplasma. Fresh Essential 8 medium was replaced daily, and cell growth was monitored. The normal morphology of HSCs is shown in Figure 2.

[0064] 2. Inducing human pluripotent stem cells to differentiate into mDA neural progenitor cells:

[0065] (1) On day 0, when human pluripotent stem cells grow to a confluence of 70-80%, confirm that the cells are in good condition and digest them with Accutase. After counting, the cells are plated on Vitronectin or Laminin coated well plates at a cell density of 400,000 cells / cm 2 Add fresh mDA neural progenitor cell induction medium.

[0066] (2) From day 1 to day 3, replace the mDA neural progenitor cell induction medium with fresh one every day.

[0067] (3) On the 4th day, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added, but without the cell mechanotransduction inhibitor Y27632, and the concentration of CHIR99021 in the culture medium was increased.

[0068] (4) On the 6th day, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added. The composition of the culture medium was the same as that used on the 4th day.

[0069] (5) On day 7, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added, but excluding SB, LDN and SAG.

[0070] (6) On day 9, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added. The composition of the culture medium was the same as that used on day 7.

[0071] 3. Induce mDA neural progenitor cells to differentiate into mDA neural precursor cells:

[0072] (1) On day 10, aspirate the culture medium added the day before and add fresh mDA neural progenitor cell induction medium.

[0073] (2) On day 11, cells were digested with Accutase for 30-40 minutes and seeded on a plate coated with 15 μg / ml poly-L-ornithine solution (PO) + 1 μg / ml Laminin + 2 μg / ml fibronectin at a seeding density of 800,000 cells / cm 2 , cultured using the same medium as on day 10.

[0074] (3) On day 12, replace the culture medium with fresh mDA neural progenitor cell induction medium, but without CHIR99021. In addition, add 10 μM DAPT to the culture medium.

[0075] (4) From day 13 to day 14, replace the neural progenitor cell induction medium with fresh one every day.

[0076] (5) Collection and cryopreservation of mDA neural progenitor cells: On day 15, cells were digested with Accutase for 20-40 minutes and then filtered through a 40 mm pore cell strainer to obtain single cells. Cells were counted, centrifuged, and resuspended in cryopreservation buffer at a density of 8 million cells / ml. The cells were then aliquoted into cryopreservation tubes. Finally, the collected cells were stored in a cryopreservation facility.

[0077] Example 2:

[0078] In this example, the mDA neural progenitor cells obtained in Example 1 were identified by immunofluorescence staining.

[0079] Using cell immunofluorescence staining, we examined the cell phenotype and confirmed that the cells obtained exhibited LMX1A+ / FOXA2+ / EN1+ characteristics. LMX1A, FOXA2, and EN1 are all unique markers of mDA neural progenitor cells. Figure 3 shows the immunofluorescence staining results of the obtained cells. As can be seen from the figure, the cells obtained in Example 1 are indeed mDA neural progenitor cells and are of high purity, with the expression percentages of the characteristic proteins LMX1A, FOXA2, and EN1 exceeding 90%. The specific immunofluorescence staining steps are as follows:

[0080] 1) Cell fixation steps:

[0081] First, remove the 12-well plate after incubation and remove the culture medium. Next, slowly add 1× PBS (pH 7.4, 1 mL / well) along the edge of the 12-well plate to wash twice. Then, slowly add 4% paraformaldehyde (PFA, 1 mL / well) along the edge of the plate and let it sit at room temperature for 15 minutes to fix the cells. Then, gently aspirate the PFA and wash three times with 1× PBS, adding 1 mL / well each time.

[0082] 2) Incubate with primary antibody:

[0083] First, 0.5% Triton X-100 (0.5 mL / well) was added to each well and incubated at 37°C for 30 minutes. Next, the Triton X-100 was removed, and 2% BSA (0.5 mL / well) was added for blocking, followed by an additional 30 minutes of incubation at 37°C. After removing the 2% BSA blocking buffer, the corresponding primary antibody was added directly to the blocking buffer and incubated overnight at 4°C. Subsequently, the primary antibody was removed, and the wells were washed three times with 1× PBS, adding 1 mL / well each time. The primary antibodies used and their associated information are as follows: Goat anti-FOXA2, from R&D, catalog #AF2400; Rabbit anti-LMX1A, from Millipore, catalog #ab10533; Mouse anti-EN1, from DSHB, catalog #4G11.

[0084] 3) Perform secondary antibody incubation:

[0085] First, add a secondary antibody diluted 1:200 in 1% BSA to each well and incubate at room temperature for 1 hour in the dark. After incubation, remove the secondary antibody and wash three times with 1× PBS, adding 1 mL / well for 5 minutes each time. Next, add 0.5 mL / well of DAPI, adjusted to a final concentration of 1 μg / mL in 1× PBS, for staining for 3 minutes. After staining, remove the DAPI and wash two times with 1× PBS, adding 1 mL / well each time. Finally, add 0.5 mL / well of 1× PBS to each well for observation and photography under a microscope. The secondary antibodies used and their related information are as follows: Alexa Fluor 555 Donkey Anti-rabbit IgG (H+L) Antibody, from Thermo Fisher, product number #A-31572; Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody, from Thermo Fisher, product number #A-21447; Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody, from Thermo Fisher, product number #R37114.

[0086] Example 3:

[0087] mDA neural progenitor cells from other sources can also be efficiently differentiated into mDA neural progenitor cells using the method provided in step S2 of the present invention. In this embodiment, mDA neural progenitor cells are obtained in two steps: first, pluripotent stem cells are differentiated into mDA neural progenitor cells using a common method, and the cell mechanotransduction signaling pathway is not regulated during the differentiation process; second, the method provided in step S2 of the present invention is used, that is, a small molecule drug is used to activate the cell mechanotransduction signaling pathway, thereby efficiently differentiating the mDA neural progenitor cells into mDA neural progenitor cells.

[0088] The specific steps are as follows:

[0089] In the first step, human pluripotent stem cells were cultured for 10 days in standard mDA neural progenitor induction medium, without modulating cellular mechanotransduction signaling pathways, to differentiate into midbrain neural floor plate cells, or mDA neural progenitors. This mDA neural progenitor induction medium consists primarily of: Neurobasal Medium (NB), 1× N2 Supplement, 1× B27 Supplement without vitamin A, 2 mM L-glutamine, 10 µM SB431542, 250 nM LDN193189, CHIR99021 (0.7 µM on D0-D3 and 7.5 µM on D4-D9), 1 µM SAG, and 10 µM Y27632. Y27632 was added on D0 only to enhance cell survival and was removed on D1. During D0-D10, the other components of the mDA neural progenitor induction medium were adjusted as shown in Figure 1. The mDA neural progenitor cell induction medium was supplemented with: TGF-β signaling pathway inhibitor SB431542, BMP signaling pathway inhibitor LDN193189, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, SHH pathway activator SAG, and ROCK inhibitor Y27632.

[0090] The specific steps are as follows:

[0091] On day 0, when human pluripotent stem cells have grown to a confluence of 70-80%, confirm that the cells are in good condition and digest them with Accutase. After counting, the cells are plated on Vitronectin- or Laminin-coated plates at a cell density of 400,000 cells / cm 2 Add fresh mDA neural progenitor cell induction medium.

[0092] From day 1 to day 3, replace the culture medium with fresh mDA neural progenitor cell induction medium every day.

[0093] On day 4, aspirate the previous culture medium and add fresh mDA neural progenitor cell induction medium. Increase the concentration of CHIR99021 in the culture medium.

[0094] On day 6, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added. The composition of the medium was the same as that used on day 4.

[0095] On day 7, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added, excluding SB, LDN and SAG.

[0096] On day 9, the previous culture medium was aspirated and fresh mDA neural progenitor cell induction medium was added. The composition of the medium was the same as that used on day 7.

[0097] In the second step, the mDA neural progenitor cells were cultured for 5 days in mDA neural progenitor cell induction medium. A small molecule drug was then added to activate the cell's mechanotransduction signaling pathway, further inducing efficient differentiation of the mDA neural progenitor cells into mDA neural progenitor cells. The mDA neural progenitor cell induction medium consists primarily of the following ingredients: NB, vitamin A-free B27 supplement, 2mM L-glutamine, 20ng / ml BDNF, 20ng / ml GDNF, 0.2mM ascorbic acid, 1ng / ml TGF-β3, 0.5mM cAMP, 10µM DAPT, and 3µM CHIR99021. The specific composition of the mDA neural progenitor cell induction medium should be adjusted at different time stages as shown in Figure 1. mDA neural progenitor cell induction medium is supplemented with: neurotrophic factors (BDNF and GDNF), induction factors ascorbic acid, cAMP, DAPT, TGF-β3, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, and cell mechanotransduction pathway activator PY-60.

[0098] The specific steps are as follows:

[0099] 7) On day 10, aspirate the culture medium added the day before and add fresh mDA neural progenitor cell induction medium (supplemented with the cell mechanotransduction signaling pathway activator PY-60).

[0100] 8) On day 11, cells were digested with Accutase for 30-40 minutes and seeded on a 15µg / ml poly-L-ornithine solution (PO) + 1µg / ml laminin + 2µg / ml fibronectin-coated plate at a seeding density of 800,000 cells / cm 2 , cultured using the same medium as on day 10.

[0101] 9) On Day 12, replace the culture medium with fresh mDA neural progenitor cell induction medium, but without CHIR99021. In addition, add 10µM DAPT to the medium.

[0102] 10) From Day 13 to Day 14, replace the neural progenitor cell induction medium with fresh one every day.

[0103] 11) Harvest and cryopreserve mDA neural progenitor cells: On day 15, digest the cells with Accutase for 20-40 minutes and filter through a 40µm pore filter to isolate single cells. Count the cells, centrifuge, and resuspend in cryopreservation buffer at a density of 8 million cells / mL. Aliquot the cells into cryovials. Store the harvested cells in a cryogenic storage facility.

[0104] Example 4:

[0105] In this example, the mDA neural progenitor cells obtained in Example 1 were characterized by flow cytometry and yield, and quantitatively compared with a control group. Cell phenotypes were assessed using flow cytometry, following standard flow cytometry procedures. Figure 4 shows the flow cytometric results of the obtained cells. As shown, the cells obtained in Example 1 were indeed mDA neural progenitor cells and had the highest purity, with a 98.72% percentage of cells double-positive for the characteristic proteins LMX1A and FOXA2, and a 97.31% percentage of cells double-positive for the characteristic proteins FOXA2 and EN1. The mDA neural progenitor cells obtained in Example 3 also had a relatively high purity, with a 93.5% percentage of cells double-positive for the characteristic proteins LMX1A and FOXA2, and a 92.8% percentage of cells double-positive for the characteristic proteins FOXA2 and EN1. Flow cytometric results for the control group showed a 91.2% percentage of cells double-positive for the characteristic proteins LMX1A and FOXA2, and a 90.81% percentage of cells double-positive for the characteristic proteins FOXA2 and EN1. The antibodies used are as follows: Rabbit anti-LMX1A from Millipore, catalog number #ab10533; PE Mouse anti-Human FoxA2 from BD, catalog number #561589; Rabbit anti-EN1 from Invitrogen, catalog number #PA5-14149; and Goat anti-Rabbit IgG H&L (APC) Pre-adsorbed from Abcam, catalog number #ab130805.

[0106] In the control group, mechanotransduction signaling pathways were not manipulated during the induction and differentiation of midbrain dopaminergic neural progenitor cells. The steps were as follows: First, human pluripotent stem cells were cultured in mDA neural progenitor cell induction medium for 10 days to differentiate into midbrain neural floor plate cells, or mDA neural progenitor cells. This mDA neural progenitor induction medium consists of the following components: Neurobasal Medium (NB), 1× N2 Supplement, 1× B27 Supplement without Vitamin A, 2mM L-glutamine, 10µM SB431542, 250nM LDN193189, CHIR99021 (0.7µM on Days 0-3; 7.5µM on Days 4-9), and 1µM SAG. The specific components of the mDA neural progenitor induction medium should be adjusted at different time points as shown in Figure 1. The mDA neural progenitor cell induction medium contains the following: TGF-β signaling pathway inhibitor SB431542, BMP signaling pathway inhibitor LDN193189, GSK-3β inhibitor and WNT signaling pathway activator CHIR99021, and SHH pathway activator SAG. In the second step, the mDA neural progenitor cells are cultured in the mDA neural progenitor cell induction medium for 5 days to promote their further differentiation into mDA neural progenitor cells. The mDA neural progenitor cell induction medium is primarily composed of NB, vitamin A-free B27 supplement, 2mM L-glutamine, 20ng / ml BDNF, 20ng / ml GDNF, 0.2mM ascorbic acid, 1ng / ml TGF-β3, 0.5mM cAMP, 10µM DAPT, and 3µM CHIR99021. The specific composition of the mDA neural progenitor cell induction medium should be adjusted according to Figure 1 at different time stages. The mDA neural progenitor cell induction medium is supplemented with: neurotrophic factors (BDNF and GDNF), induction factors ascorbic acid, cAMP, DAPT, TGF-β3, and GSK-3β inhibitor, namely WNT signaling pathway activator CHIR99021. Specific operation steps refer to Example 1.

[0107] The specific flow cytometry steps are as follows:

[0108] 1) Cell fixation steps:

[0109] First, rinse dopamine neural progenitor cells cultured in a 12-well plate with 1x DPBS (0.5 mL / well). Next, add 0.5-1 mL of Accutase digestion solution and digest at 37°C for 3-5 minutes until the cells detach. Then, dilute the digestion solution 2-4 times with DPBS and centrifuge at 200 × g. The digestion solution and DPBS are then removed. Finally, the cell pellet is fixed with 1 mL of cold 90% methanol.

[0110] 2) Perform primary antibody incubation:

[0111] First, centrifuge the cell sample at 200 × g for 2 minutes at room temperature and remove the supernatant. Then, add 5 mL of PBS to the cell sample and gently shake to mix. Centrifuge again at 200 × g for 3 minutes, remove the supernatant, add FACS buffer, and gently shake to evenly disperse the cells. Adjust the cell concentration to 2 × 10 7 100 μL / mL. Next, add 50 μL of sample cells to each flow cytometer tube and 50 μL of the corresponding primary antibody. Gently shake five times to mix, then incubate at room temperature in the dark for 30 minutes, gently shaking every 10 minutes. After incubation, add 2 mL of FACS buffer to each tube and mix thoroughly using a vortex mixer.

[0112] 3) Perform secondary antibody incubation:

[0113] Centrifuge at 200 × g for 2 minutes at room temperature. Remove the supernatant and add 50 μL of the corresponding secondary antibody to each tube. Gently shake five times to mix, then incubate at room temperature for 15 minutes in the dark. After incubation, add 300 μL of FACS buffer to each tube and mix thoroughly using a vortex mixer. Finally, use the corresponding channel of a flow cytometer to determine the positive expression rate of each marker.

[0114] Next, combining the results of cell counting with a cell counter and flow cytometry, the target cell yields of Example 1, Example 3, and the control group were calculated and compared. The yield is the ratio of the number of target cells (i.e., dopaminergic neural precursor cells) obtained to the number of starting human pluripotent stem cells. The dopaminergic neural precursor cell count was calculated based on the ratio of FOXA2 and EN1 double positivity as determined by flow cytometry. The results are shown in Figure 5. As can be seen, Example 1 had the highest target cell yield, approximately 23-fold; Example 3 had the second highest yield, approximately 18-fold; and the control group had the lowest target cell yield, approximately 15-fold.

[0115] Example 5:

[0116] In this example, the mDA neural progenitor cells obtained in Example 1 were induced to terminally differentiate into mature mDA neurons and their phenotypes were identified.

[0117] mDA neural progenitor cells further differentiate into mature mDA neurons:

[0118] Differentiation of the cells was induced according to the method described in Example 1 until day 15. The cells were then digested with Accutase and processed into a single-cell suspension. Next, the cells were plated onto cell culture plates according to the procedure described in Example 1 on day 11 and cultured until day 25. During this period, fresh neural progenitor cell induction medium was replaced daily.

[0119] Immunofluorescence staining identification:

[0120] Mature mDA neurons should be able to co-express TH and FOXA2. To verify this, these neurons can be identified using immunofluorescence staining technology. The specific immunofluorescence staining steps are the same as those described in Example 2. Figure 6 shows the immunofluorescence staining results of the terminally differentiated cells obtained. As can be seen from the figure, the cells obtained in Example 1 are indeed mDA neural precursor cells, and they can continue to terminally differentiate into mature dopaminergic neurons. After terminal differentiation, the expression percentages of TH and FOXA2, the characteristic proteins of mature dopaminergic neurons, are both above 90%. The primary antibodies used include: Rabbit anti-TH (from PelFreez, product number #P40101-150) and Goat anti-FOXA2 (from R&D, product number #AF2400).

[0121] In summary, our results demonstrate that cells differentiated from pluripotent stem cells using the induction medium and methods of the present invention do express mDA neural progenitor cell-specific proteins and have the potential to further differentiate into mature mDA neurons. This further confirms that the cells obtained in Example 1 are indeed highly pure mDA neural progenitor cells.

[0122] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the essence and principles of the present invention should be considered as equivalent replacement methods and included in the scope of protection of the present invention.

[0123] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates. Industrial Applicability

[0124] It is easy for those skilled in the art to know from the above description that the technical solution of the present invention is suitable for industrial manufacturing and use in production and life, so the present invention has industrial applicability.

Claims

1. A method for inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells, characterized in that: The following steps are included: Step S1, spreading a neural progenitor cell culture medium containing pluripotent stem cells on a cell culture plate, and performing directed differentiation culture to obtain dopaminergic neural progenitor cells; the neural progenitor cell culture medium contains a cell mechanical transduction pathway inhibitor; Step S2, spreading the neural precursor cell culture fluid containing dopaminergic neural progenitor cells on a cell culture plate, and performing directed differentiation culture to obtain dopaminergic neural precursor cells; the neural precursor cell culture fluid contains a cell mechanical conduction pathway activator.

2. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 1, characterized in that: The neural progenitor cell culture fluid comprises: neural cell basal culture medium, N2 supplement, B27 supplement without vitamin A, L-glutamine, TGF-β signaling pathway inhibitor, BMP signaling pathway inhibitor, GSK-3β inhibitor, i.e. WNT signaling pathway activator, sonic hedgehog pathway activator, cell mechanical conduction pathway inhibitor; wherein, the concentration of L-glutamine is 1-5mM; the concentration of the TGF-β signaling pathway inhibitor is 1-15μM; the concentration of the BMP signaling pathway inhibitor is 100-500nM; the concentration of the GSK-3β inhibitor, i.e. WNT signaling pathway activator, is 0.5-9µM; and the concentration of the cell mechanical conduction signaling pathway inhibitor is 5-15µM.

3. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 2, characterized in that: The TGF-β inhibitor is SB431542 or RepSox; the BMP signaling pathway inhibitor is LDN193189 or DMH-1; the GSK-3β inhibitor is CHIR99021 or BIO; the cell mechanical transduction signaling pathway inhibitor is Rho kinase inhibitor Y27632 or myosin II inhibitor Blebbistatin.

4. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 2, characterized in that: The concentration of L-glutamine was 2mM; the concentration of the TGF-β inhibitor was 10μM; the concentration of the BMP signaling pathway inhibitor was 250nM; the concentration of the GSK-3β inhibitor was 0.7μM, which was later increased to 7.5µM; and the concentration of the cell mechanical transduction signaling pathway inhibitor was 10µM.

5. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 2, characterized in that: The sonic hedgehog pathway activator is SAG at a concentration of 0.5-2 μM, SHH protein at a concentration of 100-600 ng / mL, or Purmorphamine at a concentration of 1-10 μM.

6. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 5, characterized in that: The sonic hedgehog pathway activator is SAG at a concentration of 1 μM, SHH protein at a concentration of 500 ng / mL, or Purmorphamine at a concentration of 5 μM.

7. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 1, characterized in that: The neural progenitor cell culture medium containing the pluripotent stem cells that have been blown into single cells is spread on the coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 35-39°C and 3-7% CO2 for 5-12 days, wherein the culture medium is replaced every 1-3 days.

8. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 1, characterized in that: The neural progenitor cell culture medium containing the pluripotent stem cells that have been blown into single cells is spread on the coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 37° C. and 5% CO 2 for 10 days, wherein the culture medium is replaced every 2 days.

9. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 1, characterized in that: The neural precursor cell culture fluid comprises: neural cell basal culture medium, B27 supplement, L-glutamine, nutrient factors required for the growth of dopaminergic neural progenitor cells, L-ascorbic acid, TGF-β3, GSK-3β inhibitor, i.e., WNT signaling pathway activator, activator cAMP, DAPT, cell mechanical conduction pathway activator, wherein the concentration of L-glutamine is 1-5 mM; the nutrient factors required for the growth of dopaminergic neural progenitor cells include brain-derived neurotrophic factor and neurotrophic factor derived from glial cell line; the concentration of brain-derived neurotrophic factor is 10-50 ng / mL, and the concentration of neurotrophic factor derived from glial cell line is 10-50 ng / mL; the concentration of L-ascorbic acid is 0.1-1 mM; the concentration of TGF-β3 is 0.5-5 ng / mL; the concentration of GSK-3β inhibitor, i.e., WNT signaling pathway activator, is 2-4 µM CHIR99021; the concentration of the activator cAMP is 0.2-2mM; the concentration of the DAPT is 1-15µM; the concentration of the cell mechanical transduction pathway activator is 1-20µM.

10. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 9, characterized in that: The GSK-3β inhibitor, i.e., WNT signaling pathway activator, is CHIR99021 or BIO; the cell mechanical transduction pathway activator is Hippo-YAP / Taz signaling pathway activator PY-60 or RhoA activator lysophosphatidic acid.

11. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 9, characterized in that: The concentration of L-glutamine is 2mM; the concentration of brain-derived neurotrophic factor is 20ng / mL; the concentration of glial cell line-derived neurotrophic factor is 20ng / mL; the concentration of L-ascorbic acid is 0.2mM; the concentration of TGF-β3 is 1ng / mL; the concentration of GSK-3β inhibitor, i.e. WNT signaling pathway activator, is 3µM; the concentration of activator cAMP is 0.5mM; the concentration of DAPT is 10µM; and the concentration of cell mechanical transduction pathway activator is 10µM.

12. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 9, characterized in that: The neural progenitor cell culture medium containing dopaminergic neural progenitor cells that have been blown into single cells is spread on the coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 35-39°C and 3-7% CO2 for 3-8 days, wherein the culture medium is replaced every 1-3 days.

13. The method of inducing pluripotent stem cells to differentiate into midbrain dopaminergic neural precursor cells according to claim 9, characterized in that: The neural progenitor cell culture medium containing dopaminergic neural progenitor cells that have been blown into single cells is spread on the coated cell culture plate, and the dopaminergic neural progenitor cells are obtained by directed differentiation culture at 37° C. and 5% CO 2 for 5 days, wherein the culture medium is replaced once a day.

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