A method for inducing neural stem cells to differentiate into dopaminergic neurons

Abstract

The application discloses an induction method for directional differentiation of neural stem cells into dopaminergic neurons, comprising the following steps: S1, activation and adhesion of NSCs; S2, directional induction of midbrain precursor cells; S3, directional induction of dopaminergic neurons; and S4, synaptic function maturation induction. The induction method for directional differentiation of neural stem cells into dopaminergic neurons can significantly improve the directional efficiency and lineage specificity of the midbrain precursor cells: by adding salvianolic acid B and icariin, and in cooperation with SHH, FGF8 and other induction factors, the midbrain lineage-specific signal pathway can be efficiently activated, the positive rate of the midbrain precursor cell marker LMX1A is increased to more than 76%, which is far better than 49% of the existing system, and abnormal differentiation of non-midbrain lineages is effectively inhibited.

Description

Technical Field

[0001] This invention relates to the field of neuron induction technology, specifically to a method for inducing neural stem cells to differentiate into dopaminergic neurons. Background Technology

[0002] Parkinson's disease is a common neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, leading to insufficient dopamine secretion and subsequently causing a series of clinical symptoms such as motor dysfunction. Cell replacement therapy is considered one of the most promising strategies for treating Parkinson's disease. Among these strategies, the directed differentiation of neural stem cells (NSCs) into functionally mature dopaminergic neurons is a key technical pathway for obtaining the seed cells needed for transplantation.

[0003] Existing technologies mostly rely on exogenous recombinant protein factors such as SHH and FGF8 to regulate differentiation, but the induction system has significant defects: low midbrain progenitor cell orientation efficiency, poor lineage specificity, low cell survival rate, high apoptosis rate, insufficient subsequent dopaminergic neuron shaping efficiency and functional maturity, poor process tolerance and insufficient stability, making it difficult to meet the needs of clinical applications. Summary of the Invention

[0004] In order to overcome the deficiencies in the prior art, this invention provides a method for inducing neural stem cells to differentiate into dopaminergic neurons with high process tolerance, good stability, and high directed differentiation rate.

[0005] This invention first provides a method for inducing the directed differentiation of neural stem cells into dopaminergic neurons, comprising the following steps: S1NSCs Activation and Adhesion After thawing and washing the frozen NSCs, the cell density was adjusted. The culture plate was coated with PLL (10 μg / mL) at 4°C overnight. The next day, it was washed with sterile PBS, dried, and then incubated with laminin (5 μg / mL) at 37°C. The NSCs with adjusted density were seeded into the coated culture plate and cultured. After 24 hours, the basal medium was replaced to ensure good cell adhesion. S2 brain progenitor cell directed induction The basal culture medium was supplemented with SHH (300-350 ng / mL), FGF8 (100-150 ng / mL), and CHIR99021 (5-7 μM), along with a combination of natural substances: salvianolic acid B (5-20 μM) and icariin (20-100 ng / mL). Salvianolic acid B was dissolved in sterile physiological saline containing 5% hydroxypropyl-β-cyclodextrin (HP-β-CD), and icariin was dissolved using sterile PBS buffer (pH 7.4) with ultrasonication. Both stock solutions were sterilized by filtration through a 0.22 μm filter membrane. The culture medium was changed every 2 days, and the culture was carried out for 5 days. S3 dopaminergic neuron stereotyping induction Replace the basal medium with BDNF (30-35 ng / mL), GDNF (20-25 ng / mL), and SC144 (3-5 μM); change the medium every 2 days and incubate for 7 days. S4 synaptic maturation induction Replace the basal culture medium with NT-3 (15-20 ng / mL) and enkephalin (0.5-10 μM), while retaining BDNF and GDNF at half the concentration; change the culture medium every 3 days, and culture for 14 days to complete the induction culture of dopaminergic neurons.

[0006] This invention also provides the following optimization scheme: Preferably, in step S4, homologous astrocytes are added for Transwell co-culture.

[0007] Preferably, in step S1, the cell density is adjusted to 1×10⁻⁶. 5 cells / mL.

[0008] Preferably, laminin (5 μg / mL) is added in step S1 and incubated at 37°C for 2-3 hours.

[0009] Preferably, in step S1, the culture plate is placed in an incubator at 37°C and 5% CO2 for incubation.

[0010] Preferably, in step S2, the ultrasonic power for ultrasonic-assisted dissolution is 100-200W, and the time is 5-8 minutes.

[0011] Preferably, the final concentration of HP-β-CD in step S2 is ≤0.5%.

[0012] Preferably, in step S2, SHH is 300-350 ng / mL, FGF8 is 100-150 ng / mL, and CHIR99021 is 5 μM.

[0013] Preferably, in step S3, BDNF is 30 ng / mL, GDNF is 20 ng / mL, and SC144 is 3 μM.

[0014] Preferably, in step S4, NT-3 is 15 ng / mL and enkephalin is 0.5 μM.

[0015] The beneficial effects of this invention are: The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons in this invention significantly improves the directing efficiency and lineage specificity of midbrain progenitor cells: by adding salvianolic acid B and icariin, which work synergistically with inducing factors such as SHH and FGF8, the lineage-specific signaling pathways of the midbrain can be efficiently activated, increasing the positive rate of the midbrain progenitor cell marker LMX1A to over 76%, which is far superior to the 49% of the existing system, while effectively inhibiting abnormal differentiation of non-midbrain lineages.

[0016] The method for inducing neural stem cells to differentiate into dopaminergic neurons in this invention can improve cell survival rate and reduce the risk of apoptosis during the induction process: Tanshinone B can scavenge reactive oxygen species generated during the induction process, and Icariin can synergistically promote cell proliferation and survival. The two work together to stabilize the cell survival rate at over 91%, which solves the defect of the prior art where the cell survival rate is less than 85%, reduces the loss of precursor cells, and ensures the overall induction efficiency.

[0017] The natural substances used in the method for inducing the directed differentiation of neural stem cells into dopaminergic neurons of this invention are safe and controllable, reducing the risks of clinical application: Danshensu B and icariin have a purity of ≥98%, good biocompatibility, and the induction effect is enhanced under cell protection by precisely controlling the concentration, while not affecting the core pathway. This avoids the cytotoxicity risks caused by excessive use of chemically synthesized reagents and improves the safety of cell therapy. Attached Figure Description

[0018] Figure 1 This is a Synapsin-1 detection diagram from Embodiment 1 of the present invention; Figure 2 This is a Synapsin-1 detection graph for the control group of this invention; Figure 3 This is a Homer 1 detection diagram from Embodiment 1 of the present invention; Figure 4 This is a Homer 1 detection graph for the control group of the present invention. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.

[0020] This invention first provides a method for inducing the directed differentiation of neural stem cells into dopaminergic neurons, specifically including the following steps: S1NSCs Activation and Adhesion Neural stem cells (NSCs) frozen in liquid nitrogen were removed and rapidly thawed in a 37°C water bath for 1-2 minutes, with gentle shaking of the cryovials to ensure even heating. After thaw, 3 volumes of basal culture medium containing Neurobasal-A medium, 2% B27 serum-free supplement, and 1% GlutaMAX + 100 U / mL penicillin-streptomycin were added for serial dilution. The cells were then centrifuged at 800 rpm at room temperature for 5 minutes, the supernatant was discarded, and the cells were washed twice. After washing, the cells were resuspended in basal culture medium, counted using a cell counting chamber, and the cell density was adjusted to 1 × 10⁶ cells / mL. 5 Cells / mL; Simultaneously, culture plate coating treatment was performed: 6-well culture plates were selected, and 1 mL of 10 μg / mL poly-L-lysine (PLL) solution was added to each well. The plates were placed in a 4°C refrigerator for overnight coating (≥12 hours). The next day, the culture plates were removed and washed 3 times per well with sterile PBS buffer, allowing them to stand for 1 minute after each wash. The washing solution was discarded, and the plates were placed in a clean bench to air dry for approximately 30 minutes. Then, 1 mL of 5 μg / mL laminin solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 2 hours. The adjusted NSCs suspension was seeded into the coated culture plates at a rate of 2 mL per well and statically cultured at 37°C, 5% CO2, and 95% humidity for 24 hours. After culturing, the old culture medium was completely discarded, and 2 mL of fresh basal culture medium was replaced in each well. Cell adhesion was observed under a microscope to ensure a cell adhesion rate ≥80%.

[0021] PLL, as a cationic polymer, can be adsorbed onto the surface of the culture plate via electrostatic interactions, thereby adsorbing subsequent laminin (a component of the extracellular matrix). Laminin can bind to integrin receptors on the surface of NSCs, activating intracellular adhesion signaling pathways and promoting cell adhesion and spreading; 1×10 5 A cell density of 1 / mL ensures adequate cell contact, preventing excessive density from causing nutrient competition or insufficient density from affecting cell survival. A 37°C, 5% CO2 environment maintains normal cell metabolic activity, and 24-hour medium changes remove metabolic waste secreted by cells after resuscitation, further enhancing cell viability. Double coating with PLL and laminin increases cell adhesion by ≥20% compared to single coating and maintains the undifferentiated state of NSCs.

[0022] S2 brain progenitor cell directed induction Using the basal culture medium as the stock solution, various inducing factors and natural substances were added to prepare the induction medium: the concentration of SHH was controlled at 300-350 ng / mL, the concentration of FGF8 at 100-150 ng / mL, and the concentration of CHIR99021 at 5-7 μM. Simultaneously, a combination of natural substances was added: 5-20 μM of salvianolic acid B and 20-100 ng / mL of icariin. The natural substances were pretreated: salvianolic acid B powder was dissolved in sterile physiological saline containing 5% hydroxypropyl-β-cyclodextrin (HP-β-CD) to prepare a 10 mM stock solution. During dissolution, the solution was vortexed for 10 minutes to ensure complete dissolution, and the entire process was performed in the dark. Icariin powder was dissolved in sterile PBS buffer (pH 7.4) to prepare a 100 μg / mL stock solution. Dissolution was aided by sonication at 100W power for 5 minutes, with cooling in an ice-water bath during sonication to prevent overheating. All stock solutions were sterilized by filtration through a 0.22 μm aqueous filter membrane and stored at low temperature. When using, they were diluted to the target concentration according to the ratio. The old culture medium of well-adhered NSCs in S1 was discarded, and 2 mL of freshly prepared induction medium was added to each well. The cells were then incubated in an incubator at 37°C, 5% CO2, and 95% humidity. The induction medium was changed every 2 days during the incubation period. Before each change, the cells were gently washed once with sterile PBS buffer. After each change, the cells were incubated for a total of 5 days.

[0023] Sonic Hedgehog (SHH) can activate the downstream Gli signaling pathway, inducing NSCs to differentiate into midbrain basal plate progenitor cell lineages; FGF8 can synergistically regulate the expression of midbrain-specific genes (such as LMX1A and FOXA2) with SHH, further clarifying midbrain lineage orientation; CHIR99021, as a Wnt / β-catenin pathway activator, can stabilize intracellular β-catenin, promote its nuclear binding to target genes, and enhance the proliferation and differentiation of midbrain progenitor cells; Tanshinone B can enhance its inductive activity by activating the SHH pathway, while scavenging intracellular reactive oxygen species (ROS) and reducing apoptosis during differentiation; Icariin can synergistically activate the MAPK / ERK pathway with FGF8, promoting the proliferation of midbrain progenitor cells; HP-β-CD encapsulates Tanshinone B through a hydrophobic cavity structure, improving its water solubility and avoiding precipitation; Ultrasonic dissolution enhances dissolution efficiency by mechanically vibrating and disrupting the intermolecular forces of icariin. This invention employs SHH+FGF8+CHIR99021 synergistic induction, which improves the positive rate of midbrain progenitor cells compared to single-factor induction; the introduction of natural substance combinations not only enhances induction efficiency but also avoids the toxicity of chemical solubilizers, thereby improving cell survival rate.

[0024] S3 dopaminergic neuron stereotyping induction Discard the old induction medium used in the final stage of S2, gently wash the cells once with sterile PBS buffer, and then replace it with freshly prepared induction medium: using the basal medium as the stock solution, add BDNF at a concentration of 30-35 ng / mL, GDNF at a concentration of 20-25 ng / mL, and SC144 at a concentration of 3-5 μM. After adding each factor, gently vortex for 30 seconds to ensure uniform mixing. Use the induction medium immediately after preparation, adding 2 mL to each well and incubating in an incubator at 37℃, 5% CO2, and 95% humidity. Change the induction medium every 2 days during the culture period. Each time, discard the old medium and add fresh induction medium directly. The total culture time should be controlled at 7 days.

[0025] SC144, as an Ascl1 inducer, promotes the expression of the basic helix-loop-helix (bHLH) transcription factor Ascl1. Ascl1 can directly bind to the promoters of dopaminergic neuron-specific genes (such as TH and DAT), driving their expression and guiding midbrain progenitor cells to differentiate into dopaminergic neurons. BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor) can bind to TrkB and Ret receptors on the surface of neurons, respectively, activating the PI3K / Akt and MAPK pathways, inhibiting apoptosis, and promoting the initial maturation and neurite growth of neurons. This invention specifically adds the Ascl1 inducer SC144, which improves the dopaminergic neuron differentiation rate compared to traditional induction regimens without SC144. BDNF and GDNF work synergistically to significantly reduce the apoptosis rate during differentiation.

[0026] S4 synaptic maturation induction Discard the old induction medium from the final S3 stage, gently wash the cells once with sterile PBS buffer, and replace with freshly prepared maturation medium: using the basal medium as the stock solution, add NT-3 at a concentration of 15-20 ng / mL and enkephalin at a concentration of 0.5-1.0 μM, while retaining BDNF and GDNF at half the concentrations of the S3 stage, i.e., BDNF 15-17.5 ng / mL and GDNF 10-12.5 ng / mL. After thoroughly mixing all factors, add 2 mL to each well; preferably, use the Transwell co-culture process for homologous astrocytes: select a 0.4 μm pore size Transwell chamber, and culture homologous astrocytes at a rate of 5 × 10⁶ cells / well. 4Cells / well were seeded at a density in the upper chamber of the Transwell and cultured for 24 hours to allow them to adhere. Then, the Transwell chamber was placed in a 6-well culture plate seeded with induced cells. The lower chamber contained mature culture medium and induced cells for co-culture. During the culture period, the mature culture medium was changed every 3 days. When changing the culture medium, the upper chamber of the Transwell was removed first, the old culture medium in the lower chamber was discarded, fresh mature culture medium was added, and then the upper chamber was put back. The total culture time was strictly controlled to 14 days. After the culture was completed, the induction of dopaminergic neurons was completed.

[0027] NT-3 (neurotrophin-3) can bind to TrkC receptors on the surface of neurons, promoting the formation of neuronal branching and presynaptic vesicles; enkephalin, as an endorphin-like neurotransmitter, can regulate the release of neurotransmitters from the presynaptic membrane, promote the expression and maturation of postsynaptic membrane receptors, and thus enhance synaptic transmission function; halving the concentrations of BDNF and GDNF can avoid excessive neuronal proliferation caused by high concentrations of trophic factors, focusing on the regulation of synaptic maturation; homologous astrocytes can secrete neurotrophic factors (such as NGF) and extracellular matrix components (such as chondroitin sulfate proteoglycan), which diffuse into the lower chamber through the 0.4 μm pore size Transwell membrane, simulating the in vivo neural microenvironment, promoting the functional integration of dopaminergic neurons with surrounding cells, and improving synaptic maturation; this invention specifically adds synaptic maturation-related factors, improving synaptic maturation compared to schemes without NT-3 and enkephalin; Transwell co-culture can simulate the in vivo microenvironment, improving synaptic integration efficiency compared to single neuron culture, and avoiding contamination caused by direct cell contact.

[0028] The above is a detailed description of the present invention. The following are embodiments of the present invention.

[0029] Example 1 1. Activation and Adhesion of S1 NSCs: Remove the cryovials of NSCs stored in liquid nitrogen and place them in a 37°C water bath within 10 seconds. Gently agitate for 1.5 minutes to complete thawing. Add 3 volumes of basal culture medium, followed by a serial dilution of Neurobasal-A + 2% B27 + 1% GlutaMAX + 100 U / mL penicillin-streptomycin. Centrifuge at 800 rpm for 5 minutes at room temperature, discard the supernatant, and wash twice. Resuspend the cells in basal culture medium, count them, and adjust the density to 1×10⁻⁶ cells / mL. 5 Cells / mL; Take a 6-well culture plate, add 1 mL of 10 μg / mL LPL L1 solution to each well, coat at 4℃ for 12 hours, wash 3 times with sterile PBS the next day, air dry for 30 minutes, add 1 mL of 5 μg / mL laminin solution to each well, incubate at 37℃ and 5% CO2 for 2 hours; Inoculate 2 mL of NSCs suspension with adjusted density in each well, place in an incubator for static culture for 24 hours, completely discard the old culture medium, replace with 2 mL of fresh basal culture medium, and observe under a microscope that the cell adhesion rate reaches 85%.

[0030] 2. Directional induction of brain progenitor cells in S2: Preparation of induction medium 1: Using basal medium as the stock solution, add SHH (final concentration 320 ng / mL), FGF8 (final concentration 120 ng / mL), and CHIR99021 (final concentration 6 μM), along with a combination of natural substances (tanshinone B (final concentration 10 μM) and icariin (final concentration 50 ng / mL); Pretreatment of natural substances: Tanshinone B dry powder was prepared into a 10 mM stock solution using sterile physiological saline containing 5% HP-β-CD, and vortexed for 10 minutes. Dissolve the icariin powder in the solution while avoiding light throughout the process. Prepare a 100 μg / mL stock solution using sterile PBS buffer (pH 7.4). Sonicate at 100W for 5 minutes (cool in an ice-water bath). Both stock solutions are sterilized by filtration through a 0.22 μm filter membrane and stored at 4℃ for 20 hours before use. Discard the old culture medium from stage S1 and add 2 mL of induction medium 1 to each well. Incubate in an incubator and replace the induction medium 1 on days 3 and 5 (wash gently once with sterile PBS before replacement). The total incubation period is 5 days.

[0031] 3. S3 dopaminergic neuron typesetting induction: Discard the old culture medium from the S2 stage, wash once with sterile PBS, and replace 2 mL of induction medium 2 (basal medium + 32 ng / mL BDNF + 22 ng / mL LDDNF + 4 μM MSC144) in each well. Incubate in an incubator and replace induction medium 2 on days 2, 4, and 6 of typesetting induction, for a total of 7 days of culture.

[0032] 4. S4 Synaptic Functional Maturation Induction: Discard the old culture medium from the S3 stage, wash once with sterile PBS, replace 2 mL of maturation medium in each well, and add basal medium + 18 ng / mL NT-3 + 0.8 μM enkephalin + 16 ng / mL BDNF + 11 ng / mL LGDNF; take a Transwell chamber and inoculate 5 × 10⁶ cells into the upper chamber. 4 Cells / well homologous astrocytes were cultured for 24 hours and then co-cultured with induced cells in 6-well culture plates. The maturation medium was changed on days 3, 6, 9, and 12 of maturation induction. When changing the medium, the upper chamber of the Transwell was removed first. The total culture time was 14 days to complete the induction.

[0033] Example 2 Only the following process parameters are changed; the remaining steps are the same as in Example 1: 1. S2 induction medium 1: SHH final concentration 300 ng / mL, FGF8 final concentration 100 ng / mL, CHIR99021 final concentration 5 μM, salvianolic acid B final concentration 5 μM, icariin final concentration 20 ng / mL; 2. S3 induction medium 2: BDNF final concentration 30 ng / mL, GDNF final concentration 20 ng / mL, SC144 final concentration 3 μM; 3. S4 maturation medium: NT-3 final concentration 15 ng / mL, enkephalin final concentration 0.5 μM, BDNF 15 ng / mL after halving, GDNF 10 ng / mL after halving.

[0034] Example 3 Only the following process parameters are changed; the remaining steps are the same as in Example 1: 1. S2 induction medium 1: SHH final concentration 350 ng / mL, FGF8 final concentration 150 ng / mL, CHIR99021 final concentration 7 μM, salvianolic acid B final concentration 20 μM, icariin final concentration 100 ng / mL; 2. S3 induction medium 2: BDNF final concentration 35 ng / mL, GDNF final concentration 25 ng / mL, SC144 final concentration 5 μM; 3. S4 maturation medium: NT-3 final concentration 20 ng / mL, enkephalin final concentration 1.0 μM, BDNF 17.5 ng / mL after halving, GDNF 12.5 ng / mL after halving.

[0035] Example 4 Only the following process parameters are changed; the remaining steps are the same as in Example 1: 1. S2 induction medium 1: SHH final concentration 340 ng / mL, FGF8 final concentration 140 ng / mL, CHIR99021 final concentration 6.5 μM, Tanshinone B final concentration 18 μM, Icariin final concentration 80 ng / mL; 2. S3 induction medium 2: BDNF final concentration 31 ng / mL, GDNF final concentration 21 ng / mL, SC144 final concentration 3.5 μM; 3. S4 maturation medium: NT-3 final concentration 16 ng / mL, enkephalin final concentration 0.6 μM, BDNF 15.5 ng / mL after halving, GDNF 10.5 ng / mL after halving.

[0036] control group Without the addition of salvianolic acid B and icariin, the other induction conditions and culture parameters were completely consistent with those in Example 1.

[0037] Detection experiment The following indicators were measured for Examples 1-4 and the control group: 1. LMX1A positivity rate (S2 stage): Detected using immunofluorescence staining combined with flow cytometry. After the S2 stage culture, cells were collected, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, blocked with 5% BSA, incubated with LMX1A primary antibody overnight at 4°C, incubated with fluorescent secondary antibody at room temperature for 1 hour, and after nucleus staining with DAPI, the percentage of LMX1A-positive cells was counted by flow cytometry. Each group was set up with 3 replicates, and the average value was taken.

[0038] 2. Cell viability (S2 stage): The trypan blue exclusion assay was used. S2 stage cells were collected, resuspended in sterile PBS, and then mixed with 0.4% trypan blue solution at a 1:1 volume ratio. The mixture was incubated at room temperature for 5 min, and the cells were counted under an inverted microscope. The percentage of unstained surviving cells out of the total number of cells was the cell viability. Three replicates were performed for each group, and the average value was taken.

[0039] 3. TH positivity rate (S3 stage): Quantitative analysis was performed using immunofluorescence staining combined with laser confocal microscopy. After the S3 stage culture, cell slides were fixed, permeabilized, and blocked. TH primary antibody was added and incubated overnight at 4°C. Secondary fluorescent antibody was added and incubated at room temperature for 1 hour. After DAPI staining of the nuclei, the slides were mounted. Five fields of view were randomly selected under a laser confocal microscope to count the percentage of TH-positive cells in the total cell count. Three replicates were performed for each group, and the average value was taken.

[0040] 4. Synapsin-1 / Homer 1 staining detection (S4 stage): Immunofluorescence staining combined with laser confocal microscopy imaging was used to detect the cells in Example 1 and the control group. The staining procedure was as follows: After the S4 stage induction was completed, the cells were collected, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, blocked with 5% BSA, and then incubated with Synapsin-1 primary antibody and Homer 1 primary antibody at 4°C overnight. The next day, fluorescently labeled secondary antibody was added and incubated at room temperature for 1 hour. After DAPI staining of the nuclei, the cells were mounted and imaged with fluorescence.

[0041] 5. Increase rate of cell branching (S4 stage): After the S4 stage culture, dopaminergic neurons were labeled by TH immunofluorescence staining, and the complete morphology of the cells was photographed by laser confocal microscopy. The branches of TH-positive cells were counted, and the average number of branches in each group was calculated. The increase rate of each group was calculated based on the average number of branches in the control group. The formula is: (average number of branches in each group - average number of branches in the control group) / average number of branches in the control group × 100%.

[0042] The following test results were obtained after the test.

[0043] In Example 1, the concentrations of 10 μM salvianolic acid B and 50 ng / mL icariin achieved an optimal synergistic effect with the inducing factors. Salvianolic acid B specifically enhanced the activation efficiency of the SHH-Gli pathway while simultaneously scavenging small amounts of reactive oxygen species (ROS) generated during induction, preventing oxidative damage to cells. Furthermore, it did not compete with SHH for receptor-related signaling molecules due to excessive concentration. Icariin at 50 ng / mL synergistically activated the MAPK / ERK pathway with FGF8, promoting the proliferation and survival of midbrain progenitor cells. Its concentration met the synergistic requirements with FGF8 without increasing the cellular metabolic load due to excessive concentration. The optimal concentrations of these two natural substances enhanced the induction effect without affecting the core pathway, laying a high-quality progenitor cell foundation for subsequent differentiation. Example 1, through the matching and synergistic activation of components at each stage, constructed an optimal regulatory network, representing the most effective process. This invention, by adding salvianolic acid B and icariin to the midbrain progenitor cell directed induction step, can stably and efficiently achieve the directed differentiation of neural stem cells into dopaminergic neurons.

[0044] By testing Example 1 and the control group, a detection map of Synapsin-1 and Homer-1 was obtained. Figure 1 This is an image of Synapsin-1 from Example 1. Figure 2 This is an imaging image of Synapsin-1 in the control group. Figure 3 This is an image of Homer 1 in Example 1. Figure 2 The image shows the Homer 1 imaging of the control group. As can be seen from the image, in the imaging of Example 1, the number of colocalization signals of Synapsin-1 and Homer 1 is the largest and the fluorescence intensity is the strongest. Moreover, the colocalization signals are mainly distributed around TH-positive cells, indicating that the functional synaptic connections formed between the transplanted candidate cells and surrounding cells are the richest. In contrast, the detection image of the control group shows only a few scattered colocalization bright spots with weak fluorescence intensity and poor correlation with TH-positive cells.

[0045] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An induction method for directing differentiation of neural stem cells into dopaminergic neurons, characterized by: Includes the following steps: S1NSCs Activation and Adhesion After thawing and washing the frozen NSCs, the cell density was adjusted. The culture plate was coated with PLL (10 μg / mL) at 4°C overnight. The next day, it was washed with sterile PBS, dried, and then incubated with laminin (5 μg / mL) at 37°C. The NSCs with adjusted density were seeded into the coated culture plate and cultured. After 24 hours, the basal medium was replaced to ensure good cell adhesion. S2 brain progenitor cell directed induction The basal culture medium was supplemented with SHH (300-350 ng / mL), FGF8 (100-150 ng / mL), and CHIR99021 (5-7 μM), along with a combination of natural substances: salvianolic acid B (5-20 μM) and icariin (20-100 ng / mL). Salvianolic acid B was dissolved in sterile physiological saline containing 5% hydroxypropyl-β-cyclodextrin (HP-β-CD), and icariin was dissolved using sterile PBS buffer (pH 7.4) with ultrasonication. Both stock solutions were sterilized by filtration through a 0.22 μm filter membrane. The culture medium was changed every 2 days, and the culture was carried out for 5 days. S3 dopaminergic neuron stereotyping induction Replace the basal medium with BDNF (30-35 ng / mL), GDNF (20-25 ng / mL), and SC144 (3-5 μM); change the medium every 2 days and incubate for 7 days. S4 Synaptic Functional Maturation Induction Replace the basal culture medium with NT-3 (15-20 ng / mL) and enkephalin (0.5-10 μM), while retaining BDNF and GDNF at half the concentration; change the culture medium every 3 days, and culture for 14 days to complete the induction culture of dopaminergic neurons.

2. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S4, homologous astrocytes are added for Transwell co-culture.

3. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: The cell density was adjusted to 1 x 10 5 cells / mL in step S1.

4. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S1, add laminin (5 μg / mL) and incubate at 37°C for 2-3 hours.

5. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S1, the culture plate is placed in an incubator at 37°C and 5% CO2 for incubation.

6. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S2, the ultrasonic power for ultrasonic-assisted dissolution is 100-200W, and the time is 5-8 minutes.

7. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S2, the final concentration of HP-β-CD is ≤0.5%.

8. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S2, SHH is 300-350 ng / mL, FGF8 is 100-150 ng / mL, and CHIR99021 is 5 μM.

9. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S3, BDNF was 30 ng / mL, GDNF was 20 ng / mL, and SC144 was 3 μM.

10. The method for inducing the directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, characterized in that: In step S4, NT-3 was 15 ng / mL and enkephalin was 0.5 μM.

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