Method for inducing directional differentiation of stem cells by neural factors and preparation and application thereof

Abstract

The invention discloses a method for inducing directional differentiation of stem cells by neural factors as well as a preparation and application thereof, and the preparation is a liquid preparation and comprises the following components: BDNF with the concentration of 25-75 ng / mL, GDNF with the concentration of 20-30 ng / mL, NT-3 with the concentration of 5-10 ng / mL and TSA with the concentration of 100-150 nM. The preparation for inducing the directional differentiation of the stem cells through the neural factors can remarkably improve the directional differentiation efficiency of the neural stem cells to neurons, the purity of the neurons is at a high level, the cell viability is good, target cells with mature functions can be efficiently induced, and a new thought is provided for cell treatment of Parkinson's disease.

Description

Technical Field

[0001] This invention relates to the field of neural factors, specifically to a method for inducing directed differentiation of stem cells using neural factors, its formulation, and its applications. Background Technology

[0002] The directed differentiation of stem cells into functional neurons, especially dopaminergic neurons, is one of the core technologies in neuroregenerative medicine, and it holds significant potential in the treatment of neurodegenerative diseases. Currently, methods for inducing stem cell differentiation using neurotrophic factors have made some progress, but key technological bottlenecks remain. Existing methods for directed stem cell differentiation suffer from low differentiation efficiency, insufficient proportion of dopaminergic neurons, and poor cell functional maturity. Furthermore, due to the lack of epigenetic regulation, differentiation stability and batch-to-batch consistency are difficult to guarantee.

[0003] Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Current treatments are difficult to reverse the disease progression and have significant limitations. Drug therapy, often represented by levodopa, can temporarily supplement dopamine and alleviate motor symptoms, but long-term use can lead to serious side effects such as dyskinesia and end-of-dose symptoms, and cannot stop the continuous death of neurons. Surgical methods, such as deep brain stimulation, can improve motor function, but are only suitable for patients in the late stages and carry risks of trauma and postoperative complications. Therefore, further research is needed on methods for the directed differentiation of stem cells into functional neurons. Summary of the Invention

[0004] In order to overcome the deficiencies in the prior art, this invention provides a method for inducing the directed differentiation of stem cells by neurotrophic factors, as well as its formulation and application, which can effectively improve the symptoms of Parkinson's disease in mice.

[0005] The present invention first provides a formulation for inducing the directed differentiation of stem cells by neural factors. The formulation is a liquid formulation and its components include: BDNF at a concentration of 25-75 ng / mL, GDNF at a concentration of 20-30 ng / mL, NT-3 at a concentration of 5-10 ng / mL, and TSA at a concentration of 100-150 nM.

[0006] This invention also provides the following optimization scheme:

[0007] Preferably, its components include: BDNF at a concentration of 50 ng / mL, GDNF at a concentration of 20 ng / mL, NT-3 at a concentration of 10 ng / mL, and TSA at a concentration of 100 nM.

[0008] Liquid formulations are preferably prepared and used immediately, and stored at 4°C for no more than 6 hours; TSA is preferably dissolved in DMSO, and the final concentration should preferably be below 0.15% to avoid cytotoxicity.

[0009] BDNF is brain-derived neurotrophic factor, GDNF is glial cell-derived neurotrophic factor, NT-3 is neurotrophic factor-3, TSA is trichostatin A, and DMSO is dimethyl sulfoxide.

[0010] This invention also discloses the following scheme: a method for directing the differentiation of stem cells using the above-mentioned neural factor-induced stem cell-directed differentiation preparation, comprising the following steps:

[0011] S1 reconstitutes BDNF, GDNF, and NT-3 lyophilized and rehydrated them into a high-concentration mother liquor.

[0012] When reconstituted, add the solvent slowly and mix gently to avoid vigorous shaking that could cause protein denaturation; the mother liquor is best stored at -80°C after being dispensed to avoid repeated freeze-thaw cycles.

[0013] S2 adds high-concentration stock solutions of BDNF, GDNF, and NT-3, and TSA stock solution to the basal culture medium to prepare a formulation of a specified concentration;

[0014] The order of addition is to add the neurotrophic factor stock solution first, followed by the TSA stock solution, to reduce the impact of DMSO on protein activity; after preparation, gently invert and mix to avoid generating bubbles.

[0015] S3: Add 1 mL of the preparation to the NSCs medium, label it, and then put it back into the incubator;

[0016] Before adding the formulation, confirm that the NSCs have adhered to the wall and are in good condition with a survival rate of ≥90%; the labels should clearly distinguish the groups to avoid confusion.

[0017] S4 discards 500 μL of old culture medium at specified intervals and adds 500 μL of freshly prepared formulation.

[0018] When changing the medium, the pipette should be moved slowly along the well wall to avoid direct impact on the cells; fresh preparations should be prepared on the same day to ensure factor activity.

[0019] Cell viability and neuronal differentiation efficiency were assessed 14 days after S5.

[0020] Each group should have at least 3 replicates to ensure data reliability; the incubation time of the primary antibody and the light-protected conditions of the secondary antibody should be strictly controlled during immunofluorescence staining.

[0021] Preferably, in step S1, BDNF, GDNF, and NT-3 are reconstituted in PBS containing 0.1% BSA and lyophilized.

[0022] Preferably, the formulations with the specified concentrations in step S2 are: BDNF at a concentration of 25-75 ng / mL, GDNF at a concentration of 20-30 ng / mL, NT-3 at a concentration of 5-10 ng / mL, or TSA at a concentration of 100-150 nM.

[0023] Preferably, the resuscitation and vaccination of NSCs are performed before step S3.

[0024] Preferably, the resuscitation and vaccination steps for NSCs are as follows:

[0025] Remove the frozen NSCs (commercial neural stem cells) from the liquid nitrogen tank, rapidly thaw them in a 37°C water bath, add 5-10 mL of basal culture medium, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. The water bath thaw time should not exceed 2 minutes to avoid overheating and damage to the cells. It is recommended to coat the well plates with Poly-L-Lysine in advance to enhance the cell adhesion ability.

[0026] Resuspend cells in basal culture medium, count them, and adjust the density to 5 × 10⁶ cells / mL. 4 cells / mL;

[0027] Add 1 mL of cell suspension to each well of the plate, preferably 5 × 10⁻⁶ cells / well. 4 Cells / wells are placed in an incubator for incubation.

[0028] Preferably, the specified interval in step S4 is 4 days, 7 days, or 10 days.

[0029] Preferably, cell viability is detected using the CCK-8 assay in step S5.

[0030] This invention also discloses the application of the above-mentioned neurotrophic factor-induced stem cell-directed differentiation formulation in the preparation of Parkinson's disease treatment drugs.

[0031] The beneficial effects of this invention are:

[0032] The neural factor-induced stem cell-directed differentiation formulation of this invention significantly improves the efficiency of neural stem cell-directed differentiation into dopaminergic neurons, achieving high levels of neuronal purity and dopaminergic ratio, and exhibiting good cell viability. It can efficiently induce functionally mature target cells, providing a new approach for cell therapy of Parkinson's disease. This formulation effectively improves the pathological state of the substantia nigra region in Parkinson's disease model mice. Compared with the model group, the treatment group showed a significant increase in the number of TH-positive neurons in the substantia nigra region, with more intact morphology, denser distribution, and improved continuity of the red fluorescent band. It can promote the survival and repair of damaged dopaminergic neurons, reduce the degree of neuronal loss, and provide a new and effective approach for the treatment of Parkinson's disease, showing promising application prospects. Attached Figure Description

[0033] Figure 1 This is a microscopic comparison of cell morphology between the formulation A group and the control group A group used to test the effect of TSA in this invention.

[0034] Figure 2Immunofluorescence staining images of the substantia nigra region in the normal group, model group, and treatment group under a fluorescence microscope in a Parkinson's disease treatment experiment. Detailed Implementation

[0035] 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.

[0036] The method for directing stem cell differentiation using the neurotrophic factor-induced stem cell differentiation formulation of the present invention includes the following steps:

[0037] S1 reconstitutes BDNF, GDNF, and NT-3 lyophilized and rehydrated them into a high-concentration mother liquor.

[0038] The reconstitution of BDNF, GDNF, and NT-3 for lyophilization involves reconstitution with PBS containing 0.1% BSA to a stock solution of 10 μg / mL, then aliquoting the stock solution into 10 μL / tubes and storing at -80°C.

[0039] S2 adds high-concentration stock solutions of BDNF, GDNF, and NT-3, and TSA stock solution to the basal culture medium to prepare a formulation of a specified concentration;

[0040] The basal culture medium used is Neurobasal TM +2% B27 +1% double antibiotics; first take the culture medium, then add BDNF, GDNF, NT-3 stock solution in sequence, and finally add TSA stock solution and DMSO. Gently invert 10 times to avoid generating air bubbles.

[0041] S3: Add 1 mL of the preparation to the NSCs medium, label it, and then put it back into the incubator;

[0042] Using a 1 mL pipette, slowly aspirate the original culture medium along the well wall, reserving approximately 100 μL to prevent cell drying. Add 1 mL of freshly prepared working solution to each well and gently agitate the culture plate to distribute the liquid evenly. Incubate at 37°C in a 5% CO2 incubator, and label the groups and dates.

[0043] S4 discards 500 μL of old culture medium at specified intervals and adds 500 μL of freshly prepared formulation.

[0044] On days 4, 7, and 10 after intervention, step S4 was performed, with 500 μL of supernatant slowly aspirated from each well, and 500 μL of freshly prepared working solution from the same group added after pre-warming to 37°C.

[0045] Observe cell morphology before changing the medium. Normally differentiated cells will show slender protrusions; the presence of numerous shrunken cells suggests factor toxicity. After changing the medium on day 7, cell density can be examined under a microscope. If the cells are overly proliferating, appropriate dilution is necessary. After the final medium change, reduce the number of times the incubator is opened and closed to avoid temperature fluctuations affecting differentiation.

[0046] Cell viability and neuronal differentiation efficiency were assessed 14 days after S5.

[0047] Cell viability assay (CCK-8 assay): Discard the culture medium, add 500 μL of culture medium containing 10% CCK-8 to each well, incubate at 37°C in the dark for 2 hours, take 100 μL of supernatant to a 96-well plate, measure the absorbance at 450 nm using a microplate reader, and calculate the viability rate = (OD value of experimental group / OD value of control group) × 100%.

[0048] Immunofluorescence staining was used to detect neuronal differentiation efficiency: After washing three times with PBS, the sample was fixed with 4% paraformaldehyde at room temperature for 30 minutes, followed by three more washes with PBS (5 minutes each). The sample was permeated with 0.3% Triton X-100 / PBS at room temperature for 15 minutes, then blocked with 5% BSA for 1 hour. NeuN antibody (1:1000) + TH antibody (1:800) were added, and the sample was incubated overnight in a humidified chamber at 4°C. After washing three times with PBS, the sample was incubated with Alexa Fluor 488 / 594 secondary antibody (1:1000) at room temperature in the dark for 1 hour. The nuclei were stained with DAPI (1:5000) for 10 minutes, and the sample was mounted with an anti-fluorescence quenching mounting medium. Five fields of view / wells were randomly selected using a fluorescence microscope, and the neuronal purity was calculated as (NeuN...). + Cell count / DAPI + Cell count) × 100%, Dopaminergic ratio = (TH + Cell count / NeuN + (cell count) × 100%.

[0049] The above is a detailed description of the present invention. The following are embodiments of the present invention.

[0050] Optimal proportion experiment

[0051] Prepare the following five groups of neurotrophic factor-induced stem cell-directed differentiation formulations according to step S1.

[0052] Group BDNF (ng / mL) GDNF (ng / mL) NT-3 (ng / mL) TSA(nM) Group A 50 20 10 100 Group B 25 20 10 100 Group C 50 30 10 100 Group D 50 20 5 100 Group E 50 20 10 150

[0053] Then, following steps S2-S5 above, cell viability and neuronal differentiation efficiency were tested, yielding the following data table:

[0054] Five sets of test data tables

[0055]

[0056] Group A: 50 ng / mL BDNF + 20 ng / mL LGFNF + 10 ng / mL NT-3 + 100 nMTSA is the optimal ratio for the synergistic effect of the four factors, ensuring high cell survival and efficiently inducing NSCs to differentiate into dopaminergic neurons. Therefore, Group A was selected as the optimal ratio.

[0057] TSA Effects Experiment

[0058] The formulation was prepared according to the ratio of Group A, and then a control group (Group A without TSA) was set up: 50 ng / mL BDNF+

[0059] 20 ng / mL GDNF + 10 ng / mL NT-3, both groups were operated on according to steps S1-S4. On day 7, cell morphology was observed under a microscope. Figure 1 The left side represents group A, and the right side represents the control group of group A. Figure 1 We can clearly see that the neurons in the control group A have insufficient and sparse protrusions, which is far less effective than the neuronal differentiation in the control group A.

[0060] Parkinson's mouse experiment

[0061] I. Preparation of Experimental Materials

[0062] Animals: SPF-grade male C57BL / 6 mice, 8-10 weeks old, weighing 22-25g, 18 mice, randomly divided into 3 groups of 6 mice each:

[0063] Normal group: No model construction, no treatment intervention;

[0064] Model group (PD): MPTP-induced Parkinson's model, with solvent injection;

[0065] Treatment group: MPTP induction model, injection of group A preparation: 50 ng / mL BDNF + 20 ng / mL LGDNF +

[0066] 10 ng / mL NT-3 + 100 nM TSA.

[0067] Reagents and instruments:

[0068] MPTP: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, dissolved in physiological saline, concentration 2 mg / mL; Group A formulation; perfusion fixative: 4% paraformaldehyde (PFA), 0.9% physiological saline; tissue processing reagents: 20% sucrose, 30% sucrose; immunofluorescence reagents: TH antibody (1:1000, dopaminergic neuron marker), Alexa Fluor 594 labeled secondary antibody (1:2000), DAPI staining solution (1:5000).

[0069] II. Experimental Procedure

[0070] Phase 1: Animal Adaptation (1-7 days)

[0071] All mice were placed in an SPF-grade housing environment at a temperature of 22±1℃, with a 12-hour light-dark cycle, and free access to food and water for 7 days to reduce stress response.

[0072] Phase 2: Parkinson's Disease Model Construction (8-14 days)

[0073] Normal group: 10 mL / kg of 0.9% saline was injected intraperitoneally daily for 7 consecutive days.

[0074] Model group and treatment group: MPTP 30mg / kg was injected intraperitoneally daily for 7 consecutive days, calculated by body weight. MPTP should be freshly prepared and stored away from light. After injection, the activity status of mice was observed to ensure the appearance of typical PD symptoms such as tremor and bradykinesia.

[0075] Phase 3: Pharmaceutical Intervention (15-21 days)

[0076] Preparation for stereotactic injection:

[0077] Mice were anesthetized by inhalation of 2% isoflurane, fixed in a stereotaxic apparatus, their head hair was shaved, and after disinfection with povidone-iodine, the skin was incised to expose the anterior fontanelle.

[0078] Injection coordinates in the substantia nigra region: 3.2mm posterior to the anterior fontanelle, 1.5mm lateral, depth 4.5mm, bilateral injection.

[0079] Group injection:

[0080] Normal group and model group: 5 μL of solvent: ACSF + 0.1% DMSO was injected into the substantia nigra region on each side at an injection rate of 0.5 μL / min. The needle was left in place for 5 minutes and then slowly withdrawn.

[0081] Treatment group: 5 μL of the preparation was injected into each side of the substantia nigra region, with the same injection parameters as above.

[0082] Phase 4: Feeding and Observation

[0083] After surgery, mice were housed individually to observe their diet and activity. After the wounds healed in 3-5 days, their weight and spontaneous activity were recorded daily to ensure there was no infection or serious complications.

[0084] Phase 5: Material Sampling and Tissue Processing

[0085] Cardiac perfusion fixation:

[0086] Mice were anesthetized intraperitoneally with 10 mL / kg of 4% chloral hydrate, the chest cavity was cut open to expose the heart, the needle was inserted into the left ventricle, the right atrium was cut open, and about 50 mL of 0.9% physiological saline was rapidly perfused until the liver turned white, and then about 100 mL of 4% paraformaldehyde was perfused until the mouse limbs became stiff.

[0087] Brain Removal and Dehydration:

[0088] The brain was quickly removed by decapitation and placed in 4% paraformaldehyde for fixation for 24 hours. Then it was transferred to a 20% sucrose solution at 4°C. After the brain tissue settled to the bottom, it was replaced with a 30% sucrose solution at 4°C and dehydrated until the brain tissue settled to the bottom.

[0089] Slice preparation:

[0090] Brain tissue was sectioned along the coronal plane using a cryostat. The thickness of continuous sections of the substantia nigra was approximately 10 μm. Sections containing intact substantia nigra were selected from the area 3.0-3.4 mm posterior to the anterior fontanelle. These sections were mounted on poly-L-lysine-coated slides, air-dried at room temperature, and then stored at -20°C for later use.

[0091] Stage 6: Immunofluorescence staining of the substantia nigra region

[0092] Rewarming and sealing:

[0093] The sections were removed from -20℃, allowed to warm to room temperature for 30 min, and washed three times with PBS for 5 min each time.

[0094] Add PBS containing 5% BSA + 0.3% Triton X-100 and block at room temperature for 1 hour.

[0095] Primary antibody incubation:

[0096] Discard the blocking solution, add TH antibody (1:1000 diluted in 1% BSA-PBS), and incubate overnight at 4°C for 12-16 hours in a humidified chamber.

[0097] Secondary antibody and nucleochromatosis:

[0098] Wash 3 times with PBS, 5 min each time, add Alexa Fluor 594-labeled secondary antibody (1:2000 dilution), and incubate at room temperature in the dark for 1 h;

[0099] After washing three times with PBS, add DAPI staining solution (1:5000), incubate in the dark for 10 min, and then wash twice with PBS.

[0100] Covering and photography:

[0101] The slides were mounted with anti-fluorescence quenching mounting medium, and the substantia nigra region was observed under a fluorescence microscope. Three slides were randomly selected from each group, and three fields of view were photographed from each slide, with a focus on photographing the compact portion of the substantia nigra. Figure 2 From left to right, the groups are: normal group, model group, and treatment group. Figure 2 From this we can see that,

[0102] In the normal group, the number of TH red-positive neurons in the substantia nigra were abundant, dense, and morphologically intact, exhibiting continuous and dense red fluorescent bands in the images. In the model group, the number of TH red-positive neurons in the substantia nigra was significantly reduced, approximately 30% of the normal group, and their morphology was fragmented; the red fluorescent bands in the images were broken, with only a small number of positive cells remaining. In the treatment group, the number of TH red-positive neurons in the substantia nigra was increased compared to the model group, approximately 50-60% of the normal group, with more intact morphology; the continuity of the red fluorescent bands in the images was improved, and the density of positive cells was increased. The results from the treatment group indicate that this preparation can effectively improve the damage status of dopaminergic neurons in the substantia nigra of Parkinson's disease model mice. Compared with the model group, the number of TH positive neurons in the substantia nigra of the treatment group was significantly increased, from approximately 30% of the normal group to 50-60%, and the neurons had more intact morphology. This suggests that the preparation in group A can partially reverse the neurological damage in Parkinson's disease model mice by promoting the survival and repair of dopaminergic neurons in the substantia nigra, providing a new approach for the treatment of Parkinson's disease.

[0103] 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. A formulation for inducing directed differentiation of stem cells by neural factors, characterized in that, The formulation is a liquid formulation, and its components include: BDNF at a concentration of 25-75 ng / mL, GDNF at a concentration of 20-30 ng / mL, NT-3 at a concentration of 5-10 ng / mL, and TSA at a concentration of 100-150 nM.

2. The formulation for inducing directed differentiation of stem cells by neural factors according to claim 1, characterized in that, Its components include: BDNF at a concentration of 50 ng / mL, GDNF at a concentration of 20 ng / mL, NT-3 at a concentration of 10 ng / mL, and TSA at a concentration of 100 nM.

3. A method for directed differentiation of stem cells using the agent for inducing directed differentiation of neural factors as described in claim 1, characterized in that: Includes the following steps: S1 reconstitutes BDNF, GDNF, and NT-3 lyophilized and rehydrated them into a high-concentration mother liquor. S2 adds high-concentration stock solutions of BDNF, GDNF, and NT-3, and TSA stock solution to the basal culture medium to prepare a formulation of a specified concentration; S3: Add 1 mL of the preparation to the NSCs medium, label it, and then put it back into the incubator; S4 discards 500 μL of old culture medium at specified intervals and adds 500 μL of freshly prepared formulation; Cell viability and neuronal differentiation efficiency were assessed 14 days after S5.

4. The method for directed differentiation of stem cells according to claim 3, characterized in that: In step S1, BDNF, GDNF, and NT-3 were reconstituted in PBS containing 0.1% BSA and lyophilized.

5. The method for directed differentiation of stem cells according to claim 3, characterized in that: The formulations with the specified concentrations mentioned in step S2 are: BDNF at a concentration of 25-75 ng / mL, GDNF at a concentration of 20-30 ng / mL, NT-3 at a concentration of 5-10 ng / mL, and TSA at a concentration of 100-150 nM.

6. The method for directed differentiation of stem cells according to claim 3, characterized in that: The resuscitation and vaccination of NSCs are performed before step S3.

7. The method for directed differentiation of stem cells according to claim 6, characterized in that: The resuscitation and vaccination steps for NSCs are as follows: Remove the frozen NSCs from the liquid nitrogen tank, rapidly thaw them in a 37°C water bath, add 5-10 mL of basal culture medium, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend cells in basal culture medium, count them, and adjust the density to 5 × 10⁶ cells / mL. 4 cells / mL; Add 1 mL of cell suspension (i.e., 5 × 10⁶ cells per well) to each well of the plate. 4 Cells (wells) are placed in an incubator for incubation.

8. The method for directed differentiation of stem cells according to claim 3, characterized in that: The specified interval number in step S4 is 4 days, 7 days, or 10 days.

9. The method for directed differentiation of stem cells according to claim 3, characterized in that: In step S5, the CCK-8 assay was used to detect cell viability.

10. The use of the agent of claim 1 for inducing the directed differentiation of stem cells by neural factors in the preparation of a drug for treating Parkinson's disease.

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