A method for inducing human induced pluripotent stem cells to differentiate into dopaminergic neuronal progenitor cells

Abstract

This invention relates to the technical field of stem cell induction differentiation and transplantation therapy, specifically to a method for inducing human induced pluripotent stem cells to differentiate into dopaminergic neuronal progenitor cells, comprising the following steps: S1, human induced pluripotent stem cells are induced to differentiate into primitive neuroepithelial cells under the action of SB431542, DMH1, SHH, FGF8b, Purmorphamine, and CHIR99021; S3, intermediate primitive neuroepithelial cells are continuously induced to differentiate using BDNF, GDNF, AA, N2, B27 (without added vitamin A), cAMP, and GlutaMax to obtain dopaminergic neuronal progenitor cells. The dopaminergic neuronal progenitor cells obtained using the method provided by this invention can survive for a long time in the brain microenvironment of Parkinson's rats and have a significant repair effect on rat motor dysfunction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stem cell induction differentiation and transplantation treatment, in particular to a method for inducing human induced pluripotent stem cells to differentiate into dopaminergic neuronal progenitor cells. BACKGROUND

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, about 1-2% of the elderly population over 65 years old worldwide are Parkinson's disease patients, and the prevalence is expected to double by 2030. The main pathological change is the massive death of dopaminergic neurons (DANs) in the nigrostriatal pathway of the midbrain, leading to a decrease in dopamine synthesis in the brain, and causing a series of neurological dysfunction, which has become one of the hotspots in the treatment of neurological diseases. The current main treatment methods for PD, including drug treatment such as L-dopa (L-DOPA) and surgical treatment such as deep brain stimulation, focus on symptom relief, cannot regenerate damaged neurons, and cannot fundamentally treat Parkinson's disease.

[0003] In recent years, cell transplantation to replace lost dopamine neurons (DA neurons) to restore neural transmission and protect residual DA neurons has become an important means of treating PD. DA neuronal progenitor cells isolated from human ventral midbrain (VM) can survive in the brain of PD patients and play a repair role. However, the number of human fetal VM tissue cells is limited, which hinders its clinical application. Therefore, it is crucial to find a method to obtain dopaminergic neuronal progenitor cells. SUMMARY

[0004] To solve the above technical problems, the present application provides a method for inducing human induced pluripotent stem cells to differentiate into dopaminergic neuronal progenitor cells, and the obtained dopaminergic neuronal progenitor cells can survive for a long time in the brain microenvironment of Parkinson's rats and have obvious repair effect on the motor dysfunction of rats, providing factual basis and theoretical basis for the treatment of related neurodegenerative diseases.

[0005] The first object of the present application is to provide a method for inducing pluripotent stem cells to differentiate into dopaminergic neuronal progenitor cells, comprising the following steps:

[0006] S1, in vitro culture of human induced pluripotent stem cells: resuscitating human induced pluripotent stem cell lines and culturing in vitro;

[0007] S2, inducing human induced pluripotent stem cells to differentiate into primitive neuroepithelial cells under the action of additives SB431542, DMH1, SHH, FGF8b, Purmorphamine and CHIR99021;

[0008] S3 uses additives BDNF, GDNF, AA, N2, B27, cAMP and GlutaMax to continuously induce differentiation of intermediate primitive neuroepithelial cells to obtain dopaminergic neuronal progenitor cells.

[0009] Furthermore, in S1, the culture process is as follows: cells are seeded on mTeSR TM Basal Medium was added to bring the final concentration of Y27632 to 10 μM. The old medium was discarded the next day and replaced with new mTeSR. TM Culture in Basal Medium, change the medium every 2 days, and then passage after 2 changes.

[0010] Furthermore, in S2, the specific induction differentiation process is as follows: after the human induced pluripotent stem cell clones obtained in S1 proliferate to a confluence of 30%, they are then supplemented with mTeSR containing 10mM DMH1, 10mM SB431542, SHH, FGF8b, and Purmorphamine. TM Cultured on Basal Medium until day 3, then added CHIR99021 and cultured until day 5. Then the medium was changed to neural induction medium (NIM) and cultured until day 7. Then SHH and Purmorphamine were removed and cultured until day 11. Then the medium was changed to neural differentiation medium (NEM) and cultured until day 13 to obtain primitive neural epithelial cells.

[0011] The neural induction medium NIM is DMEM / F12 medium supplemented with 1% N2, 10mM DMH1, 200ng / mL SHH, 2μM Purmorphamine, and 3μM CHIR99021.

[0012] Further, in S2, the neural differentiation medium NEM is a neurobasal medium supplemented with 1% N2, 2% B27 (without vitamin A), 10 ng / ml BDNF, 10 ng / ml GDNF, 200 μM AA, TGF-β, 1 μM cAMP, 1% GlutaMax and 3 μM CHIR99021.

[0013] Furthermore, in S3, the specific induction differentiation process is as follows: Type IV collagenase is added to the obtained primitive neuroepithelial cells for digestion, then the Type IV collagenase is discarded, the cells are collected, and they are cultured in Neurobasal medium until day 28 to obtain dopaminergic neuron progenitor cells.

[0014] The Neurobasal medium culture medium is prepared by adding 1% N2, 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, 200 μM AA, 1 ng / mL TGF-β, 1 μM cAMP, and 1% GlutaMax to the Neurobasal medium.

[0015] Furthermore, in S3, the digestion conditions are: 5% CO2, 37°C, and 30 seconds of digestion.

[0016] Furthermore, in S3, cells are pipetted into single cells before adherent culture.

[0017] Furthermore, in S3, the amount of type IV collagenase added is 700 μL.

[0018] Furthermore, in S3, during the adherent culture process, the culture medium is replaced with fresh medium every 2 days.

[0019] Furthermore, in S1-S3, all culture media and additives are of clinical grade.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. In the first stage, the present invention induces intermediate cells for 12 days by SB431542, DMH1, SHH, FGF8b, Purmorphamine and CHIR99021, which reduces the risk of tumorigenicity due to activation of pluripotency; in the second stage, the intermediate cells are induced until day 28 to obtain high-purity dopaminergic neuronal progenitor cells (93.3-96.7%).

[0022] 2. This invention employs dual SMAD inhibition (exposure to DMH1 and SB431542) and shortens the treatment time to 6 days to enhance the differentiation of iPSCs into midbrain DAPs. The selective bone morphogenetic protein (BMP) inhibitor DMH1 replaces LDN193189, significantly reducing the percentage of cells expressing pluripotency markers OCT4 and Nanog, thereby lowering the risk of tumorigenicity due to pluripotency activation. Simultaneously, it significantly increases the expression of the neural progenitor marker PAX6, promoting conversion to neural progenitor cells. SB431542 is a TGF-β / SMAD pathway inhibitor, effectively inhibiting TGFβ1-induced cell migration and invasion, and promoting Sox2 and myc-dependent function to facilitate reprogramming.

[0023] 3. Compared with the traditional 2D monolayer culture method, our improved method is more effective. It utilizes Purmorphamine to activate the Sonic Hedgehog (SHH) signaling pathway for specific differentiation into basal plate cells and CHIR99021 (a GSK3 inhibitor) to activate the WNT pathway, promoting midbrain cell differentiation and inducing a higher proportion and yield of DAPs (Th). + FOXA2 + LMX1A + and NURR1 + Cells accounted for 93.3%-96.7%, significantly higher than the approximately 85% of FOXA2 in similar studies to date. + / TUJ1 + Positive cell rate.

[0024] Notably, in the first stage of induction, CHIR99021, by inhibiting GSK3-β and activating the β-catenin / c-Jun / WNT signaling pathway, is a key factor in inducing LMX1A co-expression, promoting the neurogenic conversion of iPSC-derived midbrain neurons to DA neurons. Furthermore, Purmorphamine, as an SHH receptor agonist, strongly activates the Sonic Hedgehog (SHH) signaling pathway in combination with recombinant SHH, inducing FOXA2 / LMX1A co-expression and improving mDA neuron survival. FGF8, essential for the generation of dopamine neurons, improves the distribution of mDA neurons and reduces the number of hypothalamic nucleus neurons. BDNF promotes iPSC differentiation into neural progenitor cells, and subsequently into mature neurons, by activating the Wnt / β-catenin and ERK5 signaling pathways. cAMP upregulates TH synthesis, promoting neuronal differentiation / survival. TGF-β promotes neurite growth and neuronal survival. Additionally, dual SMAD inhibition ensures the generation of DAP cells expressing FOXA2 / LMX1A1.

[0025] 4. The method for efficiently differentiating human induced pluripotent stem cells into midbrain dopaminergic neuron progenitor cells provided by this invention can obtain midbrain dopaminergic neuron progenitor cells, providing a guarantee for the replenishment of neurons after damage and loss, and also laying the foundation for using stem cell transplantation to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0026] 5. This invention effectively verified the survival, migration, and differentiation of transplanted dopaminergic neuronal progenitor cells in the host brain by detecting tyrosine hydroxylase expression in brain slices using HE staining and immunohistochemistry. Apomorphine-induced rotation experiments, open field experiments, water maze experiments, and fatigue rotarod experiments effectively demonstrated that the transplanted dopaminergic neuronal progenitor cells have a significant therapeutic effect on Parkinson's disease rats. The dopaminergic neuronal progenitor cells obtained using the method provided in this invention can survive long-term in the brain microenvironment of Parkinson's rats and have a significant repair effect on motor dysfunction in rats, laying the foundation for stem cell transplantation to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a roadmap for inducing human induced pluripotent stem cells to differentiate into dopaminergic neuronal progenitor cells in an embodiment of the present invention;

[0029] Figure 2 The following are the morphological changes of cells at various stages during the induction of human induced pluripotent stem cells into dopaminergic neuronal progenitor cells: A is human induced pluripotent stem cells, B is primitive neural epithelial cells induced for 12 days, C is dopaminergic neuronal progenitor cells induced for 28 days, and D is mature dopaminergic neuronal cells induced for 70 days.

[0030] Figure 3 Immunofluorescence images (600×) of primitive neural epithelial cells obtained from the induced differentiation of human induced pluripotent stem cells to day 12; A1: SOX2 expression; A2: Nestin expression; A3: DAPI-labeled nuclei; A4: Overlay of the first three images; B: Statistical graph of positive cell expression levels; C1: PAX6 expression; C2: Nestin expression; C3: DAPI-labeled nuclei; C4: Overlay of the first three images; D: Statistical graph of positive cell expression levels.

[0031] Figure 4Immunofluorescence images (600×) of dopaminergic neuronal progenitor cells obtained from human induced pluripotent stem cell differentiation to day 28, used to identify their in vitro differentiation potential. A1 shows the expression of TH, a marker of dopaminergic neurons; A2 shows the expression of Tuj1, a marker of microtubule protein; A3 shows DAPI-labeled nuclei; A4 is a superimposed image of the first three images. B1 shows the expression of LMX1A, a marker of dopaminergic neurons; B2 shows the expression of Tuj1, a marker of microtubule protein; B3 shows DAPI-labeled nuclei; B4 is a superimposed image of the first three images. C1 shows the expression of FOXA2, a marker of dopaminergic neurons; C2 shows the expression of Tuj1, a marker of microtubule protein; C3 shows DAPI-labeled nuclei; C4 is a superimposed image of the first three images. D1 shows the expression of NURR1, a marker of dopaminergic neurons; D2 shows the expression of Tuj1, a marker of microtubule protein; D3 shows DAPI-labeled nuclei; D4 is a superimposed image of the first three images. E is a statistical graph of positive cell expression levels.

[0032] Figure 5 To detect DA concentration in the culture supernatant of induced dopaminergic neuronal progenitor cells and mature dopaminergic neurons, Figure A shows the ELISA method for detecting DA concentration in the culture supernatant of HiPSCs, DAPs, mDA (50 days), and mDA (70 days); Figure B shows the high performance liquid chromatography (HPLC) method for demonstrating DA secretion in the culture supernatant of DAPs cells. Figure 5 B);

[0033] Figure 6 The following figures illustrate behavioral assessments following the transplantation of dopaminergic neuronal progenitor cells into a Parkinson's disease model rat. Figure A1 shows the overlay of two sites: 6-OHDA lesion and DAP cell transplantation. Figure A2 shows the brain coordinates of the first transplantation site (AP-4.4mm, ML-1.2mm, DV-7.8mm), and Figure A3 shows the brain coordinates of the second transplantation site (AP-4.0mm, ML-0.8mm, DV-8.0mm). Figure B shows the APO-induced rotation experiment; Figure C shows the fatigue rotarod experiment; Figure D shows the open field experiment; and Figure E shows the water maze experiment.

[0034] Figure 7 Statistical graphs for APO-induced rotation experiments and open field experiments; A is the statistical graph for APO-induced rotation experiments, and B is the statistical graph for open field experiments;

[0035] Figure 8 The charts are statistical graphs for the fatigue rotator bar experiment and the water maze experiment; A is the statistical graph for the fatigue rotator bar experiment; B is the statistical graph for the water maze experiment.

[0036] Figure 9Immunofluorescence image (100×) showing the survival of transplanted cells in the host brain; the first row shows the expression of dopaminergic neuron marker protein TH, the second to fourth rows show the expression of dopaminergic neuron marker proteins LMX1A, FOXA2 and NURR1, and the fifth row shows the expression of neuronal microtubule protein marker Tuj1.

[0037] Figure 10 Immunofluorescence image (100×) showing the differentiation potential of transplanted cells in the host brain; the first row shows the expression of PSD95, a marker protein of glutamatergic neurons; the second row shows the expression of GABA, a marker protein of γ-aminobutyric acid (GABA) neurons; and the third row shows the expression of GFAP in astrocytes.

[0038] Figure 11 This diagram illustrates the synaptic connections between transplanted cells and endogenous cells in the host brain. Transplanted cells can express the synaptic protein marker synapsin and form synaptic connections with endogenous cells in the host brain (first row shows 200× fluorescently stained cell morphology; second row shows 600× fluorescently stained cell morphology). Detailed Implementation

[0039] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0040] Example 1

[0041] This embodiment provides a method for differentiating human induced pluripotent stem cells into dopaminergic neuronal progenitor cells.

[0042] I. Experimental Materials

[0043] 1. Reagents

[0044] SB431542 (a potent ALK5 inhibitor) was purchased from StemGent; DMH1 (a BMP inhibitor) was purchased from Tocris Bioscience; SHH (sound hedgehog factor) and FGF8b (human fibroblast growth factor 8b) were purchased from R&D systems; Purmorphamine (a smooth receptor agonist) was purchased from Millipore; and CHIR99021 (a GSK-3 inhibitor) was purchased from Selleckchem.

[0045] 2. Culture medium

[0046] mTeSR TM Basal Medium, NIM medium, and NEM medium (Neurobasal medium) were all purchased from Gibco. The media used at different stages were: (1) Induction of human induced pluripotent stem cells into primitive neural epithelial cells: mTeSR TM Basal Medium (Gibco) supplemented with 10mM DMH1, 10mM SB431542, SHH (200ng / mL), FGF8b (100ng / mL), and Purmorphamine (2μM) was cultured for 3 days. Then, 3μM CHIR99021 was added to this medium and cultured for 5 days. Next, the medium was changed to neural induction medium NIM (DMEM / F12 supplemented with 1% N2, 10mM DMH1, 200ng / mL SHH, 2μM Purmorphamine, and 3μM CHIR99021) and cultured for 7 days. SHH and Purmorphamine were removed and cultured for 11 days. Subsequently, the medium was changed to neural differentiation medium (NEM) supplemented with 1% N2 (serum-free), 2% B27 (without vitamin A), BDNF (brain-derived neurotrophic factor) (10ng / mL), GDNF (glial cell-derived neurotrophic factor) (10ng / mL), and 200μM Ascorbic acid. Primitive neural epithelial cells were obtained by culturing the cells in 1 ng / mL TGF-β, 1 μM cAMP (cyclic adenosine 3',5'-monophosphate), 1% GlutaMax, and 3 μM CHIR99021 for 13 days.

[0047] (2) Induction of primitive neuroepithelial cells into dopaminergic neuronal progenitor cells: Neurobasal medium (Gibco) was cultured for 28 days with 1% N2 (serum-free additive), 2% B27 (no vitamin A), 10 ng / mL BDNF (brain-derived neurotrophic factor), 10 ng / mL GDNF (glial cell-derived neurotrophic factor), 200 μM Ascorbic acid (AA), 1 ng / mL TGF-β, 1 μM cAMP (cyclic adenosine 3',5'-monophosphate), and 1% GlutaMax.

[0048] 3. Cells used for differentiation induction: The somatic cell type used for transdifferentiation induction is human induced pluripotent stem cells.

[0049] II. Experimental Methods

[0050] 1. In vitro culture of human induced pluripotent stem cells (hiPSCs)

[0051] Human induced pluripotent stem cell lines (hiPSCs) were revived and seeded into Matrigel collagen-coated T25 culture flasks for 1 hour, using mTeSR. TM Basal Medium was added to bring the final concentration of Y27632 to 10 μM. The medium was discarded the next day and replaced with mTeSR. TM Culture in Basal Medium, change the medium every 2 days, and then passage after 2 changes.

[0052] 2. Induction of human induced pluripotent stem cells (hiPSCs) into dopaminergic neuronal progenitor cells (DAPs)

[0053] (1) Human induced pluripotent stem cells (hiPSCs) are induced into primitive neural epithelial cells (NESCs):

[0054] After human induced pluripotent stem cell (hiPSC) clones proliferate to approximately 30% confluence, mTeSR TM BasalMedium supplemented with 10mM DMH1, 10mM SB431542, SHH (200ng / mL), FGF8b (100ng / mL), and Purmorphamine (2μM) was cultured for 3 days. Then, 3μM CHIR99021 was added to this medium and cultured for 5 days. Next, the medium was changed to neural induction medium NIM (DMEM / F12 supplemented with 1% N2, 10mM DMH1, 200ng / mL SHH, 2μM Purmorphamine, and 3μM CHIR99021) and cultured for 7 days. SHH and Purmorphamine were removed and cultured for 11 days. Finally, the medium was changed to neural differentiation medium NEM (Neurobasal medium supplemented with 1% N2, 2% B27, BDNF (10ng / mL), GDNF (10ng / mL), 200μM AA, 1ng / mL TGF-β, 1μM cAMP, 1% GlutaMax, and 3μM Glutamax). (CHIR99021) cultured to day 13, at this stage the cells are induced to exhibit a rosette-like structure of primitive neuroepithelial cells, thus obtaining primitive neuroepithelial cells.

[0055] (2) Primitive neural epithelial cells (NESCs) are induced into dopaminergic neuronal progenitor cells (DAPs):

[0056] Discard the old culture medium, add 700 μL of type IV collagenase to the induced primitive neuroepithelial cells (NESCs), and incubate at 37°C with 5% CO2 for 30 seconds. Discard the type IV collagenase, collect the cells in 15 mL centrifuge tubes using a cell scraper, and seed them as single cells onto coverrslips coated with Poly-l-ornithine hydrobromide (PLO) / Laminin (Gibco) (PLO-Laminin). Replace the culture medium with Neurobasal medium, adding 1% N2, 2% B27 (without vitamin A), BDNF (10 ng / mL), GDNF (10 ng / mL), 200 μM AA, 1 ng / mL TGF-β, 1 μM cAMP, and 1% GlutaMax. Culture until day 28, changing the medium every 2 days. Dopaminergic neuronal progenitor cells (DAPs) are obtained.

[0057] III. Experimental Results

[0058] 1. Human induced pluripotent stem cells (hiPSCs) are induced into primitive neural epithelial cells (NESCs).

[0059] The first stage involves chemically inducing the production of intermediate cells (primitive neural epithelial cells). Human induced pluripotent stem cells were induced to produce rosette-like structures of primitive neural epithelial cells on day 13 by administering 10mM DMH1, 10mM SB431542, 200ng / mL SHH, 100ng / mL FGF8b, 2μM Purmorphamine, and 3μM CHIR99021. Figure 2 B).

[0060] 2. Primitive neural epithelial cells (NESCs) are induced into dopaminergic neuronal progenitor cells (DAPs).

[0061] The second stage involved digesting the intermediate cells (primitive neuroepithelial cells) obtained in the first stage with type IV collagenase, then dispersing the cells into single cells and seeding them onto PLO-Laminin-coated coverlips for adherent culture. Induction continued until day 28 in a medium containing 10 ng / mL BDNF, 10 ng / mL GDNF, 200 μM AA, 1 ng / mL TGF-β, 1 μM cAMP, and 1% GlutaMax to obtain dopaminergic neuronal progenitor cells. Figure 2 C).

[0062] IV. Functional Identification of Dopaminergic Neuron Progenitor Cells Obtained in Example 1

[0063] 1. Analysis of protein marker staining in primitive neural epithelial cells (NESCs)

[0064] Immunofluorescence staining of NESCs for Nestin, SOX2, and PAX6 revealed that Nestin, SOX2, and PAX6 are all markers of primitive neural epithelial cells. Figure 3 Immunofluorescence staining results showed that the induced NESCs were able to highly express the primitive neuroepithelial cell markers SOX2, Nestin, and PAX6. Figure 3 Positive cells accounted for 91.3-92.8%, exhibiting distinct characteristics of primitive neuroepithelial cells.

[0065] 2. Protein marker staining analysis of DAPs cells.

[0066] Immunofluorescence staining of DAPs for TH, FOXA2, LMX1A, NURR1, and Tuj1 was performed. TH, FOXA2, LMX1A, and NURR1 are markers of dopamine neurons, while Tuj1 is a marker of microtubule protein. Figure 4 The induced DAPs showed high expression of DAP markers TH, FOXA2, LMX1A, NURR1, and Tuj1, and the induced dopaminergic neuronal progenitor cells also showed high expression of DAP markers TH, FOXA2, LMX1A, and NURR1. Figure 4 Positive cells accounted for 93.3-96.7%.

[0067] 3. Detection of DA concentration in cell culture supernatant of DAPs and mDAs

[0068] DAPs were further induced into mature mDA-producing neurons for 70 days. The concentration of DA in the cell culture supernatant of HiPSCs, DAPs, mDA (50 days), and mDA (70 days) was detected by ELISA. Figure 5 As shown in Figure A, the DA concentration in the cell culture supernatant of DAPs, mDA (50 days), and mDA (70 days) was significantly increased compared to the HiPSCs group (P<0.05). HPLC chromatography confirmed a significant increase in DA secretion in the cell culture supernatant of DAPs. Figure 5 B).

[0069] V. Identification of in vivo survival and differentiation capacity of DAPs

[0070] 1. Construction of PD Model

[0071] SPF-grade SD rats, weighing approximately 180–220 g, were selected and anesthetized by intraperitoneal injection of 3% pentobarbital. After adequate anesthesia, the SD rats were fixed on a stereotaxic platform. The brain skin was routinely disinfected, the surgical area was incised, and the periosteum was scraped away. Stereotactic localization was performed using a brain stereotaxic apparatus, with the anterior fontanelle as the standard reference point. The coordinates were selected as follows: ① AP - 4.4 mm, ML - 1.2 mm, DV - 7.8 mm; ② AP - 4.0 mm, ML - 0.8 mm, DV - 8.0 mm. Figure 6 A). Using a Hamilton microinjector, slowly inject 4 μL of 6-OHDA at each point for 8 minutes, leave the needle in place for 8 minutes, then slowly withdraw the needle, suture the brain skin, administer intraperitoneal injections for 3 consecutive days postoperatively to prevent postoperative infection, and observe postoperative recovery daily.

[0072] 2. Behavioral testing

[0073] (1) APO-induced rotation experiment: Two weeks after stereotyping, rats were injected intraperitoneally with apomorphine (0.5 mg / kg). Rats that successfully modeled the model exhibited sniffing, tail pressing, stiffness, and rotational behavior with the healthy hind limb as a fulcrum, bending the body towards the healthy side and connecting the head and tail. Figure 6 B) The standard for successful modeling is 210r / 30min.

[0074] (2) Fatigue Rotating Bar Test

[0075] The rotor speed was set to 10 rpm, and the time was set to 30 minutes. The rat's time on the rotor was measured. When the rat fell, the system automatically paused and recorded the time on the rotor. To ensure the accuracy of the experimental data, each rat was tested three times.

[0076] (3) Open field test: An open field box measuring 100×100×40 cm (length×width×height) was used, with a camera placed above the center grid. The computer was turned on, and the open field test tracking and recording system was opened. The camera automatically captured and recorded the rat's activities in the open field box.

[0077] (4) Morris Water Maze Experiment: The Morris water maze mainly consists of an automatic video recording and analysis system and a circular pool. A camera connected to a computer is located above the pool. The pool is 170cm in diameter and 80cm high. There is a 10cm diameter circular platform in the pool, which is placed in the middle of the third quadrant. Before the experiment, clean water is poured into the pool, and the water temperature is controlled at about 22℃. The water level is about 1-2cm below the platform. The rat is placed on the opposite side of the platform. The camera above can automatically capture and record its activities. The time from when the rat enters the water until it finds the circular platform and climbs onto it is recorded as the platform-finding time.

[0078] 3. Stereotactic transplantation of DAPs into a Parkinson's disease model rat for treatment

[0079] Pre-transplantation staining of DAPs: Collect cells in good growth condition into centrifuge tubes, digest with trypsin, and count after digestion into single cells, ensuring that the number of cells injected per rat is approximately 1 × 10⁻⁶. 5 Then prepare the cell staining solution: 5 μL CM-DiI dye + 1 mL DMEM / F12 medium. Add the prepared dye to the cells, mix well, and place in a carbon dioxide incubator for 30-40 minutes. Take it out every 10 minutes to mix it by pipetting. Keep out of light throughout the process.

[0080] Treatment of Parkinson's disease model rats with stereotactic transplantation of DAPs: Selected PD rat models with successful modeling, and transplanted cells into the right forebrain medial tract of PD rats using the same method as modeling.

[0081] 4. Tissue sampling

[0082] Two weeks after transplantation, rats were anesthetized by intraperitoneal injection of 3% pentobarbital. The rats were then fixed to a dissecting board, and the abdominal cavity was opened sequentially. Hemostatic forceps were used to tighten the mediastinum and all organs below the chest that contribute to fluid circulation. The thoracic cavity was opened to fully expose the heart. A perfusion needle was inserted into the aortic arch through the left ventricle, and the right atrial appendage was quickly cut open. Approximately 200 mL of physiological saline was infused until the outflowing blood became clear and the liver turned white. Then, approximately 200 mL of 4% paraformaldehyde was used for perfusion and fixation. After fixation, the brain was immediately dissected, and the brain tissue was fixed in 4% paraformaldehyde for approximately 4 hours. Afterward, it was dehydrated with 25% sucrose and frozen at -80°C. For sectioning, the rat brain tissue was removed from the -80°C freezer, embedded in OCT embedding medium, and frozen. Serial coronal sections with a thickness of 12-20 μm were then prepared and stored at -20°C for later use.

[0083] 5. Immunofluorescence

[0084] After removing the sections from -20°C, they were left at room temperature for 10 minutes. Then, immunofluorescence staining (cells stained with the red dye CM-DiI) was performed to observe the survival and differentiation ability of the transplanted cells in vivo.

[0085] 6. Results

[0086] Transplanted induced neuronal progenitor cells (DAPs) significantly improved cognitive and motor behavioral impairments in Parkinson's disease model rats. At weeks 2 and 8 post-DAP transplantation, APO-induced rotational behavior analysis showed a significant reduction in circling behavior in the transplantation group rats (P<0.05). Figure 7 A). Moreover, the total open field distance of the transplanted rats was significantly increased compared to the model group rats, and they were more active and excited (P<0.05). Figure 7 B and Figure 6D). Furthermore, compared to the model group, the transplanted rats showed a significant increase in rotarod time during the fatigue rotarod experiment (P<0.05). Figure 6 C and Figure 8 A). In the water maze experiment, the transplanted rats took significantly longer to find the platform than the model rats (P<0.05). Figure 6 E and Figure 8 B). Furthermore, the behavioral improvement in week 8 was more significant than that in week 2.

[0087] Immunofluorescence staining revealed that transplanted DAPs cells could differentiate into various functional neurons in the host brain, expressing dopaminergic neuron marker TH, GABAergic neuron marker GABA, glutamatergic neuron postsynaptic membrane marker PSD95, mature neuron marker and astrocyte marker GFAP. An in vivo differentiation capacity assessment diagram of dopaminergic neuron progenitor cells was obtained. Figure 9 and Figure 10 These results indicate that transplanted DAPs can differentiate into various functional neurons and astrocytes in the host brain, playing a role in damage repair.

[0088] The transplanted cells can survive in the host brain and express the presynaptic membrane marker protein synapsin. Figure 11 It then forms synaptic connections with its own neurons and functions accordingly.

[0089] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0090] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for inducing differentiation of human induced pluripotent stem cells into dopaminergic neuronal progenitor cells, characterized by, The method comprises the following steps: S1, in vitro culture of human induced pluripotent stem cells: resuscitate human induced pluripotent stem cell lines and culture in vitro; S2, S1, after the human induced pluripotent stem cell clones obtained by culture in S1 and S2 were proliferated to a fusion degree of 30%, the medium was changed to mTeSR1 medium added with 10 mM DMH1, 10 mM SB431542, 200 ng / mL SHH, 100 ng / mL FGF8b, 2 μM Purmorphamine, and the cells were cultured for 3 days, then the medium was changed to neural induction medium NIM, and the cells were cultured for 7 days, then the SHH and Purmorphamine were removed, and the cells were cultured for 11 days, then the medium was changed to neural differentiation medium NEM, and the cells were cultured for 13 days, at this stage, the cells were induced to present rosette-like structure of primitive neuroepithelial cells, and the primitive neuroepithelial cells were obtained. TM Basal Medium on the 3rd day, 3 μM CHIR99021 was continuously added and the cells were cultured for 5 days, then the medium was changed to neural induction medium NIM and the cells were cultured for 7 days, then the SHH and Purmorphamine were removed and the cells were cultured for 11 days, and then the medium was changed to neural differentiation medium NEM and the cells were cultured for 13 days, at this stage, the cells were induced to present rosette-like structure of primitive neuroepithelial cells, and the primitive neuroepithelial cells were obtained. The neural induction medium NIM is DMEM / F12 medium added with 1% N2, 10 mM DMH1, 200 ng / mL SHH, 2 μM Purmorphamine and 3 μM CHIR99021; The neural differentiation medium NEM is Neurobasal medium added with 1% N2, 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, 200 μM AA, 1 ng / mL TGF-β, 1 μM cAMP, 1% GlutaMax and 3 μM CHIR99021; S3, in the obtained primitive neural epithelial cells, collagenase type IV is added for digestion, then the collagenase type IV is discarded, the cells are collected, adherent culture is carried out in Neurobasal medium culture solution to the 28th day, and dopaminergic neuron progenitor cells are obtained; The Neurobasal medium culture solution is Neurobasal medium added with 1% N2, 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, 200 μM AA, 1 ng / mL TGF-β, 1 μM cAMP and 1% GlutaMax.

2. The method of claim 1, wherein, In S1, the culture process is: cells are seeded in mTeSR TM Basal Medium medium, add Y27632 to make its final concentration 10 μM, the next day the old culture medium is discarded and replaced with mTeSR TM The new Basal Medium culture medium is continued to be cultured, and the culture solution is replaced every 2 days, and after being replaced twice, the passage can be carried out.

3. The method of claim 1, wherein, In S3, the digestion condition is: 5% CO2, 37℃, and digestion for 30 s.

4. The method of claim 1, wherein, In S3, the single cells are obtained by blowing before adherent culture.

5. The method of claim 1, wherein, In S3, the amount of collagenase type IV added is 700 μL.

6. The method of claim 1, wherein, In S3, the fresh culture solution is replaced every 2 days during adherent culture.

7. The method according to any one of claims 1 to 6, characterized in that, In S1-S3, all the culture media and additives are of clinical grade.

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