Step-by-step differentiation protocol for treating Parkinson's disease by stem cells

Abstract

The invention provides an efficient and repeatable in-vitro differentiation protocol, and human pluripotent stem cells are differentiated into functional midbrain dopaminergic neurons by accurately controlling sequential activation of SHH and FGF8 signal pathways. The protocol comprises five key differentiation stages: neural precursor induction, midbrain mode establishment, midbrain dopaminergic precursor amplification, terminal differentiation and maturation. Experiments prove that after the midbrain dopaminergic neuron obtained by the method is transplanted into a Parkinson's disease model animal body, the survival rate exceeds 80%, and the midbrain dopaminergic neuron can be effectively integrated into a host neural network and improve dyskinesia. According to the differentiation scheme, the purity (gt; gt) of mDA neurons is remarkably improved; 85%) and functional maturity, and a reliable cell source is provided for cell replacement treatment of Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine and cell therapy, specifically relating to a method for sequentially activating the SHH and FGF8 signaling pathways to induce human pluripotent stem cells (hiPSCs) to differentiate into midbrain dopaminergic neurons (mDA) in stages, and its application in the treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Current drug treatments can only alleviate symptoms and cannot stop disease progression. Cell replacement therapy, which involves transplanting exogenous dopaminergic neurons to replenish lost neurons, is considered one of the most promising treatment strategies.

[0003] Traditional differentiation methods have several key problems: (1) low differentiation efficiency and insufficient proportion of mDA neurons; (2) strong cellular heterogeneity, which may contain tumorigenic undifferentiated cells; (3) low survival rate after transplantation (<50%); and (4) poor functional integration ability. These problems mainly stem from the imprecise regulation of key signaling pathways in midbrain development.

[0004] The SHH and FGF8 signaling pathways play a central role in midbrain development, but existing technologies have failed to accurately simulate their dynamic changes. This invention solves the aforementioned technical challenge by systematically optimizing the temporal activation of these two pathways. Summary of the Invention

[0005] This invention aims to provide a highly efficient and stable in vitro differentiation method for midbrain dopaminergic neurons (mDA neurons) to address the key challenge of low cell survival rates in current cell transplantation therapies. Simultaneously, it significantly improves the purity of differentiated mDA neurons by reducing cellular heterogeneity. Furthermore, this method strives to obtain functional neurons that are mature enough to effectively integrate into the host neural network, ultimately providing standardized cell products for the clinical treatment of Parkinson's disease. The following is a detailed explanation of the technical solution and its advantages.

[0006] The core of this invention lies in discovering and establishing the precise timing activation modes of the SHH and FGF8 signal paths:

[0007] 1. Neural induction period (D0-D5):

[0008] Neural progenitors were induced by dual SMAD inhibition (LDN193189+SB431542) without activating SHH / FGF8.

[0009] Neural induction is a crucial initial stage in the differentiation of human pluripotent stem cells into midbrain dopaminergic neurons. The core objective of this stage is to efficiently convert undifferentiated pluripotent stem cells into neural epithelial progenitor cells by precisely regulating signaling pathways, while simultaneously inhibiting the emergence of non-neural fate cells.

[0010] 1.1 Initial Cell State Preparation

[0011] Before differentiation initiation, human pluripotent stem cells (hPSCs) should be maintained in an undifferentiated state in mTeSR1 medium, with a seeding density controlled at 1.0 ± 0.2 × 10^5 cells / cm². 2 Cells can only proceed to differentiation after immunofluorescence assays confirm that the positive rates of OCT4 and NANOG expression are >95% and the positive rate of SSEA-4 is >90%. Cell confluence should reach 70-80%, at which point cells exhibit typical colony morphology with clear edges and a high nucleocytoplasmic ratio.

[0012] 1.2 Optimization of Culture Medium Composition

[0013] The basal medium was DMEM / F12 (Gibco, 11330032) supplemented with 1× non-essential amino acids (NEAA, Gibco 11140050) and 1× N2 supplement (Gibco 17502048). Key inducing factors included:

[0014] LDN193189 (Stemgent, 04-0074): 100 nM, selectively inhibits the BMP signaling pathway;

[0015] SB431542 (Tocris, 1614): 10 μM, specifically inhibits the TGF-β / Activin / Nodal pathway;

[0016] The pH of the culture medium should be maintained at 7.2-7.4, and the osmotic pressure at 290±5 mOsm / kg. Freshly prepared culture medium should be used daily. Before replacing the medium, the petri dishes should be equilibrated at 37℃ and 5% CO2 for at least 30 minutes.

[0017] 1.3 Morphological Dynamics

[0018] Significant changes in cell morphology can be observed within 24 hours of the initiation of differentiation:

[0019] D0-D1: The edges of cell colonies begin to become blurred, and the intercellular spaces slightly increase;

[0020] D2-D3: Obvious epithelial-mesenchymal transition (EMT) features appear, with cells gradually changing from polygonal to spindle-shaped;

[0021] D4-D5: Formation of typical neuroepithelial-like structures, exhibiting a "rose wreath" arrangement;

[0022] Under an inverted microscope, a uniform layer of neuroepithelial membrane should form by day 5, approximately 3-5 cell layers thick. Extensive cell shedding or the formation of cystic structures indicates that the induction conditions need optimization.

[0023] 1.4 Expression profiles of molecular markers

[0024] Monitoring the expression dynamics of key genes using qRT-PCR and immunofluorescence staining:

[0025] D2: PAX6 expression begins to be upregulated (approximately 15-20 times), and SOX1 expression increases by 5-8 times;

[0026] D3-D4: Expression of the neuroepithelial marker N-Cadherin (CDH2) was significantly enhanced, with clear membrane localization;

[0027] D5: Formation of continuous neuroepithelial-like structures, with a PAX6 positivity rate of 85±5% and a SOX1 positivity rate of 75±7%;

[0028] Simultaneously, it is necessary to monitor the downregulation of pluripotency markers:

[0029] OCT4 expression should decrease to 10-15% of the initial level by day 3;

[0030] NANOG expression should be <5% of the initial level at day 5.

[0031] 1.5 Key Parameter Control

[0032] To ensure induction efficiency, the following parameters must be strictly controlled:

[0033] Cell density: Initial density less than 0.8 × 10⁻⁶ 5 / cm 2 This can lead to incomplete induction, exceeding 1.2 × 10⁻⁶. 5 / cm 2 It can easily cause central necrosis;

[0034] Temperature fluctuations: The incubator temperature should be kept stable at 37±0.2℃. Temperatures exceeding 37.5℃ will significantly reduce induction efficiency.

[0035] Oxygen partial pressure: Optimal results can be obtained under normal culture conditions (20% O2), without the need for hypoxia treatment;

[0036] Culture medium replacement: It must be changed daily. Delayed medium replacement will lead to increased cell death (>15%).

[0037] 1.6 Quality Control Standards

[0038] The following quality standards must be met during D5 to proceed to the next stage:

[0039] Cell viability >95% (as determined by Calcein-AM / PI double staining); PAX6+ / SOX1+ double positive cell proportion >80%; OCT4 positive cell proportion <2%; no obvious spontaneous differentiation areas (such as squamous epithelial-like or fibroblast-like cell aggregations).

[0040] 1.7 Common Problem Solutions

[0041] The following problems and solutions may be encountered in actual operation:

[0042] Low induction efficiency: Check the activity of growth factors. LDN193189 and SB431542 are recommended to be aliquoted and stored at -80℃ to avoid repeated freeze-thaw cycles.

[0043] Extensive cell shedding: This is usually caused by insufficient inoculation density or unstable pH of the culture medium.

[0044] Heterogeneous differentiation: The presence of SOX17+ endoderm cells indicates insufficient BMP inhibition, and the LDN concentration can be appropriately increased to 150 nM.

[0045] The key to success in this stage lies in establishing a uniform population of neural progenitor cells, laying the foundation for subsequent midbrain specialization. Through the standardized procedures described above, high-quality mesodermal neural progenitor cells can be obtained within 5 days, whose transcriptomic characteristics are highly similar to those of the developing neural plate.

[0046] 2. Midbrain pattern establishment period (D6-D12):

[0047] D6-D9: Low concentration of SHH activation (0.1-0.3μM Purmorphamine) + FGF8 (20-50ng / ml) induces midbrain progenitors;

[0048] D10-D12: High concentrations of SHH (0.5-1μM) + FGF8 (50-100ng / ml) enhance the ventral midbrain properties;

[0049] The midbrain pattern establishment period is a critical stage that determines the specificity of differentiated cell regions. By precisely regulating the spatiotemporal activation of the SHH (Sonic Hedgehog) and FGF8 (Fibroblast Growth Factor 8) signaling pathways, neural progenitor cells can be directed to differentiate into midbrain dopaminergic neuron precursors.

[0050] 2.1 Initial Conditions

[0051] Before proceeding to this stage, it is necessary to ensure that the neural progenitor cells of D5 meet the following criteria:

[0052] PAX6 positivity rate ≥80%; SOX1 positivity rate ≥75%; cells exhibit typical neuroepithelial-like structure with no obvious fibroblast-like cell contamination; cell viability >95% (Calcein-AM / PI detection);

[0053] The culture medium was switched from DMEM / F12+N2 during the neural induction phase to Neurobasal+B27 (Gibco, 17504044) to support the survival and specialization of neuronal precursors.

[0054] 2.2 Sequential Activation Strategy for Signal Path

[0055] The core of this stage lies in the gradient regulation of SHH and FGF8, simulating the spatiotemporal dynamics of embryonic midbrain development:

[0056] 2.2.1D6-D9: Preliminary Specialization of Midbrain Precursor

[0057] SHH pathway activation: Ventralization was induced using a low concentration of Purmorphamine (SHH agonist, 0.2±0.05μM) to avoid premature high concentrations of SHH leading to non-midbrain fate (such as basal forebrain neurons);

[0058] FGF8 Supplement: Adding recombinant human FGF8b (20-50 ng / ml, R&D Systems, 423-F8) promotes midbrain-hindbrain boundary specialization.

[0059] Key morphological changes:

[0060] D7-D8: Cells gradually aggregate into clusters, forming neural tube-like structures;

[0061] D9: Otx2 (midbrain marker) expression was significantly upregulated (qRT-PCR detection, >20-fold increase);

[0062] 2.2.2D10-D12: Specialized enhancement of the ventral midbrain

[0063] Increased SHH concentration: Purmorphamine increased to 0.8±0.1 μM, promoting ventral midbrain fate (future substantia nigra).

[0064] FGF8 concentration optimization: FGF8b was increased to 80±10 ng / ml, further stabilizing midbrain properties.

[0065] Key molecular markers:

[0066] The positive rate of LMX1A (a key transcription factor for midbrain dopamine neurons) should reach 60±8% on day 12.

[0067] FOXA2 (ventral midbrain marker) and LMX1A co-expression rate >50%

[0068] If GBX2 (a marker in the hindbrain) is expressed, it indicates that the FGF8 concentration is too high and needs to be adjusted to 50-70 ng / ml.

[0069] 2.3 Optimization of Culture Conditions

[0070] Medium replacement frequency: Replace with fresh medium every 48 hours to avoid growth factor degradation affecting induction efficiency.

[0071] Cell density control: Maintain at 1.5-2 × 10⁻⁶ 5 cells / cm 2 Too low a density leads to uneven signal response, while too high a density can easily form necrotic centers.

[0072] Oxygen tension: Standard culture conditions (20% O2) are sufficient; hypoxia treatment is not required.

[0073] 2.4 Molecular Validation and Quality Control

[0074] Monitoring key gene dynamics using immunofluorescence and qRT-PCR:

[0075] D8: The proportion of Otx2+ cells should be >70%, and En1 (midbrain-hindbrain boundary marker) should begin to be expressed.

[0076] D10: The proportion of Foxa2+ cells should be >50%, and the expression of Nkx6.1 (non-midbrain ventral marker) should be <5%.

[0077] D12: Ideally, the proportion of LMX1A+ / FOXA2+ double positive cells should be ≥60%, and PAX6 expression should be reduced to <10% (indicating successful exit from the forebrain fate).

[0078] 2.5 Common Problems and Solutions

[0079] Problem 1: Insufficient ventralization (low proportion of FOXA2+ cells)

[0080] Possible causes: Insufficient SHH activity or low cell density.

[0081] Countermeasures: Increase Purmorphamine to 1 μM, or check cell seeding density.

[0082] Question 2: Ectopic hindbrain differentiation (appearance of GBX2+ cells)

[0083] Possible causes: FGF8 concentration is too high or the treatment time is too long.

[0084] Countermeasures: Shorten the FGF8 exposure time (D6-D10 only), or reduce the concentration to 50 ng / ml.

[0085] Question 3: Necrosis at the center of cell clusters

[0086] Possible causes: cells are too densely packed or the medium is not changed sufficiently.

[0087] Countermeasures: Mechanically disperse large cell clusters, or switch to suspension culture (optimization using a rotating bioreactor).

[0088] 2.6 Stage Completion Standards

[0089] The following conditions must be met during D12 to proceed to the next stage:

[0090] 1. Phenotypic criteria: LMX1A+ / FOXA2+ double positivity ≥60%; Otx2+ proportion ≥80%; residual PAX6+ cells <10%;

[0091] 2. Survival rate: >90% (proportion of PI-negative cells);

[0092] 3. Uncontaminated differentiation: Nkx2.1 (basal forebrain marker) or GBX2 positive cells <3%.

[0093] This stage successfully specialized neural progenitors into midbrain dopaminergic neuronal progenitors by precisely simulating the dynamic changes in developmental signaling pathways, laying the foundation for subsequent expansion and terminal differentiation.

[0094] 3. Precursor amplification phase (D13-D20):

[0095] Removing FGF8 and maintaining moderate SHH (0.3-0.5 μM) + Wnt activation (CHIR99021, 3 μM) promotes the proliferation of mDA precursors;

[0096] The precursor expansion phase is a crucial stage for establishing a stable, scalable population of midbrain dopaminergic neuron precursor cells. The main goal of this phase is to achieve moderate expansion of precursor cells while maintaining cell region specificity, and to avoid fate shifts or genomic instability caused by excessive proliferation.

[0097] 3.1 Key Adjustments for Phase Transition

[0098] Starting from day 13, the following important adjustments need to be made to the culture medium system:

[0099] FGF8 removal: Completely stop adding FGF8b to prevent the maintenance of hindbrain characteristics due to continuous activation;

[0100] Adjustment of SHH concentration: Purmorphamine concentration was reduced to 0.4±0.05 μM to maintain a moderate ventralization signal;

[0101] Activation of the Wnt pathway: CHIR99021 (3.0±0.2μM, Tocris 4423) was added to mimic the natural Wnt signaling during midbrain development;

[0102] The culture medium will still be Neurobasal+B27, but the following new components need to be added:

[0103] Ascorbic acid (50 μg / ml, Sigma A4544): Promotes precursor cell survival;

[0104] Laminin (1 μg / ml, Sigma L2020): Improves cell adhesion;

[0105] Penicillin-streptomycin (1×, Gibco 15140122): to prevent contamination;

[0106] 3.2 Cell dynamic changes and expansion characteristics

[0107] 3.2.1 Morphological Evolution

[0108] D13-D15: Cell clusters gradually loosen, and radially arranged protrusions appear;

[0109] D16-D18: Forms typical neurosphere-like structures with a diameter controlled between 100-200 μm;

[0110] D19-D20: Cell density increases, requiring appropriate passage (1:2 fractionation recommended);

[0111] 3.2.2 Proliferation Kinetic Parameters

[0112] Population doubling time: 38±4 hours;

[0113] Total amplification fold: 5.2 ± 0.8 times (D13-D20);

[0114] Mitotic index: 12 ± 3% as determined by pH3 immunostaining.

[0115] 3.3 Regulation of expression of key molecular markers

[0116] The following biomarkers were monitored using multiparameter flow cytometry:

[0117] Proliferation markers: Ki67 positivity rate should be maintained at 60±8% at D15; Nestin expression should be persistently positive (>90%).

[0118] Region-specific markers: LMX1A expression remained at 65±7%; FOXA2 expression increased slightly to 70±6%; CORIN (ventral midbrain progenitor marker) began to be expressed (reaching 30±5% at D18);

[0119] Differentiation inhibitory factor: HES5 expression should be maintained at a medium-high level (Ct value 24-26) as detected by qPCR.

[0120] 3.4 Optimization of Culture Conditions

[0121] 3.4.1 Physical Parameter Control

[0122] Oxygen tension: Maintain 20% O2, no hypoxia required;

[0123] CO2 concentration: strictly controlled at 5.0±0.2%;

[0124] Humidity: >95% to prevent culture medium evaporation;

[0125] Mechanical stimulation: Gently shake (50 rpm, 5 minutes) every 24 hours to prevent excessive cell aggregation.

[0126] 3.4.2 Culture medium replacement strategy

[0127] Fluid replacement frequency: 60% of the volume should be replaced every 48 hours;

[0128] Fresh addition: Add CHIR99021 (3μM) and ascorbic acid each time the medium is changed;

[0129] pH monitoring: Maintain at 7.3±0.1 (adjust immediately if outside the range).

[0130] 3.5 Key Quality Control Indicators

[0131] The following parameters need to be monitored daily:

[0132] 3.5.1. Cell state:

[0133] Survival rate >92% (trypan blue exclusion method); apoptotic cell ratio <5% (Annexin V assay).

[0134] 3.5.2. Purity Index:

[0135] The percentage of LMX1A+ / FOXA2+ double positivity is >65%; the positivity rate of non-specific markers (such as PAX6, GBX2) is <3%.

[0136] 3.5.3. Genetic stability:

[0137] G-banding karyotype analysis confirmed no major abnormalities; mitochondrial membrane potential detection (JC-1 staining) confirmed metabolic health.

[0138] 3.6 Common Problems and Optimization Solutions

[0139] Question 1: Insufficient proliferation (expansion fold < 3)

[0140] Possible causes: (1) Decreased activity of CHIR99021 (it is recommended to aliquot and store, and avoid repeated freeze-thaw cycles); (2) Cell density is too low (maintain 1-2×10⁻⁶ cells / year). 5 / cm 2 ).

[0141] Solution: Freshly prepare CHIR99021; add bFGF (10ng / ml) to temporarily promote proliferation.

[0142] Question 2: Premature differentiation (appearance of TH+ cells)

[0143] Possible causes: Low SHH concentration; insufficient ascorbic acid

[0144] Solution: Increase Purmorphamine to 0.5 μM; increase ascorbic acid to 75 μg / mL.

[0145] Question 3: Excessive cell aggregation

[0146] Possible causes: Uneven coating of laminin; insufficient mechanical disturbance.

[0147] Solution: Optimize the coating conditions (37℃ for at least 2 hours); increase the shaking frequency to once every 12 hours.

[0148] 3.7 Stage Completion Standards

[0149] The following standards should be met at D20:

[0150] 1) Quantity standards: The total number of cells reaches 5 ± 0.5 times the initial amount; the survival rate is >90%.

[0151] 2) Quality indicators: LMX1A+ / FOXA2+ / CORIN+ triple positivity >55%; Ki67 positivity rate decreased to 40±5% (indicating the beginning of cell cycle exit).

[0152] 3) Safety: The karyotype test was normal, and no abnormalities such as ectopic or inverted karyotypes were found; the sterility test, endotoxin test, and mycoplasma test were all negative.

[0153] This phase successfully established an expandable midbrain dopaminergic precursor cell bank, whose transcriptomic characteristics are highly similar to those of developing human midbrain precursor cells (Pearson correlation coefficient > 0.85), providing a high-quality cell source for subsequent terminal differentiation. A key breakthrough was achieved by strictly controlling the balance of Wnt and SHH signaling, maintaining region specificity while expanding the cell bank.

[0154] 4. Terminal differentiation phase (D21-D35):

[0155] SHH was completely removed, and BDNF (20 ng / ml), GDNF (20 ng / ml), TGF-β3 (1 ng / ml), dbcAMP (0.5 mM), and DAPT (10 μM) were added to promote maturation.

[0156] Terminal differentiation is a crucial stage in the transformation of proliferating mesencephalic progenitor cells into functional dopaminergic neurons. During this stage, precise regulation of microenvironmental factors guides cells out of the cell cycle, establishing a mature neuronal phenotype and developing complete electrophysiological characteristics.

[0157] 4.1 Key Shifts in the Initiation of Differentiation

[0158] The following core adjustments will be implemented starting with D21:

[0159] Mitosis signal removal: Complete removal of CHIR99021 and Purmorphamine;

[0160] The neurotrophic factor combination was supplemented with: BDNF (20±2 ng / ml, PeproTech 450-02); GDNF (20±2 ng / ml, PeproTech 450-10); TGF-β3 (1.0±0.2 ng / ml, R&D Systems 243-B3).

[0161] Differentiation promoters: dbcAMP (0.5±0.05mM, Sigma D0627); DAPT (10±1μM, Tocris 2634) inhibited Notch signaling;

[0162] The culture medium was adjusted to Neurobasal-A medium supplemented with: B27 antioxidant-free formulation (Gibco A1895601); ascorbic acid (100±10μg / ml); glutamine (2mM).

[0163] 4.2 Cell Morphology and Structural Development

[0164] 4.2.1 Typical morphological evolution

[0165] D21-D23: Cell bodies shrink, with diameter decreasing to 10-15 μm; primary neurites extend to 50-100 μm in length, and distinct growth cone structures appear.

[0166] D24-D28: Secondary branching occurs, exhibiting a typical bipolar / multipolar morphology; synaptophysin is expressed punctately.

[0167] D29-D35: Mature neuronal networks are established; axon length can reach 300-500μm; dendritic spines begin to form.

[0168] 4.2.2 Cytoskeleton Reorganization

[0169] Immunofluorescence revealed that βIII-tubulin (TUJ1) fibers gradually became denser; microtubule-associated protein 2 (MAP2) was specifically distributed in dendrites; and neurofilament protein (NF-H) was polarly distributed in axons.

[0170] 4.3 Dynamic Expression of Molecular Markers

[0171] 4.3.1 Acquisition of Dopaminergic Characteristics

[0172] D21-D25: Tyrosine hydroxylase (TH) expression begins (approximately 15±3% at D23); Aromatic amino acid decarboxylase (AADC) expression begins;

[0173] D26-D30: The proportion of TH+ cells rapidly increased to 60±7%; dopamine transporter (DAT) membrane localization;

[0174] D31-D35: TH+ / TUJ1+ double positivity reached 85±5%; Vesicular monoamine transporter 2 (VMAT2) functional expression.

[0175] 4.3.2 Subtype-specific markers

[0176] A9 subtype characteristics (substantia nigra compacta): GIRK2 positivity rate approximately 65±8%; CALBINDIN negative;

[0177] A10 subtype characteristics (ventral tegmental area): The positive rate of CALBINDIN is approximately 35±6%.

[0178] 4.4 Development of Electrophysiological Characteristics

[0179] Patch-clamp technique was used to record (D30-D35): (1) Resting membrane potential: -55±3mV; (2) Action potential amplitude: 85±10mV; (3) Sodium current density: 120±15pA / pF; (4) Spontaneous discharge frequency: 0.5-2Hz; (5) Postsynaptic current detection rate: >70%.

[0180] 4.5 Optimization of Culture Conditions

[0181] 4.5.1 Physical Parameters

[0182] Matrix optimization: double coating with poly-D-lysine (50 μg / ml) + laminin (5 μg / ml);

[0183] Inoculation density: 1.5 × 10⁻⁶5 / cm 2 (Too low a level leads to insufficient synapse formation);

[0184] Medium replacement strategy: Replace 50% of the culture medium every 48 hours for the first 10 days; after D30, replace 30% of the culture medium every 72 hours. 4.5.2 Environmental control

[0185] Temperature fluctuation: <±0.3℃

[0186] CO2 stability: 5.0 ± 0.1%

[0187] Humidity control: >95% to prevent osmotic pressure changes

[0188] 4.6 Functional Verification

[0189] 4.6.1 Neurotransmitter Release

[0190] HPLC detection (D35):

[0191] Basal dopamine release: 5.2 ± 0.8 ng / 10 6 cells / h;

[0192] High potassium stimulation (50 mM) induced release: an increase of 3.5 ± 0.6 times;

[0193] Reuptake efficiency: >75% (Nomifensine inhibition assay).

[0194] 4.6.2 Synaptic function

[0195] Co-culture experiment (1:3 ratio with striatal neurons):

[0196] Synaptic vesicle circulation detection (FM1-43 staining positive);

[0197] Colocalization analysis of postsynaptic density protein 95 (PSD95);

[0198] Functional connectivity formation rate: 62±8%.

[0199] 4.7 Quality Control Standards

[0200] At D35, the following should be satisfied:

[0201] 1) Phenotypic criteria: TH+ / TUJ1+ double positivity >85%; MAP2+ neuron proportion >90%;

[0202] 2) Functional criteria: Action potential evoked success rate >80%; Dopamine release >4 ng / 10 6 cells / h;

[0203] 3) Safety: Residual proliferating cells (Ki67+) <1%; no abnormal karyotype, no translocation or inversion observed.

[0204] 4.8 Key Considerations

[0205] 1) Control of oxidative stress: Add superoxide dismutase (50 U / ml) starting from D25; maintain the antioxidant state of the culture medium (containing 0.5×B27 standard formula);

[0206] 2) Metabolic support: Sodium pyruvate (1mM) enhances energy metabolism; lactate levels are maintained within the range of 2-4mM;

[0207] 3) Microbial control: Weekly mycoplasma testing (PCR method); antibiotics only used for the first two weeks.

[0208] This phase successfully yielded midbrain dopaminergic neurons with complete functional characteristics. Their molecular features, electrophysiological properties, and neurotransmitter release capabilities are highly similar to those of adult midbrain dopaminergic neurons, providing an ideal cell product for cell therapy of Parkinson's disease. Through phased signal withdrawal and neurotrophic support, a differentiation efficiency of >85% and good functional maturity were achieved.

[0209] 5. Functional Maturity Stage (D36-D50):

[0210] Add a neuromodulator (such as Valproic acid 0.5mM) and an antioxidant (such as Vitamin E 10μM).

[0211] The functional maturation phase is a crucial stage in ensuring that the differentiated midbrain dopaminergic neurons (mDA) reach a fully functional state. This stage optimizes electrical activity, synapse formation, and metabolic homeostasis, enabling neurons to acquire the typical characteristics of adult mDA neurons, including stable spontaneous rhythmic firing, a mature synaptic network, and a sustained capacity for neurotransmitter synthesis.

[0212] 5.1 Key Adjustments to the Culture System

[0213] 5.1.1 Optimization of Culture Medium Composition

[0214] The basal culture medium was replaced with BrainPhys TM Neuron culture medium (STEMCELL Technologies 05790), supplemented with the following components:

[0215] (1) Neurotrophic factors: BDNF (20±1ng / ml), GDNF (20±1ng / ml), CDNF (10±1ng / ml, R&DSystems 5427-CD);

[0216] (2) Nervous activity modulators: Valproic acid (0.5±0.05mM, Sigma P4543), Forskolin (5±0.5μM, Tocris 1099);

[0217] (3) Antioxidant system: Vitamin E (10±1μM, Sigma T3251), Glutathione (5±0.5mM, Sigma G6013).

[0218] 5.1.2 Matrix Environment Enhancement

[0219] Micromode culture system was used: microfluidic channel culture (channel width 50 μm).

[0220] Matrix composition: poly-L-ornithine (0.1 mg / ml), laminin (10 μg / ml), fibronectin (2 μg / ml) 5.2 Electrophysiological properties matured

[0221] 5.2.1 Evolution of Action Potential Parameters

[0222] Patch-clamp recordings (n≥30 cells / time point):

[0223] parameter D36-D40 D41-D45 D46-D50 Resting potential -52±3mV -58±2mV -62±2mV Threshold potential -38±4mV -42±3mV -45±2mV Peak potential amplitude 82±8mV 88±6mV 92±5mV Post-hyperpolarization 12±3mV 15±2mV 18±2mV Discharge frequency 1.2±0.5Hz 2.0±0.6Hz 2.5±0.7Hz

[0224] 5.2.2 Ion Channel Development

[0225] qPCR detection showed changes in the expression of key channel subunits:

[0226] Nav1.3 (SCN3A): decreased to 30 ± 5% of D35.

[0227] Nav1.6 (SCN8A): Increased to 3.2 ± 0.5 times D35.

[0228] Kv4.3 (KCND3): Increased to 2.8 ± 0.4 times that of D35.

[0229] 5.3 Synaptic Network Establishment

[0230] 5.3.1 Synaptic Structure Development

[0231] Quantitative analysis using confocal microscopy:

[0232] (1) Synaptic density (Synaptophysin+ / PSD95+puncta):

[0233] D36: 12±3 / 100μm dendrites; D50: 28±5 / 100μm dendrites

[0234] (2) Thickness of the postsynaptic dense region:

[0235] D40: 25±3nm (measured by electron microscopy); D50: 42±5nm

[0236] 5.3.2 Functional connectivity verification

[0237] Microelectrode array (MEA) recording:

[0238] (1) Network burst activity: D40: occasional (<0.1 / min); D50: regular (2.5±0.8 / min)

[0239] (2) Synchronicity Index (SI): increased from 0.15±0.05 on D36 to 0.38±0.07 on D50. 5.4 Metabolic characteristics maturation

[0240] 5.4.1 Energy Metabolism Conversion

[0241] Seahorse analysis shows:

[0242] (1) Basal oxygen consumption rate (OCR):

[0243] D36: 85±8pmol / min; D50: 120±10pmol / min

[0244] (2) ECAR: decreased to 60±7% of D36.

[0245] 5.4.2 Dopamine metabolic homeostasis

[0246] HPLC-MS / MS detection:

[0247] Dopamine content: 15±2 pg / cell

[0248] DOPAC / DA ratio: 0.18 ± 0.03

[0249] Synthetic enzyme activity:

[0250] TH activity: 12±2 nmol DOPA / mg protein / h

[0251] AADC activity: 8±1nmol DA / mgprotein / h

[0252] 5.5 Fine-tuning of cultivation conditions

[0253] 5.5.1 Physical Parameters

[0254] Mechanical stimulation: periodic stretching (5% strain, 0.1 Hz); fluid shear force (0.2 dyne / cm). 2 )

[0255] Gaseous environment: O2 maintained at 18-20%, CO2 at 5.0±0.1%;

[0256] Real-time pH monitoring (7.35±0.05)

[0257] 5.5.2 Fluid Replacement Strategy

[0258] Replace 50% of the culture medium every 72 hours;

[0259] Gradual nutritional supplementation:

[0260] D36-D40: Supplement with 2× amino acids

[0261] D41-D50: Supplement with 1× amino acid + 3× vitamins

[0262] 5.6 Functional Verification Standard

[0263] 5.6.1 Electrophysiological qualification standards

[0264] Action potential amplitude > 85mV; input resistance > 500MΩ; spontaneous discharge frequency 1.5-3.5Hz; success rate of inducing postsynaptic current > 75%.

[0265] 5.6.2 Biochemical indicator thresholds

[0266] TH immunofluorescence intensity >5000 AU (normalized); DAT membrane localization rate >80%; dopamine release >6 ng / 10 6 cells / h.

[0267] 5.7 Phase Completion Assessment

[0268] For D50, all of the following criteria must be met:

[0269] 1) Morphological criteria: intact neuronal polarity (axon / dendritic ratio >3:1); synaptic density >25 / 100μm.

[0270] 2) Functional standards: Synchronous network activity frequency > 2 / min; Dopamine reuptake efficiency > 80%.

[0271] 3) Stability criteria: TH expression fluctuation <15% for 5 consecutive days; cell death rate <1% / day

[0272] This stage, by simulating the mechanical and electrochemical signals of the in vivo microenvironment, enabled in vitro differentiated mDA neurons to achieve functional maturity comparable to adult neurons. Transcriptome analysis showed that the gene expression profile of D50 cells had 89±4% similarity to primary cultured human midbrain dopamine neurons (Pearson correlation coefficient), providing an ideal functional cell product for clinical transplantation.

[0273] Technical effect

[0274] 1. mDA neuron purity: 85-92% (TH+ / TUJ1+)

[0275] 2. 6-month survival rate after transplantation: 82.3 ± 5.7%

[0276] 3. Electrophysiological characteristics: >80% of cells exhibit mature action potentials and spontaneous discharge.

[0277] 4. Dopamine release: 5.2 ± 0.8 ng / 10^6 cells / h

[0278] 5. Improvement in animal behavior: Rotational asymmetry improved by 78.5 ± 9.2%.

[0279] originality

[0280] 1. Precise control of timing signal paths

[0281] This invention establishes for the first time a dynamic gradient activation mode for the SHH and FGF8 pathways. By adjusting the concentration in stages (e.g., gradually increasing SHH from 0.2 μM to 0.8 μM and then decreasing it to 0.4 μM), it accurately simulates the natural signal changes during embryonic midbrain development. Compared to the traditional continuous stimulation method, this strategy significantly improves the region specificity of midbrain dopaminergic neurons (mDA) (Otx2+ > 90%, non-target region markers < 3%).

[0282] 2. Coordinated removal strategy of Wnt pathway and SHH

[0283] During the precursor expansion phase (D13-D20), a novel combination of moderate concentrations of SHH (0.4 μM) and Wnt for activation (CHIR99021, 3 μM) was employed to maintain precursor proliferation capacity while avoiding excessive ventralization. Complete withdrawal of SHH during the terminal differentiation phase (after D21) was then demonstrated for the first time to promote A9 subtype (substantia nigra pars compacta) specific maturation (GIRK2+ ratio reaching 65±8%).

[0284] 3. Electro-chemical-mechanical coordinated regulation during the functional maturity stage

[0285] Mechanical stretching (5% strain) and microfluidic culture were introduced during the D36-D50 stage, combined with Valproic acid (0.5mM) and CDNF (10ng / ml), to achieve for the first time synchronous firing of in vitro neuronal networks (2.5±0.8 bursts / min), with synaptic density (28±5 / 100μm) close to the in vivo level.

[0286] 4. Optimization of cryopreservation and resuscitation adaptability

[0287] The innovative discovery is that D35 cells, after being pretreated with hypoxia (1% O2, 24h) and then cryopreserved, have a recovery survival rate of 88.3% (vs. 72% with conventional cryopreservation), and TH expression is maintained at >95%, solving the industry problem of long-term storage of cell products.

[0288] Beneficial effects

[0289] 1. Ultra-high differentiation efficiency and purity

[0290] The purity of mDA neurons reaches 85-92% (TH+ / TUJ1+), far exceeding existing technologies (usually 60-75%).

[0291] The A9 / A10 subtype ratio is controllable (7:3), which is better than the random differentiation of traditional methods (4:6).

[0292] 2. Breakthrough improvement in post-transplant survival rate

[0293] The 6-month survival rate in the animal model was 82.3 ± 5.7% (reported in the literature is usually <60%), mainly attributed to:

[0294] Adding CDNF during terminal differentiation (to reduce apoptosis)

[0295] Hypoxia pretreatment before cryopreservation (to enhance stress resistance)

[0296] 3. Functional maturity has significantly improved.

[0297] Electrophysiological characteristics: Spontaneous firing frequency of 2.5±0.7Hz, which is very close to that of adult substantia nigra neurons (2-4Hz).

[0298] Dopamine release: 5.2 ± 0.8 ng / 10 6 The number of cells / h reached 90% of the level of primary neurons.

[0299] 4. Standardization and scalability

[0300] At the pilot-scale of the 10-layer cell factory, the batch-to-batch TH+ cell ratio difference was <5% (CV value 3.8%), meeting GMP production requirements.

[0301] Even after replacing animal-derived matrix components (such as Synthemax II), the differentiation efficiency remains >80%, reducing clinical risks.

[0302] 5. Broad-spectrum applicability

[0303] Five iPS cell lines (including those derived from Parkinson's patients) were successfully validated, with the TH+ ratio fluctuating by only 6.3%, demonstrating that the protocol is insensitive to genetic background.

[0304] The functional recovery rate of cryopreserved cells after thawing is >93%, which facilitates the establishment of clinical-grade cell banks.

[0305] Overall advantages: This approach surpasses existing technologies in differentiation efficiency, cell survival, functional maturation, and clinical translation adaptability, providing the first cell replacement therapy for Parkinson's disease that combines high efficiency and standardization potential. Attached Figure Description

[0306] Figure 1 Differentiation process timeline diagram

[0307] Figure 2 SHH / FGF8 concentration gradient optimization results

[0308] Figure 3 : Markers at different stages express dynamics

[0309] Figure 4 Cell survival and integration after transplantation

[0310] Figure 5 Behavioral improvement curve Detailed Implementation

[0311] Example 1: Step-by-step differentiation process

[0312] 1. hPSC culture: hPSCs were kept in an undifferentiated state in mTeSR1 medium at a density of 1×10⁻⁶ at the time of inoculation. 5 / cm 2

[0313] 2. Neural induction (D0-D5):

[0314] Basic culture medium: DMEM / F12 + NEAA + N2 supplement

[0315] Add: LDN193189 (100 nM) + SB431542 (10 μM)

[0316] Change the fluid daily; neuroepithelial-like structures form on day 5.

[0317] 3. Midbrain pattern establishment (D6-D12):

[0318] D6-D9: Add Purmo (0.2μM) + FGF8 (30ng / ml)

[0319] D10-D12: Purmo(0.8μM)+FGF8(80ng / ml)

[0320] Morphological changes: Otx2+ (>90%), a midbrain marker, is present.

[0321] 4. Precursor amplification (D13-D20):

[0322] When the culture medium was changed to Neurobasal + B27 + CHIR (3μM) + Purmo (0.4μM), cell proliferation was 5-7 times, and Lmx1a+ / Foxa2+ double positivity was >85%.

[0323] 5. Terminal differentiation (D21-D35):

[0324] Remove Purmo and CHIR

[0325] Add neurotrophic factor combination

[0326] The proportion of TH+ cells reached 60% on day 28 and 85% on day 35.

[0327] 6. Mature functionality (D36-D50):

[0328] Add neuronal activity enhancer

[0329] Formation of complex neural synaptic networks

[0330] Example 2: Animal transplantation experiment

[0331] 1. Cell preparation: Collect cells at day 35 and prepare a single-cell suspension (5 × 10⁻⁶ cells / mL). 4 / μl)

[0332] 2. Animal model: SD rats with unilateral 6-OHDA lesions

[0333] 3. Transplantation procedure:

[0334] Stereotactic injection into the striatum (AP+1.0, ML±2.5, DV-4.5)

[0335] 2μl per spot, 4 spots in total

[0336] The control group was injected with an equal volume of PBS

[0337] 4. Result Evaluation:

[0338] One month post-transplantation: TH+ cell survival rate 83.7 ± 6.2%

[0339] At 6 months: survival rate 82.3±5.7%, no tumor formation observed.

[0340] Behavioral improvement: Apomorphine-induced twirl decreased by 78.5%.

[0341] Electrophysiological recordings show functional synaptic connections

[0342] Example 3: Key Parameter Optimization

[0343] Determine the optimal parameter combination through orthogonal experiments:

[0344] 1. SHH concentration gradient: 0.1-1 μM Purmo

[0345] 2. FGF8 time window: D6-D12 is optimal

[0346] 3. Timing of Wnt activation: Premature activation leads to heterotopic differentiation; D13 activation is optimal.

[0347] 4. SHH Removal Timing: Removing SHH at D21 will yield the optimal A9 / A10 subtype ratio (7:3).

[0348] Example 4: Small-scale preclinical validation (laboratory level)

[0349] Experimental Objective: To verify the reproducibility of the differentiation protocol under standard laboratory conditions and the basic characteristics of the cell products. Materials and Methods:

[0350] 1. Cell source: iPS cell line HDFa-iPS01 (ATCC ACS-1011) was used.

[0351] 2. Cultivation equipment:

[0352] 37℃, 5% CO2 incubator (Thermo Scientific Heracell 150i)

[0353] Biosafety cabinet (Baker SG403)

[0354] 3. Differentiation scale: Cultured in 6-well plates, initial cell quantity 2×10⁶ 5 / hole

[0355] 4. Key steps:

[0356] Change the culture medium strictly according to the prescribed time sequence (error < 1 hour).

[0357] Daily recording of cell morphology (Nikon Eclipse Ti microscope)

[0358] Flow cytometry was performed at D15, D30, and D45 (BD FACSAria III).

[0359] Quality control:

[0360] Three repeat holes are set for each batch.

[0361] 10% of the samples were randomly selected for external karyotype analysis.

[0362] Reserve 10% of the cells for cryopreservation backup (programmed cooling to -150℃).

[0363] result:

[0364] Batch-to-batch differences (n=6):

[0365] TH+ cell percentage: 83.5% ± 3.2% (CV = 3.8%)

[0366] Survival rate: 91.2% ± 2.1%

[0367] Consistency of electrophysiological parameters:

[0368] Resting potential: -61.3±2.7mV

[0369] Action potential amplitude: 89.5 ± 6.2 mV

[0370] Example 5: Pilot Production Validation (Pre-GMP Study)

[0371] Experimental objective: To evaluate the stability of the protocol during large-scale culture.

[0372] Materials and Methods:

[0373] 1. Cultivation system:

[0374] Multilayer cell factory (Corning 10 layers, surface area 6320 cm²) 2 )

[0375] Closed automated culture system (CompacT SelecT)

[0376] 2. Increased scale:

[0377] Starting cell count: 5 × 10 7

[0378] Final yield: 2.1-2.4×10 8 TH+ cells

[0379] 3. Process control:

[0380] Online monitoring: DO (>80% saturation), pH (7.35±0.05)

[0381] Daily sampling and testing: Glucose (maintain 2-4 mM), lactate (<6 mM) Key adjustments:

[0382] Stirring speed: 35±2 rpm (D0-D20), reduced to 25 rpm (after D21); Irrigation rate: 0.5 RV / day before D15, 1.5 RV / day after D30.

[0383] result:

[0384] Amplification effect analysis:

[0385] The difference in metabolic parameters was <15% (compared to the well plate control).

[0386] TH expression level difference: +7.2% ± 3.1% (possibly due to improved hydrodynamics) Production cost estimate:

[0387] Every 10 6 TH+ cell line consumables cost: $82.50 ± $5.30

[0388] Example 6: Validation of therapeutic effect in animal models

[0389] Experimental Objective: To evaluate the therapeutic effect of cell products in a Parkinson's disease model. Materials and Methods:

[0390] 1. Animal models:

[0391] 6-OHDA unilateral injury SD rats (n=30)

[0392] Rotational behavior baseline: >7 revolutions / minute (apomotorine induction)

[0393] 2. Transplantation protocol:

[0394] Cell dose: 1×10 5 TH+ cells / μl

[0395] Injection site: striatum (AP+1.0, ML±2.8, DV-4.5)

[0396] Injection volume: 3 μl / point, 2 points / side

[0397] 3. Evaluation Methods:

[0398] Behavioral Science: Weekly Rotation Test

[0399] Imaging: microPET ([18F]FDOPA uptake)

[0400] Histology: Samples were taken 1 / 3 / 6 months post-transplantation.

[0401] Key parameters:

[0402] Immunosuppressive regimen: Cyclosporine A (10 mg / kg / day) Cell pretreatment: Hypoxia preconditioning (1% O2, 24 h) Results:

[0403] Behavior improvement:

[0404] Rotation reduction at 8 weeks: 76.3% ± 8.2%

[0405] Duration of efficacy: ≥6 months

[0406] Cell survival:

[0407] 1 month: 83.5% ± 6.7%

[0408] 6 months: 79.2% ± 7.3%

[0409] Evidence of functional integration:

[0410] The host neuron's axonal projection into the transplant area increased 3.2 times, and the synaptic density recovered to 68% ± 9% of the normal side.

[0411] Example 7: Validation of Cryopreservation and Thawing (New)

[0412] Experimental Objective: To evaluate the quality stability of cell products after cryopreservation. Materials and Methods:

[0413] 1. Cryopreservation plan:

[0414] Cryopreservation timing: D35 (after terminal differentiation is complete)

[0415] Cryopreservation solution: 90% FBS + 10% DMSO

[0416] Procedure: -1℃ / min to -80℃, then transfer to liquid nitrogen. 2. Detection time points:

[0417] Thawing after 1 / 3 / 6 months of cryopreservation

[0418] Post-resuscitation assessment after 72 hours of culture

[0419] 3. Evaluation Indicators:

[0420] Survival rate (trypan blue + Calcein-AM)

[0421] TH expression (flow cytometry)

[0422] Electrophysiological function (patch clamp)

[0423] result:

[0424] Cryopreservation stability:

[0425] 6-month survival rate: 88.3% ± 3.1% (vs. fresh 92.1% ± 2.3%)

[0426] TH expression maintenance rate: 95.5% ± 4.2%

[0427] Function recovery:

[0428] Action potential parameter difference <10%

[0429] Dopamine release: 93% ± 7% before freezing

[0430] Example 8: Validation of suitability for different cell lines

[0431] Experimental objective: To verify the universality of the protocol for multiple iPS cell lines.

[0432] Materials and Methods:

[0433] 1. Test cell lines (n=5):

[0434] Healthy donor sources: HDFa-iPS01, BC1-iPS03

[0435] Parkinson's disease patients originate from: PD-iPS12 (SNCA mutation) and PD-iPS25 (LRRK2 mutation).

[0436] Commercialization series: ND41865 (Coriell)

[0437] 2. Standardized training:

[0438] Uniform vaccination density (1.2×10⁻⁶) 5 / cm 2 )

[0439] Synchronous Differentiation Timeline

[0440] 3. Difference Analysis:

[0441] RNA-seq comparison of key gene expression

[0442] Electrophysiological parameter statistics

[0443] result:

[0444] Differences in differentiation efficiency:

[0445] TH+ ratio range: 81.3%-87.6%

[0446] Patient-derived cells delayed by 12-18 hours

[0447] Mutation-specific characteristics:

[0448] SNCA mutant line: α-synuclein expression is 1.8 times higher; LRRK2 mutant line: neuronal neurite length is reduced by 15%.

[0449] Example 9: Alternative Matrix Test

[0450] Experimental objective: To evaluate animal-free culture protocols

[0451] Materials and Methods:

[0452] 1. Test matrix:

[0453] Traditional group: Matrigel (Corning 354230)

[0454] Alternative group:

[0455] Synthemax II (Corning 3535)

[0456] CELLstart(ThermoA1014201)

[0457] 2. Culture parameters:

[0458] Same cell source (HDFa-iPS01)

[0459] Parallel differentiation in 3 batches

[0460] 3. Key areas of testing:

[0461] Differentiation synchronicity (time series of biomarker expression)

[0462] Survival rate after transplantation (mice striatum transplantation)

[0463] result:

[0464] Differentiation efficiency:

[0465] TH+ ratio: Matrigel 84.2% vs Synthemax 80.7%

[0466] Maturity differences: MAP2 expression was similar (p>0.05)

[0467] Clinical application advantages:

[0468] Batch-to-batch variation reduced by 32%

[0469] Endotoxin level <0.1 EU / ml (meets pharmacopoeia standards)

[0470] All data from the examples were derived from at least three independent replicate experiments. The statistical analysis was performed using ANOVA with Tukey's post-hoc test, and p < 0.05 was considered statistically significant.

[0471] This invention establishes a precise developmental signal regulation pattern, achieving efficient differentiation of midbrain dopaminergic neurons, solving a key bottleneck problem in transplantation therapy, and has significant clinical application value.

Claims

1. A method for temporally activating the SHH and FGF8 signaling pathways to induce human pluripotent stem cells to differentiate into midbrain dopaminergic neurons, characterized in that, Includes the following steps: Neural induction phase (D0-D5): Neural progenitor cells were induced in a medium containing LDN193189 (100 nM) and SB431542 (10 μM); Midbrain pattern establishment period (D6-D12): D6-D9: Add Purmorphamine (0.1-0.3 μM) and FGF8b (20-50 ng / ml); D10-D12: Increase Purmorphamine to 0.5-1 μM and FGF8b to 50-100 ng / ml; Pre-amplification phase (D13-D20): FGF8b was removed, Purmorphamine (0.3-0.5 μM) was maintained, and CHIR99021 (3 μM) was added; Terminal differentiation phase (D21-D35): Purmorphamine and CHIR99021 were removed, and BDNF (20 ng / ml), GDNF (20 ng / ml), TGF-β3 (1 ng / ml), dbcAMP (0.5 mM) and DAPT (10 μM) were added; Functional maturation period (D36-D50): Neuronal maturation is promoted under conditions containing valproic acid (0.5 mM), CDNF (10 ng / ml), and mechanical stretch (5% strain).

2. The method according to claim 1, characterized in that, During the midbrain pattern establishment period (D6-D12), the concentration of Purmorphamine was 0.2±0.05μM and the concentration of FGF8b was 30±5ng / ml in the D6-D9 stage; and the concentration of Purmorphamine was 0.8±0.1μM and the concentration of FGF8b was 80±10ng / ml in the D10-D12 stage.

3. The method according to claim 1, characterized in that, The culture medium for the terminal differentiation phase (D21-D35) is a Neurobasal-A supplemented with B27 without antioxidants, and further contains ascorbic acid (100 μg / ml) and glutamine (2 mM).

4. The method according to claim 1, characterized in that, The functional maturity period (D36-D50) is conducted in a microfluidic channel system, with periodic fluid shear force (0.2 dyne / cm) applied. 2 ).

5. A population of midbrain dopaminergic neurons prepared by the method according to any one of claims 1-4, characterized in that: The proportion of tyrosine hydroxylase (TH) positive cells is ≥85%; The survival rate ≥80% 6 months after transplantation; Spontaneous discharge frequency 2-4Hz, dopamine release ≥5ng / 10 6 cells / h.

6. The midbrain dopaminergic neuron population according to claim 5, characterized in that, The ratio of GIRK2+ (A9 subtype) to CALBINDIN+ (A10 subtype) cells in the neuronal population was approximately 7:

3.

7. A method for cryopreserving midbrain dopaminergic neurons as described in claims 5-6, characterized in that, Includes the following steps: Cells were collected at D35 and pretreated with hypoxia (1% O2, 24h); After cooling to -80°C using a cryopreservation solution (90% FBS + 10% DMSO) at a rate of -1°C / min, the solution was transferred to liquid nitrogen. TH expression was maintained at a rate of ≥95% after resuscitation, and electrophysiological function was restored at a rate of ≥90%.

8. A cell preparation for the treatment of Parkinson's disease, comprising the midbrain dopaminergic neurons as described in claims 5-6, and a pharmaceutically acceptable carrier, wherein the cell concentration is 1 × 10⁻⁶. 5 -5×10 5 TH+ cells / μl.

9. The cell preparation according to claim 8, characterized in that, The formulation is injected into the patient's striatum via stereotactic injection, with a single injection volume of 2-5 μl and a cell survival rate of ≥80%.

10. The use of the method according to any one of claims 1-4 in the preparation of a cell therapy product for Parkinson's disease. illustrate Claim 1 is an independent claim, covering the core technical features of the entire step-by-step differentiation method. Claims 2-4 are dependent claims, further defining key parameters (concentration, culture system, etc.). Claims 5-6 protect the cell products obtained by this method and their properties. Claim 7 addresses the key derivative technology of cell cryopreservation. Claims 8-9 cover the clinical application protocol. Claim 10 specifies the application scenarios of the method.

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