Induction method of dopaminergic neural stem cells

Abstract

The invention discloses an induction method of dopaminergic neural stem cells, and relates to the technical field of cell culture and neurobiology, and the induction method comprises the following steps: inoculating neural stem cells into an induction culture medium, culturing under the conditions of 37 DEG C and 5% CO2, and carrying out induced differentiation for 6-14 days to obtain dopaminergic neurons; the induction culture medium takes DMEM / F12 as a basic culture medium, and contains an effective amount of the following components: human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, an epidermal growth factor (EGF) and a fibroblast growth factor (FGF). The induction culture medium and the method have the advantages that the survival of neural stem cells can be effectively supported; and the gene is guided to directionally differentiate towards the dopaminergic neuron lineage. The method is clear in operation flow and stable in differentiation process, the expression of the characteristic marker of the dopaminergic neuron can be detected in the obtained cell population, and a stable and reliable cell source is provided for related scientific research.

Description

Technical Field

[0001] This invention relates to the fields of cell culture and neurobiology, specifically to a method for inducing dopaminergic neural stem cells. Background Technology

[0002] Dopaminergic neurons are a type of neuron responsible for synthesizing and releasing the neurotransmitter dopamine. Their dysfunction or loss is closely related to neurological diseases such as Parkinson's disease. Dopaminergic neural stem cells refer to neural stem cells with the potential to differentiate into dopaminergic neurons. Their successful induction and expansion in vitro are a key foundation for disease model research, drug screening, and the development of cell replacement therapies.

[0003] Currently, existing methods for inducing neural stem cells to differentiate into dopaminergic neurons often suffer from problems such as unstable differentiation efficiency, insufficient maturity of the obtained neurons, or unclear functional characteristics.

[0004] To address the aforementioned problems, a method with clearly defined components, a stable induction process, and the ability to efficiently generate functional dopaminergic neurons was designed to solve the technical issues raised above. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for inducing dopaminergic neural stem cells, which solves the technical problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for inducing dopaminergic neural stem cells, comprising the following steps: seeding neural stem cells in an induction culture medium and culturing them at 37°C and 5% CO2 for 6-14 days to induce differentiation and obtain dopaminergic neurons; wherein the induction culture medium is based on DMEM / F12 and contains effective amounts of the following components: human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, epidermal growth factor EGF, fibroblast growth factor FGF, connective tissue growth factor CTGF, β-nerve growth factor B-NGF, vascular endothelial growth factor VEGF, soybean trypsin inhibitor, L-glutathione, oleic acid, Tween 80, phosphatidic acid, sodium selenite, total saponins of Panax notoginseng, resveratrol, lithium chloride, cholesterol, WNT3a, glutamine, laminin, fibronectin, hyaluronic acid, α2 macroglobulin, angiotensin II, and erythropoietin.

[0007] Preferably, the concentration range of each component in the induction culture medium is as follows: 0–100 ng / mL human insulin, 2 nM–200 nM progesterone, 0.05 μg / mL–0.5 μg / mL hydrocortisone, 50 nM–100 nM dexamethasone, 0.1 nM–1 nM estradiol, 0.1 ng / mL–1 μg / mL transferrin, 1–20 ng / mL LEGF, 1–20 ng / mL LFGF, 1–100 ng / mL CTGF, 20–200 ng / mL β-NGF, 1–100 ng / mL VEGF, 0.01%–10% (v / v) soybean trypsin inhibitor, 0.1–10 μg / mL L-glutathione, 0.1–100 μg / mL oleic acid, 0. 0.05%–0.5% (v / v) Tween 80, 10–600 μg / mL phosphatidic acid, 2–100 nM sodium selenite, 1–100 ng / mL total saponins of Panax notoginseng, 1–100 ng / mL resveratrol, 0.1 mM–100 mM lithium chloride, 0.01–10 mg / mL cholesterol, 0.1–100 ng / mL WNT3a, 1 mM–10 mM glutamine, 50 ng / mL–500 ng / mL laminin, 1–100 ng / mL fibronectin, 0.1%–50% (v / v) hyaluronic acid, 1–100 μg / mL α2 macroglobulin, 100–700 nmol / L angiotensin II, 0.001–0.01 U / L erythropoietin.

[0008] Preferably, the concentrations of each component in the induction medium are: 10 μg / mL human insulin, 20 nM progesterone, 0.4 μg / mL hydrocortisone, 100 nM dexamethasone, 10 nM estradiol, 100 ng / mL transferrin, 10 ng / m LEGF, 10 ng / m LFGF, 20 ng / mL CTGF, 20 ng / mL β-NGF, 10 ng / mL VEGF, 0.1% (v / v) soybean trypsin inhibitor, 2 μg / m L-glutathione, 5 μg / mL oleic acid, 0 0.01% (v / v) Tween 80, 50 μg / mL phosphatidic acid, 30 nM sodium selenite, 100 ng / mL total saponins of Panax notoginseng, 100 ng / mL resveratrol, 1 mM lithium chloride, 5 mg / mL cholesterol, 25 ng / mL WNT3a, 2 mM glutamine, 100 ng / mL laminin, 40 ng / mL fibronectin, 0.1% (v / v) hyaluronic acid, 2 μg / mL α2 macroglobulin, 200 nmol / L angiotensin II, 0.005 U / L erythropoietin.

[0009] Preferably, before inducing differentiation, the neural stem cells are expanded and cultured using a proliferation medium; the proliferation medium is based on DMEM / F12 and contains effective amounts of human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, EGF, FGF, CTGF, β-NGF, VEGF, soybean trypsin inhibitor, L-glutathione, oleic acid, Tween 80, phosphatidic acid, sodium selenite, total saponins of Panax notoginseng, resveratrol, lithium chloride, cholesterol, WNT3a, and glutamine.

[0010] Beneficial effects This invention provides a method for inducing dopaminergic neural stem cells. The induction culture medium and method provided by this invention can effectively support the survival of neural stem cells and guide their directed differentiation into dopaminergic neuronal lineages. The method has a clear operation procedure, a stable differentiation process, and the expression of characteristic markers of dopaminergic neurons can be detected in the obtained cell population, providing a stable and reliable cell source for related scientific research. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the structure of a method for inducing dopaminergic neural stem cells according to the present invention. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0013] Please see Figure 1 This invention provides a technical solution: a method for inducing dopaminergic neural stem cells, comprising the following steps: seeding neural stem cells in an induction culture medium, culturing them at 37°C and 5% CO2, and inducing differentiation for 6-14 days to obtain dopaminergic neurons; the induction culture medium is based on DMEM / F12 and contains effective amounts of the following components: human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, epidermal growth factor EGF, fibroblast growth factor FGF, connective tissue growth factor CTGF, β-nerve growth factor B-NGF, vascular endothelial growth factor VEGF, soybean trypsin inhibitor, L-glutathione, oleic acid, Tween 80, phosphatidic acid, sodium selenite, total saponins of Panax notoginseng, resveratrol, lithium chloride, cholesterol, WNT3a, glutamine, laminin, fibronectin, hyaluronic acid, α2 macroglobulin, angiotensin II, and erythropoietin.

[0014] In this embodiment, the concentration range of each component in the induction culture medium is further configured as follows: 0–100 ng / mL human insulin, 2 nM–200 nM progesterone, 0.05 μg / mL–0.5 μg / mL hydrocortisone, 50 nM–100 nM dexamethasone, 0.1 nM–1 nM estradiol, 0.1 ng / mL–1 μg / mL transferrin, 1–20 ng / mL LEGF, 1–20 ng / mL LFGF, 1–100 ng / mL CTGF, 20–200 ng / mL β-NGF, 1–100 ng / mL VEGF, 0.01%–10% (v / v) soybean trypsin inhibitor, 0.1–10 μg / mL L-glutathione, and 0.1–100 μg / mL oil. Acid, 0.005%–0.5% (v / v) Tween 80, 10–600 μg / mL phosphatidic acid, 2–100 nM sodium selenite, 1–100 ng / mL total saponins of Panax notoginseng, 1–100 ng / mL resveratrol, 0.1 mM–100 mM lithium chloride, 0.01–10 mg / mL cholesterol, 0.1–100 ng / mL WNT3a, 1 mM–10 mM glutamine, 50 ng / mL–500 ng / mL laminin, 1–100 ng / mL fibronectin, 0.1%–50% (v / v) hyaluronic acid, 1–100 μg / mL α2 macroglobulin, 100–700 nmol / L angiotensin II, 0.001–0.01 U / L erythropoietin.

[0015] In this embodiment, the concentrations of each component in the induction culture medium are further configured as follows: 10 μg / mL human insulin, 20 nM progesterone, 0.4 μg / mL hydrocortisone, 100 nM dexamethasone, 10 nM estradiol, 100 ng / mL transferrin, 10 ng / m LEGF, 10 ng / m LFGF, 20 ng / mL CTGF, 20 ng / mL β-NGF, 10 ng / mL VEGF, 0.1% (v / v) soybean trypsin inhibitor, 2 μg / m L-glutathione, and 5 μg / mL... Oleic acid, 0.01% (v / v) Tween 80, 50 μg / mL phosphatidic acid, 30 nM sodium selenite, 100 ng / mL total saponins of Panax notoginseng, 100 ng / mL resveratrol, 1 mM lithium chloride, 5 mg / mL cholesterol, 25 ng / mL WNT3a, 2 mM glutamine, 100 ng / mL laminin, 40 ng / mL fibronectin, 0.1% (v / v) hyaluronic acid, 2 μg / mL α2 macroglobulin, 200 nmol / L angiotensin II, 0.005 U / L erythropoietin.

[0016] In this embodiment, the neural stem cells are further amplified and cultured using a proliferation medium before induction of differentiation. The proliferation medium is based on DMEM / F12 and contains effective amounts of human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, EGF, FGF, CTGF, β-NGF, VEGF, soybean trypsin inhibitor, L-glutathione, oleic acid, Tween 80, phosphatidic acid, sodium selenite, total saponins of Panax notoginseng, resveratrol, lithium chloride, cholesterol, WNT3a, and glutamine.

[0017] Its detailed connection method is a well-known technology in this field. The following mainly introduces the working principle and process, and the specific work is as follows.

[0018] Example 1: Proliferation and Culture of Neural Stem Cells This embodiment details the process of culturing neural stem cells using the aforementioned proliferation medium.

[0019] First, prepare the neural stem cell proliferation medium. Using commercially available DMEM / F12 medium as the basal solution, under aseptic conditions, supplement it with the following components to reach the specified final concentrations: 10 μg / mL human insulin, 20 nM progesterone, 0.4 μg / mL hydrocortisone, 100 nM dexamethasone, 10 nM estradiol, 100 ng / mL transferrin, 10 ng / mL epidermal growth factor (EGF), 10 ng / mL fibroblast growth factor (FGF), 20 ng / mL connective tissue growth factor (CTGF), 20 ng / mL... The medium contained the following components: β-nerve growth factor (B-NGF) / mL, vascular endothelial growth factor (VEGF) 10 ng / mL, soybean trypsin inhibitor 0.1% (v / v), 2 μg / mL L-glutathione, 5 μg / mL oleic acid, Tween 80 0.01% (v / v), 50 μg / mL phosphatidic acid, 30 nM sodium selenite, 100 ng / mL total saponins of Panax notoginseng, 100 ng / mL resveratrol, 1 mM lithium chloride, 5 mg / mL cholesterol, 25 ng / mL WNT3a, and 2 mM glutamine. After all components were added, the medium was thoroughly mixed and stored at 4°C for later use, and should be used within two weeks.

[0020] The cells used in the experiment were third-generation neural stem cells differentiated from human induced pluripotent stem cells (iPSCs) using a standard induction procedure. After thawing the frozen cells, they were resuspended in the prepared proliferation medium and cultured at a density of 1.5 × 10⁶ cells / cm². 5 Cells were seeded at a density of [number] cells per cell type into T-25 cell culture flasks coated with poly-L-lysine. The culture flasks were then placed in a cell culture incubator at 37°C, 5% carbon dioxide, and saturated humidity for static culture.

[0021] During culture, the culture medium was completely replaced every two days to provide fresh nutrients to the cells and remove metabolic waste. Daily microscopic observation revealed that the cells exhibited the typical clonal cluster growth pattern of neural stem cells, with uniform cell morphology, clear boundaries, and good refractive properties. By day four, the cell coverage area (confluence) on the culture surface exceeded 80%. At this point, cell digestion and passage were performed, and trypan blue staining showed cell viability exceeding 95%. Immunofluorescence staining of the cultured cells revealed positive expression of the characteristic neural stem cell protein NESTIN, with over 98% of cells showing positive expression, confirming that this proliferation medium effectively maintained the undifferentiated state of neural stem cells and supported their stable expansion.

[0022] Example 2: Induction and Differentiation of Dopaminergic Neurons This embodiment details the process of directing the differentiation of neural stem cells into dopaminergic neurons using the aforementioned induction culture medium.

[0023] First, prepare the dopaminergic neuron differentiation induction medium. Based on the proliferation medium formula used in Example 1 above, additionally add the following key differentiation induction components to achieve the specified final concentrations: 100 ng / mL laminin, 40 ng / mL fibronectin, 0.1% hyaluronic acid (v / v), 2 μg / mL α2 macroglobulin, 200 nmol / L angiotensin II, and 0.005 U / L erythropoietin. Thoroughly mix the supplemented medium, store it at 4°C protected from light, and use it within one week.

[0024] The differentiation induction experiment began with human neural stem cells that had been expanded and cultured according to the method described in Example 1, were in the logarithmic growth phase, and had a confluence of more than 80%. Before induction, the cells were digested and prepared into a single-cell suspension.

[0025] To provide a suitable growth interface for neuronal differentiation, cell culture plates were pre-coated with PBS solution containing 100 ng / mL laminin and 40 ng / mL fibronectin. After incubation for two hours, the coating solution was aspirated and the cells were ready for use. The prepared neural stem cell suspension was seeded at an appropriate density into the coated culture dishes, and sufficient amounts of the aforementioned differentiation-inducing medium were added. The cells were then returned to the incubator and induced to differentiate at 37°C and 5% CO2.

[0026] During differentiation, the induction medium was changed every two days to ensure the activity of inducing factors and a continuous supply of nutrients. Differentiation continued until day 10. During this period, significant morphological changes were observed: the cell bodies gradually shrank, became brighter, and extended into slender neuron-like processes, forming a complex network structure.

[0027] After differentiation, the cultured cells were fixed and identified by immunofluorescence staining. Staining with an antibody targeting tyrosine hydroxylase (TH), a characteristic marker of dopaminergic neurons, revealed that a significant proportion of cells were TH-positive and co-expressed the neuronal marker β-III-tubulin (TUJ1).

[0028] The results indicate that the induction culture medium and method described in this embodiment can successfully induce neural stem cells to differentiate into cells with a dopaminergic neuron phenotype.

[0029] It should be noted that in this paper, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

Claims

1. A method for inducing a dopaminergic neural stem cell, characterized by, The method comprises the following steps: inoculating neural stem cells into an induction medium, and culturing at 37 DEG C and 5% CO2 for 6-14 days to obtain dopaminergic neurons; the induction medium is a DMEM / F12-based medium containing effective amounts of the following components: human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF), connective tissue growth factor (CTGF), beta nerve growth factor (B-NGF), vascular endothelial growth factor (VEGF), soybean trypsin inhibitor, L-glutathione, oleic acid, Tween 80, phosphatidic acid, sodium selenite, panax notoginseng saponins, resveratrol, lithium chloride, cholesterol, WNT3a, glutamine, laminin, fibronectin, hyaluronic acid, alpha 2 macroglobulin, angiotensin II and erythropoietin.

2. The method of claim 1, wherein the method is for inducing a dopaminergic neural stem cell. The concentration ranges of the components in the induction medium are as follows: 0-100 ng / mL human insulin, 2 nM-200 nM progesterone, 0.05 μg / mL-0.5 μg / mL hydrocortisone, 50 nM-100 nM dexamethasone, 0.1 nM-1 nM estradiol, 0.1 ng / mL-1 μg / mL transferrin, 1-20 ng / mL EGF, 1-20 ng / mL FGF, 1-100 ng / mL CTGF, 20-200 ng / mL B-NGF, 1-100 ng / mL VEGF, 0.01%-10% (v / v) soybean trypsin inhibitor, 0.1-10 μg / mL L-glutathione, 0.1-100 μg / mL oleic acid, 0.005%-0.5% (v / v) Tween 80, 10-600 μg / mL phosphatidic acid, 2-100 nM sodium selenite, 1-100 ng / mL panax notoginseng saponins, 1-100 ng / mL resveratrol, 0.1 mM-100 mM lithium chloride, 0.01-10 mg / mL cholesterol, 0.1-100 ng / mL WNT3a, 1 mM-10 mM glutamine, 50 ng / mL-500 ng / mL laminin, 1-100 ng / mL fibronectin, 0.1%-50% (v / v) hyaluronic acid, 1-100 μg / mL alpha 2 macroglobulin, 100-700 nmol / L angiotensin II, 0.001-0.01 U / L erythropoietin.

3. The method of claim 2, wherein the method is for inducing a dopaminergic neural stem cell. , the concentration of each component in the induction medium is: 10 μg / mL human insulin, 20 nM progesterone, 0.4 μg / mL hydrocortisone, 100 nM dexamethasone, 10 nM estradiol, 100 ng / mL transferrin, 10 ng / mL EGF, 10 ng / mL FGF, 20 ng / mL CTGF, 20 ng / mL B-NGF, 10 ng / mL VEGF, 0.1% (v / v) soybean trypsin inhibitor, 2 μg / mL L-glutamine, 5 μg / mL oleic acid, 0.01% (v / v) Tween 80, 50 μg / mL phosphatidyl acid, 30 nM sodium selenite, 100 ng / mL total saponins of Panax notoginseng, 100 ng / mL resveratrol, 1 mM lithium chloride, 5 mg / mL cholesterol, 25 ng / mL WNT3a, 2 mM glutamine, 100 ng / mL laminin, 40 ng / mL fibronectin, 0.1% (v / v) hyaluronic acid, 2 μg / mL α2 macroglobulin, 200 nmol / L angiotensin II, 0.005 U / L erythropoietin.

4. The method of claim 1, wherein the method is for inducing a dopaminergic neural stem cell. Before induction differentiation, the neural stem cells are amplified and cultured by using a proliferation culture medium; the proliferation culture medium is a DMEM / F12-based culture medium and contains effective amounts of human insulin, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, EGF, FGF, CTGF, B-NGF, VEGF, soybean trypsin inhibitor, L-glutamine, oleic acid, Tween 80, phosphatidyl acid, sodium selenite, total saponins of Panax notoginseng, resveratrol, lithium chloride, cholesterol, WNT3a and glutamine.

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