Cholinergic neuron differentiation method and kit based on traditional Chinese medicine ingredients

Abstract

A method and kit for differentiating cholinergic neurons based on traditional Chinese medicine ingredients is disclosed. The method comprises: using a first culture medium containing a small molecule agent to induce human induced pluripotent stem cells to differentiate into neuroectodermal stage cells; using a second culture medium containing a Hedgehog pathway activator to induce the neuroectodermal cells to differentiate into medial ganglionic eminence cells; and using a third culture medium containing traditional Chinese medicine ingredients to induce the medial ganglionic eminence cells to differentiate into mature cholinergic neurons. The traditional Chinese medicine ingredients include polydatin, paeoniflorin, salidroside, and stilbene glycoside. This method is highly efficient and low-cost while being practical. Cell morphology and marker gene expression confirm that the method results in targeted and efficient differentiation into cholinergic neurons. This provides a more convenient, rapid, and effective cell source solution for various cholinergic neuron-based research.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and in particular relates to a cholinergic neuron differentiation method and a kit based on traditional Chinese medicine components. Technical Background

[0002] Currently, common neurodegenerative diseases include Alzheimer's disease (AD), dementia with Lewy bodies (DLB), Parkinson's disease (PD), etc., which can seriously affect the daily life of patients, increase family burden and social costs, and more importantly, the current drug treatment for them only stays at the "treating the symptoms" level. Therefore, a large number of studies on the targeted antagonism or inhibition of the pathogenesis of Alzheimer's disease have emerged, and the research fields involved have also deepened and broadened. In this process, the defect of cholinergic neurons located at the base of the brain has become one of the more recognized pathological mechanisms, and the cultivation of a large number of cholinergic neurons for neural replacement therapy has become a new consideration. Neurons differentiated from mesenchymal stem cells have problems such as large individual differences and unstable efficacy; using human embryonic stem cells as the source of differentiation has ethical controversies; and human induced pluripotent stem cells have a wide range of sources, avoiding ethical controversies and immune rejection problems.

[0003] Numerous studies have explored the cholinergic differentiation of human induced pluripotent stem cells (iPS). A general differentiation strategy has been established: sequentially forming the neural ectoderm (NE), medial ganglionic eminence (MGE), and basal forebrain cholinergic neurons (BFCN). While seemingly comprehensive, the details of the differentiation protocol, such as the timing of each stage, the selection of culture systems, the identification and addition of key active substances, experimental cost control, and the choice of characterization and analytical methods, vary widely in research, resulting in a mature, efficient, and relatively standardized differentiation protocol yet to be established. Summary of the Invention

[0004] Based on this, the main purpose of the present invention is to provide a cholinergic neuron differentiation method and kit based on traditional Chinese medicine ingredients, and to develop an efficient and low-cost cholinergic neuron differentiation method using human induced pluripotent stem cells.

[0005] To achieve the above objectives, as one aspect of the present invention, a highly efficient method for differentiating cholinergic neurons based on traditional Chinese medicine ingredients is provided. The method specifically comprises the following steps:

[0006] Inducing human induced pluripotent stem cells to differentiate into neuroectodermal cells using a first culture medium containing a small molecule preparation, wherein the small molecule preparation includes a TGF-β pathway inhibitor, a BMP pathway inhibitor, and an mTOR pathway activator;

[0007] using a second culture medium containing a Hedgehog pathway activator to induce differentiation of neuroectodermal cells into medial ganglionic eminence cells;

[0008] A third culture medium containing traditional Chinese medicine ingredients is used to induce the medial ganglionic eminence cells to differentiate into mature cholinergic neurons, wherein the traditional Chinese medicine ingredients include polydatin, paeoniflorin, salidroside and stilbene glycoside.

[0009] As another aspect of the present invention, a cholinergic neuron differentiation kit based on traditional Chinese medicine ingredients is provided, comprising:

[0010] A first culture medium comprising a small molecule preparation for inducing human induced pluripotent stem cells to differentiate into neuroectodermal cells, wherein the small molecule preparation comprises a TGF-β pathway inhibitor, a BMP pathway inhibitor, and an mTOR pathway activator;

[0011] a second culture medium containing a Hedgehog pathway activator for inducing differentiation of neuroectodermal cells into medial ganglionic eminence cells; and

[0012] The third culture medium containing traditional Chinese medicine ingredients is used to induce the differentiation of medial ganglionic eminence cells into mature cholinergic neurons, wherein the traditional Chinese medicine ingredients include polydatin, paeoniflorin, salidroside and diphenylglycoside.

[0013] Compared with the prior art, the cholinergic neuron differentiation method and kit based on traditional Chinese medicine ingredients of the present invention have at least one or part of the following beneficial effects:

[0014] This method uses human induced pluripotent stem cells as a starting point. By changing the culture system at different developmental stages, the cells promote differentiation to different developmental stages by either stimulating or inhibiting different signaling pathways and exerting neuroprotective effects, ultimately inducing differentiation into mature neurons. This not only avoids the ethical controversies surrounding embryonic stem cells, but also ensures that the physiological state of the resulting cholinergic neurons is more consistent with that of the human body, making it easier to conduct research on human pathology or biology.

[0015] In the final maturation stage, the present invention adds traditional Chinese medicinal plant extracts such as polydatin, paeoniflorin, salidroside, and diphenylethylene glycoside. Based on the role of the Chinese medicinal ingredients in different signal pathways, a combination study is conducted. It is found that through the synergistic effect of these Chinese medicinal ingredients, brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) can be replaced. The price is low, but it also has a neurotrophic effect, promoting the differentiation and maturation of cholinergic neurons.

[0016] In the cholinergic neurons finally obtained by the present invention, high expression of related enzymes and proteins such as choline acetyltransferase (ChAT), microtubule-associated protein-2 (MAP2), microtubule protein (βIII-tubulin), and vesicular acetylcholine transporter (VAChT) can be detected, as well as high expression of cholinergic neuron-specific related genes such as CHAT, MAP2, and TUBB3. Combined with the results of cell morphological observation, it was determined that cholinergic neurons with good growth status, consistent morphological structure, and expression of corresponding genes and proteins were obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A This is a diagram showing the neural differentiation of cells at the neuroectoderm stage in an embodiment of the present invention;

[0018] Figure 1B This is a diagram showing the neural differentiation of cells at the medial ganglionic eminence stage in an embodiment of the present invention;

[0019] Figure 1C This is a diagram showing the neural differentiation of cholinergic cells at the mature stage in an embodiment of the present invention;

[0020] Figure 2A This is a graph showing changes in marker gene expression at the neuroectoderm stage obtained through fluorescence quantitative PCR testing in an embodiment of the present invention;

[0021] Figure 2B This is a graph showing changes in marker gene expression at the medial ganglionic eminence stage obtained through fluorescence quantitative PCR testing in an embodiment of the present invention;

[0022] Figure 2C This is a graph showing changes in the expression of marker genes in the cholinergic maturation stage obtained through fluorescence quantitative PCR detection in an embodiment of the present invention;

[0023] Figure 3A The results of immunofluorescence staining of cholinergic neurons using vesicular acetylcholine transporter (VAChT) antibody in the control differentiation protocol in the examples of the present invention are shown;

[0024] Figure 3B The results of immunofluorescence staining of cholinergic neurons using vesicular acetylcholine transporter (VAChT) antibody in the differentiation protocol with 0.5 μM resveratrol added in the examples of the present invention;

[0025] Figure 3C The results of immunofluorescence staining of cholinergic neurons using a vesicular acetylcholine transporter (VAChT) antibody in the differentiation protocol with the addition of 0.5 μM resveratrol and 1 μM valproic acid in the examples of the present invention are shown;

[0026] Figure 4This is a graph showing changes in the expression of marker genes in the cholinergic maturation stage using four traditional Chinese medicine ingredients at different mixed concentrations in the examples of the present invention. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0028] According to the embodiments of the present invention, the use of human induced pluripotent stem cells to differentiate cholinergic neurons has the following advantages: (1) It avoids the ethical controversy that may occur when using embryonic stem cells, and at the same time, the range of reprogrammed cells that can be selected is wide and not limited. (2) It has low immunogenicity, which can reduce the problem of immune rejection during clinical application and expand the possibility of application. (3) It also retains the multidirectional differentiation potential and unlimited proliferation ability of embryonic stem cells, which can better reflect the real situation of the human body. When using human induced pluripotent stem cells, a detailed differentiation scheme is proposed, which includes, for example, making the cell growth enter different stages, determining what kind of additives can promote directional differentiation, provide specific nutritional requirements for cells, and studying the characterization methods of different stages, so as to obtain cholinergic neurons with good growth status, consistent morphological structure, and expression of corresponding genes and proteins.

[0029] Specifically, according to some embodiments of the present invention, a method for differentiating cholinergic neurons based on traditional Chinese medicine ingredients is provided, which specifically includes the following steps S1 to S3.

[0030] In step S1, a first culture medium containing a small molecule preparation is used to induce human induced pluripotent stem cells to differentiate into neuroectodermal cells, wherein the small molecule preparation includes a TGF-β pathway inhibitor, a BMP pathway inhibitor, and an mTOR pathway activator;

[0031] In step S2, a second culture medium containing a Hedgehog pathway activator is used to induce the neuroectoderm cells to differentiate into medial ganglionic eminence cells;

[0032] In step S3, a third culture medium containing Chinese medicinal ingredients is used to induce the medial ganglionic eminence cells to differentiate into mature cholinergic neurons, wherein the Chinese medicinal ingredients include polydatin, paeoniflorin, salidroside and stilbene glycoside.

[0033] According to an embodiment of the present invention, by adjusting the additives in the culture medium corresponding to different developmental stages, different signaling pathways are promoted or inhibited, and a neuroprotective effect is exerted. Then, by inducing differentiation, the cells sequentially pass through the neuroectoderm stage and the medial ganglionic eminence stage, and finally develop into mature cholinergic neurons under the promotion of neurotrophic substances. The neurons specifically express related proteins such as acetylcholine transferase (ChAT), microtubule-associated protein (MAP2), microtubule protein (βIII-tubulin), and vesicular acetylcholine transporter (VAChT), as well as the corresponding genes CHAT, MAP2, and TUBB3.

[0034] In some embodiments, in step S1, in order to differentiate human induced pluripotent stem cells to form neural ectoderm, the optional first culture medium also includes DMEM / F12 culture medium, vitamin C magnesium phosphate (64 mg / L), sodium selenate (14 μg / L), transferrin (10.7 mg / L), insulin (19.4 mg / L), and sodium bicarbonate (543 mg / L) to meet the nutritional needs of neural ectoderm cell differentiation.

[0035] In some embodiments, the TGF-β pathway inhibitor is SB431542 (at a concentration of 5 to 10 μM, preferably 5 μM, 8 μM, 10 μM, and most preferably 10 μM), the BMP pathway inhibitor is LDN193189 (at a concentration of 5 to 500 nM, preferably 5 nM, 50 nM, 100 nM, 300 nM, 500 nM, and most preferably 500 nM), and the mTOR pathway activator is valproic acid (VPA) (at a concentration of 0.05 to 100 μM, preferably 50 nM, 100 nM, 1 μM, 10 μM, 100 μM, and most preferably 1 μM). Based on the fact that these pathway regulators can promote the directed differentiation of human induced pluripotent stem cells to the neural ectoderm, the added mTOR pathway activator VPA can activate the expression of the neural differentiation-related gene Ngn1, thereby improving the efficiency of induced differentiation.

[0036] In some embodiments, in step S2, in order to allow the cells to further differentiate into the medial ganglionic eminence structure after forming the neural ectoderm, the optional second culture medium also includes 97% Neurobasal medium + 1% N2 supplement + 2% B-27 supplement to meet the nutritional needs of the differentiation of the medial ganglionic eminence cells.

[0037] In some embodiments, the added Hedgehog pathway activator is Purmorphamine (at a concentration of 0.5 to 2 μM, preferably 0.5 μM, 1 μM, 1.5 μM, 2 μM, and most preferably 1.5 μM), which can bind to the Smo membrane protein receptor, promote its phosphorylation, further promote neural differentiation, and form the medial ganglionic eminence structure.

[0038] In some embodiments, in step S3, in order to further differentiate the medial ganglionic eminence cells into mature cholinergic neurons, the optional third culture medium further includes 97% Neurobasal medium, 1% N2 supplement, and 2% B-27 supplement.

[0039] In some embodiments, the medial ganglionic eminence is continued to be cultured until the cholinergic neurons mature, and neurotrophic substances are needed to promote the survival and differentiation of cholinergic neurons. Considering that the commonly used brain-derived neurotrophic factor BDNF and glial cell-derived neurotrophic factor GDNF are relatively expensive, traditional Chinese medicinal plant extracts such as polydatin, paeoniflorin, salidroside, and diphenylethylene glycoside are added instead. They are inexpensive but also have neurotrophic effects and promote the differentiation and maturation of cholinergic neurons.

[0040] In some embodiments, polydatin (at a concentration of 0.01 to 10 μM, preferably 10 nM, 100 nM, 1 μM, 10 μM, and most preferably 100 nM) can activate the Nrf2 / ARE signaling pathway, scavenge oxygen free radicals, and exert an antioxidant neuroprotective effect. At the same time, it can downregulate Toll-like receptors (TLR-2) and inhibit the NF-kB signaling pathway to reduce the expression of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6. In addition, it can also inhibit neuronal apoptosis by coupling the Caspase family and the Bcl-2 family.

[0041] In some embodiments, paeoniflorin (at a concentration of 0.01 to 10 μM, preferably 10 nM, 100 nM, 1 μM, 10 μM, and most preferably 100 nM) can also play a neuroprotective role by downregulating reactive oxygen species (ROS) and resisting oxidative stress. On this basis, it can also promote the secretion and expression of nerve growth factor (NGF), thereby promoting the differentiation of cholinergic neurons, enhancing nutrient metabolism in neuronal cell bodies, and promoting the synthesis of microtubules and microfilament proteins, as well as microtubule phosphorylation.

[0042] In some embodiments, salidroside (at a concentration of 0.01 to 10 μM, preferably 10 nM, 100 nM, 1 μM, 10 μM, and most preferably 100 nM) reduces the rate of neuronal apoptosis by mediating the PI3K / AKT pathway, while scavenging excess intracellular oxygen free radicals and inhibiting calcium ion overload. More importantly, it can increase the expression levels of brain-derived neurotrophic factor BDNF and nerve growth factor NGF in cells, promoting the differentiation of cholinergic neurons while exerting neurotrophic and protective effects.

[0043] In some embodiments, diphenylethylene glycosides (at a concentration of 0.01 to 10 μM, preferably 10 nM, 100 nM, 1 μM, 10 μM, and most preferably 100 nM) can effectively reduce the level of reactive oxygen species in cells and reduce nerve damage; inhibit the activation of NF-kB, inhibit the secretion of proinflammatory cytokines, and reduce the apoptosis of nerve cells; and promote the secretion of neurotrophic factors such as BDNF to further induce the differentiation and survival of cholinergic neurons.

[0044] In some embodiments, polydatin, paeoniflorin, salidroside, and stilbene glycoside are mixed at their respective appropriate concentrations and added to the culture system. Based on the synergistic effect of these four Chinese herbal ingredients, they can replace brain-derived neurotrophic factor BDNF and glial cell-derived neurotrophic factor GDNF, and play the roles of neurotrophy, neuroprotection, improving neuron survival rate, and promoting cholinergic neuron differentiation.

[0045] In some embodiments, resveratrol (at a concentration of 0.1 to 1 μM, preferably 100 nM, 300 nM, 500 nM, and 1 μM, with a most preferred concentration of 500 nM) is added to the culture system comprising the first, second, and third culture media. This can provide neuroprotective effects, including antioxidant and anti-inflammatory effects. On the one hand, it reduces the expression levels of cellular reactive oxygen species (ROS) and malondialdehyde (MDA), and on the other hand, it can reduce the expression of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α by inhibiting the NF-kB pathway. More importantly, it can also promote neural differentiation and effectively improve the efficiency of cholinergic neuron differentiation.

[0046] In some embodiments, the time for inducing human induced pluripotent stem cells to differentiate into neuroectodermal cells is 9 to 12 days, preferably 9, 10, 11, or 12 days, and more preferably 10 days. In some embodiments, the time for inducing neuroectodermal cells to differentiate into medial ganglionic eminence cells is 15 to 17 days, preferably 15, 16, or 17 days, and more preferably 15 days. In some embodiments, the time for inducing medial ganglionic eminence cells to differentiate into mature cholinergic neurons is 13 to 15 days, preferably 13, 14, or 15 days, and more preferably 14 days.

[0047] In a more specific embodiment, it takes 10 days to culture human induced pluripotent stem cells to the neural ectoderm stage. Based on this, it takes another 15 days to continue to differentiate and culture to the medial ganglionic eminence. From the 26th day, the cells begin to enter the maturation stage. After 38 days, they are fully mature, and the morphology, gene, and protein expression are all consistent with cholinergic neurons.

[0048] In some embodiments, detection and analysis of gene levels, protein levels, metabolic levels, and enzyme activities are performed at different landmark stages to gain a more comprehensive and in-depth understanding of the cholinergic neuron differentiation process.

[0049] In some embodiments, before step S1, the method further includes step S0: maintaining and culturing human induced pluripotent stem cells.

[0050] In some embodiments, in step S0, in order to maintain and culture a certain number of human induced pluripotent stem cells in good condition, the maintenance medium used comprises mTeSR TM Plus basal medium and mTeSR TM Plus 5X Supplement.

[0051] Specifically, according to some embodiments of the present invention, a cholinergic neuron differentiation kit based on traditional Chinese medicine ingredients is provided for implementing the above-mentioned cholinergic neuron differentiation method. The above-mentioned kit specifically comprises:

[0052] A first culture medium comprising a small molecule preparation for inducing human induced pluripotent stem cells to differentiate into neuroectodermal cells, wherein the small molecule preparation comprises a TGF-β pathway inhibitor, a BMP pathway inhibitor, and an mTOR pathway activator;

[0053] a second culture medium containing a Hedgehog pathway activator for inducing differentiation of neuroectodermal cells into medial ganglionic eminence cells; and

[0054] The third culture medium containing traditional Chinese medicine ingredients is used to induce the differentiation of medial ganglionic eminence cells into mature cholinergic neurons, wherein the traditional Chinese medicine ingredients include polydatin, paeoniflorin, salidroside and diphenylglycoside.

[0055] It should be noted that the first culture medium, the second culture medium and the third culture medium in the above-mentioned kit have been described in the above-mentioned method for differentiating cholinergic neurons based on traditional Chinese medicine ingredients and will not be described in detail here.

[0056] The technical solutions of the present invention are further described below by means of specific examples in conjunction with the accompanying drawings. It should be noted that the following specific examples are merely illustrative and the scope of protection of the present invention is not limited thereto. The drugs or reagents used in the following examples are all commercially available or homemade by known preparation methods. The methods used in the following examples, such as the immunofluorescence staining method, are well known in the art and can be carried out according to the description in textbooks or relevant literature and will not be described in detail.

[0057] Example 1:

[0058] The method provided by the present invention is used to differentiate cholinergic neurons, which specifically includes the following steps:

[0059] The cells used in this example were obtained from the Chinese Academy of Sciences Cell Bank / Stem Cell Technology Platform SCSP-1301, numbered DYR0100, and the maintenance medium was mTeSR IM Plus (STEMCELL TECHNOLOGIES, 100-0274) and used with 5X Supplement (STEMCELL TECHNOLOGIES, 100-0275). The matrix glue on the bottom of the dish is Vitronectin XF. TM (STEMCELL TECHNOLOGIES, 07180). After 4-5 days of culture, cells can be passaged once when the confluence reaches 90%, or single cells can be plated.

[0060] When human induced pluripotent stem cells covered more than 90% of the bottom of the dish, they were digested to single cells using TryPLE (Gibco, 12604021), centrifuged, and then lysed with mTeSR containing ROCK inhibitor Y27632 (MedChemExpress, HY-10071). IM Plus resuspended, and finally 2x10 5 The cells were seeded at a density of 10 cells / well in a 24-well plate.

[0061] One day after seeding, the culture medium was changed to differentiation medium and differentiation induction was started. The first culture medium was used, including: DMEM / F12 medium, E6 medium containing vitamin C magnesium phosphate (64 mg / L), sodium selenate (14 μg / L), transferrin (10.7 mg / L), insulin (19.4 mg / L), and sodium bicarbonate (543 mg / L), in combination with TGF-β pathway inhibitor SB431542 (10 μM) (Selleck, S1067), BMP pathway inhibitor LDN193189 (500 nM) (Selleck, S7507), mTOR pathway activator valproic acid VPA (1 μM) (Stemgent TM , 04-0007), to differentiate human induced pluripotent stem cells to the neuroectodermal stage. The medium was changed daily, with 500 μL of culture medium per well.

[0062] Starting on day 11, the first culture medium was replaced with a second culture medium containing 97% Neurobasal (Gibco, 21103049), 1% N2 (Gibco, 17502001), 2% B-27 supplement (Gibco, 17504044), and 1.5 μM Hedgehog pathway activator Purmorphamine (MedChemExpress, HY-15108). The medium was changed daily and cultured continuously for up to day 25. At this point, the cells will form the medial ganglionic eminence.

[0063] After the medial ganglionic eminence is formed, culture is continued using a third culture medium. Specifically, the composition of the culture medium is maintained unchanged: 97% Neurobasal, 1% N2, and 2% B-27 supplement. However, additional neurotrophic factor alternatives such as BDNF and GDNF, including polydatin (MedChemExpress, HY-N0120A), paeoniflorin (MedChemExpress, HY-N0293), salidroside (MedChemExpress, HY-N0109), and stilbene glycoside (MedChemExpress, HY-N0652), are added. All four substances are mixed and added to the culture system at a concentration of 100 nM until mature cholinergic neurons are formed.

[0064] The cell morphologies at the neuroectoderm stage, medial ganglionic eminence stage and cholinergic neuron maturation stage were observed under a microscope. Figures 1A to 1C They are neural differentiation diagrams at different cell differentiation stages in this embodiment, such as Figures 1A to 1CAs shown, it can be found that the cell morphology changes during the differentiation process. The most obvious change observed under the microscope is the change in the length and strength of the neurofilaments. In this process, the neurofilaments continue to lengthen, their strength gradually increases, and the connections between them become increasingly complex. The cell morphology is consistent with the corresponding different differentiation stages, and the individual differences between the differentiated cholinergic neurons are small.

[0065] Example 2:

[0066] The operation was similar to that of Example 1, except that, in Example 2, resveratrol (500 nM) was added to the first culture medium, the second culture medium, and the third culture medium, respectively.

[0067] Comparative Examples 1 to 3:

[0068] The operation is similar to that of Example 2, except that:

[0069] In comparative example 1 (+resveratrol), the mTOR pathway activator valproic acid (VPA) was not added to the culture system;

[0070] In comparative example 2 (control group), mTOR pathway activators valproic acid (VPA) and resveratrol were not added to the culture system.

[0071] For Example 2, Comparative Examples 1 and 2, after culturing to the neural ectoderm stage, medial ganglionic eminence, and cholinergic neuron maturation, TRNzol total RNA extraction reagent (TIANGEN, DP424) was used to extract the cellular RNA at this stage, and then reverse transcribed to form cDNA according to Fastking RT Kit (TIANGEN, KR116). SYBR Green dye was then used to perform PCR to detect the expression of related genes PAX6, SOX1, FOXG1, NKX2-1, DLX2, ISLET1, CHAT, MAP2, and TUBB3. The primers were designed based on the human genome transcript information published by NCBI, and the product length was controlled between 80-120 bp, the GC content was between 40% and 60%, the Tm value was between 58-62°C, and the primer length itself was between 18-22 bp. To detect the above genes, the primer sequences used are shown in Table 1 below:

[0072] Table 1

[0073] Gene Upstream primer Downstream primer PAX6 5'-TCCGTTGGAACTGATGGAGT-3' 5'-GTTGGTATCCGGGGACTTC-3' SOX1 5'-AATACTGGAGACGAACGCCG-3' 5'-AACCCAAGTCTGGTGTCAGC-3' FOXG1 5'-TGGACGCAGACCTTGAGAAC-3' 5'-GGGCACCTTTACTACGAATGC-3' NKX2-1 5'-AGCACACGACTCCGTTCTC-3' 5'-GCCCACTTTCTTGTAGCTTTCC-3' DLX2 5'-GCCTCAACAACGTCCCTTACT-3' 5'-TCACTATCCGAATTTCAGGCTCA-3' ISLEt1 5'-GCGGAGTGTAATCAGTATTTGGA-3' 5'-GCATTTGATCCCGTACAACCT-3' CHAT 5'-CAGCCCTGCCGTGATCTTT-3' 5'-TGTAGCTGAGTACACCAGAGATG-3' MAP2 5'-CTCAGCACCGCTAACAGAGG-3' 5'-CATTGGCGCTTCGGACAAG-3' TUBB3 5'-AGGTGATCAGTGATGAACATGG-3' 5'-GAGGAACATATTTGCCACCTGT-3'

[0074] The reaction conditions of fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 30 seconds; 95°C for 3 seconds; 60°C for 30 seconds; 95°C for 15 seconds; 60°C for 60 seconds; and 40 cycles.

[0075] Fluorescence quantitative PCR was used to detect marker genes of neuroectoderm, medial ganglionic eminence, and mature cholinergic neurons. Figures 2A to 2C The following are the changes in the expression levels of marker genes at different cell differentiation stages in this embodiment. According to the test results, Figures 2A to 2C It can be seen that after adding the mTOR pathway activator valproic acid VPA and resveratrol, the expression levels of marker genes in each stage were significantly improved.

[0076] For Example 2 and Comparative Examples 1 and 2, after culturing until the cholinergic neurons matured, immunofluorescence staining of the cholinergic neurons was performed using vesicular acetylcholine transporter (VAChT) antibodies. Figures 3A to 3C The immunofluorescence staining results of different cell differentiation stages in this embodiment and the comparative example are respectively as follows: Figures 3A to 3C After adding the mTOR pathway activator valproic acid VPA and resveratrol, mature cholinergic neurons were successfully obtained.

[0077] Example 3:

[0078] The operation is similar to that of Example 1, except that, in Example 3, the mixed concentrations of the four Chinese medicinal ingredients are adjusted to 10 nM, 100 nM, 1 μM, and 10 μM, respectively.

[0079] Comparative Example 3:

[0080] The difference from the operation type of Example 1 is that the four traditional Chinese medicine components were not added, that is, the concentration was 0 μM.

[0081] After the cholinergic neurons were cultured to maturity, the same fluorescence quantitative PCR detection method as in Example 2 was used to detect changes in the expression levels of the marker genes in this example. Figure 4 The effects of the four Chinese herbal ingredients at different mixed concentrations on the differentiation of cholinergic neurons are shown in FIG. Figure 4 As shown in the figure, it can be found that the expression level of marker genes is the highest at a concentration of 100 nM, and 100 nM is the optimal concentration.

[0082] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for differentiating cholinergic neurons based on traditional Chinese medicine ingredients, comprising the following steps: Inducing human induced pluripotent stem cells to differentiate into neuroectodermal cells using a first culture medium containing a small molecule preparation, wherein the small molecule preparation includes a TGF-β pathway inhibitor SB431542, a BMP pathway inhibitor LDN193189, and an mTOR pathway activator valproic acid, the concentration of SB431542 is 5-10 μM, the concentration of LDN193189 is 5-500 nM, and the concentration of valproic acid is 0.05-100 μM, and the first culture medium also includes DMEM / F12 medium, 64 mg / L magnesium ascorbyl phosphate, 14 μg / L sodium selenate, 10.7 mg / L transferrin, 19.4 mg / L insulin, and 543 mg / L sodium bicarbonate; Inducing the neuroectoderm cells to differentiate into medial ganglionic eminence cells using a second culture medium containing a Hedgehog pathway activator, Purmorphamine, at a concentration of 0.5 to 2 μM, wherein the second culture medium further comprises 97% Neurobasal medium, 1% N2 supplement, and 2% B-27 supplement; A third culture medium containing traditional Chinese medicine ingredients is used to induce the medial ganglionic eminence cells to differentiate into mature cholinergic neurons, wherein the traditional Chinese medicine ingredients include polydatin, paeoniflorin, salidroside and stilbene glycoside, the concentration of polydatin is 0.01~10 μM, the concentration of paeoniflorin is 0.01~10 μM, the concentration of salidroside is 0.01~10 μM, and the concentration of stilbene glycoside is 0.01~10 μM, and the third culture medium also includes 97% Neurobasal medium, 1% N2 supplement and 2% B-27 supplement.

2. The method for differentiating cholinergic neurons according to claim 1, wherein: The concentration of SB431542 was 10 μM; The concentration of LDN193189 was 500 nM; The concentration of valproic acid was 1 μM.

3. The method for differentiating cholinergic neurons according to claim 1, wherein: The concentration of purmorphamine was 1.5 μM.

4. The method for differentiating cholinergic neurons according to claim 1, wherein: The concentration of polydatin was 100 nM; the concentration of paeoniflorin was 100 nM; the concentration of salidroside was 100 nM; and the concentration of stilbene glycoside was 100 nM.

5. The method for differentiating cholinergic neurons according to claim 1, wherein: Resveratrol at a concentration of 0.1-1 μM is further added to the first culture medium, the second culture medium, and the third culture medium respectively.

6. The method for differentiating cholinergic neurons according to claim 5, wherein: The concentration of resveratrol was 500 nM.

7. The method for differentiating cholinergic neurons according to claim 1, wherein: The time for inducing human induced pluripotent stem cells to differentiate into neuroectoderm cells is 9 to 12 days.

8. The method for differentiating cholinergic neurons according to claim 1, wherein: The time for inducing human induced pluripotent stem cells to differentiate into neuroectoderm cells is 10 days.

9. The method for differentiating cholinergic neurons according to claim 1, wherein: The differentiation time of inducing the neuroectoderm cells to differentiate into medial ganglionic eminence cells is 15 to 17 days.

10. The method for differentiating cholinergic neurons according to claim 1, wherein: The differentiation time of inducing the neuroectoderm cells to differentiate into medial ganglionic eminence cells is 15 days.

11. The method for differentiating cholinergic neurons according to claim 1, wherein: The differentiation time of inducing the medial ganglionic eminence cells to differentiate into mature cholinergic neurons is 13 to 15 days.

12. The method for differentiating cholinergic neurons according to claim 1, wherein: The differentiation time for inducing the medial ganglionic eminence cells to differentiate into mature cholinergic neurons is 14 days.

13. A cholinergic neuron differentiation kit based on traditional Chinese medicine ingredients, comprising: A first culture medium comprising a small molecule preparation for inducing human induced pluripotent stem cells to differentiate into neuroectodermal cells, wherein the small molecule preparation includes a TGF-β pathway inhibitor SB431542, a BMP pathway inhibitor LDN193189, and an mTOR pathway activator valproic acid, the concentration of SB431542 being 5-10 μM, the concentration of LDN193189 being 5-500 nM, and the concentration of valproic acid being 0.05-100 μM, and the first culture medium also comprising DMEM / F12 medium, 64 mg / L magnesium ascorbyl phosphate, 14 μg / L sodium selenate, 10.7 mg / L transferrin, 19.4 mg / L insulin, and 543 mg / L sodium bicarbonate; a second culture medium containing a Hedgehog pathway activator, Purmorphamine, for inducing the neuroectoderm cells to differentiate into medial ganglionic eminence cells, wherein the concentration of Purmorphamine is 0.5 to 2 μM, and the second culture medium further comprises 97% Neurobasal medium, 1% N2 supplement, and 2% B-27 supplement; and A third culture medium containing traditional Chinese medicine ingredients is used to induce the medial ganglionic eminence cells to differentiate into mature cholinergic neurons, wherein the traditional Chinese medicine ingredients include polydatin, paeoniflorin, salidroside and stilbene glycoside, the concentration of polydatin is 0.01~10 μM, the concentration of paeoniflorin is 0.01~10 μM, the concentration of salidroside is 0.01~10 μM, and the concentration of stilbene glycoside is 0.01~10 μM. The third culture medium also includes 97% Neurobasal medium, 1% N2 supplement and 2% B-27 supplement.

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