Induced differentiation and identification method for obtaining nerve cells in different stages from mouse embryonic stem cells

Abstract

The invention discloses an induced differentiation and identification method for obtaining nerve cells in different stages from mouse embryonic stem cells, and relates to the technical field of biology. The induced differentiation and identification method comprises the following steps: inoculating mouse embryonic stem cells into a culture container, and carrying out pluripotent maintenance culture by adopting a pluripotent maintenance culture medium; after the pluripotency maintenance culture is completed, discarding the culture medium, washing, and adding a nerve induced differentiation culture medium for nerve induced differentiation culture; in the process of nerve induced differentiation culture, gene marker and protein marker detection is carried out to obtain nerve cells in different stages. According to the induced differentiation and identification method provided by the invention, efficient and controllable differentiation of the mouse embryonic stem cells to the neural pedigree can be realized through standardized sequential control and a multi-dimensional verification system. According to the invention, an efficient and reliable cell source and a technical platform are provided for the research of a neurodevelopment mechanism and the construction of a neurodegenerative disease model.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for inducing differentiation and identifying neural cells at different stages obtained from mouse embryonic stem cells. Background Technology

[0002] The core objective of differentiating embryonic stem cells into neural cells is to obtain functional neural stem cells, neurons, and glial cells by mimicking the in vivo neural development environment, thereby meeting the needs of disease modeling, drug screening, and cell therapy. Existing technologies are mainly divided into the following four categories:

[0003] (1) Spontaneous differentiation: By removing key factors that maintain pluripotency (such as LIF), embryoids are formed to simulate the embryonic development environment, and neural progenitor cells are induced to differentiate spontaneously.

[0004] (2) Targeted induction: Using small molecule compounds (such as RA, CHIR99021) or growth factors (such as bFGF, Noggin) to precisely regulate signaling pathways (such as BMP / SMAD inhibition, Wnt activation) to directly guide mESC to neural fate conversion;

[0005] (3) Gene editing technology: By knocking out differentiation-inhibiting genes (such as Ptbp1) or overexpressing neural transcription factors (such as Ngn2, Ascl1), it can be rapidly programmed into a specific neuronal subtype (such as dopaminergic neurons);

[0006] (4) Three-dimensional engineering model: Combined with biological scaffolds or organoid culture, to simulate the in vivo neural microenvironment.

[0007] While existing technologies have achieved preliminary control over neural differentiation through multi-dimensional regulatory systems, they still face several core bottlenecks. For example, the identification system is incomplete; most methods only detect a single biomarker, lacking dynamic monitoring of stage-specific biomarkers. Differentiation stages are ambiguous, lacking temporal verification of multi-stage molecular biomarkers, leading to high cellular heterogeneity, and the expression dynamics of the oligodendrocyte marker gene Olig2 have not been fully studied. Identification methods are limited; existing technologies mostly use single-gene expression analysis, lacking combined verification of mRNA (RT-qPCR) and protein (immunofluorescence), making it difficult to accurately define cell differentiation stages. Co-culture with feeder cells is required, resulting in poor cell adhesion. Standardization of experimental conditions is insufficient; different laboratories use different culture medium formulations (e.g., B27 concentration, insulin dosage), making it difficult to reproduce experimental results. The core deficiency of existing methods lies in the lack of a standardized system covering the entire process of "pluripotency maintenance—neural induction—stage segmentation—molecular verification," leading to low efficiency in obtaining neural cells and uncontrollable stages. Summary of the Invention

[0008] The purpose of this invention is to provide a method for inducing differentiation and identifying neural cells at different stages obtained from mouse embryonic stem cells, thereby addressing the problems existing in the prior art. The induction differentiation and identification method provided by this invention, through standardized temporal control and a multi-dimensional verification system, can achieve efficient and controllable differentiation of mouse embryonic stem cells into neural lineages.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] The present invention provides a culture medium for inducing mouse embryonic stem cells to differentiate into neural structures, the culture medium comprising a pluripotency maintenance culture medium and a neural differentiation induction culture medium;

[0011] The pluripotency maintenance medium was prepared by adding the following components to Glasgow's MEM at final concentrations: 10% fetal bovine serum, 1% 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, 1 μM PD0325901 and 3 μM CHIR-99021;

[0012] The neural differentiation-inducing medium was prepared by adding the following components to a final concentration based on Neurobasal medium and DMEM / F-12 medium at a volume ratio of 1:1: 1% B-27 serum-free supplement, 0.5% N-2 supplement, 2mM GlutaMax supplement, 0.1mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 50μM bovine serum albumin, and 20μM insulin.

[0013] The present invention also provides the application of the above-mentioned culture medium for inducing mouse embryonic stem cells to differentiate into neural cells in the construction of a cell model of neural cells using mouse embryonic stem cells.

[0014] The present invention also provides the application of the above-mentioned culture medium for inducing mouse embryonic stem cells to differentiate into neural pathways in the induction of mouse embryonic stem cells to differentiate into neural pathways.

[0015] This invention also provides a method for inducing differentiation and identifying neural cells at different stages obtained from mouse embryonic stem cells, comprising the following steps:

[0016] Mouse embryonic stem cells were seeded into culture containers and pluripotency was maintained in culture using pluripotency maintenance medium.

[0017] After the pluripotency maintenance culture is completed, the culture medium is discarded, and after washing, neural induction differentiation culture medium is added for neural induction differentiation culture.

[0018] During the neural induction differentiation culture process, gene markers and protein markers were detected to obtain neural cells at different stages;

[0019] The pluripotency maintenance medium was prepared by adding the following components to Glasgow's MEM at final concentrations: 10% fetal bovine serum, 1% 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, 1 μM PD0325901 and 3 μM CHIR-99021;

[0020] The neural differentiation-inducing medium was prepared by adding the following components to a final concentration based on Neurobasal medium and DMEM / F-12 medium at a volume ratio of 1:1: 1% B-27 serum-free supplement, 0.5% N-2 supplement, 2mM GlutaMax supplement, 0.1mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 50μM bovine serum albumin, and 20μM insulin.

[0021] Furthermore, the pluripotency maintenance culture time is 24 hours.

[0022] Furthermore, the culture conditions for maintaining pluripotency are 37°C and 5% CO2.

[0023] Furthermore, the neural induction differentiation culture time is 16 days.

[0024] Furthermore, during the neural induction differentiation culture process, the culture medium is changed every 2 days.

[0025] Furthermore, the gene markers include embryonic stem cell pluripotency marker genes, neural stem cell marker genes, neuronal marker genes, oligodendrocyte marker genes, and astrocyte marker genes;

[0026] The embryonic stem cell pluripotency marker genes include Oct4 and Nanog;

[0027] The neural stem cell marker genes include Pax6, Sox1, and Nestin;

[0028] The neuronal marker gene includes Tuj1;

[0029] The oligodendrocyte marker genes include CNPase and Oligo2;

[0030] The astrocyte marker gene includes S100β.

[0031] Furthermore, the protein markers include embryonic stem cell pluripotency marker proteins, NSC marker proteins, neuronal marker proteins, oligodendrocyte marker proteins, and astrocyte marker proteins.

[0032] The embryonic stem cell pluripotency marker protein includes OCT4;

[0033] The neural stem cell marker proteins include SOX1 and NESTIN;

[0034] The neuronal marker protein includes TUJ1;

[0035] The oligodendrocyte marker proteins include CNPASE;

[0036] The astrocyte marker protein includes S100β.

[0037] The present invention discloses the following technical effects:

[0038] This invention achieves efficient and controllable differentiation of mouse embryonic stem cells (mESCs) into neural lineages through a standardized time-series control and multi-dimensional validation system (gene expression, protein detection, and staged collection). The main technical effects are as follows:

[0039] (1) Precise phased induction control based on time sequence

[0040] Differentiation timing optimization: A standardized 16-day differentiation timing control system based on culture medium was constructed (pluripotency maintenance phase → neural induction differentiation phase), shortening the neural induction initiation time to 24 hours (compared to 48 hours in traditional methods), accelerating cell exit from pluripotency, and significantly improving differentiation efficiency. Three key stages were systematically defined: the pluripotency exit stage, the neural progenitor cell formation stage, and the lineage specialization stage.

[0041] Pluripotency exit phase (days 1-3): The expression of pluripotency marker genes (Oct4, Nanog) and protein (OCT4) decreases rapidly, indicating that cells have exited the undifferentiated state;

[0042] Neural progenitor cell formation stage (days 4-5): The expression levels of neural stem cell marker genes (Pax6, Sox1, Nestin) and proteins (SOX1, NESTIN) reach their peak simultaneously, indicating efficient synchronous acquisition of neural progenitor cell populations;

[0043] Lineage specialization phase (days 6-16): Neuronal marker Tuj1 (days 5-10), oligodendrocyte marker CNPase / Olig2 (days 4 / 8), and astrocyte marker S100β (from day 8 onwards) are expressed sequentially, achieving the temporal differentiation of neurons and glial cells.

[0044] (2) Multidimensional verification and high-purity cell acquisition

[0045] Synergistic monitoring of gene and protein expression: The mRNA and protein expression trends of key biomarkers are highly consistent (e.g., both Sox1 mRNA and SOX1 protein are highly expressed in the NSC stage), effectively avoiding the false positive risk of single-indicator detection. The four stages of neural induction are systematically divided, providing a stable cell source and theoretical basis for neural cell function research and disease model construction.

[0046] Improved cell type purity: High-purity neurons, oligodendrocytes, and astrocytes can be obtained at the differentiation endpoint, with purity significantly better than traditional methods, providing a stable cell source for disease modeling.

[0047] (3) Core technology innovation and advantages

[0048] Culture medium optimization: The 2i / LIF system was used for pretreatment to maintain the homogeneity of mESCs, and the timing regulation was achieved by combining it with the neural induction phase, which significantly improved the differentiation synchronization.

[0049] Staged simultaneous cell acquisition: Through staged acquisition, neural stem cells, neurons, oligodendrocyte markers and astrocytes can be acquired simultaneously in a single differentiation system to meet the needs of multi-scenario research.

[0050] (4) Application value

[0051] Model building: Staged high-purity cells are suitable for neurodegenerative disease models, drug screening, and transplantation therapy research.

[0052] Integrated advantages: Through time-series control, multidimensional validation and culture medium optimization, the problems of asynchronous differentiation and mixed biomarkers in traditional methods have been solved, providing a standardized technical platform for the production and mechanism research of neural cells.

[0053] In summary, the method of this invention combines high efficiency (shortened induction time), controllability (clear stage division), and reliability (small identification deviation), integrating the advantages of comprehensive coverage of differentiation sequence, cell purity, verification system, and efficiency improvement, laying the foundation for neurodevelopment research and regenerative medicine applications. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 The image shows the results of PCR detection of a highly conserved 16S rRNA coding region fragment in the mycoplasma genome; the bands from left to right are, respectively, a 1000bp DNA marker, a positive control, a negative control, and a sample (mESC culture supernatant).

[0056] Figure 2 Figure 1 shows the RT-qPCR results of changes in mRNA expression levels of the mESC marker gene; where A represents Oct4 expression level in scheme 1; B represents the relative gene expression change of Nanog in scheme 1; C represents the relative gene expression change of Oct4 in scheme 2; D represents the relative gene expression change of Nanog in scheme 2; E represents the relative gene expression change of Oct4 in scheme 3; F represents the relative gene expression change of Nanog in scheme 3; G represents the relative gene expression change of Oct4 in scheme 4; and H represents the relative gene expression change of Nanog in scheme 4.

[0057] Figure 3 The image shows the RT-qPCR results of changes in mRNA expression levels of marker genes during mESC and its neural differentiation (days 0-16); where A represents the relative gene expression changes of Oct4; B represents the relative gene expression changes of Nanog; C represents the relative gene expression changes of Pax6; D represents the relative gene expression changes of Sox1; E represents the relative gene expression changes of Nestin; F represents the relative gene expression changes of Tuj1; G represents the relative gene expression changes of CNPase; H represents the relative gene expression changes of Olig2; and I represents the relative gene expression changes of S100β.

[0058] Figure 4 Immunofluorescence images of mESCs and their marker protein OCT4 during neural differentiation (days 0-16); DAPI staining shows cell nuclei in blue; OCT4 shows in green; Marge is an overlay image;

[0059] Figure 5 Immunofluorescence images of mESCs and their marker protein SOX1 during neural differentiation (days 0-16); DAPI staining shows cell nuclei in blue; SOX1 shows in green; Marge is an overlay image;

[0060] Figure 6 Immunofluorescence images of mESCs and their marker protein NESTIN during neural differentiation (days 0-16); DAPI staining shows cell nuclei in blue; NESTIN in red; Marge is an overlay image;

[0061] Figure 7 Immunofluorescence images of mESCs and their marker protein TUJ1 during neural differentiation (days 0-16); DAPI staining shows cell nuclei in blue; TUJ1 shows in red; Marge is an overlay image;

[0062] Figure 8 Immunofluorescence images of mESCs and their marker protein CNPase during neural differentiation (days 0-16); DAPI staining shows cell nuclei in blue; CNPase shows in red; Marge is an overlay image;

[0063] Figure 9 Immunofluorescence images of mESCs and their marker protein S100β during neural differentiation (days 0-16); DAPI staining shows cell nuclei in blue; S100β shows in red; Marge is an overlay image. Detailed Implementation

[0064] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0065] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0066] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0067] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0068] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0069] This invention provides a standardized and reproducible method for inducing differentiation and identifying mouse embryonic stem cells (mESCs). This method optimizes culture conditions (such as culture medium composition and induction time) to achieve sequential and phased induction of differentiation from mESCs to neural stem cells (NSCs), neurons, oligodendrocytes, and astrocytes (e.g., NSCs after approximately day 4-5, neurons after day 5, astrocytes after day 8, and oligodendrocytes after day 10). Simultaneously, this invention establishes a multi-dimensional molecular marker identification system covering nine key genes (Oct4, Nanog, Pax6, Sox1, Nestin, Tuj1, CNPase, Olig2, S100β) and six key proteins (OCT4, SOX1, NESTIN, TUJ1, CNPase, S100β). Combined analysis (RT-qPCR + immunofluorescence) dynamically monitors the differentiation process and accurately defines cell types at each stage, significantly improving the accuracy of differentiation status identification. Details are as follows:

[0070] Example 1

[0071] 1. Experimental Materials and Methods

[0072] 1.1 Experimental Materials

[0073] 1.1.1 Cell lines

[0074] 129 mouse embryonic stem cells (hereinafter referred to as mESC) (MUAES-01001) were purchased from Cyagen Biosciences.

[0075] 1.1.2 Main experimental reagents and consumables

[0076] 1.1.2.1 Main reagents related to mESC culture (see Table 1)

[0077] Table 1. Main reagents related to mESC culture

[0078]

[0079]

[0080] 1.1.2.2 Key reagents for neural differentiation culture (see Table 2)

[0081] Table 2. Main reagents related to neural induction differentiation culture

[0082]

[0083] 1.1.2.3 Major reagents related to RNA and mRNA extraction (see Table 3)

[0084] Table 3. Main reagents related to total RNA extraction and mRNA isolation and purification

[0085]

[0086] 1.1.2.4 Major reagents related to RT-qPCR (see Table 4)

[0087] Table 4. Main reagents related to RT-qPCR

[0088]

[0089] 1.1.2.5 Key reagents related to immunofluorescence (see Table 5)

[0090] Table 5. Main reagents related to immunofluorescence

[0091]

[0092]

[0093] 1.2 Experimental Methods

[0094] 1.2.1 Optimization of mESC Pluripotency Maintenance Culture

[0095] 1.2.1.1 Culture medium preparation

[0096] Four culture medium preparation schemes were developed, and the final pluripotency maintenance culture medium was determined by screening cell morphology and pluripotency marker gene expression.

[0097] Pluripotency maintenance medium protocol 1: Prepared using Glasgow's MEM (Gibco) by adding the following components to final concentrations: 10% (v / v) fetal bovine serum, 1% (v / v) 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% (v / v) penicillin-streptomycin solution, and 1000 Units / mL recombinant mouse leukemia inhibitory factor protein (Table 6). After mixing, the prepared medium was filtered through a disposable sterile vacuum filter and stored at 4°C.

[0098] Table 6 Components of the pluripotency maintenance medium for Scheme 1

[0099]

[0100] Pluripotency Maintenance Medium Protocol 2: Prepared using Glasgow's MEM (Gibco) by adding the following components to final concentrations: 10% (v / v) fetal bovine serum, 1% (v / v) 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% (v / v) penicillin-streptomycin, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, and 1 μM PD0325901 (Table 7). After mixing, the prepared medium was filtered through a disposable sterile vacuum filter and stored at 4°C.

[0101] Table 7 Components of the pluripotency maintenance medium for Scheme 2

[0102]

[0103] Pluripotency Maintenance Medium Protocol 3: Prepared using Glasgow's MEM (Gibco) by adding the following components to final concentrations: 10% (v / v) fetal bovine serum, 1% (v / v) 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% (v / v) penicillin-streptomycin solution, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, and 3 μM CHIR-99021 (Table 8). After mixing, the prepared medium was filtered through a disposable sterile vacuum filter and stored at 4°C.

[0104] Table 8. Components of the pluripotency maintenance culture medium for Scheme 3

[0105]

[0106] Pluripotency Maintenance Medium Protocol 4: Prepared using Glasgow's MEM (Gibco) by adding the following components to final concentrations: 10% (v / v) fetal bovine serum, 1% (v / v) 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% (v / v) penicillin-streptomycin solution, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, 1 μM PD0325901, and 3 μM CHIR-99021 (Table 9). After mixing, the prepared medium was filtered through a disposable sterile vacuum filter and stored at 4°C.

[0107] Table 9 Components of the pluripotency maintenance medium for Scheme 4

[0108]

[0109] 1.2.1.2 Cell resuscitation

[0110] (1) Coating the culture flask with 0.1% gelatin: Add 0.1% gelatin to the culture flask to cover the entire bottom surface. Place the culture flask with gelatin in the incubator and discard the gelatin after 30 minutes.

[0111] (2) Place the culture medium in a 37°C bath and add 10 mL of culture medium to a 15 mL centrifuge tube.

[0112] (3) Take out the frozen mESC and put it in a -80℃ freezer. After 2 minutes, take it out and place it in 37℃. Shake it quickly to thaw it.

[0113] (4) Use a pipette to transfer the cell cryopreservation suspension into a centrifuge tube containing culture medium.

[0114] (5) Centrifuge the cell suspension at 250×g for 5 min. Remove the supernatant, add 2 mL of culture medium to the cell pellet, and gently pipette to mix the cell pellet.

[0115] (6) Convert mESC 5×10 4 pcs / cm 2 Inoculate the cells into culture flasks coated with 0.1% gelatin and add an appropriate amount of mESC medium. Gently shake in a cross-shaped motion to distribute the cells evenly. Incubate at 37°C, 5% CO2, and saturated humidity.

[0116] 1.2.1.3 Mycoplasma Detection

[0117] (1) Sample preparation: Take 100 μL of cell culture supernatant and transfer it to a centrifuge tube. Incubate at 95°C for 5 min. Then, briefly centrifuge at 250 × g for 5 s to allow cell debris particles to precipitate.

[0118] (2) PCR reaction loading: The total PCR reaction system was set to 25 μL. The reaction systems for samples, positive control and negative control are shown in Table 10. All components were mixed before loading.

[0119] Table 10 PCR Reaction System

[0120]

[0121]

[0122] (3) PCR amplification: Place all test tubes into the PCR instrument, set the parameters of the thermal cycling program and perform PCR (Table 11).

[0123] Table 11 PCR Reaction Conditions

[0124]

[0125] (4) Agarose gel electrophoresis: Prepare a 1.2% standard agarose gel and add each PCR product sequentially to the gel sample wells. Stop electrophoresis after migration of 2.5 cm (run at 100V for 25 min), and observe the experimental results using a gel imaging system.

[0126] 1.2.1.4 Cell medium exchange

[0127] (1) During the culture process, the cell culture medium is changed regularly (every 24 hours apart). The cell culture supernatant in the T25 culture flask is aspirated, 5 mL of PBS is added, and the flask is gently shaken to wash away the dead cells that have not adhered to the wall. Then the PBS is discarded.

[0128] (2) After repeating twice, add 5 mL of fresh normal culture medium along the wall of the culture flask, and finally put the T25 culture flask into an incubator with 37℃, 5% CO2 and saturated humidity for incubation.

[0129] 1.2.1.5 Cell Counting and Passaging

[0130] (1) When the cell confluence reaches 80-90%, remove the cell culture supernatant.

[0131] (2) After washing twice with 5 mL of PBS, add 1 mL of Trypsin-EDTA to a T25 culture flask, mix well, and digest on a hot plate at 37°C for 1 min.

[0132] (3) Observe the cells until most of the cells become round and fall off. Add 5 mL of culture medium to stop the digestion reaction. After the cells are uniformly pipetted, transfer them to a new 15 mL centrifuge tube and centrifuge at 250×g for 5 min. Discard the supernatant.

[0133] (4) Cell counting: Add 10 mL of culture medium and gently pipette the cells until they are evenly suspended. Take 50 μL of the cell suspension and mix it with an equal volume of trypan blue. Count the cells using a hemocytometer.

[0134] (5) Cell passage: Add 10 mL of culture medium and pipette the cells until they are evenly suspended. Then transfer the 10 mL cell suspension to two or more T25 culture flasks and finally place the culture flasks in an incubator at 37°C, 5% CO2 and saturated humidity.

[0135] 1.2.1.6 Cell Collection and Cryopreservation

[0136] (1) When the cells reach 80-90% confluence, discard the culture supernatant and wash the cells twice with PBS.

[0137] (2) Add Trypsin-EDTA to the culture flask and digest at 37°C for 2 min. When most of the cells become round and fall off, add 5 mL of fresh culture medium to stop the digestion.

[0138] (3) Cell collection: After the cells are pipetted until homogeneous, transfer them to centrifuge tubes, centrifuge at 250×g for 5min, discard the supernatant, wash the cells 3 times with PBS, and store the precipitated cell clusters at -80℃.

[0139] (4) Cell cryopreservation: After the cells are homogenized by pipetting, transfer them to centrifuge tubes and centrifuge at 250×g for 5 min. Remove the supernatant. Add 1.5 mL of cell cryopreservation solution, pipette and aspirate evenly, and transfer to cryopreservation tubes. Place them in a cryopreservation box and freeze them in an ultra-low temperature freezer at -80℃. After 24 h, store them in liquid nitrogen.

[0140] 1.2.2 Neural Induction Differentiation

[0141] 1.2.2.1 Preparation of Neural Differentiation Induction Culture Medium

[0142] mESC neural differentiation-inducing medium was prepared by mixing Neurobasal medium and DMEM / F-12 medium at a volume ratio of 1:1, and then adding the following components to a final concentration: 1% (v / v) B-27 serum-free supplement, 0.5% (v / v) N-2 supplement, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% (v / v) penicillin-streptomycin solution, 50 μM bovine serum albumin, and 20 μM insulin (Table 12). The prepared medium was filtered through a disposable sterile vacuum filter and stored at 4°C.

[0143] Table 12 Components of Neural Differentiation Induction Culture Medium

[0144]

[0145] 1.2.2.2 mESC neural induction differentiation

[0146] (1) mESC with 5×10 4 The cells were seeded into culture flasks at a density of 1 cell / cm² and cultured in pluripotency maintenance medium at 37°C and 5% CO₂ for 24 hours. After this period, the cell density reached the conditions for neural differentiation, and the pluripotency maintenance medium was discarded. The cells were washed three times with PBS to remove unattached dead cells, impurities, and small factors that inhibit cell differentiation.

[0147] (2) Add 5 mL of neural differentiation induction medium and change the medium every 2 days.

[0148] (3) Continue induction for 16 days and collect cells according to the stages (differentiation days 0-16).

[0149] 1.2.3 RT-qPCR detection of changes in mESC and marker gene mRNA expression levels during neural differentiation

[0150] RT-qPCR was used to determine the relative expression changes of mESC and related genes (internal reference Gapdh) during neural differentiation, including mESC pluripotency marker genes (Oct4, Nanog), neural stem cell (NSC) marker genes (Pax6, Sox1, Nestin), neuronal marker gene (Tuj1), oligodendrocyte marker genes (CNPase, Olig2) and astrocyte marker gene (S100β). qPCR primer sequences were designed using Primer Premier 6.

[0151] 1.2.3.1 Total RNA Extraction

[0152] To obtain high-quality total RNA, it is necessary to create a ribonuclease-free environment to avoid contamination. This is achieved using RNaseZap. TM RNase Decontamination Solution prevents RNase contamination in the environment.

[0153] (1) Take the cell cluster out of the -80℃ freezer, add 1mL of Trizol reagent, blow and aspirate the cell cluster to form a lysed cell suspension, transfer it to a sterile 1.5mL centrifuge tube and incubate at room temperature for 5min.

[0154] (2) Add 0.2 mL of chloroform to each 1 mL of Trizol reagent, tighten the centrifuge tube cap, shake vigorously for 15 s, and incubate at room temperature for 3 min after it has been fully emulsified.

[0155] (3) When the sample is centrifuged at 12000×g for 15 min at 4℃, the liquid will separate into three layers. The mixture will separate into a red lower organic phase, an intermediate layer, and a colorless upper aqueous phase.

[0156] (4) Tilt the centrifuge tube at 45° and transfer the upper aqueous phase into a sterile, enzyme-free 1.5mL centrifuge tube. Avoid contamination from other layers during the transfer of the aqueous phase. Store the middle and lower organic phases at -80°C for subsequent protein extraction.

[0157] (5) Add 0.5 mL of isopropanol to the aqueous phase, mix well, incubate at room temperature for 10 min, and then incubate at -20℃ for 2 h to precipitate RNA.

[0158] (6) Place at room temperature for 5 min, centrifuge at 12000×g at 4℃ for 10 min, aspirate the supernatant, leaving only RNA particles.

[0159] (7) Slowly add 1 mL of 75% ethanol along the centrifuge tube, gently invert the tube to wash the RNA particles, and then centrifuge at 7500×g for 5 min at 4°C.

[0160] (8) Discard the supernatant and vacuum or air dry the RNA particles for 10 minutes until the RNA particles are semi-transparent.

[0161] (9) Add 60 μL of RNase-Free ddH2O to the centrifuge tube and blow and aspirate until the RNA is completely dissolved.

[0162] (10) The RNA concentration was determined using an ultra-micro UV-Vis spectrophotometer and then used for downstream applications or stored at -80°C.

[0163] 1.2.3.2 RNA Reverse Transcription

[0164] (1) Take 100 ng of total RNA and thaw it on ice.

[0165] (2) Genomic DNA removal reaction (10 μL) (Table 13), 42℃ water bath for 5 min.

[0166] Table 13 Genomic DNA Removal Reaction System (10 μL)

[0167]

[0168]

[0169] (3) Continue the reverse transcription reaction (20 μL) to obtain cDNA. After mixing, immediately incubate at 37°C for 15 min, then heat shock at 85°C for 5 s, and continue downstream experiments or store at -20°C (Table 14).

[0170] Table 14 Reverse transcription system (20 μL)

[0171]

[0172] 1.2.3.3 qPCR

[0173] (1) Prepare the qPCR reaction system (20 μL) on ice according to the following components (Table 15).

[0174] Table 15 PCR Reaction System

[0175]

[0176] (2) After gentle mixing, perform qPCR reaction (Table 16).

[0177] Table 16 PCR Reaction Conditions

[0178]

[0179] (3) Data export and analysis, using 2 -△△Ct The relative expression level of the target gene relative to the internal reference gene (β-ACTIN) was calculated.

[0180] 1.2.4 Immunofluorescence detection of changes in marker protein expression during mESC neural differentiation

[0181] Immunofluorescence was used to determine the protein expression changes of mESCs and related genes during their neural differentiation, including mESC pluripotency marker protein (OCT4), NSC marker proteins (SOX1, NESTIN), neuronal marker protein (TUJ1), oligodendrocyte marker protein (CNPASE), and astrocyte marker protein (S100β).

[0182] (1) Prepare 1% BSA: Mix 0.5g of bovine serum albumin with 50mL of PBS.

[0183] (2) Prepare 0.2% Triton TM X-100: Take 260 μL of Triton. TM Mix X-100 with 13 mL of 1% BSA.

[0184] (3) Prepare antibodies: Dilute the primary and secondary antibodies with 1% BSA according to the dilution ratio in the antibody instructions.

[0185] (4) After sterilizing the coverslip, place it into a six-well plate and inoculate cells.

[0186] (5) Once the cell density reaches the required level, discard the cell culture medium, add 2 mL of PBS and wash 3 times, 3 min each time.

[0187] (6) Fix with 2 mL of 4% paraformaldehyde for 15 min.

[0188] (7) Wash three times with 2 mL of PBS, 5 min each time. Add 2 mL of 0.2% Triton solution. TM Incubate with X-100 for 10 minutes.

[0189] (8) Wash 3 times with 2 mL of PBS, 5 min each time. Block in 1% BSA at room temperature for 1 h.

[0190] (9) Wash 3 times with 2 mL of PBS, 5 min each time. Add 500 μL of primary antibody and incubate overnight at 4°C.

[0191] (10) Discard the primary antibody and wash three times with 2 mL of PBS, 5 min each time. Add 500 μL of secondary antibody and incubate in the dark for 1 h.

[0192] (11) Discard the secondary antibody and wash three times with 2 mL of PBS, 10 min each time. Add 100 μL of DAPI and incubate at room temperature for 3 min.

[0193] (12) Discard the DAPI staining solution, add 2 mL of PBS and wash 3 times, 5 min each time.

[0194] (13) Remove the coverslip and place it on absorbent paper to dry in the dark.

[0195] (14) After drying, place it upside down on a glass slide with glycerin and fluorescence quencher added. Seal the coverslip with nail polish to prevent it from drying out. Observe under a laser confocal microscope.

[0196] 2 Experimental Results

[0197] 2.1mESC Mycoplasma test negative

[0198] To ensure that mESCs were not contaminated with mycoplasma, PCR was used for verification. The results showed that the highly conserved 16S rRNA coding region of the mycoplasma genome was not detected in the mESC culture supernatant. Figure 1 The result indicates a negative mESC mycoplasma test, which can be used for subsequent research.

[0199] 2.2 Determination of mESC pluripotency through culture

[0200] To optimize the mESC pluripotency maintenance medium, four medium preparation schemes were developed. The changes in mESC pluripotency marker gene (Oct4 and Nanog) mRNA levels relative to the internal control (Gapdh) were detected by RT-qPCR. The results showed that scheme four was the optimal pluripotency maintenance medium, while schemes one, two, and three all led to a decrease in cell pluripotency. Figure 2 In the subsequent maintenance of pluripotency, option four was chosen.

[0201] 2.3 Changes in marker gene expression during mESC neural induction differentiation

[0202] Using mESCs as the research object, a 16-day neural differentiation induction protocol was used to culture mESCs to obtain cells at different neural differentiation stages, i.e., cells from day 0 to day 16. As expected, mESCs differentiated in the correct direction. The changes in the mRNA levels of marker genes relative to the internal control (Gapdh) during mESC neural differentiation were detected by RT-qPCR. The results showed that the mESC pluripotency-related gene Oct4 significantly decreased starting from day 3. Figure 3 Similarly, the mESC pluripotency-related gene Nanog decreased significantly starting on day 1. Figure 3(B) indicates that ESCs have begun to differentiate; the expression of the NSC-related gene Pax6 increases significantly with differentiation, reaching its peak on day 4, and then gradually decreases due to further differentiation into neurons and other neural cell types. Figure 3 (C); The expression of the NSC-related gene Sox1 significantly increases with differentiation, reaching its peak on day 4. Subsequently, as it further differentiates into neurons and other neural cell types, its expression gradually decreases, then stabilizes and remains significantly higher than that in mESCs, indicating the presence of NSCs throughout the entire differentiation process. Figure 3 (D); The expression of the NSC-related gene Nestin increases significantly with differentiation, reaching its peak on day 5, and then gradually decreases due to further differentiation into other neural cell types. Figure 3 (E); The expression of the neuron-related gene Tuj1 shows a trend of first decreasing and then increasing as differentiation progresses. Figure 3 (Middle F); oligodendrocyte-related genes CNPase and Oligo2 significantly increased from day 10 and day 4, respectively, as differentiation progressed. Figure 3 (G and H); the astrocyte-associated gene S100β increases significantly from day 4 onwards as differentiation progresses (G and H); Figure 3 (I). The above results demonstrate, through changes in marker gene expression, that mESCs and cells at different stages of neural-induced differentiation were successfully obtained.

[0203] 2.4 Changes in marker gene protein levels during mESC neural induction differentiation

[0204] To further determine the differentiation of mESCs into neural cells, immunofluorescence was used to measure the expression changes of mESCs and related proteins during neural differentiation. The results showed that the mESC pluripotency-related protein OCT4 significantly decreased after culture medium replacement and its fluorescence disappeared by day 5. Figure 4 This indicates that ESCs have begun to differentiate; the expression and fluorescence intensity of the NSC-related protein SOX1 significantly increased as differentiation progressed, reaching its peak on day 5. Subsequently, due to further differentiation into neurons and other neural cell types, the fluorescence gradually decreased but remained persistent, further demonstrating the presence of NSCs throughout the entire differentiation process. Figure 5 The NSC-related protein NESTIN showed fluorescence from day 3 to day 16 of differentiation, indicating the presence of NSCs. Figure 6 Neuron-associated protein TUJ1 appears from day 5 to day 16 of differentiation. Figure 7 Oligodendrocyte-associated protein CNPase increased significantly starting from day 8. Figure 8 Astrocyte-associated protein S100β increased significantly starting from day 8. Figure 9The above results demonstrate the successful acquisition of mESCs and cells at different stages of neural-induced differentiation through changes in marker protein levels.

[0205] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium for inducing mouse embryonic stem cells to differentiate into neural pathways, characterized in that, The culture medium includes a pluripotency maintenance medium and a neural differentiation induction medium; The pluripotency maintenance medium was prepared by adding the following components to Glasgow's MEM at final concentrations: 10% fetal bovine serum, 1% 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, 1 μM PD0325901 and 3 μM CHIR-99021; The neural differentiation-inducing medium was prepared by adding the following components to a final concentration based on Neurobasal medium and DMEM / F-12 medium at a volume ratio of 1:1: 1% B-27 serum-free supplement, 0.5% N-2 supplement, 2mM GlutaMax supplement, 0.1mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 50μM bovine serum albumin, and 20μM insulin.

2. The application of the culture medium as described in claim 1 for inducing mouse embryonic stem cells to differentiate into neural cells in the construction of a neural cell-like cell model using mouse embryonic stem cells.

3. The application of the culture medium as described in claim 1 for inducing mouse embryonic stem cells to differentiate into neural pathways in the induction of mouse embryonic stem cells to differentiate into neural pathways.

4. A method for inducing differentiation and identifying neural cells at different stages obtained from mouse embryonic stem cells, characterized in that, Includes the following steps: Mouse embryonic stem cells were seeded into culture containers and pluripotency was maintained in culture using pluripotency maintenance medium. After the pluripotency maintenance culture is completed, the culture medium is discarded, and after washing, neural induction differentiation culture medium is added for neural induction differentiation culture. During the neural induction differentiation culture process, gene markers and protein markers were detected to obtain neural cells at different stages; The pluripotency maintenance medium was prepared by adding the following components to Glasgow's MEM at final concentrations: 10% fetal bovine serum, 1% 100×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 1000 Units / mL recombinant mouse leukemia inhibitory factor protein, 1 μM PD0325901 and 3 μM CHIR-99021; The neural differentiation-inducing medium was prepared by adding the following components to a final concentration based on Neurobasal medium and DMEM / F-12 medium at a volume ratio of 1:1: 1% B-27 serum-free supplement, 0.5% N-2 supplement, 2mM GlutaMax supplement, 0.1mM 2-mercaptoethanol, 1% penicillin-streptomycin solution, 50μM bovine serum albumin, and 20μM insulin.

5. The method for inducing differentiation and identification according to claim 4, characterized in that, The pluripotency maintenance culture time is 24 hours.

6. The method for inducing differentiation and identification according to claim 5, characterized in that, The culture conditions for maintaining pluripotency were 37°C and 5% CO2.

7. The method for inducing differentiation and identification according to claim 4, characterized in that, The neural induction differentiation culture time was 16 days.

8. The method for inducing differentiation and identification according to claim 4, characterized in that, During the neural induction differentiation culture process, the culture medium is changed every 2 days.

9. The method for inducing differentiation and identification according to claim 4, characterized in that, The gene markers include embryonic stem cell pluripotency marker genes, neural stem cell marker genes, neuronal marker genes, oligodendrocyte marker genes, and astrocyte marker genes. The embryonic stem cell pluripotency marker genes include Oct4 and Nanog; The neural stem cell marker genes include Pax6, Sox1, and Nestin; The neuronal marker gene includes Tuj1; The oligodendrocyte marker genes include CNPase and Oligo2; The astrocyte marker gene includes S100β.

10. The method for inducing differentiation and identification according to claim 4, characterized in that, The protein biomarkers include embryonic stem cell pluripotency marker proteins, NSC marker proteins, neuronal marker proteins, oligodendrocyte marker proteins, and astrocyte marker proteins. The embryonic stem cell pluripotency marker protein includes OCT4; The neural stem cell marker proteins include SOX1 and NESTIN; The neuronal marker protein includes TUJ1; The oligodendrocyte marker proteins include CNPASE; The astrocyte marker protein includes S100β.

Images