Construction method for induced differentiation of intestinal neural crest cells into glial precursor cells and application

Abstract

The invention belongs to the technical field of stem cell culture, and discloses a construction method and application for inducing and differentiating intestinal neural crest cells into colloidal precursor cells, the construction method comprises the following steps: S11, culturing the intestinal neural crest cells for 4 days to form neurospheres, sucking out supernatant containing the suspended neurospheres, centrifuging, discarding the supernatant, digesting with pancreatin, and collecting the supernatant; adding a DMEM / F12 culture medium to stop digestion, centrifuging, and discarding supernatant; s12, resuspending the neural spheres by using a colloidal precursor cell induction culture medium, inoculating the neural spheres into a cell culture bottle, and culturing in an incubator containing 5% of CO2 at 37 DEG C; and S13, after culturing for 16 hours, taking out the cells from the incubator to obtain the colloidal precursor cells. According to the method disclosed by the invention, intestinal neural crest cells are induced and differentiated into glial precursor cells and are further induced and differentiated into neurons, tool cells are provided for treatment of enteric nervous system diseases, a new tool is provided for understanding development and diseases of an enteric nervous system, and meanwhile, possibility is provided for developing a new treatment method.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cell culture, and relates to a construction method and application of inducing intestinal neural crest cells to differentiate into glial precursor cells. Background Art

[0002] The intestine is one of the most important organs in the human body. One of the keys to the realization of its functions and the regulation of its interactions with other organs of the body is the enteric nervous system. Disorders of the enteric nervous system are closely related to many intestinal-related diseases, such as congenital megacolon caused by the lack of neurons in the distal colon. Therefore, it is very important to effectively restore and rebuild the enteric nervous system in clinical treatment.

[0003] The development of the enteric nervous system involves the differentiation of enteric nerves from progenitor cells to glial cells and neurons. These differentiation processes are determined in the embryonic period. Existing tools for studying and treating the enteric nervous system, especially embryonic development, include cell lines, stem cells, and organoids. Cell lines such as N-2a and SY5Y are easy to culture and convenient for in vitro experiments, but these cell lines are tumor-derived and usually show abnormal metabolic behavior, making them difficult to use in cell therapy. Mesenchymal stem cells have differentiation potential, but there are still many challenges in their clinical application, such as low transplant survival rate and difficulty in in vivo differentiation. Enteric neural crest cells (ENCCs) can self-renew and differentiate into neurons and glial cells to rebuild the nervous system, but there are some bottlenecks in ENCCs transplantation, including insufficient proliferation and migration before transplantation, a large number of apoptosis during short-term transplantation, and ineffective reconstruction of neural circuits. Treatment of ENCCs with cytokines, drugs, and signaling pathway regulators to improve these side effects has also failed to completely repair the function of the enteric nervous system. The current hot topic of research, pluripotent stem cells hESC, are ethically controversial, while iPSC is more ethically acceptable, but there is a potential risk of tumor development whether derived from hESC cells or iPSC cells. In addition, allogeneic transplantation using hESC- or iPSC-derived ENCCs requires lifelong use of immunosuppressants to reduce rejection and graft-versus-host disease, but this can induce HSCR-related, recurrent, and potentially fatal enterocolitis. Intestinal organoids are three-dimensional structures cultured in vitro from tissues and pluripotent stem cells, containing a variety of cell types, which can simulate corresponding organs in terms of cell type, spatial structure, and physiological function. However, the physiological characteristics and genetic fidelity and stability of organoids are still unclear, and they lack important physiological processes in the body as well as vascular, lymphatic, and neural functions. It can be seen that the current cellular tools for studying intestinal neural development have certain limitations, and more effective tools are urgently needed to make up for the deficiencies in the study and treatment of intestinal neural development.

[0004] In the early stage, the research team discovered a group of glial progenitor cells with bidirectional potential through single sequencing. These cells can differentiate into both neurons and glial cells. Other studies have also shown that glial progenitor cells have the potential to develop into neurons. This indicates that glial progenitor cells are a good tool for the potential treatment of enteric nervous system diseases. They exist in normal intestinal cells and have the potential to differentiate into neurons. They do not require additional cell transplantation and can differentiate into neurons through self-induced differentiation, thereby treating enteric neurological diseases such as Hirschsprung's disease.

[0005] Previous articles used in vitro induction of iPSCs, differentiation into neurons, and then cell transplantation to treat congenital megacolon. The process was complicated and there were risks such as immune rejection. However, using the intestine's own glial precursor cells eliminated the tedious in vitro induction and cell transplantation process. There are no neurons in the distal part of megacolon, but there are glial precursor cells. The method of using glial precursor cells in vivo to transdifferentiate into neurons is groundbreaking and has no ethical controversy, gene editing, and immune rejection risks. It provides a new theoretical basis and treatment method for the in vivo treatment of megacolon. Summary of the invention

[0006] The present invention proposes a construction method and application for inducing the differentiation of enteric neural crest cells into glial precursor cells. The method induces the differentiation of enteric neural crest cells into glial precursor cells, and further induces the differentiation into neurons, providing tool cells for the treatment of enteric nervous system diseases, and providing new tools for understanding the development and diseases of the enteric nervous system, and also providing the possibility for developing new treatment methods.

[0007] In order to achieve the above purpose, this application adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for inducing the differentiation of enteric neural crest cells into glial precursor cells, comprising the following steps:

[0009] S11: After the enteric neural crest cells were cultured for 4 days to form neurospheres, the supernatant containing the suspended neurospheres was aspirated, centrifuged, the supernatant was discarded, digested with trypsin, DMEM / F12 medium was added to stop digestion, centrifuged, and the supernatant was discarded;

[0010] S12: Resuspend the neurospheres with glial precursor cell induction medium, inoculate them into cell culture flasks, and culture them in an incubator at 37°C with 5% CO2;

[0011] S13: After culturing for 16 hours, the cells are taken out from the incubator to obtain the glial precursor cells.

[0012] In the above technical scheme, the preparation method of the glial precursor cell induction medium is: add 1% N2 additive, 2% B27 additive, 1% L-glutamine, 10ng / ml FGF2, 10ng / ml NRG1, and 3uM CHIR99021 to Neurobasal medium, mix well, and filter with a 0.22um sterile filter to obtain the glial precursor cell induction medium.

[0013] In a second aspect, the present invention provides the glial precursor cells obtained by the above-mentioned construction method for use in inducing differentiation into neurons.

[0014] In the above technical solution, the specific method of inducing glial precursor cells to differentiate into neurons comprises the following steps:

[0015] S21: centrifuging the glial precursor cells, discarding the supernatant, resuspending the cells with neuron induction medium, inoculating the cells into a cell culture flask, and culturing them in an incubator at 37° C. containing 5% CO 2 ;

[0016] S22: After culturing for 32 hours, the cells are taken out from the incubator to obtain the neuronal cells.

[0017] In the above technical scheme, the preparation method of the neuron induction medium is: add 1% N2 additive, 2% B27 additive, 1% L-glutamine, 1% NEAA additive, 10ng / ml BDNF, 10ng / ml GDNF, 200uM Ascorbic acid, 1mM cAMP to Neurobasal medium, mix well and filter with a 0.22um sterile filter to obtain the neuron induction medium.

[0018] In a third aspect, the present invention provides a method for inducing enteric neural crest cells to differentiate into neurons, comprising the following steps:

[0019] S31: Inducing the differentiation of enteric neural crest cells into glial precursor cells, the specific method is as follows:

[0020] S311: After the enteric neural crest cells were cultured for 4 days to form neurospheres, the supernatant containing the suspended neurospheres was aspirated, centrifuged, the supernatant was discarded, digested with trypsin, DMEM / F12 medium was added to stop digestion, centrifuged, and the supernatant was discarded;

[0021] S312: resuspend the neurospheres with glial precursor cell induction medium, inoculate them into cell culture flasks, and culture them in an incubator at 37°C with 5% CO2;

[0022] S313: After culturing for 16 hours, taking out the cells from the incubator to obtain the glial precursor cells;

[0023] The preparation method of the glial precursor cell induction medium is as follows: 1% N2 additive, 2% B27 additive, 1% L-glutamine, 10ng / ml FGF2, 10ng / ml NRG1, and 3uM CHIR99021 are added to Neurobasal medium, and the mixture is mixed and filtered through a 0.22um sterile filter to obtain the glial precursor cell induction medium;

[0024] S32: Inducing the glial precursor cells to differentiate into neurons, specifically by:

[0025] S321: centrifuging the glial precursor cells, discarding the supernatant, resuspending the cells with a neuron induction medium, inoculating the cells into a cell culture flask, and culturing them in an incubator at 37° C. containing 5% CO 2 ;

[0026] S322: After culturing for 32 hours, taking out the cells from the incubator to obtain the neuronal cells;

[0027] The preparation method of the neuron induction medium is as follows: 1% N2 additive, 2% B27 additive, 1% L-glutamine, 1% NEAA additive, 10ng / ml BDNF, 10ng / ml GDNF, 200uM Ascorbic acid, 1mM cAMP are added to Neurobasal medium, and the mixture is mixed and filtered through a 0.22um sterile filter to obtain the neuron induction medium.

[0028] In a fourth aspect, the present invention provides the use of the neurons obtained by the above-mentioned construction method in the preparation of drugs, wherein the drugs are used to treat enteric nervous system diseases, and the drugs contain the neurons to promote the growth of distal colon neurons by cell transplantation.

[0029] In the above technical solution, the enteric nervous system diseases include Hirschsprung's disease and its homologous diseases.

[0030] The beneficial effects of the present invention are as follows: the method of the present invention comprises isolating fetal rat intestinal cells, and using the fetal rat intestinal cells to culture enteric neural crest cells; inducing differentiation of enteric neural crest cells to obtain glial precursor cells with the potential to differentiate into neurons. The present invention enriches glial precursor cells in the intestine that can differentiate into neurons. The glial precursor cells have the potential to differentiate into neurons, are a new practical tool for studying the enteric nervous system, and are helpful in treating children with congenital megacolon, improving the distal aganglion cells in children with congenital megacolon, and improving intestinal peristalsis, and have important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1is a culture morphology diagram of ENCCs on the 4th day of culture in an embodiment of the present invention;

[0032] Figure 2 is an immunofluorescence identification diagram of ENCCs on the 4th day of culture in an embodiment of the present invention;

[0033] Figure 3 is a flow cytometry identification diagram of ENCCs at 0th and 4th day of culture in an embodiment of the present invention;

[0034] Figure 4 In the embodiment of the present invention, the optimal time for inducing glial precursor cells is determined;

[0035] Figure 5 It is the flow cytometry result of the change of glial precursor cell marker with culture time in the embodiment of the present invention;

[0036] Figure 6 It is the RT-qPCR result of the change of neuronal marker with culture time in the embodiment of the present invention; wherein blue represents the initial cells after ENCCs culture for 4 days, red represents the cells after 16 hours of induction with glial precursor cell induction medium, green represents the cells after 16 hours of induction with glial precursor cell induction medium and then induced with neuron induction medium for 32 hours, and purple represents the cells after 1 day of induction with glial precursor cell induction medium and then 1 day of induction with neuron induction medium. DETAILED DESCRIPTION

[0037] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0038] Example 1 Method for constructing glial precursor cells

[0039] 1. Extraction, culture and identification of primary mouse ENCCs

[0040] (1) Adult C57 mice older than 8 weeks old were selected and caged together at a male:female ratio of 2:1 at 8 pm the previous day. The plugs were checked at 8 am the next day and mice with plugs were placed together separately, marked as E0.5d. After 14 days, i.e., E14.5d, samples were prepared.

[0041] (2) Prepare sterile dissecting instruments, a clean bench, a 0.22 μm sterile filter, and prepare the digestion solution and culture medium of fetal rat enteric neural crest cells in advance. The preparation method of the digestion solution of fetal rat enteric neural crest cells is as follows: Dispase II (Sigma, concentration 2 mg / ml), collagenase IV (Sigma, concentration 0.5 mg / ml), and DNAse I (Sigma, concentration 5 mg / ml) are added to DMEM / F-12 (Gibco) basal culture medium, mixed and filtered through a 0.22 um sterile filter for use; the preparation method of fetal rat enteric neural crest cell culture medium is as follows: N2 supplement (Gibco, concentration 1%), B27 supplement (Gibco, concentration 2%), glutamine (Gibco, concentration 1%), EGF (Peprotech, 10 ng / ml), bFGF (Peprotech, 10 ng / ml), and double antibody (Gibco, concentration 1%) are added to DMEM / F-12 (Gibco) basal culture medium, mixed by pipetting and filtered through a 0.22 um sterile filter for use.

[0042] (3) Prepare two pregnant mice at E14.5 days old, kill them by cervical dislocation, spray them with alcohol, and place them on the operating table. Quickly dissect the fetuses and place them in PBS in an ice bath (keep them on ice the whole time). Remove the intestinal tissue of the fetuses under a dissecting microscope and place them in DMEM / F-12 culture medium in an ice bath (keep them on ice the whole time). After all the intestinal tissue of the fetuses is removed, transfer it to the cell operating table.

[0043] (4) Wash the intestinal tissue with pre-cooled DMEM / F-12 medium (keep on ice throughout the process), cut the intestinal tissue into 0.05-0.1 cm pieces on ice, resuspend the cut intestinal tissue with DMEM / F-12 medium, transfer to a 15 ml centrifuge tube, and centrifuge at 800 r / min at 4°C for 5 min.

[0044] (5) Discard the supernatant and add 10 ml of digestion solution (prepared freshly for use) to resuspend the cells. Mix well and digest at 37°C for 30 min, mixing every 5 min.

[0045] (6) Add an equal volume of DMEM / F-12 medium to terminate digestion, filter with a 40 μm cell filter, transfer to a 50 ml centrifuge tube, and centrifuge at 1000 r / min at 4°C for 5 min.

[0046] (7) Discard the supernatant, add 5 ml of DMEM / F-12 medium to resuspend the cells and wash the cells, and centrifuge at 1000 r / min at 4°C for 5 min.

[0047] (8) Discard the supernatant, add ENCCs culture medium to resuspend the cells, count the cells, and inoculate them into cell culture flasks at a density of 5 x 10^5 cells / ml. Add 5 ml of cell suspension to each flask and culture them in an incubator at 37°C with 5% CO2.

[0048] (9) On the second day, add 3 ml of ENCCs culture medium (recorded as culture day 1), and change half of the medium every 3 days thereafter. Observe under a microscope every day, take photos and save them, and record the changes in growth morphology.

[0049] like Figure 1 Shown is the culture morphology of ENCCs on the 4th day of culture.

[0050] 2. Inducing ENCCs to differentiate into glial precursor cells

[0051] (1) Prepare glial progenitor cell induction medium: Add 1% N2 (Gibco), 2% B27 (Gibco), 1% L-glutamine (Gibco), 10ng / ml FGF2 (Peprotech), 10ng / ml NRG1 (MCE), and 3uM CHIR99021 (MCE) to Neurobasal medium (Gibco), mix well, and filter with a 0.22um sterile filter for later use. Prepare the medium just before use and use it immediately.

[0052] (2) After the ENCCs extracted above were cultured for 4 days to form neurospheres, the supernatant containing the suspended neurospheres was aspirated, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, digested with 1 ml of trypsin for 1 min, and then 1 ml of basal culture medium was added to stop digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the neurospheres were resuspended in 5 ml of glial precursor cell induction medium. The cells were counted and seeded into a cell culture flask at a density of 1 x 10^5 cells / ml, and cultured in an incubator at 37°C containing 5% CO2.

[0053] (3) After 16 h of culture, the cells were removed from the incubator and flow cytometry was performed to verify the content of glial precursor cells.

[0054] Example 2 Inducing differentiation of glial precursor cells into neurons

[0055] The specific method is:

[0056] (1) Prepare neuronal induction medium: Add 1% N2 (Gibco), 2% B27 (Gibco), 1% L-glutamine (Gibco), 1% NEAA (Gibco), 10 ng / ml BDNF (Peprotech), 10 ng / ml GDNF (Peprotech), 200 uM Ascorbic acid (Sigma), and 1 mM cAMP (Sigma) to Neurobasal medium (Gibco), mix well, and filter through a 0.22 um sterile filter for later use.

[0057] (2) ENCCs were cultured with glial progenitor cell induction medium for 16 h, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended with 5 ml of neuronal induction medium, inoculated into a cell culture flask, and cultured at 37°C in an atmosphere containing 5% CO2.

[0058] (3) After 32 h of culture, the cells were removed and flow cytometry was performed to verify the neuronal cell content.

[0059] Example 3 Identification of extracted ENCCs and induced glial precursor cells and neurons

[0060] 1. Immunofluorescence staining of primary mouse ENCCs

[0061] (1) Place a cell slide in a 12-well plate, add 500 ul of poly-lysine (0.1 mg / ml) to each well, so that the poly-lysine covers the cell slide at the bottom of the culture plate, and incubate in a 37°C incubator for 1 h;

[0062] (2) Take out the poly-lysine-coated 12-well plate, aspirate the poly-lysine solution with a pipette, wash it three times with sterile PBS, and set aside;

[0063] (3) The cultured ENCCs were transferred to a 15 ml centrifuge tube, centrifuged at 800 rpm for 5 min, the supernatant was discarded, the culture was resuspended with ENCCs culture medium, and inoculated into a poly-lysine-coated 12-well plate, placed in a 37°C incubator, and cultured for 1 h until the ENCCs adhered to the wall;

[0064] (4) Take out the 12-well plate, discard the culture medium, wash twice with PBS, add 400 μl of 4% paraformaldehyde and fix on a shaker for 30 min;

[0065] (5) Aspirate and discard paraformaldehyde, and wash with PBS on a shaker three times, 5 min each time;

[0066] (6) Aspirate and discard PBS, and block with 5% BSA solution (containing 0.3% Triton X-100) prepared in PBS at room temperature for 1 h;

[0067] (7) Aspirate the BSA solution and wash with PBS on a shaker three times, 5 min each time;

[0068] (8) Aspirate and discard PBS, add primary antibody P57 diluted in PBS NTR (ab52987, abcam) and Nestin (ab81462, abcam), incubated overnight at 4°C;

[0069] (9) Take the cells out of the 4°C refrigerator and place them on a shaker. After returning to room temperature (about 10 min), recover the primary antibody and wash them three times with PBS, each time for 5 min.

[0070] (10) Aspirate and discard PBS, add fluorescent secondary antibody CoraLite488-conjugated Goat Anti-Rabbit IgG (H+L) (SA00013-2, Proteintech) and Cy3-labeled goat anti-rat IgG (GB21302, Servicebio) diluted in PBS, and incubate at room temperature in the dark for 1 h;

[0071] (11) Aspirate and discard the secondary antibody, and wash with PBS three times, 5 min each time;

[0072] (12) Aspirate PBS, add DAPI stain to counterstain the cell nucleus, and incubate in the dark for 5 min;

[0073] (13) Aspirate and discard DAPI, and wash with PBS three times, 5 min each time;

[0074] (14) Take out the cell slide from the well plate, wait for it to dry slightly, attach the cell slide to a glass slide, seal the slide with an anti-fluorescence quencher sealant (G1401, Servicebio), and observe and collect images under a fluorescence microscope.

[0075] like Figure 2 The figure shows the immunofluorescence identification of ENCCs on the 4th day of culture; two markers (P75 NTR and Nestin) co-label enteric neural crest cells in neurospheres.

[0076] 2. Identification of primary mouse ENCCs by flow cytometry

[0077] (1) After digestion and resuspension of ENCCs, centrifuge at 1400 rpm, 4°C for 5 min, discard the supernatant, wash the cells once with PBS (containing 1% BSA), and add 1×10 7 Resuspend the cells in 500ul PBS (containing 1% BSA) at a concentration of cells / mL;

[0078] (2) For each tube of blank control, isotype control, and test sample, 100uL of the above resuspended cells were placed in a centrifuge tube. No antibody was added to the blank control tube, and 0.5uL of FVS dye (564406, BD bioscience) was added to the isotype control and test tubes, respectively. The tubes were gently mixed and incubated at 4°C in the dark for 20 min. The tubes were gently mixed every 10 min, and the tubes were centrifuged at 1400 rpm, 4°C for 5 min, and the supernatant was removed.

[0079] (3) Resuspend the cells in 100uL PBS (containing 1% BSA). No antibody is added to the blank control tube, and 3uL P75 NTR (130-128-624, Miltenyi), 3ul HNK1 (359610, Biolegend) antibody, used to label ENCCs, incubated at 4°C in the dark for 30min, and gently mixed once every 10min;

[0080] (4) Add 100uL PBS (containing 1% BSA), centrifuge at 1400rpm, 4°C for 5min, wash 1-2 times, resuspend in 500uL PBS (containing 1% BSA) and add to the flow tube, filter and detect on the flow cytometer.

[0081] like Figure 3 The figure shows the flow cytometry analysis of fetal rat enteric neural crest cells at 0 and 4 days of culture. + CD271 + The cell ratio also showed statistically significant difference (P<0.001). NTR+ HNK1 + The proportion of cells increased from 1.98% on day 0 to 4.50%, indicating that after 4 days of culture, the proportion of enteric neural crest cells (P75 NTR+ HNK1 + ) significantly increased.

[0082] 3. Identification of glial precursor cells by flow cytometry

[0083] The experimental steps were the same as in 2, using antibodies FABP7 (ab279649, Abcam) and SOX10 (sc-365692, Santa Cruz Biotechnology), and using fluorescent secondary antibodies DyLight 405, Goat Anti-Rabbit IgG (A23120, Abbkine) and Dylight 649, Goat Anti-Mouse IgG (A23610, Abbkine) to label glial precursor cells. Figure 4 As shown in the figure, it was found that when glial precursor cells were cultured in the induction medium for 16 h, FABP7 +and SOX10 + Therefore, it is believed that 16 hours of induction of glial precursor cells is the best induction condition, which can better induce ENCCs into glial precursor cells.

[0084] like Figure 5 The results are shown in the flow cytometry results of the changes of glial progenitor cell markers over the culture time. It can be seen that the content of glial progenitor cells in ENCCs is 5.56%. After 16 hours of induction with glial progenitor cell induction medium, FABP7 + SOX10 + The content of double-positive cells was the highest, accounting for 8.54%. As the induction time of glial progenitor cells increased, the content of glial progenitor cells gradually decreased, indicating that the induction of glial progenitor cells for 16 h was the key to induce FABP7. + SOX10 + The optimal time for glial precursor cells can significantly increase the content of glial precursor cells.

[0085] 4. RT-PCR Identification of Neuronal Content

[0086] The initial cells of ENCCs cultured for 4 days (ENCCs 4d), the glial progenitor cells induced by glial progenitor cell induction medium for 16 hours on the basis of ENCCs (ENCCs 4d+16h), the cells of glial progenitor cells induced by neuron induction medium for 32 hours (ENCCs 4d+16h+32h), and the cells induced by glial progenitor cell induction medium for 1 day and then by neuron induction medium for 1 day (ENCCs 4d+1d+1d) were collected respectively. It was found that the neurons content was highest when ENCCs were induced by glial progenitor cell induction medium for 16 hours and then by neuron induction medium for 32 hours.

[0087] like Figure 6 The results of RT-qPCR of neuronal markers changing with culture time are shown; blue represents the initial cells after 4 days of ENCCs culture, red represents the cells after 16 hours of induction with glial progenitor cell induction medium, green represents the cells after 16 hours of induction with glial progenitor cell induction medium and then 32 hours of induction with neuron induction medium, and purple represents the cells after 1 day of induction with glial progenitor cell induction medium and then 1 day of induction with neuron induction medium. It was found that after 16 hours of glial progenitor cell induction and 32 hours of neuron induction, the neuronal cell content of ENCCs increased significantly, and was significantly higher than that of the group without neuron induction or the group with 1 day of glial progenitor cell induction and 1 day of neuron induction, indicating that neuron induction is effective, and the best neuron induction result can be achieved by inducing glial progenitor cells for 16 hours and then neuron induction for 32 hours.

[0088] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for inducing the differentiation of enteric neural crest cells into glial precursor cells, characterized in that: The following steps are involved: S11: After the enteric neural crest cells were cultured for 4 days to form neurospheres, the supernatant containing the suspended neurospheres was aspirated, centrifuged, the supernatant was discarded, digested with trypsin, DMEM / F12 medium was added to stop digestion, centrifuged, and the supernatant was discarded; S12: Resuspend the neurospheres with glial precursor cell induction medium, inoculate them into cell culture flasks, and culture them in an incubator at 37°C with 5% CO2; S13: After culturing for 16 hours, the cells are taken out from the incubator to obtain the glial precursor cells.

2. The construction method according to claim 1, characterized in that: The preparation method of the glial precursor cell induction medium is as follows: 1% N2 additive, 2% B27 additive, 1% L-glutamine, 10ng / ml FGF2, 10ng / ml NRG1, and 3uM CHIR99021 are added to Neurobasal medium, and the mixture is mixed and filtered through a 0.22um sterile filter to obtain the glial precursor cell induction medium.

3. The glial precursor cells obtained by the construction method according to any one of claims 1 to 2 are used to induce differentiation into neurons.

4. The application according to claim 3, characterized in that: The specific method of inducing glial precursor cells to differentiate into neurons comprises the following steps: S21: centrifuging the glial precursor cells, discarding the supernatant, resuspending the cells with neuron induction medium, inoculating the cells into a cell culture flask, and culturing them in an incubator at 37° C. containing 5% CO 2 ; S22: After culturing for 32 hours, the cells are taken out from the incubator to obtain the neuronal cells.

5. The application according to claim 4, characterized in that: The preparation method of the neuron induction medium is as follows: 1% N2 additive, 2% B27 additive, 1% L-glutamine, 1% NEAA additive, 10ng / ml BDNF, 10ng / ml GDNF, 200uM Ascorbic acid, 1mM cAMP are added to Neurobasal medium, and the mixture is mixed and filtered through a 0.22um sterile filter to obtain the neuron induction medium.

6. A method for inducing the differentiation of enteric neural crest cells into neurons, characterized in that: The following steps are involved: S31: Inducing the differentiation of enteric neural crest cells into glial precursor cells, the specific method is: S311: After the enteric neural crest cells were cultured for 4 days to form neurospheres, the supernatant containing the suspended neurospheres was aspirated, centrifuged, the supernatant was discarded, digested with trypsin, DMEM / F12 medium was added to stop digestion, centrifuged, and the supernatant was discarded; S312: Resuspend the neurospheres with glial precursor cell induction medium, inoculate them into cell culture flasks, and culture them in an incubator at 37°C with 5% CO2; S313: After culturing for 16 hours, taking out the cells from the incubator to obtain the glial precursor cells; The preparation method of the glial precursor cell induction medium is as follows: 1% N2 additive, 2% B27 additive, 1% L-glutamine, 10ng / ml FGF2, 10ng / ml NRG1, and 3uM CHIR99021 are added to Neurobasal medium, and the mixture is mixed and filtered through a 0.22um sterile filter to obtain the glial precursor cell induction medium; S32: Inducing the glial precursor cells to differentiate into neurons, specifically by: S321: centrifuging the glial precursor cells, discarding the supernatant, resuspending the cells with a neuron induction medium, inoculating the cells into a cell culture flask, and culturing them in an incubator at 37° C. containing 5% CO 2 ; S322: After culturing for 32 hours, taking out the cells from the incubator to obtain the neuronal cells; The preparation method of the neuron induction medium is as follows: 1% N2 additive, 2% B27 additive, 1% L-glutamine, 1% NEAA additive, 10ng / ml BDNF, 10ng / ml GDNF, 200uM Ascorbic acid, 1mM cAMP are added to Neurobasal medium, and the mixture is mixed and filtered through a 0.22um sterile filter to obtain the neuron induction medium.

7. Use of the neuron obtained by the construction method of claim 6 in the preparation of medicines, characterized in that: The medicine is used for treating enteric nervous system diseases. The medicine contains the neurons and promotes the growth of neurons in the distal part of the colon by cell transplantation.

8. The application according to claim 7, characterized in that: The enteric nervous system diseases include Hirschsprung's disease and its homologous diseases.

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