APPLICATION OF TGF-B INHIBITOR IN INDUCING NEURAL STEM CELLS AND ORGANOID FORMATION

Abstract

Provided is a new use of a TGF-β small molecule inhibitor in the field of neuroregeneration, which can be used for the in vitro regeneration and directed differentiation of various nerve cells and brain-like organs. By adding same to a set of basal media having clear chemical compositions. pluripotent stem cells can be induced into adult cells derived from a variety of neural stem cells, and the number of induced nerve cells and the size of organoids can be greatly increased. The induction system provided in the present invention expands new functions of a single small molecule in the field of ectodermal cell induction and differentiation and at the same time avoids the use of B27 and other serum substitutes, thereby completely avoiding the potential risks caused by the presence of animal-derived components in cell culture processes, and greatly expanding the clinical prospects of a variety of nerve cell transplantations.

Claims

1. An application of a TGF-β inhibitor in the formation of induced neural stem cells and organoids, wherein the TGF-β inhibitor is 4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide.

2. The application of claim 1, wherein the TGF-β inhibitor is added to the basal medium to form a neural stem cell inducing medium.

3. The application of claim 2, wherein the basal medium consists of Dulbecco's modified Eagle/F12 medium, minimum essential medium non-essential amino acids, sodium chloride, sodium selenite, insulin and recombinant human transferrin.

4. The application of claim 3, wherein the basal medium consists of Dulbecco's modified Eagle/F12 medium, 1% minimum essential medium non-essential amino acids, 0.1 g/L-0.8 g/L sodium chloride, 13.6 μg/L sodium selenite, 20 ng/ml-42 μg/ml insulin and 50 ng/ml-180 ng/ml recombinant human transferrin.

5. The application of claim 2, wherein the concentration of the TGF-β inhibitor ranges from 10 nM to 100 μM.

6. The application of claim 5, wherein the concentration of the TGF-β inhibitor is 12.5 μM.

7. The application of claim 6, wherein the basal medium contains 0.5 g/L sodium chloride, 13.6 μg/L sodium selenite, 22 μg/ml insulin and 100 ng/ml recombinant human transferrin.

8. The application of claim 2, wherein the formation of the neural stem cells includes the following step: pluripotent stem cells are adherently cultured in the neural stem cell inducing medium.

9. The application of claim 8, wherein the pluripotent stem cells are mammalian pluripotent stem cells.

10. The application of claim 9, wherein the pluripotent stem cells are human pluripotent stem cells.

11. The application of claim 8, wherein the adherent culture is performed in the presence of a basement membrane preparation.

12. The application of claim 11, wherein the basement membrane preparation is one or a combination of two or more of Matrigel, laminin, and vitronectin.

13. The application of claim 8, wherein the neural stem cells differentiate into neurons, and the neurons are selected from one or more of the following groups: pain receptor neurons, photoreceptor neurons, and dopaminergic neurons.

14. Application of the neural stem cells obtained by the step as described in claim 8 in the preparation of drugs for treating nerve injury.

15. The application of claim 1, wherein the formation of the organoids includes the following steps: (1) Suspension cell masses of pluripotent stem cells culture are cultured by adding 10 μM ROCK inhibitor Y-27632 to the neural stem cell inducing medium as described in claim 7; (2) The next day, replace the culture medium with the neural stem cell inducing medium as described in claim 7, and replace the fresh culture medium every day until the 10th day: (3) On the 10th day, replace the culture medium with the basic culture medium as described in claim 3, and add 2% B27 cell culture additive to it, and then culture until the 120th day.

16. Application of the organoids obtained by the step as described in claim 15 in drug screening of nervous system diseases.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0047] FIG. 1 Comparison of differences between the method of the invention (LY inducing method) and the control method (LSB inducing method) in the induction process of neural stem cells. FIG. 1a shows the neural rosette structure induced by a variety of small molecule inhibitors (LSB inducing method); FIG. 1b shows the neural rosette structure produced by neural stem cell induction (LY inducing method) using LY2157299 alone; FIG. 1c shows the difference in the number of neural stem cells obtained by the two methods.

[0048] FIG. 2 Screening results of core components of basic culture medium in the method of the invention. FIG. 2a shows the effect of different concentrations of sodium chloride on the osmotic pressure of the culture system. Excessive concentration will lead to osmotic pressure exceeding cell tolerance. FIG. 2b shows the effect of different concentrations of insulin on cell growth (Cyquant experiment). Insulin concentration was positively correlated with cell viability. FIG. 2c shows the effect of different concentrations of recombinant human serum albumin on cell growth (Cyquant experiment). The concentration of recombinant human serum albumin was positively, correlated with cell viability.

[0049] FIG. 3 molecular identification of the cells obtained by by the present method (LY inducing method) versus the control method (LSB inducing method). FIG. 3a represents the reconstruction of the structure of the neural rosette after in vitro passage of neural stem cells obtained by using the LSB inducing method; FIG. 3b represents that neural stem cells obtained by using the LSB inducing method express the specific marker Pax6; FIG. 3c represents that the neural stem cells obtained by using the LSB inducing method expressed the specific marker Nestin; FIG. 3d represents the channel integration picture of FIGS. 3a-3c; FIG. 3e represents the reconstruction of the structure of the neural rosette after in vitro passage of neural stem cells obtained by using the LY inducing method; FIG. 3f represents neural stem cells obtained by using LY inducing method expressed specific marker Pax6; FIG. 3g represents that the neural stem cells obtained by using the LY inducing method expressed the specific marker Nestin; FIG. 3h represents the channel integration picture of FIGS. 3e-3g; FIGS. 3i-3k show the difference in expression of neural stem cell markers during neural stem cell formation using Q-PCR comparing the LY inducing method with the LSB control inducing method.

[0050] FIG. 4 different concentrations of LY2157299 were used for chemical induction of NSCs. FIG. 4a shows neural stem cells obtained by using a multi molecular control (LSB inducing method, designated CK); FIG. 4b shows chemical induction using LY2157299 at 0 nM; FIG. 4c shows chemical induction using LY2157299 at 20 nM; FIG. 4d shows chemical induction using LY2157299 at 12.5 μM; FIG. 4e shows chemical induction using LY2157299 at 25 μM; FIG. 4f shows the regulation of Pax6 by different concentrations of LY2157299 during induction compared using Q-PCR analysis; FIG. 4g shows the regulation of apoptosis gene CASP3 using Q-PCR analysis comparing different concentrations of LY2157299 during the inducing process.

[0051] FIG. 5 differentiation ability identification of neural stem cells obtained by LY inducing method. FIGS. 5a-5c show induced neural stem cells obtained through LY2157299 as well as further differentiation of the resulting nociceptor neurons; FIG. 5A shows that nociceptor neurons derived by this method express the nociceptor neuron specific marker SCN11A; FIG. 5b shows that nociceptor neurons derived by this method express the nociceptor neuron specific marker Nestin; FIG. 5c shows the integration of two fluorescence channels in FIG. 5a and FIG. 5b; FIGS. 5d-5f show the induced neural stem cells Obtained by LY2157299 along with further differentiation of the resulting dopaminergic neurons; FIG. 5d shows that the dopaminergic neurons obtained by this method express the specific marker Pitx3 of mature dopaminergic neurons; FIG. 5e shows that dopaminergic neurons derived by this method express the mature dopaminergic neuron specific marker TH; FIG. 5f shows the integration of two fluorescence channels in FIG. 5d and FIG. 5e; FIGS. 5g-5i show the induced neural stem cells obtained through LY2157299 along with further differentiation of the resulting photoreceptor neurons; FIG. 5g shows that the photoreceptor neurons obtained by this method express the photoreceptor neuron specific marker OPSIN; FIG. 5h shows that the photoreceptor neurons derived by this method express the photoreceptor neuron specific marker CRX; FIG. 5I shows the integration of two fluorescence channels in FIG. 5g and FIG. 5h.

[0052] FIG. 6 MHC related gene expression analysis of neural stem cells obtained by LY inducing method. FIG. 6a-6d indicate that neural stem cells obtained with LY2157299 were significantly lower than human induced pluripotent stem cells in the expression of CD4, HLA-A, HLA-C, HLA-F and HLA-DPB1, respectively.

[0053] FIG. 7: Detection of HLA-DR antigens of neural stem cells induced by LY2157299 using flow cytometry. FIG. 7a is a negative control without antibody staining, and FIG. 7b is the HLA-DR detection result of neural stein cells induced by LY2157299. FIG. 7a-1 shows the use of SSC and FSC channels to exclude cell debris and other particulate impurities and determine the cell populations for analysis by gating; FIGS. 7a-2 and 7a-3 show the use of blank fluorescence channels and cell morphology to analyze cells respectively; FIG. 7a-4 shows the use of two blank fluorescence channels to analyze cells; FIG. 7b-1 shows the use of SSC and FSC channels to exclude cell debris and other particulate impurities and determine the cell populations for analysis by gating; FIG. 7b-2, 7b-3 indicate that cells were analyzed using CD45 PerCP (Abcam, cat. No. ab157309), HLA-DR FITC (CST, cat. No. 54126) channels and cell morphology, respectively; FIG. 7b-4 shows the analysis of cells using two fluorescent channels CD45 PerCP and HLA-DR FITC. The results showed that the HLA-DR antigen expression of the neural stem cells induced by LY2157299 was negative.

[0054] FIG. 8 LY2157299 can induce the generation of cerebral organoids. FIG. 8a shows the cerebral organoids formed by the LSB inducing method (D50); FIG. 8b shows the cerebral organoids formed by the LY inducing method (D50); FIG. 8c shows a comparison of the spontaneous discharge numbers of cerebral organoids prepared by different methods by using a multichannel electrode system; FIG. 8d shows a comparison of the spontaneous firing frequency of cerebral organoids prepared by different methods by using a multichannel electrode system; FIG. 8e shows the diameter of organoids prepared by different methods (n=10); FIG. 8f-8g show the analysis of gene expression differences obtained by different organoid preparations by using Q-PCR.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention will be father described below with reference to the accompanying drawings and specific embodiments, and the protection content of the present invention is not limited to the following embodiments. It should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are all included in the present invention, and the appended claims and any equivalents thereof are within the protection scope of the present invention. In the present specification and claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The experimental methods that do not specify specific conditions in the following examples are the general knowledge and common knowledge of those skilled in the art, or according to the conditions suggested by the manufacturer. Unless otherwise specified, all materials and reagents used in the examples are commercially available products.

EXAMPLES

Example 1: Preparation of Neural Stem Cell Inducing Medium

[0056] The formula of neural inducing basal medium (hereinafter referred to as NouvNeu001): Dulbecco's modified Eagle/F12 medium (DMEM/F12), 1% minimum essential medium non-essential amino acids (MEM non-essential amino acids, Thermo Fisher, Cat. No.: 11140076), sodium chloride (0.5 g/L), sodium selenite (13.6 μg/L), insulin (22 μg/mL), recombinant human transferrin (100 ng/ml).

[0057] The neural stem cell inducing medium used in the present invention is prepared by adding 55 nM-24 μM LY2157299 (Selleck, S2230) to NouvNeu001 basal medium, and the final concentration is preferably 300 nM, 500 nM, 700 nM, 900 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM or 22 μM, most preferably 12.5 μM. The above-mentioned medium is referred to as the LY inducing medium for short, and the experimental group using this medium is referred to as the LY inducing method below.

[0058] The medium used for the control experiments is prepared by supplementing NouvNeu001 basal medium with 100 nM LDN-193189 (Selleck, S2618) and 10 μM SB431542 (Selleck, S1067). The above-mentioned medium is referred to as the LSB inducing medium for short, and the experimental group using this medium is referred to as the LSB inducing method hereinafter.

Example 2: Induction and Identification of Neural Stem Cells

[0059] 2.1 Chemical Induction of Neural Stem Cells:

[0060] Human pluripotent stem cells include embryonic pluripotent stem cells, such as the H9 cell line and human induced pluripotent stem cells. Wherein, the human induced pluripotent stem cells used in the present invention are obtained from the reprogramming of CD34.sup.+ cells, which refers to “reprogramming culture medium and reprogramming induced pluripotent stem cell culture method” (the method in the patent ZL201910050800.7).

[0061] Human pluripotent stem cells are used to coat T25 cell culture flasks with Matrigel (STEMCELL Technologies), and then placed in a 37° C. incubator for more than one hour after plating. Follow 1×10.sup.6 Cells were seeded in T25 flasks for expansion and passage.

[0062] For neural induction, use 50 μg/mL poly-lysine (SIGMA, Cat. No.: P6407) to coat a 6-well culture plate, and incubate at 37° C. for more than 3 hours after plating; then use 5 μg/mL Laminin (SIGMA ALDRICH, Cat. No.: 12020) for further coat, and then place in a 37° C. incubator to incubate for more than 3 hours. When pluripotent stem cells reached 70% coverage, they were digested with EDTA at 37° C. for 5 min, and DMEM was used to stop cell digestion. Cells are washed and centrifuged at 2×10.sup.5 the proportions of each flask and are re-seeded in T25 plates. Neural induction is performed using the medium of the present invention in combination with the neural inducing compound, and the medium is changed every day until the neural stem cell rosette is formed. As shown in FIG. 1a and FIG. 1b, compared with the multi-molecule inducing method (LSB induction method), the single-molecule inducing method used in the present invention (LY inducing method) formed small and uniform neural rosette structures. This result shows that compared with the multi-molecule inducing method used in the control, the single-molecule inducing method provided by this method produces more uniform induction results, while the rosette structures produced by the control experiment have different sizes. And in the same culture period, there are still some parts that do not form a neural rosette structure in the control group. Therefore, the single-molecule inducing method provided by this method can induce pluripotent stem cells to form neural rosette structures more efficiently and uniformly.

[0063] 2.2 Concentration Identification of Key Components in the Inducing System:

[0064] The Cyquant assay was used to perform the quantitative detection of cell viability, thus investigating the effect of LY2157299 on the physiological state of the cells during long-term culture of the cells; Among them, the LSB method was used as the control group. 96-well light-tight cell culture plates were coated; after the coating was complete, cells were seeded separately at 5×10.sup.4 cell number per well and three sets of parallel replicates were set (the average of three sets was calculated as data) to repeat the procedure of example 2.1. Wherein, the culture procedures described previously were incubated at 37° C. with 5% CO.sub.2. Samples were taken on days 1, 5, 10, 15 and 20 for cell viability assay using CyQuant Kit (Invitrogen, cat. No: X12223). The aforementioned assay was operated according to the instructions with a Spectramax i3 Multi-Mode Microplate Reader (VWR, model number: ID3-STD) for data reading. The results are shown in FIG. 1c: CyQuant assay showed that LY2157299 induced more number of neural cells compared with LSB method, which illustrated that LY2157299 possessed more proliferative activity during the inducing process.

[0065] The CyQuant assay was also used to perform quantitative assays of cell viability, thereby investigating the effect of different concentrations of recombinant insulin and recombinant serum albumin on the physiological state of the cells during long-term culture, and the results are shown in FIG. 2b and FIG. 2c: Both recombinant insulin and recombinant serum albumin concentrations were positively correlated with the rate of cell proliferation. Combined with cost factors, the range of recombinant insulin and recombinant serum albumin used by the present invention falls within the range of optimal use.

[0066] A fully automated freezing point osmometer (FM-8P, Shanghai Yida Instruments Co., Ltd.) was used to detect the effects of different concentrations of sodium chloride on the osmolality of the culture system, and the specific operations were enrolled in the product instructions (FM-8P, Shanghai Yida Instruments Co., Ltd.). The test results are detailed in FIG. 2a. The results illustrate a linear increase with the concentration of sodium chloride and the osmotic pressure of the culture system. However, when the concentration reached 1 μg/mL, the osmolality of the culture system already exceeded the cell culture limit of 320 mOsm/kg. The above experimental results show that the concentration of sodium chloride used in the present invention is the one most suitable for cell culture.

[0067] 2.3 Fluorescence Immunoassay of Markers of Neural Stem Cells:

[0068] The neural stem cells obtained from the LY inducing method and LSB inducing method, respectively, were identified by immunofluorescence staining, and then the cells were fixed with 4% paraformaldehyde for 40 min at room temperature and subsequently washed twice with DPBs buffer; after permeabilization of the cells with 0.1% Triton X-100 for 5 min, they were washed twice with DPBs buffer; Cells were then incubated overnight at 4° C. with DPBs buffer containing 10% horse serum and 0.1% Triton X-100; Cells were then washed with DPBs buffer and primary antibody was diluted in DPBs butler containing 2% horse serum and 0.1% Triton X-100 and incubated for 2 hours at 37° C.; After the cells were washed with DPBs buffer, the secondary antibody was diluted with DPBs buffer containing 2% horse serum and 0.1% Triton X-100, and the cells were incubated at 37° C. for 2 h. Then the cells were washed three times and photographed using Leica DMi8. The antibody usage details are shown in Table 1 and the results are shown in FIG. 3a-3h. The result shows that, compared with the LSB induction method, the LY inducing method enables the generation of neural rosette structures from pluripotent stem cells and the cells can express the neural stem cell markers Pax6 and Nestin.

TABLE-US-00001 TABLE 1 antibodies used for immunofluorescence staining of cells primary antibody secondary antibody Concen- Concen- antibody tration antibody tration Anti-PAX6-antibody 1:200 Goat anti rabbit IgG (H + L), 1:1000 (Abcam) Alexa Fluor 488 (Invitrogen A11034) Anti-Nestin antibody 1:400 Donkey anti mouse IgG 1:1000 (Millipore) (H + L), Alexa Fluor 568 (Invitrogen A11004)

[0069] 2.4 Transcriptional Level Identification of Neural Stem Cells:

[0070] Transcriptional changes of different marker genes during induction from pluripotent stem cells to neural stem cells were detected by using Q-PCR. The induced pluripotent stem cells used in this example are neural stem cells derived by LY inducing method and LSB inducing method, respectively. Neural stem cells were prepared as described previously using Rneasy Mini or Micro Kit (QIAGEN) for total RNA extraction, in which 1 mg RNA was used to synthesize cDNA with superscript III First-Strand Synthesis System (Invitrogen).

[0071] SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used to perform labeling and reactions for Quantitative PCR, with beta-Actin as internal reference. All the above data were analyzed with the delta-Ct method. Each set of trials was tested using three sets of replicates, and variance statistics were performed. The primer sequences used to identify the encoding genes for the different cell markers are shown in Table 2, The results are shown in FIG. 3i-3k: the neural stem cells obtained by the LY inducing method and the LSB inducing method both expressed the neural stem cell-specific markers Sox2, Pax6 and Nestin, and the LY inducing method showed an increase in the expression of NSCs markers compared to the LSB inducing method.

[0072] Through the above method, different concentrations of LY2157299 in NouvNeu001 basal medium were added for concentration screening, and the results are shown in FIG. 4b-4e: FIG. 4b shows that the absence of LY2157299 will result in a failure of the cells to differentiate into ectodermal cells; FIG. 4c shows that using 20 nm LY-2157299 for chemical induction, a mixed population of cells including neural cells was obtained; FIG. 4d shows that chemical induction with 12.5 μM LY2157299 resulted in a uniform cell population, and the cells had typical neural stem cell morphology, similar to the control (4a); FIG. 4e shows that using 25 μM LY2157299 chemical induction, a mixed population of neural cells was obtained, while some cells formed longer axons and cells showed signs of stress with intracellular vacuoles. From the above results, it is known that the differentiation is not complete under the low concentration condition, and many different types of cells can be seen. However, neural stem cells obtained in high concentration condition are easily differentiated and cell stress appears. Transcript levels further demonstrate that the concentrations used herein are optimal (FIG. 4f-4g); FIG. 4f shows that the concentration of LY2157299 is positively correlated with Pax6 expression; FIG. 4g shows that the concentration of LY2157299 is positively correlated with apoptosis. The above results indicate either too low or too high concentrations of LY2157299 adversely affect neural induction.

TABLE-US-00002 TABLE 2 Different marker gene primers during the induction of pluripotent stem cells to neural stem cells Gene Primer sequences SEQ ID NO. SOX2-F CATGCAGGTTGACACCGTTGG SEQ ID NO: 1 SOX2-R ATGGATTCTCGGCAGACTGATTCA SEQ ID NO: 2 PAX6-F TCTTTGCTTGGGAAATCCG SEQ ID NO: 3 PAX6-R CTGCCCGTTCAACATCCTTAG SEQ ID NO: 4 CASP3-F GGAAGCGAATCAATGGACTCTGG SEQ ID NO: 5 CASP3-R GCATCGACATCTGTACCAGACC SEQ ID NO: 6 Nestin-F TCAAGATGTCCCTCAGCCTGGA SEQ ID NO: 7 Nestin-R TGGCACAGGTGTCTCAAGGGTAG SEQ ID NO: 8 ACTIN-F TCCCTGGAGAAGAGCTACGA SEQ ID NO: 9 ACTIN-R AGCACTGTGTTGGCGTACAG SEQ ID NO: 10

Example 3: Identification of Differentiation Function of Neural Stem Cells

[0073] The neural stem cells obtained by the LY inducing method in Example 2 were subjected to directed differentiation in NouvNeu001 basal medium. For neuronal differentiation, use 50 μg/mL polylysine (SIGMA ALDRICH, Cat. No.: P6407) to coat a 6-well culture plate, and place the plate in a 37° C. incubator and incubate for more than 3 hours until cells are seeded.

[0074] 3.1 Nociceptor Neuron Differentiation:

[0075] The neural stem cells obtained in example 2 were seeded at a density of 1×10.sup.5 the ratio per flask was inoculated in T25 flasks, and 3 μM CHIR99021 (Selleck, Cat. No.: S2924), 10 μM SU5402 (Tocris, Cat. No.: 3300/1), 10 μM DAPT (Selleck, Cat. No.: S2215) were added to NouvNeu001 basal medium. Fresh medium was replaced every 3 days until day 21. The above cultures were incubated at 37° C. with 5% CO.sub.2. The experimental results demonstrate that the resulting neural stem cells in example 2 have axonal structures upon differentiation and are able to express the nociceptor neuronal markers SCN11A and Nestin (FIG. 5a-5c). FIG. 5a shows that nociceptor neurons derived by further differentiation of induced neural stem cells obtained through LY2157299 express the nociceptor neuron specific marker SCNI11A; FIG. 5b shows that nociceptor neurons derived by this method express the nociceptor neuron specific marker Nestin; FIG. 5c shows the integrated pictures of two kinds of fluorescence channels in FIGS. 5a and 5b.

[0076] 3.2 Induction of Dopamine Neuron Cells:

[0077] The neural stem cells obtained in example 2 were seeded at a density of 1×10.sup.5 the ratio per flask was inoculated in T25 flasks, and 1 μM Purmorphamine (Sellek, Cat. No.: S3042) and 1 ng/mL TGF-β3 (Novoprotein, Cat. No.: CJ44) were added to NouvNeu001 basal medium to culture neural stem cells. Fresh medium was replaced every 3 days until day 30. The above cultures were incubated at 37° C. with 5% CO.sub.2. Experimental results showed that the resulting neural stem cells in example 2 had axonal structures upon differentiation and could express the mature dopaminergic neuron specific markers Tyrosine Hydroxylase (TH) and Pitx3 (FIG. 5d-S1). FIG. 5d shows that dopaminergic neurons obtained by LY2157299 induced neural stem cells to further differentiate and the differentiation products can express mature dopaminergic neuron specific marker Pitx3; FIG. 5e shows that dopaminergic neurons derived by this method express the mature dopaminergic neuron specific marker TH; FIG. 5f shows the integrated pictures of two kinds of fluorescence channels in FIGS. 5d and 5e.

[0078] 3.3 Photoreceptor Neuron Differentiation:

[0079] The neural stem cells obtained in example 2 were seeded at a density of 1×10.sup.5 the ratio per flask was inoculated in T25 flasks, and 0.5 μM retinyl acetate (SIGMA ALDRICH, Cat. No.: R7782) was added to NouvNeu001 basal medium. Fresh medium was replaced every 3 days until day 24. The above cultures were incubated at 37° C. with 5% CO.sub.2. Experimental results demonstrated that the resulting neural stem cells in example 2 had axonal structures and could express the photoreceptor neuronal markers OPSIN and CRX after differentiation (FIG. 5g-5i). FIG. 5g shows that photoreceptor neurons obtained by LY2157299 induced neural stem cells to further differentiate and the differentiation products can express the photoreceptor neuron specific marker OPSIN; FIG. 5h shows that photoreceptor neurons derived by this method express the photoreceptor neuron specific marker CRX; FIG. 5i shows the integrated pictures of two kinds of fluorescence channels in FIGS. 5g and 5h.

[0080] After induction and differentiation, the cells were harvested and identified by immunofluorescence staining: cells were fixed with 4% paraformaldehyde at room temperature for 40 minutes, washed twice with DPBS buffer; then permeabilized with 0.1% Triton X-100 for 5 minutes, and buffered with DPBS The cells were then washed twice with DPBS buffer containing 10% horse serum and 0.1% Triton X-100 at 4° C. overnight; the cells were washed with DPBS buffer and then washed with 2% horse serum and 0.1% Triton X-100. The secondary antibody was diluted with 100 DPBS buffer, incubated at 37° C. for 2 hours, washed three times, and photographed with Leica Dmi8. Antibody usage details are shown in Table 3.

TABLE-US-00003 TABLE 3 Antibodies used for directed differentiation primary antibody secondary antibody Concen- Concen- antibody tration antibody tration Anti-SCN11A- 1:200 Goat anti rabbit IgG(H + L), 1:1000 antibody Alexa Fluor 488 (Invitrogen (Abcam) A11034) Anti-Tuj antibody 1:200 Donkey anti mouse IgG(H + L), 1:1000 (Millipore) Alexa Fluor 568 (Invitrogen A11004) Anti- Tyrosine 1:400 Donkey anti mouse IgG(H + L), 1:1000 Hydroxylase- Alexa Fluor 568 (Invitrogen antibody A11004) (Santa Cruz) Anti-Pitx3 antibody 1:200 Goat anti rabbit IgG(H + L), 1:1000 (Invitrogen Alexa Fluor 488 (Invitrogen 38-2850) A11034) Anti-Opsin antibody 1:200 Goat anti rabbit IgG(H + L), 1:1000 (Millipore) Alexa Fluor 568 (Invitrogen A11011) Anti-CRX antibody 1:100 Donkey anti mouse IgG(H + L), 1:1000 (Novusbio) Alexa Fluor 488 (Invitrogen A21202)

[0081] The above results indicated that the neural stem cells obtained by LY inducing method had the differentiation ability of a variety of neurons.

Example 4: Effect of LY2157299 on MHC Formation

[0082] 4.1 Q-PCR Detection:

[0083] The major histocompatibility complex (MHC) genes of neural stem cells induced by LY2157299 was detected by Q-PCR method, and pluripotent stem cells (iPS) were used as control. The primer sequences are shown in Table 4, and test results are shown in FIG. 6: the MHC associated genes CD4, HLA-A, HLA-C, hla-f, and HLA-DPB1 were expressed at extremely low levels in neural stem cells obtained by LY2157299 induction compared with human pluripotent stem cells.

TABLE-US-00004 TABLE 4 Primers of immune system related genes Gene Primer sequences SEQ ID NO. CD4-F CAAGCAGAGCGGATGTCTCAGAT SEQ ID NO: 11 CD4-R AATTGTAGAGGAGGCGAACAGGAG SEQ ID NO: 12 HLA-A-F TGTGGTGGTGCCTTCTGGAGAG SEQ ID NO: 13 HLA-A-R ACTGCTTGCAGCCTGAGTGTAAC SEQ ID NO: 14 HLA-C-F CGCTTCATCTCAGTGGGCTACG SEQ ID NO: 15 HLA-C-R CCGTCCTCGCTCTGGTTGTAGTA SEQ ID NO: 16 HLA-F-F AACCTGCTCCGCCGCTACAA SEQ ID NO: 17 HLA-F-R TCCTCTGCATATTCCTCTGCCTCA SEQ ID NO: 18 HLA-DPB1- TCATCTGTGGAGTGGGCATCTTCA SEQ ID NO: 19 F HLA-DPB1- AGCAACTTCTTGGCAGCAGTATCC SEQ ID NO: 20 R ACTIN-F TCCCTGGAGAAGAGCTACGA SEQ ID NO: 9 ACTIN-R AGCACTGTGTTGGCGTACAG SEQ ID NO: 10

[0084] 4.2 Flow Cytometry Detection:

[0085] The HLA-DR antigens of neural stem cells induced by LY2157299 were detected by flow cytometry. HLA-DR is an MHC class II molecule whose expression is critical for the antigen-presenting function of cells and plays a key role in adoptive immune responses. The induced neural stem cells obtained in Example 2 were digested with EDTA, washed with DPBS, centrifuged to remove the supernatant, and 10.sup.6 Cells were resuspended in 100 μL of diluted primary antibody (CD45 PerCP, Abeam, Cat. No. ab157309; HLA-DR FITC, CST, Cat. No. 54126), incubated on ice for 1 hour in the dark, centrifuged to remove supernatant, and cells were resuspended in 500 μL of antibody dilution buffer (CST) and analyzed on a flow cytometer. Samples incubated without primary antibody were used as controls. The results are shown in FIG. 7, FIG. 7a is a negative control, FIG. 7b is the HLA-DR detection result of the neural stem cells obtained by LY2157299 induction. The result shows that the neural stem cells obtained by the LY inducing method do not express HLA-DR. This result is consistent with the result of Example 4.1.

Example 5: Effect of LY2157299 on Organoid Formation

[0086] Organoid culture was performed with human induced pluripotent stem cells. Organoid inducing medium was formulated as follows: NouvNeu basal medium supplemented with 12.5 μM LY2157299. Induced pluripotent stem cells were digested to single cells by Accutase, seeded in U-bottom-ultra-low attachment 96-well plates at a ratio of 9000 cells/150 mL of brain organoid inducing medium per well. 10 μM ROCK inhibitor Y-27632 (Selleck, S1049) was added, and the cells were placed in a 37° C., 5% carbon dioxide cell incubator for 24 hours (Panasonic, model: MCO-18AC). Y-27632-free fresh organoid inducing medium was changed the following day until day 10. Day 10 the organoids were transferred to ultra-low-attachment 6-well plates and the medium was changed to NouvNeu001 basal medium supplemented with 2% B27 cell culture additive. The culture was conducted at 37° C. in a 5% CO.sub.2 cell culture incubator on a horizontal rotator at 80 rpm until day 120.

[0087] The control experiment was carried out according to the published method (Clair B et. al, Nat Methods. 2019, November; 16 (11): 1169-1175). N2B27 was used as the basic medium for organ like culture. The cerebral organoid inducing medium for control experiment was formulated as follows: The N2B27 basal medium was supplemented with 10 μM SB-431542, 100 nM LDN-193189 and 2 μM XAV-939. The rest of the operations are as above. The method is abbreviated as the N2B27 method in the invention and illustrations.

[0088] Organoids were photographed by Leica Dmi8. The brain organoids were photographed and their diameters were measured. The results are shown in FIG. 8a, FIG. 8b, and FIG. 8e.

[0089] Induced brain organoids were subjected to the detection of spontaneous cell firing signals using multi-channel electrodes. Brain organoids induced for 120 days were digested with 10% trypsin/EDTA at 37° C. for 5-8 minutes. A 96-well MEA system multi-channel electrode plate (AXION Biosystem, US) was taken with 100 ng/mL poly-1-lysine (Sigma-Aldrich, P4707) for coating, placed in 37° C., 5% carbon dioxide cell incubator (Panasonic, model: MCO-18AC) for 12 hours. Take out the poly-1-lysine coating MEA multi-channel electrode plate, aspirated poly-1-lysine, washed with sterilized water 3 times, and used a PBS solution containing 3 μg/mL gelatin (Laminin, SIGMA ALDRICH, Cat. No.: 12020) as the nerve cell coating matrix, added to the MEA multi-channel electrode plate, placed in a 37° C., 5% carbon dioxide cell incubator for 3 hours. After the MEA multi-channel electrode plate was coated, the plate was seeded with digested organoid mix cells following the 5.0×10.sup.5 cells per well. Place the seeded MEA multi-channel electrode plate in the MEA chamber, adjust the cell culture conditions in AxIS Navigator 2.0.2 software to 37° C., 5% carbon dioxide, and run for 10 minutes until the chamber environment is stable. Spontaneous cell firing signals were recorded with AxIS Navigator 2.0.2 software (AXION Biosystem, US). The experimental results showed that the neurons induced by the NouNeu system exhibited good electrophysiological activity. When a multichannel motor system was used to detect the cell spontaneous discharge, we can obtain the following conclusion by comparing the discharge number per cell unit time (FIG. 8C) and the average discharge rate (FIG. 8D): compared with the N2B27 control inducing method, the LY inducing method has more active electrophysiological activity.

[0090] Using Q-PCR to detect the transcriptional changes of different marker genes after the formation of brain organoids: The total RNA of brain organoids obtained by different methods for 120 days and the control of human induced pluripotent stem cells were respectively used Rneasy Mini or Micro Kit (QIAGEN) to extract. 1 mg of RNA was used to synthesize cDNA by using the SuperScript III First-Strand Synthesis System (Invitrogen). SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used for labeling and reaction of Quan-titative PCR, and Beta-actin was used as internal reference. All data were analyzed using the delta-Ct method. Three replicates were used for each group of experiments, and variance statistics were performed. The primer sequences used to identify the encoding genes for the different cell markers are shown in Table 5. Both brain organoids formed by LY-inducing method and N2B27 controlling group could produce glial cells, neural progenitors, and neurons (FIGS. 8f-8g).

TABLE-US-00005 TABLE 5 Primers used for brain organoid detection Q-PCR Gene Primer sequences SEQ ID NO. EN1-F GGACAATGACGTTGAAACGCAGCA SEQ ID NO: 21 EN1-R AAGGTCGTAAGCGGTTTGGCTAGA SEQ ID NO: 22 SOX2-F CATGCAGGTTGACACCGTTGG SEQ ID NO: 1 SOX2-R ATGGATTCTCGGCAGACTGATTCA SEQ ID NO: 2 GFAP-F CTGGAGAGGAAGATTGAGTCGC SEQ ID NO: 23 GFAP-R ACGTCAAGCTCCACATGGACCT SEQ ID NO: 24 CASP3-F GGAAGCGAATCAATGGACTCTGG SEQ ID NO: 5 CASP3-R GCATCGACATCTGTACCAGACC SEQ ID NO: 6 ACTIN-F TCCCTGGAGAAGAGCTACGA SEQ ID NO: 9 ACTIN-R AGCACTGTGTTGGCGTACAG SEQ ID NO: 10

[0091] Taken together, LY2157299 can induce the generation of cerebral organoids, which have electrophysiological functions and the expression of key genes. In addition, the cerebral organoids formed by the LY inducing method had a larger diameter and had more active electrophysiological functions compared to the N2B27 control inducing method.

[0092] All documents mentioned herein are incorporated by reference in their entirety in this application. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the modifications of these equivalent forms also fall within the scope defined by the claims of the present application.