Preparation method and application of brain-like organ model with neural crest cell group

Abstract

The invention belongs to the field of biological medicine, and relates to a preparation method and application of a brain-like organ model with a neural crest cell group. The neural crest class group contained in the brain-like organ prepared by the method is very similar to the in-vivo neural crest class group, the neural crest class group with higher quality is obtained, and a very good model is provided for researching neural ridge related neurodevelopment diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method for preparing a brain organoid model with a neural crest cell population and its application. Background Art

[0002] Kabuki syndrome is a rare autosomal dominant genetic disorder characterized by congenital multi-system developmental defects. Patients with this disorder have varying degrees of facial abnormalities, heart problems, and intellectual problems, indicating neural crest and neural development abnormalities. However, there is a lack of treatment for this disease, and its molecular mechanism is also unclear. One type of Kabuki syndrome (KS1) accounts for approximately 70% and is caused by mutations in KMT2D. KMT2D belongs to the histone methyltransferase family and methylates histone H3 at lysine 4 (H3K4me) through its SET domain. Existing Kabuki models include heterozygous mice, zebrafish, and 2D human cell models. However, the complexity of human brain development cannot be compared with animal models and single 2D cell models. In 2013, a major breakthrough was achieved in the field of stem cells, where stem cells differentiated in three-dimensional space could form brain organoids under specific induction, and the developmental process was highly similar to that of the human brain. 1 However, current brain organoids can only simulate three-dimensional neural development and cannot simultaneously simulate the neural crest development process. Therefore, there is an urgent need for a brain organoid model with a neural crest cell population to simulate Kabuki and neural crest-related neurodevelopmental diseases. Summary of the Invention

[0003] In some embodiments, the present invention provides a method for preparing a brain organoid model, the method comprising the step of contacting stem cells with a stem cell culture medium supplemented with SB431542 or a salt thereof, and LDN193189 or a salt thereof, wherein the brain organoid is a brain organoid with a neural crest cell population.

[0004] In some embodiments, the stem cells are selected from embryonic stem cells or induced pluripotent stem cells.

[0005] In some embodiments, the method further comprises: adding Matrigel to the embryoid bodies.

[0006] In some embodiments, the Matrigel encapsulates the embryoid bodies.

[0007] In some embodiments, the added Matrigel is in a liquid state.

[0008] In some embodiments, the method further comprises: adding the embryoid bodies to a groove and then adding Matrigel.

[0009] In some embodiments, the method further includes: culturing embryonic stem cells or induced pluripotent stem cells for culturing brain organoids on feeder cells.

[0010] In some embodiments, the method comprises the following steps: (1) culturing embryonic stem cells or induced pluripotent stem cells on feeder cells; (2) inoculating the embryonic stem cells or induced pluripotent stem cells into a low-attachment culture plate for culturing to obtain embryoid bodies; a stem cell medium containing LDN193189 and SB431542 is used during the culturing process; (3) contacting the embryoid bodies with Matrigel so that the Matrigel wraps the embryoid bodies; (4) adding a medium to culture the embryoid bodies wrapped with Matrigel to obtain brain organoids.

[0011] In some embodiments, the method comprises the following steps: (a) culturing embryonic stem cells or induced pluripotent stem cells on feeder cells; (b) digesting the embryonic stem cells or induced pluripotent stem cells; (c) resuspending the cells with a medium containing bFGF and Y27632, and inoculating the cells into a low-attachment culture plate; (d) replacing the medium with a stem cell medium containing LDN193189 and SB431542 for culturing; (e) changing the medium to a neuroepithelial induction medium to obtain embryoid bodies; (f) adding Matrigel to the embryoid bodies so that the Matrigel wraps the embryoid bodies; (g) culturing the embryoid bodies wrapped in Matrigel with a first neural differentiation medium; (h) placing the embryoid bodies into a second neural differentiation medium and culturing in an orbital shaker incubator to obtain brain organoids.

[0012] In some embodiments, in step (a), the embryonic stem cells or induced pluripotent stem cells are cultured on feeder cells for 3 to 8 passages.

[0013] In some embodiments, in step (a), the embryonic stem cells or induced pluripotent stem cells are cultured on feeder cells for 4 passages.

[0014] In some embodiments, in step (a), the embryonic stem cells or induced pluripotent stem cells are resuspended in a medium; then the embryonic stem cells or induced pluripotent stem cells are inoculated into a culture plate seeded with feeder for culturing; the components of the medium include DMEM / F12, KSR, NEAA, GlutaMax, β-Me and bFGF.

[0015] In some embodiments, in step (a), the components of the medium include 10 - 30% v / v KSR, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, 0.01 - 0.5 mM β-Me, 5 - 20 ng / mL bFGF.

[0016] In some embodiments, in step (a), the culture medium components include 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, and 10 ng / mL bFGF.

[0017] In some embodiments, in step (c), the culture medium is KSR medium.

[0018] In some embodiments, in step (c), the components of the KSR medium include DMEM / F12, KSR, GlutaMAX, NEAA, β-Me, bFGF, and Y27632.

[0019] In some embodiments, in step (c), the components of the KSR medium include 10 - 30% v / v KSR, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, 0.01 - 0.5 mM β-Me, 1 - 10 ng / mL bFGF, and 20 - 80 μM Y27632.

[0020] In some embodiments, in step (c), the components of the KSR medium include 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 4 ng / mL bFGF, and 50 μM Y27632.

[0021] In some embodiments, the reagents used in the method for preparing the organoid model of the brain according to the present invention cannot contain WNT inhibitors. In some embodiments, in step (d), the components of the stem cell culture medium containing LDN193189 and SB431542 include DMEM-F12, KSR, NEAA, GlutaMax, β-Me.

[0022] In some embodiments, in step (d), the stem cell culture medium containing LDN193189 and SB431542 does not contain bFGF.

[0023] In some embodiments, in step (d), the components of the stem cell culture medium containing LDN193189 and SB431542 further include 10 - 30 v / v KSR, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, and 0.01 - 0.5 mM β-Me.

[0024] In some embodiments, in step (d), the components of the stem cell culture medium containing LDN193189 and SB431542 include 0.1 μM LDN193189, 10 μM SB431542, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, and 0.1 mM β-Me.

[0025] In some embodiments, the concentrations of LDN193189 and SB431542 are 25 - 100 nM and 1 - 20 μM, respectively.

[0026] In some embodiments, the concentrations of LDN193189 and SB431542 are 25 - 80 nM and 1 - 15 μM, respectively.

[0027] In some embodiments, the concentrations of LDN193189 and SB431542 are 25 - 60 nM and 1 - 10 μM, respectively.

[0028] In some embodiments, in step (e), the components of the neuroepithelial induction medium include the components: DMEM-F12, N2, NEAA, GlutaMax, and heparin.

[0029] In some embodiments, in step (e), the components of the neuroepithelial induction medium include 0.5 - 5% v / v N2, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, and 0.5 - 5 μg / mL heparin.

[0030] In some embodiments, in step (e), the components of the neuroepithelial induction medium include 1% N2, 1% NEAA, 1% GlutaMax, and 1 μg / mL heparin.

[0031] In some embodiments, in step (f), after a translucent circle appears around the outer surface of the embryoid body sphere, Matrigel is added.

[0032] In some embodiments, in step (f), the Matrigel is in liquid form.

[0033] In some embodiments, in step (f), the embryoid body is transferred onto the sealing film of a specific groove, the excess culture medium is discarded, liquid Matrigel is added, and it is placed at 25 - 40 °C for 15 - 30 min.

[0034] In some embodiments, the components of the first neural differentiation medium include: DMEM-F12, Neurobasal, insulin, N2, B27, NEAA, and GlutaMax.

[0035] In some embodiments, the components of the first neural differentiation medium further include Penicilin-streptomycin.

[0036] In some embodiments, the B27 is B27 without vitamin A.

[0037] In some embodiments, the components of the first neural differentiation medium include: 10-80% v / v Neurobasal, 0.5-6% v / v insulin, 0.1-1% v / v N2, 0.1-10% v / v B27 [without Vitamin A], 0.1-5% v / v NEAA, and 0.1-10% v / v GlutaMax.

[0038] In some embodiments, the components of the first neural differentiation medium include 0.1-10% v / v Penicilin-streptomycin.

[0039] In some embodiments, the components of the first neural differentiation medium include: 50% v / v Neurobasal, 2.5% v / v insulin, 0.5% v / v N2, 1% v / v B27 [without Vitamin A], 0.5% v / v NEAA, 1% v / v GlutaMax.

[0040] In some embodiments, the components of the first neural differentiation medium further include 1% v / v Penicilin-streptomycin.

[0041] In some embodiments, the components of the second neural differentiation medium include DMEM-F12, Neurobasal, insulin, N2, B27 (with Vitamin A), NEAA, GlutaMax.

[0042] In some embodiments, the components of the second neural differentiation medium further include Penicilin-streptomycin.

[0043] In some embodiments, the components of the second neural differentiation medium include: 10-80% v / v Neurobasal, 0.5-6% v / v insulin, 0.1-5% v / v N2, 0.1-10% v / v B27 (with Vitamin A), 0.1-5% v / v NEAA, and 1-10% v / v GlutaMax.

[0044] In some embodiments, the components of the second neural differentiation medium include DMEM-F12, 50% Neurobasal, 2.5% insulin, 0.5% N2, 1% B27 (with Vitamin A), 0.5% NEAA, and 1% GlutaMax.

[0045] In some embodiments, the components of the second neural differentiation medium further include 1% Penicilin-streptomycin.

[0046] In some embodiments, step (e) is carried out after the 6th day of step (c4).

[0047] In some embodiments, step (d) is carried out after the 4th day of step (c).

[0048] In some embodiments, step (h) is carried out after the 4th day of step (g).

[0049] In some embodiments, the brain organoid is a mammalian brain organoid.

[0050] In some embodiments, the mammal is a human.

[0051] In some embodiments, the present invention provides a brain organoid model that uses SB431542 or a salt thereof, and LDN193189 or a salt thereof, and the brain organoid is a brain-like organoid having a neural crest cell population.

[0052] In some embodiments, the present invention provides a brain organoid model obtained by the described method.

[0053] In some embodiments, the present invention provides the use of a composition in the preparation of a brain organoid model, the composition including SB431542 or a salt thereof, and LDN193189 or a salt thereof, and the brain organoid is a brain-like organoid having a neural crest cell population.

[0054] In some embodiments, the present invention provides a culture medium, characterized by containing the matrix components of an embryonic stem cell or induced pluripotent stem cell culture medium, and LDN193189 or a salt thereof, and SB431542 or a salt thereof.

[0055] In some embodiments, the present invention provides a method for screening a drug for treating a neurodevelopmental disease or a brain disease, which includes the step of contacting a candidate drug with the described brain organoid model.

[0056] In some embodiments, the present invention provides in vitro uses of the brain organoids in drug discovery screening, toxicity assays, drug or gene expression studies of brain or nerve diseases, studies on the mechanisms of brain or nerve injury and repair; studies on pathogenic mechanisms; studies on the etiology of brain cancer; or uses in recombinant gene expression.

[0057] In some embodiments, the present invention provides the use of the brain organoids in the preparation of drugs for treating neurological disorders, neurological diseases or nerve or brain diseases, or the use in the preparation of drugs for regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Immunofluorescence identification of brain organoids. Upper panel: Protein expression of SOX2 (red) and TUJ1 (green); Lower panel: Protein expression of PAX6 (red) and FOXG1 (green).

[0059] Figure 2 Immunofluorescence identification of neural crest markers in brain organoids. Upper panel: Protein expression of SOX10 (red); Lower panel: Protein expression of P75 (red).

[0060] Figure 3 scRNA-seq data analysis results of brain organoids cultured with DSI induction until D21 and D30 under the feeder system. (A) UMAP plot showing cell types; (B) Proportions of three neural crest cell populations in brain organoid samples at two time points; (C-D) Distribution of brain organoid cells at two time points on the UMAP plot. GABAergic Neuron: GABAergic neuron; Glutamate Neuron: Glutamate neuron; Melanocyte, melanin; NC-Mesenchyme, neural crest-mesenchyme;

[0061] NC-Migratory: Neural crest-migrating; NC-Sensory: Neural crest-sensory neuron; Radial Glia (Cycling): Radial glia (in cell cycle); Radial Glia (Non-Cycling): Radial glia (not in cell cycle); Roof Plate: Roof plate.

[0062] Figure 4 Marker gene expression of three neural crest cell populations. Among them, LUM, COL3A1, and TWIST1 are markers of mesenchymal cells; POU4F1, NEUROD1, and NEUROD4 are markers of sensory neurons; SOX10 and ETS1 are markers of migrating neural crest.

[0063] Figure 5For the comparison of our scRNA-seq data with other data. (A) Projection results of early mouse neural crest development data on our UMAP; (B) Correlation between cell types in our brain organoids and cell types in human early embryos; NC 1: sensory neuron; NC2: schwann; NC3: mesenchyme; NP: neural progenitor. (C-D) Results of the proportion of neural crest cell clusters in our brain organoids and the correlation with neural crest cell clusters in human early embryos in the published brain organoid articles. NC 1: sensory neuron; NC2: schwann; NC3: mesenchyme; PNS: peripheral nervous system; NC: neural crest; Mes: mesenchyme; CP&Mes: choroid plexus&mesenchymal-like cells; Sensory: sensory neuron.

[0064] Figure 6 For the cell type distribution of + / - DSI in the Lancaster feeder-free system. (A) UMAP plot showing cell types; (B) Proportion of three neural crest cell clusters in two brain organoid samples; (C-D) Distribution of brain organoid cells in two samples on the UMAP plot.

[0065] Figure 7 For the morphological change diagram of brain organoids. Above: without dual smad; Below: with dual smad.

[0066] Figure 8 For the immunofluorescence staining diagram of brain organoids. Above: DS: Dorsomorphin + SB431542; Below: LS: LDN193189 + SB431542. Detailed implementation mode

[0067] The technical solutions of the present invention are further described below through specific embodiments. The specific embodiments do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0068] In the case of any conflict between any definition set forth below and any document incorporated herein by reference, the definition set forth shall prevail.

[0069] As used herein, the terms "comprising", "having", "containing", "including" and other similar forms, and their grammatical equivalents, have equivalent meanings and are open-ended, i.e., one or more items following any of these words do not mean an exhaustive listing of that item or items, nor do they mean limited to the listed item or items. For example, a composition "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain not only components A, B, and C, but also may include one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and their grammatical equivalents encompass the disclosure of embodiments "consisting essentially of" or "consisting of".

[0070] As used herein, and as embodied in the appended claims, the singular forms "a", "or", and "the" include plural referents unless the context clearly dictates otherwise.

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry).

[0072] As used herein, the term "induced pluripotent stem cell" refers to a pluripotent stem cell that is artificially derived from a non-pluripotent cell. The non-pluripotent cell may be a cell with lower self-renewal and differentiation potential than a pluripotent stem cell. The cell with lower potential may be, but is not limited to, a somatic stem cell, a tissue-specific progenitor cell, a primary or secondary cell. A significant advantage of the present method is that it can generate induced pluripotent stem cells from any somatic cell including an aged cell or a senescent cell, which was previously considered non-inducible to pluripotency due to its aged phenotype.

[0073] As used herein, the term "organoid" refers to: a three-dimensional cell aggregate that forms in vitro (outside the body) and is similar to a biological tissue. In the context of an organoid, "similar to a biological tissue" or "similar to a biological tissue" means that the anatomical structure of the organoid is similar to the anatomical structure of the biological tissue. The cells that make up the organoid are, for example, mostly cells with the ability to differentiate and proliferate. The cultured cells that make up the organoid can be differentiated into various cell types according to known methods. The cultured cells that make up the organoid can, for example, be differentiated into multiple cell types by including an additive or removing an additive in the culture medium for culturing. The cultured cells that make up the organoid can, for example, be induced to differentiate by culturing in a culture medium for culturing that does not contain the additive of the present invention. An organoid can contain a part of differentiated cells or cells without the ability to differentiate. An organoid can, for example, be manufactured according to known cell culture methods. An organoid can, for example, be manufactured by maintaining specified cells in a culture medium for culturing that contains the additive of the present invention in a cell culture device.

[0074] As used herein, the term "feeder cells" or "feeder layer" is a term used to describe the co-culture of one type of cell with a second type of cell to provide an environment in which the second type of cell can grow, because the feeder cells provide growth factors and nutrients for the support of the second cell type. Optionally, the feeder cells are derived from different species as they support the cells. For example, certain human cell types, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. When co-cultured with other cells, the feeder cells are usually inactivated by irradiation or treatment with an anti-mitotic dissociating agent such as mitomycin C to prevent them from growing too large for the cells they support. Without limiting the foregoing, specific feeder cell types can be human feeder cells, such as human skin fibroblasts or human embryonic stem cells. Another type of feeder cell can be mouse embryonic fibroblasts (mEF).

[0075] As used herein, the term "induced pluripotent stem cell (iPSC)", which is also commonly abbreviated as iPS cell or iPSC cell, refers to a type of pluripotent stem cell that is artificially derived from non-pluripotent cells, such as adult somatic cells, by inducing the "forced" expression of certain genes. hiPSC refers to human iPSC.

[0076] The terms "human embryonic stem cell", "hES cell" and "hESC" refer to cells that are derived from, obtainable from or originated from a human embryo or blastocyst, which are self-renewing and pluripotent or totipotent and have the ability to give rise to all cell types present in a mature animal. Human embryonic stem cells (hESC) can be isolated from, for example, human blastocysts obtained from pre-implantation embryos in the human body, in vitro fertilized embryos or single-cell human embryos expanded to the blastocyst stage.

[0077] The term "embedding" refers to wrapping matrigel around the outer layer of the sphere. "EB" refers to embryoid body.

[0078] In the examples of this article, the single-cell related methods are as follows:

[0079] (1) For single-cell RNA sequencing (scRNA-seq), according to the user guide of the manufacturer, a 3'-library was constructed using the Chromium Single Cell Kit v3 (10X GENOMICS). The amplified and purified library was quantified on a Qubit 2.0 (Invitrogen) using the Quant-iT dsDNA Assay Kit, High Sensitivity (Thermo Fisher), and sequenced on an Illumina HiSeq Xten platform (Illumina) using PE 150 (paired-end sequencing, 150 bp read length).

[0080] (2) Align the FASTQ files of scRNA-seq sequencing results to the human reference genome (GRCh38) using STAR software (v.2.7) and generate the relevant cell-gene count matrix. Analyze the unique molecular identifier (UMI) counts using the Python package Scanpy (v.1.6.0). We removed low-quality cells based on the percentage of mitochondrial genes, gene counts, and the number of detected genes. Finally, cells with an average of approximately 2500 genes were used for subsequent analysis, demonstrating the complexity of the library. Evaluate the impact of droplets that may contain more than one cell using Scrublet software (v.0.1), with an expected doublet rate of 0.05 and a score threshold of 0.5. The UMI counts per cell were normalized by the total expression, multiplied by a scaling factor of 10000, and log-transformed. Identify highly variable genes (HVGs) using the function highly_variable_genes, with min_mean set to 0.0125, max_mean set to 3, and min_disp set to 0.5. Implement the scale function to eliminate the variation caused by the differences in total UMI per cell. To further attenuate data noise and reduce the gene expression space, we merged the scRNA-seq datasets and performed PCA on the scaled data of variable genes. Use the harmonypy Python package (v.0.0.5) to eliminate batch effects across datasets. Briefly, cells in the top 50 PCA vector spaces were selected, and an iterative algorithm was applied to calibrate dataset-specific variations. Finally, this adjustment projected the cells into a shared embedding, in which the cells were grouped by cell type rather than dataset-specific conditions. This embedding was used for downstream analysis and visualization. Calculate the neighborhood graph of cells using the Leiden clustering algorithm with the number of neighborhood points set to 10. Visualize using uniform manifold approximation and projection (UMAP).

[0081] (3)Integrative analysis of organoids and early human embryos (CS12-16) was performed using the harmonypy package (v.0.0.5). Briefly, neural progenitor, neuron, and neural crest cell subtypes (i.e., sensory, schwann, and mesenchyme) were extracted from the human embryonic ectoderm clusters. To accelerate the calculation speed, half of the cells in each cluster were selected, totaling 17,406 cells. To validate the neural crest subtypes in the organoid scRNA-seq data, we integrated the organoid data with the mouse neural crest dataset using the harmonypy package. In the organoids, mesenchymal, migratory, and sensory neurons were annotated as neural crest subtypes. The ten nearest neighbor cells of mouse neural crest cells corresponding to the organoid cells were calculated in the PCA space and projected onto the UMAP of the organoid dataset.

[0082] (4)To calculate the proportion and correlation of neural crest subtypes, the neural crest subtype characteristic markers were visualized in the UMAP of the organoid dataset. Cell types were annotated based on the original identity or the expression pattern of neural crest characteristic markers. Based on the expression pattern of cross-cell type-specific ectodermal markers in the human embryonic ectoderm, the Pearson correlation between human and organoid neural crest or neural subtype clusters was calculated and shown as a heatmap.

[0083] In this article, Feeder refers to feeder layer cells.

[0084] In this article, the low-attachment 96-well plate, U-bottom ultralow attachment plates, 96well ((Corning, cat. no. CLS7007).

[0085] In this article, the low-attachment 24-well plate, Ultralow attachment plates, 24well (Corning, cat. no. CLS3473).

[0086] In this article, D4 and D6 refer to Day 4 and Day 6 respectively, and so on; the starting time is counted as Day 1 from the time when the stem cells are digested and inoculated into the low-attachment culture plate suspension balls.

[0087] In the examples of this article, CF-1 feeder cells refer to "mouse embryonic fibroblasts".

[0088] In the examples of this article, the embryonic stem cells (hES) used are derived from HN4 of Hainan Medical College.

[0089] In the examples of this article, the sources of each component are shown in the following table:

[0090] Component Manufacturer Source and Catalog Number Component GlutaMax Gibco Cat#35050-061 NEAA Gibco Cat#11140-050 bFGF ORYZOGEN Cat#HYC005MO1 KSR Gibco Cat#10828-028 β-Me Sigma Cat#M3148 Y27632 CHEMLEADER Cat#C14357 Matrigel BD Bioscience Cat#354277 Heparin Sigma Cat#H3149 LDN193189 Targetmol Cat#T6158 SB431542 Selleckchem Cat#S1067 DMEM-F12 Gibco Cat#11330-032 Neurobasal Gibco Cat#21103-049 Insulin Sigma Cat#I9278 N2 Gibco Cat#17502048 B27 [without Vitamin A] Gibco Cat#12587-010 B27 [with Vitamin A] Gibco Cat#17504-044

[0091] In the embodiments of the present invention, KSR refers to KnockOut Serum Replacement (KSR), a serum substitute.

[0092] Lancasterprotocol refers to the method disclosed in the article published by Lancaster, M.A. et al [1].

[0093] In the embodiments of the present invention, DSI refers to Dual SMAD inhibitor (DSI), which means that two small molecules, LDN193189 and SB431542, are added simultaneously.

[0094] Example 1: hES Cell Culture in Feeder System

[0095] One day before passage, coat a 6-well plate with 0.1% gelatin and place it at 37 °C for 30 min. Take out CF-1 feeder cells from the liquid nitrogen tank, quickly dissolve them in a 37 °C water bath, add 10% FBS medium (10% v / v FBS, 1% v / v NEAA, 1% v / v GlutaMax, DMEM), and centrifuge at 250 g for 5 min. Discard the supernatant, resuspend with 10% FBS (10% v / v FBS, 1% v / v NEAA, 1% v / v GlutaMax, DMEM) medium, and seed them into the 6-well plate pre-coated with 0.1% gelatin at a density of 450,000 cells per well, then culture in a 37 °C incubator. The next day, take out the cells to be passaged, wash them once with DMEM / F12, add 1 mg / mL Collagenase IV, digest at 37 °C for 5 min, discard 1 mg / mL Collagenase IV, wash three times with DMEM / F12, add KSR medium (DMEM / F12, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 10 ng / mL bFGF) to resuspend, gently scrape the cells with a pipette tip or Pasteur pipette, and passage them to the 6-well plate pre-seeded with feeder cells at a ratio of 1:6, then culture in a 37 °C incubator. Change the medium every day until the cell density reaches 70%-80%, and then perform the next passage according to the above method.

[0096] Example 2: Organoid Culture in Feeder System

[0097] The hES cells were cultured on CF-1 feeder for 4 passages. When they reached a density of 70-80%, they were washed once with DPBS and digested with 1 mg / mL Dispase until the clones curled up and only adhered to the middle. Then, they were gently tapped to make the clones fall off. The Dispase solution was discarded, and the cells were resuspended in KSR medium (DMEM / F12, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 4 ng / mL bFGF) containing 4 ng / mL bFGF. After sedimentation for 1 min, the supernatant was discarded, and the operation was repeated once. Then, 1 mL of 0.05% trypsin / EDTA was added and digested at 37 °C for 2 min. 1 mL of trypsin inhibitor was added, and the cells were centrifuged at 270 g for 5 min.

[0098] The supernatant was discarded, and the cells were resuspended in KSR medium (DMEM / F12, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 4 ng / mL bFGF and 50 μM Y27632) containing 4 ng / mL bFGF and 50 μM Y27632. The cells were counted and seeded into a low-attachment 96-well plate, 9000 cells per well. The medium was changed every other day until D6. After D4, the medium was changed to KSR medium without bFGF (DMEM-F12, 0.1 μM LDN193189, 10 μM SB431542, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me).

[0099] On D6, the cells were transferred into a low-attachment 24-well plate using a 1 mL pipette tip with the tip cut off, and then cultured in neural epithelium induction medium (DMEM-F12, 1% v / v N2, 1% v / v NEAA, 1% v / v GlutaMax, 1 μg / mL heparin) until the outer surface of the EB spheres became translucent.

[0100] Matrigel was dissolved on ice 1-2 h in advance. After it was completely dissolved, the EB spheres with the outer surface becoming translucent were transferred onto a sealing film with pre-pressed grooves using a 200 μL pipette tip with the tip cut off (the sealing film needed to be sterilized by ultraviolet irradiation in advance). The excess medium was discarded, 30 μL of Matrigel was added, and it was placed at 37 °C for 20 min.

[0101] Gently blow down the EB spheres with neural differentiation medium I (DMEM-F12, 50% v / v Neurobasal, 2.5% v / v insulin, 0.5% v / v N2, 1% v / v B27 [without Vitamin A], 0.5% v / v NEAA, 1% v / v GlutaMax, 1% v / v Penicilin-streptomycin), transfer them to a 6-cm culture dish for culture, change the medium every other day. After culturing for 4 days, transfer them to an orbital shaker incubator for continuous culture, and change to neural differentiation medium II (DMEM-F12, 50% v / v Neurobasal, 2.5% v / v insulin, 0.5% v / v N2, 1% v / v B27 [with Vitamin A], 0.5% v / v NEAA, 1% v / v GlutaMax, 1% v / v Penicilin-streptomycin), change the medium every 2 - 3 days to obtain brain organoids.

[0102] Example 3: Detection of Neural Crest Cell Types in Organoid Model

[0103] 3.1 Immunofluorescence analysis

[0104] Take the brain organoids cultured to D35 for cryosectioning and immunofluorescence, and it is found that multiple neural tube structures with high specific expression of SOX2, TUJ1, PAX6, and FOXG1 are formed inside (as Figure 1 shown), and neural crest markers SOX10 and P75 (as Figure 2 shown).

[0105] 3.2 scRNA-seq data analysis

[0106] Take the brain organoids induced and cultured to D21 and D30 for scRNA-seq data analysis. As Figure 3 A, Figure 4 shown, it is found that there are neural crest cell populations in our brain organoids, and there are 3 subpopulations, including migratory neural crest (NC-Migratory), mesenchymal cells derived from neural crest (NC-Mesenchyme), and sensory neuron cells (NC-Sensory). The cell distribution at two time points and the changes in the 3 neural crest populations can be seen as follows: at D21, the proportion of migratory neural crest is larger; at D30, the proportions of mesenchyme and sensory neurons increase ( Figure 3 B - D). This result is consistent with the law of neural crest development.

[0107] 3.3 Comparative example 1

[0108] We compared our data with the published data related to neural crest development and brain-like data. The projection results of cells related to early neural crest development in mice on our UMAP showed that sensory neurons, mesenchymal cells, and migrating neural crest were consistent with the positions of our cell clusters ( Figure 5 A). The correlation results between the cell types in our brain-like and those in human early embryos also indicated that we were highly consistent with the corresponding cell types in human early embryos, and the correlation of the three neural crest subpopulations reached about 0.9 ( Figure 5 B). These results suggest that the neural crest populations contained in our brain-like are very similar to those in vivo.

[0109] In addition, compared with the neural crest populations in the published brain-like articles, the proportion of neural crest population cells in our brain-like is larger, and the correlation with the neural crest populations in vivo is higher, indicating that our brain-like has higher-quality neural crest populations ( Figure 5 C-D).

[0110] Figure In 5D, Lancaster protocol: refers to the brain organoid culture system based on the Lancaster 2014 Nature protocol; Dual SMAD INH: represents the addition of dual SMAD inhibitors (LDN193189 and SB431542) during the culture process; WNT INH: represents the addition of a WNT signaling pathway inhibitor. Example 4: Reproducing the Result of Increased Neural Crest Ratio by Adding DSI in Feeder-Free System

[0111] Figure 6

[0112] Meanwhile, we used a feeder-free system for culturing brain organoids. The specific method is as follows: On the premise of the culturing method of the Feeder system in Example 1, the step of removing Feeder cells was omitted to culture hES cells (referred to as "hES cultured in the feeder-free system"). When the density of hES cells cultured in the feeder-free system reached 80%, the cells were washed with 1 mL of DPBS without calcium and magnesium, and then the DPBS was discarded. Then, 600 μl of 0.5 mM EDTA solution was added to the cell well. The cells were placed back in the incubator for 4 minutes. The EDTA solution was gently aspirated away without disturbing the clones, and 1 mL of Accutase was added and the cells were placed back in the incubator and left standing for another 4 minutes.

[0113] 1 mL of mTeSR (MTeSR TM 1 serum-free medium) + Y27632 (10 μM) medium was added to detach all the clones, and then this 2 mL of mixed liquid was transferred to a 15 mL centrifuge tube and pipetted and mixed until the clones formed single cells. After taking an appropriate amount of the sample for counting, another 3 mL of mTeSR (MTeSR TM 1 serum-free medium) + Y27632 (10 μM) medium was added and mixed. Centrifugation was performed at 270 g for 5 min at room temperature while completing the counting.

[0114] After centrifugation, discard the supernatant, and resuspend the cell pellet with an appropriate amount of KSR medium (DMEM / F12, 20% KSR, 1% NEAA, 1% GlutaMax, 0.1 mM β-Me, 4 ng / mL bFGF, and 50 μM Y27632) containing 4 ng / mL bFGF and 50 μM Y27632. Seed the cells into a low-attachment 96-well plate at a density of 9000 cells / well, and then place it back into the incubator for static culture.

[0115] The subsequent steps and the use of relevant media are the same as those in Example 2. Specifically as follows: Change the medium every other day, that is, on the second day, change the KSR medium (DMEM / F12, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 4 ng / mL bFGF, and 50 μM Y27632) containing 4 ng / mL bFGF and 50 μM Y27632. On the fourth day, change the KSR medium containing dual smad inhibitor (DMEM-F12, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 10 μM SB431542, 0.1 μM LDN193189).

[0116] On D6, use a 1 mL pipette tip with the tip cut off to transfer the EB spheres in the low-attachment 96-well plate to a 15 mL centrifuge tube. After natural sedimentation, discard all the supernatant. Resuspend the EB spheres with neural epithelium induction medium (DMEM-F12, 1% v / v N2, 1% v / v NEAA, 1% v / v GlutaMax, 1 μg / mL heparin), and then transfer them to a low-attachment 24-well plate at a density of 8 - 16 EB spheres / well. After culturing for about 2 - 3 days, until the outer surface of the EB spheres becomes translucent, the next step of embedding Matrigel can be carried out.

[0117] Dissolve Matrigel on ice 1 - 2 h in advance. After it is completely dissolved, use a 200 μL pipette tip with the tip cut off to transfer the EB spheres to the sealing film with pre-pressed grooves (the sealing film needs to be sterilized by ultraviolet light in advance). Discard the excess medium, add 30 μL of Matrigel, and place it at 37 °C for 20 min.

[0118] Gently pipette the EB spheres with neural differentiation medium I (DMEM-F12, 50% v / v Neurobasal, 2.5% v / v insulin, 0.5% v / v N2, 1% v / v B27 [without Vitamin A], 0.5% v / v NEAA, 1% v / v GlutaMax, 1% v / v Penicillin-streptomycin), transfer them to a 6-cm culture dish for cultivation, change the medium every other day. After culturing for 4 days, transfer them to an orbital shaker incubator for continued cultivation, and change to neural differentiation medium II (DMEM-F12, 50% v / v Neurobasal, 2.5% v / v insulin, 0.5% v / v N2, 1% v / v B27 [with Vitamin A], 0.5% v / v NEAA, 1% v / v GlutaMax, 1% v / v Penicillin-streptomycin), change the medium every 2 - 3 days to obtain brain organoids.

[0119] The biggest difference in culturing brain organoids between the feeder-free system and the feeder system lies in the different hES culture systems of the starting cells, resulting in different steps of digesting into single cells before the hanging spheres: the feeder-free system does not require discarding the feeder; the feeder-free system uses EDTA + accuase for digestion, while the feeder system uses dispase, type IV collagenase, and trypsin, etc. for digestion. The stability of brain organoid culture in the feeder-free system is not as good as that in the feeder system.

[0120] Single-cell transcriptome analysis shows that in the feeder-free system, without adding DSI, the proportion of neural crest lineage cells is reduced ( Example 5: Feeder-Free Dual SMAD System vs. Dual SMAD-Added System ), highlighting the important function of DSI in this system.

[0121] Figure 7

[0122] The experimental method of the group with the dual smad system added in this example is the same as that in Example 2. The group without the dual smad system is the same as the group with the dual smad system added except that dual smad is not added.

[0123] Taking pictures and observing the brain organoids under the bright field of a microscope shows that compared with the case of adding Dual SMAD inhibitor (DSI), the induction success rate of brain organoids in the Feeder system without adding DSI drops significantly, presenting a vacuolated state ( Example 6: Effect of Replacing DUAL SMAD Small Molecules on the Development of Brain Organoids ), suggesting that adding DSI promotes the induction efficiency of the neuroectoderm, including the neural crest cell types differentiated from the neuroectoderm.

[0124] Figure 8

[0125] The experimental procedure of this example is the same as that of Example 2, except that LDN193189 (LS) in the DUAL SMAD small molecule is replaced with dorsomorphin (DS), and it becomes Figure 8 the HN4-DS group in

[0126] Replacing LDN193189 (LS) with dorsomorphin (DS) weakens neural differentiation. Immunofluorescence ( Figure 8 ) shows a decrease in the expression of the neural marker TUJ1, indicating the importance of LDN193189.

[0127] References:

[0128] 1. Lancaster, M.A. & Knoblich, J.A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 2014; 9: 2329 - 2340.

[0129] 2. Soldatov, R. et al. Spatiotemporal structure of cell fate decisions in murine neural crest. Science 2019; 364.

[0130] 3. Bhaduri, A. et al. Cell stress in cortical organoids impairs molecular subtype specification. Nature 2020; 578: 142 - 148.

[0131] 4. Velasco, S. et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 2019; 570: 523 - 527.

[0132] 5. Xiang, Y., et al. Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration. Cell Stem Cell 2017; 21: 383-398.e387.

[0133] 6. Kanton, S., et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 2019; 574: 418-422.

[0134] 7. He, Z., et al. Lineage recording in human cerebral organoids. Nat. Methods 2022; 19: 90-99.

[0135] 8. Sloan, S. A., et al. Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells. Neuron 2017; 95: 779-790.e776.

[0136] 9. Trujillo, C. A., et al. Complex Oscillatory Waves Emerging from Cortical Organoids Model Early Human Brain Network Development. Cell Stem Cell 2019; 25: 558-569.e557.

Claims

1. A method for preparing an organoid model of the brain, characterized in that, The method includes contacting stem cells with a stem cell culture medium, the stem cell culture medium being added with SB431542 or its salt, and LDN193189 or its salt, and the brain organoid being a brain-like organoid having a neural crest cell population.

2. The method according to claim 1, characterized in that, The stem cells are selected from embryonic stem cells or induced pluripotent stem cells; Preferably, the method further includes: adding Matrigel to the embryoid bodies; Preferably, the Matrigel wraps the embryoid bodies; The Matrigel is a liquid; Preferably, the method further includes: adding the embryoid bodies into a groove and then adding Matrigel; Preferably, the method further includes: culturing embryonic stem cells or induced pluripotent stem cells for culturing brain organoids on feeder cells; Preferably, the method includes the following steps: (1) Culturing embryonic stem cells or induced pluripotent stem cells on feeder cells; (2) Inoculating the embryonic stem cells or induced pluripotent stem cells into a low-attachment culture plate for culturing to obtain embryoid bodies; A stem cell culture medium containing LDN193189 and SB431542 is used during the culturing process; (3) Contacting the obtained embryoid bodies with Matrigel to make the Matrigel wrap the embryoid bodies; (4) Adding a culture medium to culture the embryoid bodies wrapped with Matrigel to obtain brain organoids; Preferably, the method includes the following steps: (a) Culturing embryonic stem cells or induced pluripotent stem cells on feeder cells; (b) Digesting the embryonic stem cells or induced pluripotent stem cells; (c) Resuspending the cells with a culture medium containing bFGF and Y27632 and inoculating the cells into a low-attachment culture plate; (d) Replacing the culture medium with a stem cell culture medium containing LDN193189 and SB431542 for culturing; (e) Changing the culture medium to a neuroepithelial induction medium to obtain embryoid bodies; (f) Adding Matrigel to the embryoid bodies so that the Matrigel wraps the embryoid bodies; (g) Culturing the embryoid bodies wrapped in Matrigel with a first neural differentiation medium; (h) Placing the embryoid bodies into a second neural differentiation medium and culturing them in an orbital shaker incubator to obtain brain-like organoids; Preferably, in step (a), the embryonic stem cells or induced pluripotent stem cells are cultured on feeder cells for 3 to 8 passages; Preferably, in step (a), the embryonic stem cells or induced pluripotent stem cells are cultured on feeder cells for 4 passages; Preferably, in step (a), the embryonic stem cells or induced pluripotent stem cells are resuspended in a culture medium; then the embryonic stem cells or induced pluripotent stem cells are inoculated into a culture plate seeded with feeder cells for culturing; the components of the culture medium include DMEM / F12, KSR, NEAA, GlutaMax, β-Me and bFGF; Preferably, in step (a), the components of the culture medium include 10 - 30% v / v KSR, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, 0.01 - 0.5 mM β-Me, 5 - 20 ng / mL bFGF; Preferably, in step (a), the culture medium components include 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, and 10 ng / mL bFGF; Preferably, in step (c), the culture medium is KSR medium; Preferably, in step (c), the components of the KSR medium include DMEM / F12, KSR, GlutaMAX, NEAA, β-Me, bFGF, and Y27632; Preferably, in step (c), the components of the KSR medium include 10 - 30% v / v KSR, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, 0.01 - 0.5 mM β-Me, 1 - 10 ng / mL bFGF, and 20 - 80 uM Y27632; Preferably, in step (c), the components of the KSR medium include 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, 0.1 mM β-Me, 4 ng / mL bFGF, and 50 uM Y27632.

3. The method according to claim 2, wherein In step (d), the stem cell culture medium containing LDN193189 and SB431542 does not contain bFGF; Preferably, the concentrations of LDN193189 and SB431542 are 25 - 100 nM and 1 - 20 μM, respectively; Preferably, the concentrations of LDN193189 and SB431542 are 25 - 80 nM and 1 - 15 μM, respectively; Preferably, the concentrations of LDN193189 and SB431542 are 25 - 60 nM and 1 - 10 μM, respectively; Preferably, in step (d), the components of the stem cell culture medium containing LDN193189 and SB431542 further include DMEM-F12, KSR, NEAA, GlutaMax, and β-Me; Preferably, in step (d), the components of the stem cell culture medium containing LDN193189 and SB431542 further include 10 - 30 v / v KSR, 0.1 - 5% v / v NEAA, 0.1 - 5% v / v GlutaMax, and 0.01 - 0.5 mM β-Me; Preferably, in step (d), the components of the stem cell culture medium containing LDN193189 and SB431542 include 0.1 μM LDN193189, 10 μM SB431542, 20% v / v KSR, 1% v / v NEAA, 1% v / v GlutaMax, and 0.1 mM β-Me; Preferably, in step (e), the components of the neuroepithelial induction medium include the components: DMEM-F12, N2, NEAA, GlutaMax, and heparin; Preferably, in step (e), the components of the neuroepithelial induction medium include 0.5-5% v / v N2, 0.1-5% v / v NEAA, 0.1-5% v / v GlutaMax, and 0.5-5 μg / mL heparin; Preferably, in step (e), the components of the neuroepithelial induction medium include 1% v / v N2, 1% v / v NEAA, 1% v / v GlutaMax, and 1 μg / mL heparin; Preferably, in step (f), after a translucent circle appears around the outer surface of the embryoid body sphere, Matrigel is added; Preferably, in step (f), the Matrigel is in liquid form; Preferably, in step (f), the embryoid body is transferred onto the sealing film of a specific groove, the excess medium is discarded, liquid Matrigel is added, and it is placed at 25-40 °C for 15-30 min; Preferably, the components of the first neural differentiation medium include: DMEM-F12, Neurobasal, insulin, N2, B27, NEAA, and GlutaMax; Preferably, the components of the first neural differentiation medium further include Penicilin-streptomycin; Preferably, the B27 is B27 without vitamin A; Preferably, the components of the first neural differentiation medium include: 10-80% v / v Neurobasal, 0.5-6% v / v insulin, 0.1-1% v / v N2, 0.1-10% v / v B27[without Vitamin A], 0.1-5% v / v NEAA, and 0.1-10% v / v GlutaMax; Preferably, the components of the first neural differentiation medium further include 0.1-10% v / v Penicilin-streptomycin; Preferably, the components of the first neural differentiation medium include: 50% v / v Neurobasal, 2.5% v / v insulin, 0.5% v / v N2, 1% v / v B27[without Vitamin A], 0.5% v / v NEAA, 1% v / v GlutaMax; Preferably, the components of the first neural differentiation medium further include 1% v / v Penicilin-streptomycin; Preferably, the components of the second neural differentiation medium include DMEM-F12, Neurobasal, insulin, N2, B27(with Vitamin A), NEAA, GlutaMax; Preferably, the components of the second neural differentiation medium further include Penicilin-streptomycin; Preferably, the B27 contains vitamin A; Preferably, the components of the second neural differentiation medium include 10-80% v / v Neurobasal, 0.5-6% v / v insulin, 0.1-5% v / v N2, 0.1-10% v / v B27 (with Vitamin A), 0.1-5% v / v NEAA, and 0.5-10% v / v GlutaMax; Preferably, the components of the second neural differentiation medium include DMEM-F12, 50% Neurobasal, 2.5% insulin, 0.5% N2, 1% B27, 0.5% NEAA, and 1% GlutaMax; Preferably, the components of the second neural differentiation medium further include 1% Penicilin-streptomycin; Preferably, step (d) is carried out after the 4th day of step (c); Preferably, step (e) is carried out after the 6th day of step (c); Preferably, step (h) is carried out after the 4th day of step (g); Preferably, the cerebral organoid is a mammalian cerebral organoid; Preferably, the mammal is a human.

4. A brain organoid model, characterized in that, It uses SB431542 or its salt, and LDN193189 or its salt, and the cerebral organoid of the brain is a brain organoid with a neural crest cell population.

5. A brain organoid model, characterized in that, Obtained by the method according to any one of claims 1-3; Preferably, the cerebral organoid is a mammalian cerebral organoid; Preferably, the mammal is a human.

6. Use of a composition in the preparation of an organoid model of the brain, characterized in that, The composition includes SB431542 or its salt, and LDN193189 or its salt, and the cerebral organoid of the brain is a brain organoid with a neural crest cell population.

7. A culture medium, characterized in that, It contains matrix components of an embryonic stem cell or induced pluripotent stem cell medium, and LDN193189 or its salt, and SB431542 or its salt.

8. A method for screening a drug for treating a neurodevelopmental disorder or a brain disease-related disorder, which includes the step of contacting a candidate drug with the cerebral organoid model according to claim 4 or 5.

9. The in vitro use of the cerebral organoid according to claim 4 or 5 in drug discovery screening, toxicity determination, drug or gene expression research of brain or nerve diseases, research on the mechanism of brain or nerve injury and repair; research on the pathogenic mechanism; research on the etiology of brain cancer; or, the use in recombinant gene expression.

10. The application of the cerebral organoid according to claim 4 or 5 in the preparation of a drug for treating a neurological disorder, a neurological disease, or a nerve or brain disease, or the application in the preparation of a drug for regenerative medicine.

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