Preparation, sorting and application of LGE / CGE organ and GABAergic neuron

Abstract

The invention provides preparation, sorting and application of LGE / CGE organoid and GABAergic neurons, and belongs to the field of brain organoid differentiation and GABAergic neurons. The preparation method of the LGE / CGE type organ comprises the following steps: S1, performing embryoid formation and initial culture on the 0th-4th day; s2, performing nerve induction and matrigel embedding on the 5th-11th day; s3, culturing the brain organoid on the 12th day to the 19th day; s4, dynamically culturing and screening the brain organoid on the 20th-35th day; and S5, carrying out differentiation and collection on LCOs of the LGE / CGE type organs after the 35th day. According to the method, the LGE / CGE organs containing the high-purity GABA energy neurons are obtained through differentiation from the brain organs for the first time, the high-purity LGE type GABA energy neurons and the high-purity CGE type GABA energy neurons are obtained through a specific sorting method, and the method has the deep application potential in the fields of disease model construction, medicine research and development and the like.

Description

Technical Field

[0001] The present invention relates to the field of brain organoid differentiation and GABAergic neurons, and in particular to the preparation, sorting and application of LGE / CGE organoids and GABAergic neurons. Background Art

[0002] GABAergic neurons are the primary inhibitory neurons in the central nervous system (CNS). They regulate neural circuit activity by releasing gamma-aminobutyric acid (GABA), maintaining the balance between excitation and inhibition. They play a crucial role in key brain functions such as motor control, cognition, and epilepsy prevention. During brain development, GABAergic neurons primarily originate from the ganglionic eminences (GEs), including the medial (MGE), lateral (LGE), and caudal (CGE) ganglionic eminences. GABAergic neuron dysfunction is closely associated with a variety of neurological disorders, including epilepsy, Huntington's disease, Parkinson's disease, schizophrenia, and Dravet syndrome.

[0003] Transplantation of human fetal GABAergic neurons has demonstrated good efficacy and safety in animal models and clinical trials for the treatment of epilepsy and Huntington's disease. Recently, MGE-derived GABAergic neurons derived from pluripotent stem cells (PSCs) were used in an ongoing Phase I / II clinical trial for the treatment of refractory mesial temporal lobe epilepsy, further highlighting the translational potential of PSC-derived GABAergic neurons in the treatment of neurological diseases.

[0004] The Sonic Hedgehog (SHH) signaling pathway plays a key role in the development of GABAergic neurons, particularly in specifying precursor cells in the MGE and regulating their differentiation into GABAergic neurons. In contrast, the lineage specification process of GABAergic neurons in the LGE and CGE is more complex, involving multiple signaling pathways, including SHH, WNT, and FGF8. Currently, there are many methods to differentiate PSCs into LGE and CGE type GABAergic neurons by using different signaling molecules or their combinations (such as SHH, WNT inhibitors, retinoic acid RA, and activin A). However, the efficiency of these differentiation methods still varies greatly and needs further optimization.

[0005] Furthermore, in the human fetal brain, the LGE and CGE populations themselves exhibit heterogeneity: LGE neurons can develop into striatal D1 and D2 medium spiny neurons (MSNs) and olfactory bulb interneurons, while CGE neurons can give rise to cortical and subcortical interneurons. In most studies, the identification of PSC-derived LGE or CGE-type GABAergic neurons relies primarily on specific markers, such as the LGE neuronal markers MEIS2 and FOXP2, and the CGE neuronal markers CALB2 and NR2F2 (also known as COUP-TFII). Therefore, it remains unclear to what extent PSC-derived GABAergic neurons can recapitulate the neuronal subtype composition of the human fetal LGE and CGE.

[0006] Currently, two-dimensional (2D) culture systems remain the primary method for obtaining PSC-derived GABAergic neurons. In contrast, three-dimensional (3D) cultured brain organoids have demonstrated significant advantages in simulating early human brain development, capable of reproducing the complex cellular diversity, spatial organization, and microenvironment of the human brain in vitro, providing an alternative approach for obtaining PSC-derived GABAergic neurons.

[0007] In addition to improving differentiation efficiency, developing effective enrichment methods is also crucial for obtaining highly pure neurons and their subpopulations. Enrichment techniques based on specific cell surface markers, such as magnetic bead sorting (MACS) and flow cytometry (FACS), have been widely used to purify various cell types. Notably, the surface marker CD90 (also known as Thy1) has been used to isolate neurons from cortical spheres derived from human pluripotent stem cells (PSCs), while CLSTN2 and PTPRO have been used to purify PSC-derived midbrain dopaminergic neurons. The surface marker ERBB4 has been used to enrich for PSC-derived MGE-type GABAergic neurons, thereby increasing their proportion. However, because ERBB4 is expressed in MGE, CGE, and LGE-type GABAergic neurons, its use may lead to the contamination of other GABAergic neuronal populations. Furthermore, there is currently a lack of specific surface markers for enriching LGE-type or CGE-type GABAergic neurons, making it challenging to obtain highly purified versions of these neurons. Summary of the Invention

[0008] To address the aforementioned issues with existing technologies, the present invention provides methods for preparing, sorting, and applying LGE / CGE organoids and GABAergic neurons. This invention, for the first time, differentiates LGE / CGE organoids containing highly purified GABAergic neurons from cerebral organoids. Furthermore, through a specific sorting method, high-purity LGE- and CGE-type GABAergic neurons are obtained, respectively. These methods hold great potential for application in disease modeling and drug development.

[0009] The technical solutions of the present invention are as follows:

[0010] The present invention first provides a method for preparing LGE / CGE organoids (LCOs), comprising the following steps:

[0011] S1. Embryoid body formation and initial culture: On days 0-4, H9 embryonic stem cells (ESCs) were digested and aggregated to form embryoid bodies (EBs), initiating early differentiation.

[0012] S2. Neural induction and Matrigel embedding: On days 5-11, EBs are guided to differentiate toward a neural fate using neural induction medium (NI medium), and Matrigel embedding provides a three-dimensional scaffold to promote the formation of neuroepithelial structures, i.e., early brain organoids.

[0013] S3. Cerebral organoid culture: On days 12-19, the early-stage cerebral organoids formed after embedding are transferred to a larger space and cultured in NI medium to promote their further development and growth.

[0014] S4. Dynamic Cultivation and Screening of Brain Organoids: From days 20 to 35, brain organoids are dynamically cultured in BOD medium to simulate the in vivo environment and promote the development of complex brain regions. Qualified brain organoids containing cerebral cortical structures are screened.

[0015] S5. Differentiation and collection of LGE / CGE organoids (LCOs): After day 35, qualified brain organoids form microspheres (BOMSs) on their surface by budding, which are then collected to obtain the LGE / CGE organoids (LCOs).

[0016] Preferably, the digestion in step S1 uses EDTA, the culture medium used on day 0 is Essential 8 supplemented with ROCK inhibitor Y-27632, and the culture medium used on days 1-4 is Essential 8 culture medium without Y-27632.

[0017] Preferably, the components of the neural induction NI medium in step S2 include: DMEM / F12, N-2 supplement, MEM-NEAA and heparin; the EBs are cultured in the NI medium for 5-7 days; the Matrigel embedding is to embed the EBs in NI medium containing 25% Matrigel on the 8th to 9th day, and gradually add NI medium; and culture in NI medium on the 9th to 11th day.

[0018] Preferably, the components of the BOD culture medium in step S4 include: DMEM / F12, insulin, GlutaMAX, MEM-NEAA, 2-mercaptoethanol, N-2 supplement and B-27 supplement; the dynamic culture is cultured on a shaker at 37°C and 60rpm, and the medium is replaced by half every 2-3 days.

[0019] Preferably, the LGE / CGE organoids LCOs obtained in step S5 are aggregated by gravity sedimentation and stored in a culture flask containing BOD medium. The culture is continued on a shaker at a speed of 60-100 rpm until the 110th day, and the medium is replaced by half every 2-3 days.

[0020] The present invention also provides a method for sorting LGE / CGE type GABAergic neurons, comprising the following steps:

[0021] M1. Prepare single cell suspension: Use TrypLE enzyme to digest and disaggregate the BOMSs collected in step S5 of claim 1 into single cells in preparation for subsequent labeling and sorting;

[0022] M2, Fc receptor blocking and CXCR4 antibody labeling: resuspend single cells in BOD medium, incubate with Fc receptor blocking solution at room temperature for 5-15 minutes, and then stain with human CXCR4 antibody for 10-20 minutes on ice;

[0023] M3, magnetic bead binding and washing: After staining, wash the cells with BOD medium and then use MojoSort TM Incubate with anti-PE nanomagnetic beads for 10-20 minutes and wash to remove unbound antibodies and magnetic beads;

[0024] M4, MACS sorting and fraction collection: CXCR4-positive cells bound to magnetic beads were separated using a Miltenyi Biotec sorting column using a magnetic field, and unsorted cells, CXCR4-positive cells, and CXCR4-negative cell fractions were collected separately.

[0025] Preferably, the human CXCR4 in step M2 is PE-labeled, with clone number 12G5.

[0026] The present invention also provides an application of LGE / CGE organoids (LCOs) in establishing a Huntington's disease model. The CRISPR / Cas9 gene editing method is used to construct H9 embryonic stem cells expressing a mutant HTT gene. LGE / CGE organoids (LOCs) expressing wild-type (i.e., initial H9) and mutant HTT genes are differentiated and prepared according to the above preparation method. The Huntington's disease model is constructed by performing GABAergic neuron pathological phenotype analysis and neuronal activity detection on the LOCs.

[0027] The present invention also provides another use of LGE / CGE organoids (LCOs) in preparing drugs for nervous system diseases, wherein the drugs are prepared by dissociating the prepared LCOs into single cells, or by mixing the sorted LGE-type GABAergic neurons with pharmaceutical excipients.

[0028] Preferably, the neurological disease comprises Huntington's disease.

[0029] The beneficial technical effects of the present invention are:

[0030] 1. The present invention provides a method for generating LGE / CGE organoids (also known as BOMSs) or LGE / CGE-type GABAergic neurons without the need for specific exogenous signal induction. This method is simple to operate, can simultaneously achieve differentiation of cerebral organoids and LGE / CGE organoids, and can be easily produced in batches.

[0031] 2. The purity of LGE / CGE GABAergic neurons in the LGE / CGE organoids prepared by the present invention is high, accounting for more than 90%. The LGE / CGE GABAergic neurons are similar to human embryonic LGE / CGE GABAergic neurons at the transcriptional level and are approximately at the 12-13 gestational week stage. The LGE / CGE organoids well reproduce the GABAergic neurons and their subtypes in human embryonic LGE / CGE.

[0032] 3. Based on single-cell sequencing analysis, this study discovered that CGE-type GABAergic neurons in LGE / CGE organoids specifically express the CXCR4 gene, suggesting potential applications for enriching these neurons. This study leverages the expression of CXCR4 on the surface of CGE-type GABAergic neurons and, using CXCR4 surface markers and antibodies, established a novel MACS sorting method for enriching CGE-type GABAergic neurons from LGE / CGE organoids (CXCR4+ >90%).

[0033] 4. The LGE neurons in the LGE / CGE organoids obtained by the present invention have the potential to differentiate into striatal MSNs projection neurons, and have application value in Huntington's model construction, pathogenesis research, and cell therapy. The CGE neurons enriched from the LGE / CGE organoids in the present invention highly express CXCR4, and have application value in the study of CGE neuron migration mechanisms and epilepsy cell therapy. The LGE / CGE organoids obtained by the present invention each account for approximately half of the LGE and CGE neurons, and as an ideal in vitro developmental model, have application value in the study of the fate determination of LGE and CGE neurons. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is an analysis of the products of each step in the differentiation of brain organoids into neurospheres (BOMSs) in Test Example 1.

[0035] Figure 2 The expression of each product of BOMSs in test example 1 during continuous culture.

[0036] Figure 3 Transcriptome analysis of BOMSs and brain organoids (BOs) in Test Case 2.

[0037] Figure 4 This is the functional analysis of GABAergic neurons in BOMS in test example 3.

[0038] Figure 5 Single-cell RNA sequencing analysis of BOMSs and brain organoids in Test Example 4.

[0039] Figure 6 This is the single-cell transcriptome analysis of BOMSs and GABAergic neurons in brain-like organs in Test Example 5.

[0040] Figure 7 Purification of CXCR4 from LCOs (BOMSs) in Test Example 6 + CGE inhibitory neurons.

[0041] Figure 8 The distribution diagram of SOX2+ / PAX6+ neurons in the non-cortical area of ​​the brain organoids based on the prior art is cited for Test Example 1.

[0042] Figure 9 For Test Example 4, single-cell RNA sequencing analysis of brain organoids (BOs) of the prior art and LGE / LGE organoids (BOMSs) of the present invention was cited.

[0043] Figure 10 The UMAP visualization of the prior art cited in Test Example 5 shows the expression of LGE, CGE, and MGE type GABAergic neuron marker genes. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to the accompanying drawings and embodiments. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, rather than all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, all raw materials used in the present invention are purchased from the market.

[0046] Example 1: Culture and collection of LGE / CGE organoids (LCOs)

[0047] This example provides a method for preparing LGE / CGE organoids (LCOs), and the specific steps are as follows:

[0048] Step S1: Embryoid formation and initial culture (Days 0-4): Human embryonic stem cells (H9 ESCs) are digested and aggregated to form embryoid bodies (EBs) to initiate early differentiation. The specific steps are as follows:

[0049] Day 0: Human H9 ESCs were digested with 0.5 mM EDTA and seeded into non-treated 6-well plates to form embryoid bodies (EBs). The culture medium was Essential 8 (E8) supplemented with 5 μM ROCK inhibitor Y-27632.

[0050] Day 1: Use a 1 mL pipette to gently pipette to resuspend EBs and replace with fresh E8 medium without Y-27632.

[0051] Days 1-4: EBs were cultured in E8 medium with half the medium changed daily.

[0052] Step S2: Neural Induction and Matrigel Embedding (Days 5-11): Neural Induction (NI) medium is used to guide EBs to differentiate into neural fates, and Matrigel embedding provides a three-dimensional scaffold to promote the formation of neuroepithelial structures (i.e., early brain organoids). The specific steps are as follows:

[0053] Day 5: Replace E8 medium with neural induction (NI) medium (components: DMEM / F12, N-2 supplement (1X), MEM-NEAA (1X), heparin (2 μg / mL).

[0054] Days 5-7: EBs were cultured in NI medium with half the medium changed daily.

[0055] Day 8: EBs were embedded in NI medium containing 25% Matrigel (0.5-0.7 mL / well of a 12-well plate), incubated at 37°C for 4-6 hours to allow solidification, and then 2 mL of NI medium was slowly added to cover.

[0056] Day 9: Gently add another 2 mL of NI medium to each well of the 12-well plate.

[0057] Days 9-11: Brain organoids (where structures begin to form) are cultured in NI medium with half the medium changed daily.

[0058] Step S3: Cerebral organoid culture (Days 12-19): Transfer the early-stage cerebral organoids formed after embedding to a larger space (6-well plate) and continue culturing in NI medium to promote their further development and growth. The specific steps are as follows:

[0059] Day 12: Gently transfer the brain organoids to a new 6-well plate using a 1 mL pipette.

[0060] Days 12-19: Continue culturing the brain organoids in NI medium, changing half of the medium daily. Before changing the medium, gently blow up the brain organoids that are attached to the wall with a 1 mL pipette to avoid prolonged adherence to the wall.

[0061] Step S4: Dynamic culture and screening of brain organoids (Days 20-35): Transfer the brain organoids to BOD medium and place them in a shaker for dynamic culture to simulate the in vivo environment and promote the development of complex brain regions. Qualified organoids containing cerebral cortical structures are screened. The specific steps are as follows:

[0062] Day 20: Transfer the brain organoids to a T25 culture flask and replace the culture medium with brain organoid differentiation (BOD) medium (ingredients: DMEM / F12, insulin (2.5 μg / mL), GlutaMAX (1X), MEM-NEAA (0.5X), 2-mercaptoethanol (3.5 μL / L), N-2 supplement (1X), B-27 supplement (1X)).

[0063] Culture conditions: Place the T25 flask in a shaker at 37°C and shake continuously at 60 rpm.

[0064] Medium change: Change half of the medium every 2-3 days (using BOD medium).

[0065] Screening (Days 20-35): During this period, healthy brain organoids containing cerebral cortical structures are screened for subsequent steps (LCOs formation). The screening method includes: transferring the brain organoids to a 6-well plate with a 10 mL pipette; observing under a stereomicroscope (in a sterile environment) and identifying brain organoids with cortical structures (with typical structural features that are easy to identify); removing and discarding organoids without cortical structures with a pipette; and transferring qualified brain organoids to a T75 culture flask for continued culture to complete the brain organoid screening.

[0066] Step S5: LGE / CGE organoid (LCO) harvesting and maintenance (approximately after day 35): Budding microspheres (BOMSs, i.e., LGE / CGE organoid LCOs) that naturally detach from the surface of qualified brain organoids are collected and maintained in dynamic culture conditions for a long time. The specific steps are as follows:

[0067] Cultivation and medium exchange of brain organoids: After approximately 35 days of differentiation, half the medium should be exchanged every 2-3 days. Due to the small size of BOMSs, the culture bottle should be upright and left to stand for about 2 minutes before medium exchange to allow the BOMSs and brain organoids to sink to the bottom. Then, some of the supernatant culture medium should be gently removed and half the medium should be exchanged to avoid loss and increase the number of BOMSs collected.

[0068] BOMSs formation and shedding: After approximately 35 days of differentiation, qualified brain organoids will form tiny spherical structures (BOMSs) by "budding" on the surface. These BOMSs are then identified as LGE / CGE organoids (LCOs) and eventually fall off naturally from the surface of the mother brain organoids.

[0069] BOMS collection: BOMS are significantly smaller than brain organoids and are separated and collected by gravity sedimentation (after standing, the brain organoids sink to the bottom first, followed by the BOMS, and the supernatant containing the BOMS is aspirated). Collection is typically done 1-2 times per week. It should be noted that due to the small size of BOMS, when changing the medium, the culture flask should be left to stand for approximately 2 minutes to allow both the BOMS and brain organoids to sink to the bottom. The medium should then be partially removed and a half-volume medium exchange performed to minimize loss and maximize the amount of BOMS collected.

[0070] BOMSs maintenance culture: The collected BOMSs (LCOs) were transferred to a new T25 culture flask and cultured with BOD medium at 37°C and 60 rpm on a shaker.

[0071] BOMSs medium change: Change half of the medium every 2-3 days (using BOD medium).

[0072] Test Example 1: Process testing and analysis of neurospheres generated by differentiating brain organoids

[0073] The operation process of Example 1 and the microstructure of the products of each step are as follows Figure 1 As shown, (A) is a schematic diagram of the process of differentiating brain organoids (BOs) to produce BOMSs. (B) is a bright field image showing the budding of BOMSs on the surface of brain organoids, scale bar: 100μm. Arrow: BOMSs. (C) is a bright field image showing BOMSs falling off from brain organoids. Scale bar: 100μm, arrow: BOMSs falling off from BOs. As can be seen from the figure, bud-like structures appeared on the surface of brain organoids 35 days after inducing human embryonic stem cells (ESCs) to differentiate into brain organoids. Subsequently, these small buds naturally fell off from the brain organoids to form microspheres, which the inventors called "BOMSs"

[0074] Figure 1(D) Bright field images of BOMSs at different differentiation time points. BOMSs were harvested on differentiation day 35 and cultured individually. Scale bar: 100 μm. Figure 1 (E) Quantitative analysis of the size of BOMSs at different differentiation culture time points. Statistical analysis was performed using an unpaired t-test. The red line represents the mean. Sample size n = 116 (day 41), 186 (day 60), 78 (day 79), and 127 (day 110). **** indicates p < 0.0001, and ns indicates p > 0.05 (no statistical difference). As can be seen from the figure, between days 35 and 65 of differentiation, brain organoids can continuously generate BOMSs, which can be harvested every 3-4 days, and BOMSs can be cultured individually for up to 110 days ( Figure 1 A, D, and E). As the culture time increased, the size of BOMSs increased significantly, reaching its maximum on the 79th day of differentiation, with an average diameter of approximately 300 μm ( Figure 1 D and E).

[0075] Figure 1 (F) CTIP2, TBR2, and SOX2 staining results for brain organoids and BOMSs. Scale bar: 50 μm. (G) PAX6 and TUJ1 staining results for brain organoids and BOMSs. Scale bar: 50 μm. As can be seen from the figure, when BOMSs and brain organoids at the early differentiation stage were stained with the NPC markers SOX2 and PAX6, the intermediate progenitor cell marker TBR2, and the neuronal markers CTIP2 and TUJ1, brain organoids derived from human H9 ESCs have a SOX2+ / PAX6+ ventricular zone (VZ), a TBR2+ subventricular zone (SVZ), a CTIP2+ cortical plate (CP), and TUJ1+ neurons. Although SOX2+, PAX6+, CTIP2+, and TUJ1+ cells are also present in BOMSs, TBR2+ intermediate progenitor cells are rarely observed in BOMSs. Furthermore, unlike brain organoids, no clear VZ, SVZ, and CP hierarchical structures were observed in BOMSs. These results suggest that BOMSs differ significantly from brain organoids in terms of cellular composition and structure.

[0076] Figure 1 (H) SOX2 and MAP2 staining results of brain organoids and BOMSs, scale bar: 50 μm. As can be seen from the figure, SOX2+ / CTIP2+ double-positive cells and PAX6+ / TUJ1+ double-positive cells are widely present in BOMSs, while these are almost absent in the cortical region of brain organoids ( Figure 1F and G). This suggests that the neuronal composition of BOMSs and brain organoids is significantly different, with a significantly higher proportion of SOX2+ or PAX6+ neurons in BOMSs. In addition, co-staining of SOX2 with the mature neuronal marker MAP2 also showed that a large number of SOX2+ / MAP2+ double-positive cells were present in BOMSs at day 45 of differentiation, and MAP2 expression was significantly higher than that in brain organoids ( Figure 1 H). In summary, the inventors developed a novel differentiation method to generate neurospheres (BOMSs) containing SOX2+ and PAX6+ neurons from brain organoids.

[0077] SOX2 and PAX6 are classic NPC markers that play a key role in maintaining NPC characteristics and regulating neuronal differentiation. When NPCs initiate neuronal differentiation during cerebral cortex development, the expression of SOX2 and PAX6 is usually downregulated. This phenomenon has also been confirmed during the differentiation of brain organoids ( Figure 1 F and G).

[0078] To observe whether the expression of SOX2 and PAX6 in BOMSs persists during differentiation, the inventors performed immunofluorescence staining analysis of SOX2, MAP2, and NeuN in BOMSs and brain organoids on day 75 or 78 of differentiation. Figure 2 (A) SOX2 and MAP2 staining results in BOMSs and cerebral organoids (BOs). Scale bar: 50 μm. (B) SOX2 and NeuN staining results in BOMSs and cerebral organoids. Scale bar: 50 μm. As can be seen in the figure, MAP2+ and NeuN+ neurons in BOMSs still express SOX2, which is different from the cortical neurons in cerebral organoids, which almost no longer express SOX2.

[0079] Figure 2 (C) is the PAX6 and MAP2 staining results of BOMSs and brain organoids, scale bar: 50μm. (D) is the PAX6 and NeuN staining results of BOMSs and brain organoids, scale bar: 50μm. As can be seen from the figure, in BOMSs, MAP2+ and NeuN+ neurons also show PAX6 positivity, which is obviously different from the situation in brain organoids where cortical neurons hardly express PAX6. These results further indicate that the neurons in BOMSs are SOX2+ and PAX6+, which makes them different from traditional cortical neurons in molecular characteristics. It is worth noting that SOX2+ / PAX6+ neurons were also observed in non-cortical areas of brain organoids ( Figure 8 ), suggesting that BOMSs are likely to be differentiated from non-cortical regions of brain organoids.

[0080] Figure 2 (E) NeuN expression in BOMSs and brain organoids + 、SOX2 + NeuN + and PAX6 + NeuN + Statistical analysis of cells, statistical analysis was performed using unpaired t-test; n = 8 images; **** indicates p < 0.0001. (F) and (G) are the expression of SOX2 (F) and PAX6 in BOMSs and brain organoids.

[0081] (G) Statistical analysis of relative protein abundance using unpaired t-test; **** indicates p < 0.0001. Figures E and E share sample labels for BOMSs and brain organoids. Quantitative analysis showed that the proportions of NeuN+, SOX2+ / NeuN+, and PAX6+ / NeuN+ neurons in BOMSs were 92.93%, 69.42%, and 64.41%, respectively, significantly higher than those in the cortical regions of brain organoids ( Figure 2 E), indicating that the number of neurons in BOMSs is significantly higher than that in brain organoids. In addition, the expression levels of SOX2 and PAX6 proteins in neurons of BOMSs are significantly lower than those in NPCs of brain organoids ( Figure 2 A, C, E, and F), indicating that the expression of SOX2 and PAX6 is downregulated during neuronal differentiation in BOMSs.

[0082] (H) GFAP, Olig2, and MAP2 staining results of BOMSs and brain organoids, scale bar: 50μm. As can be seen from the figure, on day 110 of differentiation, the inventors performed immunofluorescence staining analysis of GFAP, a marker for astrocytes, and Olig2, a marker for oligodendrocytes, on BOMSs and brain organoids, respectively. The results showed that GFAP+ and Olig2+ glial cells were only observed in brain organoids, while no glial cells were observed in BOMSs, indicating that BOMSs have a limited ability to generate glial cells in long-term culture. In summary, the inventors confirmed that BOMSs are mainly composed of neurons expressing low levels of SOX2 and PAX6.

[0083] Test Example 2: Expression test of GABAergic neurons in BOMSs

[0084] To further identify neurons in BOMSs at the transcriptional level, the inventors performed bulk RNA sequencing (bulk RNA-seq) analysis on BOMSs and brain organoids on day 60 of differentiation. Figure 3 shown.

[0085] Figure 3 (A) Schematic diagram of bulk RNA sequencing and data analysis. (B) FPKM values ​​of neural progenitor cell, neuron, astrocyte, and oligodendrocyte marker genes in BOMSs and cerebral organoids. Statistical analysis was performed using an unpaired t-test; results are presented as mean ± SD; n = 3 replicates; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns: not significant difference. (C) Principal component analysis (PCA) shows the transcriptional similarity between BOMSs and human primary neurons. (D) Volcano plot showing differentially expressed genes (DEGs) between BOMSs and cerebral organoids. Genes with an absolute average fold change > 1.5 and p < 0.05 are highlighted in blue and red. (E) Kyoto Encyclopedia of Genes and Genomes pathway analysis of upregulated and downregulated DEGs in BOMSs. (F) FPKM values ​​of excitatory and inhibitory neuron marker genes in BOMSs and cerebral organoids. Statistical analysis was performed using an unpaired t-test; results are presented as mean ± standard deviation; n = 3 replicates; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. (G) GABA and MAP2 staining results in BOMSs and brain organoids. Scale bar: 50 μm.

[0086] First, the inventors detected the expression of major neural cell marker genes in BOMSs and brain organoids, including neural progenitor cells, neurons, astrocytes, and oligodendrocytes ( Figure 3 B). The results showed that brain organoids mainly expressed neuronal marker genes (such as BCL11B, MAP2, MAPT, STMN2, SV2A, SYN1, SYP and TUBB3), neural progenitor cell marker genes (such as GLI3, HES5, MKI67, NES, NOTCH1, VIM, PAX6 and SOX2), and some astrocyte marker genes (such as FGFR3, NFIA and SLC1A3) ( Figure 3 B). Compared with brain organoids, BOMSs mainly express neuronal marker genes, and the expression abundance of several genes is significantly higher than that in brain organoids ( Figure 3 B). Although the expression levels of most neural progenitor cell marker genes in BOMSs were significantly lower than those in brain organoids, there was no significant change in SOX2 gene expression, and PAX6 gene expression was slightly upregulated ( Figure 3 B), which is consistent with the previous immunofluorescence staining results, that is, BOMSs are mainly composed of SOX2 + and PAX6 + Neuronal composition ( Figure 2Furthermore, principal component analysis (PCA) results showed that the transcriptome of BOMSs was closer to that of human primary neurons ( Figure 3 C) This further demonstrates at the transcriptome level that BOMSs are mainly regulated by SOX2. + and PAX6 + Neuronal composition.

[0087] Neurons can be divided into multiple subtypes based on the neurotransmitters they release, including glutamatergic, GABAergic, cholinergic, dopaminergic, 5-hydroxytryptaminergic, etc. Since the inventors have determined that BOMSs are mainly composed of SOX2 + and PAX6 + Neuronal composition, next, the inventors wanted to know which subtype the neurons in BOMSs belong to. To this end, the inventors identified differentially expressed genes (DEGs) between BOMSs and brain organoids based on the following criteria: 1) differential fold change (BOMSs / BOs) > 1.5; 2) p value < 0.05. A total of 3,543 differentially expressed genes were identified, of which 1,476 were upregulated in BOMSs and 2,067 were downregulated in BOMSs ( Figure 3 D) KEGG pathway enrichment analysis showed that the downregulated DEGs in BOMSs were significantly enriched in pathways related to cell proliferation, such as “cell cycle” and “DNA replication” ( Figure 3 E), which is consistent with the lack of neural progenitor cells in BOMSs ( Figure 3 B). In contrast, the upregulated DEGs were significantly enriched in neuronal function-related pathways such as “synaptic vesicle cycle” and “action potential” ( Figure 3 E), which is highly consistent with the result that the number of neurons in BOMSs is greater ( Figure 2 E).

[0088] Of particular note, the upregulated DEGs in BOMSs were significantly enriched in the “GABAergic synapse” pathway ( Figure 3 E), suggesting that BOMSs may be mainly composed of GABAergic neurons. This conclusion is further supported by the following results: compared with brain organoids, the expression levels of GABAergic neuron marker genes (such as GAD1, GAD2, SLC6A1, GABBR1 and GABBR2) in BOMSs were significantly increased, while the expression of excitatory neuron marker genes (such as NEUROD2, NEUROD6, SLC17A7 and TBR1) was significantly decreased ( Figure 3 F). γ-Aminobutyric acid (GABA) is a specific marker of GABAergic neurons. Immunofluorescence staining results show that GABA is widely present in BOMSs. +neurons, while only small amounts of GABA were observed in the cortical regions of brain organoids + Neuron ( Figure 3 G). Taken together, these results indicate that BOMSs are primarily composed of GABAergic neurons.

[0089] Test Case 3: Functional Analysis of GABAergic Neurons in BOMS

[0090] GABAergic neurons release inhibitory neurotransmitters, namely gamma-aminobutyric acid (GABA), through their synapses, reducing neuronal excitability and maintaining the balance of neural circuits. To evaluate whether GABAergic neurons in BOMSs have good neuronal activity, the inventors performed immunostaining on BOMSs differentiated for 88 days and detected the presynaptic neuron marker SYN1 and the GABAergic neuron marker GAD1 (i.e., GAD67). The results are as follows: Figure 4 shown.

[0091] Figure 4 (A) Immunofluorescence staining of GAD67 (GAD1) ​​and SYN1 in BOMSs. Scale bar: 10 μm. (B) Immunofluorescence staining of astrocytes from brain organoids (BOs) (GFAP and MAP2). Scale bar: 20 μm. (C) Na+ / -β-actin-1 (A) and β-actin-2 (B) in BOMS neurons. + and K + Current results (n = 4). EF) Evoked action potentials in BOMS neurons (n ​​= 4). (GI) Inhibitory postsynaptic potentials (IPSPs). (HI) Quantitative analysis of the amplitude (H) and inter-event interval (I) of IPSPs (n = 4). Data are expressed as mean ± standard deviation (SD).

[0092] GAD1 is a key enzyme that converts glutamate into GABA, and its protein is highly enriched in the synapses of GABAergic neurons. As expected, a large amount of SYN1 was expressed in BOMSs. + synaptic sites, which are associated with GAD1 + Signals highly overlapped ( Figure 4 A), indicating that there are abundant inhibitory synapses in BOMSs. This is consistent with the previous transcriptome analysis results, that is, the "GABAergic synaptic pathway" is significantly enriched in BOMSs ( Figure 3 E).

[0093] Since astrocytes play a key role in the maturation and function of neurons, co-culture with neurons is widely used to promote the maturation of pluripotent stem cell-derived neurons. The inventors isolated and enriched astrocytes from long-term adherent brain organoids. Immunostaining confirmed that these astrocytes highly expressed GFAP and low expressed MAP, and had a typical astrocyte morphology ( Figure 4 B). Subsequently, the inventors co-cultured BOMSs neurons with the above-mentioned astrocytes for three weeks to promote the maturation of neurons for subsequent electrophysiological experiments. Under voltage step stimulation (-40mV to +40mV), BOMSs neurons showed obvious inward Na + Current and outward K + Current( Figure 4 CD), indicating that its sodium and potassium channels are fully functional, providing the necessary physiological basis for the generation of action potentials. Whole-cell patch clamp experiments showed that BOMS neurons can generate action potentials when injected with step current, and the firing frequency is positively correlated with the intensity of the injected current ( Figure 4 EF). In addition, BOMSs neurons also exhibit inhibitory postsynaptic potentials (IPSPs) ( Figure 4 GI), further confirming that GABAergic neurons have established functional synaptic connections through GABA release. In summary, these results indicate that BOMSs neurons can further differentiate into functional GABAergic neurons.

[0094] Test Example 4: Analysis of GABAergic Neuron Subpopulation Composition in BOMSs

[0095] GABAergic neurons can be further divided into multiple subtypes based on their developmental origin, including the MGE (medial ganglion area), LGE (lateral ganglion area), and CGE (caudal ganglion area). The MGE primarily generates cortical interneurons expressing parvalbumin (PV) and somatostatin (SST); the LGE generates olfactory bulb interneurons and striatal projection neurons (MSNs); and the CGE generates cortical interneurons expressing calretinin (CR), vasoactive intestinal peptide (VIP), and reelin.

[0096] To further analyze the composition of GABAergic neuron subpopulations in BOMSs and brain organoids, the inventors performed single-cell RNA sequencing (scRNA-seq) analysis on BOMSs and brain organoids differentiated for 69 days. Figure 5 and Figure 9 shown.

[0097] Figure 5(A) Visualization of cell clusters and cell annotation results in BOMSs and brain organoids (BOs) using Unified Mapping (UMAP) dimensionality reduction analysis. (B) Population proportions of different cell types in each sample. (C) UMAP plot showing the expression distribution of marker genes. (D) Heatmap showing the top 200 differentially expressed genes (DEGs) in each cell type. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs in each cell type. (F) Integrated analysis of single-cell data from BOMSs and BOs with single-cell data from the human ganglionic eminence (GE, gestational weeks 9–18).

[0098] Figure 9 (A) Quality control of scRNA-seq datasets from BOMSs and BOs. Each dot represents a single cell. Cells with fewer than 800 genes per cell (nGene) or a mitochondrial gene percentage exceeding 5% were excluded from subsequent analysis. (B) UMAP visualization of cells from BOMSs and BOs. (C-D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (C) and Gene Ontology (GEN) enrichment analysis of the top 500 differentially expressed genes (DEGs) in the cluster annotated as "other cells." The dashed line indicates the statistically significant enrichment threshold (p < 0.05).

[0099] After quality control ( Figure 9 A) After that, a total of 28,194 cells were obtained and analyzed for transcriptional profiles, including 12,616 cells from BOMSs and 15,578 cells from brain organoids. Through UMAP dimensionality reduction cluster analysis, the inventors identified multiple different cell populations ( Figure 5 A. Figure 9 B), and annotated based on specific marker genes, including: NPC marker genes MKI67, GLI3, VIM; neuron marker genes: DCX, STMN2, BCL11B; excitatory neuron marker genes: NEUROD2, NEUROD6, TBR1; GABAergic neuron marker genes: GAD1, GAD2, SLC6A1 ( Figure 5 AC). BOMSs are mainly composed of GABAergic neurons, while brain organoids are mainly composed of NPCs, excitatory neurons and inhibitory neurons ( Figure 5 A and B). Notably, compared with brain organoids, GABAergic neurons predominated in BOMSs, accounting for 90.3% of the total cell number ( Figure 5 AB), further confirming that BOMSs are mainly composed of GABAergic neurons.

[0100] Subsequently, the inventors performed differentially expressed gene (DGE) analysis to identify genes that are specifically highly expressed in each cell type. The results showed that GABAergic neurons highly expressed a series of key genes related to the development and function of inhibitory neurons, including ERBB4, GAD1, SLC32A1, SP8, DLX5, DLX6, GABRA2 and GABRB1 ( Figure 5 D). The inventors found that a group of cells expressing high levels of cell stress-related genes (such as XBP1, STC2, GADD45B, SERP1, DDIT3, and HERPUD1) were classified as "other cells," suggesting that these cells may be in an unhealthy state and were therefore excluded from subsequent analyses ( Figure 5 D. Figure 9 CD). KEGG pathway enrichment analysis of the top 500 DEGs showed that both GABAergic and excitatory neurons were enriched in neural function-related pathways; NPCs were mainly enriched in cell proliferation-related pathways; GABAergic synaptic pathways were significantly enriched only in GABAergic neurons ( Figure 5 E). In addition, the inventors integrated and compared the scRNA-seq data with the single-cell transcriptome data of the human fetal basal ganglia (GE) region and found that BOMSs and NPCs, GABAergic neurons, and excitatory neurons in brain organoids corresponded well to GE progenitor cells, GABAergic neurons, and excitatory neurons in fetal GE data, respectively. Figure 5 F). This result further verifies the accuracy of the inventors' annotation of cell types.

[0101] Test Example 5: Analysis of BOMSs Identified as LGE / CGE Organoids

[0102] To identify the main subtypes of GABAergic neurons in BOMSs and brain organoids, the inventors performed UMAP cluster analysis on GABAergic neurons from both. Figure 6 (A) shown. Figure 6 (A) Cluster analysis of GABAergic neurons in BOMSs and brain organoids (BOs) using UMAP, and annotation of cell subpopulations based on marker gene expression.

[0103] Figure 6 (A) shows that five independent cell populations were finally identified. Next, the inventors annotated these cell populations based on the marker genes of MGE, LGE, and CGE type GABAergic neurons. PAX6, as a classic LGE / CGE type GABAergic neuron marker gene, is widely expressed in all five cell populations. In contrast, marker genes of MGE type GABAergic neurons (such as NKX2-1, LHX6, LHX8) are not expressed in all cell populations (see Figure 10 A. Figure 10 UMAP visualization shows the expression of genes that represent markers of LGE, CGE, and MGE GABAergic neurons. (A) is a marker for MGE GABAergic neurons. (B) is a marker for LGE GABAergic neurons (differentiation potential of striatal MSNs). (C) is a marker for LGE GABAergic neurons (differentiation potential of olfactory bulb interneurons). (D) is a marker for CGE GABAergic neurons. Each dot represents a single cell, and the color indicates the gene expression level (blue: high expression; gray: low expression). This result indicates that GABAergic neurons in BOMSs and brain organoids are primarily of the LGE / CGE type, rather than the MGE type.

[0104] Further analysis showed that four cell populations were annotated as LGE-type neurons, including LGE-1(MEIS2+,TSHZ1 + )、LGE-2(MEIS2 + ,SIX3 + )、LGE-3(MEIS2 + ,EBF1 + ) and LGE-4(MEIS2 + ,CHD7 + These neuronal subpopulations also co-express several characteristic genes of LGE-type GABAergic neurons, such as PBX3, ERBB4, FOXP2, FOXP1, DLX5, DLX2, GSX2, etc. ( Figure 10 BC). Figure 6 (B) UMAP expression profile of marker genes at the single-cell level. (C) Analysis of the proportion of each GABAergic neuron subtype in different samples.

[0105] The fifth cell group was annotated as CGE-type GABAergic neurons, showing low MEIS2 expression and high expression of CGE marker genes, including NR2F2, CALB2, NFIB, SCGN, PCDH9, etc. ( Figure 6 AB, Figure 10 D) These results indicate that inhibitory neurons in BOMSs and brain organoids are primarily composed of LGE and CGE-type GABAergic neurons.

[0106] It is worth noting that although PAX6 and SOX2 are expressed in all LGE and CGE cell subsets, their expression distributions are significantly different. PAX6 is more highly expressed in LGE-1 and LGE-2, while SOX2 is more strongly expressed in LGE-2, LGE-4, and CGE cell populations ( Figure 6AB). This suggests that PAX6 and SOX2 genes may play an important regulatory role in the fate determination of LGE / CGE type GABAergic neuron subtypes. In addition, the composition of GABAergic neuron subpopulations in BOMSs and brain organoids is significantly different. Brain organoids contain all five clusters, mainly including LGE-2 (28.1%), LGE-3 (25.2%) and LGE-4 (33.8%). BOMSs are mainly composed of LGE-1 (32.9%), LGE-2 (13.9%) and CGE (49.5%), while LGE-4 is extremely rare, accounting for only 0.1% ( Figure 6 C). Based on the above results, the inventors defined BOMSs as LGE / CGE organoids (LCOs).

[0107] Human fetal LGE mainly contains four subpopulations with different differentiation potentials, namely D1 medium spiny neurons (MSNs, TSHZ1 + )、D1 MSNs(EBF1 + )、D2 MSNs(SIX3 + ) and olfactory bulb interneurons. The inventors also identified four LGE-type GABAergic neuron subpopulations in BOMSs and brain organoids ( Figure 6 A). The inventors next tested whether GABAergic neurons in BOMSs and brain organoids could recapitulate the major LGE-type GABAergic neuron subtype in human fetuses.

[0108] Figure 6 (D) Heatmap showing the top 200 differentially expressed DEGs enriched in each GABAergic neuron subtype. (E) Prediction of the differentiation potential of each GABAergic neuron subtype in BOMSs and BOs, based on the results of studies on human fetal GABAergic neurons. (F) Gene Ontology (GO) functional enrichment analysis of DEGs. (G) Integrated analysis of the BOMS single-cell data from this study with single-cell data from human fetal LGE / CGE GABAergic neurons (gestational age weeks 9-18, GW09-18).

[0109] As can be seen from the figure, the inventors performed differentially expressed gene (DGE) analysis to identify genes that are highly expressed in each LGE / CGE type GABAergic neuron subpopulation ( Figure 6 D) Based on the published marker genes of human fetal LGE GABAergic neuron subpopulations, the inventors confirmed that the LGE-type GABAergic neurons in LCOs and brain organoids well reproduced the four major subpopulations of human fetal LGE-type GABAergic neurons ( Figure 6D and E). LGE-1 subpopulation cells highly expressed marker genes such as TSHZ1, FOXP2, PBX3, and ERBB4, showing a tendency towards D1 MSN (TSHZ1 + ) differentiation potential ( Figure 6 B, D, and E, Figure S3B). LGE-2 subpopulation cells highly expressed SIX3, ZFHX4, CXCL12, FOXP1, and PENK, indicating that they have the potential to target D2 MSNs (SIX3 + ) differentiation potential ( Figure 6 B, D and E, Figure 10 B) LGE-3 subpopulation cells highly express EBF1, PDYN, TAC1, ISL1, and ZNF503, indicating their migration to D1 MSN (EBF1 + ) differentiation tendency ( Figure 6 B, D and E, Figure 10 B). LGE-4 subpopulation cells highly express CHD7, DLX5, ARX, DLX2, and GSX2, suggesting that they may differentiate into olfactory bulb interneurons ( Figure 6 B, D and E, Figure 10 C). Consistent with the above results, Gene Ontology (GO) analysis showed that genes highly expressed in LGE-1, LGE-2, and LGE-3 subpopulations were significantly associated with striatal functions, such as motor behavior and pain perception ( Figure 6 F); while genes highly expressed in LGE-4 subpopulation cells are significantly associated with olfactory bulb development and olfactory bulb interneuron differentiation ( Figure 6 F). These results indicate that LGE-type GABAergic neurons in LCOs and brain organoids closely mimic the major subpopulation of LGE-type GABAergic neurons in human fetuses.

[0110] In contrast, CGE-type GABAergic neurons in LCOs and brain organoids highly expressed a series of CGE-type GABAergic neuron markers, including NR2F2, CALB2, VIP, CXCR4, NFIB, PROX1, SCGN, PCDH9, and RELN (Reelin) ( Figure 6 B and D, Figure 10 D), which indicates that they have a + VIP + and Reelin + Differentiation potential of cortical inhibitory neurons ( Figure 6 E). GO analysis further supported this finding, and genes highly expressed in CGE subpopulation cells were significantly associated with the differentiation of GABAergic interneurons in the cerebral cortex ( Figure 6 F).

[0111] To further evaluate the developmental stage of LCOs, the inventors performed a PCA analysis on the developmental trajectory of human fetal LGE / CGE from gestational weeks 9 to 18 (GW09-GW18) reported in the literature. The results showed that the developmental stage of LCOs roughly corresponds to the 12th to 13th gestational weeks (GW12-13) of human LGE / CGE development ( Figure 6 G). Taken together, these findings demonstrate that GABAergic neurons in LCOs and cerebral organoids accurately recapitulate the major subpopulations of human fetal LGE / CGE-type GABAergic neurons. Notably, LCOs are primarily composed of LGE-type GABAergic neurons with striatal differentiation potential and CGE-type GABAergic neurons with cortical interneuron differentiation potential.

[0112] Example 2: Isolation of LGE / CGE GABAergic Neurons

[0113] Since the proportions of CGE-type and LGE-type GABAergic neurons in LCO are roughly equal ( Figure 6 A and C), the inventors selected and optimized methods for enriching these two types of cells. Antibody-based sorting techniques, including magnetic bead cell sorting (MACS) and flow cytometry sorting (FACS), are widely used for cell enrichment. However, there is currently no method specifically for enriching LGE-type or CGE-type GABAergic neurons. To address this problem, the inventors evaluated the feasibility of isolating LGE-type or CGE-type GABAergic neurons from LCO, and the results are as follows. Figure 7 shown.

[0114] Figure 7 (A) Identification of genes encoding cell membrane proteins specifically expressed in LGE or CGE type GABAergic neurons. (B) UMAP dimensionality reduction analysis shows the cell types of the human embryonic ganglionic eminence (GE). Cell population annotation is based on the expression of existing marker genes. (C) UMAP shows the expression distribution of the CXCR4 gene in the human GE region during development. (D) UMAP shows the expression of the CXCR4 gene in LCOs (BOMSs). (E) Schematic diagram showing the enrichment of CXCR4 from LCOs using magnetic bead sorting (MACS) technology. + Method for CGE type GABAergic neurons. (F) Flow cytometry confirmed the expression of CXCR4 protein on the cell surface. (G) Flow cytometry verified the expression of CXCR4- and CXCR4 + (H) shows the effect of cell separation on total LCO cells, CXCR4- subsets and CXCR4 + CXCR4 in subpopulations +The ratio of cells was quantitatively analyzed. Statistical analysis was performed using an unpaired t-test; data are presented as mean ± standard deviation; n = 3; ****p < 0.0001. (I) UMAP analysis of the expression of MEIS2 in LCOs. (J) LCOs, CXCR4- subsets, and CXCR4 + MEIS2 staining results for cell subpopulations. Scale bar: 20 μm. (K) Quantitative analysis of MEIS2 protein levels in different cell populations shown in Figure J. Statistical analysis was performed using an unpaired t-test; red lines indicate the mean; ****p < 0.0001, **p < 0.01.

[0115] The inventors first analyzed the subcellular localization of proteins encoded by differentially expressed genes (DEGs) in each GABAergic neuron subpopulation and identified some proteins specifically expressed on the cell membrane of LGE-type or CGE-type GABAergic neurons ( Figure 7 A). It is noteworthy that the proteins encoded by PTGFR, CXCR4, NTN4, and LHFPL2 genes are mainly expressed on the cell membrane of CGE neurons, while the proteins expressed by genes such as CNTN3, OPN3, UNC5C, and CDH10 are specifically expressed on the cell membrane of LGE GABAergic neurons ( Figure 7 A). Among them, the CXCR4 gene attracted the attention of the inventors because it is expressed in human fetal CGE and MGE type GABAergic neurons ( Figure 7 B and C), and plays a key role in the migration of neurons to specific cortical layers. In addition, the CXCR4 gene is also specifically expressed in CGE-type GABAergic neurons in LCOs ( Figure 7 A and D), and commercial antibodies with good performance are available, making it an ideal target for enriching CGE-type GABAergic neurons from LCOs.

[0116] Next, the inventors used anti-CXCR4 antibody (Clone 12G5) combined with MACS technology to try to enrich CXCR4 from LCOs. + CGE-type GABAergic neurons ( Figure 7 E). As expected, CXCR4 + CXCR4- cells were successfully isolated ( Figure 7 F and G). In CXCR4 + The proportion of CXCR4+ cells in the LCOs group increased significantly to 90.88%, while it decreased significantly to 7.02% in the CXCR4- group. + The proportion of cells was 28.87% ( Figure 7 FH). These results indicate that the inventors have successfully enriched CXCR4 from LCOs. +CGE-type GABAergic neurons.

[0117] Since the number of CGE-type GABAergic neurons is reduced, LGE-type GABAergic neurons are expected to be enriched in the CXCR4- group. As mentioned above, MEIS2 is a classic marker gene for LGE-type GABAergic neurons. In LCOs, it is highly expressed in LGE-type neurons and lowly expressed in CGE-type neurons ( Figure 7 1) In order to verify the above hypothesis, the inventors + Immunofluorescence staining was performed for the levels of MEIS2 protein in the CXCR4-, CXCR4-, and LCO total cell populations. As expected, the level of MEIS2 protein in the CXCR4- group was significantly higher than that in the total cell population, while the CXCR4 + group was significantly lower than the total group ( Figure 7 J, K). These results indicate that LGE-type GABAergic neurons were effectively enriched in the CXCR4- group.

[0118] In combination with the above operations and tests, the inventors provide a method for isolating GABAergic neurons using magnetic bead sorting (MACS), the specific steps of which are as follows:

[0119] M1. Use TrypLE to digest the BOMSs obtained in step S5 of Example 1 into single cells for MACS sorting.

[0120] M2. The cells were resuspended in BOD medium and incubated with Fc receptor blocking solution at room temperature for 10 minutes, and then stained with human CXCR4 antibody (PE-labeled, clone 12G5) on ice for 15 minutes.

[0121] After staining, cells were washed with BOD medium and then stained with MojoSort on ice. TM Incubate with anti-PE nanomagnetic bead antibody for 15 minutes.

[0122] M4, then the cells were loaded into MS sorting columns (Miltenyi Biotec) and MACS sorted using a QuadroMACS sorter. Finally, unsorted cells, CXCR4-positive cells, and CXCR4-negative fraction cells were collected.

[0123] Example 3: Application of LCOs or LGE / CGE type GABAergic neurons

[0124] From the above operations and tests, it can be seen that the inventors have provided a new method for LGE / CGE organoid differentiation. In the absence of additional signal induction, LGE / CGE organoids are spontaneously differentiated from brain organoids, which better simulates the developmental process of the LGE / CGE brain region in vivo, and provides a new human cell research platform for studying the developmental process of LGE / CGE-type GABAergic neurons and their regulatory mechanisms.

[0125] In terms of development, the differentiation pathways of LGE and CGE GABAergic neurons are relatively similar, so what specific signals determine the differentiation fate of the two still needs more research. In addition, the current research on the development of LGE / CGE type neurons is mainly carried out in animal models. Due to species differences, the relevant regulatory mechanisms may be different, so the use of human cell models to carry out relevant research can form an effective complement. Therefore, the inventors provide an application of LGE / CGE organoids in determining the fate of LGE and CGE type GABAergic neurons. The specific operation is to find potential signal pathways or transcription factors that regulate the differentiation fate of LGE and CGE type neurons based on the single-cell sequencing analysis results of LGE / CGE organoids, and then verify and analyze the mechanism by which these signals or transcription factors regulate the differentiation fate of LGE and CGE type neurons by adding inhibitors or activators of the relevant signal pathways to the differentiation culture medium, or knocking out the relevant genes through gene editing, combined with the above-mentioned test and analysis methods.

[0126] The second application provided by the present invention provides a new platform for constructing new human disease models. Huntington's disease is a neurodegenerative disease caused by mutations in the HTT gene. The CRISPR / Cas9 gene editing method is used to construct H9 embryonic stem cells expressing the mutant HTT gene. LGE / CGE organoids (LOCs) expressing wild-type and mutant HTT genes (wherein the wild-type is the initial H9, i.e., the control without gene editing; the mutant refers to the HTT mutant gene introduced into the control H9 using the CRISPR / Cas9 gene editing method) are differentiated and prepared according to the preparation method described in claim 1. The Huntington's model is constructed by analyzing the GABAergic neuron pathological phenotype and detecting neuronal activity in the LOCs.

[0127] From the above tests, it can be seen that the LGE neurons in the LGE / CGE organoids have the potential to differentiate into striatal MSN neurons, and Huntington's disease is a neurodegenerative disease caused by HTT gene mutations. During its development, the striatal MSN neurons are the first to develop lesions. The results of preclinical and clinical trials show that cell transplantation therapy is an effective method for treating Huntington's disease. Therefore, the third application of the present invention is to use a new method for differentiation and enrichment of LGE neurons to prepare a drug with Huntington's disease cell therapy effect. Since the drug adopts cell therapy, it is necessary to dissociate the prepared LCOs into single cells, or to mix the sorted LGE GABAergic neurons with artificial cerebrospinal fluid (aCSF, artificial cerebrospinal fluid) to prepare an injection drug. Huntington's disease is treated by injecting the cell injection into the striatum of the brain.

[0128] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing LGE / CGE organoids (LCOs), characterized in that: The following steps are involved: S1. Embryoid body formation and initial culture: On days 0-4, H9 embryonic stem cells (ESCs) were digested and aggregated to form embryoid bodies (EBs) to initiate early differentiation. S2. Neural induction and Matrigel embedding: On days 5-11, EBs are guided to differentiate toward a neural fate using neural induction medium (NI medium), and Matrigel embedding provides a three-dimensional scaffold to promote the formation of neuroepithelial structures, i.e., early brain organoids. S3. Cerebral organoid culture: On days 12-19, the early-stage cerebral organoids formed after embedding are transferred to a larger space and cultured in NI medium to promote their further development and growth. S4. Dynamic Cultivation and Screening of Brain Organoids: From days 20 to 35, brain organoids are dynamically cultured in BOD medium to simulate the in vivo environment and promote the development of complex brain regions. Qualified brain organoids containing cerebral cortical structures are screened. S5. Differentiation and collection of LGE / CGE organoids (LCOs): After day 35, qualified brain organoids form microspheres (BOMSs) on their surface by budding, which are then collected to obtain the LGE / CGE organoids (LCOs).

2. The preparation method according to claim 1, characterized in that The digestion in step S1 employed EDTA, and the culture medium used on day 0 was Essential 8 supplemented with the ROCK inhibitor Y-27632. The culture medium used on days 1-4 was Essential 8 without Y-27632.

3. The preparation method according to claim 1, characterized in that The components of the neural induction NI medium in step S2 include: DMEM / F12, N-2 supplement, MEM-NEAA and heparin; the EBs are cultured in the NI medium for 5-7 days; the Matrigel embedding is to embed the EBs in NI medium containing 25% Matrigel on days 8-9, and gradually add NI medium; and culture in NI medium on days 9-11.

4. The preparation method according to claim 1, characterized in that The components of the BOD culture medium in step S4 include: DMEM / F12, insulin, GlutaMAX, MEM-NEAA, 2-mercaptoethanol, N-2 supplement and B-27 supplement; the dynamic culture is cultured on a shaker at 37°C and 60 rpm, and half the medium is replaced every 2-3 days.

5. The preparation method according to claim 1, characterized in that The LGE / CGE organoids (LCOs) obtained in step S5 were aggregated by gravity sedimentation and stored in a culture flask containing BOD medium. Culture was continued on a shaker at 60-100 rpm until day 110, with half the medium replaced every 2-3 days.

6. A method for sorting LGE / CGE type GABAergic neurons, characterized in that: The following steps are involved: M1. Prepare single cell suspension: Use TrypLE enzyme to digest and disaggregate the BOMSs collected in step S5 of claim 1 into single cells in preparation for subsequent labeling and sorting; M2, Fc receptor blocking and CXCR4 antibody labeling: resuspend single cells in BOD medium, incubate with Fc receptor blocking solution at room temperature for 5-15 minutes, and then stain with human CXCR4 antibody for 10-20 minutes on ice; M3, magnetic bead binding and washing: After staining, wash the cells with BOD medium and then use MojoSort TM Incubate with anti-PE nanomagnetic beads for 10-20 minutes and wash to remove unbound antibodies and magnetic beads; M4, MACS sorting and fraction collection: CXCR4-positive cells bound to magnetic beads were separated using a Miltenyi Biotec sorting column using a magnetic field, and unsorted cells, CXCR4-positive cells, and CXCR4-negative cell fractions were collected separately.

7. The sorting method according to claim 6, characterized in that: The human CXCR4 in step M2 is PE-labeled and has a clone number of 12G5.

8. A use of LGE / CGE organoids (LCOs) in establishing a Huntington's disease model, characterized in that: The CRISPR / Cas9 gene editing method was used to construct H9 embryonic stem cells expressing the mutant HTT gene. LGE / CGE organoids (LOCs) expressing wild-type and mutant HTT genes were differentiated and prepared according to the preparation method described in claim 1. The GABAergic neuron pathological phenotype and neuronal activity of the LOCs were analyzed to construct a Huntington's disease model.

9. Use of LGE / CGE organoids (LCOs) in the preparation of drugs for nervous system diseases, characterized in that: The drug is prepared by dissociating the LCOs prepared in claim 1 into single cells, or mixing the LGE type GABAergic neurons obtained by sorting in claim 6 with pharmaceutical excipients.

10. The use according to claim 9, characterized in that The neurological disease includes Huntington's disease.

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