A method for culturing organoids derived from human and non-human primate subventricular zone neural stem cells

Abstract

The application belongs to the technical field of regenerative medicine, and discloses a kind of organoid culture method derived from human and non-human primate subventricular zone neural stem cells. By optimizing the medium formula and regulating transcription factors, the organoid is successfully induced to differentiate into CRABP1-expressing neuronal precursors. These cells express TAC3, and the application overcomes the limitations of existing technologies in simulating primate-specific neural lineages and complex brain structures, providing a breakthrough in vitro model for studying neural development, neurological diseases and drug development. The established system not only provides a new tool for studying the development of primate nervous system and disease mechanism, but also provides a reliable experimental platform and transplantable cell source for developing targeted drugs, constructing advanced disease models and conducting cell replacement therapy.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine technology, and more specifically, relates to an organoid culture method derived from neural stem cells in the subventricular region of human and non-human primates. Background Technology

[0002] Neural stem cells (NSCs) are cells with self-renewal and multi-lineage differentiation potential, capable of differentiating into neurons, astrocytes, and oligodendrocytes. In the adult mammalian brain, NSCs are mainly located in two neurogenesis regions: the subgranular area (SGZ) of the dentate gyrus of the hippocampus and the subventricular area (SVZ) of the paraventricular region. Existing research has largely focused on culturing NSCs derived from rodent or human forebrain tissues. For example, cells isolated from forebrain tissue are used to form neurospheres under the influence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2), and subsequently induced to differentiate into neurons or glial cells under conditions such as the addition of brain-derived neurotrophic factor (BDNF). These methods have made some progress in neural stem cell research, providing fundamental tools for in vitro neurogenesis studies.

[0003] However, existing technologies have significant limitations in the construction of organoids from neural stem cells (NSCs) derived from the SVZ in primates, especially adult primates. First, the source of NSCs is not clearly defined. Existing methods mostly use "forebrain tissue" as the source, which includes both the hippocampus and SVZ, making it difficult to distinguish the specific origin of the cells. Although there are reports of hippocampal-derived neurospheres, no literature has yet demonstrated the ability to obtain neurospheres independently from the SVZ of adult primates. This makes it impossible to distinguish whether they possess SVZ-specific neurogenesis potential. Second, existing methods lack resolution in neuronal subtype differentiation. Most existing reports only detect a small number of common neuronal or glial cell markers, without clearly identifying cortical neurons or primate-specific neuronal subtypes. This means that although these methods can induce a broad group of neurons, the lack of representative marker molecules for specific neuronal subtypes prevents further confirmation of whether the differentiation direction is a specific type of neuron, thus limiting their application in simulating higher cognitive functions and primate-specific diseases. Third, the culture systems have insufficient purity and activity. Traditional methods, such as neurosphere culture, often result in reduced purity due to contamination with non-NSC cells (such as fibroblasts or glial cells). There are two traditional methods: adherent culture and suspension culture, each with its advantages and disadvantages. Adherent culture easily introduces non-NSCs like fibroblasts, leading to reduced cell purity; while suspension culture is prone to contamination with red blood cells and impurities in the early stages of culture, and can cause loss of neural stem cells when changing the culture medium. Current reports indicate that the purity of initially isolated NSCs is often below 80%, requiring further purification. These limitations significantly restrict the accuracy of subsequent studies on specific neuronal subtypes, drug screening, and cell therapy, especially when studying specific cell types (such as CRABP1-expressing neuronal precursors).

[0004] The primate SVZ can generate a class of primate-specific CRABP1 neurons. These neurons express a neurokinin, TAC3 (tachykinin precursor 3), which is closely related to neural development and nervous system function. TAC3 is a neurokinin that plays a crucial role in the primate and human nervous systems, regulating neuronal migration and differentiation through the NK3 receptor, especially in the generation of primate-specific inhibitory neurons (such as striatal neurons). Its receptor is expressed in both neural progenitor cells and mature neurons, regulating neuronal migration and differentiation. The primate-specific existence of CRABP1 neurons reflects the complexity and evolutionary characteristics of human brain function. If in vitro culture systems can induce CRABP1-expressing neurons, it will provide an important tool for studying primate-specific neurobiology, the pathology of nervous system diseases, and the mechanisms of neural cell regeneration. CRABP1-specific neurons have multifaceted functions in the nervous system, including reproductive regulation, inhibitory neurotransmission, and possible motor neuron subtype designation, and may also be involved in neural functions such as habit formation, pain perception, and emotion. They are particularly important in primates, reflecting the complexity and evolutionary characteristics of brain function.

[0005] The CRABP1 gene is selectively expressed in neurons of a few brain regions adjacent to the SVZ, such as the striatum, but not in the vicinity of the hippocampal dentate gyrus. This can serve as an important indicator in our SVZ organoid culture process, verifying its SVZ origin. However, there are currently no reports on primate SVZ organoid culture, and no evidence that existing methods can induce the acquisition of CRABP1⁺ neuronal precursor cells in vitro. This directly results in the current system being unable to meet the needs of studying primate-specific neurogenesis mechanisms and disease models. Summary of the Invention

[0006] Based on the aforementioned deficiencies in the existing technology, the present invention first provides an organoid culture method derived from neural stem cells in the subventricular zone (SVZ) of human and non-human primates.

[0007] A second objective of this invention is to provide an application of organoids derived from neural stem cells in the subventricular zone (SVZ) of human and non-human primates.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] An organoid culture method derived from neural stem cells in the subventricular zone (SVZ) of human and non-human primates includes the following steps:

[0010] S1. Tissue Acquisition and Pretreatment: SVZ tissue was obtained from adult monkeys and pretreated for later use.

[0011] S2. Tissue mincing: Remove the meninges, cut the remaining tissue into small pieces, and enzymatically hydrolyze: 0.025% trypsin-EDTA, 37°C for 10 minutes, then enzymatically hydrolyze with a mixture of papain and DNase I at 37°C for 10 minutes.

[0012] S3. Centrifugation and resuspension: After dissociation, add an equal volume of neural stem cell culture medium to the tissue suspension and mix gently. Then centrifuge, remove the supernatant, and add fresh neural stem cell culture medium.

[0013] S4. Cell grinding and filtration: Filtration, seeding the filtrate into pre-treated well plates, adding neural stem cell culture medium to each well, and culturing at 37°C and 5% CO2 for 24 hours.

[0014] S5. Impurity removal and culture: After washing the cells, collect the cells, centrifuge, discard the supernatant, and resuspend them in fresh neural stem cell culture medium for continued suspension culture.

[0015] S6. Neurosphere monoclonalization: Cell growth status is monitored in real time. Cells that proliferate rapidly and can expand upward to form clonal spheres are initially identified as being of neural stem cell origin. When the diameter of the clonal spheres reaches 100-200μm, the neurospheres are digested into single cells, and each single-cell neurosphere is independently seeded into a low-adhesion plate for suspension culture.

[0016] S7. Replace the neural stem cell culture medium with the basic induction medium: containing 1 μM retinoic acid and 5 μM retinoic acid, and culture at 37°C and 5% CO2 for 7-14 days, changing the medium every 2-3 days.

[0017] Several key technical bottlenecks exist in the existing literature regarding the culture of human neural stem cells (NSCs): First, the tissue origin of NSCs is unclear, often being a mixture of human forebrain tissue, including the hippocampal dentate gyrus and the subventricular zone (SVZ), and there is currently a lack of methods for independently isolating and culturing neurospheres from the SVZ region. Second, the types of neurons obtained are unclear; only a few major neuronal markers have been detected, failing to identify cortical neurons or primate-specific neuronal subtypes. Third, traditional culture methods (such as adherent or suspension culture) suffer from low purity; the initially isolated NSCs are often mixed with other cell types (such as fibroblasts and glial cells), affecting the accuracy and reliability of subsequent applications. Fourth, existing reports are mostly based on NSCs derived from the hippocampal dentate gyrus, whose differentiation potential is mainly limited to this brain region itself, lacking the ability to generate cortical neurons.

[0018] To address the aforementioned shortcomings, this invention makes the following breakthroughs:

[0019] 1. Targeted solutions to the problem of unclear cell origin

[0020] This invention directly selects the SVZ region of adult primates (including humans and cynomolgus monkeys) as the tissue source, avoiding the contamination caused by using the "whole forebrain" as material in existing technologies. By combining tissue isolation and cell culture methods, the obtained NSCs are ensured to have a clear and stable source, avoiding interference with the function and differentiation potential of hippocampal cells.

[0021] 2. Optimize the separation and purification process of NSC.

[0022] Building upon the advantages and disadvantages of existing adherent and suspension cultures, this invention proposes a combined strategy of "short-term adherence + low-adhesion amplification." First, short-term adherent culture promotes NSC attachment and removes non-specific cells; then, the purified cells are transferred to low-adhesion conditions for neurosphere amplification. This method effectively removes impurities such as erythrocytes, fibroblasts, and glial cells, improving NSC purity and activity, resulting in more uniform and stable neurosphere formation, and ensuring the reliability and reproducibility of subsequent differentiation induction.

[0023] 3. Establish a primate-specific differentiation direction regulation system.

[0024] By precisely controlling the components of the culture medium, such as growth factors and signaling molecules, this invention has, for the first time, successfully induced inhibitory neuronal precursor cells expressing CRABP1 in vitro, filling a technological gap in this field. These cells are closely related to the TAC3 neuropeptide and can reflect the unique neuronal migration and differentiation characteristics of primates, overcoming the challenge of obtaining primate-specific neural precursors using existing technologies.

[0025] 4. Verify differentiation potential and specific biomarkers

[0026] This invention not only yielded high-purity, highly active NSCs, but also further confirmed that these cells can differentiate into cortical neurons in a neurosphere state, and co-express CRABP1 and cortical neuron-specific markers. This result indicates that the obtained organoids possess a clearly defined subtype differentiation capacity, distinguishing them from existing studies at the "pan-neuronal" level.

[0027] This invention improves the extraction method of neural stem cells by first rapidly adhering to the culture medium to reduce contamination from non-NSC cells, and then cultivating neural spheroids. The neural spheroids of a certain volume are then subjected to stepwise digestion with low-concentration trypsin and papain to reduce enzyme damage to the cells themselves. Finally, cells isolated from individual clonal spheroids are cultured in suspension to form monoclonal neurospheres. This method significantly improves the purity and viability of isolated and purified NSCs. Furthermore, the culture dishes used in this experiment are pretreated with polyornithine and laminin, enabling the isolated adult primate SVZ cells to form organoids and maintain their proliferation and differentiation potential in vitro.

[0028] Specifically, the improvements of this invention over existing cultivation methods are reflected in the following aspects:

[0029] 1. Optimization of Digestive Enzyme Combination: Traditional methods typically use 0.05%-0.25% trypsin-EDTA for prolonged digestion (15-30 minutes), which easily leads to cell damage and decreased viability. We used a screened low concentration of 0.025% trypsin-EDTA for 5 minutes, followed by DNase I (7.5 mg / ml) and papain (40 U / ml) for an additional 10 minutes of digestion. This combined digestion method not only shortens the digestion time but also reduces cell damage and improves cell survival rate and quality by using lower concentrations of trypsin and milder papain.

[0030] 2. Optimization of adhesion culture: Digested cells were seeded in pretreated 24-well plates and cultured at 37°C and 5% CO2 for 24 hours. Healthy NSCs adhered to the plates more quickly. The old culture medium was removed, and the cells were washed with PBS before adding fresh culture medium.

[0031] 3. Isolation of monoclonal cells: After culturing for 7-14 days, proliferating cells are observed and labeled in real time. Fibroblasts (forming a monolayer) and NSCs (forming monoclonal spheres) are distinguished by the direction of cell proliferation. When the clonal spheres reach a certain volume, each clonal sphere is digested into a single cell using the soft digestive enzyme Accumax.

[0032] 4. Low adhesion culture: Digested single cells are seeded into 48-well plates, with each well corresponding to one clone sphere, and labeled and named. This not only ensures that the best clones are selected through subsequent screening based on stemness and differentiation ability, but also obtains multiple high-purity clones.

[0033] Preferably, in the above method, the suspension culture operation in S5 is as follows: the culture medium is changed every two days, and the culture is continued for 7-14 days.

[0034] Preferably, in the above method, the basic induction medium described in S7 further contains 20 ng / mL BDNF and +20 ng / mL NT-3.

[0035] The present invention also provides the application of organoids derived from human and non-human primate subventricular zone (SVZ) neural stem cells obtained by the above method as research models for nervous system development and diseases.

[0036] The present invention also provides the application of organoids derived from human and non-human primate subventricular zone (SVZ) neural stem cells obtained by the above method in the development of drugs for nervous system diseases.

[0037] This invention addresses the technical bottlenecks in existing adult primate neural stem cell (NSC) organoid research, including mixed cell origins, insufficient purity, unclear differentiation lineages, and lack of primate-specific neural precursors. It proposes for the first time a complete, reproducible, and controllable system for the culture and differentiation of organoids derived from the subventricular zone (SVZ) of adult primates. The key inventive technical points of this invention include:

[0038] 1. Construction of a primate SVZ-specific NSC isolation system: This invention strictly selects the SVZ region of adult primates (including humans and cynomolgus monkeys) as the sole tissue source. Combining precise anatomical localization, gentle mechanical dissociation, and combined enzymatic digestion, it ensures that the NSCs have a clear origin and possess the unique lineage potential of the SVZ. The obtained NSCs maintain high activity and primate-specific differentiation direction in subsequent culture, laying the foundation for the subsequent induction of CRABP1 neuronal precursors.

[0039] 2. Combination digestive enzyme optimization strategy to reduce cell damage and improve survival rate: The combination of 0.025% trypsin-EDTA short digestion (5 minutes) with DNase I (7.5 mg / ml) and papain (40 U / ml) for mild digestion not only shortens the digestion time, but also reduces the damage of protein hydrolysis to cell membranes and intercellular connections, significantly improving cell survival rate and purity, reducing the proportion of non-NSC cells in subsequent culture, and improving cloning efficiency.

[0040] 3. A dual-phase culture strategy of short-term adherence enrichment + low-adhesion amplification: Short-term adhesion first promotes rapid attachment of NSCs and removes impurity cells, followed by amplification under low-adhesion conditions to form neurospheres. This achieves a dynamic balance between purification and amplification, solving the problem of difficulty in simultaneously achieving purity and proliferation efficiency in either single adherent or suspension culture. Neurospheres exhibit uniform morphology and well-maintained stemness, significantly improving the stability and controllability of subsequent induced differentiation.

[0041] 4. Single-clone sphere selection and individualized culture system: The proliferation of neurospheres is observed in real time. Single clones with standard morphology are selected and dissociated into single cells using the soft digestive enzyme Accumax. Each single cell is then suspended and cultured in an independent well. This solves the problem of large heterogeneity among clones and difficulty in accurately analyzing differentiation potential caused by traditional population culture. It enables parallel expansion and stemness screening of different clone lines, resulting in multiple high-purity and high-activity neural stem cell lines suitable for subsequent functional verification and drug sensitivity analysis.

[0042] 5. Precise induction of differentiation direction based on transcription factor regulation: By adjusting the composition of the induction culture medium (including 1 μM retinoic acid and 5 μM forscolin) and the microenvironment conditions (37 ℃, 5% CO2), the expression levels of key transcription factors CRABP1 and LHX8 are precisely regulated, guiding NSCs to differentiate into CRABP1⁺ neuronal precursors. For the first time, primate-specific CRABP1⁺ inhibitory neuronal precursors are obtained in vitro, filling a technological gap in this field and providing a specific disease model and drug target validation platform.

[0043] This invention forms a complete process for high-purity isolation, fine culture, and lineage-specific induction of NSCs derived from SVZ in adult primates. It breaks through the limitations of existing technologies in cell source, differentiation lineage, stemness maintenance, and neuronal subtype generation, and provides a novel tool and reusable experimental platform for primate neurobiology, regenerative medicine, and precision drug development.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention marks the first successful isolation and culturing of organoids with three-dimensional structures from SVZs in adult primates (humans and cynomolgus monkeys), filling a gap in in vitro research on neurogenesis in adult primates. Existing techniques primarily rely on rodent embryonic tissues (e.g., G-Peroxin et al., 2013) or human induced pluripotent stem cells (iPSCs, e.g., Lam et al., 2019; Li et al., 2017) to generate neurospheres or organoids. These methods cannot effectively simulate the complex microenvironment of adult primate SVZs, and iPSC-derived organoids tend to resemble the embryonic state, lacking the mature phenotype of adult tissues. This invention, by directly isolating cells from adult primate SVZs to generate organoids, provides a model closer to the biology of the adult human brain, offering a reliable platform for studying the dynamic behavior of neural stem cells (such as proliferation, migration, and differentiation).

[0046] This invention significantly improves the purity, viability, and differentiation efficiency of NSCs through a purification technique involving adhesion followed by suspension, and optimized basal and three-dimensional organoid culture. It enables the long-term maintenance of the self-proliferation and differentiation capacity of SVZ-derived organoids in the adult primate brain, maximally mimicking the growth and development environment of neural progenitor cells in vivo. Simultaneously, it screens for new transcription factors, allowing cultured cells to differentiate into the desired cell types. While existing technologies can induce various types of neurons, they cannot induce primate-specific functional neurons such as CRABP1 neurons.

[0047] The organoids of this invention provide an ideal model for studying neurodegenerative diseases involving SVZ or CRABP1 neurons, such as Alzheimer's disease and Parkinson's disease. Because the organoids are derived directly from adult primate tissue, they can more accurately simulate the pathological state of the adult brain, superior to organoids derived from iPSCs (e.g., Lancaster et al., 2013). Furthermore, organoids can be used to screen drugs targeting neural stem cells or neurons, accelerating the development of treatments for neurodegenerative diseases. In the prior art, rodent models or iPSC organoids have limitations in simulating primate-specific disease mechanisms. The three-dimensional structure of organoids preserves complex intercellular interactions, provides a more realistic drug response testing platform, and offers a more reliable primate model than rodent models, overcoming this deficiency. Attached Figure Description

[0048] Figure 1 A schematic diagram of the SVZ-NSC isolation, culture, and analysis process;

[0049] Figure 2 The process of isolation, culture and subculturing of SVZ-NSC; scale bar, 40 μm;

[0050] Figure 3 For the identification of SVZ-NSC neural stem cells; (AE) Immunofluorescence staining of neurospheres using neural stem cell (NSC) markers (NESTIN, SOX2, HMGB2), neuroblastoma markers (DCX), and proliferation markers (MKI67); (FH) Double immunostaining of neurospheres using MKI67 and NESTIN; white arrows indicate cells co-expressing both markers;

[0051] Figure 4 The SVZ-NSCs exhibit typical neural stem cell differentiation potential; (A) Immunostaining of TAC3 in neurospheres shows partial expression in some cells; white arrows indicate TAC3-positive cells; (BE) Immunofluorescence analysis of lineage-specific markers on neural stem cells from SVZ after differentiation under different induction conditions: early neuron marker TUJ1 (B), mature neuron marker MAP2 (C), astrocyte marker GFAP (D), and oligodendrocyte marker CNP (E); scale bar, 40 μm;

[0052] Figure 5The SVZ-NSCs express the stem cell marker NES, the proliferation marker MKI67, and CRABP1; UMAP visualization of cultured cells, stained by the expression of selected marker genes; NES is a neural progenitor marker; MKI67 is a proliferating cell marker; CRABPP1 is a TAC3 lineage marker; color scales represent log-normalized values ​​of gene expression levels;

[0053] Figure 6 The optimized curve of retinoic acid concentration gradient during differentiation induction and the time curve of CRABP 1% under the final optimized conditions (7, 14, 21 days). Detailed Implementation

[0054] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0055] Example 1: Optimization of the Differentiation Induction System

[0056] I. Acquisition of the neurosphere

[0057] 1. Preparation of Neural Stem Cell Culture Medium: First, prepare 50 mL of basal culture medium, including DMEM / F12, 1X streptomycin, 25 μg / mL insulin, 50 μg / mL transferrin, 1.28 ng / mL progesterone, 16 ng / mL putrescine, and 0.52 μg / mL sodium selenite. Then, add 20 ng / mL recombinant mouse epidermal growth factor (R&D Systems), 20 ng / mL recombinant human bFGF (R&D Systems), 2 µg / mL heparin, 1% N2, 1% B27, and 1X primary cell antibiotic to the basal culture medium. (Store the prepared culture medium at 4°C, preheat to 37°C before use, and ensure aseptic operation throughout the process).

[0058] 2. Tissue Acquisition and Pretreatment: Using sterile scalpels and surgical scissors, brain tissue was harvested from adult monkeys. The SVZ tissue was quickly cut open and placed in pre-cooled Hank's balanced salt solution. The SVZ tissue was rinsed 5 times with Hank's balanced salt solution and placed in a 10cm culture dish containing 10mL of neural stem cell culture medium. It was then cultured for 30 minutes in a 5% CO2, 37°C cell culture incubator.

[0059] 3. Meningeal removal and tissue fragmentation: Under a stereomicroscope, the meninges were removed, and the remaining tissue was cut into small pieces. The tissue was then subjected to the following enzymatic hydrolysis: 0.025% trypsin-EDTA, hydrolyzed at 37°C for 10 minutes; followed by a mixture of papain (40 U / mL) and DNase I (7.5 mg / mL), hydrolyzed at 37°C for 10 minutes. During the hydrolysis process, the tissue was gently shaken every 10 minutes to promote dissociation.

[0060] 4. Centrifugation and resuspension: After dissociation, add an equal volume of neural stem cell culture medium to the tissue suspension and mix gently. Then, centrifuge at 180g / min for 5 minutes, remove the supernatant, and add fresh neural stem cell culture medium.

[0061] 5. Cell grinding and filtration: Gently pipette the tissue using a Pasteur pipette, then filter it through a 70μm cell filter. Seed the filtrate into pretreated (polyornithine + laminin) 24-well plates, add 2mL of neural stem cell culture medium to each well, and incubate at 37°C and 5% CO2 for 24 hours to promote the adhesion of non-NSC cells (such as fibroblasts and glial cells).

[0062] 6. Impurity removal and culture: After washing once with PBS, collect the cells, centrifuge at 180×g for 5 minutes, discard the supernatant, and resuspend in fresh neural stem cell culture medium for continued suspension culture. Change the culture medium every two days and continue culturing for 7-14 days.

[0063] 7. Observation of neurosphere growth: Monitor cell growth status in real time. Cells that proliferate rapidly and can expand upward to form clonal spheres can be preliminarily identified as originating from neural stem cells. When the diameter of the clonal spheres reaches 100-200μm, proceed to the next step of processing.

[0064] 8. Neurosphere monoclonalization: Neurospheres are digested into single cells using a gentle dissociation enzyme (such as Accumax). Each neurosphere is individually seeded into a low-adhesion 48-well plate and numbered for tracking amplification.

[0065] II. Differentiation of the neurosphere

[0066] Induction culture: Replace the NSC medium (neural stem cell medium) with the basic induction medium: DMEM / F12, 1% B27, 1% N2, 20 ng / mL BDNF, 10 ng / mL NT-3, 20 ng / mL GDNF, 0.5 mM dbcAMP, 1 μM retinoic acid (RA) and 5 μM forskolin, 1 x Penicillin / Streptomycin, and culture at 37°C and 5% CO2 for 7-14 days, changing the medium every 2-3 days.

[0067] The optimization process used the percentage of CRABP1-positive cells (CRABP1%) as the primary quantitative indicator, combined with cell viability and morphological observation for comprehensive evaluation. All experiments were conducted at 7, 14, and 21 days to ensure dynamic tracking of the induction effect. First, the retinoic acid concentration gradient was optimized. In the preliminary experiment, we set 0, 1, 3, and 5 μM retinoic acid concentrations as the initial screening range. At 7 days, all three groups of cells survived normally; at 14 days, the 1 μM group showed CRABP1+ (approximately 20%); at 21 days, the 1 μM group maintained stable cell counts, while the ≥3 μM group showed significant apoptosis. Therefore, in the formal experiment, we used 0.5-2 μM as a finer gradient range for further comparison, ultimately confirming 1 μM as the optimal concentration.

[0068] Next, we compared cAMP pathway agonists. Based on the optimal concentration of retinoic acid (1 μM), we compared three common cAMP pathway agonists: Forskolin, 8-Br-cAMP, and IBNX. The results showed that forskolin (5 μM) could increase CRABP1+ to the highest level (approximately 20%), 8% under 8-Br-cAMP, and 5% under IBNX. Forskolin was superior to other candidate drugs in maintaining cell morphology and growth status.

[0069] Finally, we explored the supplementation of neurotrophic factors. Under the basal conditions of retinoic acid (1 μM) + forscoringin (5 μM), we added BDNF (20 ng / mL), GDNF (10 ng / mL), NT-3 (20 ng / mL), and combinations thereof. We found that adding any one of these factors individually increased CRABP1+ (approximately 5-8%). The combined addition of BDNF and NT-3 showed the best effect (approximately 25%), and appropriate amounts of neurotrophic factors could further improve induction efficiency. Based on the above optimization experiments, we finally determined the optimal induction system to be 1 μM retinoic acid + 5 μM forscoringin, at 37 ℃, 5% CO2, and 20% O2, with optional supplementation of BDNF (20 ng / mL) + NT-3 (20 ng / mL) to further enhance efficiency. In adult primate SVZ organoids, CRABP1% stably reached approximately 25-30% after 14 days, and cell viability remained at 75-80%, providing a reliable basis for subsequent applications.

[0070] Example 2: In vitro induction to obtain CRABP1⁺ neuronal precursor cells

[0071] I. Acquisition of the neurosphere

[0072] 1. Preparation of Neural Stem Cell Culture Medium: First, prepare 50 mL of basal culture medium, including DMEM / F12, 1X streptomycin, 25 μg / mL insulin, 50 μg / mL transferrin, 1.28 ng / mL progesterone, 16 ng / mL putrescine, and 0.52 μg / mL sodium selenite. Then, add 20 ng / mL recombinant mouse epidermal growth factor (R&D Systems), 20 ng / mL recombinant human bFGF (R&D Systems), 2 µg / mL heparin, 1% N2, 1% B27, and 1X primary cell antibiotic to the basal culture medium. (Store the prepared culture medium at 4°C, preheat to 37°C before use, and ensure aseptic operation throughout the process).

[0073] 2. Tissue Acquisition and Pretreatment: Using sterile scalpels and surgical scissors, brain tissue was harvested from adult monkeys. The SVZ tissue was quickly cut open and placed in pre-cooled Hank's balanced salt solution. The SVZ tissue was rinsed 5 times with Hank's balanced salt solution and placed in a 10cm culture dish containing 10mL of neural stem cell culture medium. It was then cultured for 30 minutes in a 5% CO2, 37°C cell culture incubator.

[0074] 3. Meningeal removal and tissue fragmentation: Under a stereomicroscope, the meninges were removed, and the remaining tissue was cut into small pieces. The tissue was then subjected to the following enzymatic hydrolysis: 0.025% trypsin-EDTA, hydrolyzed at 37°C for 10 minutes; followed by a mixture of papain (40 U / mL) and DNase I (7.5 mg / mL), hydrolyzed at 37°C for 10 minutes. During the hydrolysis process, the tissue was gently shaken every 10 minutes to promote dissociation.

[0075] 4. Centrifugation and resuspension: After dissociation, add an equal volume of neural stem cell culture medium to the tissue suspension and mix gently. Then, centrifuge at 180g / min for 5 minutes, remove the supernatant, and add fresh neural stem cell culture medium.

[0076] 5. Cell grinding and filtration: Gently pipette the tissue using a Pasteur pipette, then filter it through a 70μm cell filter. Seed the filtrate into pretreated (polyornithine + laminin) 24-well plates, add 2mL of neural stem cell culture medium to each well, and incubate at 37°C and 5% CO2 for 24 hours to promote the adhesion of non-NSC cells (such as fibroblasts and glial cells).

[0077] 6. Impurity removal and culture: After washing once with PBS, collect the cells, centrifuge at 180×g for 5 minutes, discard the supernatant, and resuspend in fresh neural stem cell culture medium for continued suspension culture. Change the culture medium every two days and continue culturing for 7-14 days.

[0078] 7. Observation of neurosphere growth: Monitor cell growth status in real time. Cells that proliferate rapidly and can expand upward to form clonal spheres can be preliminarily identified as originating from neural stem cells. When the diameter of the clonal spheres reaches 100-200μm, proceed to the next step of processing.

[0079] 8. Neurosphere monoclonalization: Neurospheres are digested into single cells using a gentle dissociation enzyme (such as Accumax). Each neurosphere is individually seeded into a low-adhesion 48-well plate and numbered for tracking amplification.

[0080] II. Differentiation of the neurosphere

[0081] Induction culture: Replace the NSC medium (neural stem cell medium) with the basic induction medium: DMEM / F12, 1% B27, 1% N2, 20 ng / mL BDNF, 10 ng / mL NT-3, 20 ng / mL GDNF, 0.5 mM dbcAMP, 1 μM retinoic acid (RA) and 5 μM forskolin, 1 x Penicillin / Streptomycin, and culture at 37°C and 5% CO2 for 7-14 days, changing the medium every 2-3 days.

[0082] Fixation: Remove the differentiation-matched sampler from the 48-well plate. Gently wash the cells twice with 1 ml of PBS (pH 7.4) for 5 minutes each time, using 1 ml of PBS per well. After washing, add 1 ml of 4% PFA to each well and fix at room temperature for 30 minutes. Wash three times with 1 ml of PBS for 5 minutes each time to remove the fixative.

[0083] Permeation: Add 1 ml of 0.1% Triton X-100 (dissolved in PBS) to each well and incubate at room temperature for 10-15 minutes to allow permeation through the cell membrane. Wash three times with PBS for five minutes each time to remove the permeabilizing agent.

[0084] Blocking: Prepare blocking buffer: dissolve 5% normal goat serum (donkey) or 5% BSA in PBS. Add 1 ml of blocking buffer to each well and incubate at room temperature for 30-60 minutes to prevent non-specific antibody binding.

[0085] Primary antibody incubation: Prepare the primary antibody mixture according to the manufacturer's instructions, selecting antibody combinations that avoid species cross-linking (e.g., mouse antibody for Tuj1, rabbit antibody for MAP2). Add 500 μl of monoclonal antibody mixture to each well, covering the cell surface. Place the six wells in a humidified chamber and incubate overnight at 4°C (or at room temperature for 2 hours). Wash three times with 1 ml PBS for 5 minutes each time to remove unbound primary antibody.

[0086] Secondary antibody incubation: Prepare secondary antibody mixtures (Alexa Fluor 488-labeled goat anti-mouse IgG (for Tuj1 and GFAP): 1:500-1:1000; Alexa Fluor 594-labeled goat anti-rabbit IgG (for MAP2 and S100β): 1:500-1:1000; Alexa Fluor 647-labeled goat anti-rabbit IgG (for CRABP1): 1:500-1:1000). Add 500 μl of the secondary antibody mixture to each well and incubate at room temperature in the dark for 1 hour. Wash three times with 1 ml PBS for 5 minutes each time to remove unbound secondary antibodies. Prepare 1 ml of DAPI solution (1:1000 diluted in PBS) and incubate at room temperature for 5-10 minutes. Wash twice with PBS for 5 minutes each time.

[0087] III. Results Observation

[0088] Mounting and imaging: Use Fluoromount-G to mount the slides. Add 50–100 μL to each well and cover with a 22×22 mm coverslip. Cure at room temperature in the dark for 1 hour.

[0089] When using confocal microscopy imaging, set the following excitation / emission wavelengths:

[0090] Tuj1, S100β: 488 / 500–550nm; MAP2, GFAP: 594 / 600–650nm; CRABP1: 647 / 660–730nm; DAPI: 358 / 461nm.

[0091] Data analysis: Image analysis software was used to quantify the proportion of positive cells and record the distribution and intensity of specific marker expression.

[0092] Under these conditions, the synergistic effect of small molecule signaling pathways significantly activated the expression of transcription factors CRABP1 and LHX8, thereby achieving precise regulation of differentiation direction at the molecular level. Ultimately, inhibitory neuronal precursors expressing CRABP1⁺ were successfully induced, and their origin and differentiation potential were confirmed through molecular marker detection and functional validation. This approach not only outperforms other alternative conditions mentioned above but also fills the gap in existing technologies that cannot induce primate-specific CRABP1 neuronal precursors.

Claims

1. A method for culturing organoids derived from Cynomolgus monkey subventricular zone neural stem cells, characterized by, The method comprises the following steps: S1, tissue acquisition and pretreatment: SVZ tissue is obtained from adult monkeys and pretreated for use; S2, tissue chopping: the dura mater is peeled off, and the remaining tissue is chopped into small pieces, and then enzymolysis is performed: 0.025% trypsin-EDTA is used for enzymolysis at 37°C for 10 minutes, and then a mixed solution of papain and DNase I is used for enzymolysis at 37°C for 10 minutes; S3, centrifugation and resuspension: after dissociation, an equal volume of neural stem cell culture medium is added to the tissue suspension, and then centrifuged, the supernatant is removed, and fresh neural stem cell culture medium is added; S4, cell grinding and filtration: filtration, and then the filtrate is inoculated into a pre-treated well plate, and neural stem cell culture medium is added to each well, and then cultured at 37°C and 5% CO2 for 24 hours; S5, impurity removal and culture: after washing the cells, the cells are collected, centrifuged, the supernatant is discarded, and the cells are resuspended in fresh neural stem cell culture medium for suspension culture; S6, neural sphere monoclonalization: the growth state of the cells is monitored in real time, and the cells that proliferate rapidly and can expand upward to form a clonal sphere are preliminarily judged to be derived from neural stem cells, and when the diameter of the clonal sphere reaches 100-200 μm, the neural sphere is digested into single cells, and each single cell neural sphere is inoculated into a low-adhesion well plate for suspension culture; S7, the neural stem cell culture medium is replaced with a basic induction culture medium: containing 1 μM retinoic acid and 5 μM, and cultured at 37°C and 5% CO2 for 7-14 days, and the culture medium is replaced every 2-3 days.

2. The method of claim 1, wherein the organoid culture of cerebrum sub-ventricular zone neural stem cells of Cercopithecus aethiops is characterized by, The operation of the suspension culture in S5 is: the culture medium is replaced every two days, and the culture is continued for 7-14 days.

3. The method of claim 1, wherein the organoid culture derived from the subventricular zone neural stem cells of the Cynomolgus monkey is characterized by, The basic induction culture medium in S7 further contains 20 ng / mL BDNF and +20 ng / mL NT-3.

4. Application of the organoids derived from the subventricular zone neural stem cells of Macaca fascicularis obtained by the method of any one of claims 1 to 3 as a research model of nervous system development and diseases.

5. Application of the organoids derived from the subventricular zone neural stem cells of Macaca fascicularis obtained by the method of any one of claims 1 to 3 in the development of drugs for nervous system diseases.

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