Use of brain organoid model for study of simulating brain development damage caused by environmental pollutants in embryonic period

Abstract

The present invention relates to the technical field of environmental exposure prevention and treatment. Disclosed is a use of a brain organoid model for a study of simulating brain development damage caused by environmental pollutants in an embryonic period. The brain organoid model at least comprises progenitor cells (SOX2+cells), neurons (MAP2+cells), deep-layer neurons (TBR1+cells) and immature neurons (TUJ1+cells). The simulated exposure of a brain organoid to environmental pollutants constructed in the present invention and subsequent mechanism exploration and therapeutic intervention studies are a promising method for studying the impact of environmental pollutants on human health. Compared with a traditional toxicology testing method, the technology has multiple advantages, helping researchers to better understand the impact of environmental related diseases on human health, and facilitating developing new strategies for preventing and treating these diseases.

Description

Application of a brain organoid model for simulating damage to brain development caused by environmental pollutants during embryonic period Technical Field

[0001] The present invention belongs to the technical field of environmental exposure prevention and treatment, and specifically relates to the application of a brain organoid model for simulating the damage of environmental pollutants to brain development during the embryonic period. Background Art

[0002] Studying the effects of environmental exposures on the human body relies heavily on technology, from monitoring devices that measure air and water quality to sophisticated imaging techniques that enable researchers to visualize the effects of environmental toxins on tissues and organs. Despite ongoing technological advances, several challenges remain in studying the effects of environmental exposures on the human body. These include: Limitations of biomonitoring: While biomonitoring can provide valuable information about exposure levels, it does not always provide a clear link between exposure and health outcomes. This is because different people may respond differently to the same level of exposure, depending on factors such as age, genetics, and overall health. Limited availability of environmental sensors: While environmental sensors can provide real-time data on pollutant levels, they are not widely available or affordable to researchers. This may limit their use in areas with high levels of pollution. Ethical considerations: Some techniques used to study the effects of environmental exposures on the human body, such as exposing people to environmental toxins, raise ethical concerns. Ensuring that research is conducted ethically and responsibly is crucial. Lack of longitudinal studies: Many studies on the effects of environmental exposures on the human body are cross-sectional, meaning they provide feedback on exposure and health outcomes at only a single point in time. Longitudinal studies that track individuals over time can provide more information about the long-term effects of environmental exposures. Difficulty attributing health outcomes to environmental exposures: Attributing specific health outcomes to environmental exposures can be challenging because many factors can influence health. This can make it difficult to identify causal relationships and develop effective interventions to reduce exposure and prevent adverse health effects. Overall, while technology has advanced research into the effects of environmental exposures on the human body, several challenges and limitations remain that need to be addressed. Further research is needed to develop new technologies and methods to better understand the complex relationships between environmental exposures and human health. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the present invention provides an application of a brain organoid model for simulating the damage of environmental pollutants to brain development during the embryonic period. The disease model of the present invention is more accurate and predictive, has lower cost, and requires less animal testing. It can be grown in large quantities and screened using high-throughput methods, allowing researchers to quickly and effectively test the effects of various environmental toxins on organoid behavior.

[0004] In order to achieve the above object, the technical solution adopted by the present invention includes:

[0005] In a first aspect, the present invention provides an application of a brain organoid model for simulating the damage of environmental pollutants to brain development during the embryonic period.

[0006] Furthermore, in the above technical solution, the brain organoid model has a cerebral cortex structure, and the brain organoid model at least includes: histocytes: SOX2 + Cells, neurons: MAP2 + Cells, deep neurons: TBR1 + cells and immature neurons: TUJ1 + cell.

[0007] Furthermore, in the above technical solution, the embryonic period is the human embryonic period.

[0008] Furthermore, in the above technical solution, the environmental pollutants include pollutants generated by production, construction or activities that are harmful to life or the ecological environment.

[0009] Furthermore, in the above technical solution, the environmental pollutants include: endocrine disrupting chemicals (EDCs), pharmaceuticals and personal care products (PPCPs), perfluorinated compounds (PFCs), brominated flame retardants (BRPs), and drinking water disinfection by-products (DBPs).

[0010] Furthermore, in the above technical solution, the method for constructing the brain organoid model includes: culturing human embryonic stem cells in a serum-free human embryonic stem cell culture medium to form embryoid bodies; then adding a ROCK inhibitor, and after a certain period of differentiation, the embryoid bodies are differentiated into cortical organoids; then changing to a neural differentiation culture medium without vitamin A for culturing.

[0011] Furthermore, in the above technical solution, the human embryonic stem cells are human embryonic stem cell line H9.

[0012] Furthermore, in the above technical solution, the ROCK inhibitor is Y27632.

[0013] Furthermore, in the above technical solution, the concentration of the added ROCK inhibitor is 50-60 μM.

[0014] Furthermore, in the above technical solution, the certain differentiation time is 10-12 days.

[0015] The present invention mainly relates to methods for exposure to environmental pollutants and selection of damage indicators, including:

[0016] a) Expose pollutants to brain organoid models (exposure concentrations can be predicted based on pollutant levels in air and water, allowing for the establishment of concentration gradients to investigate the threshold concentrations that cause damage to brain development);

[0017] b) Characterize apoptosis markers in brain organoid growth to determine whether environmental exposures affect the growth and development of various cells in the developing brain;

[0018] c) Morphological characterization of whether the brain organoids are damaged as a whole, and preliminary estimation of whether environmental pollutants have an impact on brain structure;

[0019] d) Detecting damage to key cell groups during brain organoid growth using neural and glial cell markers to identify specific cell populations and determine whether environmental pollutants have potential impacts on brain development;

[0020] e) High-throughput sequencing to explore the mechanisms of damage. For example, using transcriptome sequencing to identify differentially expressed genes and their enrichment in specific pathways provides a starting point for understanding the underlying mechanisms of damage. Further examination of potential sites that may be found in these key genes will allow for the development of agonists and inhibitors of these potential targets to reverse the pathological modulation of environmental pollutants.

[0021] Beneficial effects:

[0022] The present invention found that the brain organoid model constructed by the present invention is more suitable for studying the effects of environmental pollutants on embryonic brain development than animal models. The brain organoid model is very similar to the cell clusters in the developing cerebral cortex, which is believed to play a key role in evolving the size and complexity of the human cortex. Unlike rodent models, the human embryonic cerebral cortex contains a large number of specialized outer radial glial cells (oRGCs) in the outer subventricular zone (oSVZ). In brain organoids, this key feature of human brain development can be replicated. Brain organoids reproduce the developmental process of neuronal marker expression in the six cortical layers, which show a developmental trajectory similar to that of the human cortex. Compared with traditional research methods, this disease model helps researchers better understand the impact of environmental toxins on human health, and through this research result, corresponding diagnosis and treatment methods can be formulated.

[0023] The present invention utilizes a new paradigm of brain organoid models to simulate human embryonic exposure to environmental exposure substances, which has several advantages. The latest technologies used in brain organoid exposure research include the culture and maturation of brain organoids, the selection of exposure substances for simulated exposure (high throughput), characterization using advanced imaging technology, and high-throughput sequencing screening methods. This new paradigm of brain organoids simulating environmental exposure is more accurate and predictive; it is less expensive and requires less animal testing; it can be grown in large quantities and screened using high-throughput methods, allowing researchers to quickly and effectively test the effects of multiple environmental toxins on organoid behavior. In short, the brain organoid-based exposure model is the most advanced method for studying the effects of environmental toxins on human tissues and organs. Compared with traditional methods, it has several advantages, including higher accuracy and predictability, lower costs, high-throughput screening, and personalized medical approaches.

[0024] 1. Research results:

[0025] 1) More Accurate and Predictive: Brain organoids are three-dimensional structures that closely mimic the structure and function of the embryonic human brain. Their cell population structure and overall function more closely resemble the human brain than rodents. Simulating exposure of organoids to environmental toxins can provide a more accurate and predictive model of the effects of these toxins on human tissues and organs than traditional two-dimensional cell cultures.

[0026] 2) High-throughput screening: Organoids can be grown in large quantities and screened using high-throughput methods, allowing researchers to quickly and efficiently test the effects of multiple environmental toxins on organoid behavior.

[0027] 3) Personalized medicine: Organoids can be derived from patient-specific stem cells, allowing a personalized medicine approach to study the effects of environmental toxins on human tissues and organs.

[0028] 4) Improve reproducibility: Organoid-based testing can improve reproducibility by controlling environmental factors and batches. Traditional toxicology testing methods may be affected by factors such as differences between individual animals.

[0029] 5) Improved understanding of disease mechanisms: Organoids can be used to study the impact of environmental toxins on the development and progression of disease. By exposing organoids to environmental toxins, researchers can gain deeper insights into the underlying mechanisms of disease development through methods such as genetic sequencing and identify potential targets for therapeutic intervention.

[0030] 6) Development of New Therapies: Organoids can be used to test the efficacy and safety of new drugs and treatments for environmentally related diseases. By using organoids to screen potential drug candidates, researchers can identify the most promising treatments for further testing and development.

[0031] 2. Cost reduction:

[0032] 1) Less need for animal testing: Organoid-based assays can be cheaper than traditional animal models used to study the effects of environmental toxins. This can also reduce the need for animal testing, which is a major ethical concern.

[0033] 2) Reduce time and resources: Simulating material exposure with organoids can significantly reduce the time and resources required for toxicology testing. Traditional toxicology testing methods are time-consuming and expensive, while organoid-based assays can quickly and efficiently screen for environmental toxins.

[0034] 3. Ethical aspects:

[0035] 1) Safer for researchers: Traditional methods for studying the effects of environmental toxins on human tissues and organs can be harmful to researchers. Organoids are grown outside the body, reducing the risk of exposure to toxic substances.

[0036] 2) Reduce or even replace animal experiments, reducing the need for animal testing.

[0037] 3) Brain organoids can simulate the process of brain development during embryonic development, detect the effects of environmental exposure intervention on brain development, and study its mechanisms, which cannot be carried out in real experiments.

[0038] Overall, modeling environmental exposures in brain organoids is a promising approach for studying the impacts of environmental toxins on human health. This technique offers several advantages over traditional toxicology testing methods and has the potential to improve our understanding of environmentally related diseases and contribute to the development of new strategies for their prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG1 is an embodiment of the present invention.

[0040] Figure 2 shows a white-light image of a brain organoid under a microscope according to one embodiment of the present invention. The white-light image shows that the organoid is in good and stable growth. It also shows an image of the organoid after being treated with environmental pollutants, which directly demonstrates the impact of environmental pollutants on the growth and development of the brain organoid.

[0041] Figure 3 shows the frozen sections of the cultured organoids and the immunofluorescence experiments. As shown in the figure, brain organoids include at least histocytes (SOX2 + cells), neurons (MAP2 + cells), deep neurons (TBR1 + cells) and immature neurons (TUJ1 + cells) that is, it has a relatively complete cerebral cortex structure, forming a "miniature landscape" of brain development similar to the embryonic period.

[0042] FIG4 shows an embodiment of the present invention showing immunofluorescence staining of frozen sections of brain organoids after environmental pollutant intervention, including growth and apoptosis-related markers. These indicators characterize the levels of cell growth and apoptosis during the growth and development of brain organoids;

[0043] Figure 5 shows gene expression at the transcriptome level;

[0044] Figure 6 shows the differentially enriched gene pathways. DETAILED DESCRIPTION

[0045] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0046] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Instead, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0047] The human embryonic stem cells described in the embodiments of the present invention are the human embryonic stem cell line H9. The H9 cell line used in the present invention is an established, mature, and commercialized cell line. The present invention does not involve the steps of obtaining embryonic stem cells from human embryos or blastocysts, nor does it involve the use of human embryos for industrial or commercial purposes.

[0048] The brain organoid model constructed using the present invention is used to simulate the damage of environmental pollutants to brain development during the embryonic period. The results obtained are only used as intermediate results for preventing or treating diseases. The brain organoid model of the present invention can only be used to evaluate the impact of environmental pollutants on the embryonic brain, and is not used as a basis for diagnosing and treating certain diseases.

[0049] Example 1 Cultivation and Preparation of Brain Organoid Models

[0050] When the specific experimental temperature is not specified in the examples of the present invention, the experimental temperature is room temperature (20-25°C). The sources of the reagents used in the examples of the present invention are as follows:

[0051] DMEM / F12 medium was purchased from GIBCO, USA, Neurobasal medium was purchased from GIBCO, USA, cell culture additive Glutamax was purchased from Thermo Fisher Scientific, USA, non-essential amino acids NEAAs were purchased from Thermo Fisher Scientific, USA, Rho-associated protein kinase (ROCK) inhibitor Y27632 was purchased from Stem Cell Technologies, Canada, cell culture additive B-27 without vitamin A was purchased from ThermoFisher Scientific, USA, penicillin-streptomycin was purchased from SIGMA, USA, and insulin was purchased from MedChemExpress, USA.

[0052] The culture medium used in the embodiments of the present invention was prepared independently, and its components included: 0.48 parts by volume of DMEM / F12 medium, 0.48 parts by volume of Neurobasal medium, 0.01 parts by volume of cell culture additive Glutamax, 0.01 parts by volume of non-essential amino acids NEAAs, 0.01 parts by volume of cell culture additive B27, 0.01 parts by volume of penicillin-streptomycin, and 0.0025 parts by volume of insulin.

[0053] Human embryonic stem cell line H9 was seeded on a cell culture plate coated with Matrigel (Corning, New York, USA) and incubated at 37°C in 5% CO2, passaged once every four days, and cultured in mTeSR plus serum-free medium. To induce neuroectoderm derivation in a 3D culture system, human embryonic stem cell line H9 was cultured at 9×10 3 The cells were plated at a density of 10 cells / well in an ultra-low juxtaposed 96-well plate to form embryoid bodies (EBs). A high concentration (50 μM) of ROCK inhibitor Y27632 was added to promote the formation of embryoid body EBs. The culture medium was changed every other day until the 10th day of differentiation. After inducing neural differentiation through 3D culture during this period, EBs were allowed to differentiate into cortical organoids (HCOs), which can mimic the cortical tissue of the embryonic brain. On the 10th day, a 5 mL pipette was used to transfer the EBs from the ultra-low attachment 96-well plate to an ultra-low attachment 6-well plate, and the culture medium was replaced with a neural differentiation medium without vitamin A. On the 18th day, neural differentiation medium was used for neural maturation and long-term culture. A brain organoid model was constructed.

[0054] The brain organoid model obtained by the above culture was frozen and sliced ​​and subjected to immunofluorescence experiment. As shown in Figure 3, the brain organoid model includes at least histocytes (SOX2 + cells), neurons (MAP2 +cells), deep neurons (TBR1 + cells) and immature neurons (TUJ1 + cells), that is, it has a relatively complete cerebral cortex structure and is histologically closer to natural brain tissue than animal models, forming a good in vitro disease model.

[0055] Example 2 Effects of Environmental Pollutants on Brain Organoid Models

[0056] In order to verify that the brain organoid model constructed in Example 1 can provide a reliable model for studying environmental pollutants, the present invention simulates the exposure environment by adding environmental pollutants (taking PM2.5 as an example) to the brain organoid model culture medium, studies the inhibitory effects of environmental pollutants on the brain organoid model, and detects the proliferation and apoptosis levels at the cellular level.

[0057] First, the present invention used immunofluorescence to analyze the expression of the proliferation marker Ki67. The untreated control showed significant expression of the proliferation marker Ki67. However, after intervention with environmental pollutants, Ki67 expression was observed to be suppressed in brain organoid cells, as shown in Figure 4. In addition, TUNEL staining analysis was performed to confirm the inhibitory effect of environmental pollutants on the brain organoid model. The results showed that environmental pollutants significantly inhibited the growth of brain organoids at the cellular level. To investigate the mechanism by which environmental pollutants affect human cortical brain organoids, transcriptional analysis was performed on brain organoids before and after exposure to different concentrations of environmental pollutants (Figures 5 and 6). Figure 5 shows that a large number of genes were downregulated after intervention with environmental pollutants. Figure 6 shows that some growth and development-related pathways were significantly downregulated after intervention with environmental pollutants, again demonstrating the impact of environmental pollutant nanoplastics on brain organoids. Sequencing results showed that a large number of genes were regulated at least 2-fold in brain organoids. This difference indicates that the duration of treatment has a significant impact on the gene expression profile within the organoids. After intervention with environmental pollutants, genes regulating cell proliferation were significantly downregulated in brain organoids.

[0058] Example 3 High-throughput screening of drugs that reverse pathological regulation of environmental pollutants

[0059] Based on the above-mentioned use of transcriptome sequencing technology to identify differentially expressed genes and the enrichment of these genes in specific pathways, we further examined the key genes in these pathways and conducted high-throughput screening of agonists and inhibitors of corresponding potential targets to develop drugs that reverse the pathological regulation of environmental pollutants.

[0060] Brain organoids for drug screening are mixed with culture medium corresponding to the specific exposure period (in this case, exposure mimicking the maturation phase, using the maturation medium formulation described above). The mixture is then seeded into multiple 96-well culture plates (depending on the number of drugs being screened) using a multichannel pipette or serial pipette, ensuring that the volume of the organoid and culture medium mixture in each well is equal. Test drugs (agonists or inhibitors of target genes, or other potential therapeutic targets. For example, the WNT pathway, the agonist SKL2001 and the inhibitor LF3 of β-Catenin, the most significantly downregulated gene, could be used as target drugs) are added to the 96-well plate, with one drug per well. Three to five replicate wells are set up for each drug. A blank control, a control group treated with environmental pollutants without drug (either no substance or the same volume of drug solvent as in the test wells), and a control group treated with environmental pollutants plus drug are also included. After two weeks of incubation, the organoids are observed under light microscopy and evaluated by immunofluorescence to identify the optimal drug for blocking environmental pollutant-induced brain developmental pathology. This method can achieve high-throughput screening of target drugs, so as to find therapeutic drugs more quickly and reduce the impact of environmental pollutants on human health.

[0061] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. Application of a brain organoid model for simulating the damage of environmental pollutants to brain development during the embryonic period.

2. The use according to claim 1, characterized in that: The brain organoid model has a cerebral cortex structure, and the brain organoid model at least includes: histocytes: SOX2 + Cells, neurons: MAP2 + Cells, deep neurons: TBR1 + Cells and immature neurons: TUJ1 + cell.

3. The use according to claim 1, characterized in that: The embryonic period is the human embryonic period.

4. The use according to claim 1, characterized in that: The environmental pollutants include pollutants generated by production, construction or activities that endanger life or the ecological environment.

5. The use according to claim 4, characterized in that: The environmental pollutants include: endocrine disruptors, pharmaceuticals and personal care products, perfluorinated compounds, brominated flame retardants, and drinking water disinfection by-products.

6. The use according to any one of claims 1 to 4, characterized in that: The method for constructing the brain organoid model comprises: culturing human embryonic stem cells in a serum-free human embryonic stem cell culture medium to form embryoid bodies; then adding a ROCK inhibitor, and after a certain period of differentiation, the embryoid bodies are differentiated into cortical organoids; and then replacing the culture medium with a neural differentiation medium without vitamin A to obtain the model.

7. The use according to claim 6, characterized in that: The human embryonic stem cells are human embryonic stem cell line H9.

8. The use according to claim 6, characterized in that: The ROCK inhibitor is Y27632.

9. The use according to claim 6, characterized in that: The ROCK inhibitor was added at a concentration of 50-60 μM.

10. The use according to claim 6, characterized in that: The certain differentiation time is 10-12 days.

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