Brain organoid containing optic vesicles generated on basis of h9 induction and eye-brain integrated culture method therefor

Abstract

A brain organoid containing optic vesicles generated on the basis of H9 induction and an eye-brain integrated culture method therefor. H9 embryonic stem cells are used to induce the generation of a brain organoid which contains optic vesicles and has primitive visual fields. On the basis of an established system for culturing a brain organoid containing optic vesicles, and by means of microscopic imaging in combination with antibodies against specific markers related to early retinal development and photosensitive cell development and maturation, the structure and function of the brain organoid containing optic vesicles are identified.

Description

H9-based induction of brain organoids containing optic vesicles and their eye-brain fusion culture method Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for producing brain-like organs containing optic vesicles based on H9 induction and culturing the eye-brain fusion thereof. Background Art

[0002] Human brain organoids are miniature brains cultured from induced pluripotent stem cells (iPSCs) in a medium that mimics the developmental environment of the brain. They possess cellular and structural characteristics of the human brain and can replicate fetal brain developmental pathways, providing a promising model for studying development, disease, and drug effects. This opens up unprecedented opportunities for the transplantation of brain organoids to treat neurological disorders. Retinal organoids (ROs) are cell aggregates formed by specialized culture and differentiation of embryonic and induced pluripotent stem cells (ESCs). Their structure and function resemble those of corresponding retinal tissue. They were first described in 2012, and since 2014, several studies have shown that retinal organoid transplantation can be used to treat retinitis pigmentosa, retinal epithelial dysfunction, age-related macular degeneration, and improve visual function. During normal embryonic development, the retinal primordium originates on both sides of the diencephalon in the forebrain region. During the development of the eye embryo, a double-layered cup-shaped structure is formed, extending forward from the optic disc and ultimately giving rise to the retina. This structure can sense light and send signals to other areas of the brain at the same time, so constructing a culture based on human brain organoids to induce the production of optic vesicles is of great research significance.

[0003] Optic vesicle organoids have a wide range of applications in biology and medicine. In terms of retinal development, observing and analyzing the development of optic vesicle organoids derived from pluripotent stem cells can provide a better understanding of the formation and developmental mechanisms of retinal tissue. Through the eye-brain fusion culture mechanism, retinal ganglion cells form extensive axonal projections in the forebrain region, opening up a new avenue for in vitro research on human embryonic brain and eye development within a single organoid. In terms of disease modeling, the optic vesicle organoid can be used to establish disease-related models to study the causes and pathogenesis of retinal diseases. In terms of drug screening and evaluation, by studying the effects of drugs on the optic vesicle organoid, researchers can evaluate the therapeutic efficacy and safety of drugs for retinal diseases, providing new ideas and methods for drug development. In terms of environmental toxicology, the optic vesicle organoid can also be used as a model to analyze the impact of the environment on embryonic brain and eye development, such as atmospheric pollutants, soil pollutants, water pollutants, radiation pollution, pesticide pollution, viruses, etc. In terms of regenerative medicine, the optic vesicle organoid also provides retinal organ-related marker cells (such as retinal progenitor cells, retinal ganglion cells, retinal pigment epithelial cells, amacrine cells, photoreceptor precursor cells, visual restoration proteins, etc.), which can be used to explore the ability of optic vesicle organoids to promote the repair and regeneration of damaged retinal tissue. In summary, optic vesicle brain organoids are a promising technology with broad applications, providing new insights and approaches for the research and treatment of visual system-related diseases. However, studies on the culture of optic vesicles derived from H9-derived human brain organoids have yet to be reported. Summary of the Invention

[0004] In order to solve the deficiencies of the above technical solutions, the purpose of the present invention is to provide an eye-brain fusion culture method based on H9 induction to produce brain-like organs containing optic vesicles.

[0005] Another object of the present invention is to provide a brain organoid containing optic vesicles obtained by the above-mentioned culture method.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] A method for culturing eye-brain fusion based on H9-induced generation of brain organoids containing optic vesicles, comprising the following steps:

[0008] Step 1: Cultivating H9 embryonic stem cells and differentiating them into three-dimensional brain organoids containing optic vesicles, including the following steps:

[0009] H9 embryonic stem cells were cultured in mTeSR1 medium until the cell confluence reached 70%. Single-cell suspensions were prepared and neural induction medium containing 10 mM Y27632 was added. The cells were seeded into well plates and the medium was replaced by half every day with neural induction medium without 10 mM Y27632. Neurospheres were formed from 0 to 5 days of culture. Neurosphere medium was added and the cells were cultured statically. The medium was replaced by half every day with neurosphere medium until the 6th to 7th day of culture. Early optic vesicle organoid culture medium was added on the 8th day. Mature optic vesicle organoid culture medium was added on the 10th day and the cells were rotated and cultured. The medium was replaced by half every week with mature optic vesicle organoid culture medium until the cells differentiated into three-dimensional brain organoids containing optic vesicles.

[0010] Step 2: Continue culturing the brain organoids described in step 1 until they develop strongly colored areas, and perform immunostaining and imaging on the brain organoids marked with forebrain patterns and visual field norms to verify the structural function and neural development of the brain organoids.

[0011] In step 1, the neural induction medium comprises 83% DMEM / F-12, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 50 μM β-Mercaptoethanol, 10 μM SB431542, and 2 μM XAV939.

[0012] In step 1, the neurosphere culture medium is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 1.5 nM BMP-4, and 0.1% Matrigel.

[0013] In step 1, the early optic vesicle organoid culture medium is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100nMol Retinol acetate, 0.5% MEM-NEAA, 1% Glutamax supplement, 0.5μM Dorsomorphin, 1% Penicillin-Streptomycin, 0.1% Matrigel, 50μM β-Mercaptoethanol, 2.5μM SB431542, and 1.5nM BMP-4.

[0014] In step 1, the culture medium for the mature optic vesicle brain organoid is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100 nMol Retinol acetate, 0.5% MEM-NEAA, 1% Glutamax supplement, 0.5 μM Dorsomorphin, 1% Penicillin-Streptomycin, 0.1% Ascorbic acid, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 10 ng / ml BDNF, and 10 ng / ml CNTF.

[0015] The brain organoid containing optic vesicles obtained by the above culture method.

[0016] The advantages and beneficial effects of the present invention are:

[0017] 1. The present invention uses H9 embryonic stem cells to induce the production of a brain organoid containing an optic vesicle with a primitive visual field. Based on the established optic vesicle brain organoid culture system, microscopic imaging is combined with antibodies to specific markers related to early retinal development and photoreceptor cell maturation to identify the structure and functionality of the brain organoid containing the optic vesicle.

[0018] 2. This invention innovatively integrates retinal structures and brain organoids, which is beneficial for studying the interaction between the retina and the forebrain, providing an in vitro model for studying brain and eye development and diseases, and offering a new approach for studying the effects of drugs and environmental exposures on the development of the human embryonic brain and eyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is a light microscopic image of a brain organoid containing an optic vesicle;

[0020] Figure 2 shows the immunofluorescence comparison of brain organoids containing optic vesicles, where (A) is forebrain patterning molecules (FOXG1) and neural progenitor cells (SOX2), and (B) is for retinal progenitor cells (VSX2);

[0021] Figure 3 is an immunofluorescence comparison of brain-like organs containing optic vesicles, among which (A) is optic nerve progenitor cells (PAX6), (B) is the molecule that determines the fate of photoreceptors in the primitive visual field area (RAX), (C) is neuronal precursor cells (DCX), (D) is amacrine cells (HUC / D), (E) is retinal ganglion cells (BRN3), retinal epithelial cells (MITF), (F) is cone and rod photoreceptor transcription factor (CRX), (G) is visual recovery protein (RECOVERIN), and (H) is photoreceptor transcription factor (OTX2). DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below with reference to specific embodiments.

[0023] Example 1

[0024] A method for culturing eye-brain fusion based on H9-induced generation of brain organoids containing optic vesicles, comprising the following steps:

[0025] Step 1, culturing H9 embryonic stem cells and differentiating them into three-dimensional brain organoids containing optic vesicles, comprising the following steps:

[0026] H9 embryonic stem cells were cultured in mTeSR1 medium, revived and digested with EDTA, and then subcultured until the cell confluence reached 70%. The cells were digested with Accutase enzyme for 10 min in a 37°C incubator to prepare a single-cell suspension. After cell counting, neural induction medium (83% DMEM / F-12, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 50 μM β-Mercaptoethanol, 10 μM SB431542, 2 μM XAV939, 10 mM Y27632 (to promote cell aggregation when seeding)) was added and the cells were seeded at 10,000 cells / well in ultra-low adhesion V-bottom 96-well plates with 100 μL per well. The plates were plated daily without 10 mM The medium was replaced by half of the Y27632 neural induction medium (other components remained unchanged) for 0-5 days to form neurospheres. The neurospheres were then transferred to low-adhesion 3-5 cm culture dishes and cultured statically with neurosphere medium (48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.1% Matrigel, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 1.5 nM BMP-4). The medium was replaced by half of the neurosphere medium every day until the 6th-7th day. On the 8th day, optic vesicle organoid culture medium containing retinol acetate (48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.1% Matrigel, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 1.5 nM BMP-4) was added. supplement, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin-Streptomycin, 0.1% Matrigel, 100nMol Retinol acetate, 0.5μM Dorsomorphin, 50μM β-Mercaptoethanol, 2.5μM SB431542, 1.5nM BMP-4, and on day 10, mature optic vesicle brain organoid culture medium (48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin-Streptomycin, 0.1% Ascorbic acid, 100 nM Retinol acetate, 0.5 μM Dorsomorphin, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 10 ng / ml BDNF, 10 ng / ml CNTF) were transferred to a shaker for long-term rotational culture. The medium was replaced with half the volume of mature optic vesicle organoid culture medium weekly until differentiation into three-dimensional optic vesicle-containing organoids was achieved.

[0027] Step 2: Continue culturing the brain organoids described in step 1 for 20 to 30 days. Pigment deposition will appear on one side of the brain organoids. Around the 30th day, a strongly pigmented area will develop. Obtain light microscopic images of the brain organoids (to observe the morphology at different culture periods and the area of ​​pigment deposition). Immunostain and image the brain organoids marked with forebrain patterns and visual field specifications. Use the marked FOXG1 and SOX2 regions as forebrain region molecules, and the RAX and VSX2 regions as the original visual field regions (RAX is the retinal and proneural fold homeobox transcription factor, and VSX2 is the retinal progenitor cell). , as well as early retinal development-related markers, optic nerve progenitor cells (PAX6), neuronal precursor cells (DCX), amacrine cells (HUC / D), optic ganglion cells (BRN3), retinal epithelial cells (MITF), cone and rod photoreceptor transcription factors (CRX and OTX2), and visual recovery protein (RECOVERIN). As shown in Figures 1 to 3, it can be seen that the neural development of the brain organoids containing the optic vesicles is in good condition, as well as the status of retinal development and photoreceptor cell development in the brain organoids containing the optic vesicles, verifying that the structural function and neural development of the brain organoids containing the optic vesicles are good.

[0028] The above is an exemplary description of the present invention. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by other skilled in the art without expending creative labor falls within the scope of protection of the present invention.

Claims

1. A method for culturing eye-brain fusion based on H9-induced brain organoids containing optic vesicles, characterized in that: The following steps are involved: Step 1: Cultivating H9 embryonic stem cells and differentiating them into three-dimensional brain organoids containing optic vesicles, including the following steps: H9 embryonic stem cells were cultured in mTeSR1 medium until the cell confluence reached 70%. A single-cell suspension was prepared and neural induction medium containing 10 mM Y27632 was added. The cells were seeded into well plates and the medium was replaced by half every day with neural induction medium not containing 10 mM Y27632. Neurospheres were formed after culturing for 0-5 days. Neurosphere medium was added and the cells were cultured statically. The medium was replaced by half every day with the neurosphere medium until the 6-7th day. On the 8th day, early optic vesicle organoid medium was added. On the 10th day, mature optic vesicle organoid medium was added and the cells were rotated and cultured. The medium was replaced by half every week with mature optic vesicle organoid medium until the cells differentiated into three-dimensional brain organoids containing optic vesicles. Step 2: Continue culturing the brain organoids described in step 1 until they develop strongly colored areas, and perform immunostaining and imaging on the brain organoids marked with forebrain patterns and visual field norms to verify the structural function and neural development of the brain organoids.

2. The culture method according to claim 1, wherein In step 1, the neural induction medium comprises 83% DMEM / F-12, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 50 μM β-Mercaptoethanol, 10 μM SB431542, and 2 μM XAV939.

3. The culture method according to claim 1, wherein In step 1, the neurosphere culture medium is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 1.5 nM BMP-4, and 0.1% Matrigel.

4. The culture method according to claim 1, wherein In step 1, the early optic vesicle organoid culture medium is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100nMol Retinol acetate, 0.5% MEM-NEAA, 1% Glutamax supplement, 0.5μM Dorsomorphin, 1% Penicillin-Streptomycin, 50μM β-Mercaptoethanol, 2.5μM SB431542, 1.5nM BMP-4, and 0.1% Matrigel.

5. The culture method according to claim 1, wherein In step 1, the culture medium for the mature optic vesicle brain organoid is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100 nMol Retinol acetate, 0.5% MEM-NEAA, 1% Glutamax supplement, 0.5 μM Dorsomorphin, 1% Penicillin-Streptomycin, 50 μM β-Mercaptoethanol, 2.5 μM SB431542, 0.1% Ascorbic acid, 10 ng / ml BDNF, and 10 ng / ml CNTF. 6 . A brain organoid containing optic vesicles obtained by the culture method according to claim 1 .

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