H9-based induction of brain organoids containing optic vesicles and eye-brain fusion culture methods

Abstract

The present invention discloses a brain organoid containing optic vesicles induced by H9 and a method for culturing the eye-brain fusion thereof. The present invention uses H9 embryonic stem cells to induce a brain organoid containing optic vesicles with a primitive visual field, and based on the established optic vesicle brain organoid culture system, uses microscopic imaging combined with antibodies for specific markers related to early retinal development and photoreceptor cell maturation to identify the results and functions of the brain organoid containing optic vesicles.

Description

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 and eye-brain fusion culture based on H9 induction. Background Art

[0002] Human brain organoids are miniature brains cultured in a culture medium that simulates the brain development environment using induced pluripotent stem cells (iPSCs). They have the cellular and structural characteristics of the human brain. They can replicate the fetal brain development path, provide a good model for studying development, disease and drug effects, and provide unprecedented opportunities for brain organoid transplantation to treat neurological diseases. Retinal organoids (RO) are cell aggregates formed by specific culture differentiation of embryonic stem cells and induced pluripotent stem cells. Their structure and function are similar to the corresponding retinal tissue. They were first studied in 2012. After 2014, many studies have found 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 from both sides of the diencephalon in the forebrain region. It is a double-layered cup-shaped structure formed during the development of the eye embryo. It develops forward from the optic disc and eventually develops into 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, by observing and analyzing the development process of optic vesicle organoids induced by pluripotent stem cells, we can better understand the formation and development mechanism 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 way for in vitro research on human embryonic brain and eye development in a single organoid; in terms of disease modeling, optic vesicle organoids 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 optic vesicle organoids, researchers can evaluate the therapeutic effects and safety of drugs on retinal diseases, providing new ideas and methods for drug development; in terms of environmental toxicology, optic vesicle organoids can also be used as a model for analyzing the effects 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, optic vesicle organoids also provide 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.), and optic vesicle organoids can be used to explore the research on promoting the repair and regeneration of damaged retinal tissue. In conclusion, optic vesicle brain organoids are a technology with broad application prospects, which can provide new ideas and methods for the research and treatment of visual system-related diseases. However, there are no reports on the culture of optic vesicles based on H9-induced human brain organoids. Summary of the invention

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

[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 induction to produce brain organoids containing optic vesicles, comprising the following steps:

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

[0009] H9 embryonic stem cells were cultured in mTeSR1 medium until the cell fusion reached 70%, and a single cell suspension was prepared. A neural induction medium containing 10 mM Y27632 was added and the cells were seeded in a well plate. The medium was replaced by half of the neural induction medium without 10 mM Y27632 every day. The cells were cultured for 0 to 5 days to form neurospheres. Neurosphere medium was added and static culture was carried out. The medium was replaced by half of the neurosphere medium every day until the 6th to 7th day. The early optic vesicle organoid culture medium was added on the 8th day. The mature optic vesicle organoid culture medium was added on the 10th day for rotation culture. The medium was replaced by half of the mature optic vesicle organoid culture medium every week until the cells differentiated into three-dimensional brain organoids containing optic vesicles.

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

[0011] In step 1, the neural induction medium is 83% DMEM / F-12, 15% Knockout serum replacement, 1% MEM-non-essential amino acids (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 (medium), 0.4% N2 supplement (supplement), 1% B27 supplement without vitamin A (supplementwithout vitamin A), 0.5% MEM-non-essential amino acids (NEAA), 1% Glutamax supplement (supplement), 1% Penicillin / Streptomycin (Penicillin / Streptomycin), 50μM β-mercaptoethanol (β-Mercaptoethanol), 2.5μM SB431542, 1.5nM bone morphogenetic protein-4 (BMP-4), and 0.1% Matrigel.

[0013] In the 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-non-essential amino acids (NEAA), 1% Glutamax supplement, 0.5μM Dorsomorphin, 1% Penicillin-Streptomycin, 50μM β-mercaptoethanol, 2.5μM SB431542, 1.5nM bone morphogenetic protein-4 (BMP-4), and 0.1% Matrigel.

[0014] In the step 1, the culture medium of the mature optic vesicle brain organoid is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100nMol retinol acetate, 0.5% MEM-non-essential amino acids (NEAA), 1% Glutamax supplement, 0.5μM Dorsomorphin, 1% Penicillin-Streptomycin, 50μM β-Mercaptoethanol, 2.5μM SB431542, 0.1% ascorbic acid, 10ng / ml brain-derived neurotrophic factor (BDNF), and 10ng / ml ciliary neurotrophic factor (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 a brain organoid containing optic vesicles with primitive visual fields. Based on the established optic vesicle brain organoid culture system, microscopic imaging is combined with antibodies for specific markers related to early retinal development and photoreceptor cell maturation to identify the results and functionality of the brain organoid containing optic vesicles.

[0018] 2. The present invention innovatively integrates retinal structure and brain organoids, which is conducive to studying the interaction between the retina and the forebrain, providing an in vitro model for studying the development and diseases of the brain and eyes, and providing a new approach for studying the effects of drugs and environmental exposure on the development of the human embryonic brain and eyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a light microscopic image of a brain organoid containing an optic vesicle;

[0020] Figure 2 Immunofluorescence comparison of brain organoids containing optic vesicles, including (A) forebrain patterning molecules (FOXG1) and neural progenitor cells (SOX2), and (B) retinal progenitor cells (VSX2);

[0021] Figure 3 This is an immunofluorescence comparison of brain organoids containing optic vesicles, including (A) optic nerve progenitor cells (PAX6), (B) primitive visual field region molecules that determine photoreceptor fate (RAX), (C) neuronal precursor cells (DCX), (D) amacrine cells (HUC / D), (E) optic ganglion cells (BRN3), retinal epithelial cells (MITF), (F) cone and rod photoreceptor transcription factors (CRX), (G) visual recovery protein (RECOVERIN), and (H) photoreceptor transcription factor (OTX2). DETAILED DESCRIPTION

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

[0023] Example 1

[0024] A method for culturing eye-brain fusion based on H9 induction to produce 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, and after cell recovery and EDTA digestion, they were subcultured until the cell confluence reached 70% after subculture. Accutase enzyme was used to digest the cells in a 37°C incubator for 10 minutes to prepare a single cell suspension. After cell counting, neural induction medium (83% DMEM / F-12, 15% Knockout serum replacement, 1% MEM-non-essential amino acids (NEAA), 1% Glutamax supplement, 50μM β-mercaptoethanol, 10μM SB431542, 2μM XAV939, 10mMY27632 (to promote cell aggregation when seeding)) was added, and the cells were seeded in an ultra-low adhesion V-bottom 96-well plate at a ratio of 10,000 cells / well and 100μL per well. The cells were washed once a day without 10mM The neural induction medium of Y27632 (other components remain unchanged) was replaced with half of the medium for 0-5 days to form neurospheres, and the neurospheres were transferred to low-adhesion 3-5 cm culture dishes and neurosphere medium (48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-non-essential amino acids (NEAA), 1% Glutamax supplement, 1% Penicillin / Streptomycin, 50 μM β-mercaptoethanol, 2.5 μM SB431542, 1.5 nM bone morphogenetic protein-4 (BMP-4), 0.1% Matrigel), static culture, half of the medium was replaced with the neurosphere culture medium every day, and culture was continued until the 6th to 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, 100nMol retinol acetate, 0.5% MEM-non-essential amino acids, 1% Glutamax supplement, 0.5μM doxormorphine, 1% penicillin-streptomycin, 50μM β-mercaptoethanol, 2.5μM SB431542, 0.1% Matrigel, 1.5nM bone morphogenetic protein-4) was added, and on the 10th day, mature optic vesicle organoid culture medium (48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100nMol retinol acetate, 0.5% MEM-non-essential amino acids (NEAA), 1% Glutamax supplement, 0.5μM Dorsomorphin, 1% Penicillin-Streptomycin, 50μM β-Mercaptoethanol, 2.5μM SB431542, 0.1% Ascorbic acid, 10ng / ml brain-derived neurotrophic factor (BDNF), 10ng / ml ciliary neurotrophic factor (CNTF), transferred to a shaker for long-term rotation culture, and the medium was replaced by half of the mature optic vesicle brain organoid culture medium every week until it differentiated into a three-dimensional brain organoid containing optic vesicles. .

[0027] Step 2, continue to culture the brain organoids described in step 1 from the 20th to the 30th day, pigment deposition will appear on one side of the brain organoid, and around the 30th day, a strong pigmentation 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, RAX and VSX2 regions as original visual field regions (RAX is the retinal and proneural fold homeobox transcription factor, VSX2 is the retinal progenitor cell), and 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), visual recovery protein (RECOVERIN), such as Figure 1-Figure 3 As shown, 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 present invention is described above by way of example. 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 those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for culturing eye-brain fusion based on H9 to induce brain organoids containing optic vesicles, characterized in that: The following steps are involved: Step 1, culturing H9 embryonic stem cells and differentiating them into three-dimensional brain organoids containing optic vesicles, comprising the following steps: H9 embryonic stem cells were cultured in mTeSR1 medium until the cell fusion reached 70%, a single cell suspension was prepared, a neural induction medium containing 10 mM Y27632 was added, the cells were inoculated in a well plate, half of the medium was replaced with a neural induction medium not containing 10 mM Y27632 every day, the cells were cultured for 0 to 5 days to form neurospheres, neurosphere medium was added, the cells were statically cultured, half of the medium was replaced with the neurosphere medium every day, the cells were cultured until the 6th to 7th day, the early optic vesicle brain organoid medium was added on the 8th day, the mature optic vesicle brain organoid medium was added on the 10th day for rotational culture, and half of the medium was replaced with the mature optic vesicle brain organoid medium every week until the cells were differentiated into three-dimensional brain organoids containing optic vesicles; the neural induction medium was 83% DMEM / F-12, 15% Knockout serum substitute, 1% MEM-non-essential amino acids, 1% Glutamax supplement, 50 μM β-mercaptoethanol, 10 μM SB431542, 2 μM XAV939; 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-non-essential amino acids, 1% Glutamax supplement, 1% penicillin / streptomycin, 50μM β-mercaptoethanol, 2.5μM SB431542, 1.5nM bone morphogenetic protein-4, 0.1% Matrigel; the pre-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-non-essential amino acids, 1% Glutamax supplement, 0.5μM doxorubicin, 1% penicillin-streptomycin, 50μM β-mercaptoethanol, 2.5μM SB431542, 1.5nM bone morphogenetic protein-4, 0.1% Matrigel; the mature optic vesicle brain organoid culture medium is 48% DMEM / F-12, 48% Neurobasal medium, 0.4% N2 supplement, 1% B27 supplement, 100nMol retinol acetate, 0.5% MEM-non-essential amino acids, 1% Glutamax supplement, 0.5μM doxorubicin, 1% penicillin-streptomycin, 50μM β-mercaptoethanol, 2.5μM SB431542, 0.1% ascorbic acid, 10ng / ml brain-derived neurotrophic factor, 10ng / ml ciliary neurotrophic factor; Step 2, continue to culture the brain organoids described in step 1 until they develop strong colored areas, perform immunostaining and imaging on the brain organoids marked with forebrain patterns and visual field norms, and verify the structural function and neural development of the brain organoids.

2. A brain organoid containing optic vesicles obtained by the culture method according to claim 1.

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