US-12624339-B2 - Brain organoid containing optic vesicles generated based on H9 induction and eye-brain fusion culture method

Abstract

A brain organoid containing optic vesicles generated based on H9 induction and an eye-brain fusion culture method thereof are provided. H9 embryonic stem cell induction is used to generate a brain organoid containing optic vesicles with primitive visual field. Based on an established optic vesicle brain organoid culture system, microscopic imaging is combined with specific marker antibodies related to early retinal development and photoreceptor cell maturation to structurally and functionally identify the brain organoid containing optic vesicles.

Inventors

• Liqun Chen
• Rui Hu
• Dong Ming

Assignees

• TIANJIN UNIVERSITY

Dates

Publication Date

20260512

Application Date

20250604

Priority Date

20240129

Claims (1)

1. 1 . An eye-brain fusion culture method for generating a three-dimensional brain organoid containing optic vesicles through the induction of H9 embryonic stem cells, comprising the following steps: A) culturing the H9 embryonic stem cells in a feeder-free cell culture and maintenance medium until a cell confluence reaches 70%, B) preparing a single cell suspension, C) removing the feeder-free cell culture and maintenance medium and adding a neural induction medium containing 10 mM Y27632, inoculating in an orifice plate, changing half of the medium with a neural induction medium without 10 mM Y27632 each day for 0-5 days to form neurospheres, wherein the neural induction medium comprises 83% DMEM/F-12, 15% serum replacement supplement, 1% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 50 μM ß-mercaptoethanol, 10 μM SB431542, and 2 μM XAV939; D) removing the neural induction medium and adding a neurosphere medium, and changing half of the medium with the neurosphere medium each day until a 6-7 th day, wherein the neurosphere medium comprises 48% DMEM/F-12, 48% neuronal cell culture medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 1% penicillin/streptomycin, 50 μM ß-mercaptoethanol, 2.5 M SB431542, 1.5 nM bone morphogenetic protein-4, and 0.1% extracellular matrix; E) removing the neurosphere medium and adding an early-stage optic vesicle brain organoid medium on an 8th day, wherein the early-stage optic vesicle brain organoid medium comprises 48% DMEM/F-12, 48% neuronal cell culture medium, 0.4% N2 supplement, 1% B27 supplement, 100 nMol retinol acetate, 0.5% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 0.5 μM Dorsomorphin, 1% penicillin-streptomycin, 50 μM ß-mercaptoethanol, 2.5 μM SB431542, 1.5 nM bone morphogenetic protein-4, and 0.1% extracellular matrix; F) removing the early-stage optic vesicle brain organoid medium adding a mature-stage optic vesicle brain organoid medium on a 10 th day and culturing under rotational culture, and changing half of the medium with the mature-stage optic vesicle brain organoid medium every week until the three-dimensional brain organoid containing the optic vesicles is formed; wherein the mature-stage optic vesicle brain organoid medium comprises 48% DMEM/F-12, 48% neuronal cell culture medium, 0.4% N2 supplement, 1% B27 supplement, 100 nMol retinol acetate, 0.5% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 0.5 μM Dorsomorphin, 1% penicillin-streptomycin, 50 μM ß-mercaptoethanol, 2.5 μM SB431542, 0.1% ascorbic acid, 10 ng/ml brain-derived neurotrophic factor, and 10 ng/ml ciliary neurotrophic factor; G), continuing culture of the brain organoid obtained in step 1 until pigment deposition occurs on one side, and H) performing immunostaining and imaging on the brain organoid marked by a forebrain pattern and a visual field specification to verify a structural function and neural development of the brain organoid.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of PCT/CN2025/084058, filed Mar. 21, 2025, which claims priority of Chinese Patent Application No. 202410121193.X, filed on Jan. 29, 2024. The entire contents of International Patent Application PCT/CN2025/084058 and Chinese Patent Application No. 202410121193.X are incorporated herein by reference. TECHNICAL FIELD The present disclosure relates to the field of biomedical technology, and in particular relates to a brain organoid containing optic vesicles generated based on H9 induction and an eye-brain fusion culture method. BACKGROUND Human brain organoids are miniature brains cultured in a culture medium that simulates the brain developmental environment using induced pluripotent stem cells (iPSCs). Human brain organoids exhibit cellular and structural characteristics of the human brain and may replicate fetal brain development pathways, providing excellent models for studying development, diseases, and medicine efficacy, and offering unprecedented opportunities for brain organoid transplantation for the treatment of neurological diseases. Retinal organoids (ROs) are cell aggregates formed by the differentiation of embryonic stem cells and induced pluripotent stem cells through specific culture, with a structure and function similar to the corresponding retinal tissue. First reported in 2012, subsequent studies after 2014 have shown that retinal organoid transplants may be used to treat retinitis pigmentosa, retinal epithelial dysfunction, and age-related macular degeneration, improving visual function. During normal embryonic development, the retinal primordium originates from the diencephalon on both sides of the forebrain. A bilayer cup-shaped structure is formed during the development of the eye embryo, which develops forward from the optic disc and eventually develops into the retina. This structure may perceive light and transmit signals to other brain regions. Therefore, constructing a culture for generating optic vesicles based on human brain organoid induction holds significant research value. Optic vesicle brain organoids have broad applications in biology and medicine. In retinal development, observing and analyzing the developmental process of pluripotent stem cell induced development of optic vesicle brain organoids may enhance understanding of retinal tissue formation and developmental mechanisms. The eye-brain fusion culture mechanism enables retinal ganglion cells to form extensive axonal projections in the forebrain region, opening new avenues for in vitro studies of human embryonic brain and eye development within a single organoid. In terms of disease modeling, optic vesicle brain organoids may be used to establish disease-related models for studying the etiology and pathogenesis of retinal disorders. In terms of medicine screening and evaluation, researchers may assess the therapeutic effects and safety of medicines on retinal diseases by studying the effects of medicines on the optic vesicle brain organoids, providing new ideas and methods for medicine development. In terms of environmental toxicology, such optic vesicle brain organoids may also be used as models to analyze 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 the field of regenerative medicine, optic vesicle brain organoids also provide marker cells related to retinal organs (such as retinal progenitor cells, optic ganglion cells, retinal pigment epithelial cells, amacrine cells, photoreceptor precursor cells, visual restorative proteins, etc.), which may be used to explore and promote the repair and regeneration ability of damaged retinal tissue. In summary, optic vesicle brain organoids represent a technology with wide-ranging applications, offering new insights and methods for the study and treatment of visual system-related diseases. However, research on the culture for generating optic vesicles of brain organoids based on H9 induction has not yet been reported. SUMMARY To address the shortcomings of the aforementioned technical solutions, an objective of the present disclosure is to provide an eye-brain fusion culture method for generating a brain organoid containing optic vesicles based on H9 induction. Another objective of the present disclosure is to provide a brain organoid containing optic vesicles obtained through the aforementioned culture method. The objectives of the present disclosure are achieved through the following technical solutions. An eye-brain fusion culture method for generating a brain organoid containing optic vesicles based on H9 induction includes the following steps: step 1, culturing H9 embryonic stem cells to differentiate into a three-dimensional brain organoid containing optic vesicle, including following steps:culturing the H9 embryonic stem cells in an MTESR™ 1 medium