The Study Of Human Embryonic Retinal Development And Human Retinal Organoids Differentiation

Vision is the most important sense-perceptions for human.The retina,as the starting point for vision perception,is responsible for converting light signals into electrical signals to form image vision and non-image visual information.Studies have shown that retinal degenerative diseases such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD)can cause irreversible death of photoreceptors and eventually lead to blindness,which has a serious impact on the normal life of human beings,but the circuit between other types of cells in the retina and the neural circuit from the retina to the visual cortex remain intact.Therefore,transplantation of human fetal retinae can improve patients’ vision to some extent.However,due to the scarcity of human embryonic retinae that can be used for transplantation therapy and the constraints of ethical issues,the treatment of retinal degenerative diseases has been greatly limited.The establishments of the in vitro culture system of human pluripotent stem cells(hPSCs)and the development of differentiation methods of hPSCs-derived retinal cells provide a new source of transplanted cells and new strategies for the treatment of retinal degenerative diseases.Retinal organoids(ROs)derived from hPSCs have been demonstrated to recapitulate the cell types and development order of human retinae at the gene and protein expression level,regarded as an in vitro model to study human retinal development and disorders.However,little is known about the similarities and differences of chromatin accessibility and transcriptional regulation features between human retinal and RO development.In addition,recent multi-omics studies of human retinae have revealed the histone modification and transcriptional features of the developing retina,but the chromatin accessible features during human retinal development haven’t been studied.Therefore,comparative studies of chromatin accessibility and transcriptional regulation features of human retinae and ROs would promote the understanding of the developmental mechanism of human retinae,and lay the foundation for the improvement of ROs differentiation systems and the clinical applications of ROs for retinal degenerative diseases in the future.Chromatin accessibility refers to the degree of the physical contact between the large molecules(such as DNase)and chromatin DNA in the nucleus,that is,the openness of chromatin structure,which is determined by the binding of nucleosomes and other chromatin binding factors to DNA.During cell development and fate determination,chromatin accessibility is closely related to the regulation of gene expression and regulated by multiple epigenetic characteristics such as DNA methylation,histone modification,non-coding RNA,and chromatin remodeling,which affect the combination of binding factors(such as RNA polymerase II and transcription factors)and corresponding cis-regulatory elements.With the development of high-throughput sequencing technologies,assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq)has been proven to be a sensitive,robust,and easy-to-use method for studying the chromatin accessibility since its invention.It has become a mainstream method in the research field of chromatin accessibility,which can be used to study the open chromatin regions,nucleosome binding positions,and transcription factors enrichment and occupancy.In this thesis,we collected ATAC-seq and RNA-seq data of human fetal retinae samples and ROs samples at different development time points,and systematically analyzed the chromatin accessibility and transcriptional dynamics during human fetal retinal and RO development.Our results revealed that the developing human retina exhibited a unique pattern of chromatin and transcriptional dynamics correlated with retinogenesis,corresponding to the three major developmental stages of early cell proliferation,mid-term neurogenesis,and late retinal differentiation.ROs recapitulated the chromatin and transcriptional features of human retinogenesis to a large extent,but different chromatin features were also found.Next,we conducted an integrated analysis of the ATAC-seq and RNA-seq data to reconstruct the transcriptional regulatory network during human retinal and RO development,and demonstrated the similarity between human retinal and RO regulatory networks.Notably,NFIB and THRA were identified as new regulatory factors necessary for human retinal development.In order to verify the regulatory function of NFIB and THRA in human retinal development,we conducted gene manipulation experiments on ROs,which systematically and comprehensively establishing a rigorous closed-loop research system of "discovery of molecular mechanisms of human retinal development-verification through gene manipulation of ROs".The research system solves the difficulty in directly performing genetic manipulation and functional verification on human retinae,and the verification also indicats that ROs are a good in vitro model for human retinal study.Finally,since histone modification is one of the most important features of chromatin,we collected existing data to compare and cross-analyze the histone modifications and chromatin features between the developing human and mouse retina.The analysis revealed a bivalent state of H3K4me3 and H3K27me3 modifications in human retinal neurogenesis but not in murine,suggesting a more dedicated and complicated epigenetic regulation of the human genome during the neurogenesis,which may also become potential targets for the treatment of retinal development or aging diseases.Taken together,our systematic research on chromatin signatures and transcriptional dynamics during retinal development provided a comprehensive view of the chromatin and gene expression profiles that accompany the murine,human retinal and RO development,filling the gaps in the field of studies on the epigenetic features of human retinal development and the comparison of the epigenetic features between human and mouse retinal development.Based on these data,we established a developmental temporal-correlation roadmap of both transcriptional and epigenetic features between the human retinae and ROs,demonstrating the similarities and differences of the epigenetic and transcriptional dynamics between human retinal and RO development.The similarities of development and the gene manipulation experiments on ROs have proved that ROs can be used as an in vitro model for studying human retinal development.Moreover,the differences in the human retinal and RO development found in this study provided an invaluable data source and research foundation for improving RO differentiation system under the guidance of human retinogenesis.Thereby ROs can better simulate the types of retinal cells and the layered structure of human retinae in vivo,and become a better in vitro model of human retinae for clinical applications.In general,our work has laid the solid foundation for the goal of preparing ROs,which are highly similar to human retinas and utilizing ROs as cell or organ sources for transplanting therapies to treat retinal diseases in the future clinical application.We firmly believe that by continuously solving various problems and challenges before clinical application,regenerative medicine technologies such as stem cells and organoids can provide effective treatment for different kinds of human tissue and organ diseases including retinal degenerative diseases in the near future,supporting the better life and health-care for human beings.