声明
摘要
Abstract
Table of contents
ABBREVIATIONS
PROJECT Ⅰ Accelerated photoreceptor differentiation of hiPSC-derived retinal organoids by contact co-culture with retinal pigment epithelium
Chapter 1 Introduction
1．1 Stem cell culture
1．2 Classification and sources of stem cells
1．3 Generation of induced pluripotent stem cells(iPSCs)
1．4 Application of iPSCs
1．5 Organoid culture
1．6 Applications of Organoids
1．7 Retinal organoids(ROs)
1．8 Retinal organoid based disease models
1．9 Challenges in stem cell differentiation into retinal organoids and their solutions
1．10 Retinal pigment epithelium
1．11 RPE Culture
1．12 Function of RPE in maintaining photoreceptor
Chapter 2 Materials and methods
2．1 Key resources and primers
2．2 Ethics statement
2．3 hiPSCs culture
2．4 Retinal organoid differentiation
2．5 Primary culture of mouse RPE
2．6 Co-culture method
2．7 Conditioned media
2．8 Immunofluorescent assay
2．9 Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)
2．10 Western blot analysis
2．11 Quantification and statistical analysis
Chapter 3 Results
3．1．3 hiPSC-derived ROs mimic human retina at early developmental stages
3．1．4 hiPSC-derived ROs mimic human retina at late developmental stages
3．2．1 Primary culturing of mouse retinal pigment epithelium
3．2．3 Co-culture duration of ROs with RPE
3．3．1 ROs-RPE co-culture at early stages of ROs differentiation
3．3．2 ROs-RPE interaction enhanced early photoreceptor differentiation in ROs
3．4 Effects of ROs-RPE co-culture upon other early-born retinal markers
3．5 Effects of conditioned media on photoreceptor progenitors of ROS
3．6．1 ROs-RPE co-culture at later stages of ROs differentiation
3．6．2 Participation of RPE in accelerated differentiation of photoreceptors at later stages of ROs
3．6．3 Effects of ROs-RPE co-culture upon other early and late-born photoreceptors markers
3．7 Effect of ROs-RPE co-cultu re on H9 cells derived ROs at early and later stages of differentiation
Graphical abstract
Chapter 4 Discussion
4．1 Discussion
Conclusions and future directions
References
PROJECTⅡ NRL-/- retinal organoids revealed novel mechanism of human NRL in repressing cone genesis
Summary
Chapter 1 Introduction
1．1 Retina of mammals
1．2 Structure of the mammalian eye
1．3 Structure and functions of the neural retina
1．4 Cell biology of vision
1．5 Photo-transduction
1．6 The visual cycle
1．7 Disc renewal
1．8 Retinal development
1．9 Regulations of retinal neurogenesis
1．10 Differentiation of retinal neurons
1．1 1 Photoreceptor specification
1．12 Determination of cone opsin expression patterns
1．13 Cell lineage specificity of neural retina
1．14 Background
1．15 Aims
Chapter 2 Materials and methods
2．1 Key resources
2．2 Gene Knockout
2．3 CRISPR Cloning
2．4 Transformation of CRISPR Clone in bacterial cells
2．7 Fluorescence activated cell sorting (FACS) of Transfected Cells
2．8 CRISPR/Cas9-Mediated Deletion and off-target detection
2．9 hiPSCs culture
2．10 Retinal organoid differentiation
2．11 Immunofluorescent assay
2．12 Real-time quantitative reverse transcription polymerase chain reaction and RNA-seq
2．13 ATAC sequencing
2．14 RNA-seq analysis
2．15 Quantification and statistical analysis
Chapter 3 Results
3．2 Generation of NRL-/- ROs from BC1-eGFP hiPSCs
3．3 Comparison of transcriptome analysis of wild type and NRL-/-ROs derived from hiPSCs
3．4 RNA seq of wild type and NRL-/- organoids derived from hiPSCS
3．5 Prediction and checking of off-targets sites by RNA sequencing
3．7 RNA analysis to find out the important TF/gene need to be used to improve the enrichment of cones Nrl-/- mice versus human NRL-/- ROs
3．8 Experiment verification of new findings
Chapter 4 Discussion
4．1 Discussion
References
Acknowledgment
Publications
中国科学技术大学;
