运用人类人工诱导多能干细胞衍生的视网膜类器官研究感光细胞分化中的视网膜色素上皮细胞和NRL基因的作用
发布时间：2023-04-19 19:35
视觉的形成依赖于光感受器细胞与视网膜色素上皮(Retinal pigment epithelium,RPE)之间功能的相互协调作用,这种稳态的破坏可能导致失明。因此,RPE与视网膜之间的相互作用在发育阶段以及成年视网膜的正常功能中至关重要。成熟的人视网膜是中枢神经系统种一种复杂而精细的感觉器官,缺乏再生能力。因此,对人视网膜的任何损伤都可能导致视力下降甚至失明。日常生活中,视网膜直接暴露在强光下或者受到有害因素的影响都会容易导致光感受器细胞退化和死亡。正常生理功能的RPE能够通过多种途径来减轻这些有害因素对视网膜功能的影响。此外,RPE对于视网膜外层的正常生理至关重要,具有对脱落的光感受器细胞外段的吞噬作用以及神经营养和血管营养类型生长因子的分泌功能。源自人诱导多能干细胞(hiPSC)的视网膜类器官(Retinal organoid,RO)重构了人视网膜的三维结构,模仿了人类视网膜的发育,并为临床前视网膜移植提供了细胞来源。然而,体外RO-RPE共培养能否促进RO中光感受器细胞的分化成熟仍然未知。在本文中,我们在体外成功地通过连续的诱导过程将hiPSC分化成为RO。在分化过程中,RO的...
【文章页数】：164 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
PROJECT Ⅰ
Chapter 1 Introduction
1.1 Stem cell culture
1.2 Classification and sources of stem cells
1.2.1 Stem cells classification according to their origin
1.2.1.1 Embryonic Stem Cells (ESCs)
1.2.1.2 Embryonic Germ Stem Cells
1.2.1.3 Fetal stem cells
1.2.1.4 Infant stem cell
1.2.1.4.1 Umbilical cord stem cells
1.2.1.4.2 Wharton's jelly
1.2.1.5 Adult stem cell
1.2.1.6 Mesenchymal stem cells
1.2.1.7 Hematopoietic stem cells
1.2.1.8 Neural Stem Cells
1.2.1.9 Gastrointestinal stem cells
1.2.1.10 Epidermal stem cells
1.2.1.11 Hepatic stem cells
1.2.1.12 Pancreatic stem cells
1.2.1.13 Cancer stem cells
1.2.2 Types of stem cells according to their differentiation
1.2.2.1 Totipotent stem cells
1.2.2.2 Pluripotent stem cells
1.2.2.3 Multipotent stem cells
1.2.2.4 Unipotent stem cell
1.2.2.5 Oligopotent stem cells
1.3 Generation of induced pluripotent stem cells (iPSCs)
1.4 Application of iPSCs
1.4.1 Disease modeling
1.4.2 Regenerative medicine
1.4.3 Drug discovery
1.5 Organoid culture
1.6 Applications of Organoids
1.6.1 Developmental biology
1.6.2 Disease pathology of infectious disease
1.6.3 Drug toxicity and efficacy testing
1.6.4 Personalized medicine
1.6.5 Organoid and tumor microenvironment
1.7 Retinal organoids (ROs)
1.8 Retinal organoid based disease models
1.9 Challenges in stem cell differentiation into retinal organoids and theirsolutions
1.9.1 Time
1.9.2 Maturation
1.9.3 Lack of extra-retinal structures
1.9.4 Genetic variations
1.9.5 Scalability and automation
1.9.6 Mimicking complexity
1.10 Retinal pigment epithelium
1.10.1 Trans-epithelial Transport
1.10.2 Transport from Blood to Photoreceptors
1.10.3 Transport from Sub-retinal Space to Blood
1.10.4 Absorption of Light and Protection against Photo-oxidation
1.10.5 Visual Cycle
1.10.6 Phagocytosis
1.10.7 Secretion
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
3.1.1 Generation of hiPSC-derived photoreceptors using 2D/3D differentiation
3.1.2 hiPSC-derived ROs mimic human retina
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
3.2.1 Primary culturing of mouse retinal pigment epithelium
3.2.2 Effects of the time period upon the morphological features of RPE culture
3.2.3 Co-culture duration of ROs with RPE
3.3
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
3.6.1 ROs-RPE co-culture at later stages of ROs differentiation
3.6.2 Participation of RPE in accelerated differentiation of photoreceptors atlater stages of ROs
3.6.3 Effects of ROs-RPE co-culture upon other early and late-bornphotoreceptors markers
3.7 Effect of ROs-RPE co-culture on H9 cells derived ROs at early and laterstages of differentiation
Graphical abstract
Chapter 4 Discussion
4.1 Discussion
Conclusions and future directions
References
PROJECT Ⅱ
Summary
Chapter 1 Introduction
1.1 Retina of mammals
1.2 Structure of the mammalian eye
1.2.1 External layer
1.2.2 Intermediate layer
1.2.3 Internal layer
1.3 Structure and functions of the neural retina
1.3.1 Retinal Cellular organization
1.3.2 Retinal neuron types
1.3.2.1 Photoreceptors
1.3.2.2 Bipolar cells
1.3.2.3 Horizontal cells
1.3.2.4 Amacrine cells
1.3.2.5 Ganglion cells
1.3.2.6 Muller glia
1.4 Cell biology of vision
1.4.1 Structure of photoreceptors
1.4.2 Rod outer segments
1.4.3 Cone outer segment
1.4.4 Cell soma
1.4.5 Synaptic terminals
1.5 Photo-transduction
1.6 The visual cycle
1.6.1 Rod visual cycle
1.6.2 Cone visual cycle
1.7 Disc renewal
1.8 Retinal development
1.8.1 Early eye development
1.8.1.1 Morphogenesis of the early eye
1.8.1.2 Genetic regulators of eye morphogenesis
1.8.2 Role of soluble factors in early eye morphogenesis
1.9 Regulations of retinal neurogenesis
1.9.1 Overview of histogenesis in the retina
1.9.2 Role of Notch signaling in retinal neurogenesis
1.10 Differentiation of retinal neurons
1.10.1 Retinal ganglion cells
1.10.2 Horizontal cells
1.10.3 Amacrine cells
1.10.4 Bipolar cells
1.10.5 Muller glia
1.11 Photoreceptor specification
1.12 Determination of cone opsin expression patterns
1.13 Cell lineage specificity of neural retina
1.13.1 Dominant Transcriptional model of photoreceptor differentiation
1.13.2 Diverse progenitors intrinsically give rise to specified progeny
1.13.3 The existence of retinoic acid signaling components in eye during development
1.13.4 Role of Retinoic acid during morphogenesis of eye
1.13.5 Role of retinoic acid during photoreceptor differentiation
1.14 Background
1.15 Aims
Chapter 2 Materials and methods
2.1 Key resources
2.2 Gene Knockout
2.2.1 CRISPR Design
2.3 CRISPR Cloning
2.3.1 Annealing of each pair of protospacer oligos (Cas9 expression cassette)
2.3.2 Digestion of backbone vector
2.3.3 Ligation of annealed oligos into pX330
2.4 Transformation of CRISPR Clone in bacterial cells
2.5 Preparation of CRISPR/Cas9 plasmid
2.6 CRISPR/Cas9 plasmid transfections (Nucleofection)
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 andRNA-seq
2.13 ATAC sequencing
2.14 RNA-seq analysis
2.15 Quantification and statistical analysis
Chapter 3 Results
3.1 Generation of hiPSC-NRL-/-derived photoreceptors
3.2 Generation of NRL-/- ROs from BC1-eGFP hiPSCs
3.3 Comparison of transcriptome analysis of wild type and NRL-/- ROs derivedfrom 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
3.6 RNA analysis to find out the important TF/gene need to be used to improvethe enrichment of cones Nrl-/- mice versus human NRL-/- ROs
3.7 Experiment verification of new findings
Chapter 4 Discussion
4.1 Discussion
References
Acknowledgment
Publications
本文编号：3794086
【文章页数】：164 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
PROJECT Ⅰ
Chapter 1 Introduction
1.1 Stem cell culture
1.2 Classification and sources of stem cells
1.2.1 Stem cells classification according to their origin
1.2.1.1 Embryonic Stem Cells (ESCs)
1.2.1.2 Embryonic Germ Stem Cells
1.2.1.3 Fetal stem cells
1.2.1.4 Infant stem cell
1.2.1.4.1 Umbilical cord stem cells
1.2.1.4.2 Wharton's jelly
1.2.1.5 Adult stem cell
1.2.1.6 Mesenchymal stem cells
1.2.1.7 Hematopoietic stem cells
1.2.1.8 Neural Stem Cells
1.2.1.9 Gastrointestinal stem cells
1.2.1.10 Epidermal stem cells
1.2.1.11 Hepatic stem cells
1.2.1.12 Pancreatic stem cells
1.2.1.13 Cancer stem cells
1.2.2 Types of stem cells according to their differentiation
1.2.2.1 Totipotent stem cells
1.2.2.2 Pluripotent stem cells
1.2.2.3 Multipotent stem cells
1.2.2.4 Unipotent stem cell
1.2.2.5 Oligopotent stem cells
1.3 Generation of induced pluripotent stem cells (iPSCs)
1.4 Application of iPSCs
1.4.1 Disease modeling
1.4.2 Regenerative medicine
1.4.3 Drug discovery
1.5 Organoid culture
1.6 Applications of Organoids
1.6.1 Developmental biology
1.6.2 Disease pathology of infectious disease
1.6.3 Drug toxicity and efficacy testing
1.6.4 Personalized medicine
1.6.5 Organoid and tumor microenvironment
1.7 Retinal organoids (ROs)
1.8 Retinal organoid based disease models
1.9 Challenges in stem cell differentiation into retinal organoids and theirsolutions
1.9.1 Time
1.9.2 Maturation
1.9.3 Lack of extra-retinal structures
1.9.4 Genetic variations
1.9.5 Scalability and automation
1.9.6 Mimicking complexity
1.10 Retinal pigment epithelium
1.10.1 Trans-epithelial Transport
1.10.2 Transport from Blood to Photoreceptors
1.10.3 Transport from Sub-retinal Space to Blood
1.10.4 Absorption of Light and Protection against Photo-oxidation
1.10.5 Visual Cycle
1.10.6 Phagocytosis
1.10.7 Secretion
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
3.1.1 Generation of hiPSC-derived photoreceptors using 2D/3D differentiation
3.1.2 hiPSC-derived ROs mimic human retina
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
3.2.1 Primary culturing of mouse retinal pigment epithelium
3.2.2 Effects of the time period upon the morphological features of RPE culture
3.2.3 Co-culture duration of ROs with RPE
3.3
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
3.6.1 ROs-RPE co-culture at later stages of ROs differentiation
3.6.2 Participation of RPE in accelerated differentiation of photoreceptors atlater stages of ROs
3.6.3 Effects of ROs-RPE co-culture upon other early and late-bornphotoreceptors markers
3.7 Effect of ROs-RPE co-culture on H9 cells derived ROs at early and laterstages of differentiation
Graphical abstract
Chapter 4 Discussion
4.1 Discussion
Conclusions and future directions
References
PROJECT Ⅱ
Summary
Chapter 1 Introduction
1.1 Retina of mammals
1.2 Structure of the mammalian eye
1.2.1 External layer
1.2.2 Intermediate layer
1.2.3 Internal layer
1.3 Structure and functions of the neural retina
1.3.1 Retinal Cellular organization
1.3.2 Retinal neuron types
1.3.2.1 Photoreceptors
1.3.2.2 Bipolar cells
1.3.2.3 Horizontal cells
1.3.2.4 Amacrine cells
1.3.2.5 Ganglion cells
1.3.2.6 Muller glia
1.4 Cell biology of vision
1.4.1 Structure of photoreceptors
1.4.2 Rod outer segments
1.4.3 Cone outer segment
1.4.4 Cell soma
1.4.5 Synaptic terminals
1.5 Photo-transduction
1.6 The visual cycle
1.6.1 Rod visual cycle
1.6.2 Cone visual cycle
1.7 Disc renewal
1.8 Retinal development
1.8.1 Early eye development
1.8.1.1 Morphogenesis of the early eye
1.8.1.2 Genetic regulators of eye morphogenesis
1.8.2 Role of soluble factors in early eye morphogenesis
1.9 Regulations of retinal neurogenesis
1.9.1 Overview of histogenesis in the retina
1.9.2 Role of Notch signaling in retinal neurogenesis
1.10 Differentiation of retinal neurons
1.10.1 Retinal ganglion cells
1.10.2 Horizontal cells
1.10.3 Amacrine cells
1.10.4 Bipolar cells
1.10.5 Muller glia
1.11 Photoreceptor specification
1.12 Determination of cone opsin expression patterns
1.13 Cell lineage specificity of neural retina
1.13.1 Dominant Transcriptional model of photoreceptor differentiation
1.13.2 Diverse progenitors intrinsically give rise to specified progeny
1.13.3 The existence of retinoic acid signaling components in eye during development
1.13.4 Role of Retinoic acid during morphogenesis of eye
1.13.5 Role of retinoic acid during photoreceptor differentiation
1.14 Background
1.15 Aims
Chapter 2 Materials and methods
2.1 Key resources
2.2 Gene Knockout
2.2.1 CRISPR Design
2.3 CRISPR Cloning
2.3.1 Annealing of each pair of protospacer oligos (Cas9 expression cassette)
2.3.2 Digestion of backbone vector
2.3.3 Ligation of annealed oligos into pX330
2.4 Transformation of CRISPR Clone in bacterial cells
2.5 Preparation of CRISPR/Cas9 plasmid
2.6 CRISPR/Cas9 plasmid transfections (Nucleofection)
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 andRNA-seq
2.13 ATAC sequencing
2.14 RNA-seq analysis
2.15 Quantification and statistical analysis
Chapter 3 Results
3.1 Generation of hiPSC-NRL-/-derived photoreceptors
3.2 Generation of NRL-/- ROs from BC1-eGFP hiPSCs
3.3 Comparison of transcriptome analysis of wild type and NRL-/- ROs derivedfrom 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
3.6 RNA analysis to find out the important TF/gene need to be used to improvethe enrichment of cones Nrl-/- mice versus human NRL-/- ROs
3.7 Experiment verification of new findings
Chapter 4 Discussion
4.1 Discussion
References
Acknowledgment
Publications
本文编号：3794086
本文链接：https://www.wllwen.com/shoufeilunwen/yxlbs/3794086.html
教材专著
