運用人類人工誘導多能干細胞衍生的視網(wǎng)膜類器官研究感光細胞分化中的視網(wǎng)膜色素上皮細胞和NRL基因的作用
發(fā)布時間：2023-04-19 19:35
視覺的形成依賴于光感受器細胞與視網(wǎng)膜色素上皮(Retinal pigment epithelium,RPE)之間功能的相互協(xié)調作用,這種穩(wěn)態(tài)的破壞可能導致失明。因此,RPE與視網(wǎng)膜之間的相互作用在發(fā)育階段以及成年視網(wǎng)膜的正常功能中至關重要。成熟的人視網(wǎng)膜是中樞神經系統(tǒng)種一種復雜而精細的感覺器官,缺乏再生能力。因此,對人視網(wǎng)膜的任何損傷都可能導致視力下降甚至失明。日常生活中,視網(wǎng)膜直接暴露在強光下或者受到有害因素的影響都會容易導致光感受器細胞退化和死亡。正常生理功能的RPE能夠通過多種途徑來減輕這些有害因素對視網(wǎng)膜功能的影響。此外,RPE對于視網(wǎng)膜外層的正常生理至關重要,具有對脫落的光感受器細胞外段的吞噬作用以及神經營養(yǎng)和血管營養(yǎng)類型生長因子的分泌功能。源自人誘導多能干細胞(hiPSC)的視網(wǎng)膜類器官(Retinal organoid,RO)重構了人視網(wǎng)膜的三維結構,模仿了人類視網(wǎng)膜的發(fā)育,并為臨床前視網(wǎng)膜移植提供了細胞來源。然而,體外RO-RPE共培養(yǎng)能否促進RO中光感受器細胞的分化成熟仍然未知。在本文中,我們在體外成功地通過連續(xù)的誘導過程將hiPSC分化成為RO。在分化過程中,RO的...
【文章頁數(shù)】：164 頁
【學位級別】：博士
【文章目錄】：
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
本文編號：3794086
【文章頁數(shù)】：164 頁
【學位級別】：博士
【文章目錄】：
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
本文編號：3794086
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