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AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

ABSTRACT: Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.

SUBMITTER: Garita-Hernandez M

PROVIDER: S-EPMC7036814 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

Garita-Hernandez Marcela M Routet Fiona F Guibbal Laure L Khabou Hanen H Toualbi Lyes L Riancho Luisa L Reichman Sacha S Duebel Jens J Sahel Jose-Alain JA Goureau Olivier O Dalkara Deniz D

International journal of molecular sciences 20200203 3

Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AA ...[more]

PMID: 32028585

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Project description:Background Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated. Methods A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes. Results In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix. Conclusions This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02651-9.

Dataset Information

AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

Publications

AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

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