Dynamic full-field optical coherence tomography module adapted to commercial microscopes allows longitudinal in vitro cell culture study.

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility.

Interface self-referenced dynamic full-field optical coherence tomography.

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as an invaluable live label-free and non-invasive imaging modality able to image subcellular biological structures and their metabolic activity within complex 3D samples. However, D-FFOCT suffers from fringe artefacts when imaging near reflective surfaces and is highly sensitive to vibrations. Here, we present interface Self-Referenced (iSR) D-FFOCT, an alternative configuration to D-FFOCT that takes advantage of the presence of the sample coverslip in between the sample and the objective by using it as a defocused reference arm, thus avoiding the aforementioned artefacts.

Bankable human iPSC-derived retinal progenitors represent a valuable source of multipotent cells.

Retinal progenitor cells (RPCs) are the source of all retinal cell types during retinogenesis. Until now, the isolation and expansion of RPCs has been at the expense of their multipotency. Here, we report simple methods and media for the generation, expansion, and cryopreservation of human induced pluripotent stem-cell derived-RPCs (hiRPCs).

Modeling PRPF31 retinitis pigmentosa using retinal pigment epithelium and organoids combined with gene augmentation rescue.

Mutations in the ubiquitously expressed pre-mRNA processing factor (PRPF) 31 gene, one of the most common causes of dominant form of Retinitis Pigmentosa (RP), lead to a retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed in PRPF31 patients. Retinal organoids and retinal pigment epithelial (RPE) cells derived from human-induced pluripotent stem cells (iPSCs) provide potential opportunities for studying human PRPF31-related RP.

Dynamic full-field optical coherence tomography allows live imaging of retinal pigment epithelium stress model.

Retinal degenerative diseases lead to the blindness of millions of people around the world. In case of age-related macular degeneration (AMD), the atrophy of retinal pigment epithelium (RPE) precedes neural dystrophy. But as crucial as understanding both healthy and pathological RPE cell physiology is for those diseases, no current technique allows subcellular in vivo or in vitro live observation of this critical cell layer.

Reproducing diabetic retinopathy features using newly developed human induced-pluripotent stem cell-derived retinal Müller glial cells.

Muller glial cells (MGCs) are responsible for the homeostatic and metabolic support of the retina. Despite the importance of MGCs in retinal disorders, reliable and accessible human cell sources to be used to model MGC-associated diseases are lacking. Although primary human MGCs (pMGCs) can be purified from post-mortem retinal tissues, the donor scarcity limits their use.

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells.

Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies.

Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids.

Optical coherence tomography offers astounding opportunities to image the complex structure of living tissue but lacks functional information. We present dynamic full-field optical coherence tomography as a technique to noninvasively image living human induced pluripotent stem cell-derived retinal organoids. Coloured images with an endogenous contrast linked to organelle motility are generated, with submicrometre spatial resolution and millisecond temporal resolution, creating a way to identify specific cell types in living tissue via their function.

[Retinal organoids as a new tool for understanding and treating retinal diseases].

Generation of retinal organoids from pluripotent stem cells represents an important advance in the study of retinal development and offer new perspectives for the study of retinal diseases missing suitable animal models. Understanding the key stages of retinal development in vertebrates enabled to design protocols to generate self-organized three-dimensional structures derived from pluripotent stem cells and containing all retinal cell types. In addition to their application in basic research, such as the characterization of molecular and cellular mechanisms in retinal pathophysiology, these miniature organs also open up encouraging prospects in the field of cell therapy or the screening of therapeutic molecules, although some obstacles remain to be overcome.

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

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.

Generation of human induced pluripotent stem cell lines from a patient with ITM2B-related retinal dystrophy and a non mutated brother.

Human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of a patient affected with an autosomal dominant retinal dystrophy carrying the mutation c.782A>C, p.Glu261Ala in ITM2B and from an unaffected brother.

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests.

Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells.

The reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) has broad applications in regenerative medicine. The generation of self-organized retinal structures from these iPSCs offers the opportunity to study retinal development and model-specific retinal disease with patient-specific iPSCs and provides the basis for cell replacement strategies. In this study, we demonstrated that the major type of glial cells of the human retina, Müller cells, can be reprogrammed into iPSCs that acquire classical signature of pluripotent stem cells.

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids.

Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids.

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing.

Noninvasive gene delivery to foveal cones for vision restoration.

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques.

Establishment of an induced pluripotent stem (iPS) cell line from dermal fibroblasts of an asymptomatic patient with dominant PRPF31 mutation.

A human iPS cell line was generated from fibroblasts of a phenotypically unaffected patient from a family with PRPF31-associated retinitis pigmentosa (RP). The transgene-free iPS cells were generated with the human OSKM transcription factors using the Sendai-virus reprogramming system. iPS cells contained the expected c.

Generation of an induced pluripotent stem cell (iPSC) line from a patient with autosomal dominant retinitis pigmentosa due to a mutation in the NR2E3 gene.

A human iPSC line was generated from fibroblasts of a patient affected with autosomal dominant Retinitis Pigmentosa (RP) carrying the mutation p.Gly56Arg in the NR2E3 gene. The transgene-free iPSCs were generated with the human OSKM transcription factors using the Sendai-virus reprogramming system.

Otx2-Genetically Modified Retinal Pigment Epithelial Cells Rescue Photoreceptors after Transplantation.

Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease.

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells.

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF-α.

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14 blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human-induced pluripotent stem cell-derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF-α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others.

Production of Retinal Cells from Confluent Human iPS Cells.

Human induced pluripotent stem (hiPS) cells could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. Although much progress has been made in the differentiation of pluripotent stem cells towards different retinal lineages, the production of retinal cells from hiPS cells for therapeutic approaches require the development of easy and standardized protocols. In this chapter, we describe a simple and effective protocol for retinal differentiation of hiPS cells bypassing embryoid body formation and the use of exogenous molecules and substrates.