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Abstract 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. We demonstrate here that RPE cells carrying PRPF31 mutations present important morphological and functional changes and that PRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level of PRPF31 expression may explain the defective phenotypes of PRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes by PRPF31 gene augmentation provide the proof of concept for future therapeutic strategies.

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. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.

Summary: 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. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation. : Gagliardi and colleagues show the possibility of isolating a homogeneous and functional population of photoreceptors from human induced pluripotent stem cells by targeting of a single surface antigen, CD73. Transplanted human cells are able to be incorporated into a host retina, generating mostly cone photoreceptors. Keywords: retina, photoreceptor, organoids, cell sorting, neurodegeneration, transplantation, iPSC, cell therapy

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. These Müller glial cell-derived iPSCs were able to differentiate toward retinal fate and generate concomitantly retinal pigmented epithelial cells and self-forming retinal organoid structures containing retinal progenitor cells. Retinal organoids recapitulated retinal neurogenesis with differentiation of retinal progenitor cells into all retinal cell types in a sequential overlapping order. With a modified retinal maturation protocol characterized by the presence of serum and high glucose levels, our study revealed that the retinal organoids contained pseudolaminated neural retina with important features reminiscent of mature photoreceptors, both rod and cone subtypes. This advanced maturation of photoreceptors not only supports the possibility to use 3D retinal organoids for studying photoreceptor development but also offers a novel opportunity for disease modeling, particularly for inherited retinal diseases.

Abstract 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). Thawed and passed hiRPCs maintained biochemical and transcriptional RPC phenotypes and their ability to differentiate into all retinal cell types. Specific conditions allowed the generation of large cultures of photoreceptor precursors enriched up to 90% within a few weeks and without a purification step. Combined RNA-seq analysis between hiRPCs and retinal organoids identified genes involved in developmental or degenerative retinal diseases. Thus, hiRPC lines could provide a valuable source of retinal cells for cell-based therapies or drug discovery and could be an advanced cellular tool to better understand retinal dystrophies.

Sight is a major sense for human and visual impairment profoundly affects quality of life, especially retinal degenerative diseases which are the leading cause of irreversible blindness worldwide. As for other neurodegenerative disorders, almost all retinal dystrophies are characterized by the specific loss of one or two cell types, such as retinal ganglion cells, photoreceptor cells, or retinal pigmented epithelial cells. This feature is a critical point when dealing with cell replacement strategies considering that the preservation of other cell types and retinal circuitry is a prerequisite. Retinal ganglion cells are particularly vulnerable to degenerative process and glaucoma, the most common optic neuropathy, is a frequent retinal dystrophy. Cell replacement has been proposed as a potential approach to take on the challenge of visual restoration, but its application to optic neuropathies is particularly challenging. Many obstacles need to be overcome before any clinical application. Beyond their survival and differentiation, engrafted cells have to reconnect with both upstream synaptic retinal cell partners and specific targets in the brain. To date, reconnection of retinal ganglion cells with distal central targets appears unrealistic since central nervous system is refractory to regenerative processes. Significant progress on the understanding of molecular mechanisms that prevent central nervous system regeneration offer hope to overcome this obstacle in the future. At the same time, emergence of reprogramming of human somatic cells into pluripotent stem cells has facilitated both the generation of new source of cells with therapeutic potential and the development of innovative methods for the generation of transplantable cells. In this review, we discuss the feasibility of stem cell-based strategies applied to retinal ganglion cells and optic nerve impairment. We present the different strategies for the generation, characterization and the delivery of transplantable retinal ganglion cells derived from pluripotent stem cells. The relevance of pluripotent stem cell-derived retinal organoid and retinal ganglion cells for disease modeling or drug screening will be also introduced in the context of optic neuropathies.

Behavioral analyses of mice intoxicated by the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) have generated conflicting results. We therefore analyzed the relationship between behavioral changes, loss of monoamine levels, and loss of dopaminergic cell bodies in groups of mice intoxicated with acute or subchronic MPTP protocols. Despite a higher degree of neuronal loss in the mice intoxicated using subchronic protocols, dopamine loss was severe and homogeneous in the striatum in all groups. Dopamine levels were less severely reduced in the frontal cortex in the three groups of MPTP-intoxicated mice. Norepinephrine and serotonin levels in the striatum were decreased only in the mice intoxicated with the acute protocol. The most surprising result was that the mice intoxicated with the subchronic protocols were more active than the saline-treated mice. As reported in rats with dopamine depletion in the prefrontal cortex, the hyperactivity observed in our mice could be due to the reduced dopamine levels detected in this structure.