Lise Morizur

Herardot Elise

Monville Christelle
email: cmonville@istem.fr

Ben M'

Barek Karim
email: kbenmbarek@istem.fr

Human Pluripotent Stem Cells: a Toolbox to Understand And Treat Retinal Degeneration

Keywords: Human pluripotent stem cells, Retinopathies, Cell therapy, Disease modeling, Age-related Macular Degeneration, Retinitis Pigmentosa

Introduction

Composed of different neural layers, the retina converts light inputs into electrical signals, which are then transmitted to the brain. Photoreceptors (PRs), cones and rods, are the primary cells involved in light detection. Rods are sensitive to dim light whereas cones detect specific wavelengths allowing color vision. The electrical signal generated by PRs is further processed by other neurons (bipolar, amacrine, horizontal cells) and transmitted to the brain through ganglion cells. A number of retinal diseases, such as Age-related Macular Degeneration and Retinitis Pigmentosa, leads to the degeneration of PRs which ultimately results in vision impairment and in some cases in blindness. Although highly informative, animal models are not able to recapitulate all the pathological hallmarks of these pathologies and human diseased tissues are difficult to obtain. Development of differentiation protocols to generate retinal cells from human pluripotent stem cells (hPSCs) has considerably broaden the access to human cellular models of retinal degeneration. They offer renewed opportunities to better understand the underlying pathological mechanisms and develop new therapeutics. In addition, retinal tissues derived from healthy hPSCs also represent a promising source for regenerative medicine. This review will discuss these different aspects of the use of hPSCs to understand and treat retinal disorders.

Retinal degenerative diseases 2.1. Age-related Macular Degeneration

After uncorrected refractive errors and cataracts, AMD is the third leading cause of moderate or severe vision loss worldwide [START_REF] Flaxman | Global causes of blindness and distance vision impairment 1990-2020: a systematic review and meta-analysis[END_REF]. Projections for 2040 are alarming with an estimated number of people affected with all stages of the disease of 288 million [START_REF] Wong | Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis[END_REF]. Interestingly, the prevalence of all stages of AMD is higher (12.3%) in people with European ancestry than Asian (7.4%) or African (7.5%) populations [START_REF] Wong | Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis[END_REF]. The geographically isolated population of Timor-Leste has an even lower prevalence for AMD -less than 0.5% -suggesting the influence of genetic context on disease appearance [START_REF] Morrison | Ancestry of the Timorese: age-related macular degeneration associated genotype and allele sharing among human populations from throughout the world[END_REF]. AMD is classified according to the stage of the disease based on ophthalmic examinations. Early or intermediate AMD is characterized by the presence in the macula of medium drusen (diameter between 63 and 125µm; early AMD) or large drusen (larger than 125µm) and changes in retinal pigmentation (intermediate AMD) [START_REF] Mitchell | Age-related macular degeneration[END_REF].

Late AMD is defined by the presence of macular choroidal neovascularisation (wet AMD, corresponding to 10-20% of cases) or macular atrophy (dry AMD, corresponding to 80-90% of cases) [START_REF] Mitchell | Age-related macular degeneration[END_REF][START_REF] Al-Khersan | Innovative therapies for neovascular age-related macular degeneration[END_REF]. Early AMD is usually asymptomatic [START_REF] Mitchell | Age-related macular degeneration[END_REF]. Initial symptoms are distortion of central vision progressing towards scotomas in the central visual field. In wet AMD, new vessels develop in the macular area and are susceptible to leak and generate hemorrhages, damaging the macula [START_REF] Handa | A systems biology approach towards understanding and treating non-neovascular age-related macular degeneration[END_REF]. Vision loss can be fast and severe in this context. Vision loss in dry AMD is more progressive with the degeneration of the retinal pigment epithelium (RPE) and subsequent atrophy of macular retina.

AMD is caused by a combination of genetic and environmental factors. The major risk factor is age. The prevalence of all stages of AMD is of 3.5% between the ages of 55 and 59 years but rises to 27.3% after 85 years in the European population [START_REF] Colijn | Prevalence of Age-Related Macular Degeneration in Europe: The Past and the Future[END_REF]. Smoking is a critical modifiable risk factor in the development of late AMD [START_REF] Mitchell | Smoking and the 5-year incidence of agerelated maculopathy: the Blue Mountains Eye Study[END_REF]. Other environmental risks include sunlight exposition, diet, alcohol and hypertension. Among the genes associated with increased risk for AMD, complement factor H (CFH) and age-related maculopathy susceptibility 2/ hightemperature requirement factor A1 (ARMS2/HTRA1) polymorphisms are to date the most important [START_REF] Handa | A systems biology approach towards understanding and treating non-neovascular age-related macular degeneration[END_REF]. Several mechanisms are suggested to play a role in AMD such as inflammation, defective regulation of the complement system, oxidative stress, metabolic dysfunctions, accumulation of lipids and extracellular matrix alterations [START_REF] Handa | A systems biology approach towards understanding and treating non-neovascular age-related macular degeneration[END_REF][START_REF] Hussain | Understanding the complexity of the matrix metalloproteinase system and its relevance to age-related diseases: Age-related macular degeneration and Alzheimer's disease[END_REF][START_REF] Brown | Metabolomics and Age-Related Macular Degeneration[END_REF]. However, the exact pathological mechanisms are still not fully understood. Accumulation of proteins and lipids in the Bruch's membrane is suspected to promote inflammation and affect the circulation of nutrients and liquids, damaging the RPE. In addition, RPE degeneration in dry AMD may precipitate the secondary loss of PRs.

No treatment is currently available for the dry form of AMD. The most effective clinical treatment for wet AMD patients is anti-VEGF therapy delivered at regular intervals to prevent worsening eyesight. Although response to therapy is not systematic, some patients experience a moderate improvement in visual acuity [START_REF] Al-Khersan | Innovative therapies for neovascular age-related macular degeneration[END_REF]. Patient compliance to treatment protocol could be one of the reasons for incomplete response as injections are frequent (once a month or every 2 months) and expensive. Diet supplementation with zinc and antioxidants in AMD patients has also been suggested to decrease the risk of progression to late AMD [START_REF]Age-Related Eye Disease Study Research G (2001) A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8[END_REF].

Retinitis Pigmentosa

RP is a heterogeneous group of inherited retinal dystrophies, which affects 1.5 million of patients worldwide [START_REF] Verbakel | Non-syndromic retinitis pigmentosa[END_REF]. The inheritance of RP is autosomal-recessive in 50-60% of all cases and linked to chromosome X in 5-15% of patients [START_REF] Pfeiffer | Persistent remodeling and neurodegeneration in late-stage retinal degeneration[END_REF][START_REF] Hartong | Retinitis pigmentosa[END_REF]. Syndromic forms of RP account for 20-30% of cases, including Usher's and Bardet-Biedl syndromes (the most frequent ones) [START_REF] Hartong | Retinitis pigmentosa[END_REF].

The ophthalmic eye fundus examination reveals the classical clinical triad of RP: bone spicule pigmentation, which may correspond to melanin deposits from RPE cells that have migrated into the retina, attenuation of retinal vessels that could be secondary to PR loss and waxy pallor of the optic nerve likely due to the presence of glial cells [START_REF] Verbakel | Non-syndromic retinitis pigmentosa[END_REF]. While clinical manifestations vary according to the underlying genetic defect, RP patients typically experience night blindness -which may not be noticed by patients at early stages -followed by a progressive reduction of the visual field (tunnel vision). RP patients also report seeing flashes of light (photopsia) which worsen under stress [START_REF] Bittner | A survey of photopsias in self-reported retinitis pigmentosa: location of photopsias is related to disease severity[END_REF][START_REF] Bittner | Photopsias are related in part to perceived stress and positive mood in retinitis pigmentosa[END_REF]. Central vision is usually preserved until the later stages of the disease. The median age of symptom appearance is 29year old [START_REF] Iftikhar | Classification of disease severity in retinitis pigmentosa[END_REF]. Of note, the age of onset as well as the clinical manifestation and progression of the disease vary depending on the mutated gene in addition to genetic background and environmental factors. Most patients with RP are legally blind by 40-year old due to severe visual field alterations [START_REF] Hartong | Retinitis pigmentosa[END_REF].

The primary cellular defect is localized in rods or, for about 5% of cases, in the RPE [START_REF] Ben | Stem Cell-Based RPE Therapy for Retinal Diseases: Engineering 3D Tissues Amenable for Regenerative Medicine[END_REF]. At advanced stages of the disease, optical coherence tomography shows that the ganglion cell layer and inner nuclear layer are relatively well preserved while the PR layer is completely depleted. However, a retinal remodeling occurs following the complete loss of PRs leading after decades of degeneration to rewiring, glial hypertrophy and global cell death [START_REF] Pfeiffer | Persistent remodeling and neurodegeneration in late-stage retinal degeneration[END_REF]. This important aspect of the pathology suggests that the retinal circuitry does not degenerate immediately after loss of PRs. RP patients could thus be eligible to PR cell transplantation during a specific time window. To date, a vast number of disease-causing variants in more than 80 genes for RP have been identified (https://sph.uth.edu/retnet/sum-dis.htm). Most of these genes encode proteins that play a role in the photo-transduction cascade, the visual cycle, ciliary structures and transport, RNA splicing as well as intracellular trafficking [START_REF] Verbakel | Non-syndromic retinitis pigmentosa[END_REF][START_REF] Hartong | Retinitis pigmentosa[END_REF]. However, disease-causing mutations are still unknown in 30% of non-syndromic and 50% of autosomal-dominant RP patients [START_REF] Verbakel | Non-syndromic retinitis pigmentosa[END_REF][START_REF] Hartong | Retinitis pigmentosa[END_REF].

Few therapeutic options are available to treat RP. The only approved treatment is a gene therapy for patients with RPE65 gene mutations (2% of RP cases, Luxturna®) [START_REF] Ben | Cell Therapy for Retinal Dystrophies: From Cell Suspension Formulation to Complex Retinal Tissue Bioengineering[END_REF]. This therapy was shown to be safe and induced an improvement in navigational abilities and light sensitivity. The effect was found maximal at 30-day post-injection and stable for at least 4 years [START_REF] Maguire | Efficacy, Safety, and Durability of Voretigene Neparvovec-rzyl in RPE65 Mutation-Associated Inherited Retinal Dystrophy: Results of Phase 1 and 3 Trials[END_REF]. Vitamin A palmitate as well as docosahexaenoic acid (DHA, an omega-3 fatty acid present in oily fish) supplementations are recommended by some clinicians. While still debated, a few studies reported that such nutritional treatments might delay vision degradation [START_REF] Hartong | Retinitis pigmentosa[END_REF][START_REF] Rayapudi | Vitamin A and fish oils for retinitis pigmentosa[END_REF][START_REF] Zhao | Vitamins and Mineral Supplements for Retinitis Pigmentosa[END_REF]. Approximately 40-50% of individuals with RP develop cataracts that further impair their visual acuity. These patients can benefit from cataract surgery with significant visual gain [START_REF] Bayyoud | Long-term clinical results after cataract surgery with and without capsular tension ring in patients with retinitis pigmentosa: a retrospective study[END_REF][START_REF] Auffarth | Complicated cataracts in various forms of retinitis pigmentosa. Type and incidence[END_REF]. Finally, night vision devices (ie. specific goggles) that amplify light can be used by patients to improve their mobility [START_REF] Hartong | Improved mobility and independence of night-blind people using night-vision goggles[END_REF][START_REF] Ikeda | Night-vision aid using see-through display for patients with retinitis pigmentosa[END_REF].

Generation of retinal cells from human pluripotent stem cells

The development of physiologically relevant cellular models is a key step towards modeling the complexity of retinal diseases. Indeed, it is essential to accurately capture the biological complexity of the retina in vitro to better understand the underlying pathological mechanisms and ultimately find new therapeutics. The different cell sources to generate such models include early postnatal PR precursors from animal models, human fetal and adult primary cells or tissues, and more recently hPSCs -including both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) (reviewed in [START_REF] Ben | Cell Therapy for Retinal Dystrophies: From Cell Suspension Formulation to Complex Retinal Tissue Bioengineering[END_REF]). hPSCs have gained great interest from the research community owing to their unlimited self-renewal potential and their ability to differentiate into any cell type of the adult body. First derived in 1998, hESCs are isolated from the inner cell mass of human blastocyst-stage embryos [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF].

Sharing the same key properties, hiPSCs are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by the forced expression of a cocktail of pluripotency factors [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF]. Besides their use in disease modeling studies, they also represent a renewable cell source that enable the derivation of large numbers of functional retinal cells for regenerative medicine.

Eye development

Substantial progress has been made over the last 15 years in the development of robust protocols to differentiate both hESCs and hiPSCs into retinal cells by mimicking in vitro the developmental steps occurring in the human embryo. The vertebrate retina is derived from the neuroectoderm, initially through the concomitant inhibition of Activin/Nodal, bone morphogenetic protein (BMP) and Wnt/β-catenin signaling [START_REF] Smith | Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm[END_REF][START_REF] Chambers | Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling[END_REF][START_REF] Fuhrmann | Wnt signaling in eye organogenesis[END_REF]. Shortly after gastrulation, the anterior neural plate is specified into the eye field in which cells co-express a unique combination of transcription factors called the eye-field transcription factors (EFTFs). Then the eye field evaginates from the diencephalon to form bilateral optic vesicles containing retinal stem cells that will give rise to the optic stalk, RPE and neural retina (NR) [START_REF] Fuhrmann | Eye morphogenesis and patterning of the optic vesicle[END_REF]. These retinal stem cells commit towards a specific cell lineage after invagination into the bi-layered optic cup depending on their initial regionalization: while the NR originates from the inner layer, the outer layer gives rise to the RPE [START_REF] Zhao | Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells[END_REF]. Within the naive NR, retinal progenitor cells (RPCs) give rise in a temporal and conserved order to the different retinal cell types, including PR precursors, to form the mature retina with its characteristic laminar cytoarchitecture. This differentiation process is tightly regulated by exogenous signals coming from adjacent tissues and cell-intrinsic factors, such as Wnt, fibroblast growth factor (FGF), BMP, Notch, sonic hedgehog (SHH), retinoic acid (RA) and activin A signalings [START_REF] Zhao | Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells[END_REF]. Levering on developmental studies, the overall strategy is to expose hPSCs to signaling molecules in a time and dose dependent manner along the differentiation process to generate the retinal cell type of interest. For an in-depth description of differentiation protocols, we refer the reader to excellent review articles [START_REF] Leach | Concise Review: Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients With Ocular Disease[END_REF][START_REF] Llonch | Organoid technology for retinal repair[END_REF][START_REF] Gagliardi | Photoreceptor cell replacement in macular degeneration and retinitis pigmentosa: A pluripotent stem cell-based approach[END_REF].

Derivation of RPE cells

Both hESCs and hiPSCs have the potency to differentiate into RPE cells that are equally effective in protecting the retina from degeneration following transplantation into the subretinal space of a RP rodent model [START_REF] Riera | Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies[END_REF]. A summary of selected differentiation protocols to generate RPE cells from hPSCs is listed in Table S1.

Initial studies demonstrated the spontaneous differentiation potential of hPSCs into RPE cells upon FGF2 withdrawal from the culture medium [START_REF] Klimanskaya | Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics[END_REF][START_REF] Buchholz | Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells[END_REF][START_REF] Ferguson | Deprivation of bFGF Promotes Spontaneous Differentiation of Human Embryonic Stem Cells into Retinal Pigment Epithelial Cells[END_REF][START_REF] Ben | Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration[END_REF]. After a few weeks in culture, pigmented areas begin to emerge and cells acquire a distinctive cobblestone morphology. These pigmented patches are manually isolated and amplified to obtain a pure population of RPE cells (Figure 1). However, such "spontaneous" protocol is highly operator-dependent, time-consuming (2-3 months) and does not allow for the production of large cell banks. To overcome these limitations, studies have focused on directing RPE differentiation by adding signaling molecules that mimic developmental cues known to be important in RPE development and specification at critical time points. Early work by the group of Masayo Takahashi reported that hPSCs exposed to Wnt and Nodal antagonists using a SFEB (serum-free floating culture of embryoid body-like aggregates) system gave rise to 30-35% of cells positive for the RPE characteristic transcription factors PAX6 and MITF after 6 weeks [START_REF] Osakada | Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells[END_REF][START_REF] Hirami | Generation of retinal cells from mouse and human induced pluripotent stem cells[END_REF]. Two other studies showed that addition of nicotinamide and Activin A, a member of the TGF-β superfamily, promoted the differentiation of hPSCs to neural and subsequently to RPE fate with 80% of pigmented cells after 8 weeks in culture [START_REF] Idelson | Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells[END_REF][START_REF] Kokkinaki | Human induced pluripotent stem-derived retinal pigment epithelium (RPE) cells exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression pattern similar to native RPE[END_REF]. Based on previous research [START_REF] Idelson | Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells[END_REF][START_REF] Koh | A 2020 vision for healthy people[END_REF][START_REF] Lamba | Efficient generation of retinal progenitor cells from human embryonic stem cells[END_REF], Buchholz et al. further combined use of retinal inducing factors (Noggin, DKK1, insulin-like growth factor-1 (IGF-1), FGF2) and known RPE differentiation factors (nicotinamide, Activin A, FGFR/VEGFR inhibitor SU5402, and vasoactive intestinal peptide -VIP) [START_REF] Buchholz | Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium[END_REF]. Addition of these factors at specific times led to the conversion of approximately 80% of the cells to an RPE phenotype in only 14 days, as evidenced by the expression of pigmentation marker PMEL17 [START_REF] Buchholz | Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium[END_REF]. Although these directed protocols considerably increased the yield of RPE cells from hPSCs compared to that obtained with the spontaneous method, mechanical enrichment of pigmented cells during the course of the differentiation was still required to obtain a homogenous RPE cell population. The same team improved the protocol by addition of CHIR99021, an activator of the Wnt canonical pathway.

It improved significantly the efficiency of RPE derivation from hESCs to 97%, bypassing the necessity of manual enrichment [START_REF] Leach | Canonical/beta-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells[END_REF]. However, it is important to note that this protocol did not significantly reduce the differentiation duration from that of the "spontaneous" method as RPE cells were banked after more than 100 days in culture [START_REF] Leach | Canonical/beta-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells[END_REF]. More recently, our group developed a simplified protocol in which hESCs were treated with nicotinamide, Activin A and CHIR99021 in a sequential manner to obtain highly enriched and functional RPE cells within 84 days [START_REF] Regent | Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions[END_REF].

As a step toward clinical use in regenerative medicine, differentiation protocols were optimized to obtain RPE cells: i) in conditions using a good manufacturing practice (GMP)compliant production process and ii) in large numbers to treat the millions of patients affected by RPE-associated retinal degeneration (Figure 1). Some studies addressed these bottlenecks by developing xeno-free/ feeder-free protocols [START_REF] Reichman | Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions[END_REF][START_REF] Choudhary | Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage[END_REF][START_REF] Reyes | Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model[END_REF][START_REF] Pennington | Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate[END_REF][START_REF] Vaajasaari | Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells[END_REF]. The development of directed differentiation protocols has fostered the implementation of automated systems to upscale the production process and increase its robustness. The use of a modular platform was reported for the long-term maintenance and passaging of hiPSCs and suggested the potential to differentiate cells to retinal lineages, including RPE [START_REF] Crombie | Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells[END_REF]. Matsumoto et al.

further demonstrated the feasibility of the automated culture of hiPSC-derived RPE cell sheets in a closed automated cell-culture system, removing the need for operational skills and facilities [START_REF] Matsumoto | Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine[END_REF]. We recently achieved a fully automated process allowing the large-scale production of RPE cells from hPSCs, considerably upscaling the production capacity as more than 16 billion of mature and functional RPE cells could now be produced within 12 weeks with only one round of production [START_REF] Regent | Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions[END_REF].

Derivation of photoreceptors

A summary of selected differentiation protocols to generate PRs from hPSCs is presented in Table S2.

Pioneering work from the group of Thomas Reh demonstrated the capacity of hESCs to differentiate in vitro into PRs (Figure 2) [START_REF] Lamba | Efficient generation of retinal progenitor cells from human embryonic stem cells[END_REF]. Using a combination of the eye field inducer IGF-1, BMP signaling inhibitor Noggin and Wnt/β-catenin signaling pathway antagonist Dkk1, they were able to direct hESCs into 80% of PAX6+/VSX2+ retinal progenitors after 3 weeks in culture [START_REF] Fuhrmann | Wnt signaling in eye organogenesis[END_REF]. However, differentiation of these RPCs into PRs was infrequent with only 12% of CRXpositive PR precursors and less than 0.01% of S-Opsin-or Rhodopsin-positive mature PRs [START_REF] Fuhrmann | Wnt signaling in eye organogenesis[END_REF].

In two other studies, Osakada et al. cultured hPSCs as embryoid bodies using a SFEB system in presence of Wnt and Nodal antagonists for 20 days before switching them to adherent culture (3D/2D protocol) with the addition of factors known to be involved in rod genesis (RA, taurine, FGF2, Shh) [START_REF] Osakada | Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells[END_REF][START_REF] Osakada | In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction[END_REF]. Twenty percent of CRX-positive cells were obtained, giving rise later to approximately 8.5% of Rhodopsin-positive mature PRs by day 200 [START_REF] Osakada | Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells[END_REF][START_REF] Osakada | In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction[END_REF]. It was also the first demonstration that inhibition of Notch signaling in vitro with the γ-secretase inhibitor DAPT induced the cell cycle exit of proliferating RPCs and a concomitant increase in early born ganglion cells and CRX-positive PR precursors [START_REF] Osakada | Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells[END_REF]. Using a similar 3D/2D approach, Meyer and collaborators showed that hPSCs could differentiate into early and late PR phenotypes without the need of exogenous factors in a sequence and time course highly reminiscent of normal retinal development [START_REF] Meyer | Modeling early retinal development with human embryonic and induced pluripotent stem cells[END_REF]. This protocol was further refined into a 3D/2D/3D method to isolate neural rosette-containing cell colonies maintained as cellular aggregates in suspension [START_REF] Meyer | Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment[END_REF].

Using this technique, a subpopulation of aggregates developed optic vesicle characteristics and differentiated toward the PR lineage with 56% of CRX-positive cells by day 80 [START_REF] Meyer | Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment[END_REF].

Although these studies successfully recapitulated the main steps of retinal development in vitro, key structural and functional features of PRs, i.e. the presence of outer-segment discs and light sensitivity, were still lacking.

Considerable progress came with the first production of self-organized bi-layered optic cup structures from hESCs that then differentiated into a multilayered neural retina containing retinal cells, including cones and rods (Figure 2) [START_REF] Nakano | Self-formation of optic cups and storable stratified neural retina from human ESCs[END_REF]. Importantly, this study was the first to report the formation of PRs with reasonable development of inner segments and connecting cilia [START_REF] Nakano | Self-formation of optic cups and storable stratified neural retina from human ESCs[END_REF]. Based on the protocol developed by Meyer et al. [START_REF] Meyer | Modeling early retinal development with human embryonic and induced pluripotent stem cells[END_REF][START_REF] Meyer | Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment[END_REF], the group of Valeria Canto-Soler more recently demonstrated that hiPSCs could also self-organize into retinal cups that generate a fully laminated 3D retinal tissue in presence of serum, RA and taurine [START_REF] Zhong | Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs[END_REF].

Noteworthy, it was the first evidence that PRs reached an advanced stage of maturation in vitro as evidenced by some level of photosensitivity in a limited number of cells and the presence of a minority of PRs with outer-segment discs after 27 weeks of differentiation [START_REF] Zhong | Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs[END_REF].

Although introduction of 3D organoid technology has dramatically improved the generation of mature PRs, current protocols are still hindered by variability issues. A recent study by Capowski et al. generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time in an attempt to develop a staging system to reduce inconsistencies in cultures [START_REF] Capowski | Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines[END_REF]. Three consistent morphological stages of hPSCderived retinal organoid development were distinguished by light microscopy, each one corresponding to a specific cellular composition and lamination relative to the timeline of differentiation [START_REF] Capowski | Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines[END_REF].

For future clinical use of hPSC-derived PRs, recent studies have focused on adapting these research-grade protocols to GMP-grade under feeder-free and xeno-free conditions [START_REF] Reichman | Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions[END_REF][START_REF] Choudhary | Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage[END_REF][START_REF] Wiley | cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness[END_REF]. While functional RPE cells are currently derived from hESCs with high yield and purity using GMP-compliant differentiation protocols, challenges remain for PR production. This is notably due to extremely long culturing times required to obtain mature PRs in vitro (>100 days), operator-dependent manual enrichment of neural retina / optic cup structures as well as heterogeneous cellular composition of organoids. Different selection strategies have been developed to enrich PRs from retinal cultures. A screen of human retinal samples and hPSCderived retinal organoids against a large panel of human monoclonal antibodies using a high throughput flow cytometry approach identified PR-specific cell surface markers [START_REF] Lakowski | Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-Derived Retinal Organoids and Fetal Retinae[END_REF]. Double negative CD29/SSEA-1 selection increased the enrichment of CRX and Recoverin positive cells from 16.5% to 61% from day 200 retinal organoid cultures [START_REF] Lakowski | Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-Derived Retinal Organoids and Fetal Retinae[END_REF]. In addition, a cone biomarker panel combining CD markers for positive cone selection (CD26, CD147, CD133) and negative selection of undesirable cells (SSEA-1) successfully enriched L/S-opsin cones from fetal retina and 17-18 week-old hESC-derived retinal samples to 30% and 50% respectively [START_REF] Welby | Isolation and Comparative Transcriptome Analysis of Human Fetal and iPSC-Derived Cone Photoreceptor Cells[END_REF]. The group of Olivier Goureau also showed that CD73 targeting by magnetic-activated cell sorting (MACS), previously described for the enrichment of mouse PSC-derived PRs [START_REF] Eberle | Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina[END_REF], was an effective strategy to isolate a transplantable population of PRs from hiPSC-derived retinal organoids at day 120 [START_REF] Gagliardi | Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids[END_REF].

Age-related macular degeneration

As a multifactorial and late onset disease, AMD is by nature difficult to model. The genetic contribution to the disease is well established with CFH and ARMS2/HTRA1 variants being the most strongly associated [START_REF] Handa | A systems biology approach towards understanding and treating non-neovascular age-related macular degeneration[END_REF]. In a recent genome-wide association study, 52 independent risk variants distributed across 34 loci were also identified [START_REF] Fritsche | A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants[END_REF]. However, their exact roles are still largely unknown. Thus, the global strategy is to evaluate the implication of the different risk alleles associated to AMD, focusing on the most affected cell type (ie. RPE cells). Thereafter, we will only focus on AMD models linked to CFH and ARMS2/HTRA1 variants.

AMD hiPSC derived RPE (hiPSC-RPE) cells carrying the ARMS2/HTRA1 high-risk genotype did not present an obvious diseased phenotype but were found similar to 1-year old wildtype monkey RPE [START_REF] Yang | Validation of genome-wide association study (GWAS)-identified disease risk alleles with patientspecific stem cell lines[END_REF]. As lipofuscin fluorophore A2E accumulate with age in normal RPE cells, hiPSC-RPE cells were treated with A2E for 10 days. In that context, hiPSC-RPE cells from AMD patients homozygous for the high-risk haplotype exhibited a reduced protein level and activity of superoxide dismutase 2 (SOD2), suggesting a compromised antioxidant capacity [START_REF] Yang | Validation of genome-wide association study (GWAS)-identified disease risk alleles with patientspecific stem cell lines[END_REF]. This was confirmed following exposure to blue light in order to generate oxidative stress [START_REF] Yang | Validation of genome-wide association study (GWAS)-identified disease risk alleles with patientspecific stem cell lines[END_REF]. The same results were also obtained by an exposure to H2O2 [START_REF] Golestaneh | Repressed SIRT1/PGC-1alpha pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration[END_REF]. In addition, this last study identified a dysfunction of the NAD-dependent deacetylase sirtuin1 (SIRT1) and peroxisome proliferator activated receptor gamma coactivator 1-alpha (PGC-1α) pathway that is suggested to alter mitochondrial biogenesis and remodeling causing increased reactive oxygen species (ROS) production [START_REF] Golestaneh | Repressed SIRT1/PGC-1alpha pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration[END_REF]. The group of Sally Temple further characterized the effect of the homozygous ARMS2/HTRA1 risk genotype and evidenced a significantly higher expression of complement and inflammatory factors similarly to what is observed in AMD patients [START_REF] Saini | Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration[END_REF][START_REF] Kauppinen | Inflammation and its role in age-related macular degeneration[END_REF] (Figure 3). hiPSC-RPE cells carrying the high-risk Y402H polymorphism in CFH gene did not exhibit obvious morphological or functional defects (RPE markers, secretion of PEDF, transepithelial resistance, phagocytosis potential). A deeper characterization revealed signs reminiscent of AMD, including increased inflammation, cellular stress, accumulation of lipid droplets and deposition of drusen-like deposits [START_REF] Hallam | An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure[END_REF] (Figure 3). SOD2 gene expression level was, interestingly, also reduced in hiPSC-RPE cells carrying the high risk allele. When CFH gene is overexpressed, hiPSC-RPE cells exposed to oxidized lipids are protected from cell death mediated by a caspase dependent apoptosis process [START_REF] Borras | CFH exerts anti-oxidant effects on retinal pigment epithelial cells independently from protecting against membrane attack complex[END_REF]. Taken together, these results suggest that the CFH gene function in response to oxidative stress is altered by the high-risk Y402H polymorphism.

Retinitis Pigmentosa

hPSC technology has also been used over the last decade as a model platform to study RP, the most common form of hereditary retinal disorder (Table 1). In the first report of an in vitro disease model, the group of Masayo Takahashi derived hiPSCs from RP patients carrying known causative mutations in the RP1 axonemal microtubule associated protein (RP1), Pim-1 kinase associated protein (RP9), peripherin 2 (PRPH2) or rhodopsin (RHO) gene [START_REF] Jin | Modeling retinal degeneration using patient-specific induced pluripotent stem cells[END_REF][START_REF] Jin | Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling[END_REF]. As observed in RP, patient-derived rod PRs from all mutations selectively degenerated in vitro.

These cells expressed markers for endoplasmic reticulum stress (RHO mutation) and oxidative stress (RP9 mutation) [START_REF] Jin | Modeling retinal degeneration using patient-specific induced pluripotent stem cells[END_REF][START_REF] Jin | Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling[END_REF]. hiPSCs derived from a RP patient harboring a different RHO mutation (E181K) in another study reproduced these findings [START_REF] Yoshida | The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa[END_REF]. This pathological phenotype was reverted to normal in PRs after correction of the expression of RHO using a helper-dependent adenoviral vector [START_REF] Yoshida | The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa[END_REF]. However, it should be noted that the gene correction was performed in hiPSCs, not in derived retinal cells. It is unclear whether this rescue could be also obtained in retinal cells and thus be relevant to gene therapy in RP patients.

Disease Mutation

Cell In an innovative approach, the exome sequencing of an individual with sporadic RP led to the identification of a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK), a protein normally expressed in the inner segments, cell bodies, and axons of rods and cones [START_REF] Tucker | Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa[END_REF]. hiPSCs were then generated from a RP patient homozygous for the Alu insertion and a non-MAK-associated RP patient as control. Retinal cells derived from the hiPSCs of the RP patient with the MAK Alu insertion presented a loss of a tissue-specific MAK splice variant normally found in retinal precursors [START_REF] Tucker | Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa[END_REF]. The same group later used a similar strategy to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP [START_REF] Tucker | Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa[END_REF]. USH2A mutations were associated to an upregulation of GRP78 and GRP94 indicative of protein misfolding and subsequent ER stress in hiPSC-derived PR precursors [START_REF] Tucker | Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa[END_REF]. When grafted in mice's subretinal space, these patient's cells were able to develop and mature as PRs. This suggests that the mutation may not interfere with the normal development of the retina but rather with the survival of PRs later in life. Such patients may therefore be amenable to preventive therapeutic strategies before the onset of degeneration. TRNT1, a CCA-adding enzyme which belongs to the tRNA nucleotidyltransferase/poly(A) polymerase family functions by adding CCA to the 3′ end of all tRNA precursors. A mutation in this gene was expected to cause protein misfolding and ER stress. However, it was not the case in hiPSCderived retinal organoids from RP patients with mutations in TRNT1 [START_REF] Sharma | Patient-specific induced pluripotent stem cells to evaluate the pathophysiology of TRNT1-associated Retinitis pigmentosa[END_REF]. The cells rather exhibited a defect in autophagy as evidenced by an accumulation of the microtubuleassociated protein LC3-II and elevated levels of oxidative stress [START_REF] Sharma | Patient-specific induced pluripotent stem cells to evaluate the pathophysiology of TRNT1-associated Retinitis pigmentosa[END_REF].

Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) and RP2 gene account respectively for 70-90% and 10-20% of X-linked RP (XLRP) leading to rapid vision loss in boys and young men [START_REF] Bird | X-linked retinitis pigmentosa[END_REF][START_REF] Hardcastle | Mutations in the RP2 gene cause disease in 10% of families with familial Xlinked retinitis pigmentosa assessed in this study[END_REF][START_REF] Breuer | A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa[END_REF]. Using a 3D retinal differentiation protocol, Megaw et al. generated

PRs from hiPSCs carrying a RPGR mutation to investigate its role in PR maintenance and its molecular pathogenesis mechanisms [START_REF] Megaw | Gelsolin dysfunction causes photoreceptor loss in induced pluripotent cell and animal retinitis pigmentosa models[END_REF]. This group identified an increased actin polymerization in the PR connecting cilia, resulting in rhodopsin mislocalisation to the inner segment and ultimately in cell stress and degeneration [START_REF] Megaw | Gelsolin dysfunction causes photoreceptor loss in induced pluripotent cell and animal retinitis pigmentosa models[END_REF]. CRISPR/Cas9-mediated hiPSC gene editing correction of the mutation rescued both PR structure and electrophysiological properties in retinal organoids [START_REF] Deng | Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients[END_REF]. Mutations in the RP2 gene is the second most common cause of XLRP. In accordance with early studies hinting at the central role of RP2 in vesicle trafficking and cilia function, Schwarz et al. characterized the phenotype of hiPSC-RPE cells from an RP2 patient that lost the RP2 protein expression. These cells displayed mislocalization of the intraflagellar transport protein IFT20 and a disrupted Golgi cohesion [START_REF] Schwarz | Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells[END_REF]. They later showed in hiPSC-derived 3D optic cups that the RP2 protein, along with the small GTPase ARL3, were key regulators of the trafficking of ciliary tip kinesins [START_REF] Schwarz | Arl3 and RP2 regulate the trafficking of ciliary tip kinesins[END_REF]. Interestingly, the use of translational read-through inducing drugs caused an increase in functional RP2 protein levels, reversing the observed disease phenotypes in hiPSC derived retinal cells [START_REF] Schwarz | Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells[END_REF][START_REF] Schwarz | Arl3 and RP2 regulate the trafficking of ciliary tip kinesins[END_REF].

While the primary cellular defect in RP patients is in most cases localized in PRs, abnormalities in the RPE are also reported in about 5% of cases [START_REF] Ben | Stem Cell-Based RPE Therapy for Retinal Diseases: Engineering 3D Tissues Amenable for Regenerative Medicine[END_REF]. For instance, mutations in Membrane

Frizzled-related Protein (MFRP), a RPE-specific type II transmembrane protein similar to WNTbinding frizzled proteins, cause autosomal recessive RP. To investigate disease mechanisms, hiPSC-RPE cells were derived from two patients with MFRP mutations. These cells had altered actin polymerization, abnormal morphology with less pigments, mislocalized pigment distribution, as well as loss of clear cellular boundaries and cell-to-cell contacts [START_REF] Li | Gene therapy in patient-specific stem cell lines and a preclinical model of retinitis pigmentosa with membrane frizzled-related protein defects[END_REF].

Furthermore, reintroducing normal MFRP expression with AAV vector therapy directly in hiPSC-RPE cells reversed the pathological phenotype [START_REF] Li | Gene therapy in patient-specific stem cell lines and a preclinical model of retinitis pigmentosa with membrane frizzled-related protein defects[END_REF]. This result suggests that gene therapy could be used to correct disease-causing mutations in RPE cells.

One of the most important functions of RPE cells is their ability to phagocyte shed PR outer segments (POS), a process vital for proper retinal function. Indeed, defects in RPE phagocytosis lead to an accumulation of cellular debris in the subretinal space ultimately resulting in progressive degeneration of PRs, as seen in the RCS rat, a widely used model for recessively inherited retinal degeneration [START_REF] Cruz | Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat[END_REF]. Individuals with mutations in MER receptor tyrosine kinase (MERTK), the human orthologue of the RCS rat retinal dystrophy gene, are affected with severe and progressive RP [START_REF] Gal | Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa[END_REF]. MERTK-signaling in the retinal pigment epithelium was later shown to be essential for efficient phagocytosis of POS by RPE cells [START_REF] Feng | Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells[END_REF]. In accordance with these observations, hiPSC-RPE cells from individuals harboring mutations in the MERTK gene were unable to mediate the engulfment of POS [START_REF] Ramsden | Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs[END_REF]100]. The use of translational readthrough inducing drugs were able to restore MERTK expression and phagocytosis function [START_REF] Ramsden | Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs[END_REF].

Screening strategies for drug discovery

Besides enabling in-depth mechanistic studies of disease phenotypes in vitro, the development of robust differentiation protocols of hPSCs into retinal cells has proven instrumental in the development of high-throughput drug screening strategies to identify novel therapeutic agents. Drug discovery has relied for a long time on molecular target-based approaches, also called "reverse pharmacology" [101]. Such hypothesis-driven strategy relies on the identification of a molecular target whose activity or expression is directly associated to a disease pathogenesis. Compounds are then screened for their ability to bind and modulate the target. On the contrary, a phenotypic approach does not require prior understanding of the molecular mechanism of action but rather focuses on identifying compounds capable of reversing a phenotype thought to be representative of the disease. The Similarly, Yoshida et al. screened for agents able to protect rods from the accelerated cell loss induced by the rhodopsin mutation [START_REF] Yoshida | The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa[END_REF]. hiPSC-derived PRs were treated with molecules that could modify ER stress-related pathways. In such context, rhodopsin-related cell loss was suppressed, with a concomitant reduction of ER stress and apoptosis markers, after treatment with mTOR inhibition (rapamycin, PP242), AMP kinase activation (AICAR), apoptosis signalregulating kinase 1 [ASK1] inhibition (NQDI-1), or suppression of protein synthesis (salubrinal) [START_REF] Yoshida | The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa[END_REF]. A proof-of-principle example of a target-based screening approach was also shown in hiPSC-RPE cells from a RP patient with a MERTK nonsense mutation [START_REF] Ramsden | Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs[END_REF]. Two translational read-through inducing drugs, G418 and PTC124 demonstrated their ability to restore MERTK gene expression and full-length protein level. In addition, PTC124 functionally rescued 12% of the phagocytic function of RP hiPSC-RPE cells [START_REF] Ramsden | Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs[END_REF]. Although it is now possible to synthesize and test increasingly large compound libraries, theoretically increasing the probability of identifying novel lead compounds, more than 85% of drug development programs across all indications fail to progress from phase I clinical trials to approval [112]. This gap between lead compound identification and success in the clinic might be attributed, at least in part, to the fact that the vast majority of high-throughput screening strategies relies on assays carried out on two-dimensional (2D) cell culture models that do not adequately recapitulate human pathophysiology. Indeed, while 2D models are well suited to study cell intrinsic deficits, they ultimately fail to recapitulate the cellular microenvironment, in particular endogenous signaling and cell-cell as well as cell-extracellular matrix interactions, limiting the extent to which diseases can be modeled. To overcome these shortcomings, three-dimensional (3D) culture systems that more closely resemble the native tissue architecture are becoming part of the drug discovery toolbox. At the forefront of this effort is the generation of retinal organoids that, as discussed before, more faithfully recapitulate some aspects of the histoarchitecture and cellular composition of the developing human retina both spatially and temporally. Although highly informative, it is important to note that retinal organoids might not necessarily be suited for RPE screens as they contain rather low number of RPE cells. organoids and organ-on-a-chip, they provided a controllable vasculature-like perfusion to the 3D culture system and recapitulated the interaction of mature PR segments with RPE cells.

While retinal organoids hold promising advantages

This microfluidic retina-on-a-chip was validated for compound screening and toxicological studies by exposing the system to two molecules whose pathological side effects on the retina had been previously described, the anti-malaria drug chloroquine and the antibiotic gentamicin [115,121]. While much work remains before complex retina-like 3D in vitro systems become standards for toxicity and drug screenings, the rapid development in recent years of organoids, biomaterials and microfluidic technologies are bringing this prospect closer to realization.

Human pluripotent stem cells for cell therapy 5.1. Strategies for tissue restoration

Depending on the retinal degenerative disease and its specific stage of progression, different cell types are lost. In AMD, RPE cells stop performing their support functions and degenerate, which then triggers PR death. While the primary cellular defect in RP patients is mostly localized in PRs, dysfunctional RPE cells are also reported in about 5% of cases. Cell therapy products for AMP and RP patients should be designed accordingly to replace or supplement the remaining RPE cells and/or PRs (Figure 4). The other major aspect of tissue restoration is to determine precisely the injection site as the grafted material cannot cover the totality of the retina. Even if RP affects the peripheral retina first, the macula is ultimately damaged.

Thus, in AMD and RP, the macula is the targeted site of implantation to maximize visual recovery as visual acuity mostly rely upon this area. The second formulation approach is to deliver an already formed hPSC-RPE epithelium. This strategy drastically complicates the transport, storage and subretinal delivery. Indeed, as a living monolayer of cells, the graft can only be kept for a limited period of time in a controlled environment [128,129]. A supporting scaffold made of polymers or of biological composition is usually needed in vitro during epithelial reformation of hPSC-RPE cells to ensure that cells can be removed from the culture dish [START_REF] Ben | Stem Cell-Based RPE Therapy for Retinal Diseases: Engineering 3D Tissues Amenable for Regenerative Medicine[END_REF][130][131][132][133]. For implantation, specific devices have to be developed in order to deliver the hPSC-RPE sheet into the subretinal space [START_REF] Ben | Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration[END_REF]129,[134][135][136]. The rational leading to the development of this strategy is that RPE cells are functional only when organized as an epithelium with the secretion of cytokines [137]. In addition, the diseased environment with oxidative stress and altered Bruch's membrane could limit in vivo epithelial reformation of hPSC-RPE cells grafted as a cell suspension. Indeed, hESC-RPE are more resistant to oxidative stress, which is present in AMD and RP, when organized as monolayers [138]. Compared to cell suspension injections, the viability of hPSC-RPE cells grafted in the subretinal space of Nude rat was higher when delivered as a sheet [131]. Our group, as well as others, demonstrated that hPSC-RPE sheets improved the therapeutic visual outcomes of RCS rats compared to the same cells injected as a cell suspension [START_REF] Ben | Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration[END_REF]132].

Preliminary results of three human clinical trials using a RPE sheet formulation to treat AMD

PR cell therapy

For successful cell replacement, PR precursors need to integrate into the pre-existing neuronal network of the host retina and maturate with the formation of outer segments sensitive to light. Similarly, to RPE cells, they can either be delivered as a cell suspension or as an organized tissue.

Cell suspension injection

Pioneer studies provided evidence that stage-specific post-mitotic PR precursors from mice could integrate into the host outer nuclear layer (ONL -corresponding to the layer of PR It is important to note that all the above-mentioned studies were conducted with mouse donor cells. Recent transplantation experiments using human cells showed that material exchange was rather limited within the rodent eye as the vast majority of donor cells remained in the subretinal space and only few cells integrated into the host ONL [START_REF] Gagliardi | Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids[END_REF]157,158].

Therefore, cell suspension transplantation into primates are required to evaluate the level of material transfer, the integration of human donor cells into the ONL and their capacity to form outer segments in vivo.

When

Retinal sheet transplantation

An alternative to the injection of PRs as a cell suspension is the transplantation of retinal sheets. This approach, based on the dissection of retinal organoids in small sections, was PRs in the graft and to improve the product delivery into the eye in order to reduce rosette formation.

Future of retinal cell therapy

Both preclinical and clinical studies hint that the use of hPSC derivatives is a safe and viable therapeutic option for the treatment of retinal degenerative diseases. However, a number of important questions must be addressed before this strategy can be part of the arsenal of clinical tools, among which the challenge of transplant immune rejection, the development of more complex retinal grafts through tissue engineering and the potential combined use of optogenetic tools.

Graf rejection: immunological considerations

The success of cell replacement therapies does not rely solely on the quality and maturation of the grafted cell product but also on our understanding of the host immunological responses following transplantation. As ocular immune privilege provides the eye with immune protection, it was initially postulated that transplanted grafts into the eye would largely be protected from immune rejection. However, it is difficult to address the graft immunogenicity as hPSC derived retinal cells are usually grafted in a xenogeneic context and therefore under heavy immune suppression [START_REF] Ben | Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration[END_REF][START_REF] Gagliardi | Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids[END_REF]129,163,164,[167][168][169]. In addition, the diseased ocular microenvironment is likely to increase the risk of immune rejection. Autologous hPSC-derived cell products could overcome the rejection risk. However, such strategy is not achievable for wide-scale clinical practice. An alternative is to establish hPSC banks from "super-donors" homozygous for conserved human leucocyte antigen (HLA)

haplotypes that are representative of a specific population to enable HLA matching and therefore minimize the risk of allograft rejection [173]. Current estimates predict that a bank of hiPSCs from 100 to 150 highly selected donors would meet the needs of 50-90% of the recipient population in the UK [174,175]. However, the cost of such banks remains high.

Another promising strategy is the engineering of allogenic hPSCs to be hypoimmunogenic to both adaptive and innate immune responses using CRISPR-Cas9 gene editing. In a recent approach, hypoimmunogenic hESCs were generated after deletion of the highly polymorphic Cardiomyocytes, endothelial cells as well as smooth muscle cells derived from these cells reliably escaped immune rejection in MHC-mismatched allogenic recipients and survived longterm without the use of immunosuppression [177]. Thus, the generation of hypoimmunogenic hPSCs holds the promise of universal cell grafts from a unique cell bank.

Looking forward: scaffold-based tissue engineering

The final formulation of the cell therapy product has major impact on correct integration and polarization of the grafted material in the host cellular environment [START_REF] Ben | Cell Therapy for Retinal Dystrophies: From Cell Suspension Formulation to Complex Retinal Tissue Bioengineering[END_REF]. RGCs sensitive to light engender the loss of the sophisticated processing that occurs through the retina (from PRs to RGCs) while a small number of dormant cones remains. As significant progress is being made with the generation of more specific and improved opsins, the use of optogenetic tools could be broaden by combining stem cell therapy and optogenetics to counteract the difficulty to obtain functional PRs from hPSCs with light-sensitive outer segments [START_REF] Gagliardi | Photoreceptor cell replacement in macular degeneration and retinitis pigmentosa: A pluripotent stem cell-based approach[END_REF]. This was achieved recently in mice suggesting that this strategy may be viable

[158].

Conclusion

As highlighted in this review, hPSCs have become valuable tools to investigate the different stages of retinal degeneration and help tailor therapeutic strategies for the future. Before the complete loss of PRs, drug compounds identified through high throughput screening on RP/AMD hPSC models could be delivered to patients. When the degeneration is at an advanced stage, hPSCs differentiated into retinal cells could be transplanted into the eye to replace lost cells or to support remaining PRs through cytoplasmic exchanges. While significant roadblocks need to be addressed, the fast development of more physiologically relevant cellular models that accurately capture the biological complexity of the retina in vitro bring these expectations closer to reality. There is still a long way to go before first treatments reach patients but many phase 1/2 clinical trials have already started.

Figure 1 :

 1 Figure 1: Schematic representation of spontaneous and directed differentiation methods to obtain RPE cells from hPSCs based on selected studies.

Figure 2 :( 1 )

 21 Figure 2: Main protocols for the differentiation of hPSCs into PRs in vitro.

Figure 3 :

 3 Figure 3: Current model of pathological mechanisms underlying AMD based on hiPSC-RPE cell studies. Red arrows indicate components that are modulated by the disease (increased or decreased function). P: phosphorylation; Ac: acetylation; RPE: retinal pigment epithelium; POS: photoreceptor outer segment; ROS: reactive oxygen species.

  contribution of phenotypic screening to the discovery of new first-in-class small molecule drugs approved by the US Food and Drug Administration (FDA) between 1999 and 2008 largely exceeded that of target-based approaches [102]. A critical efficiency variable of this approach is the use of physiologically relevant cellular models that accurately capture the biological complexity of the disease state in vitro [103]. With the advent of PSC technology, hPSCderived cellular models have gained great interest from the scientific community as an alternative platform for drug screening [104].The group of Masayo Takahashi first evaluated the potential of candidate drugs in hiPSCderived PRs[START_REF] Jin | Modeling retinal degeneration using patient-specific induced pluripotent stem cells[END_REF]. As rod PRs from RP patients underwent degeneration in vitro similarly to what is observed in vivo, they assessed effects of antioxidant vitamins α-tocopherol, ascorbic acid, and β-carotene on this cellular model. Interestingly, α-tocopherol treatment significantly increased the number of rhodopsin-positive cells harboring RP9 mutation, providing proof-ofprinciple for drug screening based on the phenotype of hiPSC-derived retinal cells[START_REF] Kokkinaki | Human induced pluripotent stem-derived retinal pigment epithelium (RPE) cells exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression pattern similar to native RPE[END_REF][START_REF] Jin | Modeling retinal degeneration using patient-specific induced pluripotent stem cells[END_REF].

hPSC-based models

  of AMD were also successful in identifying disease-modulating compounds. Following the differentiation of AMD hiPSC into RPE cells, Chang et al. found that these cells displayed an abnormal accumulation of ROS. Therefore, to attenuate oxidative damage, they screened compounds to find an effective scavenger of ROS [105]. Among several dietary supplements for retinal protection and natural antioxidant compounds, the team demonstrated that curcumin effectively protected AMD hiPSC-RPE cells from H2O2-induced cell death through upregulation of several oxidative stress regulatory proteins and significant reduction of ROS production [105]. More recently, the group of Sally Temple used a panel of AMD biomarkers combined with transcriptome analysis to demonstrate that nicotinamide treatment improved AMD-related phenotypes. They suggested that this effect was mediated by the inhibition of the production of drusen proteins and VEGF, as well as by decreasing inflammatory and complement factors [72]. Recent machine learning approaches were developed to predict RPE cell functionality (transepithelial resistance, polarized VEGF secretion) [106]. Broaden use of deep neural networks applied to drug screening would be a potent alternative to identify new compounds. Bruch's membrane (BM), the extracellular matrix in which RPE cells are attached plays a critical function in AMD progression. Therefore, several strategies were developed to reproduce the interaction between BM and RPE cells [107]. In particular, non-enzymatic nitration of the extracellular matrix mimics the damages accumulated in aged BM [108]. BM is constantly attacked by matrix metalloproteinase and renewed by RPE and choroid [109]. In a similar manner, an immortalized RPE cell line (ARPE-19) was used in vitro to produce a BM-like extracellular matrix [108]. Alternatively, BM could be obtained from aged human cadavers [110]. First examples of AMD modeling demonstrated that AMD-like extracellular matrix perturbed functions of normal hiPSC-RPE cells (increased VEGF release, complement activation) [108]. Moreover, AMD hiPSC-RPE cells had reduced ability to attach and survive on nitrite modified extracellular matrix [111]. While not yet implemented in drug screening, future studies should take into account RPE matrix modifications on disease modeling as BM could affect both normal and AMD RPE cells.

  over other culture systems, several critical technical limitations still exist. The first concern is that organoids contain immature cells corresponding to a fetal stage, where PRs develop a partially mature organization and neurons display an abnormal synaptic connectivity [113, 114]. The addition of an extracellular matrix might improve connectivity and responsiveness to light [114]. Another concern is the cell death that occurs in organoid centers. Cells self-organize into 3D structures, develop a well-organized initial lamination and correct temporal production of the diverse cell types. However, cells localized in the center of the organoid will disorganize and undergo progressively cell death: this is particularly the case for retinal ganglion cells and inner cell layers [63, 113]. Another hurdle to overcome is that existing retinal organoid protocols are not able to generate a specific area of the retina such as the fovea or the macula, which is obviously a limitation when addressing macular degenerative diseases. The last limitation is related to the absence of RPE adjacent to PR. Indeed the microenvironment provided by RPE cells (nutrients, matrix, debris phagocytosis…) might be important particularly for the long culture duration required for PRs as suggested recently [115, 116]. Thus, organoids with current protocols are only a step forward that improve existing 2D models. Knowing these shortcomings, the use of retinal organoids for drug screening remains the object of intense research to overcome technical constraints. Indeed, incorporating 3D culture systems into miniaturized 384-or 1536-well formats in an automated and costeffective screening setup is still in infancy [117]. Two of the biggest challenges are the visualization of 3D structures with automated imaging systems well as the general lack of robust quantitative technologies to analyze organoids on a large scale [117, 118]. To address this need, Vergara et al. developed a screening platform that enabled accurate quantification of fluorescent reporters in complex hiPSC-derived retinal organoids, allowing for quantitative analysis of dynamics of developmental processes and cellular physiological states [118]. Of importance, technologies and readout assays that have been developed and successfully implemented in low-to high-throughput strategies in other 3D systems, for example cancerlike spheroids, could be adapted to retinal organoids [119, 120]. Adding another level of complexity, recent studies have focused on increasing the physiological relevance of hPSC-derived retinal models by juxtaposing a polarized RPE sheet to the retinal structure [115, 116]. Achberger et al. developed a microfluidic platform to create a complex multi-layer structure from hiPSCs that includes all cell types and layers present in retinal organoids in direct interplay with an RPE layer, recapitulating the complex in vivo anatomy of the human retina in vitro [115, 121]. Combining the two promising technologies,

Figure 4 : 5 . 1 . 1 .

 4511 Figure 4: Schematic representation of retinal degeneration in AMD/RP patients and associated cell therapy strategies. PR: photoreceptor; RPE: retinal pigment epithelium; AMD: Age-Related Macular Degeneration; RP: Retinitis Pigmentosa.5.1.1. RPE cell therapyRPE cells can either be delivered as a cell suspension or as an organized monolayer. The cell suspension formulation offers the advantage of a simplified logistic: cells are sent frozen to the hospital where they can be stored and thawed when desired. In addition, little manipulation is required before transplantation in the subretinal space and a simple syringe

  were published recently[133, 135, 139]. The graft monolayer was cultured over polyester[135], parylene[133] or without scaffold[139]. None of these studies reported a serious adverse event related to the grafted hPSC-RPE cells. While these studies were not designed to evaluate efficacy, first data are encouraging. In particular, Prof. Coffey and collaborators treated two severe exudative AMD patients with a hESC-RPE sheet on a polyester scaffold [135]. Such patients had lost dramatically their visual acuity due to retinal dysfunction [140]. If untreated, their retinas will degenerate and their vision permanently lost. Existing treatments consist of CNV removal and/or anti-VEGF medication that might improve the visual outcomes but the incidence of RPE rips is more frequent (17%) than without treatment (10%) [141]. These RPE rips expose Bruch's membrane and choroids and have a poor visual prognosis. The CNV removal itself does not improve visual acuity but reduce the risk of severe visual loss [142]. In Coffey's study, the CNV might have been removed unintentionally [143] and the outcomes should be taken with caution (few treated patients, no control untreated eyes or anti-VEGF treated eyes for comparison). Nevertheless, one of the two patients improved enough his visual acuity that he was able to read again [135].

  nuclei), differentiate to acquire the morphological characteristics of mature PRs and improve moderately the vision in murine models of retinal degeneration[144][145][146][147]. Altogether, these data led researchers to the conclusion that migration and integration of donor PR cells was the underlying mechanism of some visual function recovery.Five independent back-to-back studies published starting from late 2016 called for a reevaluation of this paradigm[148][149][150][151][152]. Indeed, the vast majority of transplanted PR precursors did not integrate into the host retina but rather remained in the subretinal space where they exchanged cytoplasmic material with host PRs. Following GFP positive cell grafting into the subretinal space of mice ubiquitously expressing a red fluorescent protein, cells coexpressing both reporters were found in the ONL, which indicates exchange of material[148][149][150]. In complementary experiments, Nrl-GFP mice were injected with the thymidine analogue EdU during development to independently label the nucleus and cytoplasm[149, 152]. While the majority of GFP+ donor cells in the subretinal space was EdU+, no EdU labelling was observed in GFP+ cells located in the host ONL. Thus, donor and host cells only exchanged their cytoplasmic content, not their nuclei. Gender-mismatched transplants confirmed this observation as the nuclei of GFP+ cells in the host ONL contained only the X-or Y-chromosome present in the host[148][149][150]. No polynucleated cells were observed. Such result suggests that GFP protein and/or mRNA was transmitted between donor/recipient cells. In light of these recent findings, we should reevaluate previous reports that had concluded that donors cells could integrate in the host ONL where they expressed donor proteins that were otherwise missing in the endogenous PRs of the recipient (such as rod α-Transducin, Peripherin-2 and Rhodopsin)[145, 146, 153, 154]. It can be inferred that these vision-related proteins may in fact be transferred from donor to host PRs through material exchange.The Cre/Lox system gave direct evidence for bidirectional exchange of intracellular material between donor and host cells[148][149][150]. Transplantation of donor PRs isolated from floxed reporter mice into PR-specific Cre mice showed expression of the reporter on transplanted cells in the subretinal space, revealing transfer of Cre-recombinase from host PRs to donor cells -and vice versa. Together these results demonstrate that while donor PR migration and integration do occur in rare events, the majority of transplanted cells engages in a process of material transfer through molecular mechanisms that remain to be determined. As a result, material transfer of functional proteins from murine donor cells to remaining host PRs after transplantation, rather than their structural integration, could explain partial recovery of visual function in retinal degeneration models. This material exchange appears to be specific between donor PR precursors/ host PRs. Indeed, no exchange was observed when GFP proteins were injected directly in the subretinal space or when GFP retinal progenitor cells as well as fibroblasts were transplanted[148]. This transfer is also limited to the ONL. Only one report, not confirmed by other studies, suggests that other cells types might be involved, including bipolars and Mullers [152]. The mechanisms involved in this material exchange are not elucidated but a vector is needed to mediate this intercellular communication. Several options are suggested including a fusion of donor/host plasma membranes or through the exchange of extracellular vesicles (like exosomes or microvesicles) [155]. This last proposition is attractive as such vesicles allow the transfer of mRNA / miRNA /proteins / lipids from one cell type to another with specificity [156].

  reevaluating only studies where donor PRs were transplanted in completely degenerated rodent to avoid cytoplasmic exchanges, visual gain remains unclear. For example, Rd1 mice with a degenerated ONL and zero functionality were transplanted with a GFP photoreceptor cell suspension at 10-12 weeks in 2013 [159]. The authors suggested an anatomical connection between donor rod and host bipolar cells (rod specific synaptic proteins, GFP labelling). This was correlated with pupil light responses. However, when reevaluating these results in light of material transfer, only separate analysis of cone and rod ERG responses could address each contribution to functionality. Indeed, in this model, cones could remain, even nonfunctional for a long time and it is not excluded that cones could be functionally rescued following rod transplantation and elicit a pupil light response [155]. When grafted in degenerated Rd1 mice, hESC derived photoreceptors differentiated into cones and appeared to make connection with bipolar cells as evidenced by histology, but without evaluation of visual functions [160]. Thus, in atrophic mice, donor PRs are able to make connection with host neurons but without a clear demonstration of visual functionality.

  MHC class I and class II molecules and expression of immunomodulatory factors PD-L1, HLA-G, and CD47 [176]. Accordingly, engineered vascular smooth muscle cells derived from these hESCs elicited significantly less T cell-and natural killer cell-mediated immune responses with minimal engulfment by macrophages [176]. CD47 overexpression and ablation of MHC class I and II were later found sufficient to generate hypoimmunogenic hiPSCs [177].

  Attempts at delivering single cell suspensions of hPSC-derived PRs and RPE cells have led to poor integration rate and limited visual recovery compared to their transplantation as cell sheets[START_REF] Ben | Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration[END_REF] 129,[148][149][150][163][164][165]. Importantly, PRs transplanted as cell sheets from retinal organoids formed synaptic connections and developed polarized segments[163][164][165].However, the formation of rosettes was also observed, within which outer segments of PRs were separated from the support of endogenous RPE cells[163, 164]. Levering on the clinical success of RPE sheet transplants grown on biocompatible substrates, the addition of supportive scaffolds is currently explored to deliver PRs in a more physiologically structured way. This may improve implantation and facilitate proper apical-basal polarization of the graft [178-180]. Of importance, Jung et al. recently developed a 3D biodegradable micro-structured scaffold designed to capture PRs in cup-shaped wells that support the correct orientation of axonal processes[180]. While the long-term biocompatibility and integration of such system within the host retina remains to be investigated in vivo, this scaffold-based tissue engineering approach provides an innovative way forward for the development of more complex retinal grafts. In addition, the design of bio-engineering tissues composed of a combination of layers of organized RPE cells and PRs might be a suitable graft for advanced stage of AMD and RP where RPE cells are defective and PRs lost.Combining stem cell therapy and optogeneticsOptogenetic uses light to control cells genetically modified to express an optogene encoding light-sensitive membrane proteins known as opsins[181]. The overall strategy is to convert surviving retinal neurons into light-sensitive cells and thereby functionally turn them into artificial PRs able to convert light into electrical signal to restore vision[182]. Importantly, the use of optogenes is a mutation-independent approach that can be used in a wide range of retinal degenerative conditions. Targeted electrical stimulation has been reported in dormant cone PRs lacking their light-sensitive outer segments[181, 183, 184] as well as in downstream retinal neurons such as RGCs [185-188] and bipolar cells[189][190][191]. These studies further hinted that the restored light information was transmitted to the visual cortex of formerly blind mice as they exhibited an improvement in visually guided behavioral responses. So far, two clinical trials based on this technology (targeting RGCs) are underway in patients affected with RP (clinicaltrials.gov identifiers: NCT03326336 and NCT02556736). However, rendering

Table 1 :

 1 hiPSC based models of RP and associated characteristic phenotypes.

  Similar results were reported in primates (only one animal at 1.5 year post-surgery) by measuring visually-guided saccades[166]. While the advantage of this strategy is to engraft an already formed and organized layer of PRs, additional work is needed to limit the presence of neurons other than

	evaluated after transplantation in rodents, primates and cats [161-163]. In such tissue, PRs
	are transplanted with other cell types [162-164] that might limit the connection with second
	order neurons of the recipient retina. In addition, implanted retinal sheets tend to form
	rosettes in vivo with outer segments inside, leading to a separation with RPE cells [161, 162,
	164].
	Despite these limitations, synaptic connections with host retinas and some visual recovery

were observed in rodent models of retinal degeneration, as evidenced by multi-electrode array recordings and assessment of visual behavior

[162,[164][165][166]

.

  For example, progressive loss of RPE cells as well as alteration of Bruch's membrane in AMD ultimately compromise the blood-retinal barrier, further degrading the immune privilege of the eye. Besides, the injection procedure itself could also favor transplant rejection by allowing immune cells to enter the retina. The result of this uncertainty is that the best immunosuppression strategy is still In vivo imaging and histological analysis evidenced a strong inflammatory response to MHC-mismatched allografts in a highly localized and aggressive manner by 4 days post-transplantation, continued through 3 weeks, ultimately resulting in the rejection of iPS-RPE grafts[171, 172].

	debated in ongoing and planned human clinical trials, with current strategies ranging from
	local to high systemic immunosuppression regimen [125, 133, 135].

One way to evaluate the immune response in an allogeneic context is to transplant monkey iPS-RPE allografts into the non-immune-suppressed subretinal space of monkeys. In that context, retinal tissue damage was observed in major histocompatibility complex (MHC)-mismatched monkeys but not in MHC-matched animals

[170][171][172]

.

Funding Statement

This work was supported by grants from the ANR [RebuildingRETINA: ANR-19-CE18-0004] and the Fondation pour la Recherche Medicale (Transplantation and cell therapy -PME201906008797) to KB. It was supported by NeurATRIS: A Translational Research Infrastructure for Biotherapies in Neurosciences (Investissements d'Avenir -ANR-11-INBS-0011) and INGESTEM: the National Infrastructure Engineering for Pluripotent and differentiated Stem cells (Investissements d'Avenir -ANR-11-INBS-000) to CM. I-Stem is part of the Biotherapies Institute for Rare Diseases supported by the Association Française contre les Myopathies-Téléthon.

Conflicts of Interest

CM and KB are inventors of a patent (FR3078712) related to medical devices for the preparation of retinal tissues for regenerative medicine. LM, CM and KB are inventors of a pending patent related to the automated differentiation of hPSC into RPE cells.