Sacha Reichman

Rod-Derived Cone Viability Factor Promotes Cone Survival by Stimulating Aerobic Glycolysis

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Significance Human induced pluripotent stem cells (hiPSCs) could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. The production of retinal cells from hiPSCs for personalized therapeutic approaches must comply with certain criteria, such as safety, efficiency, reproducibility, and low production cost. Here, we report a simple and scalable retinal differentiation process for the generation of retinal pigmented epithelial cells and neural retinal tissues containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This method also provides an accessible in vitro model to investigate mechanisms involved in human retinogenesis and retinal diseases. Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

The homeobox gene CHX10/VSX2 regulates RdCVF promoter activity in the inner retina

Rod-derived Cone Viability Factor (RdCVF) is a trophic factor with therapeutic potential for the treatment of retinitis pigmentosa, a retinal disease that commonly results in blindness. RdCVF is encoded by Nucleoredoxin-like 1 (Nxnl1), a gene homologous with the family of thioredoxins that participate in the defense against oxidative stress. RdCVF expression is lost after rod degeneration in the first phase of retinitis pigmentosa, and this loss has been implicated in the more clinically significant secondary cone degeneration that often occurs. Here, we describe a study of the Nxnl1 promoter using an approach that combines promoter and transcriptomic analysis. By transfection of selected candidate transcription factors, chosen based upon their expression pattern, we identified the homeodomain proteins CHX10/VSX2, VSX1 and PAX4, as well as the zinc finger protein SP3, as factors that can stimulate both the mouse and human Nxnl1 promoter. In addition, CHX10/VSX2 binds to the Nxnl1 promoter in vivo. Since CHX10/VSX2 is expressed predominantly in the inner retina, this finding motivated us to demonstrate that RdCVF is expressed in the inner as well as the outer retina. Interestingly, the loss of rods in the rd1 mouse, a model of retinitis pigmentosa, is associated with decreased expression of RdCVF by inner retinal cells as well as by rods. Based upon these results, we propose an alternative therapeutic strategy aimed at recapitulating RdCVF expression in the inner retina, where cell loss is not significant, to prevent secondary cone death and central vision loss in patients suffering from retinitis pigmentosa.

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

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two‐step xeno‐free/feeder‐free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self‐forming neuroretinal‐like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation‐compatible CD73+ photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC‐derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73+ photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP‐compliant retinal cell manufacturing protocol allowing large‐scale production and banking of hiPSC‐derived retinal cells and tissues. Stem Cells 2017;35:1176–1188

Cis-silencing of PIP5K1B evidenced in Friedreich's ataxia patient cells results in cytoskeleton anomalies

Friedreichs ataxia (FRDA) is a progressive neurodegenerative disease characterized by ataxia, variously associating heart disease, diabetes mellitus and/or glucose intolerance. It results from intronic expansion of GAA triplet repeats at the FXN locus. Homozygous expansions cause silencing of the FXN gene and subsequent decreased expression of the encoded mitochondrial frataxin. Detailed analyses in fibroblasts and neuronal tissues from FRDA patients have revealed profound cytoskeleton anomalies. So far, however, the molecular mechanism underlying these cytoskeleton defects remains unknown. We show here that gene silencing spreads in cis over the PIP5K1B gene in cells from FRDA patients (circulating lymphocytes and primary fibroblasts), correlating with expanded GAA repeat size. PIP5K1B encodes phosphatidylinositol 4-phosphate 5-kinase β type I (pip5k1β), an enzyme functionally linked to actin cytoskeleton dynamics that phosphorylates phosphatidylinositol 4-phosphate [PI(4)P] to generate phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Accordingly, loss of pip5k1β function in FRDA cells was accompanied by decreased PI(4,5)P2 levels and was shown instrumental for destabilization of the actin network and delayed cell spreading. Knockdown of PIP5K1B in control fibroblasts using shRNA reproduced abnormal actin cytoskeleton remodeling, whereas over-expression of PIP5K1B, but not FXN, suppressed this phenotype in FRDA cells. In addition to provide new insights into the consequences of the FXN gene expansion, these findings raise the question whether PIP5K1B silencing may contribute to the variable manifestation of this complex disease.

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

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14+ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human‐induced pluripotent stem cell‐derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF‐α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF‐α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age‐related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.

Expression of rod-derived cone viability factor: dual role of CRX in regulating promoter activity and cell-type specificity.

Background RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina. Methodology/Principal Findings In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5′-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5′-deletion analysis identified the human −205/+57 bp and murine −351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human −393/+27 bp and murine −195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (−393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (−77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role. Conclusions/Significance The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity.

Production of Retinal Cells from Confluent Human iPS Cells

Human induced pluripotent stem (hiPS) cells could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. Although much progress has been made in the differentiation of pluripotent stem cells towards different retinal lineages, the production of retinal cells from hiPS cells for therapeutic approaches require the development of easy and standardized protocols. In this chapter, we describe a simple and effective protocol for retinal differentiation of hiPS cells bypassing embryoid body formation and the use of exogenous molecules and substrates. In 2 weeks, confluent hiPS cells cultured in pro-neural medium can generate both retinal pigmented epithelial cells and self-forming neural retina-like structures containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This differentiation system and the resulting hiPS-derived retinal cells will also offer opportunity to study the molecular and cellular mechanisms underlying human retinal development, and the establishment of in vitro models of human retinal degenerative diseases.

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

Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.

Noninvasive gene delivery to foveal cones for vision restoration

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.