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Summary Human-induced pluripotent stem cells (hiPSCs) can be differentiated into well-structured retinal organoids. In this protocol, we successfully established 3D retinae from patient-derived hiPSCs and built the retinitis pigmentosa model in vitro. Moreover, mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene was corrected by CRISPR-Cas9 gene editing, which rescued the structure and function of the 3D retinae. For complete details on the use and execution of this protocol, please refer to Deng et al. (2018)., Graphical abstract, Highlights • Patient-specific human induced pluripotent stem cells (hiPSCs) were established. • The RPGR mutation was corrected by CRISPR/Cas9 in hiPSCs. • hiPSCs were differentiated into 3D retinal organoids., Human-induced pluripotent stem cells (hiPSCs) can be differentiated into well-structured retinal organoids. In this protocol, we successfully established 3D retinae from patient-derived hiPSCs and built the retinitis pigmentosa model in vitro. Moreover, CRISPR-Cas9-mediated correction of the retinitis pigmentosa GTPase regulator mutation rescued the structure and function of the 3D retinae.

To characterize a glycosyltransferase (UGT74AN3) from Catharanthus roseus and investigate its specificity toward cardiotonic steroids and phenolic compounds. UGT74AN3, a novel permissive GT from C. roseus, displayed average high conversion rate (> 90%) toward eight structurally different cardiotonic steroids. Among them, resibufogenin, digitoxigenin, and uzarigenin gave 100% yield. Based on LC–MS, 1H-NMR and 13C-NMR analysis, structure elucidation of eight glycosides was consistent with 3-O-β-d-glucosides. We further confirmed UGT74AN3 was permissive enough to glycosylate curcumin, resveratrol, and phloretin. The cDNA sequence of UGT74AN3 contained an ORF of 1,425 nucleotides encoding 474 amino acids. UGT74AN3 performed the maximum catalytic activity at 40 °C, pH 8.0, and was divalent cation-independent. Km values of UGT74AN3 toward resibufogenin, digitoxigenin, and uzarigenin were 7.0 µM, 12.3 µM, and 17.4 µM, respectively. UGT74AN3, a glycosyltransferase from a noncardenolide-producing plant, displayed catalytic efficiency toward cardiotonic steroids and phenolic compounds, which would make it feasible for glycosylation of bioactive molecules.

Purpose Accumulating evidence has demonstrated that excessive immunoreaction plays a prominent role in the pathogenesis of dry AMD. Toll-like receptor 3 (TLR3) can be activated by double-stranded (ds)RNA in retinal pigment epithelia and trigger an innate immunity-mediated inflammatory response. However, its role in photoreceptor cells, the effectors of AMD geographic atrophy, remains unclear. Methods The expression of TLR3 was examined in mouse retina and in a murine photoreceptor cell line (661W). Retinal structure, function, and cell death in the polyinosine-polycytidylic acid (poly I:C)-treated retina were investigated by optical coherence tomography, electroretinography (ERG), and immunostaining. Cytokine and chemokine expression as well as cell death were measured in poly I:C-exposed 661W cells and explant retinas. By comparing the RNA sequencing (seq) data of 661W cells and murine retina, we comprehensively investigated the contribution of photoreceptor in poly I:C-induced retinal immune response. Results Toll-like receptor 3 was highly expressed in the inner segment of the photoreceptor and in 661W cells. We found poly I:C induced significant retinal structural damages and impairment of ERG responses. Focal ERG demonstrated that injected and parainjected zones were functionally damaged by poly I:C. In addition, poly I:C acted on cultured photoreceptor cells directly and evoked an inflammatory response that exhibited similarities with the immune response in mouse retina. Moreover, TLR3 activation initiated cell death in murine photoreceptor cells in vivo and in vitro. Additionally, poly I:C initiated immune response in explant retinas. Conclusions We deciphered the TLR3-mediated inflammatory response in photoreceptor cells. Our findings suggested TLR3-mediated inflammatory response in photoreceptor cells may play an important role in dry AMD, offering new insights of potential treatments targeting photoreceptor immunity.

Summary Retinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE) cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence., Graphical Abstract, Highlights • HiPSC-derived 3D retinae with outer segments and electrophysiological properties • RPGR mutation results in diseased photoreceptor in patient iPSC-derived 3D retinae • Mutation correction rescues defects in photoreceptor morphology and electrophysiology • Ciliogenesis defects appear in RPGR patient-specific iPSCs, iPSC-RPE, and 3D retinae, Jin and colleagues demonstrate that patient-specific iPSC-derived 3D retinae can recapitulate disease progress of retinitis pigmentosa through presenting defects in photoreceptor morphology, gene profile, and electrophysiology, as well as the defective ciliogenesis in iPSCs, iPSC-RPE, and 3D retinae. CRISPR/Cas9-mediated gene correction can rescue not only photoreceptor structure and electrophysiological property but also observed ciliopathy.