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Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9
摘要： Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.
关键词： retinal pigment epithelial cells,CRISPR/Cas9,retinitis pigmentosa,PRPF8,induced pluripotent stem cells
更新于2025-09-23 15:23:52
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Novel Protective Effects of Cistanche Tubulosa Extract Against Low-Luminance Blue Light-Induced Degenerative Retinopathy
摘要： Background/Aims: Blue light-emitting diode light (BLL)-induced phototoxicity plays an important role in ocular diseases and causes retinal degeneration and apoptosis in human retinal pigment epithelial (RPE) cells. Cistanche tubulosa extract (CTE) is a traditional Chinese medicine with many beneficial protective properties; however, few studies have examined the ocular protective roles of CTE. In this study, we investigated the mechanisms underlying the effects of CTE on BLL-induced apoptosis in vitro and in vivo. Methods: RPE cells were applied in the current in vitro study and cell viability was determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis-related protein expression was determined by western blot analysis and immunofluorescence staining. Brown Norway rats were used to examine exposure to commercially available BLL in vivo. Hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blot assays were used to examine retinal morphological deformation. Results: CTE significantly inhibited hydrogen peroxide-, tert-butyl hydroperoxide-, sodium azide-, and BLL-induced RPE damage. Further, CTE reduced the expression of apoptotic markers such as cleaved caspase-3 and TUNEL staining after BLL exposure by inactivating apoptotic pathways, as shown via immunofluorescent staining. In addition, CTE inhibited the BLL-induced phosphorylation of c-Jun N-terminal kinase, extra signal-related kinases 1/2, and p38 in RPE cells. In vivo, the oral administration of CTE rescued 60-day periodic BLL exposure-induced decrements in retinal thickness and reduced the number of TUNEL-positive cells in the brown Norway rat model. Conclusion: CTE is a potential prophylactic agent against BLL-induced phototoxicity.
关键词： Cistanche tubulosa extract,Blue light,Retinal pigment epithelial cells,Apoptosis
更新于2025-09-10 09:29:36