Stem cell therapy in retinal diseases.

Alteration of the outer retina leads to various diseases such as age-related macular degeneration or retinitis pigmentosa characterized by decreased visual acuity and ultimately blindness. Despite intensive research in the field of retinal disorders, there is currently no curative treatment. Several therapeutic approaches such as cell-based replacement and gene therapies are currently in development. In the context of cell-based therapies, different cell sources such as embryonic stem cells, induced pluripotent stem cells, or multipotent stem cells can be used for transplantation. In the vast majority of human clinical trials, retinal pigment epithelial cells and photoreceptors are the cell types considered for replacement cell therapies. In this review, we summarize the progress made in stem cell therapies ranging from the pre-clinical studies to clinical trials for retinal disease.

Proteins Associated with Phagocytosis Alteration in Retinal Pigment Epithelial Cells Derived from Age-Related Macular Degeneration Patients.

Age-related macular degeneration (AMD) is partially characterized by retinal pigment epithelial (RPE) cell dysfunction. This study focused on phagocytosis activity and its involvement in AMD. Phagocytic activity was analyzed by flow cytometry using porcine photoreceptor outer segment (POS) and fluorescent beads in basal and under oxidative stress condition induced by Fe-NTA in fifteen hiPSC-RPE cell lines (six controls, six atrophic AMD and three exudative AMD). Oxidative stress exposure inhibited phagocytosis in the same manner for control, atrophic AMD (AMDa) and exudative AMD (AMDe) cell lines. However, altered phagocytosis in basal condition in hiPSC-RPE AMDa/e was observed compared to control cell lines. Gene expression after 3 or 24 h of POS incubation was analyzed by RNA-Seq based transcriptomic profiling. Differential gene expression was observed by RNA seq after 3 and 24 h POS exposure. We have focused on the genes involved in mTOR/PI3K-AKT/MEK-ERK pathway. We investigated differences in gene expression by analyzing the expression levels and activity of the corresponding proteins by Western blot. We showed the involvement of three proteins essential for phagocytosis activity: fak, tuberin and rictor. These findings demonstrate that hiPSC-RPE AMDa/e cells have a typical disease phenotype characterized by alteration of the main function of RPE cells, phagocytosis activity.

Use of Physiologically-Based Kinetics Modelling to Reliably Predict Internal Concentrations of the UV Filter, Homosalate, After Repeated Oral and Topical Application.

Ethical and legal considerations have led to increased use of non-animal methods to evaluate the safety of chemicals for human use. We describe the development and qualification of a physiologically-based kinetics (PBK) model for the cosmetic UV filter ingredient, homosalate, to support its safety without the need of generating further animal data. The intravenous (IV) rat PBK model, using PK-Sim®, was developed and validated using legacy in vivo data generated prior to the 2013 EU animal-testing ban. Input data included literature or predicted physicochemical and pharmacokinetic properties. The refined IV rat PBK model was subject to sensitivity analysis to identify homosalate-specific sensitive parameters impacting the prediction of Cmax (more sensitive than AUC(0-∞)). These were then considered, together with population modeling, to calculate the confidence interval (CI) 95% Cmax and AUC(0-∞). Final model parameters were established by visual inspection of the simulations and biological plausibility. The IV rat model was extrapolated to oral administration, and used to estimate internal exposures to doses tested in an oral repeated dose toxicity study. Next, a human PBK dermal model was developed using measured human in vitro ADME data and a module to represent the dermal route. Model performance was confirmed by comparing predicted and measured values from a US-FDA clinical trial (Identifier: NCT03582215, https://clinicaltrials.gov/). Final exposure estimations were obtained in a virtual population and considering the in vitro and input parameter uncertainty. This model was then used to estimate the Cmax and AUC(0-24 h) of homosalate according to consumer use in a sunscreen. The developed rat and human PBK models had a good biological basis and reproduced in vivo legacy rat and human clinical kinetics data. They also complied with the most recent WHO and OECD recommendations for assessing the confidence level. In conclusion, we have developed a PBK model which predicted reasonably well the internal exposure of homosalate according to different exposure scenarios with a medium to high level of confidence. In the absence of in vivo data, such human PBK models will be the heart of future completely non-animal risk assessments; therefore, valid approaches will be key in gaining their regulatory acceptance. Clinical Trial Registration: https://clinicaltrials.gov/, identifier, NCT03582215.

Corrigendum to "Cathepsin B pH-Dependent Activity Is Involved in Lysosomal Dysregulation in Atrophic Age-Related Macular Degeneration".

[This corrects the article DOI: 10.1155/2019/5637075.].

Cathepsin B pH-Dependent Activity Is Involved in Lysosomal Dysregulation in Atrophic Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is characterized by retinal pigment epithelial (RPE) cell dysfunction beginning at early stages of the disease. The lack of an appropriate in vitro model is a major limitation in understanding the mechanisms leading to the occurrence of AMD. This study compared human-induced pluripotent stem cell- (hiPSC-) RPE cells derived from atrophic AMD patients (77 y/o ± 7) to hiPSC-RPE cells derived from healthy elderly individuals with no drusen or pigmentary alteration (62.5 y/o ± 17.5). Control and AMD hiPSC-RPE cell lines were characterized by immunofluorescence, flow cytometry, and electronic microscopy. The toxicity level of iron after Fe-NTA treatment was evaluated by an MTT test and by the detection of dichloro-dihydro-fluorescein diacetate. Twelve hiPSC-RPE cell lines (6 AMD and 6 controls) were used for the experiment. Under basal conditions, all hiPSC-RPE cells expressed a phenotypic profile of senescent cells with rounded mitochondria at passage 2. However, the treatment with Fe-NTA induced higher reactive oxygen species production and cell death in hiPSC-RPE AMD cells than in hiPSC-RPE Control cells. Interestingly, functional analysis showed differences in lysosomal activity between the two populations. Indeed, Cathepsin B activity was higher in hiPSC-RPE AMD cells compared to hiPSC-RPE Control cells in basal condition and link to a pH more acidic in this cell population. Moreover, oxidative stress exposure leads to an increase of Cathepsin D immature form levels in both populations, but in a higher proportion in hiPSC-RPE AMD cells. These findings could demonstrate that hiPSC-RPE AMD cells have a typical disease phenotype compared to hiPSC-RPE Control cells.

hRPE cells derived from induced pluripotent stem cells are more sensitive to oxidative stress than ARPE-19 cells.

The ARPE-19 cell line is currently used as an in vitro model for retinal diseases such as age-related degeneration (AMD). However, several studies have pointed out morphological and genetic differences between ARPE-19 cells and human fetal or adult retinal pigment epithelial (hRPE) cells. This study aims to compare ARPE-19 cells to hRPE cells derived from human induced pluripotent stem cells (hiPSCs) in both normal and oxidative stress conditions induced by Fe-NTA treatment. Indeed, oxidative stress is an essential contributing factor in AMD. hiPSC obtained from peripheral venous blood samples or fibroblasts of individuals aged over 60 years were first reprogrammed to hiPSC and then differentiated into RPE cells. In contrast to ARPE-19 cells, hiPSC-RPE cells expressed ß-galactosidase activity, suggesting that only the latter display signs of senescence. Treatment with 10 mM of FeNTA induced a higher reactive oxygen species (ROS) production and increased cell death in hiPSC-RPE cells compared to ARPE-19 cells. Moreover, morphological analysis and Annexin V and Propidium iodide (PI) test suggested a necrotic cell death pattern induced by treatment in hiPSC-RPE cells that is not observed in ARPE-19 cells. Taken as a whole, our findings suggest that hiPSC-RPE cells are more sensitive to oxidative stress than ARPE-19 cells.