Pharmacological Modulation of Photoreceptor Outer Segment Degradation in a Human iPS Cell Model of Inherited Macular Degeneration

Degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is essential for vision, and studies have implicated altered POS processing in the pathogenesis of some retinal degenerative diseases. Consistent with this concept, a recently established hiPSC-RPE model of inherited macular degeneration, Best disease (BD), displayed reduced rates of POS breakdown. Herein we utilized this model to determine (i) if disturbances in protein degradation pathways are associated with delayed POS digestion and (ii) whether such defect(s) can be pharmacologically targeted. We found that BD hiPSC-RPE cultures possessed increased protein oxidation, decreased free-ubiquitin levels, and altered rates of exosome secretion, consistent with altered POS processing. Application of valproic acid (VPA) with or without rapamycin increased rates of POS degradation in our model, whereas application of bafilomycin-A1 decreased such rates. Importantly, the negative effect of bafilomycin-A1 could be fully reversed by VPA. The utility of hiPSC-RPE for VPA testing was further evident following examination of its efficacy and metabolism in a complementary canine disease model. Our findings suggest that disturbances in protein degradation pathways contribute to the POS processing defect observed in BD hiPSC-RPE, which can be manipulated pharmacologically. These results have therapeutic implications for BD and perhaps other maculopathies

Physiological Anxiety Responses with Cell Phone Separation and Subsequent Contact

An article that appeared in JASS, issue 2015Past research has shown that people separated from their cell phones exhibit physiological responses related to anxiety. These responses include an increase in the release of stress hormones leading to an increase in heart rate, perspiration, and respiration rate. Because of the strong attachment young American adults have to their cell phones, the researchers hypothesized participants would show signs of anxiety when separated from their phones. To test this, twenty participants completed a word search once with their phone in their possession, and another when their phone was out of their reach. During the latter condition, an experimenter anonymously contacted the subject first by text and then by phone call. Heart rate, respiration rate, and galvanic skin response measurements were taken continuously throughout both conditions. The results obtained indicate that the only significant response supporting the researchers’ hypothesis was the skin response data. The data obtained regarding heart rate and respiration rate was not sufficient to prove that there was a measurable physiological response to being separated from one’s phone

Functional analysis of serially expanded human iPS cell-derived

PURPOSE. To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. METHODS. Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RESULTS. RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. CONCLUSIONS. Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation. Keywords: induced pluripotent stem cell, retinal pigment epithelium, passaging D egenerative disorders that target the RPE, such as AMD, lead to blindness in large part due to loss of functional support for neighboring photoreceptors. 1 Examples of critical supportive functions of RPE include photoreceptor outer segment (POS) phagocytosis and degradation, maintenance of the outer blood-retinal barrier, secretion of paracrine factors, and regulation of ion and fluid homeostasis, among others. 2 Access to renewable sources of highly functional and expandable human RPE would be ideal for studying these cells and developing therapies to preserve or replace them. Although human fetal RPE (hfRPE) derived from donor tissue remains the culture standard, patient-specific, human-induced pluripotent stem cells (hiPSCs) have emerged as an important and ethically neutral source of RPE for disease modeling, 3-5 drug testing, 3 and transplantation. 6,7 Production of RPE from heterogeneous cultures of differentiating hiPSCs occurs with varying efficiency, often necessitating the isolation and expansion of hiPSC-RPE populations prior to further use