Figure - available from: Frontiers in Bioengineering and Biotechnology
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Activin signaling increases photoreceptor viability. In order to assess Activin A function in vitro enriched post-mitotic photoreceptor precursors were cultured alone (A), in the presence of activin A only (B), and with both activin A and the inhibitor SB-431542 (C) for 72 h. Representative images of live/dead cell staining (FDA, green, alive; PI, red, dead) are shown. Quantitative automated image analysis was carried out to determine the relative number of living cells (D) for each condition (N = 11, 16, 14 culture wells each for control, activin treated, and activin + SB groups, respectively). A median of 361 cells were counted per image (averaging 19, 000 total cells per treatment). Different letters represent a significant difference, P < 0.05.
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We have developed an accessible software tool (receptoR) to predict potentially active signaling pathways in one or more cell type(s) of interest from publicly available transcriptome data. As proof-of-concept, we applied it to mouse photoreceptors, yielding the previously untested hypothesis that activin signaling pathways are active in these cell...
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... Similar to the ARPE-19 microtissue, cells in the hESC-derived RPE-µ Ts secrete 3X more PEDF (p < 0.01) and less than 1% of VEGF (p < 0.01) than standard adherent culture (Figure 5f). Based on these secreted factor profiles, we predicted that CM from RPE-µ T cultures would increase photoreceptor survival, using a dissociated mouse retina assay [61]. After 48 hours, photoreceptor-enriched in vitro cultures demonstrated superior survival with RPE-µ T CM compared to CM collected from monolayer cultures (1.41 ± 0.11fold increase, p < 0.05) and this effect was exaggerated by 72 hours with a nearly two-fold increase in cell survival, using RPE-µ T CM (p < 0.01; Figure 5g). ...
... Photoreceptors were isolated, enriched and cultured as previously described [61]. Briefly, mouse retinas were dissected from the eyes of postnatal day 4 CD1 mice obtained through a secondary-use protocol approved by the Animal Care Committee at the University of Calgary according to IACUCC standards. ...
The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch’s membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.
