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Lei Zheng, Amanda-Jayne F. Carr, Ma'ayan Semo, Li Li Chen, Mark Hankins, Pete J. Coffey, Anthony A. Vugler; Light Responsiveness Of RPE Derived From Human Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2696.
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© ARVO (1962-2015); The Authors (2016-present)
Light is a potentially important stimulus to consider when generating retinal cell types from pluripotent human cells in vitro. Here we examine the ability of human embryonic stem cell-derived RPE (HESC-RPE) to respond to light at both the electrophysiological and molecular level.
Low passage cultures of HESC-RPE were examined for their ability to respond to light using several different approaches including Multi-Electrode Array (MEA), patch clamping, calcium imaging, quantitative RTPCR and western blot. The light delivered to stimulate HESC-RPE was either white (broad spectrum) or monochromatic.
Following exposure of HESC-RPE to white light, MEA recordings showed a clear spiking activity, which occurred within 1 second of stimulus onset. A similar response was also observed using calcium imaging to analyse light responses in individual cells. The ability of isolated HESC-RPE to respond intrinsically to light was confirmed by patch clamp recordings, which revealed a slow inward current in response to a 10 second pulse of 420nm blue light. On a molecular level, HESC-RPE exposed to 30 minutes of blue light and sampled by RTPCR after a further 30 minutes in darkness showed modulation of RPE associated genes (relative to a dark control group), including an enhancement of LRAT, IRBP and PER1 expression. Interestingly, exposure of HESC-RPE to longer wavelengths of light for 22hrs in the cell culture incubator resulted in a marked increase in a 65kDa band detected by western blot using the RPE65 antibody.
Here, we demonstrate for the first time that HESC-RPE are intrinsically light responsive. This stimulus can modulate both membrane potential and gene expression in HESC-RPE and as such, we suggest that light is an important variable to control during the culture of both HESC-RPE and other potentially light-responsive retinal cell types derived from pluripotent stem cells.
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