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Jonathan Luisi, Nick Motamedi, Wen Huang, Shuang Zhu, John Wiktorowicz, Wenbo Zhang, Massoud Motamedi; Characterizations of constitutively produced exosomes by photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):172.
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© 2017 Association for Research in Vision and Ophthalmology.
Exosomes are a class of microvesicles shown to have importance in cell-cell communication. Retina pigmental epithelial cells and astrocytes are known to release exosomes that modulate the pathophysiological responses of other retinal cells. In the CNS, emerging evidence indicates that neurons can communicate to multiple cell types in a non-canonical pathways, such as through microvesicles. It is largely unknown whether photoreceptor, as specialized neuronal cells, can also communicate with non-adjacent cells though exosomes. This study is to determine whether photoreceptor cells have the ability to produce and release exosomes.
Photoreceptor cell line 661W is a general gift from Dr. Muayyad Al-Ubaidi. It is cultured in DMEM containing 1 g/L glucose, 40 ng/ml hydrocortisone 21-hemisuccinate sodium salt, 40 ng/ml progesterone, 32 mg/ml putrescine dihydrochloride, 0.004% beta-mercaptoethanol and 10% FBS. 24 hours before collecting the media, cells were washed with PBS and incubated in culture medium containing exosome-free FBS. Microparticles were isolated from 661W conditioned medium by a stepwise ultracentrifugation gradient. After isolation the particles were profiled with a Malvern HPPS. The sizing profiles of the 661W cells were compared against those of ARPE19 cells, which are known to secrete exosomes. The contents of isolated particles were identified by Western blot with antibodies against the typical exosome markers CD63, HSP70, CD81, and CD9.
A significant yield of microparticles were isolated from both 661W and ARPE19 conditioned medium, in contrast to the control medium (cells free) where there were no detectable particles. Analysis of the particle size profiles revealed that the particles from 661W cells have a z-average of 126 nm, which is similar in size to those from ARPE19 cells (106 nm). Moreover, particles isolated from 661W cells were positive for exosome markers CD63, HSP70, and CD81.
We have identified extracellular vesicles constitutively released from photoreceptor cells as exosomes. Further comparison of quantity, size, and contents of exosomes released in the normal versus stressed state may elucidate a novel role of exosomes in the communication between photoreceptor cells and other retina cells in pathophysiological settings. The size and surface marker profiles are validation of exosome isolation protocol to allow for downstream proteomic analysis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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