Purchase this article with an account.
Srinivas Sripathi, Julien Maruotti, Karl J. Wahlin, Vinod Ranganathan, John A Fuller, Valentin Sluch, Catherine Kim, James T Handa, Cindy Berlinicke, Donald J Zack; High-throughput siRNA screening of oxidatively stressed human iPS derived RPE . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4195.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress mediated RPE damage is thought to be a key factor in the pathogenesis of age-related macular degeneration (AMD); however, the underlying mechanisms that mediate RPE degeneration by oxidative insult remain incompletely understood. The advent of induced pluripotent (iPS) and embryonic stem (ES) cell derived RPE cells enable us to investigate the oxidative stress pathways in RPE as well as screen for candidate neuroprotective compounds. To achieve these goals, we are developing an siRNA approach with ES/iPS-derived RPE cells to screen for genes that are involved in the RPE’s response to oxidative stress.
Human iPS and ES cells were cultured in mTeSR with fibroblast growth factors, followed by the differentiation media containing 15% KO serum. Differentiated cells were allowed to mature in RPE medium with B27 serum free supplement. Cells were reverse transfected with siRNA using the LipoFectamine RNA iMAX reagent for 48-72 hrs followed by the treatment with different oxidative stress inducers including tert-butyl hydroperoxide, paraquat and cigarette smoke extract for another 24-48 hrs. Cell viability was measured with a plate reader using CellTiter-Glo luminescence assay or with automated fluorescent imaging by calcein AM-staining.
The lethal doses (LD 25, 50 and 75) for RPE under different stressor conditions were determined after dissociating hiPS-RPE monolayers. Effiecient gene knockdown in the range of 70-90% was achieved by reverse siRNA transfection in hiPS-RPE cells. As an initial proof-of-principle step towards performing an expanded siRNA screen of hiPSc-RPE under oxidative stress, knockdown of the expression of the transcription factor NRF2 or its negative regulator KEAP1 was performed, and this resulted in the down or upregulation, respectively, of phase II antioxidant responsive genes, including NQO1, HO-1, and GCLM/GCLC.
High-throughput siRNA screening of hiPS/ES-derived RPE cells under oxidative stress may provide useful biological insights and help to identify potential neuroprotective drugs. Based on our ongoing siRNA and small molecule screening work we hope to identify possible target genes involved in the response of RPE cells to oxidative stress.
This PDF is available to Subscribers Only