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Julien Maruotti, John Fuller, Karl Wahlin, Valentin Sluch, Catherine Kim, Jun Wan, Kapil Bharti, Janine Davis, Sheldon S Miller, Donald J Zack; A High Throughput Screen for small molecules that promote stem cell differentiation into Retinal Pigmented Epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2672.
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Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), the layer of cells that surrounds and nourishes the neurosensory retina. RPE dysfunction can lead to photoreceptor death, and subsequent vision loss. Cell-based transplantation strategies offer the promise of being able to restore RPE cells. Human pluripotent stem cells (hPSCs) may prove suitable for this purpose: significant advances have recently been made in inducing the differentiation of hPSCs toward an RPE-like cell fate. Nevertheless, the length and efficiency of RPE generation from hPSCs are still not optimal. We therefore sought to develop a high-throughput screen aimed at finding small molecules that could improve RPE differentiation in terms of efficiency and time course.
hPSC were cultured until confluence in 384 well plate format. Thereafter, they were treated with a focused library of over 300 compounds for 10 days and analyzed by high-throughput quantitative real-time PCR for the expression of the following key RPE markers: MITF, OTX2 and PMEL17. Bioinformatic analysis was performed to identify primary hits. For hit confirmation, dose-response experiments were done folllowed by qPCR for an extended panel of RPE markers. To further assess the small molecule efficiency at generating RPE, the area of pigmented cells was measured after compound treatment.
After bioinformatic anlysis, 4 compounds were found to up-regulate both MITF and OTX2 above the 3-fold threshold, while a single compound was able to increase all 3 markers. qPCR dose-response assays confirmed that 2/5 primary hits significantly up-regulated RPE markers compared to vehicle conditions. These two compounds were BIO and Chetomin. Next we checked the ability of these small molecules to induce RPE differentiation from hPSC: after 50 days of differentiation, the area of pigmented cells following BIO or chetomin treatment was 10 and 15 times larger, respectively, compared to vehicle treatment.
In conclusion, we have developed a successful HTS qPCR approach to identify molecules that promote RPE differentiation from hPSC. We are currently studying how combinations of these new compounds may help set up faster and more efficient ways to produce RPE from hPSC.
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