Crispr/cas9-mediated genome editing of human pluripotent stem cells to advance human retina regeneration research

Destruction of the human neural retina (NR), by either disease or injury, results in irreversible loss of vision. None of the current treatments restore lost retinal tissue. Under specific exogenous stimuli during a specific developmental window, both the embryonic chick and mouse can induce NR reprogramming from the retinal pigmented epithelium (RPE), whereas adult mammals lack the ability to undergo NR regeneration following NR injury. In contrast, following NR detachment, adult newts regenerate a functional NR from the RPE without any exogenous stimuli. Subsequent to NR injury, both the newt and adult human RPE dedifferentiate, and proliferate. However, the hRPE undergoes an epithelial to mesenchymal transition (EMT) that leads to scar formation. The common initial reaction of the RPE upon NR injury in the newt and human RPE suggests that a proper stimulus could reprogram hRPE-NR. Identifying the exogenous stimuli (small molecules/growth factors) to induce the hRPE-NR reprogramming represents this dissertation’s goal. Here, we exploited genetic engineering of human induced pluripotent stem cells (hiPSCs) to design fluorescence-based reporters to identify compounds that induce RPE-NR progenitor (RPE-NRP) reprogramming. Specifically, we generated a single hiPSC transgenic line to report real-time expression of VSX2, a gene expressed by NRPs, via cyan fluorescent protein expression. We used RPE differentiated from our transgenic hiPSC line in a pilot experiment, where we identified a combination of 4 factors that induced hRPE-NRP reprogramming in this line and in three other hiPSC-derived RPE lines. Furthermore, we created an hiPSC (PGP1) that reports real-time expression of VSX2, BRN3b (a ganglion marker), and RCVRN (a photoreceptor marker) via Cerulean, eGFP, and mCherry expression, respectively. PGP1-derived RPE (PGP1.RPE) was used in high-throughput screening of two small molecule libraries. In the developmental library, we identified three compounds that induce Cerulean, including a MEK inhibitor (U0126), an mTOR inhibitor (KU0063794), and AKT activator (SC79). In the epigenetics library, we discovered 11 compounds that induce Cerulean, including 4 histone methylation or demethylation inhibitors (ML324, 5-Carboxy-8-hydroxyquinoline, MI-463 and OICR-9429), 4 Aurora kinases inhibitors (Hesperadin, Barasertib, ZM-447439, and BI-847325), an AMPK/SMAD inhibitor (Dorsomorphin), an epigenetic reader domain inhibitor (I-BRD9), and a NF-kB inhibitor ((-)-Parthenolide). Interestingly, BI-847325, a dual MEK/Aurora kinase inhibitor, induced both Cerulean and eGFP expression in PGP1.RPE. This represents the first demonstration that hRPE can reprogram into NR under specific exogenous stimuli. This work describes a novel screening strategy and serves as a first step to develop regenerative treatments to combat human retina injury.