Efficient directed differentiation of human induced pluripotent stem cells (hIPSCs) towards photoreceptor progenitors using small molecules.

Photoreceptor degeneration and death is the ultimate cause of permanent vision loss, common to
all retinal degenerative disease (RDD). Millions of people worldwide are affected with current therapies lacking long-term efficacy. Human pluripotent stem cells (hPSCs) are a limitless source cell type that can be used for development of retinal cell types. They have the potential to be used for transplantation for a host of RDDs. The efficient differentiation of hPSCs to photoreceptors in sufficient numbers at clinical grade standards, remains a challenge. This 66-day pilot study used a CRSIPR/Cas9 transgenic reporter hIPSC line to develop a directed differentiation of hiPSC to photoreceptor precursors using small molecules. Preliminary results for ICC, FACS and qPCR over five timepoints demonstrate the presence of early photoreceptor markers and genes with the activation of representative transcription factors at relevant, stage-wise time points. Live cell images at D28 indicate the activation of the mCherry tagged RCVRN gene. Further immunohistochemistry at D30 support the presence of Nr2F2 and by D46 CRX, PRDM1 and RCVRN are all highly expressed, this is followed by expression of Opsin-M and ARRESTIN at the final D66 timepoint. Flow cytometry data at D45 support activation of cerulean tagged VSX2 which was coupled with fluoresce seen under live cell microscopy. qPCR data also demonstrated expression of Nr2F2, PRDM1 and RECVRN at stage-specific time points. This initial study demonstrates the potential to generate homogenous populations of photoreceptors and their precursors without the need for selection which can be refined towards a Good Manufacturing Practice (GMP) protocol for pre-clinical study.