Development of a small molecule protocol for the differentiation of human pluripripotent stem cells (hPSCs) to photoreceptors

Photoreceptor (PhR) 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) provide a limitless source cell that can be used for differentiation of all retinal cell types. They offer potential for use in transplantation for a host of RDDs. However the development of protocols to provide efficient differentiation of hPSCs to PhRs in sufficient number of clinical grade cells, remains a challenge. This 66-day repeat study used PGP1, CRISPR/Cas9 transgenic reporter hiPSC line to optimise and repeat a protocol for directed differentiation of hiPSC to PhRs using small molecules. Preliminary results across six timepoints demonstrate the presence of cells expressing early PhR markers, along with transcription factors at relevant time points. Live cell images at D27 indicate activation of the mCherry-tagged RCVRN gene. Further immunocytochemistry supports the high expression of RCVRN at D31, and of CRX, PRDM1 and ARR by D52. This is followed by expression of OPSIN-M and ARRESTIN at D66. Flow cytometry data at D52 supports activation of mCherry-tagged RCVRN by 97.4% of culture positive for RCVRN expression. This repeated protocol demonstrates the potential to generate homogeneous populations of PhRs without the need for cell selection.
This protocol can be refined to give a Good Manufacturing Practice (GMP) protocol for pre-clinical study.