The loss of photoreceptors is a key hallmark of many incurable blinding diseases and regenerative medicine has great potentials of alleviating blindness in patients. Previous studies showed the feasibility of using transcription factors to reprogram glial cells into retinal neurons both in vitro and in vivo in rodents (1,2), providing an innovative gene-therapy approach to stimulate retinal regeneration. However, it remained unclear whether this reprogramming approach can translate to human cells. This study aims to use CRISPR activation (CRISPRa) to reprogram human Müller glia into photoreceptors (induced photoreceptors, iPH) in vitro.
As part of the Human Cell Atlas initiative (humancellatlas.org), we recently generated a human retina transcriptome atlas at single cell levels and identified the transcriptome of the major retinal cell types (1). From this dataset, we performed network topology analysis to predict transcription factors that could reprogram Müller glia into photoreceptors. To test the shortlisted transcription factors, we developed a CRISPRa pipeline (4) which allowed us to activate up to 9 transcription factors simultaneously. Using this CRISPRa platform, we have screened and identified cocktails of transcription factors that allow reprogramming of human Müller glia into iPH in vitro. qPCR and immunocytochemical analysis demonstrated that iPH expressed a panel of photoreceptor markers, including RHO and PDE6B. Subsequently we performed single cell transcriptomic to profile iPH and compared with the human retina transcriptome atlas as a benchmark, we showed that iPH reprogramming promoted transcriptomic transitions from Müller glia to photoreceptors, resulting in activation of photoreceptor markers in iPH. Our study demonstrated the feasibility of using CRISPRa to promote cell reprogramming of Müller glia into photoreceptors, providing a potential cell source for tissue engineering and regenerative medicine. Future application for in vivo reprogramming provides an exciting innovative approach to regenerate photoreceptors and restore vision in retinal degenerative diseases.