Direct reprogramming of Müller glia into photoreceptors via multiple transcription factors and small molecules: molecular mechanisms and transcriptomic analysis

Abstract Direct reprogramming, involving overexpression of transcription factors in combination with specific small-molecule treatments to induce cell fate conversion, represents a promising strategy for regenerative medicine and disease modeling. Monocistronic expression of four transcription factors ( CRX , NEUROD , RAX , and OTX2 ) has been demonstrated to induce the expression of phototransduction-associated genes and confer light responsiveness in fibroblasts. In contrast, polycistronic expression of the same four factors resulted in the upregulation of only two cone-specific genes, indicating that optimization of reprogramming conditions is required to achieve complete photoreceptor differentiation. In this study, we focused on Müller glial cells, which exhibit regenerative potential in the retina of lower vertebrates, and explored their reprogramming into photoreceptor-like cells. We thus combined polycistronic expression of the aforementioned transcription factors and a pharmacological reprogramming approach utilizing a defined cocktail of small molecules. Transcriptomic analysis revealed that transcription factor introduction alone led to the upregulation of the genes related to neuronal identity and extracellular matrix components. Notably, the combination of transcription factor expression and chemical treatment induced photoreceptor-specific gene expression. However, no genes associated with phototransduction were detected, suggesting that further refinement is required to promote full differentiation into functionally mature light-responsive photoreceptors. Overall, our findings demonstrate that immortalized Müller glia can partially activate neuronal and photoreceptor genes through reprogramming, suggesting their potential for scalable drug screening and disease modeling. Clinical trial number: not applicable.