Direct Chemical Reprogramming of Human Fibroblasts into Retinal Progenitor-like Cells for Ocular Delivery

Direct chemical reprogramming provides a potentially scalable approach for generating retinal lineage-associated cells without genetic manipulation. In this study, human Tenon's capsule fibroblasts were converted into retinal progenitor-like cells using a defined small-molecule cocktail. Retinal lineage-associated features were evaluated by immunofluorescence staining, quantitative reverse-transcription PCR, Western blot analysis, and bulk RNA sequencing, showing upregulation of neural and retinal markers, including VSX2, and transcriptomic remodeling consistent with transcriptional features associated with neuronal differentiation programs. Functional responsiveness was assessed by glutamate-evoked intracellular calcium imaging, revealing glutamate-responsive intracellular calcium dynamics in induced cells but not in parental fibroblasts. For in vivo assessment, induced cells were delivered via intravitreal transplantation in Wistar rats and subretinal transplantation in Long-Evans rats. One month after transplantation, structural and functional evaluations using optical coherence tomography, electroretinography, and histological analyses showed localized alterations in retinal structure at the subretinal injection site, while no significant differences were observed in scotopic ERG responses under the present experimental conditions. In contrast, fibroblast transplantation showed more prominent structural alterations under similar conditions. Human nuclei-positive signals were detectable in a subset of eyes, exhibiting focal and heterogeneous distribution within retinal regions at the one-month endpoint. Collectively, these suggest the induction of retinal lineage-associated molecular and functional features, with short-term functional tolerability observed in vivo under the present experimental conditions.