UC San Diego

285 million people worldwide suffer from visual impairment, primarily due to the loss of retinal ganglion cells and photoreceptors. Endogenous regeneration could be instrumental in addressing this, but experiments that attempt to induce regeneration in the popular organoid model have no straightforward way to assess whether it was successful. ASCL1 has long been identified as an important transcription factor in cell generation and regeneration. Thus, an ASCL1 fluorescent reporter was introduced into pluripotent stem cells (PSCs), giving unprecedented access to retinal progenitors. These were subsequently differentiated into retinal organoids and monitored by longitudinal live-cell imaging and histological analysis in fixed cryo-sections during the first three months of differentiation. Bulk RNA-seq was carried out on FACS-sorted ASCL1+ reporter cells to assess transcriptional changes in retinal progenitors over time. Shifts in cell-specific transcription factors informed us about timelines for the birth and maturation of specific retinal cell types, such as NRL+ and Recoverin+ photoreceptors which peaked day 60, prior to when photoreceptors are detected by immunohistochemistry. Pathways associated with retinal maturation and function, such as sensory perception of light stimuli and eye development, were more highly enriched in older organoid samples than in younger samples, demonstrating the changing identity of retinal progenitors.