Vision impairments affect over 2.2 billion people worldwide. The most physiologically accurate human retina models are Retinal Organoids (ROs) — stem cell-derived structures containing the major cell types found in the human retina. However, the current protocol for deriving ROs from stem cells is highly labor-intensive and involves a time-consuming neural induction period. In this study, we investigated an alternative differentiation protocol that could decrease the neural induction period from two weeks to four days and elucidate the essential parameters, such as seeding density, required for neural induction of ROs. Expediting the neural induction of RO progenitors can be applied to improve efficiency in the differentiation of ROs.

We characterized RO progenitors using gene expression data from qPCR and by visual inspection with bright-field microscopy. Although seeding density has historically been shown to affect gene expression in the culture of ocular cells, seeding density did not significantly (p > .05) impact the gene expression or morphology of cells at the tested time points of differentiation. Cells on day 4 (D4) of differentiation demonstrated morphology and neural gene expression (Pax6 and Lhx2) characteristic of neural cells. Additionally, transferring cells from adherent culture to suspension culture on day 2 (D2) instead of D4 of this differentiation yielded intact neural spheres with a phase-bright outer ring and defined borders after 14 days of culture. Therefore, these results indicate that our alternative differentiation protocol successfully expedited the neural induction of RO progenitors. These results will contribute to the establishment of a more efficient neural induction when generating ROs, which may expedite the production of RO-based retinal therapies.