Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids.

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.