SIX6 regulation in human optic vesicles is mediated by early microenvironmenta factors

Human pluripotent stem cells (PSCs) represent an exciting tool to investigate the mechanisms of human eye development and to build models of human retinal disease. When grown in 3D, these cells can self-organize into laminar organized retinas; however, significant variation in the size, shape and composition of individual organoids exists, and this variation can create challenges. Neither the microenvironment nor the timing of critical growth factors driving eye formation or retinogenesis are fully understood. To explore the dynamics of early retinal organoid development, we have developed a SIX6-GFP reporter line that enables the systematic optimization of early eye field development, allowing for the identification of conditions that favor optic development. We found that SIX6 expression in 3D optic structures is influenced by small molecules regulating SHH and WNT signaling in a dose and time dependent fashion. In addition, we demonstrated that hypoxic growth conditions favor SIX6 expression, and promote eye formation. The systematic optimization of these pathways leads to optic vesicles with robust SIX6 expression and global gene expression profiles consistent with a retinal identity. The use of stem cell derived human retinal organoids with a real time marker for retinogenesis, therefore, represents an important model for probing the mechanisms of early human eye development. Day 35 retina cups were pooled (n=10-12) and separated as either GFP+ (n=2) or GFP- (n=3) samples and compared to human stem cells (n=2) (total of 7 samples run for RNA-seq).