Self-formation of concentric zones of telencephalic and ocular tissues and directional retinal ganglion cell axons

Reviewer #1 (Public Review):

The authors set out to develop an organoid model of the junction between early telencephalic and ocular tissues to model RGC development and pathfinding in a human model. The authors have succeeded in developing a robust model of optic stalk(OS) and optic disc(OD) tissue with innervating retinal ganglion cells. The OS and OD have a robust pattern with distinct developmental and functional borders that allow for a distinct pathway for pathfinding RGC neurites.

This study falls short on a thorough analysis of their single cell transcriptomics (scRNAseq). From the scRNAseq it is unclear the quality and quantity of the targeted cell types that exist in the model. A comparative analysis of the scRNAseq profiles of their cell-types with existing organoid protocols, to determine a technical improvement, or with fetal tissue, to determine fidelity to target cells, would greatly improve the description of this model and determine its utility. This is especially necessary for the RGCs developed in this protocol as they recommend this as an improved model to study RGCs.

Future work targeting RGC neurite outgrowth mechanisms will be exciting.

Reviewer #2 (Public Review):

The study by Liu et al. reports on the establishment and characterization of telencephalon eye structures that spontaneously form from human pluripotent stem cells. The reported structures are generated from embryonic cysts that self-form concentric zones (centroids) of telencephalic-like cells surrounded by ocular cell types. Interestingly, the cells in the outer zone of these concentric structures give rise to retinal ganglion cells (RGCs) based on the expression of several markers, and their neuronal morphology and electrophysiological activity. Single-cell analysis of these brain-eye centroids provides detailed transcriptomic information on the different cell types within them. The single-cell analysis led to the identification of a unique cell-surface marker (CNTN2) for the human ganglion cells. Use of this marker allowed the team to isolate the stem cell-derived RGCs.

Overall, the manuscript describes a method for generating self-forming structures of brain-eye lineages that mimic some of the early patterning events, possibly including the guidance cues that direct axonal growth of the RGCs. There are previous reports on brain-eye organoids with optic nerve-like connectivity; thus, the novel aspect of this study is the self-formation capacity of the centroids, including neurons with some RGC features. Notably, the manuscript further reports on cell-surface markers and an approach to generating and isolating human RGCs.