Figure 1.

Generation of retinal organoids and identification and characterization of cell subpopulations

(A) Retinal organoids were generated from iPSCs over a period of 49 days. iPSCs were differentiated as a monolayer for the first 28 days, and then cultured in 3D as a suspension for 7 days. Resulting organoids were then plated onto Matrigel and grown until retinal ganglion cells started to project out of the organoid at 49 days. These were harvested for scRNA-seq.

(B) Uniform Manifold Approximation and Projection (UMAP) representation of cells grouped into 23 subpopulations, as identified by Louvain clustering.

(C) UMAP plot of the cell types and lineages, as determined by analysis of differentially expressed genes of individual subpopulations and trajectory analysis. RGC clusters form one branch of the trajectory. Other cell types—RPE, interneurons, photoreceptors, and lens—arise from another branch of the trajectory. The last main branch consists of differentiating RPC subpopulations. RPC, retinal progenitor cell; RGC, retinal ganglion cell; RPE, retinal pigmented epithelium.

(D) Heatmap of the mean expression of cell-type-specific gene markers across each subpopulation. Expression values have been scaled and converted to Z scores and genes have been grouped by cell type. AC, amacrine cell; BC, bipolar cell; HC, horizontal cell; MG, Müller glia; PR, photoreceptor; RPC, retinal progenitor cell; RPE, retinal pigment epithelium; RGC, retinal ganglion cell.