Single-cell analysis reveals cellular heterogeneity and limits of marker-based assessment in retinal ganglion cell-enriched organoid cultures

Human pluripotent stem cell (hPSC)-derived retinal organoids provide an in vitro system for generating retinal ganglion cells (RGCs), yet the cellular composition and developmental fidelity of RGC-enriched cultures remain insufficiently characterised. Here, we tested an RGC-enriched approach involving dissociation of hPSC-derived retinal organoids at day 40, corresponding to peak expression of RGC markers, followed by two-dimensional culture conditions intended to enrich for RGC survival. Flow cytometry was used to assess the expression of RGC markers, including POU4F, ISL1, SNCG, and THY1. Across four samples, POU4F expression ranged from 79-95%, ISL1 from 18-58%, SNCG from 22-91% and THY1 from 3-29%, indicating substantial variability between markers and samples. Single-cell RNA sequencing analysis of 73,642 cells identified multiple retinal lineages, including retinal progenitors, RGCs, photoreceptor-committed cells, amacrine and horizontal cells, and retinal pigment epithelium (RPE), as well as off-target populations comprising HOX-enriched posterior neural cells and other cell types. Cellular composition varied across samples. Transcriptomically defined RGCs accounted for 19-45% of cells across samples, with different RGC subclusters identified. These findings indicate that marker-based assessments alone may overestimate RGC identity and reveal substantial cellular heterogeneity, including partially specified RGC states and off-target populations. Accordingly, the term "RGC-enriched" is used in a relative sense based on comparison with previously published scRNA-seq-validated differentiation studies rather than indicating complete lineage purity. Interpretation of RGC enrichment should consider differences between protein- and transcript-based measurements, sampling variability between assays, and the incomplete specification of functional RGC subtypes.