Table 2.

Overview of RGC differentiation protocols from hPSCs published in the past five years (2020–2025)

Cell type	Culture format	Isolation/enrichment Method	Percentage of RGCs	Total protocol length	References
hiPSC • keratinocytes or blood cells- derived	2D adherent culture	None	Flow: • 87% POU4F+ • 93% SNCG+ • 22.5% RBPMS+ • 85.5% CD90/THY1+	36 days	[47]
hiPSC: • CHOPWT8 • CHOPWT9 • CHOPWT10	2D adherent culture	MACS using Thy1.2 microbeads	Flow (before MACS): • 58% THY1+ • 84% POU4F2+ • 12% RBPMS+ Immunocytochemistry: • 95% POU4F1+ (with MACS)	35 days	[42]
hiPSC	2D adherent culture	None	Varies between hiPSC-derived RGCs from different patients Flow: • 49.5–87% POU4F+ • 6–93% SNCG+ • 2.3–46% RBPMS+ • 42–91%CD90/THY1+	36 days	[60]
hiPSC • SIX6 risk allele • Healthy control	2D adherent culture of neural rosettes to RGCs	None	scRNA seq: • 11.6% mature RGC (58% on retinal lineage; 20% of those in mature RGC stage)	39 days	[29]
hiPSC • MT-ND4 mutation • Healthy control	3D embryoid bodies and neurospheres + 2D adherent culture of plated neurospheres	None	Not stated	27 days for embryoid bodies, neurosphere generation + 18 days for neurosphere plating to neurite outgrowth	[98]
hiPSC • healthy control; fibroblast- and Müller glia-derived	3D retinal organoids + 2D adherent culture of dissociated, enriched cells	• Dissociation of retinal organoids • MACS using human THY1 antibody	Flow: • 60% THY1⁺ cells before sorting (dissociated culture) • 78% THY1⁺ with MACS enrichment	56 days for retinal organoid generation + 7 days for RGC adherent culture	[43]
hESC: • H1	3D retinal organoids + 2D adherent culture of dissociated cells	Dissociation of retinal organoids	Immunocytochemistry: • 50% ATOH7+ • 30% SNCG+ • 30% ISL1+ • 50% HuC/D+	28 days for retinal organoid generation + 10 days for RGC adherent culture	[24]
hiPSC • TLHD2 • 201B7	3D retinal organoids + 2D adherent culture of dissociated, enriched cells	• Dissociation of retinal organoids • Two-step immunopanning	Not stated	55–65 days for retinal organoid generation + 3–5 days for RGC adherent culture	[93]
hiPSCs • corneal keratocytes-derived	3D retinal organoids + 2D adherent culture of dissociated cells	Dissociation of retinal organoids	Not stated	30 days for retinal organoid generation	[97]
hPSC: • OPTN(E50K) mutation	3D retinal organoids + 2D adherent culture of dissociated, enriched cells	• Dissociation of retinal organoids • MACS using Thy1.2	Immunocytochemistry: • 20–35% POU4F+ • 15–30% ISL1+	45 days for retinal organoid generation + up to 28 days for RGC adherent culture	[99]
hiPSC: • fibroblast-derived	3D retinal organoids + 2D adherent culture of retinal organoids	• None	scRNA-seq: 17% RGCs (based on the expression of POU4F2, ISL1, RBPMS, SNCG, GAP43, NEFL/M, ELAVL4, EOMES, and DCX)	35 days for retinal organoid generation + 14 days for retinal organoid adherent culture	[70]
hiPSC • MT-ND4 mutation • Healthy control	3D retinal organoids + 2D adherent culture of retinal organoids	• None	Not stated	35 days for retinal organoid generation + 17 days for retinal organoid adherent culture	[100]
hiPSC: • IMR90.4 • GM23720 hESC • WA09	2D adherent culture Genome-integrated transcription factors: NGN2, ATOH7, ISL1, POU4F2	• Clonal selection with zeocin	Immunocytochemistry: 88–94% POU4F2+ (tdTomato+)	6–7 days	[86]
hiPSC: • 297-1 hESC: • POU4F2-driven mCherry H7-A81	2D adherent culture Lentiviral delivery of transcription factors: NGN2	• Selection with puromycin	Immunocytochemistry: 14–20% POU4F1+	7 days	[84]
hiPSC: • NTUH-iPSC-02-02 • NTUH-iPSC-01–05	2D adherent culture Lentiviral delivery of transcription factors: ATOH7, POU4F2, and SOX4	• Selection with puromycin + zeocin	Immunocytochemistry: • 89.5% POU4F1+ • 94.1% ISL1+ 85.3% POU4F1+/ISL1+	15–20 days	[85]