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Reporter-guided photoreceptor differentiation in 2D culture using PGP1-hIPSCs: A comparable alternative to 3D
Davinia Beaver1, Adrian C Cioanca2, Nigel L Barnett1
1Clem Jones Centre for Regenerative Medicine, Faculty of Health Science & Medicine, Bond University, QLD Australia.
Purpose:
To develop and characterize a xeno-free, two-dimensional (2D) differentiation protocol for directing human induced pluripotent stem cells (hIPSCs) toward photoreceptor (PhR)-like cells, using a live-reporter system and transcriptomic analysis to evaluate lineage fidelity and maturation compared to the three-dimensional (3D) culture paradigm.
Methods:
A CRISPR/Cas9-engineered PGP1 hIPSC line expressing fluorescent reporters for retinal markers (VSX2, BRN3B, and RCVRN) was differentiated in adherent culture using chemically defined media supplemented with small molecules (T3, DAPT, taurine, and retinoic acid). Differentiation was assessed over time by immunocytochemistry, flow cytometry, reverse transcription quantitative polymerase chain reaction, ultrastructural imaging (transmission electron microscopy and scanning electron microscopy), and bulk RNA sequencing. Comparative transcriptomic analysis with 3D retinal organoid data was conducted to evaluate developmental kinetics and pathway enrichment.
Results:
The 2D protocol reproducibly generated PhR-like cells expressing PhR-associated protein markers, including CRX, NR2F2, RCVRN, THRβ OPSIN-S, OPSN-M/L, and ARR3. Flow cytometric analysis demonstrated 93.6%, 96.2%, and 70.3% of RCVRN, OPSN-M/L, and OPSN-S positive populations at day (D) 42, while up to 97.4% of cells stained positive for RCVRN by D52. Early commitment to a PhR lineage was evident by D30, supported by transcriptional profiles consistent with PhR-like ontogeny. Ultrastructural analyses revealed features of putative inner and outer segments, including developing cilia and disc-like "whorls" supported by expression of gap junction protein markers and cilium markers (TMEM138 and CX36). Bulk RNA sequencing demonstrated faithful temporal regulation of PhR gene networks and highlighted accelerated differentiation compared to 3D cultures.
Conclusions:
This pilot xeno-free 2D differentiation protocol offers a timely and scalable method for generating PhR-like cells from hIPSCs comparable to standard 3D culture systems. The results validate its downstream utility for retinal cell therapy development, high-throughput screening, and transplantation outputs pending further investigation, while supporting the transcriptome dominance model as a framework for evaluating photoreceptor fate acquisition in this culture paradigm.