Wild-type E14TG2a (MMRRC, University of California Davis, Davis, CA, USA) mES were cultured on 10-cm tissue culture plates (BD Falcon, Heidelberg, Germany) in mES medium supplemented with 10
3 U/mL LIF and 1 μM PD0325901. Cells were passaged every 2 to 3 days (70% confluence) using TrypLE Express (Invitrogen, Schwerte, Germany) and reseeded at 0.5 to 1.0 × 10
6 cells per plate. Medium was changed daily. To generate aggregates, dissociated mES were seeded at 3000 cells per 100 μL per 96-well (U-bottom, low adhesion; Lipidure Coat, NOF, White Plains, NY, USA) in retinal differentiation medium (RDM); 2% Matrigel (growth factor reduced; BD Biosciences, Heidelberg, Germany) was added on day (D) 1, defining the day the differentiation protocol started as D0. Organoids were transferred to bacterial-grade Petri dishes (Greiner Bio-One, Frickenhausen, Germany) on D9, and cultured (37°C, 5% CO
2, 20% O
2,) in retinal maturation medium (RMM). Fifty percent of medium was changed every 2 to 3 days including EC23 (0.3 μM; Tocris, Avonmouth, Bristol, UK) from D14. Organoids were transduced with AAV2/8YF Rho–green fluorescent protein (GFP)
29 (1 × 10
10 vector genomes [vg]/organoid) from D20 to 22 followed by complete media change. The number of biological replicates was defined as experiments started from separate mES preparations (N) and the technical replicates as the number of individual organoids analyzed (n). Adeno-associated virus vectors were produced and purified as previously described.
29 Genomic titers were determined as previously described
30,31 with one of the following primer pair combinations: ITR2F, 5′-GGA ACC CCT AGT GAT GGA GTT-3′ and ITRR, 5′-CGG CCT CAG TGA GCG A-3′ or GFPF, 5′-TTC TCG TTG GGG TCT TTG CTC AG-3′ and GFPR, 5′-CGA CCA CTA CCA GCA GAA CAC-3′. For more details on media composition, AAV production, retinal organoid immunohistochemistry, and RT-PCR see Supplementary Methods.