Retinoic acid

Human SH-SY5Y neuroblastoma cells were cultured for use as a model of neuronal differentiation (Figure 1A) (24 (link)). Undifferentiated SH-SY5Y cells were cultured in growth media under standard conditions (5% CO₂, 37°C) with DMEM:F12 (Sigma D6421), supplemented with 10% fetal bovine serum (FBS) (Sigma F9665), 2 mM l-glutamine and 1% non-essential amino acids. SH-SY5Y differentiations were performed in triplicate. To differentiate the SH-SY5Y cells, flasks were coated with poly-lysine. Cells were seeded at 4.4 × 10⁴ cells/cm² and grown for 24 h in standard growth medium. The growth media was then exchanged for differentiation media neurobasal medium (Thermo Fisher 21103049), 2 mM l-glutamine, 1% FBS, 10% B27 supplement (Thermo Fisher 17504044) and 10 mM retinoic acid (Sigma R2625). Media was replaced after 48 h. After 72 h of exposure to 10 mM retinoic acid the media was exchanged to differentiation media without retinoic acid and cells were allowed to further differentiate for 72 h, including a media change at 48 h. RNA was then extracted from undifferentiated and differentiated cells with Tri Reagent (Thermo Fisher 15596026) and RNeasy columns (Qiagen 74106).

The cover slips (Thermo Scientific, Brunswick, Germany) were placed in 24-well plates and coated with poly-L-ornithine (28 μg/cm², 0.1 mg/ml; Sigma-Aldrich) plus laminin for 48 h at 37 °C. cover slips were washed twice and cells plated in differentiation medium consisting of DMEM/F12-GlutaMAX medium supplemented with 4% FBS, pencillin/streptomycin, HEPES, B27-supplemented with retinoic acid (Gibco, Life Technologies).

hPSCs grown on Laminin-521 were allowed to reach ~ 90% confluence in six-well plates (Corning) under self-renewing medium conditions. Once ~ 90% confluent, hPSCs were differentiated to retinal organoids as described by Gonzalez-Cordero et al. [10 (link)] with the following modifications. Briefly, after 4–5 weeks in culture, NRVs were transferred into either ultra-low-attachment 100-mm plates (Sarstedt) or 100-ml bioreactors (Chemglass) and cultured in RDM supplemented with 10% foetal bovine serum (Gibco), 2% Glutamax (Gibco) and 100 μM taurine (Sigma-Aldrich) (RDM + F) until formation of larger retinal organoids (weeks 5–10). BioMIXdrive 3 magnetic spinners (2Mag) were used to stir the medium in the bioreactors at a constant 22 rpm throughout the full differentiation period. The medium was changed once a week from here onwards. Developing retinal organoids were cultured in RDM + F supplemented with 1 μM retinoic acid (RA) (Sigma-Aldrich) (RDM + F + RA) (weeks 10–13). From week 13 onwards, retinal organoids where cultured with RDM + F made to the previous composition but using DMEM/F12 Glutamax (Cat. No. 10565–042; Gibco) instead of DMEM high glucose and adding 1% N2 supplement (RDM90 + F + RA), and the final retinoic acid concentration in culture was reduced to 0.5 μM (weeks 13–17).