SU 5402

iPSC lines, WT1 (Female, aged 31) 33, WT2 (Male, aged 68) 34, WT3 (Female, aged 84) 34, RP (PRPF 31, c.1115_1125del11, RP11VS iPSC line) 33, and AMD (CFH, rs1061170, F181 iPSC line) 35 (derived and fully characterized) were cultured with mTeSR1 (Stem Cell Technologies, Cambridge, UK) on growth factor reduced matrigel‐coated 6‐well plates (Corning, NY). Retinal organoids were generated by dissociating cells at 90% confluence with Accutase (Thermo Fisher Scientific, Waltham, MA) and seeding 3,000–12,000 cells into each well of a 96‐well lipidure‐coated U‐bottom plate (Amsbio, Boston, MA) in 100 μl of mTeSR1 with 10 μM ROCK inhibitor (Y27632, Tocris, Bristol, UK). After 2 days, mTeSR1 + ROCK inhibitor was changed to differentiation medium, which comprised 45% IMDM (225 ml), 45% HAM's F12 (225 ml), 10% KOSR (50 ml), 1% GlutaMAX (5 ml), 1% Chemically Defined Lipid Concentrate (11905031; 5 ml), Pen/Strep (5 ml; Thermo Scientific, UK) and 1‐thioglycerol M6145 (450 μM; Sigma, UK). Medium was changed every 2 days, at day 6 BMP‐4 (1.5 nM) was added at day 6 as described in Kuwahara et al. 36 At day 18, “pooled” retinal organoids were transferred to ultra‐low attachment 6‐well plates (Corning, NY) and medium was changed to maintenance media: DMEM/F12, 10% FBS, GlutaMAX (5 ml), 1% N2, Pen/Strep (5 mL), plus 3 μM CHIR99021 (Stem Cell Technologies, Cambridge, UK), 5 μM SU5402 (Bio‐techne, Abingdon, UK). At day 24, medium was changed to maintenance media minus CHIR99021 and SU5402.
Process control experiments where performed as described above, with the following modifications in design 1: cell‐seeding number of 6,000, 9,000, or 12,000. KOSR: 5%, 10%, or 15%; chemically defined lipid concentrate: 0.5%, 1% or 1.5%; 1‐thioglycerol: 300, 450, or 600 μM; BMP4: 0.75, 1.5, or 2.25 nM. Design 2: cell number: 3,000, 5,500, or 8,000; CHIR99021: 1.5, 3, and 6 μM: SU5402: 2.5, 5, and 10 μM. BMP4: 2, 3.5, and 5 nM.
Moxifloxacin (Sigma, Cambridge, UK) was added to the culture media at concentrations of 50, 100, 300, 700, and 1000 μg/ml with an exposure time of 24 hours.

iPSCs, stably transfected with epB-NIL, an inducible expression vector containing the Ngn2, Isl1, and Lhx3 transgenes [125 (link)], were maintained in Nutristem XF/FF medium (Biological Industries, Beit-Haemek, Israel) in plates coated with hESC-qualified Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and passaged every 4–5 days with 1 mg/mL dispase.
To obtain spinal MNs, iPSCs were differentiated as previously described [61 (link)]. Briefly, hiPCSs were dissociated to single cells with Accutase (Thermo Fisher Scientific, Waltham, MA, USA) and plated in Nutristem-XF/FF medium (Biological Industries) supplemented with 10 μM rock inhibitor (Enzo Life Sciences, Pero (MI), Italy) on Matrigel coated dishes (BD Biosciences) at a density of 100,000 cells/cm². After 2 days of 1 μg/mL doxycycline (Thermo Fisher Scientific) induction in DMEM/F12 medium (Sigma-Aldrich, Milan, Italy), supplemented with 1× Glutamax (Thermo Fisher Scientific), 1× NEAA (Thermo Fisher Scientific), and 0.5× Penicillin/Streptomycin (Sigma-Aldrich), the doxycycline induction is maintained for additional 3 days in Neurobasal/B27 medium (Neurobasal Medium, supplemented with 1× B27, 1× Glutamax, 1× NEAA, all from Thermo Fisher Scientific, Waltham, MA, USA; and 0.5× Penicillin/Streptomycin, Sigma Aldrich, Milan, Italy), containing 5 μM DAPT and 4 μM SU5402 (both from Sigma-Aldrich, Milan, Italy). At day 5, cells were dissociated with Accutase and plated on Matrigel-coated dishes at a density of 100,000 cells/cm². Ten micrometers of rock inhibitor were added for the first 24 h after dissociation. Neuronal cultures were maintained in neuronal medium (Neurobasal/B27 medium supplemented with 20 ng/mL BDNF, 10 ng/mL GDNF, both from PreproTech, London, UK, and 200 ng/mL l-ascorbic acid, Sigma Aldrich, Milan, Italy) for 7 days.
Downregulation of circDLC1 was achieved by siRNA transfection at day 5 of differentiation and cells were collected after 3 days. A mix of two siRNAs targeting the back splicing junction of circDLC1 and the control siRNA scramble (see Figure S6) were used at 50 nM final concentration in presence of RNAiMax transfection reagent (Thermo Fisher, Waltham, MA, USA) following the manufacturer’s instructions.

The feeder-free hPSC-culture, SFEBq, preconditioning, d0-SAG, BMP, and induction-reversal culture methods were performed as described^{6 (link)–8 (link),41 (link)} with slight modifications. In Process 1 (maintenance culture), human iPSCs were maintained on LM511-E8 matrix (Nippi) in StemFit medium (Ajinomoto) according to a published protocol^{41 (link)} with slight modifications^{8 (link)}. The medium was changed every 1–2 days until the cells reached 70–80% confluence. iPSCs were passaged using TrypLE Select Enzyme (Thermo Fisher Scientific) and dissociated into single cells by gentle pipetting. The dissociated iPSCs were seeded at a density of 700–1700 cells/cm² and cultured in LM511-E8 matrix-coated six-well culture plates (Iwaki) containing StemFit medium with 10 µM Y-27632 (Wako)^{8 (link)}. Prior to differentiation, iPSCs were treated with SB431542 (SB; Wako) and/or smoothened agonist (SAG; Enzo Biochem) as the preconditioning step.
In Process 2 (retinal differentiation), hPSCs were treated with TrypLE Select Enzyme at 37 °C for 4–7 min, and dissociated into single cells by gentle pipetting. The dissociated iPSCs were quickly reaggregated using low-cell-adhesion 96-well plates with V-bottomed wells (Sumilon PrimeSurface plates; Sumitomo Bakelite) in differentiation medium (gfCDM+KSR) with Y-27632 and SAG (d0-SAG method). The differentiation medium was gfCDM supplemented with 10% KSR, while gfCDM alone comprised 45% Iscove’s modified Dulbecco’s medium (Gibco), 45% Hams F12 (Gibco), Glutamax, 1% Chemically Defined Lipid Concentrate (Gibco), and 450 µM monothioglycerol (Sigma-Aldrich). The day of SFEBq culture initiation was defined as day 0. On day 3, recombinant human BMP4 (R&D Systems) was added at 1.5 nM (55 ng/ml)^{7 (link)}. Thereafter, the culture medium was changed every 3–4 days to generate immature retinal tissue.
In Process 3 (induction-reversal culture), the immature retinal tissue generated from iPSCs was subjected to a two-step induction-reversal culture as follows. For the induction culture, cell aggregates on days 14–18 were transferred from 96-well plates to 90-mm non-cell-adhesive petri dishes (Sumitomo Bakelite; approximately 32–48 aggregates/90-mm dish), and cultured for 3 days in DMEM/F12-Glutamax medium (Gibco) containing 1% N2 supplement (Gibco), 3 µM CHIR99021 (GSK3 inhibitor; Wako), and 5 µM SU5402 (FGFR inhibitor; Wako). For the reversal culture (Process 3) and the maturation culture (Process 4), the cell aggregates were cultured in retina maturation medium as described (Nukaya et al. WO2019017492A1, WO2019054514A1). The medium was changed every third or fourth day to obtain 3D-retinas. Floating cell aggregates were analyzed using an inverted microscope (Keyence BZ-X810, Nikon Eclipse-Ti, or Olympus IX83).