Papain dissociation kit

Neocortex was carefully dissected from E15.5/E17.5 PGK^Cre, Rosa26^tdTomato embryos and digested in EBSS with 20 U/mL papain, 0.005% DNase (Papain dissociation kit, Worthington) for 25 min at 37 °C. Samples were then mechanically dissociated in EBSS with 0.005% DNase and cells were resuspended at 5 × 10⁴ cells per µL in Neurobasal medium (Life Technologies), supplemented with 1 × SM1 supplement (Stem Cell), 200 mM L-Glutamine and 1× penicillin–streptomycin (both from Life Technologies). Cells were seeded on scaffolds as previously described [27 (link),51 (link)]: (i) scaffolds were placed in a syringe along with a cell suspension of approximately 3 × 10⁵ cells; (ii) the plunger was introduced, and the syringe tip was closed using a 3-way valve; finally (iii) vacuum was induced by moving the plunger about 3 cm up and down until the scaffold was fully impregnated and became transparent. Scaffolds were then placed in 12-well plates. Culture medium was replaced after five days by BrainPhys supplemented with 1X SM1 supplement (both from Stem Cell).

Primary cortical neurons were isolated from cerebral cortices of embryonic day 13–14 mouse embryos (Charles River Laboratories) as previously described [93 (link)] with modifications. Briefly, pregnant female mice were euthanized between embryonic days 13 and 14, and embryonic cortices were dissected and enzymatically dissociated using a papain dissociation kit (Worthington Biochemical, LK003178). Neurons were plated on poly-D-lysine-coated 384- (Corning Life Sciences, 354,663) and 24-well plates (Falcon, 08-772-1) at densities of 1 × 10⁴ and 2.5 × 10⁵ viable neurons, respectively. The neurons were maintained at 37 °C with 5% CO₂ in Neurobasal medium (Life Technologies, 21,103–049) supplemented with 1% GlutaMAX (Thermo Fisher Scientific, 35,050–061), 2% B-27 supplement (Life Technologies, 17,504–044), and 1% penicillin/streptomycin (Life Technologies, 15,070–063). Primary cortical neurons were grown for 5 days before receiving described drug treatments and analysis.

Whole eye cups of transplanted eyes or organoids maintained in culture from the same differentiation round were dissociated with papain as described above (Papain Dissociation Kit, Worthington Biochemical Corp.; 20 U/mL). Cells were resuspended in MACS buffer and filtered through a 35 μm mesh before FACS and sequencing (method was modified based on ref. 62 (link); see Supplemental Methods for details).

CGNPs for cell culture and cell sorting were harvested from transgenic cerebella, as described previously [18 (link)]. In short, cerebella were dissected and meninges were removed. A single cell suspension was prepared with a papain dissociation kit, according to the manufacturer’s instructions (Worthington, Lakewood, NJ, USA).
For cell culturing, unsorted P0 and P7 CGNPs were resuspended in culture media (DMEM-F12 (Gibco, New York, NY, USA) supplemented with 1% N2 (Invitrogen, Waltham, MS, USA); 1.5% glucose (Invitrogen); 5 μM HEPES (Invitrogen); and 0.25 μg/mL SHH (R&D Systems, Minneapolis, MN, USA)). The cells were seeded on pre-coated plates with poly-D-lysine (100 μg/mL, Sigma) at a density of 250.000 cells per well in 24-well plates. For ionizing radiation experiments, the cells were irradiated the next morning with 0.5, 1 or 2 Gray of Y-rays from a ¹³⁷Cesium source (IBL 637 Cesium-137 Y-ray-machine). The cells were fixed at 2 h post irradiation. For reversine treatment, the cells were treated with DMSO or 250 nM of reversine (Sigma) for 48 h and subsequently fixed.
For gDNA, RNA, and protein isolations, tdTomato positive/DAPI negative cells were sorted on a Beckman Coulter MoFlo Astrios sorter (Brea, CA, USA). Cell pellets were stored at −80 °C until further processing. For the isolation of nuclei for scWGS, see below.