R2625

Spheres obtained under DMEM/F12-GF conditions were examined for their neurogenic potential by culturing single cells in neural differentiation media. Briefly, single cells were seeded at 3.5 x10⁴ cells/cm² in a 24-well plate on coated coverslips with poly-L-lysine (10 µg/ml) solution. Our protocol for elevating intracellular cAMP was modified from Deng, et al.8 (link). In addition to the primary culture conditions to generate mesenchymal neurospheres, single cells were differentiated in serum-free DMEM/F12 plus N2, α-trans retinoic acid (1 µM), R-2625, SIGMA), and forskolin (5 µM, F6886, SIGMA) by 7 days after plating (DMEM/F12-RA+FORSK). Forskolin, a phosphodiesterase inhibitor, increases cAMP levels and retinoic acid induces neural differentiation. The DMEM-GF medium was changed on day 4 with a freshly prepared medium. On day 7, the cell culture medium was removed and cells fixed with p-formaldehyde 4% in PB 0,1 M for 15 min. Once fixed, plates were maintained in PBS solution at 4 °C until immunostaining was performed.

Cell lines stably expressing each half of the split Venus fluorescent system (V1S and SV2) for testing cell-to-cell transmission of α-synuclein were generated as described previously [18 (link)]. V1S and SV2 cells were subcultured in high-glucose Dulbecco’s modified Eagle medium (DMEM; SH30243.01, Hyclone) supplemented with 10% fetal bovine serum (FBS; SH30919.03, Hyclone) and 100 units ml⁻¹ penicillin/streptomycin (GIB-15070-063, Gibco), additionally containing 200 μg ml⁻¹ G418 (11811-031, Invitrogen). Cultures were maintained at 37 °C in a humidified 5% CO₂ atmosphere, and the culture medium was replaced every 2 days.
Human neuroblastoma SH-SY5Y cells (CRL-2266, ATCC) were cultured and maintained at 37 °C in a humidified 5% CO₂ atmosphere with media exchange every 2 days. Cells were differentiated by culturing in growth medium (DMEM + 10% FBS + 100 units ml⁻¹ penicillin/streptomycin) containing 50 μM retinoic acid (R2625, Sigma Aldrich).

After a 36 h si-RNA transfection, cells were cultures in a serum-free medium for 12 h followed by culturing with mediums containing Rol (17772, 5 μg/L, Sigma-Aldrich, St. Louis, MO), atRA (R2625, 5 μmol/L, Sigma-Aldrich, St. Louis, MO), atRA and AGN193109 (SML2034, 1 μmol/L, Sigma-Aldrich, St. Louis, MO) mixture, or equivalent amounts of DMSO (Sigma -Aldrich, St. Louis, MO), respectively. Then, cells were fixed in 4% paraformaldehyde or collected for subsequent analysis 24 h later.

The Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University approved this study (2022-041, date of approval: 1 June 2022). Equal numbers of male and female Kunming mice (9–10 weeks) (Caven Biogle (Suzhou) Model Animal Research Co. Ltd., Suzhou, China) were mated overnight. The vaginal plug was examined the following day, and 25 pregnant mice were used for subsequent experiments. The day (08:00) a vaginal plug was observed was regarded as the embryonic day 0 (E0d), and 16:00 was considered E0.5d. NTD models were established as described previously (Yu et al., 2017 (link)). On E7.0d–7.25d, the mice in the NTD group (N = 18) were administered corn oil-dissolved retinoic acid (50 mg/kg of body weight) (R2625; Sigma, St. Louis, MO, USA) by one-time gavage. Mice in the control group (N = 7) were administered an equal amount of corn oil. On E16.5d, the mice were sacrificed by cervical dislocation, and embryos were taken from the uteri. NTDs were confirmed using a dissecting microscope. Brain tissues (anterior end of the neural tube) of NTD and control embryos were collected and frozen for storage.

The organoid assay was established as described previously (Ng-Blichfeldt et al., 2018 (link); Ng-Blichfeldt et al., 2019 (link); Wu et al., 2019a (link)). EpCAM⁺ cells were combined with fibroblasts at a 1:1 ratio in DMEM/F12 (10% FBS) at a density of 2 * 10⁵ cells/ml. The cell suspension was then diluted 1:1 (v/v) with Matrigel (Fisher Scientific, Landsmeer, The Netherlands) and were seeded into transwell inserts (Thermo Fischer Scientific, Waltham, United States #10421761) witin 24-well plates (100 µl/insert). Cultures were maintained in DMEM/F12 with 5% (v/v) FBS, 2 mM glutamine, antibiotics, insulin-transferrin-selenium (1x, Gibco #15290018), recombinant mouse EGF (0.025 μg/ml, Sigma #SRP3196), bovine pituitary extract (30 μg/ml, Sigma #P1476), and freshly added all-trans retinoic acid (0.01μM, Sigma #R2625) at 37°C with 5% CO2. Media was refreshed every 2–3 days. The total number of organoids per well was counted manually 14 days after seeding using a light microscope at ×20 magnification. Organoid diameter was measured at the same day using a light microscope connected to NIS-Elements software. Thereafter, organoid cultures were fixed for immunofluorescence.