Ascorbic acid

Retinal organoids between days 30 and 35 were cut into small pieces (0.1–0.5 mm) and plated on Matrigel coated culture dishes, or dissociated with Trypsin (Sigma-Aldrich, T9935) to single cells and plated on poly-D-lysine and laminin coated glass coverslips (Corning, 354087). After attachment, retinal organoid cells were cultured in RDM or BrainPhys neuronal medium (Stemcell Technologies, 05790) with SM1 (Stemcell Technologies, 05711) and N2 supplements (Thermo Fisher, 17502048), 20 ng/ml BDNF (PeproTech, 450-02), 20 ng/ml GDNF (Stemcell Technologies, 78058), 1 mM dibutyryl cyclic-AMP (Stemcell Technologies, 73882), and 200 nM ascorbic acid (Stemcell Technologies,72132) till desired time, followed by immunofluorescent labeling or electrophysiological recordings.

Induced pluripotent stem cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 10⁴ cells per cm² in primed hiPS cell medium (KSR/FGF2). After 48 h, the medium was changed daily with RPMI-based medium with B27 supplement (Gibco, ThermoFisher Scientific), 100 ng ml⁻¹ activin A (Peprotech), 1 µM CHIR99021, 1% penicillin and streptomycin for 3 days. On days 4–8, the medium was changed daily with DMEM/F12-based medium with N2 (Gibco, ThermoFisher Scientific) and B27 supplements, 0.05 mg ml⁻¹ ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol (Sigma-Aldrich), 2 µM dorsomorphin (Peprotech), 10 µM SB-431542 (Miltenyi Biotec), 1% penicillin and streptomycin. On days 9–12, the medium was changed daily with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 20 ng ml⁻¹ BMP4 (Peprotech), 0.5 µM all-trans retinoic acid (Sigma-Aldrich), 3 µM CHIR99021, 1% penicillin and streptomycin. On days 12–20, the medium was changed every other day with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 10 ng ml⁻¹ FGF10 (Stemcell Technologies), 10 ng ml⁻¹ FGF7 (Peprotech), 3 µM CHIR99021, 50 nM dexamethasone (Sigma-Aldrich), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1% penicillin and streptomycin.

Induced pluripotent stem cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 10⁴ cells per cm² in primed hiPS cell medium (KSR/FGF2). After 48 h, the medium was changed daily with RPMI-based medium with B27 supplement (Gibco, ThermoFisher Scientific), 100 ng ml⁻¹ activin A (Peprotech), 1 µM CHIR99021, 1% penicillin and streptomycin for 3 days. On days 4–8, the medium was changed daily with DMEM/F12-based medium with N2 (Gibco, ThermoFisher Scientific) and B27 supplements, 0.05 mg ml⁻¹ ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol (Sigma-Aldrich), 2 µM dorsomorphin (Peprotech), 10 µM SB-431542 (Miltenyi Biotec), 1% penicillin and streptomycin. On days 9–12, the medium was changed daily with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 20 ng ml⁻¹ BMP4 (Peprotech), 0.5 µM all-trans retinoic acid (Sigma-Aldrich), 3 µM CHIR99021, 1% penicillin and streptomycin. On days 12–20, the medium was changed every other day with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 10 ng ml⁻¹ FGF10 (Stemcell Technologies), 10 ng ml⁻¹ FGF7 (Peprotech), 3 µM CHIR99021, 50 nM dexamethasone (Sigma-Aldrich), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1% penicillin and streptomycin.

The potential of ASC to differentiate into osteoblasts and adipocytes was confirmed in monolayer culture. To induce adipogenic differentiation, cells were cultured in MesenCult™ adipogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) supplemented with MesenCult® MSC basal medium (STEMCELL Technologies, Canada). To induce osteogenic differentiation, we used MesenCult™ osteogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) in the osteogenic medium containing 10⁻⁴ M dexamethasone (STEMCELL Technologies, Canada), 1 M β-glycerophosphate (STEMCELL Technologies, Canada), and 10 mg/ml ascorbic acid (STEMCELL Technologies, Canada). Media from both cultures were replaced every 3 d for 21 d in total. The differentiation potential for adipogenesis and the formation of intracellular lipid droplets were assessed by Oil Red O staining after fixation in 10% formalin. The differentiation potential for osteogenesis was assessed by Alizarin Red S (ARS) staining after fixation in 10% formalin. To induce chondrogenic differentiation, we used a chondrogenic medium (LONZA, Switzerland). For histological analysis, pellets were embedded in paraffin and sectioned. Chondrogenic differentiation was assessed by Masson trichrome staining. Morphology and differentiation potential of ASCs are represented for one individual donor from three independent donors.

Human iPSC‐derived neural progenitor cells (NPCs) were purchased from ATCC (Manassas, USA). Information about the donors is readily available online (https://www.atcc.org/en.aspx). iPSC‐derived NPCs obtained from twp donors: ATCC‐DYS0530 from PD patient, ATCC‐BXS0117 from normal control were used. Neuronal differentiation from NPCs was accomplished by culturing on PLO/Laminin‐coated plates in neuronal differentiation medium, which was composed of DMEM/F12 + Neurobasal Medium (1:1) supplemented with N2, B27, BDNF (20 ng mL⁻¹), GDNF (20 ng mL⁻¹), NT3 (10 ng mL⁻¹), IGF (10 ng mL⁻¹), ascorbic acid (200 µm) (all from Stemcell Technologies), and dbcAMP (100 nm) (Sigma Aldrich). The neurons generated from the iPSC differentiation were both NeuN and MAP2 positive as previously described.^[24]