Ascorbic acid 2 phosphate

Rabbit bone marrow was obtained from 3- to 4-month-old New Zealand white rabbits according to a procedure approved by the Animal Care and Use Committee of Royan Institute, Tehran, Iran. First, the animals were anesthetized by intramuscular injections of 1.5 mL ketamine (100 mg/mL) and 0.5 mL xylazine (20 mg/mL). Bone marrow was aspirated under aseptic conditions from the tibia medullary canal by using a 19-gauge needle. Then, the bone marrow was mixed with Dulbecco’s modified Eagle’s medium (DMEM; Gibco) that contained 1% penicillin and streptomycin (PAN-Biotech) and 15% fetal bovine serum (FBS; Gibco) and incubated at 37 °C and 5% CO_2. The medium was changed twice a week until cells reached confluency. Passage-3 cells were used for the experiments.
To assess the osteogenic and adipogenic capability of the isolated cells, the medium culture was substituted by osteogenic medium (50 μg/mL ascorbic acid 2-phosphate [Sigma-Aldrich], 10 nM dexamethasone [Sigma-Aldrich], and 10 mM β-glycerol phosphate [Sigma-Aldrich]) and adipogenic medium (100 nM dexamethasone, 50 μg/mL ascorbic acid 2-phosphate, and 50 μg/mL indomethacin [Sigma-Aldrich]), respectively, for 21 days. Then, the cells were fixed and stained with Alizarin Red and Oil Red O (Sigma, USA) and observed by light microscopy.

A GBA1–/–hESC line was previously generated and characterized [8 (link)]. Neural precursor cells (NPCs) were generated from the hESCs using a modified dual SMAD inhibition protocol [9 (link)], as previously described [10, 11 (link)]. For neuronal differentiation, NPC were dissociated with 0.05% trypsin-EDTA (ThermoFisher, Cat. 25300054) and plated on poly-L-ornithine/laminin (PLO)-coated dishes at a density of 10,000–15,000 cells/cm2 in N2B27 medium supplemented with 100 ng/ml FGF-8 (PeproTech, Cat.100-25), 200 ng/ml sonic hedgehog (PeproTech, Cat. 100-45), and 100μM ascorbic acid 2-phosphate (Sigma, Cat. A8960-5G) for seven days. Finally, the NPCs were plated on PLO-coated plates at a density of 50,000 cells/cm² in BGAA medium, which was composed of N2B27 medium supplemented with 20 ng/ml BDNF (R&D System, Cat. 248-BDB-01M/CF), 10 ng/ml GDNF (PeproTech, Cat. 450-10), 500μM Dibutyryl-cAMP (STEMCELL Technologies, Cat. 100-0244), and 100μM ascorbic acid 2-phosphate (Sigma, Cat. A8960-5G). Neuronal cultures were maintained in BGAA medium for 6 weeks, and the cell culture medium was changed every 4 days.

Neural stem cells (NSCs) were generated and differentiated into neural cultures as described previously^{72 (link)}. For patterning, NSCs were plated on polyornithin-/laminin-coated culture flasks at 1E4 cells/cm² in DMEM/F-12 with GlutaMax (#31331093) and neurobasal medium (#21103049) supplemented with 1X B27 (ThermoFisher #12587010), 1X N2 (ThermoFisher #17502048), 50 µM 2-mercaptoethanol (ThermoFisher #31350010) and 100 ng/ml FGF-8 (Peprotech), 200 ng/ml sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma) and cultured for one week. For differentiation, the resultant progenitors were plated at 5E4 cells/cm² in basal medium supplemented with 20 ng/ml BDNF, 10 ng/ml glial cell-derived neurotrophic factor (GDNF; Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), and 100 μM ascorbic acid 2-phosphate.

pMSCs and eMSCs were induced to differentiate into osteogenic, chondrogenic, and adipogenic lineages. For osteogenic differentiation, both types of cells were cultured in osteogenic medium (DMEM [Dulbecco's modified Eagle's medium] supplemented with 10% fetal bovine serum (FBS), 50 μM ascorbic acid‐2‐phosphate, 10 mM β‐glycerophosphate, and 50 nM dexamethasone [all from Sigma‐Aldrich]). The medium was changed every 3 days. After 3 weeks, the cells were fixed in 4% paraformaldehyde and processed for Alizarin Red S and Von Kossa staining. For chondrogenic differentiation, cells were cultured in chondrogenic medium (DMEM supplemented with 10% FBS, 50 μM ascorbic acid‐2‐phosphate [Sigma‐Aldrich], and 10 ng/ml transforming growth factor [TGF]‐β1 [R&D Systems, Minneapolis, MN,
https://www.rndsystems.com]). Cells were processed for Safranin O and immunocytochemistry staining for COL2A1 and Aggrecan after 3 weeks. For adipogenic differentiation, cells were exposed to adipogenic medium (DMEM supplemented with 10% FBS, 1 μM dexamethasone, 200 μM indomethacin [Sigma‐Aldrich], 10 μg/ml insulin [Sigma‐Aldrich], and 0.5 mM methylisobutylxanthine [Sigma‐Aldrich]) for 4 weeks. The medium was changed every 3 days. After 4 weeks, the cells were processed for Oil Red O and immunocytochemistry staining of aP2 and C/EBP.

Microtissues were fabricated as described in our previous report [21] (link). CultiSpher S (Sigma) are macroporous porcine gelatin microcarriers with a diameter of 130∼380 mm and pore size of 10 mm. Microcarriers were rehydrated in Ca²⁺ and Mg²⁺ free PBS, autoclaved and rinsed once with PBS and twice with growth medium. Microcarriers were loaded at 2 mg/mL in 250-mL siliconized spinner flasks (Corning) in growth medium supplemented with 0.05 mg/ml of ascorbic acid-2-phosphate (Sigma) and 5×10⁻⁵ M of mercaptoethanol (Sigma). Cells were seeded at 5×10⁴ cells/mL using an intermittent stirring regime (3 min agitation at 30 rpm followed by 30 min settling, which was repeated for a total of 8 h). Culture was agitated at 50 rpm and maintained in growth medium for 8 days before medium was replaced with osteogenic medium consisting of DMEM supplemented with 10% FBS, 100 nM dexamethasone (Sigma), 10 mM sodium-β-glycerophosphate (Sigma), 0.1 mg/mL ascorbic acid-2-phosphate and 5×10⁻⁵ M of mercaptoethanol. Culture lasted for another 20 days and medium change was performed every 2 days.