Thermanox

iNSCs were dissociated using TryPLE select (Gibco) and replated (50,000 cells/cm²) on plastic cover slips (Thermanox; Thermo) coated with 5 μg/ml poly-L-ornithine (PLO; Sigma) and 50 μg/ml fibronectin (FN; Sigma). On the next day, the medium was replaced with neural induction medium; STEP2 medium containing 1 μM purmorphamine (Tocris) and 10 ng/ml brain-derived neurotrophic factor (BDNF; R&D) for 12–14 days.
For GABAergic neuronal induction, the cells at STEP2 were incubated with the neural induction medium for 12–14 days, followed by STEP2 medium consisting of 50 μM dbcAMP (Sigma) and 20 ng/ml BDNF for another 12–14 days.

Fifteen fertilized eggs were incubated at 37°C with 60% humidity. On day 2 of incubation 3 ml of albumen were removed using a syringe with a 21g needle. A square window was then opened in the eggshell and the window covered with Scotch^® Magic^™ tape (3M, St. Paul, MN) to prevent dehydration and contamination. At day 8 of incubation, 13 mm Thermanox^™ (Thermo Scientific^™ Nunc^™, Rochester, NY) rings were placed on the CAM under sterile conditions. 100 ng rmMCP-6 or 7 in 20μL of assay buffer (50 nM MES, 1M NaCl, pH 6.5) was placed into the Thermanox^™ rings. For controls 20 μL of assay buffer only were placed in the Thermanox^™ rings. On day 12 of incubation, the CAM was fixed in situ with 2% paraformaldehyde for 20 min. The Thermanox^™ rings with the underlying CAM were cut out and transferred to a petri dish containing PBS. Samples were stained with hematoxylin and eosin. The samples were mounted on glass slides and a minimum of five fields/sample was analyzed with a 4x objective on an Olympus BX-50 microscope (Olympus America Inc., Melville, NY) equipped with a SPOT RT3 digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).

Dental Pulp Stem Cells (DPSCs) were bought from Lonza (Lonza Group Ltd., Basel, Switzerland) and grown by using α-MEM medium (Sigma-Aldrich, St. Louis, MO, USA) added with 10% FBS, 1% penicillin/streptomycin (Euroclone S.p.A., Milan, Italy), at 37 °C and 5% CO₂ up to passage 5.
DPSCs were seeded on each titanium disc and on round shaped Thermanox (Thermo Fisher Scientific™, Waltham, MA, USA) of the same density, based on the diameter of the latter, for 1, 3, 7, 14 and 28 days. In each well of a non-tissue culture treated 24 well plate (Falcon^®, Corning Incorporated, New York, NY, USA) 1 mL of differentiation medium (DM) was added, or, rather, a complete α-MEM supplemented with osteogenic differentiating factors (dexamethasone10 nM, β‒glycerophosphate 5 mM and ascorbic acid phosphate 0.1 mM, all purchased from Sigma-Aldrich, St. Louis, MO, USA). At each established experimental time, supernatants were collected for further analyses.

The titanium discs and the control surface (Thermanox, Thermo, Waltham, MA, USA) were placed in 24-well microplates (one per well). Cloning tubes with a diameter of 0.8 cm and an area of 0.5 cm² were added on top of each disc to prevent the spheroids from rolling out of the materials. Spheroids were transferred from the culture plate to the disks using an automatic pipette with a volume of 20 µL. Each disc received 4 spheroids, placed on different quadrants of each disc surface, and 700 µL of high glucose DMEM supplemented with 10% FBS and 1% streptomycin/penicillin was added. The plates were incubated at 37 °C and 5% CO₂ for a total of 7 days, and samples were collected on days 2, 5, and 7.