Deciduous teeth were either collected locally, through the Department of Pediatric Dentistry (neurotypical controls; n = 2), or remotely, for children with AS (n = 6). Immediately following the loss of the tooth, it was placed in transportation media (DMEM/F12 50/50 mix with HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin). DPSCs were isolated and cultured according to our previously described protocol72 . Briefly, after mincing the dental pulp from inside the tooth cavity, 3 mg/mL dispase II and 4 mg/mL collagenase I were added to digest the tissue. Cells were then seeded on Poly-D-Lysine-coated 12-well plates with media containing DMEM/F12 1:1, 20% FBS, newborn calf serum (NCS), and 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep). Confluent cultures (80%) were passaged with TrypLE Express (Gibco), and neuronal differentiation was performed only on early passage cells (less than four).
DPSC lines were seeded at 20,000 cells/cm2 on Poly-D-Lysine and Matrigel-coated coverslips with DMEM/F12 1:1, 10% FBS, 10% NCS, and Pen/Strep. At 80% confluence, the neuronal differentiation protocol was followed as previously published71 (link). Briefly, epigenetic reprogramming was performed by exposing the DPSC to 10 μM 5-azacytidine (Acros Scientific) in DMEM/F12 containing 2.5% FBS and 10 ng/mL bFGF (Fisher Scientific) for 48 h. Neural differentiation was induced by exposing the cells to 250 μM IBMX, 50 μM forskolin, 200 nM TPA, 1 mM db-cAMP (Sigma Aldrich), 10 ng/mL bFGF, 10 ng/mL NGF (Invitrogen), 30 ng/mL NT-3 (Peprotech), and 1% insulin-transferrin-sodium selenite premix (ITS) (Fisher Scientific) in DMEM/F12 for 3 days. At the end of neural induction, the cells were washed with 1X PBS. Neuronal maturation was performed by maintaining the cells in Neurobasal A media (Gibco) with 1 mM db-cAMP, 2% B27, 1% N2 supplement (Gibco), 30 ng/mL NT-3, and 1X Glutamax (Gibco) for 30 days.
DPSC lines were seeded at 20,000 cells/cm2 on Poly-D-Lysine and Matrigel-coated coverslips with DMEM/F12 1:1, 10% FBS, 10% NCS, and Pen/Strep. At 80% confluence, the neuronal differentiation protocol was followed as previously published71 (link). Briefly, epigenetic reprogramming was performed by exposing the DPSC to 10 μM 5-azacytidine (Acros Scientific) in DMEM/F12 containing 2.5% FBS and 10 ng/mL bFGF (Fisher Scientific) for 48 h. Neural differentiation was induced by exposing the cells to 250 μM IBMX, 50 μM forskolin, 200 nM TPA, 1 mM db-cAMP (Sigma Aldrich), 10 ng/mL bFGF, 10 ng/mL NGF (Invitrogen), 30 ng/mL NT-3 (Peprotech), and 1% insulin-transferrin-sodium selenite premix (ITS) (Fisher Scientific) in DMEM/F12 for 3 days. At the end of neural induction, the cells were washed with 1X PBS. Neuronal maturation was performed by maintaining the cells in Neurobasal A media (Gibco) with 1 mM db-cAMP, 2% B27, 1% N2 supplement (Gibco), 30 ng/mL NT-3, and 1X Glutamax (Gibco) for 30 days.
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