Seaplaque

The chondrocyte-agarose constructs were prepared as previously described [17 (link),18 (link)]. Briefly, trypsinized chondrocytes were embedded in 2% agarose (Seaplaque, Cambrex BioScience) at a density of 2 x 10⁶ cells/mL. Constructs were then placed in 6-well culture dishes and treated for 3 weeks with BIT culture medium. The culture medium was replaced every 2 days.

On day E13.5, the pregnant mothers were injected with BrdU (50 mg/kg, Sigma) and the fetal brains were collected 1 hour later. The brain explants, with intact meninges, were embedded in low-melt agarose (3% SeaPlaque, Cambrex) and cut into 300-μm coronal sections with a vibratome (Leica). For the brain explant co-culturing experiments, DMEM/F12 media containing 10% fetal bovine serum, 2 mM glutamine, 100 mM dextrose, 100 μM penicillin/streptomycin was removed 2 days prior to the explant experiment and replaced with 1.5 ml of Neurobasal (NB) medium (Invitrogen) containing B27 supplement with or without recombinant human STI-1 (100 ng/mL, Novus Biologicals). Thus, the media was conditioned for 48 hours prior to the explant experiment. Filter inserts (0.40 μm, Millipore) in 60-mm plates (Costar) were placed in the wells containing the mAPSCs/conditioned media (mAPSCs-CM), and NL (control) or FOXC1^-/- brain explants were transferred into the inserts and cultured for 36 hours post BrdU injection. For treatment with STI-1, a stock solution of 1 mg/mL STI-1 was diluted to 100 ng/mL in NB medium. Following culturing, all explants were fixed for 30 min in 4% paraformaldehyde and cryosectioned in 10-μm increments. Sections were immunolabeled for BrdU and Ki-67 and analyzed for cell cycle exit as described above.