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Neural Induction of H9 hESC: H9 Human embryonic stem cells (hESC) (Wicell Madison, WI) were cultured on mouse embryonic fibroblasts in hESC medium (DMEM:F12, 20% Knockout Serum Replacement (KOSR), non-essential amino acids, 20 mM glutamine) (Invitrogen), 1X penicillin/streptomycin (Lonza), bFGF (Cat. No. 354060). Cell cultures were treated with Dispase (Cat. No. 354235) and cell colonies were scraped and placed in hESC medium without FGF in low adhesion plates to form embroid bodies (EB). After 4 days the EBs were plated on BD™ Matrigel-coated plates (Cat. No. 354277) and placed in neural induction medium (DMEM:F12 (Invitrogen), 1X ITS supplement (Sigma), 250 ng/ml recombinant Noggin (R&D Systems) and penicillin/streptomycin (Lonza) and cultured for an additional 15 days. Differentiation of H9-derived NSC: H9 hESC-derived NSC were differentiated in neural differentiation medium (DMEM:F12, 0.5X B27, 0.5X N2 (Invitrogen), 1X penicillin/streptomycin (Lonza), 20 ng/ml BDNF, 20ng/ml GDNF (both from Peprotech) and 0.5 mM dibutyryl cyclic AMP (Sigma) for 28 days.
Differentiation of H9-derived NSC: H9 hESC-derived NSC were differentiated in neural differentiation medium (DMEM:F12, 0.5X B27, 0.5X N2 (Invitrogen), 1X penicillin/streptomycin (Lonza), 20 ng/ml BDNF, 20ng/ml GDNF (both from Peprotech) and 0.5 mM dibutyryl cyclic AMP (Sigma) for 11 days. Culture medium was replaced with AGM™ Astrocyte Growth Medium (Lonza) for an additional 16 days.