Mouse Embryonic Stem Cell Culture:
The murine embryonic stem cell (mESC) line D3 was cultivated on mitotically inactivated primary mouse embryonic fibroblasts (MEFs).
Embryoid body formation:
About 75 drops of ES medium containing various numbers of ES cells were placed on the lids of 10 cm petri dishes filled with phosphate-buffered saline (PBS) and were cultivated as hanging drops for 2 days. EB aggregates were then transferred from the hanging drops into 6 cm bacteriological petri dishes and were further cultivated for 5 days in suspension.
Generation of ESC-derived Neural Progenitor Cells (NPCs):
Embryoid bodies (EBs) generated from mESCs by the hanging drop method were plated in Iscove’s Modified Dulbecco’s Medium containing 10% FCS on 0.1% gelatin-coated dishes. After 1 day, medium was replaced with Dulbecco's Modified Eagle Medium:Ham’s F-12 (DMEM/F-12) supplemented with 5 µg/ml insulin, 50 µg/ml transferrin, 30 nM selenium and 5 µg/ml fibronectin. Cells were cultured at 37°C and 5% CO2 for 7 days, generating NPCs.
Enrichment of NCSCs from NPCs with CD57 expression sorting:
Dissociated NPCs were plated on poly-D-lysine/fibronectin (both at 150 µg/ml) coated culture dishes and cultured in NCSC medium, consisting of 5:3 DMEM low-glucose:neurobasal medium supplemented with 20 ng/ml bFGF, 20 ng/ml IGF-1, 1% N2 supplement, 2% B27 supplement, 35 ng/ml retinoic acid, 50 µM β-mercaptoethanol and 15% chicken embryonic extract. The cells were cultured for 7 days in a hypoxia chamber adjusted to 3% oxygen. FACS sorting of low-enriched NCSC-like population for expression of CD57 generated higher-enriched NCSC-like cells. CD57 antibody (clone NK1, Abcam, Cambridge, UK) was used for FACS sorting.
Higher Enrichment of SAP-like Cells by Sorting for Expression of GD2:
Low-enriched NCSC-like population were FACS-sorted for GD2 expression to generate highly-enriched SAP-like cells. GD2 antibody (Clone 14.G2a, BD Biosciences) was used to stain the cells for 30 min, followed by FACS sorting.