For cell surface marker identification, a single cell suspension of sixth-to eighth-passage cells from trypsinized spheres was stained with anti-CD133, CD117 (c-Kit), or ABCG2 and secondary fluorescein (FITC)-or phycoerythrin (PE)-coupled antibodies (Dako, Carpinteria). Cells were fixed with 2% paraformaldehyde and were analyzed with a BD FACSCalibur apparatus (Becton, Dickinson and Company, Franklin Lakes, NJ).