Finally, we investigate whether single CSCs retained the their ability to form spheres/tumors even after long-term cultures. After the induction of secondary spheres from adherent cultures, spheres were subjected to 70 additional passages and then CD133-positive
immunoglobulin light chain were isolated by flow cytometry. From among 960 clonally isolated CD133- positive cells, 2 developed tertiary spheres that morphologically and phenotypically resembled the primary spheres and immunoreacted with nestin, musashi-1, and CD133 (Fig. 3A). Limiting dilution analysis showed that the self-renewal capacity of both tertiary CSC lines was about 26 and 10 times that of the primary and secondary spheres, respectively (Fig. 3B). Upon culture in differentiation medium, the clone-derived spheres also showed outgrowth and the
cells were positive for nestin, musashi-1, and glial and/or neuronal markers (Fig. 3C). Flow cytometric quantification indicated 2.15 (clone1) and 11.3% (clone2) of these cells were CD133-positive. Orthotopically (i.c.) transplanted tertiary spheres also recapitulated the histopathological properties of the parental tumors (Fig. 3D). Our data suggest that a small population of CSCs retained its stem cell properties even after cumulative passages under non-adherent/adherent culture conditions.