To generate NSCs, feeder-free defined medium cultured iPSC colonies were detached and cultured in suspension as EBs in a defined medium followed by adhered culture. Both iPSC lines formed neural tube-like rosette structures morphologically undistinguishable from those differentiated from hESCs (Fig. 1E). These rosette-derived cells uniformly expressed NSC markers nestin, Sox1, and musashi (Fig. 1G–1H), but not differentiated neuronal (e.g., β-III tubulin) or glial markers (GFAP or O4; data not shown). We did not observe significant differences between the two iPSC lines and the hESCs regarding the efficiency of generation of neural rosettes and NSCs. Furthermore, NSCs that were expanded in defined medium for over 10 passages maintained a normal karyotype and the expression of NSC markers Sox1 nestin and musashi. They also retained the ability to differentiate into neurons, astrocytes, and oligodendrocytes (Fig. 1I–1L). This result indicates that similar to hESCs, iPSCs adapted to defined medium differentiate into neural cells under defined conditions.
