(A) Phase-contrast image of MEFs after overnight treatment with Sox2 retrovirus in fibroblast medium.

(B) Sox2-infected cells in NSC medium with growth factors generate networks and colonies on gelatin-coated glass coverslips by 8 days after infection.

(C) Sox2-transformed colonies are positive for the NSC markers Nestin and Sox2.

(D) Fibroblasts cultured in NSC medium with growth factors but without Sox2 retroviral transduction do not generate colonies or networks.

(E) Sox2-transduced cells after 11 days have drastically different morphology from their fibroblast counterparts.

(F) After three rounds of neurosphere generation, reprogrammed cells take on the characteristic bipolar NSC morphology.

(G) After multiple passages as a monolayer, NSC-like cells are a morphologically homogenous population.

(H) Morphology of NSC-like cells stays the same over prolonged passaging, and reprogrammed cells can proliferate over 28 passages.

(I) The morphology of NSC-like cells is similar to that of wild-type cortical-derived NSCs such as the commercial cell line SCR029 (Millipore).

(J–M) For the miNSC-A21 cell line, expression of Nestin and Sox2 is similar to that of brain-derived wild-type NSCs as revealed by immunostaining.

(N and O) qRT-PCR reveals that miNSC-A21 express typical NSC markers (N) but do not express pluripotency-related genes (O). Error bars denote standard deviation of triplicate reactions.

(P–R) In suspension culture, miNSC-A21 generates neurospheres similar to wild-type NSCs and with similar efficiency (n = 3). Values are mean ± SD.

Scale bars represent 50 μm in (A), (D)–(I), and (J)–(M) and 100 μm in (B), (C), (P), and (Q).