Figure 1

Controlled derivation of neuroectodermal spheres from human embryonic stem cells. (A) Schematic showing neuroectodermal sphere (NES). By a simple medium change without an attachment step, embryoid bodies (EBs) could be differentiated to NESs harboring neuroprogenitor cells. EBs were grown in EBM for a week and then transferred to NSM supplemented with growth factors. The first subculture was performed one week later (D14) and, about two days later (D16), rosette-containing NESs appeared. The NES samples we used were D21 NESs, if not otherwise indicated. (B) Photographs of differentiating cell clumps at indicated times. Human embryonic stem cell (hESC) colonies (a) were divided into regular-sized (500 μm in length) clumps (b) using a chopper. Floating EBs at day 7 (c) are shown. NESs at day 21 have prominent rosette-like folded structures in the spheres (d). We piled up EBs and NESs in single spots before taking pictures. (C) Expression of neural stem cell (NSC) markers in NESs. NESs were allowed to attach to culture equipment and were stained either for SOX1 (a), PAX6 (b), Nestin (NES, c) and TUJ1 (d). TUJ1-positive neurites are scattered, usually around the boundaries of NES clumps (arrows). Boundaries of rosettes are indicated by dotted circles. (D) RT-PCR for various marker genes of different cell lineages. NSC marker genes are abundantly transcribed in hESC-derived NESs (right panel). Other lineage markers such as those of ESCs (Stem), mesoderm lineage cells (Meso) and endoderm lineage cells (Endo) are not preferentially expressed in NESs (left panel). β-Actin (ACTB), internal control. (E) RT-PCR analysis for markers of anterior regional identity (FB; FOXG1 and OTX2), mid-hind brain markers (MB/HB; PAX2 and EN1), and posterior CNS markers (HB; KROX20 and HOXB4). Scale bars, 200 μm in B and 100 μm in C; EBM, embryoid body medium; NSM, neurosphere medium; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; LIF, leukemia inhibitory factor.