Additional culture in N2 medium resulted in the formation of suspended spherical structures (Fig. 5B) and adherent NPCs (Fig. 5C). The adherent cell population could be cultured and passaged on reaching approximately 70% confluency. The spherical structures responded to FGF-2 stimulation with an increase in size; quartered or halved sections grew back to their original size. The ICC characterization of the expanded cell populations from the entire EB outgrowth indicated that 66% ± 6% (1070/1622, two replicates) and 55% ± 4% (939/1710, two replicates, P > 0.05) of the cells were positive for the NPC markers, nestin and musashi1, respectively (Fig. 6, A and B). In an effort to enrich NPCs, mechanical isolation of tubular structures from EB outgrowths, as seen in Figure 5A, was undertaken with subsequent culture in N2 medium containing FGF-2. ventolin inhalers
Expansion of the cell population in the spherical structures that formed was achieved by dissecting the spheres (diameter, >0.7 mm) into halves or quarters under a dissecting microscope using a scalpel blade. The expanded cell populations from mechanically harvested tubular structures showed increased percentages of cells positive for nestin (78% ± 4%, 587/749, three replicates) and musashi1 (71% ± 8%, 474/667, three replicates), respectively.
FIG. 3. Formation of monkey ES cell-derived EBs. Phase-contrast micrographs show early EBs (A) and a complex/cystic EB (B). Original magnification X40 (A) and X200 (B).
FIG. 4. Expression of markers of three germ layers in complex/cystic EBs. An en-dodermal marker, a-fetoprotein (A); mesodermal marker, cardiac troponin I (B); and an ectodermal marker, vimentin (C), are shown. The EBs also expressed the NPC markers nestin (D) and musashi! (E) and neuron-specific enolase (F). Antibody localization utilized fluorescein isothiocya-nate-labeled (A-E) or Cy5-labeled (F) second antibody. Original magnification X100.
FIG. 5. Derivation of NPCs from monkey ES cells. Tubular structures of tightly packed columnar cells in an EB outgrowth are shown (A), as are spherical structures (B) and dissociated NPCs (C). Original magnification X100 (A and B) and X200 (C).