FIG. 2.

Differentiation of SHED in vitro. (A) SHED formed sphere-like clusters when cultured in serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Most cells in the SHED-derived spheres expressed nestin (B) and βIII-tubulin (C), but not TH (D). After incubation with a cytokine cocktail for 7 days, SHED-derived spheres generated neuron-like cells with multiple neurites (E) that expressed βIII-tubulin (F), MAP2 (G), and TH (H). Nuclei were counterstained with DAPI (blue). (I) Expression of TH (68 kDa) was confirmed by western blot analysis (positive control: rat TH [60 kDa]). (J) Real-time PCR revealed that gene transcription levels of the neuronal precursor, nestin, and the early neuronal marker, βIII-tubulin, in differentiated SHED induced from SHED-derived spheres were down-regulated, while expression of the mature neuronal marker MAP2 and TH, the rate-limiting enzyme during DA synthesis, was up-regulated (n = 3). Samples were normalized to the control gene actin. Scale bars: 50 μm (A) and 20 μm (B–H). *P < 0.05; **P < 0.01.