Figure 4.

Neural differentiation of SHED. (A–H) Immunocytochemical staining depicts SHED expressing nestin, GFAP, NFM, CNPase, βIII-tubulin, GAD, and NeuN. (I) Western blot analysis confirmed that SHED expressed neural markers as described above. After 4 weeks of culture in the presence of B27 supplement, bFGF (40 ng/ml), and epidermal growth factor (20 ng/ml) (Neural Diff. +), expression levels of βIII-tubulin, GAD, and NeuN were up-regulated when compared with regular culture conditions as described in Materials and Methods (Neural Diff. −). However, expression levels of nestin, GFAP, CNPase, and NFM remained the same after the treatment. (J–O) SHED may coexpress neuronal markers including βIII-tubulin (green)/GAD (red) and βIII-tubulin (green)/NFM (red). The morphology of SHED showed elongated cell-cytoplasmic processes that sometimes coexpress neural markers (triangles) or only express individual neural marker (open arrows). (P–S) Toluidine blue (0.1%) staining depicting the altered morphology of SHED after induction with neural culture medium (P and Q, arrows). Immunopositive staining for anti-MAP2 and anti-Tau antibodies on dendrites and axon (R and S, arrows), respectively. Double-staining experiments showing βIII-tubulin-positive cells were also detected in the same field (R, triangle, green). (T–W) SHED continued to express glial cell markers including nestin (red), CNPase (red), GFAP (red), and NFM (green) by immunocytostaining.