Figure 4

In vitro differentiation of ALDH⁺/GD2⁺cells into neuro-ectodermal lineages. Monolayer cultures of Aldehyde dehydrogenase 1 (ALDH)⁺ cells obtained from BC-P007 xenografts were induced to differentiate into various cells lineages. Adipocyte was induced by incubation with dexamethasone, insulin, isobutylmethylxanthine and indomethacin for 30 days, and identified by Oil Red O staining (A. 400×). Osteocytes were induced by treatment with ascorbic acid, β-glycerophosphate and dexamethasone for 23 days and examined by Alizarin red staining (B. 400×). Chondrocytes were induced by culturing with ITS + premix, sodium pyruvate, TGF-β and dexamethasone for 15 days, and confirmed by immunofluorescence staining with anti-human collagen II antibody (C. 400×). The expression of GD2 (green) and collagen type 2 (red) in chondrocytes was detected by fluorescence-conjugated antibodies and observed under fluorescence microscope (D and E. 400×). Neuron-like cells were induced by incubation of the ALDH⁺/GD2⁺ cells from xenografted tumor of BC-P007 with retinoic acid, hydrocortisone, ITS and c-AMP, and was observed under phase contrast microscope on Day 7 (F. 400×) and Day 14 (G. 400×). Twenty-two days after culture, the neuronal lineages of these cells were examined by fluorescence microscopy with AF488 labeled antibodies against nestin, βIII-tubulin and GFAP (H. 400×). And, double staining of nestin and βIII-tubulin, βIII-tubulin and GFAP and GFAP and nestin (I - K, 1,000×) with AF488 or AF594-labeled antibodies was shown.