We next examined neural differentiation efficiencies from neurospheres into neurons and glial cells. The neurospheres were plated on fibronectin- and poly-l-ornithine-coated plates, and they were under differentiation conditions using neural culture medium containing four factors (brain-derived neurotrophic factor, glial-derived neurotrophic factor, ascorbic acid and dibutyryl-cAMP) for 10 days. The differentiated cell types were identified by immunocytochemical analysis using the neuronal markers, βIII-tubulin and MAP2 (microtubule-associated protein 2), the astrocyte markers, GFAP (glial fibrillary acidic protein) and S100B, and the oligodendrocyte markers, OLIG2 and SOX10. After 10 days, the differentiated cells expressed the neuronal marker and astrocyte marker in both the sample groups, but they did not express oligodendrocyte marker (Figure 2e and Supplementary Figure 2). These results indicate that the control and patient-derived neurospheres could differentiate into neurons and astrocytes, but not oligodendrocytes. We compared the efficiencies of neural and glial inductions between control and patient-derived neurospheres. The fraction of neurons in the total differentiated cells was significantly reduced by approximately 10% in patient-derived neurospheres (P=0.0086) when compared with that in control ones (Figure 2f). In contrast, the fraction of astrocytes in the total cells was significantly increased by approximately 12% in patient-derived neurospheres (P=0.0056; Figure 2f).
