Characterization of rfNSCs and generation of IDO expressing NSCs.

rfNSCs were cultured in two different methods in vitro. (A) rfNSCs initially formed neurospheres under suspension culture conditions and were then maintained adherently on PLO-coated plates. Scale bars, 50 μm. (B) The expression of NSC markers (Nestin, Sox2, and Musashi1) was confirmed by RT-PCR. As shown, there was no difference in the expression of NSC markers in both culture methods. (C) Undifferentiated rfNSCs were positive for Nestin. However, differentiated rfNSCs showed an increased expression of neural cell markers such as Olig2 (oligodendrocyte), GFAP (astrocyte), and NeuN (neuron). Scale bars, 50 μm. (D) Control and IDO expressing vector map scheme. IDO and EmGFP genes were inserted into the lentiviral vector, which were expressed by dual promoters, the CMV promoter and the PGK promoter, respectively. (E) After infection, the expression of EmGFP as a selection marker was observed under fluorescent microscope. rfNSCs-Cont (left) and rfNSCs-IDO (right) expressed EmGFP. Scale bars, 100 μm. The expression level of IDO was confirmed by (F) RT-PCR and (G) western blot.