NTera-2 cells are capable of differentiating into mixtures of neural and glial cells depending upon the induction conditions [33] [34] [35] . Since NTera-2 cells express high levels of Oct-4 [36, 37] , we used a lentiviral vector carrying a short hairpin (sh)RNA (Fig. 1A) to downregulate Oct-4 expression in order to develop a new protocol for cell differentiation. Three days after transduction with the shRNA lentivirus, Oct-4 expression was greatly diminished (Fig. 1B) . As shown in Fig. 1C , the cellular morphology remained the same as control NTera-2 cells at 3 days. However, dramatic morphological transformation was observed after 21 days of transduction, and cells had become flattened and enlarged, and showed a bushy morphology with numerous short and highly branched processes (Fig. 1C) . Such a cell morphology exhibited characteristics similar to protoplasmic astrocytes, distinct from ''fibrous astrocytes'' [38] . In addition to these morphological changes, the reduction in Oct-4 expression by NTera-2 cells was accompanied by a marked reduction in cell proliferation (Fig.  S1A ). The growth potential of NTera-2 cells after transduction was studied by flow cytometry, and these cells had almost ceased cell cycling with accumulations of cells in the G0/G1 phase on days 4 and 14 (40.6% and 69.1%, respectively, in Fig. S1B ). Thereafter, these cells remained mostly in the G0/G1 phase (e.g., on day 14) and underwent significant morphological changes, as described above. In addition, downregulation of Oct-4 expression often leads to upregulation of Cdx2 in ES cells [39, 40] . In NTera-2 cells, however, Cdx2 expression was not detected in RNAi-transduced cells (data not shown), indicating that this cell line may be derived from cells at a stage of embryogenesis later than the origin of the ES cell line. In addition, NTera-2 cells normally express the intermediate filament protein, nestin (Fig. 1C) , a marker of neuroglial progenitor cells. But, the expression of nestin disappeared 3 days after transduction (Fig. 1C) .