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. 2014 Apr 11;9(4):e94888. doi: 10.1371/journal.pone.0094888

Figure 4. Differentiation of hESC-derived IM cells into NPCs.

Figure 4

A. (a) Time course of the transcriptional expression of key NPCs marker SIX2 in hESC-derived NPCs. Appropriate duration of NPCs induction was determined by relatively higher transcriptional expression of SIX2 than other days. Transcription of other NPCs marker genes were examined by (b) real time RT-PCR and (c) RT-PCR in hESC-derived NPCs. Human fetal kidney (HFK) cDNA was used as positive control. Relative transcriptional levels were normalized to GAPDH, and the bars show mean ± S.E.M (n = 3). B. SIX2 expression rate was compared between hESC-derived IM cells and NPCs. Quantification of the number of cells expressing the key markers of specific differentiation stage was performed by manual counting in three randomly chosen fields. Scale bars = 200 µm. C. Several NPCs markers, including SIX2 (red), PAX2 (green), SALL1 (red) and WT1 (green) were notably detected in hESC-derived NPCs. Scale bars = 100 µm. D. Co-expression of other NPCs markers such as PAX2 (green) and SALL1 (green) with SIX2 (red) in hESC-derived NPCs in immunofluorescence. Scale bars = 100 µm. E. Immunocytochemistry for WT1 expression during NPCs induction in hESC-derivatives. WT1 expression (red) was evaluated at four different time points of NPCs induction (D0, D6, D10 and D15 of NPCs induction after IM stage). WT1-positive regions are designated by white lines. Scale bars = 100 µm.