S100A9 promotes apical-basal polarity and neural tube formation during differentiation of embryonic stem cells into neural progenitor cells

Objective To explore the role of S100A9 in the apical-basal polarity (ABP) and neural tube formation during the differentiation of embryonic stem cells (ESCs) into neural progenitor cells (NPCs) in vitro. Methods Immunofluorescence assay was used to observe the luminal arrangement, expression of Nestin and paired box protein 6 (Pax-6), and localization of S100A9, N-cadherin and Zonula occludens 1 (ZO-1) on days 1, 2, 3 and 6 of the ESCs differentiation into NPCs. In addition, the ESCs were treated with S100A9-downregulation lentivirus as well as subsequential exogenous S100A9 recombinant protein, then the localization changes of N-cadherin and ZO-1 were detected. The S100A9 expression in different time points during ESCs differentiation process was measured by Western blotting, and the effects of S100A9 on Notch1 and downstream centromere binding factor 1 (CBF1) were also tested. Results Nestin and Pax-6 were positively expressed after ESCs differentiation for 3 d, N-cadherin and ZO-1 were gathered at the apical side and presented the characteristic neural rosette structure of ABP, and the neural tubes were formed on day 6. The expression of S100A9 was upregulated significantly from day 3 after differentiation (P < 0.01) and was located on the apical side. Although ESCs were still able to differentiate into NPCs after down-regulation of S100A9, the number of neural rosettes was greatly reduced (P < 0.01), which could be partially restored by supplement of exogenous S100A9 recombinant protein (P < 0.01). Furthermore, declined S100A9 inhibited the expression of Notch 1 and downstream CBF1 during differentiation. Conclusion S100A9 may contribute to the ABP and the formation of neural lumen during the differentiation of ESCs into NPCs by regulating Notch 1/CBF1.