Isoflurane damages neuronal differentiation by repressing the self-renewal of mES cells. (A) Immunostaining of tuj1 showed the gradual serious inhibition of neural differentiation from the mES cells treated with isoflurane for 2, 4, and 6 h. Ctrl means control groups with no isoflurane. The scale bar represents 25 μm. (B) Detection of the mRNA level of neural markers, tuj1, mash1, and map2, in the group treated with isoflurane compared with the control group by qRT-PCR. Ctrl means mES cells treated without isoflurane. Isoflurane means cell treated with isoflurane for 6 h. Data shown are mean±SD (n=3). *P<0.05, **P<0.01. (C) AP detection staining showed the inhibition of mES cell self-renewal by isoflurane at 2, 4, and 6 h. Ctrl means control groups treated without isoflurane. The scale bar represents 100 μm. (D) Detection of the mRNA level of stemness makers, Oct4, Sox2, Nanog, and Lin28, in mES cells treated with isoflurane for 6 h and the control group. Ctrl means mES cells treated without isoflurane. Data shown are mean±SD (n=3). **P<0.01. (E) Immunostaining of stemness markers, Oct4, Nanog, and SSEA1, in the mES cells treated with isoflurane for 6 h and the control group. Ctrl means mES cells treated without isoflurane. The scale bar represents 100 μm. (F) Detection of the mRNA level of the markers of the mesoderm, endoderm, and glia on day 7 of neural induction. Data shown are mean±SD (n=3). *P<0.05. mES cells, mouse embryonic stem cell; AP, alkaline phosphatase. Color images available online at www.liebertpub.com/scd