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Establishing an efficient differentiation procedure is prerequisite for the cell transplantation of pluripotent stem cells. Activating fibroblast growth factor (FGF) signals and inhibiting the activin/nodal pathway are both conserved principles to direct the neural induction (NI) of developing embryos and human embryonic stem cells (hESCs). Wnt signal and OCT4 expression are critical for the hESC pluripotency; however, their roles in cell differentiation are largely unclear. We demonstrate that in the presence of FGF2 and activin inhibitor SB431542, applying a small-molecule Wnt agonist, BIO, efficiently and rapidly steers the NI of all our tested hESCs. A human induced pluripotent stem cell (iPSC), which is refractory for efficient neural conversion by FGF2, effectively differentiated to SOX1(+) cells after the BIO/SB431542/FGF2 treatment. In addition, BIO promoted cell survival and transiently sustained OCT4 expression at the early NI stage with FGF2 and SB431542. Interestingly, at the late NI stage, the OCT4 level rapidly declined in the treated hESCs and consequently initiated the formation of neural rosettes with forebrain neuron characteristics. This study illustrates the distinct effects of Wnt activation on maintaining pluripotency and committing neural lineages at the early and late NI stages of hESCs and iPSCs, respectively.

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.