EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Glycogen Synthase Kinase 3b and Activin/Nodal Inhibition in Human Embryonic Stem Cells Induces a Pre-Neuroepithelial State That is Required for Specification to a Floor Plate Cell Lineage

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3b and activin/nodal pathways in hESC induces a cellular state of SOX21/PAX62 expression, we describe as ‘‘preneuroepithelial.’’ Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA21 cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage. STEM CELLS 2012;30:2400–2411 Disclosure of potential conflicts of interest is found at the end of this article.