Glycogen Synthase Kinase 3β 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

Mark Denham,Brock J Conley,Chris Bye,Lachlan H Thompson,Jessie Leung,Mirella Dottori

doi:10.1002/stem.1204

Abstract

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 GSK3β and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6− expression, we describe as “pre-neuroepithelial.” 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. FOXA2+ 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 Cells2012;30:2400–2411

Highlights

The floor plate is a secondary organizer that secretes Sonic hedgehog (Shh) in order to pattern ventral regions of the developing neural tube [1]
We found that inhibition of both glycogen synthase kinase 3b (GSK3b) and activin/nodal pathways in human embryonic stem cells (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 upregulate expression of the floor plate marker, FOXA2, while supressing PAX6 expression to inhibit neuroepithelial fate
HESCs were cultured on mitomycin-C-treated mouse embryonic fibroblasts (MEFs) in hESC medium consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) without sodium pyruvate, supplemented with insulin/transferrin/selenium 1%, b-mercaptoethanol 0.1 mM, nonessential amino acids (NEAA) 1%, glutamine 2 mM, penicillin 25 U/ml, streptomycin 25 lg/ml, and fetal calf serum (FCS) 20% (Hyclone, Logan, UT, http://www.hyclone.com) or on mitomycin-C treated human foreskin fibroblasts (HFF) in knockout serum replacer (KSR) media consisting of www.StemCells.com

Summary

Introduction

The floor plate is a secondary organizer that secretes Sonic hedgehog (Shh) in order to pattern ventral regions of the developing neural tube [1]. Studies in chick embryos have demonstrated that exposing the dorsal neural tube to Shh signaling promotes ventralization to motor neurons but not to a floor plate fate [6], suggesting that high levels of Shh itself are not enough to confer commitment to a floor plate identity. More recently it was demonstrated in chick that in addition to requiring high levels of Shh concentration, the timing and duration of Shh signaling is critical for specifying cells toward a floor plate fate [9] These studies suggest that floor plate cells are specified by Shh in a temporarily restricted window, earlier than the neighboring neuroepithelial cells. They demonstrated that early exposure of SHH promoted floor plate fate at the expense of differentiation toward anterior neuroectoderm, and that delaying SHH treatment to cultures

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