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

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

Generating a Non-Integrating Human Induced Pluripotent Stem Cell Bank from Urine-Derived Cells

“…It had been shown that the external signaling pathways such as SMAD and GSK3β signaling are important in shaping the neural stem cell fate [38], [39]. As for the urine cell reprogramming, we reasoned that the signaling inhibitors present in the medium played critical roles in determining whether the cells become NPCs or iPSCs.…”

How to make a midbrain dopaminergic neuron

“…This also led to correctly specified and functional mDA cells capable of long-term survival without overgrowth formation, which innervated target structures and improved both drug-induced and spontaneous motor behavior in a rat unilateral 6-OHDA model of PD, similar to fetal human midbrain tissue (Grealish et al, 2014). GSK3β inhibitors have been also successfully used in other protocols to produce correctly specified mDA neurons (Denham et al, 2012;Xi et al, 2012) (Fig. 5K,L).…”

“…However, logistical and ethical difficulties in performing such studies using human fetal tissue have led to the search for more amenable cell preparations that could be standardized for quality, safety and functionality, prior to clinical use in PD. Human mDA neurons have been generated from multiple cell types in vitro, including neural stem/progenitor cells (NS/PCs) (Maciaczyk et al, 2008;Riaz et al, 2002;Ribeiro et al, 2012;Sanchez-Pernaute et al, 2001), pluripotent stem cells (PSCs) (Denham et al, 2012;Grealish et al, 2014;Kirkeby et al, 2012;Kriks et al, 2011;Xi et al, 2012) and by lineage reprogramming of somatic cells such as fibroblasts (Caiazzo et al, 2011;Kim et al, 2011;Pfisterer et al, 2011). The in vivo functional capacity of PSC-derived mDA cells has recently been found to match that of fetal tissue (Grealish et al, 2014).…”

Regulation of endogenous neural stem/progenitor cells for neural repair—factors that promote neurogenesis and gliogenesis in the normal and damaged brain

“…This is in contrast to work being performed in the embryonic stem (ES) cell and induced pluripotent stem (iPS) cell field, in which specific transcription factors are induced by addition of extrinsic factors for defined periods of time and in a specific sequence to allow production of desired neural cell types. For example, such a system has been used to generate floor plate cells which can subsequently generate mesencephalic dopaminergic neurons (Denham et al, 2012). Of course, such specific transcriptional regulation by exogenous factors is possible in the highly defined ES/iPS tissue culture environment but much more difficult in vivo , where there are many competing endogenous factors regulating NSPC function under normal conditions and even more so following neural injury.…”