Abstract
BackgroundThe in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required.Methodology/Principal FindingsIn this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation.Conclusions/SignificanceOverall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.
Highlights
Neural induction in vertebrate embryos was first described by Mangold and Spemann in 1924 [1] and results in the establishment of a neuroectodermal primordium from where the nervous system will arise
Our results show that commitment to neural fate in RHB-A occurs faster and produces a higher percentage of Sox1-GFP+ neuroepithelial progenitors (NPs), when compared to N2B27 (Fig. 1A)
We show that NPs derived from embryonic stem (ES) cells organize themselves into rosette-like structures, with an apico-basal distribution of polarity proteins similar to that described for neuroepithelial cells in the embryonic neural tube [7,8]
Summary
Neural induction in vertebrate embryos was first described by Mangold and Spemann in 1924 [1] and results in the establishment of a neuroectodermal primordium from where the nervous system will arise. The initial population of specified neuroepithelial progenitors (NPs) is known to express various pan-neural genes, like sox and sox2 [4,5] These NPs will acquire competence to produce neurons when they become part of the closing neural tube during neurulation, in a process that involves retinoid signalling from adjacent somites and the activity of proneural genes [6]. Neighbouring neuroepithelial cells signal to each other through Delta/Jagged ligands and Notch receptors, in a process that maintains a population of proliferating NPs and coordinates the timely production of neurons throughout embryonic development (reviewed in [10,11]) This unique architecture of the embryonic neural tube has transient character and disappears perinatally to give way to definitive CNS structures like the brain and spinal cord. To achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
