Abstract
ABSTRACTTwo existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES‐derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical‐like neurons that are “stalled” at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES‐derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES‐derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical‐like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes. J. Comp. Neurol. 522:2691–2706, 2014. © 2014 Wiley Periodicals, Inc.
Highlights
IntroductionNeocortical projection neurons undergo distinct molecular refinements at progenitor (Molyneaux et al, 2005; Chen et al, 2005; Chen et al, 2008; Azim et al, 2009) and post-mitotic (Weimann et al, 1999; Arlotta et al, 2005; Alcamo et al, 2008; Britanova et al, 2008; Lai et al, 2008; Joshi et al, 2008; Azim et al, 2009; Tomassy et al, 2010; Cederquist et al, 2013) stages of development
We demonstrate the utility of coordinating markers of neuronal maturation with markers of neocortical subtypes to assess the stage and extent of neocortical differentiation
Previous reports of embryonic stem (ES)-derived neocortical neuronal subtypes have assessed the presence of individual markers or, less commonly, combinations of very limited and relatively broad markers to identify neocortical subtypes (Gaspard et al, 2008; Eiraku et al, 2008; Nasu et al, 2012; Ideguchi et al, 2010; Mariani et al, 2012; Shi et al, 2012; Espuny-Camacho et al, 2013)
Summary
Neocortical projection neurons undergo distinct molecular refinements at progenitor (Molyneaux et al, 2005; Chen et al, 2005; Chen et al, 2008; Azim et al, 2009) and post-mitotic (Weimann et al, 1999; Arlotta et al, 2005; Alcamo et al, 2008; Britanova et al, 2008; Lai et al, 2008; Joshi et al, 2008; Azim et al, 2009; Tomassy et al, 2010; Cederquist et al, 2013) stages of development. Populations of ESderived neocortical-like neurons sequentially express single genes characteristic of neocortical neurons in vivo Many of these genes (e.g., Pax, Ctip, Satb2) are not specific only to the neocortex, but are expressed in other regions of the developing neural tube. Pax is differentially expressed throughout the rostro-caudal extent of the neural tube ventricular zone (Ericson et al, 1997; Osumi et al, 1997; Briscoe et al, 2000; Alaynick et al, 2011), and Ctip is expressed in striatum, olfactory bulb, and hippocampus (Leid et al, 2004; Arlotta et al, 2005; Arlotta et al, 2008)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
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
