9form a cortical plate (CP). Between the CP and SVZ, a calretinin+ and chondroitin
sulfate proteoglycan (CSPG)-rich subplate and intermediate zone are formed. At
day 70, the CP is mostly composed of Tbr1+ and Ctip2+ early-born/deep-layer neu-
rons, but by day 91, it contains a number of Satb2+ and Brn2+ late-born/superficial-
layer neurons in addition to Tbr1+ and Ctip2+ neurons (Fig. 1.3b). Remarkably,
within the cortical plate derived from human ESCs, late-born neurons (Satb2+/
Brn2+) tend to preferentially localize more superficially than early-born neurons
(Tbr1+/Ctip2+) on day 91 (Fig. 1.3b), which is reminiscent of the inside-out pattern
during fetal corticogenesis. Furthermore, after prolonged culture, one of the matura-
tion markers of neurons, CaMKII, is preferentially expressed in Tbr1+ neurons
located in the deep portion of the cortical plate on day 112, indicating the advanced
maturation of early-born neurons. Using a nonselective differentiation method, a
similar observation of the advanced stratified cortical tissue generated from human
PSCs has been reported (Lancaster et al. 2013 ). These data suggest that human
ESC-derived cortical NE spontaneously forms multilayered structures as seen in
human fetal cortex at the beginning of the second trimester. However, the ESC-
derived cortical tissues don’t have distinct layer with inside-out pattern formation as
in vivo cortex. This may suggest the necessity of not only more prolonged culture
but also extrinsic factors from the periphery, such as the meninges, cerebrospinal
fluid, and vascular networks, which is lacking in the in vitro cortical tissues (Tiberi
et al. 2012 ).
In early corticogenesis, COUP-TF1 is expressed in a caudal-high to rostral-low
gradient across the cortex (O’Leary et al. 2007 ; Zhou et al. 2001 ), whereas Sp8 is
expressed in an opposing gradient (Sahara et al. 2007 ) (Fig. 1.4a). In vivo studies
using mutant mice showed that Coup-TF1 and Sp8 have a crucial role for the caudal
and rostral specification of the cortex, respectively (Armentano et al. 2007 ;
Zembrzycki et al. 2007 ). FGF8, which is secreted from anterior neural ridge and the
commissural plate (Crossley and Martin 1995 ), is widely recognized as a key player
of cortical arealization through the regulation of expression of these transcriptional
factors (Fig. 1.4a). Indeed, overexpression of FGF8 in the mouse cortex induces a
caudal shift of graded expression of the transcriptional factors followed by the
expansion of rostral areas, while reduction of FGF8 induces a rostral shift of graded
expression of them followed by the reduction of rostral areas (Fukuchi-Shimogori
and Grove 2001 , 2003 ; Garel et al. 2003 ). With the advance of 3D culture, cortical
arealization is also recapitulated. In the case of mouse SFEBq culture, extrinsic
FGF8 treatment of ESC-derived cortical NE strongly suppresses COUP-TF1
expression. These cells express Tbx21, Reelin, and Tbr1, which is characteristic of
the olfactory bulb projection neurons in the rostral-most cortex (Yoshihara et al.
2005 ). In contrast, COUP-TF1+ cells substantially increase in number after treating
the NE with the Fgf inhibitor FGFR3-Fc. In a mouse study, ESC-derived cortical
NE gradually broke into several epithelial vesicles called neural rosettes in the
aggregate (Fig. 1.3a), which makes it difficult to study continuous tissue formation.
On the other hand, the human ESC-derived cortical structure keeps a continuous NE
structure for a long time (up to 90 days) (Eiraku et al. 2008 ; Kadoshima et al. 2013 ).
Interestingly, such continuous human ESC-derived NE frequently shows graded
1 Telencephalic Tissue Formation in 3D Stem Cell Culture