6
1.2.2 3D Cortical Tissue Generation In Vitro
Using SFEBq methods, mouse ESC-derived aggregates can be differentiated into
cortical progenitors and self-organize a stratified structure while recapitulating early
steps of corticogenesis (Eiraku et al. 2008 ; Nasu et al. 2012 ). We have also induced
human ESC-derived cortical tissue that resembles the very early cortical anlage dur-
ing the first trimester of human development (Eiraku et al. 2008 ). Moreover, recent
advances have allowed us to evaluate human PSC-derived cortical tissues in more
detail (Kadoshima et al. 2013 ). It is well recognized that the default state of neural
progenitors for regional specification along the neural AP axis is that of the rostral
forebrain, including the telencephalon and hypothalamus, and subsequently pat-
terned by caudalizing signals such as Wnt, RA, and FGF (Nordström et al. 2002 ;
Durston et al. 1998 ; Muhr et al. 1999 ; Cox and Hemmati-Brivanlou 1995 ) (Fig. 1.1a).
As seen in vivo, mouse and human ESC-derived neural progenitors can differentiate
into telencephalic precursors (Foxg1+) efficiently in the absence of caudalizing sig-
nals (Gaspard et al. 2008 ; Espuny-Camacho et al. 2013 ). Specifically, the active
inhibition of Wnt signaling by the Wnt antagonist Dkk1 or a chemical inhibitor is
effective for inducing the differentiation of telencephalic precursors (Watanabe
et al. 2005 ; Eiraku et al. 2008 ; Kadoshima et al. 2013 ). In the absence of any pat-
terning signals along the DV axis, the telencephalic precursors derived from human
ESCs tend to have dorsal identities (Pax6+ cerebral cortex), whereas in the case of
mouse cells, they tend to have more ventral identities (Gaspard et al. 2008 ). During
in vivo corticogenesis, cortical apical progenitors produce different types of neu-
rons (corresponding to different cortical layers) in a sequential manner. This sequen-
tial generation of layer-specific pyramidal neurons is controlled by intrinsic
mechanisms in cortical progenitors along a time axis (Hevner et al. 2003 ). This
sequential generation of layer-specific neurons can be seen even in primary mouse
cell culture of isolated cortical progenitors (Shen et al. 2006 ). Consistent with this,
mouse ESC-derived cortical progenitors in both 2D and 3D cultures also generate
layer-specific neurons in a sequential manner (Gaspard et al. 2008 ; Eiraku et al.
2008 ). In the case of human PSCs, it takes longer to induce neural tissues than it
does in mouse cells. Human ESC-derived telencephalic progenitors exhibit a NE
structure and start to express Foxg1 around day 18–20 (~75% of total cells). Most
of the Foxg1+ NE cells express Pax6, suggesting that they acquire cortical identities.
In the human SFEBq aggregates, Reelin+/Tbr1+ Cajal–Retzius cells (layer I neu-
rons) are born around day 35, followed by the generation of Reelin−/Tbr1+/Ctip2+
deep-layer neurons (layer V and VI neurons) around day 46, and then Satb2+/Brn2+
upper-layer neurons (layer II–IV neurons) differentiate around day 91 (Eiraku et al.
2008 ; Kadoshima et al. 2013 ), recapitulating the sequential generation observed
in vivo. Other groups have reported similar sequential generation of cortical neu-
rons in both 2D and 3D human PSC cultures (Espuny-Camacho et al. 2013 ;
Lancaster et al. 2013 ).
One of the striking aspects of in vitro human corticogenesis is the protracted
differentiation process compared with mice, which reflects the species-specific
T. Kadoshima et al.