7difference in developmental speed in vivo. Indeed, the emergence of each subtype
of cortical pyramidal neuron in vitro substantially coincides with the predicted tim-
ing of that observed in vivo (Clancy et al. 2007 ). Interestingly, such protracted
development of human PSC-derived neurons is also observed after transplantation
into the mouse brain (Espuny-Camacho et al. 2013 ). This suggests that the progeni-
tors and differentiating neurons sense and respond precisely to the differentiation
processes via an intrinsic timer.
Although the sequential generation of cortical neurons can be observed in both
2D and 3D cultures (Gaspard et al. 2008 ; Eiraku et al. 2008 ), 3D aggregate culture
has an advantage in the formation of complex tissue structures (Eiraku et al. 2008 ;
Kadoshima et al. 2013 ). In mouse SFEBq culture, cortical tissues generate a self-
organized laminar structure including four distinct zones along the apical–basal
axis: Pax6+ VZ, Tbr2+ SVZ, Tbr1+ early cortical plate, and Reelin+ marginal zone
(Eiraku et al. 2008 ) (Fig. 1.3a). In the case of human SFEBq culture, the apical side
(aPKC+) of the Foxg1+ NE is located on the surface of the aggregate at an early
phase (Kadoshima et al. 2013 ). After day 20, the structure starts to break into sev-
eral large domains and subsequently becomes apically concave. After dynamic roll-
ing morphogenesis, Foxg1+ NE generates a Pax6+/Sox2+ semispherical cortical
structure with a lumen at around day 30 (Fig. 1.3b). The cortical NE derived from
human ESCs shows a large, continuous structure surrounding a ventricle-like cavity
inside. The apical side markers are localized on the inner side facing the cavity,
while basal side markers are located outside. Pax6+ and Sox2+ cortical progenitors
form cell dense VZ on the luminal side, whereas pH 3+ progenitors undergoing
mitosis are found exclusively in its innermost part. TuJ1+ neurons occupy the zone
outside of the VZ and express markers of early cortical neurons such as Reelin,
Tbr1, and Ctip2 on day 42 (Eiraku et al. 2008 ; Kadoshima et al. 2013 ) (Fig. 1.3b).
In addition, live imaging of cortical NE derived from human ESCs shows that the
Pax6+ cortical progenitors are highly proliferative and undergo characteristic inter-
kinetic nuclear migration, typical for pseudostratified epithelium, similar to the
early embryonic cortex (Miyata et al. 2001 ). Taken together, ESC-derived cortical
NE spontaneously forms a typical apicobasal polarity as seen in early embryonic
cortex with interkinetic nuclear migration.
Generally, the prolonged culture of human ESC-derived cortical tissues leads to
oxygen and nutrient deprivation in the deep portion of the aggregates, and typically
the breakdown of cortical structure and cell death occur after about 50 days. But
when the 3D cortical tissues are cultured with 40% O 2 and nutrient supplementation
to the medium, they can grow for several months, which allows the induction of
cortical tissues that are equivalent to the second trimester of human development
(Kadoshima et al. 2013 ). The human ESC-derived cortical NE increases in thick-
ness and starts to show morphologically stratified structure on day 70 (Fig. 1.3b).
Layer marker expression patterns reveal complex zone separations in the cortical
tissues. On the luminal side, in addition to the VZ (Pax6+/Sox2+), Tbr2+ intermedi-
ate progenitors form a SVZ basally adjacent to the VZ. The superficial-most portion
is the marginal zone (MZ), which contains Reelin+ Cajal–Retzius (CR) cells and has
accumulated laminin in its surface. Beneath the MZ, cortical pyramidal neurons
1 Telencephalic Tissue Formation in 3D Stem Cell Culture