13neurons are GABAergic and express GAD65/67 (Marin et al. 2000 ; Nóbrega-
Pereira et al. 2010 ). In a mouse ES cell study, SFEBq-cultured mouse ESC aggre-
gates differentiated preferentially into cortical progenitors (Eiraku et al. 2008 ) in the
absence of exogenous Shh signals. When Shh signaling was blocked by the hedge-
hog (Hh) inhibitor cyclopamine, the majority of aggregates expressed the cortical
markers Pax6 or Tbr1, whereas few expressed ventral markers such as Gsh2,
Nkx2.1, Dlx2, and GAD67 (Danjo et al. 2011 ). However, a moderate dose of Shh
(10 nM) strongly inhibited Pax6 or Tbr1 expression, and the ESC-derived Foxg1+
domain expressed subpallial markers Gsh2, Dlx2, or GAD67. Although the MGE
marker Nkx2.1 was rarely seen in cells cultured with a moderate dose of Shh (10
nM), the aggregates treated with a higher concentration (30 nM) contained a sub-
stantial number of Nkx2.1+ cells (Danjo et al. 2011 ). When induced under these
conditions and purified by a fluorescence-activated cell sorter, telencephalic cells
efficiently differentiated into Nolz1+/Ctip2+ LGE neuronal precursors and subse-
quently, both in culture and after in vivo grafting, into DARPP32+ medium-sized
spiny neurons. Purified telencephalic progenitors treated with high doses of the
hedgehog (Hh) agonist SAG (smoothened agonist) differentiated into MGE- and
CGE-like tissues. Interestingly, in addition of Shh signaling, the efficient specifica-
tion of MGE cells requires Fgf8 signaling but is inhibited by treatment with
Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed
by Fgf8, suggesting that specific Fgf signals play different, critical roles in the posi-
tional specification of ESC-derived ventral subpallial tissues.
As in mouse ES cells, human ES cell-derived cortical tissues can also acquire a
ventral telencephalic fate after Shh treatment (Kadoshima et al. 2013 ). When the
human ESC-derived telencephalic NE was partially ventralized by a moderate level
of hedgehog agonist (30 nM smoothened agonist or SAG for days 15–21), a
substantial portion of Foxg1+ NE expressed Gsh2 (Fig. 1.5b). A mass of GAD65+
GABAergic neurons was generated underneath this LGE NE, as seen in vivo,
whereas the rest of the telencephalic NE was largely positive for the cortical NE
marker Pax6. Higher concentrations of SAG induced the MGE marker Nkx2.1 at
the cost of Pax6 and Gsh2 expression (Fig. 1.5c). Interestingly, in the human aggre-
gates, continuous epithelial tissues were retained, resulting in the generation of cor-
tical (Pax6+) and LGE (Gsh2+) domains as seen in vivo, suggesting that our improved
culture method allows self-formation of pallial–subpallial structures in sequential
order (Fig. 1.5b). Generation of pallium–subpallium in one sphere will offer the
analysis of tangential migration of GABAergic cortical interneurons using the
SFEBq method; thus, SFEBq culture relating to ventral telencephalon is an efficient
in vitro culture system for cortical interneurons and is also very interesting for not
only stem cell biology but also human developmental biology. Besides, analysis of
Shh signaling cascades on the subpallial patterning of ESC-derived telencephalic
tissues is also an interesting topic.
Deficiencies in subtypes of interneurons, and the resulting excitatory–inhibitory
imbalances, have been associated with many neurodevelopmental and neurodegen-
erative diseases, such as epilepsy, schizophrenia, autism, and Huntington’s disease
(Sigurdsson and Duvarci 2015 ; Kumar et al. 2015 ), so the recapitulation of ventral
1 Telencephalic Tissue Formation in 3D Stem Cell Culture