17To date, there have been only two reports regarding hippocampal induction from
PSCs (Yu et al. 2014a; Sakaguchi et al. 2015 ). Yu et al. first reported a protocol for
selective differentiation of DG granule neurons using human ES and iPS cells (Yu
et al. 2014a). They differentiated Prox1+ DG granule neurons by Wnt3a/BDNF
treatment; these neurons were electrically functional and were able to integrate into
endogenous DG circuitry. A recent paper further showed that titrating BMP and
Wnt exposure to neocortical tissues allowed the self-organization of dorsomedial
telencephalic tissues that include Foxg1+/Lef1+/Lhx2+ medial pallium tissues
(Sakaguchi et al. 2015 ). Interestingly, the sequential expression pattern of genetic
markers of choroid plexus (Foxg1−/Lmx1a+/TTR+), cortical hem (Foxg1−/Lmx1a+/
TTR−), and medial pallium (Foxg1+/Lhx2+/Lef1+) suggested that a self-organized
form of differentiation was occurring in the same chronological order as in in vivo
development (Fig. 1.6e). These SFEBq-induced dorsomedial telencephalic tissues
give rise to hippocampal granule (DG)- and pyramidal (CA)-like neurons through
further long-term culture. On day 61, Lef1+/Foxg1+ medial pallium-like continuous
NE portions clearly formed adjacent to the Lmx1a+ choroid plexus- and hem-like
domain, and a more basal layer of the Lef1+ NE was positive for neuropillin2 and
Zbtb20 (BTB/POZ zinc-finger family), both of which are known as entire hippo-
campal markers in the embryonic hippocampus (Chen et al. 1997 ; Xie et al. 2010 ).
IHC showed clear Zbtb20 expression in cells beneath the Lef1+ NE and weak
Zbtb20 expression in the NE. Collectively, these results demonstrate the successful
in vitro generation of hippocampal primordium-like tissue.
Furthermore, using dissociation culture, these dorsomedial telencephalic tissues
gave rise to Zbtb20+/Prox1+ granule neurons (corresponding to DG neurons) and
Zbtb20+/KA1+ pyramidal neurons (corresponding to CA3 neurons) (Fig. 1.6f), both
of which were electrically functional and positive for the maturation marker
CaMKII. Notably, by examining intracellular calcium dynamics, calcium transients
at 8 weeks after dissociation showed a synchronized pattern of network activity that
was not seen at 4 weeks after dissociation, suggesting the development of synaptic
connections and a neuronal network formation. Thus, it is suggested that the hip-
pocampal primordium tissues induced by SFEBq culture could recapitulate human
hippocampus development, allowing the generation of functional hippocampal
granule- and pyramidal-like neurons (Sakaguchi et al. 2015 ).
As mentioned above, the complex process of hippocampal development involves
cell fate determination, cell movement, dentate gyrogenesis, and circuit formations
(Yu et al. 2014b); this complexity has made it difficult to induce hippocampal tis-
sues in vitro. Thus, the discovery of a method for inducing continuous dorsomedial
structures in vitro is very intriguing. A speculated mechanism of hippocampal tissue
generation using SFEBq is as follows: (i) a small portion of hESC-derived cortical
tissue differentiates into dorsomedial tissue by culturing in the medium for choroid
plexus induction (days 18–21); (ii) despite culturing in medium based on the condi-
tions for neocortex induction from day 21, the induced dorsomedial tissue domain
functions as a dorsalizing organizer; and (iii) with the effect of this organizer, the
1 Telencephalic Tissue Formation in 3D Stem Cell Culture