26
2.1 Introduction
Over the last decade, stem cell research has revolutionized the way to investigate the
intractable neuronal diseases. iPSCs have emerged as important tools for regenera-
tive medicine against brain damage. Recent advances in the techniques that differ-
entiate iPSCs into specific types of cells shed light on a novel replacement therapy.
In addition, these techniques enabled us to establish in vitro disease models from
the patient-derived iPSCs that allow us to understand the pathophysiology related to
disease mechanisms.
We have developed three-dimensional (3D) culture systems of hPSCs for neural
induction with high efficiency. In serum-free floating culture of embryoid body-like
aggregates with quick reaggregation (SFEBq) method, robust neural differentiation
is observed (Eiraku et al. 2008 ; Wataya et al. 2008 ). As observed in the development
in vivo, neural induction is the “default pathway” of PSC differentiation (Hemmati-
Brivanlou and Melton 1997 ; Muñoz-Sanjuán and Brivanlou 2002 ; Sasai and De
Robertis 1997 ; Stern 2006 ; Weinstein and Hemmati-Brivanlou 1999 ). It occurs in
PSCs when they are cultured in the absence of external inductive signals for meso-
derm or endoderm (Chambers et al. 2009 ; Kamiya et al. 2011 ; Kawasaki et al. 2000 ;
Smukler et al. 2006 ; Tropepe et al. 2001 ; Watanabe et al. 2005 ; Ying et al. 2003 ). In
SFEBq culture, PSCs are dissociated (to minimize possible effects of the culture
substrate or matrix) and subsequently reaggregated using a low-cell-adhesion
V-bottomed 96-well culture plate. PSCs selectively differentiate into neural pro-
genitors (>90% in total cells). Inhibition of endogenous SMAD signaling with
TGF-β antagonists can promote neural induction, particularly in hPSCs (Chambers
et al. 2009 ). Within few hours in this culture, hPSCs start to form reaggregates
and to express pluripotent stem cell markers (e.g., Oct3, Nanog), which gradually
disappear by day 7. Around 14 days, the majority of hPSC-derived tissue becomes
N-cadherin+, Nestin+, Pax6+, and Sox2+ neural progenitors, and they form
neuroepithelial- like structures with apicobasal polarity (Fig. 2.1).
As in vivo, the most anterior region of the brain can be easily induced in PSC
culture, due to default differentiation tendency of naive neuroectoderm (Eiraku
et al. 2008 ; Gaspard et al. 2008 ; Muguruma and Sasai 2012 ; Sasai 2013 ; Vallier
et al. 2004 ; Wataya et al. 2008 ). As for more caudal regions, the character and iden-
tity of PSC-derived neural progenitors are specified by a combination of embryonic
positional signals, including Fgfs, Wnts, retinoic acid (RA), and Shh, along the
anteroposterior (AP) and dorsoventral (DV) axes in neural tube (Anderson and
Vanderhaeghen 2014 ; Elkabetz et al. 2008 ; Gaspard and Vanderhaeghen 2010 ;
Kirkeby et al. 2012 ; Lee et al. 2000 ; Li et al. 2005 ; Lu et al. 2016 ; Mizuseki et al.
2003 ; Su et al. 2006 ; Watanabe et al. 2005 ; Wichterle et al. 2002 ; Zeng et al. 2010 )
(Fig. 2.2). Ample evidence suggests that PSC-derived neural progenitors have the
ability to sense positional information through patterning signals. Manipulation of
the positional information by addition or inhibition of secreted signals led to estab-
lishment of reproducible differentiation protocols for cortical, striatal, midbrain
dopaminergic, spinal motor, and autonomic nervous system from hPSCs.
K. Muguruma