292.2 Self-Organization of Cerebellar Tissue from hPSCs
in 3D Culture
2.2.1 Development of Mammalian Cerebellum
In humans, the cerebellum develops over a long period of time, from the early
embryonic days to the first postnatal year. Such protracted development could be the
source of vulnerability and the cause of broad spectrum of developmental disorders.
Cellular and molecular studies on the cerebellar development have been done
mostly in model animals. However, it is not known whether the mechanisms
revealed in model animals are simply applicable to human or not. Thus, the cellular
and molecular studies in humans have been long-awaited for elucidation of func-
tions and dysfunctions of the human cerebellum.
The cerebellum is a highly ordered brain structure with several well-defined
types of cells (Fig. 2.3). The early development of the cerebellum is conserved
among amniotes including human. The initial phase of cerebellar development is
the formation of the isthmic organizer, which lies at the midbrain-hindbrain bound-
ary (MHB). The isthmic organizer secretes positional signals such as Fgf8 and
Wnt1 and controls the AP pattern of the caudal mesencephalon (midbrain)-rostral
hindbrain. Fgf8 is essential for the induction of cerebellar development, while trans-
plantation of Fgf8-soaked beads in the rostral midbrain can cause ectopic formation
of cerebellar structures (Sato et al. 2001 ). The isthmic organizer is formed and
maintained by the intricate regulatory functions of region-specific transcription fac-
tors such as En1/En2, Pax2/5/8, Otx2, and Gbx2. The expression of these factors is
under the control of Fgf8 secreted by the isthmic organizer itself (Wurst and Bally-
Cuif 2001 ), and the format of a positive feedback loop involves Fgf8 contributing to
the maintenance of the tissue’s identity. Under its inductive influence, the cerebellar
anlage arises in the dorsal region of rostral hindbrain (alar plate of rhombomere 1)
(Nakamura et al. 2005 ; Zervas et al. 2005 ).
Cerebellar cells are generated in the two distinct germinal zones in the rhombo-
mere 1. One is the ventricular zone (VZ) of the cerebellar plate, which expresses the
basic helix-loop-helix (bHLH) transcription factor Ptf1a. The Ptf1a+ progenitors
produce GABAergic neurons of the cerebellar cortex (Purkinje, basket, Golgi, and
stellate cells) and of the deep cerebellar nuclei (DCN). The other one is the upper
rhombic lip (RL), which expresses another bHLH factor, Atoh1. The Atoh1+ pro-
genitors generate cerebellar glutamatergic neurons, including granule cells and
large DCN projection neurons (Butts et al. 2014 ; Hoshino 2006 ; Martinez et al.
2013 ; Millen and Gleeson 2008 ) (Fig. 2.4). The most prominent neuron type of the
cerebellum is the Purkinje cell, with its large soma arranged in a plane (known as
the Purkinje cell layer) and an extensive, flattened dendritic tree projecting into the
molecular layer (Fig. 2.3). Purkinje cells have a central role in integrating heteroge-
neous neural inputs from the mossy and climbing fibers. The dendrites of Purkinje
cells receive synaptic input from parallel fibers of granule cells, the main excitatory
interneurons of the cerebral cortex. In humans, each Purkinje cell dendritic tree may
2 Self-Organized Cerebellar Tissue fromfiHuman Pluripotent Stem Cells andfiIts...