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4.3 Generation of Inner Ear Organoids
One of the earliest protocols for differentiation of mouse embryonic stem cells
(ESCs) into hair cell-like cells involved placement of stem cell aggregates onto an
adhesive culture surface in the presence of epidermal growth factor (EGF) and
insulin- like growth factor 1 (IGF-1) for a specified time period followed by an addi-
tion of bFGF to form inner ear progenitor cells. These progenitors were isolated and
induced toward further differentiation by removal of growth factors, which resulted
in detection of hair cell markers including Myo7a and espin (Li et al. 2003 ).
After confirmation that ESCs can be differentiated into hair cell-like cells,
attempts to generate functional/mechanosensitive hair cells followed. Cells gener-
ated in a protocol using induced pluripotent stem (iPS) cells, which included addi-
tion of IGF-1 and FGF2, displayed stereocilia and a kinocilium mimicking
endogenous vestibular hair cells. This protocol was dependent on plating stem cell-
derived otic progenitors over inactivated, fibroblast-like cells from embryonic
chicken utricles. Despite the relatively low yield of successfully derived hair cell-
like cells and the need for exogenous tissue, mechanical stimulation of these cells
elicited adaptation properties and transduction currents consistent with immature
hair cells (Oshima et al. 2010 ).
Recent insights and advances have enabled in vitro generation of inner ear sen-
sory epithelia and corresponding neural structures from mouse ESC aggregates.
This organogenesis was facilitated through the use of three-dimensional (3D) cell
culture techniques and precisely timed administration of recombinant proteins and
small molecules, with recognition of the importance of the NNE and the PPR as
precursors to otic differentiation. These aggregates subsequently develop and orga-
nize in a self-directed manner to form both mechanosensitive hair cells and corre-
sponding sensory neural components (Koehler et al. 2013 ; Koehler and Hashino
2014 ).
Organogenesis is increasingly being studied in the context of 3D floating cell
culture. In vitro organ models have been developed with 3D culture techniques
because they facilitate self-organization of cells into complex organ-like structures,
(Eiraku et al. 2008 , 2011 ; Sato et al. 2009 ; Suga 2011 ) as opposed to traditional
monolayer culture which restricts free movements of cells (Sasai et al. 2012 ). As
evidenced above, an array of intricate signaling gradients and morphological rela-
tionships takes place during organ development; 3D culture more closely models
physiologic embryogenesis and appears to facilitate self-organization of cells into
recognizable tissues (Sasai et al. 2012 ; Xinaris et al. 2015 ). This is due to the
increased freedom for morphologic changes (e.g., invagination and vesicle forma-
tion) and cellular interactions within the developing tissue (Sasai et al. 2012 ).
Organoids of neuroectodermal fate, namely, cerebral cortex and retina, have
been formed in vitro (Eiraku et al. 2008 ; Eiraku et al. 2011 ; Li et al. 2013 ). We pos-
tulated that inner ear organoids may be generated in a similar manner by driving the
ectoderm toward NNE as opposed to neuroectoderm. Detailing self-guided differ-
entiation within a 3D culture system of the functional anterior pituitary gland tissue
A.N. Elghouche et al.