67The next step was to determine the type of hair cells arising in our culture.
Co-expression of Pax2 and Sox2 has been shown to be specific to vestibular hair
cells (Warchol and Richardson 2009 ). If these cells also express calbindin 2 (Calb2)
(Fig. 4.4f), they are likely to be type II vestibular hair cells (Oesterle et al. 2008 ).
Analysis of day 20 aggregates reveals that derived hair cells are positive for Sox2/
Pax2/Calb2, indicating a homogenous population of type II vestibular hair cells.
The overall morphology of the derived vesicles with a tightly packed bilayer of hair
cells and supporting lining on the luminal surface resembles that of a vestibular end
organ such as the utricle or saccule. Additionally, we detected Brn3a+Tuj1+ sensory
neurons forming ribbon synapses with hair cells in the culture. Moreover, the num-
ber of ribbon synapses increased with time, mimicking in vivo maturation of inner
ear hair cells (Lysakowski et al. 2011 ).
4.4 Potential Applications and Limitations
The inner ear organoid we have established faithfully recapitulates the formation
and differentiation of the inner ear in vitro, thus serving as a potent model system to
study both normal and pathological development of the inner ear. Utilizing mouse
embryos for studying early inner ear development is extremely difficult due to the
size and inaccessibility of the tissues, which cannot be maintained for extended
periods of time ex vivo. The ability to generate a large amount of developmentally
synchronized inner ear tissues by the organoid system also provides an unmet
opportunity for various biochemical assays, such as chromatin immunoprecipitation-
based assays.
a bBrn3aMyo7aDAPISox2Myo7a
TUJ1DAPICalb2Myo7aDAPISox2Myo7aDAPI
d efD14 D14D20 D28c D20Calb2EspinDAPIFig. 4.4 Immunofluorescence analysis of day 14–28 inner ear organoids (a–f). Myo7a; Myosin
7a, Calb2; Calbindin 2. Scale bars: 10 μm4 Inner Ear Organoids: Recapitulating Inner Ear Development in 3D Culture