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central core of the aggregate. Between days 9 and 12, continuous evagination of
vesicles into the outer cell layer occurs (Fig. 4.3i). These vesicles, appearing as
translucent cysts, comprise cells from the presumptive OEPD epithelium that are
Ecad+ Pax2+ Pax8+ Sox2+, consistent with formation of the prosensory domain.
Treatment of aggregates for 48 hours with a Wnt inhibitor, XAV939, suppresses the
formation of prosensory and Pax2+ vesicles, suggesting the importance of Wnt sig-
naling in otic vesicle formation (Groves and Fekete 2012 ). The evagination of vesi-
cles from the aggregate is inconsistent with the observed invagination of in vivo otic
vesicles into the head mesenchyme. This may be attributed to the basal component
of the epithelium being oriented toward the superficial aspect of the aggregate as
determined by analysis of aggregates allowed to develop epidermal tissue and con-
firmed through staining for the polarity markers aPKC and laminin. Some of the
large vesicles grew to a size (Fig. 4.3j–l) comparable to typical adult mouse utricles
(~1000 μm) (Li et al. 2008 ).
4.3.5 Morphologic and Functional Analysis of Inner Ear
Sensory Epithelia
The prosensory domain of the otic vesicle further develops into the sensory epithe-
lium with Sox2+ supporting cells and Atoh1/Myo7a+ hair cells (Fig. 4.3m–p). By
day 14 of the 3D culture system described, vesicles contained Sox2+Jag1+ cells
(analogous to embryonic day 9.5 mouse otic vesicles), and, over the next 48 hours,
aggregates contained an average of 15 vesicles harboring cells resembling sensory
hair cells (Myo7a+ Sox2+ with basal nuclei and elongated apical ends) (Fig. 4.4a–b).
These cells are arrayed alongside one another with apical ends facing toward the
vesicle lumen. Further consistent with sensory epithelium formation was a layer of
Sox2+ cells representing supporting cells present at the basal aspect of the sensory
hair cell-like cells (Fig. 4.4c). Tight cell-cell junctions, which stain positive for
F-actin, were found at the luminal surface with F-actin+ espin+ stereocilia bundles
(Fig. 4.4d–e), each with a single acetylated-α-tubulin+ kinocilium and associated
basal body. Transmission electron microscopy of the kinocilium revealed an inter-
nal configuration consisting of two central microtubules surrounded by nine micro-
tubule doublets, characteristic of a kinocilium in a native sensory hair cell. The
height of these stereocilia bundles increases between day 20 and 24 to reach that of
bundles in the adult mouse utricle (Li et al. 2008 ). The hair cell identity of these
cells was further confirmed by rapid uptake of FM1–43 dye, which is specifically
taken up through mechanotransduction channels (Meyers et al. 2003 ). Moreover,
these cells displayed membrane properties comparable to those in native hair cells
in the inner ear with various voltage-dependent currents. All hair cells exhibited
outwardly rectifying potassium currents, and some cells displayed a transient
inward current consistent with calcium channel activity. Based on all these morpho-
logical, biochemical, and functional properties, it was concluded that Myo7a+ cells
in ESC-derived organoids are hair cells analogous to those found in the inner ear.
A.N. Elghouche et al.