65is apparently contingent on internal FGF signaling; this was proven when applica-
tion of the FGF inhibitor SU5402 prohibited thickened epithelial patches from
forming, though Tfap2 expression was maintained. Exogenous application of FGF2
concurrent with LDN administration was shown to significantly increase the epithe-
lial thickening compared to aggregates not treated with FGF2. Nearly all aggregates
treated with BMP4/SB/LDN/FGF2 demonstrated a thickened epithelium positive
for Gata3, Six1, and Tfap2a, which subsequently ruffled and formed ovoid vesicles
from days 6 through 8. It is based on these findings that this outer epithelium was
determined to represent the PPR.
4.3.3 Formation of the Otic Placode
Cranial sensory placodes can be distinguished by expression of various Pax tran-
scription factors; the otic placode arises from the OEPD and is defined by co-
expression of Pax2, Pax8, and Ecad. Quantitative polymerase chain reaction (qPCR)
showed increased Pax2 and Pax8 expression in aggregates treated with BMP4/SB/
LDN/FGF2 as compared to the other conditions; by day 6, these aggregates also
demonstrated cells positive for Pax8 and Ecad throughout the outer epithelial layer
consistent with formation of the OEPD. Ecad expression is important in recognition
of derived OEPD given that Pax2 and Pax8 co-expression is also found in the optic
stalk, mid-hindbrain, and kidney in addition to the otic vesicle. The extent of Pax8
expression was directly related to the FGF2 concentration. Pax8+ Ncad+ cells were
also present within the core of each aggregate, indicating mid-hindbrain tissue for-
mation in the aggregate interior. Development of βIII-tubulin+ neurons within this
region further confirmed the neural identity. Further development of the outer epi-
thelium with increased expression of Pax8 and Ecad recapitulated otic placode
induction between days 6 and 8 (Fig. 4.3g–h), indicating the importance of LDN/
FGF2 administration for in vitro otic placode formation. Pax6, which identifies the
anterior placodes, was not expressed in aggregates treated with BMP4/SB/LDN/
FGF2. Pax3 co-expression with Pax8 was noted within the aggregate interior—
Pax3 marks the trigeminal placode, which is closer toward the Pax8+ PPR. The
expression of more caudally oriented Pax proteins is potentially caused by insulin
in Knockout Serum Replacement within the media, given that insulin has been uti-
lized for mid-hindbrain induction in floating culture (Muguruma et al. 2010 ).
4.3.4 Self-Guided Differentiation to the Inner Ear Sensory
Epithelium
In order to facilitate self-organization and further differentiation of the aggregates
following otic placode induction, on day 8, aggregates are transferred to serum-free
floating culture. By day 9, the interior cell mass migrates toward the outside of
the aggregate causing the outer epithelium to become an inner epithelium lining the
4 Inner Ear Organoids: Recapitulating Inner Ear Development in 3D Culture