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(derived from that anterior aspect of the PPR) has been documented. After NNE
formation, treatment with the medium that did not contain any factors to affect tis-
sue organization and patterning facilitated self-guided differentiation. Furthermore,
the authors demonstrated that treatment of smaller stem cell aggregates with exog-
enous BMP also led to induction of NNE markers (Suga 2011 ).

4.3.1 Formation of Nonneural Ectoderm (NNE)

We recognized that epithelium similar to the definitive ectoderm (Nanog- Laminin+
E-cadherin+) is present on day 3 of ESC aggregates after an addition of Matrigel
(Fig. 4.3a–d). It was postulated that the cultured aggregates could be directed to
form tissue arising from the NNE, such as the inner ear, via induction of NNE from
this definitive ectoderm-like epithelium. As outlined above, the differentiation of
the definitive ectoderm into the neuroectoderm and NNE is mediated by BMP

D5 D8

D16 D20 D24

D24

D2 D3

D5

D1

D8

D4

D14

D10

D24

EcadAP2DAPI

GFP GFP GFPMyo7aDAPI

a b c d

e g

i

mn

f h

jkl

o p

EcadPax8DAPI

Fig. 4.3 Development of inner ear organoids in 3D culture. Morphological progression of an ESC
aggregate from day 1 to day 24 (a–e, g, i–l). Immunofluorescence for Ecad/AP2 and Ecad/Pax8 in
days 5 and 8 aggregates, respectively (f, h). Atoh1-GFP expression marks hair cells arising in
organoids (m–o). Immunofluorescence for Myosin 7a (Myo7a) confirms that Atoh1-GFP-positive
cells are indeed hair cells (p). Scale bars: 100 μm (d, h, i, l, m, o), 10 μm (p)

4 Inner Ear Organoids: Recapitulating Inner Ear Development in 3D Culture