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. 2013 Aug 8;500(7461):217-21.
doi: 10.1038/nature12298. Epub 2013 Jul 10.

Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture

Karl R Koehler  1 , Andrew M Mikosz, Andrei I Molosh, Dharmeshkumar Patel, Eri Hashino
Affiliations

Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture

Karl R Koehler et al. Nature. .

Abstract

The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm, two critical precursors during inner ear development. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture. We show that by recapitulating in vivo development with precise temporal control of signalling pathways, ESC aggregates transform sequentially into non-neural, preplacodal and otic-placode-like epithelia. Notably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Non-neural and pre-placodal ectoderm induction in 3D culture
a, Non-neural ectoderm induction strategy. de, definitive ectoderm; me, mesendoderm; nne, non-neural ectoderm; ne, neuroectoderm. b-d, Morphology of control (Ctrl), BMP, and BMP/SB aggregates. e, SB decreases the level of brachyury expression induced by BMP (n=3; **P<0.01; mean ± s.e.m.). f-h, Brachyury+ cells are less prevalent in BMP/SB aggregates. i-k, BMP/SB aggregates contain an outer AP2/Ecad+ epithelium and an interior Sox1+ and Ncad+ cell layer. l, BMP/SB aggregate composition on day 5. m, Pre-placodal ectoderm induction strategy. epi, epidermis; ppe, pre-placodal ectoderm. n-p, BMP/SB-FGF/LDN are distinguished by a thickened AP2+ epithelium absent in other conditions. Scale bars, 100 μm.
Figure 2
Figure 2. Otic induction from the pre-placodal epithelium in vitro
a, OEPD induction in mice. nc, neural crest; se, surface ectoderm. b, Pax8 and c, Pax2 mRNA expression on day 8 (n=3–4; **P<0.01, *P<0.05; mean ± s.e.m.) d-g, Pax8/Ecad expression in (d, f) BMP/SB and (e, g) BMP/SB-FGF/LDN aggregates on day 6 and 8. Arrowheads indicate vesicles. h, Day 12 BMP/SB-FGF/LDN aggregate with Pax2/Ecad+ vesicles (arrowheads). i, Pax2/Ecad+, (j) Pax2/8+, and (k) Pax8/Sox2+ vesicles invaginate from the inner-epithelium from day 9–12. l, XAV939 decreases the number of vesicles expressing Pax2/Ecad, Pax2/Pax8 and Pax8/Sox2 on day 12. (n=9 aggregates; ***P<0.001, **P<0.01; mean ± s.e.m.). m, Self-guided, inside-out rearrangement of BMP/SB-FGF/LDN aggregates and formation of otic vesicles. Scale bars, 100 μm (d, e, f, h), 50 μm (i-k), 25 μm (g).
Figure 3
Figure 3. Stem cell-derived otic vesicles generate functional inner ear hair cells
a, b, Expression of Myo7a in the E9.5 otic vesicle (a; OtV) and day 14 vesicles (b). nb, neuroblasts. c-e, Myo7a/Sox2+ hair cells (hc) with underlying Sox2+ supporting cells (sc) on day 15 (c) and 16 (d, e). f-i, Whole-mount immunofluorescence for Myo7a and Sox2 (f) and 3D reconstruction (g-i) of a vesicle in a day 20 BMP/SB-FGF/LDN aggregate. j, Vesicles display the hallmarks of inner ear sensory epithelia. k-m, F-actin (F-act) labels cell-cell junctions on the luminal surface and stereocilia bundles. m, Acetylated-a-Tubulin (Tublin) labels kinocilium and the cuticular plate. n, Transmission electron micrograph of stereocilia bundles and kinocilium (arrow). o, Distribution of stereocilia and kinocilium heights on days 20 and 24 compared to adult mouse utricle, range indicated by gray boxes (n>100 cells; ± max/min). p, Representative hair cell following 1 min FM1-43FX incubation, fixation and staining for F-actin. q, Representative epithelium preparation (inset) and hair cell during electrophysiological recordings. r, Representative voltage-current responses recorded from hair cells. The voltage protocol is shown at the top. s, Day 20 aggregate with Myo7a/Sox2+ vesicles. epi, epidermis (dashed outline). t, Number of hair cells on day 20 (n=12–16; mean ± s.e.m.). Scale bars, 250 μm (f, s, q-inset), 50 μm (d, g, h), 25 μm (a-c, e, i, k, l), 10 μm (q), 5 μm (m, p), 250 nm (n).
Figure 4
Figure 4. Stem cell-derived sensory epithelia are comparable to immature vestibular end organs
a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, Pax2 and (c) Calretinin is expressed in all Myo7a+ stem cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d-g, The structural organization of vesicles with Calretinin/Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, non-sensory epithelium. h, TuJ1+ neurons extending processes to hair cells. i, The synaptic protein SNAP-25 is localized to the basal end of hair cells. j, The post-synaptic marker synaptophysin (Synp) co-localizes with CtBP2/RIBEYE (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n>100 cells, *P<0.05, ***P<0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 μm (i), 5 μm (j).
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References

    1. Bermingham-McDonogh O, Reh TA. Regulated reprogramming in the regeneration of rensory receptor cells. Neuron. 2011;71:389–405. - PMC - PubMed
    1. Brigande JV, Heller S. Quo vadis, hair cell regeneration? Nat Neurosci. 2009;12:679–685. - PMC - PubMed
    1. Ouji Y, Ishizaka S, Nakamura-Uchiyama F, Yoshikawa M. In vitro differentiation of mouse embryonic stem cells into inner ear hair cell-like cells using stromal cell conditioned medium. Cell Death Dis. 2012;3:e314. - PMC - PubMed
    1. Oshima K, et al. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell. 2010;141:704–716. - PMC - PubMed
    1. Kondo T, et al. Tlx3 exerts context-dependent transcriptional regulation and promotes neuronal differentiation from embryonic stem cells. Proc Natl Acad Sci USA. 2008;105:5780–5785. - PMC - PubMed

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