Abstract
The inner ear sensory epithelium harbors mechanosensory hair cells responsible for detecting sound and maintaining balance. This protocol describes a three-dimensional (3D) culture system that efficiently generates inner ear sensory epithelia from aggregates of mouse embryonic stem (mES) cells. By mimicking the activations and repressions of key signaling pathways during in vivo inner ear development, mES cell aggregates are sequentially treated with recombinant proteins and small molecule inhibitors for activating or inhibiting the Bmp, TGFβ, Fgf, and Wnt signaling pathways. These stepwise treatments promote mES cells to sequentially differentiate into epithelia representing the non-neural ectoderm, preplacodal ectoderm, otic placodal ectoderm, and ultimately, the hair cell-containing sensory epithelia. The derived hair cells are surrounded by a layer of supporting cells and are innervated by sensory neurons. This in vitro inner ear organoid culture system may serve as a valuable tool in developmental and physiological research, disease modeling, drug testing, and potential cell-based therapies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Whitfield TT (2015) Development of the inner ear. Curr Opin Genet Dev 32:112–118. doi:10.1016/j.gde.2015.02.006
Warchol ME (2011) Sensory regeneration in the vertebrate inner ear: differences at the levels of cells and species. Hear Res 273(1–2):72–79. doi:10.1016/j.heares.2010.05.004
Grocott T, Tambalo M, Streit A (2012) The peripheral sensory nervous system in the vertebrate head: a gene regulatory perspective. Dev Biol 370(1):3–23. doi:10.1016/j.ydbio.2012.06.028
Wilson PA, Hemmatibrivanlou A (1995) Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376(6538):331–333. doi:10.1038/376331a0
Wilson PA, Lagna G, Suzuki A, HemmatiBrivanlou A (1997) Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer smad1. Development 124(16):3177–3184
Kwon HJ, Bhat N, Sweet EM, Cornell RA, Riley BB (2010) Identification of early requirements for preplacodal ectoderm and sensory organ development. PLoS Genet 6(9):e1001133. doi:ARTN e1001133 1371/journal.pgen.1001133
Barth KA, Kishimoto Y, Rohr KB, Seydler C, Schulte-Merker S, Wilson SW (1999) Bmp activity establishes a gradient of positional information throughout the entire neural plate. Development 126(22):4977–4987
Neave B, Holder N, Patient R (1997) A graded response to BMP-4 spatially coordinates patterning of the mesoderm and ectoderm in the zebrafish. Mech Develop 62(2):183–195. doi:10.1016/S0925–4773(97)00659-X
Harvey NT, Hughes JN, Lonic A, Yap C, Long C, Rathjen PD, Rathjen J (2010) Response to BMP4 signalling during ES cell differentiation defines intermediates of the ectoderm lineage. J Cell Sci 123(10):1796–1804. doi:10.1242/jcs.047530
Pieper M, Ahrens K, Rink E, Peter A, Schlosser G (2012) Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm. Development 139(6):1175–1187. doi:10.1242/dev.074468
Kwon HJ, Riley BB (2009) Mesendodermal signals required for otic induction: bmp-antagonists cooperate with Fgf and can facilitate formation of ectopic otic tissue. Dev Dyn 238(6):1582–1594. doi:10.1002/dvdy.21955
Reichert S, Randall RA, Hill CS (2013) A BMP regulatory network controls ectodermal cell fate decisions at the neural plate border. Development 140(21):4435–4444. doi:10.1242/dev.098707
Ahrens K, Schlosser G (2005) Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. Dev Biol 288(1):40–59. doi:10.1016/j.ydbio.2005.07.022
Brugmann SA, Pandur PD, Kenyon KL, Pignoni F, Moody SA (2004) Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor. Development 131(23):5871–5881. doi:10.1242/dev01516
Litsiou A, Hanson S, Streit A (2005) A balance of FGF, BMP and WNT signalling positions the future placode territory in the head (vol 132, pg 4051, 2005). Development 132(21):4895–4895
Schlosser G (2006) Induction and specification of cranial placodes. Dev Biol 294(2):303–351. doi:10.1016/j.ydbio.2006.03.009
McCarroll MN, Lewis ZR, Culbertson MD, Martin BL, Kimelman D, Nechiporuk AV (2012) Graded levels of Pax2a and Pax8 regulate cell differentiation during sensory placode formation. Development 139(15):2740–2750. doi:10.1242/dev.076075
Ohyama T, Mohamed OA, Taketo MM, Dufort D, Groves AK (2006) Wnt signals mediate a fate decision between otic placode and epidermis. Development 133(5):865–875. doi:10.1242/dev.02271
Sai XR, Yonemura S, Ladher RK (2014) Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination. Dev Biol 394(2):206–216. doi:10.1016/j.ydbio.2014.08.022
Koehler KR, Mikosz AM, Molosh AI, Patel D, Hashino E (2013) Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500(7461):217–221. doi:10.1038/nature12298
Koehler KR, Hashino E (2014) 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat Protoc 9(6):1229–1244. doi:10.1038/nprot.2014.100
Longworth-Mills E, Koehler KR, Hashino E (2016) Generating inner ear organoids from mouse embryonic stem cells. Methods Mol Biol 1341:391–406. doi:10.1007/7651_2015_215
Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E, Okuda S, Sekiguchi K, Adachi T, Sasai Y (2011) Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472(7341):51–U73. doi:10.1038/nature09941
Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, Wataya T, Nishiyama A, Muguruma K, Sasail Y (2008) Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3(5):519–532. doi:10.1016/j.stem.2008.09.002
Nakano T, Ando S, Takata N, Kawada M, Muguruma K, Sekiguchi K, Saito K, Yonemura S, Eiraku M, Sasai Y (2012) Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10(6):771–785. doi:10.1016/j.stem.2012.05.009
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling (vol 27, pg 275, 2009). Nat Biotechnol 27(5):485–485. doi:10.1038/nbt0509-485a
Liu XP, Koehler KR, Mikosz AM, Hashino E, Holt JR (2016) Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells. Nat Commun 7:11508. doi:ARTN 11508 1038/ncomms11508
Kriks S, Shim JW, Piao JH, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang LC, Beal MF, Surmeier DJ, Kordower JH, Tabar V, Studer L (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480(7378):547–U177. doi:10.1038/nature10648
Chambers SM, Qi YC, Mica Y, Lee G, Zhang XJ, Niu L, Bilsland J, Cao LS, Stevens E, Whiting P, Shi SH, Studer L (2012) Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol 30(7):715. doi:10.1038/nbt.2249
DeJonge RE, Liu X-P, Deig CR, Heller S, Koehler KR, Hashino E (2016) Modulation of Wnt signaling enhances inner ear organoid development in 3D culture. PLoS ONE 11(9): e0162508. doi:10.1371/journal.pone.0162508
Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453(7194):519–U515. doi:10.1038/nature06968
Oshima K, Shin K, Diensthuber M, Peng AW, Ricci AJ, Heller S (2010) Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell 141(4):704–716. doi:10.1016/j.cell.2010.03.035
Lumpkin EA, Collisson T, Parab P, Moer-Abdalla A, Haeberle H, Chen P, Doetzlhofer A, White P, Groves A, Segil N, Johnson JE (2003) Math1-driven GFP expression in the developing nervous system of transgenic mice. Gene Expr Patterns 3(4):389–395. doi:10.1016/S1567-133x(03)00089–9
Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa S, Sasai Y (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28(1):31–40. doi:10.1016/S0896-6273(00)00083–0
Wichterle H, Lieberam I, Porter JA, Jessell TM (2002) Directed differentiation of embryonic stem cells into motor neurons. Cell 110(3):385–397. doi:10.1016/S0092-8674(02)00835–8
Okada Y, Shimazaki T, Sobue G, Okano H (2004) Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol 275(1):124–142. doi:10.1016/j.ydbio.2004.07.038
Forge A, Schacht J (2000) Aminoglycoside antibiotics. Audiol Neuro-Otol 5(1):3–22. doi:10.1159/000013861
Acknowledgments
This work was supported by a National Institutes of Health grant R01 DC013294 (to E.H.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Nie, J., Koehler, K.R., Hashino, E. (2017). Directed Differentiation of Mouse Embryonic Stem Cells Into Inner Ear Sensory Epithelia in 3D Culture. In: Tsuji, T. (eds) Organ Regeneration. Methods in Molecular Biology, vol 1597. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6949-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6949-4_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6947-0
Online ISBN: 978-1-4939-6949-4
eBook Packages: Springer Protocols