title: Generating Inner Ear Organoids from Mouse Embryonic Stem Cells.
abstract: This protocol describes a three-dimensional culture method for generating inner ear sensory epithelia, which comprises sensory hair cells and a concurrently arising neuronal population. Mouse embryonic stem cells are initially plated in 96-well plates with differentiation media; following aggregation, Matrigel is added in order to promote epithelialization. A series of small molecule applications is then used over the first 14 days of culture to guide differentiation towards an otic lineage. After 16-20 days, vesicles containing inner ear sensory hair cells and supporting cells arise from the cultured aggregates. Aggregates may be analyzed using immunohistochemistry and electrophysiology techniques. This system serves as a simple and relatively inexpensive in vitro model of inner ear development.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, 980 West Walnut Street, WH-C400, Indianapolis, IN, 46202, USA.|Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.|Anatomy and Cell Biology Graduate Program, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
authors.forename: Emma
authors.initials: E
authors.lastname: Longworth-Mills
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, 980 West Walnut Street, WH-C400, Indianapolis, IN, 46202, USA.|Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
authors.forename: Karl R
authors.initials: KR
authors.lastname: Koehler
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, 980 West Walnut Street, WH-C400, Indianapolis, IN, 46202, USA. ehashino@iupui.edu.|Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, 46202, USA. ehashino@iupui.edu.|Anatomy and Cell Biology Graduate Program, Indiana University School of Medicine, Indianapolis, IN, 46202, USA. ehashino@iupui.edu.
authors.forename: Eri
authors.initials: E
authors.lastname: Hashino
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D007758:Ear, Inner / Q000166:cytology*; D006198:Hair Cells, Auditory / Q000166:cytology*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology*; D055495:Neurogenesis; D009940:Organoids / Q000166:cytology*
keywords: Cell culture techniques; Cell differentiation; Hair cell; Inner ear; Neurogenesis; Stem cell; Three-dimensional cell culture; Vestibular
references.citation: Development. 1999 Nov;126(22):4977-87
references.pmid: 10529416
references.citation: Development. 2003 Aug;130(15):3379-90
references.pmid: 12810586
references.citation: Development. 2005 Mar;132(6):1273-82
references.pmid: 15703277
references.citation: Genes Dev. 2005 Jul 1;19(13):1612-23
references.pmid: 15961523
references.citation: Development. 2005 Sep;132(18):4051-62
references.pmid: 16093325
references.citation: Development. 2006 Mar;133(5):865-75
references.pmid: 16452098
references.citation: Dev Biol. 2006 Jul 15;295(2):743-55
references.pmid: 16678814
references.citation: Development. 2008 Oct;135(20):3415-24
references.pmid: 18799542
references.citation: Cell Stem Cell. 2008 Nov 6;3(5):519-32
references.pmid: 18983967
references.citation: Nat Biotechnol. 2009 Mar;27(3):275-80
references.pmid: 19252484
references.citation: Dev Biol. 2010 Apr 15;340(2):595-604
references.pmid: 20171206
references.citation: J Cell Sci. 2010 May 15;123(Pt 10):1796-804
references.pmid: 20427322
references.citation: PLoS Genet. 2010 Sep 23;6(9):e1001133
references.pmid: 20885782
references.citation: Nature. 2011 Feb 3;470(7332):105-9
references.pmid: 21151107
references.citation: Nature. 2011 Apr 7;472(7341):51-6
references.pmid: 21475194
references.citation: Nat Protoc. 2011 Nov 10;6(12):1920-8
references.pmid: 22082986
references.citation: Nat Protoc. 2011 Dec 15;7(1):69-79
references.pmid: 22179593
references.citation: Development. 2012 Mar;139(6):1175-87
references.pmid: 22318231
references.citation: Cell Stem Cell. 2012 Jun 14;10(6):771-785
references.pmid: 22704518
references.citation: Dev Biol. 2012 Oct 1;370(1):3-23
references.pmid: 22790010
references.citation: PLoS One. 2012;7(12):e53024
references.pmid: 23300850
references.citation: Nature. 2013 Aug 8;500(7461):217-21
references.pmid: 23842490
references.citation: Development. 2013 Nov;140(21):4435-44
references.pmid: 24089471
references.citation: Nat Protoc. 2014;9(6):1229-44
references.pmid: 24784820
references.citation: Nature. 1995 Jul 27;376(6538):331-3
references.pmid: 7630398
references.citation: Mech Dev. 1997 Mar;62(2):183-95
references.pmid: 9152010
references.citation: Development. 1997 Aug;124(16):3177-84
references.pmid: 9272958