The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR–Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.
At a glance
- Sound strategies for hearing restoration. Science 344, 1241062 (2014). &
- Degenerative patterns in the aging human vestibular neuro-epithelia. Acta Otolaryngol. (Stockh.) 76, 208–220 (1973).
- New treatment options for hearing loss. Nat. Rev. Drug Discov. 14, 346–365 (2015). &
- Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline. J. Neurosci. 33, 13686–13694 (2013). , , &
- 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat. Protoc. 9, 1229–1244 (2014). &
- Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). , , , &
- Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells. Nat. Commun. 7, 11508 (2016). , , , &
- Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells. Dev. Biol. 379, 208–220 (2013). , &
- Identification of early requirements for preplacodal ectoderm and sensory organ development. PLoS Genet. 6, e1001133 (2010). , , , &
- Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation. Cell Death Differ. 23, 1347–1357 (2016). et al.
- Genetic correction of induced pluripotent stem cells from a deaf patient with MYO7A mutation results in morphologic and functional recovery of the derived hair cell-like cells. Stem Cells Transl. Med. 5, 561–571 (2016). et al.
- Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method. Neurosci. Lett. 599, 49–54 (2015). et al.
- Single-cell analysis delineates a trajectory toward the human early otic lineage. Proc. Natl. Acad. Sci. USA 113, 8508–8513 (2016). , , , &
- Inner ear hair cell-like cells from human embryonic stem cells. Stem Cells Dev. 23, 1275–1284 (2014). et al.
- Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 490, 278–282 (2012). et al.
- Anatomical and physiological development of the human inner ear. Hear. Res. 338, 9–21 (2016). &
- Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be? Reproduction 147, D1–D12 (2014). et al.
- Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat. Biotechnol. 30, 715–720 (2012). et al.
- From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes. Development 137, 1777–1785 (2010). , &
- Shaping sound in space: the regulation of inner ear patterning. Development 139, 245–257 (2012). &
- Wnt signals mediate a fate decision between otic placode and epidermis. Development 133, 865–875 (2006). , , , &
- Modulation of Wnt signaling enhances inner ear organoid development in 3D culture. PLoS One 11, e0162508 (2016). et al.
- Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011). et al.
- Identification and characterization of mouse otic sensory lineage genes. Front. Cell. Neurosci. 9, 79 (2015). , , , &
- Selective cochlear degeneration in mice lacking the F-box protein, Fbx2, a glycoprotein-specific ubiquitin ligase subunit. J. Neurosci. 27, 5163–5171 (2007). et al.
- Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear. Nat. Commun. 6, 8557 (2015). , , , &
- Ionic currents and electromotility in inner ear hair cells from humans. J. Neurophysiol. 79, 2235–2239 (1998). et al.
- Developmental acquisition of voltage-dependent conductances and sensory signaling in hair cells of the embryonic mouse inner ear. J. Neurosci. 24, 11148–11159 (2004). , &
- HCN channels expressed in the inner ear are necessary for normal balance function. J. Neurosci. 31, 16814–16825 (2011). , , &
- The function and molecular identity of inward rectifier channels in vestibular hair cells of the mouse inner ear. J. Neurophysiol. 108, 175–186 (2012). &
- Connecting the ear to the brain: molecular mechanisms of auditory circuit assembly. Prog. Neurobiol. 93, 488–508 (2011). &
- Distinctive neurophysiological properties of embryonic trigeminal and geniculate neurons in culture. J. Neurophysiol. 88, 2058–2074 (2002). , , &
- Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7, 2029–2040 (2012). et al.
- Modeling mouse and human development using organoid cultures. Development 142, 3113–3125 (2015). &
- Robust formation and maintenance of continuous stratified cortical neuroepithelium by laminin-containing matrix in mouse ES cell culture. PLoS One 7, e53024 (2012). et al.
- Production of hepatocyte-like cells from human pluripotent stem cells. Nat. Protoc. 8, 430–437 (2013). , , &
- ScaleS: an optical clearing palette for biological imaging. Nat. Neurosci. 18, 1518–1529 (2015). et al.
- Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013). et al.
- Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011). et al.
- RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). et al.
- Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015). et al.
- Supplementary Text and Figures (23,494 KB)
Supplementary Figures 1–16, Supplementary Tables 1–5 and Supplementary Data