Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells

Journal name:
Nature Biotechnology
Volume:
35,
Pages:
583–589
Year published:
DOI:
doi:10.1038/nbt.3840
Received
Accepted
Published online

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

Figures

  1. Step-wise induction of otic-placode-like epithelia.
    Figure 1: Step-wise induction of otic-placode-like epithelia.

    (a) Overview of mammalian ectoderm development in the otic placode cranial region. (b) Timeline for key events of human otic induction. Day 0 on the timeline indicates the approximate stage of development represented by hPSC ~12 d post conception. (c) Differentiation strategy for non-neural ectoderm (NNE), otic-epibranchial progenitor domain (OEPD), and otic placode induction. Potentially optional or cell-line-dependent treatments are denoted in parentheses. (d) qPCR analysis on day 2 of differentiation of WA25 cell aggregates treated with DMSO (control), 10 μM SB, or 10 μM SB + 10 ng/ml BMP4, denoted as SBB. Gene expression was normalized to undifferentiated hESCs; FC, fold change; n = 3 biological samples, two technical repeats; *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; box limits: upper/lower quartile; center line: median; whiskers: max./min. (e,f) Representative TFAP2A, ECAD, and PAX6 expression in WA25 aggregate treated with 10 μM SB (n = 15 aggregates, three experiments) or with 200 nM LDN + 10 μM SB for 6 d (n = 6 aggregates, three experiments). (g) TFAP2A, ECAD, and PAX6 expression in mND2-0 hiPSCs treated with 10 μM SB + 2.5 ng/ml BMP4 (SBB) on day 6. (h,i) Representative image of an SB-treated WA25 aggregate on day 8: live (h) and immunostained with PAX8 and TFAP2A antibodies (i). When comparing morphology in h and i, note that the outer-epithelium crumples into the aggregate core during the cryosectioning process. (j,k) Representative image of an SB-treated WA25 aggregate on day 8 after treatment with 50 ng/ml FGF-2 and 200 nM LDN (SBFL) on day 4: live (j) and immunostained with PAX8 and TFAP2A antibodies (k). (ln) WA25 SBFL-treated aggregates on day 12. The outer epithelium contains PAX8+ECAD+ cells (l) and occasional patches of PAX8+PAX2+ otic-placode-like cells (m,n). The specimens shown were treated with 25 μl of additional CDM on day 8. Images are representative of specimens obtained from at least three separate experiments. Scale bars, 100 μm (em), 50 μm (n).

  2. WNT signaling activation initiates self-organization and maturation of inner ear organoids containing vestibular-like hair cells.
    Figure 2: WNT signaling activation initiates self-organization and maturation of inner ear organoids containing vestibular-like hair cells.

    (a) Inner ear organoid induction strategy. Day 12 aggregates were embedded in Matrigel droplets to support vesicle formation. CNCC, cranial neural crest cell. (bd) Representative differential interference contrast (DIC) images showing that CHIR-treated samples (b; n = 84, seven experiments), but not DMSO (control) samples (c; n = 70, seven experiments), displayed otic-pit-like structures protruding from the outer epithelium (d). (ei) Representative immunostained cryosections showing that on days 14–35, pits and vesicles expressed otic specific markers, such as SOX10, SOX2, JAG1, PAX8, PAX2, and FBXO2. The dotted area in f indicates the epithelium. Non-otic regions of the epithelium from which vesicles arise expressed the epidermal keratinocyte marker KRT5 by day 35 (h; n = 15, three experiments). (j) By days 40–60, the aggregates contain multiple organoids and, typically, a single epidermal unit visible under DIC imaging. Inner ear organoids are distinguishable by a defined epithelium with ~25- to 40-μm apparent thickness and a lumen (j inset). The organoids labeled 1, 2, and 3 in panel j are indicated in the immunostained cryosection in panel k. (k) Inner ear organoids are typically oriented around the epidermal unit (dotted area) and contain sensory epithelia with ANXA4+PCP4+ hair cells. The luminal surface of organoids is F-actin-rich, as denoted by phalloidin staining (k″). (lo) Representative cryosections showing that hair cells are MYO7A+SOX2+, and supporting cells are SOX2+. F-actin-rich hair bundles protrude from the hair cells into the lumen (n, o; asterisks denote hair bundle location in m). (p,q) Representative cryosections showing that mND2-0 hiPSC-derived sensory epithelia have a similar morphology to WA25 hESC-derived sensory epithelia and contain PCP4+ANXA4+ hair cells. SOX10 is expressed throughout the supporting and non-sensory epithelial cell populations, but not in hair cells (p). Supporting cells express the supporting cell marker SPARCL1 (q). (r) Hair cells in organoids have ESPN+ hair bundles with a single acetylated-tubulin (TUBA4A)+ kinocilium. Images are representative of specimens obtained from at least three separate experiments. Scale bars, 200 μm (j), 100 μm (b, c, e), 50 μm (d, g, h, k, l), 25 μm (f, i, m, p), 10 μm (n, q), 5 μm (r), 2.5 μm (o).

  3. Development of an ATOH1 fluorescent reporter hESC line for tracking hair cell induction.
    Figure 3: Development of an ATOH1 fluorescent reporter hESC line for tracking hair cell induction.

    (a) ATOH1-2A-eGFP CRISPR design. The two guide RNAs (blue, with PAM sequence in red) direct Cas9n to make two nicks (red triangles) near the stop codon (underlined with pink background) of ATOH1. The resulting DNA double-strand break is repaired by the donor vector, which has a 2A-eGFP-PGK-Puro cassette and 1 kb left and right homology arms (LHA and RHA). The loxP-flanked PGK-Puro sub-cassette is subsequently removed by Cre recombinase. In ATOH1-expressing hair cells, eGFP is transcribed along with ATOH1. (bd) Representative live cell images of eGFP+ hair cells in 62- (b; combined with DIC image of the organoid epithelium) and 100-d-old inner ear organoids (c,d; 3D projected confocal images of a wholemount specimen). The asterisk in c denotes the approximate location of the hair cells in panel (d) and the dotted area of panel (d) highlights a single hair cell. Panel c is an image from Supplementary Video 3 (i.e., SVideo 3). (e) Expression of BRN3C in 140-d-old cryosectioned eGFP+ hair cells. (f) A representative 3D projection of confocal images shows expression of ESPN in the hair bundles of 100-d-old eGFP+ hair cells. Images are representative of specimens obtained from at least three separate experiments. Panel f is an image from Supplementary Video 4 (i.e., SVideo 4). Scale bars, 100 μm (c), 50 μm (b), 25 μm (e), 5 μm (d, f).

  4. hESC-derived hair cells have similar electrophysiological properties as native hair cells and form synapse-like contacts with sensory neurons.
    Figure 4: hESC-derived hair cells have similar electrophysiological properties as native hair cells and form synapse-like contacts with sensory neurons.

    (a) Family of outward rectifier potassium currents recorded from a human organoid hair cell (d64), evoked by the series of voltage steps shown below. (b) The outward currents had an activation range that was well-fitted by a Boltzmann equation (line) with voltage of half-maximal activation of −31 mV. (c) Mean (± s.e.) maximal current-voltage relationships for seven human organoid hair cells (d64–d67) and eight mouse utricle type II hair cells. For current–voltage relations, we averaged data from hair cells with large currents over 2 nA. (d) Family of rapidly activating, rapidly inactivating, inward currents evoked by the depolarizing steps shown below. (e) Family of slowly activating, non-inactivating inward currents (d64) evoked by hyperpolarizing steps, shown below. (f) Activation curve for the current family shown in e, fitted by a Boltzmann equation (line) with a voltage of half-maximal activation of −71 mV. (g) Family of membrane responses (d64) recorded in current-clamp mode, evoked by the current injection protocol shown below. (h) Membrane response (d65) to three cycles of a 5-Hz sine wave stimulus (below). (i) A representative 3D projection of eGFP+ hair cells with ESPN+ hair bundles surrounded by clusters of sensory-like neurons in a wholemount immunostained sample. Insets 1 and 2 show the two neuron morphologies observed: unipolar and bipolar (asterisks indicate neurites from other neurons). Inset 3 shows hair cell morphology and NEFH+ neurites in the sensory epithelium (asterisks indicate neuronal processes in the epithelium). Panel i is an image from Supplementary Video 5 (i.e., SVideo 5). (j) Representative confocal image of a cryosection with NEFL+ neurons innervating an organoid sensory epithelium (dotted region of interest highlighted in inset). (k) Representative cryosection showing that S100β+ Schwann-like cells associate with neuronal soma and appear to myelinate NEFL+ neuronal processes (dotted region of interest highlighted in inset). (l) Representative cryosection showing that NEFL+ neuronal processes infiltrate the epithelium and are closely associated with CTBP2+ puncta at the base of eGFP+ hair cells. (m) Representative cryosection showing that CTBP2+ puncta are co-localized with SYP+ puncta (putative synapses are highlighted in insets 1-4). (n) Summary of neurogenesis analysis. Images are representative of specimens obtained from at least three separate experiments. Scale bars, 100 μm (i), 25 μm (j, k), 10 μm (l, m), 5 μm (i insets).

Videos

  1. Video 1:
    Otic vesicles and epidermal core on day 35
  2. Video 2:
    Multi-chambered inner ear organoid viewed through the surface of a day 48 aggregate using DIC imaging.
  3. Video 3:
    Inner ear organoids with ATOH1-2A-eGFP+ hair cells (day 100 live cell imaging).
  4. Video 4:
    Multi-chambered inner ear organoid with ATOH-2A-eGFP+ hair cells in flat-mount preparation (day 100)
  5. Video 5:
    Inner ear organoid with ESPN+ eGFP+ hair cells with innervation by NEFH+ sensory-like neurons.

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Author information

  1. Present address: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.

    • Xiao-Ping Liu

Affiliations

  1. Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Karl R Koehler,
    • Jing Nie,
    • Emma Longworth-Mills,
    • Jiyoon Lee &
    • Eri Hashino
  2. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Emma Longworth-Mills
  3. Departments of Otolaryngology and Neurology, F.M. Kirby Neurobiology Center Boston Children's Hospital, and Harvard Medical School, Boston, Massachusetts, USA.

    • Xiao-Ping Liu &
    • Jeffrey R Holt
  4. Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Eri Hashino

Contributions

K.R.K. conceived, designed, and led the study, performed experiments, analyzed data, and drafted the manuscript with input from all authors. J.N. generated the ATOH1-2A-eGFP cell line, performed experiments, and wrote the manuscript. E.L.-M. performed experiments, data analysis and wrote the manuscript. X.-P.L. performed electrophysiology experiments and wrote the manuscript. J.L. performed experiments and data analysis. J.R.H. designed and analyzed electrophysiology experiments and wrote the manuscript. E.H. designed and oversaw the study and wrote the manuscript.

Competing financial interests

K.R.K. and E.H., with the Indiana University Research and Technology Corporation, have applied for a patent on the cell culture method described in this manuscript.

Corresponding authors

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Author details

Supplementary information

Video

  1. Video 1: (26.52 MB, Download)
    Otic vesicles and epidermal core on day 35
  2. Video 2: (20.55 MB, Download)
    Multi-chambered inner ear organoid viewed through the surface of a day 48 aggregate using DIC imaging.
  3. Video 3: (23.3 MB, Download)
    Inner ear organoids with ATOH1-2A-eGFP+ hair cells (day 100 live cell imaging).
  4. Video 4: (28.07 MB, Download)
    Multi-chambered inner ear organoid with ATOH-2A-eGFP+ hair cells in flat-mount preparation (day 100)
  5. Video 5: (24.93 MB, Download)
    Inner ear organoid with ESPN+ eGFP+ hair cells with innervation by NEFH+ sensory-like neurons.

PDF files

  1. Supplementary Text and Figures (23,494 KB)

    Supplementary Figures 1–16, Supplementary Tables 1–5 and Supplementary Data

Additional data