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Cited by 341Hearing, Cochlea, Tinnitus, GeneticsPluripotent Stem Cells ResearchVestibular and auditory disorders

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

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Koehler, Karl R, Nie, Jing, Longworth-Mills, Emma, Liu, Xiao-Ping, Lee, Jiyoon, Holt, Jeffrey R, Hashino, Eri·2017·Nature Biotechnology
DOI 10.1038/nbt.3840PMC PMC5462862
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Abstract

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.

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  • Co-treatment of basic-FGF and a BMP inhibitor, LDN-193189, promotes induction of CNCC-like cells that can generate mesenchymal cells.

    “In our inner ear culture system, we determined that a co-treatment of basic-FGF (FGF) and a BMP inhibitor, LDN-193189 (LDN) promotes induction of CNCC-like cells that can generate mesenchymal cells; however, we did not previously investigate dermal cell specification in the system9.”

    From Hair-bearing human skin generated entirely from pluripotent stem cells (2020) · cites this paper as [9]

    The paper reports FGF-2/LDN co-treatment and, later, cranial neural crest–like cells forming mesenchyme around otic pits (main text, page unavailable). But those cells were observed after an additional CHIR99021 treatment; the cited passages do not establish that FGF-2/LDN co-treatment itself promotes their induction. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • A critical distinction between human and mouse IEO inductions is that TBXT+ mesodermal lineage cells appear to be missing in the human IEO system, perhaps due to the higher dosage of a TGF-β pathway inhibitor used in the human protocol.

    “A critical distinction between our human and mouse IEO inductions is that TBXT+ mesodermal lineage cells appear to be missing in the human IEO system, perhaps due to the higher dosage of a TGF-β pathway inhibitor used in the human protocol”

    From Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells (2022) · cites this paper as [33]

    The paper’s discussion of non-neural conversion (page not provided) says that, unlike its mouse culture, the human culture did not induce Brachyury (TBXT)+ mesendoderm cells. The Online Methods report 10 μM of the TGF-β inhibitor SB-431542 in the human protocol, but the retrieved passages neither establish that this dose was higher than in the mouse protocol nor suggest that the dose explains the difference. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • To successfully generate skin, co-development of epidermis and dermis is required, and precise alterations of major signaling pathways (TGF-β, BMP4, and bFGF) are critical.

    “To successfully generate skin, co-development of epidermis and dermis is required, and precise alterations of major signaling pathways (TGF-β, BMP4, and bFGF) are critical”

    From Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells (2022) · cites this paper as [33]

    The paper reports using FGF-2, BMP4, and a TGF-β inhibitor to direct non-neural epithelium toward inner ear organoids, and says BMP concentration may need fine-tuning between cell lines (Abstract; page unavailable). It also reports keratinocyte-like cells, but does not establish that epidermis and dermis must co-develop to generate skin or that these signaling changes are critical for skin generation. (LLM, conf=0.79)

    Experimental — accuracy not yet measured

  • BMP activation promotes surface ectoderm induction and suppresses neuroectoderm induction.

    “TGFβ inhibition promotes ectoderm induction from pluripotent stem cells, whereas BMP activation promotes surface ectoderm (alternatively, non-neural ectoderm) induction and suppresses neuroectoderm induction9–12.”

    From Hair-bearing human skin generated entirely from pluripotent stem cells (2020) · cites this paper as [9]

  • TGFβ inhibition promotes ectoderm induction from pluripotent stem cells.

    “TGFβ inhibition promotes ectoderm induction from pluripotent stem cells, whereas BMP activation promotes surface ectoderm (alternatively, non-neural ectoderm) induction and suppresses neuroectoderm induction9–12.”

    From Hair-bearing human skin generated entirely from pluripotent stem cells (2020) · cites this paper as [9]

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References 41 · Cited by 341

Research works printed but not linked: 38

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Authors
Koehler, K. R., Nie, J., Longworth-Mills, E., Liu, X.-P., Lee, J., Holt, J. R., & Hashino, E.
Affiliations
  • Indiana University School of Medicine (US)
  • Indiana University Indianapolis (US)
  • Boston Children's Hospital (US)
Corresponding author
  • Koehler, Karl R
  • Hashino, Eri
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2017-05-01
Volume
35
Issue
6
Pages
583-589
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/nbt.3840
PMC
PMC5462862
OpenAlex
W2610506730
Cited by
341
References
41
Citations by year
2026: 30 · 2025: 47 · 2024: 33 · 2023: 41 · 2022: 47 · 2021: 50 · 2020: 38 · 2019: 30 · 2018: 19 · 2017: 6
FWCI
15.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
  • IUScholarWorks (Indiana University) (submittedVersion)
  • Purdue University Indianapolis (Indiana University) (submittedVersion)
Funders
  • National Institutes of Health (UL1TR001108, 1c06rr020128-01)
  • National Institute on Deafness and Other Communication Disorders (R03DC015624, F31DC015968, R01DC013294)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR075018)
  • National Center for Research Resources (C06-RR020128)
  • National Center for Advancing Translational Sciences
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54HD090255)
Topics
  • Life Sciences › Neuroscience › Sensory Systems › Hearing, Cochlea, Tinnitus, Genetics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Neuroscience › Neurology › Vestibular and auditory disorders
Keywords
inner ear organoids, hair cells, human pluripotent stem cells, otic vesicle, sensory epithelium, sensory neurons, TGF signaling, BMP signaling, FGF signaling, Wnt signaling, Atoh1, CRISPR/Cas9, electrophysiological properties, hearing loss, balance disorders, cell therapy, 3D cell culture
MeSH terms
Cells, Cultured, Hair Cells, Auditory, Inner, Humans, Ear, Inner, Organoids, Tissue Engineering, Organogenesis, Pluripotent Stem Cells

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