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BRAIN Publication-derived

Human Inner Ear Organoids with Cochlear Hair Cell-like Cells

Source Koehler et al. · Indiana University / Boston Children's Hospital / Harvard Medical School, USA · 10.1038/nbt.3840

👤 Koehler KR, Mikosz AM, Molosh AI, Patel D, Hashino E ⏱ 75 days 📋 5 phases 🧫 Human iPSC / Human ESC

Abstract

Koehler et al. adapted their mouse inner ear organoid protocol for human iPSCs, generating organoids containing both cochlear-type (PRESTIN+ outer, VGLUT3+ inner) and vestibular hair cells with functional mechanotransduction channels over 180 days of culture.

Cell source
Human iPSC / Human ESC
Application
Human Hearing Loss Modeling

Protocol overview

22 steps across 5 phases

hPSC maintenance and aggregate formation Pre-differentiation and Day 0–2
  1. 1 Culture human pluripotent stem cells
  2. 2 Dissociate hPSCs and form aggregates
  3. 3 Incubate aggregate formation
Non-neural ectoderm induction Day 0–12
  1. 1 Transfer aggregates to chemically defined medium with non-neural induction factors
  2. 2 Monitor aggregate epithelialization
  3. 3 Add FGF-2 and BMP inhibitor for pre-placode/OEPD induction
  4. 4 Continue SBFL incubation and add medium
  5. 5 Optional CHIR treatment for otic placode enhancement
Otic placode induction and vesicle formation Day 12–18
  1. 1 Pool and wash aggregates; embed in Matrigel droplets
  2. 2 Apply WNT activation (CHIR treatment)
  3. 3 Monitor otic pit formation and medium change
  4. 4 Transition to floating culture
Organoid maturation and hair cell development Day 18–75+
  1. 1 Culture organoids in floating or plate-based format
  2. 2 Identify mature organoid structures
  3. 3 Detect hair cell markers and sensory epithelium formation
  4. 4 Assess neurogenesis and synapse formation
  5. 5 Long-term culture and functional maturation
CRISPR-Cas9 generation of ATOH1-2A-eGFP reporter line (optional) One-time cell line engineering
  1. 1 Design and clone gRNA vectors
  2. 2 Construct donor vector
  3. 3 Transfect vectors into WA25 hESCs
  4. 4 Select and remove puromycin cassette
  5. 5 Establish and validate clonal cell lines

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Koehler et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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