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BRAIN Publication-derived

Brain Organoids for Disease Modelling (2017)

Full SOP with Academic or a plan

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

Authors: Koehler KR, Mikosz AM, Molosh AI, Patel D, Hashino E Duration: 75 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Koehler et al.. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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