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BRAIN Publication-derived

A next-generation iPSC-derived forebrain organoid model of tauopathy with tau fibrils by AAV-mediated gene transfer

Source Shimada et al., 2022 · Keio University School of Medicine · 10.1016/j.crmeth.2022.100289

👤 Hiroko Shimada, Yuta Sato, Takashi Sasaki, Aki Shimozawa, Kent Imaizumi, Tomoko Shindo, Sachiyo Miyao, Kosuke Kiyama, Takahiro Kondo, Shinsuke Shibata, Seiji Ishii, Junro Kuromitsu, Hirofumi Aoyagi, Daisuke Ito, Hideyuki Okano ⏱ 126 days 📋 7 phases 🧫 Human iPSC, Patient-Derived iPSC (familial Alzheimer's disease)

Abstract

This protocol describes the generation of forebrain organoids (FBOs) from feeder-free induced pluripotent stem cells (iPSCs) by regulating FGF2 concentration, and subsequent modeling of tauopathy through AAV-mediated delivery of mutant tau (P301L). The method produces mature 3D brain organoids that recapitulate both amyloid-beta pathology and tau fibrils, providing a platform for studying tau-related neurodegeneration and testing therapeutic candidates.

Cell source
Human iPSC, Patient-Derived iPSC (familial Alzheimer's disease)
Application
Disease modeling

Protocol overview

24 steps across 7 phases

iPSC culture and FGF2 pre-conditioning Day 0 to Day 14 (prior to differentiation)
  1. 1 Maintain feeder-free iPSCs in high-FGF2 culture
  2. 2 Reduce FGF2 concentration stepwise
Embryoid body (EB) formation and neural induction Day 0 to Day 7
  1. 1 Dissociate iPSCs into single cells
  2. 2 Apply dual SMAD inhibitors
  3. 3 Switch to induction medium
Neuroepithelium formation and EB embedding Day 7 to Day 14
  1. 1 Embed EBs in Matrigel
  2. 2 Select and isolate neuroepithelia
Forebrain organoid maturation in bioreactor Day 14 to Day 71
  1. 1 Transfer to bioreactor with differentiation medium
  2. 2 Culture with magnetic stirring
  3. 3 Increase oxygen supply from day 35
  4. 4 Switch to maturation medium at day 71
AAV injection and tau pathology induction Day 35–126 (injection at weeks 5, 8, or 13; analysis 5 weeks post-injection)
  1. 1 Select FBOs of uniform size and morphology
  2. 2 Prepare AAV solutions
  3. 3 Perform microinjection into FBO center
  4. 4 Culture injected FBOs and harvest at 5 weeks post-injection
Tissue preparation and quality control analysis Post-culture (day 84–126 or 5 weeks post-AAV injection)
  1. 1 Fix and cryoprotect FBOs
  2. 2 Embed and section FBOs
  3. 3 Perform immunohistochemistry (IHC)
  4. 4 Acquire immunofluorescence images
  5. 5 Perform thioflavin-S staining (optional)
Biochemical analysis of tau aggregation Post-culture preparation
  1. 1 Prepare soluble (RIPA) fractions
  2. 2 Prepare sarkosyl-insoluble fractions
  3. 3 Quantify protein content
  4. 4 Perform Western blot analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Shimada et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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