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BRAIN Publication-derived

Small-molecule induction of Aβ-42 peptide production in human cerebral organoids to model Alzheimer's disease associated phenotypes

Source Pavoni et al., 2018 · Service d'Etude des Prions et Infections Atypiques (SEPIA), Institut François Jacob, CEA, Université Paris-Saclay, Fontenay-aux-Roses, France · 10.1371/journal

👤 Serena Pavoni, Rafika Jarray, Ferid Nassor, Anne-Cécile Guyot, Steve Cottin, Jessica Rontard, Jacqueline Mikol, Aloïse Mabondzo, Jean-Philippe Deslys, Frank Yates ⏱ 5 days 📋 11 phases 🧫 Human iPSC

Abstract

This protocol describes the generation and chemical induction of human cerebral organoids (mini-brains) using Aftin-5 to produce elevated Aβ-42 peptide and reproduce Alzheimer's disease-associated phenotypes. Mini-brains differentiated from control iPSCs demonstrate time-dependent Aβ accumulation and, following Aftin-5 treatment, show increased Aβ42/Aβ40 ratios characteristic of sporadic Alzheimer's disease.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

30 steps across 11 phases

iPSC Maintenance and Feeder-Free Adaptation Ongoing until EB generation
  1. 1 Maintain iPSC cultures in feeder-free conditions
Embryoid Body (EB) Generation via Hanging Drop Method Day 0–2
  1. 1 Dissociate iPSC colonies and prepare cell suspension
  2. 2 Culture hanging drops on petri dish cover
  3. 3 Harvest embryoid bodies
Neural Induction Day 2 onwards (exact duration per Lancaster protocol)
  1. 1 Perform neural induction on EBs
Neural Differentiation and Organoid Formation Following neural induction through 2 months of culture
  1. 1 Embed neural induced EBs in Matrigel droplets
  2. 2 Culture organoids for expansion and regionalization
Aftin-5 Chemical Induction At 1-month and/or 2-month-old organoids
  1. 1 Prepare Aftin-5 treatment solution
  2. 2 Treat organoids with Aftin-5
  3. 3 Harvest conditioned media
Sample Preparation for Histological Analysis Upon completion of treatment or at specified timepoints
  1. 1 Fix organoids in formaldehyde
  2. 2 Prepare organoids for paraffin embedding
  3. 3 Section paraffin-embedded organoids
Immunohistochemical Analysis Upon completion of paraffin sectioning
  1. 1 Dewax and perform epitope retrieval
  2. 2 Block and wash slides
  3. 3 Incubate with primary antibodies
  4. 4 Wash and apply secondary antibody
  5. 5 Develop chromogenic signal and counterstain
  6. 6 Dehydrate and mount slides
Gene Expression Analysis via Real-Time qPCR Upon sample collection at specified timepoints
  1. 1 Extract total RNA from organoids
  2. 2 Perform reverse transcription
  3. 3 Perform real-time qPCR amplification
  4. 4 Analyze qPCR data
Western Blot Analysis of APP and PrP(C) Upon sample collection at specified timepoints
  1. 1 Prepare organoid lysates
  2. 2 Perform SDS-PAGE and transfer
Aβ Quantification by ELISA 4 days post-Aftin-5 treatment (or at specified timepoints)
  1. 1 Prepare conditioned media samples
  2. 2 Perform multiplex Aβ ELISA
  3. 3 Calculate Aβ42/Aβ40 ratio
Statistical Analysis Upon completion of all measurements
  1. 1 Organize and analyze data
  2. 2 Perform statistical significance testing

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Pavoni et al., 2018. 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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