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BRAIN Publication-derived

Detection of all adult Tau isoforms in a 3D culture model of iPSC-derived neurons

Source Miguel et al., 2019 · Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital · 10.1016/j.scr.2019.101541

👤 Laetitia Miguel, Anne Rovelet-Lecrux, Maxime Feyeux, Thierry Frebourg, Pierre Nassoy, Dominique Campion, Magalie Lecourtois ⏱ 25 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of iPSC-derived neurons within Matrigel-coated alginate capsules to generate mature cortical neurons expressing all six adult Tau (MAPT) mRNA isoforms. Using a novel quantitative fluorescent RT-PCR assay, the protocol enables simultaneous individual analysis of all adult MAPT mRNA transcripts, making it suitable for modeling tauopathies and related neurodegenerative diseases.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

37 steps across 8 phases

iPSC Maintenance and Encapsulation Day 1–7
  1. 1 Prepare Matrigel-coated culture plates
  2. 2 Maintain BC-1 iPSC line under feeder-free conditions
  3. 3 Prepare microfluidic coextrusion device
  4. 4 Perform encapsulation via microfluidic device
  5. 5 Transfer encapsulated cells to initial culture medium
Neural Induction via dual-SMAD inhibition Day 8–15
  1. 1 Initiate neural induction of iPSCs
  2. 2 Confirm neural stem cell (NSC) formation
Neural Stem Cell Differentiation into Cortical Neurons Day 16–week 25
  1. 1 Initiate NSC differentiation with dual culture medium option
  2. 2 Culture encapsulated neurons to 15 weeks of maturation
  3. 3 Culture encapsulated neurons to 20 weeks of maturation
  4. 4 Culture encapsulated neurons to 25 weeks of maturation
Sample Preparation: RNA Extraction At designated time points (15, 20, or 25 weeks)
  1. 1 Harvest and rinse capsules
  2. 2 Disintegrate alginate capsule
  3. 3 Wash dissociated cells
  4. 4 Extract total RNA
Sample Preparation: Protein Extraction and Dephosphorylation At designated time points (25 weeks primary end point)
  1. 1 Harvest and rinse capsules
  2. 2 Disintegrate alginate capsule
  3. 3 Wash dissociated cells
  4. 4 Lyse cells and prepare protein extract
  5. 5 Clarify lysate
  6. 6 Quantify protein concentration
  7. 7 Dephosphorylate protein sample
Analysis of Neuronal Maturation by RT-PCR At designated time points (15, 20, 25 weeks)
  1. 1 Perform reverse transcription
  2. 2 Set up PCR reaction
  3. 3 Analyze PCR products by gel electrophoresis
Analysis of Adult MAPT Isoforms by Fluorescent RT-PCR At designated time points (15, 20, 25 weeks)
  1. 1 Perform reverse transcription
  2. 2 Set up fluorescent PCR reaction
  3. 3 Run PCR products on capillary electrophoresis analyzer
  4. 4 Analyze electropherograms
Western Blotting for Tau Protein Isoforms At 25 weeks maturation (or designated time point)
  1. 1 Prepare dephosphorylated protein sample
  2. 2 Prepare SDS-PAGE gel
  3. 3 Load samples and standard
  4. 4 Perform SDS-PAGE electrophoresis
  5. 5 Transfer proteins to nitrocellulose membrane
  6. 6 Perform immunoblotting
  7. 7 Detect signal with chemiluminescence
  8. 8 Image and analyze blot

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Miguel et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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