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BRAIN Publication-derived

Cortical 2D Cell Cultures for Disease Modelling with Enhanced Maturation

Full SOP with Academic or a plan

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

Authors: Laetitia Miguel, Anne Rovelet-Lecrux, Maxime Feyeux, Thierry Frebourg, Pierre Nassoy, Dominique Campion, Magalie Lecourtois Duration: 25 days 8 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Miguel et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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