Skip to content
Back to browse
BRAIN Publication-derived

Frontotemporal Dementia Cerebral Organoids

Full SOP with Academic or a plan

Source Glasauer et al., 2022 · University of California, Santa Barbara, Neuroscience Research Institute · 10.1016/j.stemcr.2022.07.011

Authors: Stella M.K. Glasauer, Susan K. Goderie, Jennifer N. Rauch, Elmer Guzman, Morgane Audouard, Taylor Bertucci, Shona Joy, Emma Rommelfanger, Gabriel Luna, Erica Keane-Rivera, Steven Lotz, Susan Borden, Aaron M. Armando, Oswald Quehenberger, Sally Temple, Kenneth S. Kosik Duration: 96 days 7 phases Cell source: Human iPSC from MAPT mutation carriers and isogenic controls

Abstract

This protocol generates human cerebral organoids (hCOs) from iPSC lines carrying pathogenic MAPT tau mutations (V337M and R406W) and isogenic controls to model early molecular events in frontotemporal dementia. Single-cell transcriptomics and lipidomics reveal that MAPT mutations cause upregulation of the cholesterol biosynthesis pathway in astrocytes and elevated cholesterol and precursor metabolites, identifying cholesterol dyshomeostasis as an early event in tau-driven neurodegeneration.

Cell source
Human iPSC from MAPT mutation carriers and isogenic controls
Application
Disease modeling of frontotemporal dementia (FTD) and tau-related neurodegeneration

Protocol overview

33 steps across 7 phases

Phase 1 · iPSC Preparation and Culture Continuous maintenance prior to organoid differentiation
  1. a Culture iPSC lines in feeder-free conditions
  2. b Passage iPSCs weekly using enzymatic dissociation
Phase 2 · Organoid Initiation in AggreWell™800 Day 0-1
  1. a Prepare AggreWell™800 plates with organoid formation medium
  2. b Pre-treat iPSCs with ROCK inhibitor
  3. c Dissociate iPSCs to single-cell suspension
  4. d Distribute cells into AggreWell™800 and form aggregates
  5. e Incubate iPSC aggregates for 24 hours
Phase 3 · Guided Differentiation (Days 1-24) Day 1-24
  1. a Transfer organoids to ultra-low attachment plates with Medium A
  2. b Daily medium changes with Medium A (Days 1-5)
  3. c Switch to Medium B on Day 6
  4. d Physically separate fused organoids during culture (Days 1-24)
  5. e Transition to Medium C on Day 25
Phase 4 · Maturation and Long-Term Culture (Days 25–onwards) Day 25-96+ (typically 4-8 months)
  1. a Continue Medium C feeding (Days 25-42)
  2. b Switch to Medium D from Day 43 onwards
  3. c Maintain long-term culture and periodic inspection
  4. d Harvest organoids for analysis at desired timepoints
Phase 5 · Single-Cell RNA Sequencing (Drop-seq) At designated timepoints (4-8 months)
  1. a Dissociate organoids to single-cell suspension using papain
  2. b Quench papain and collect dissociated cells
  3. c Count cells and assess viability
  4. d Prepare cells for drop-seq at correct concentration
  5. e Perform drop-seq using FlowJEM microfluidic device
  6. f Amplify cDNA and prepare Nextera libraries
  7. g Sequence libraries on Illumina Nextseq500
Phase 6 · Immunohistochemistry (HMGCS1/GFAP Co-labeling) At designated timepoints (typically 6 months)
  1. a Fix organoids in paraformaldehyde
  2. b Embed and cryosection organoids
  3. c Block and co-label HMGCS1 and GFAP
  4. d Stain nuclei and image
  5. e Quantify HMGCS1-positive astrocytes
Phase 7 · Lipidomics (Sterol Analysis by LC-MS) At designated timepoints (e.g., 4 and 7 months)
  1. a Snap-freeze individual organoids
  2. b Homogenize organoids
  3. c Perform BUME lipid extraction
  4. d Set up and run LC-MS analysis
  5. e Process and analyze LC-MS data

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Glasauer et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence