Skip to content
← Back to browse
BRAIN Publication-derived

Microglia-containing cerebral organoids derived from induced pluripotent stem cells for the study of neurological diseases

Source Hong et al., 2023 · Western University of Health Sciences, College of Veterinary Medicine, Pomona, CA · 10.1016/j.isci

👤 Yiling Hong, Xu Dong, Lawrence Chang, Chen Xie, Mariann Chang, Jose S. Aguilar, Jimmy Lin, Juncheng Lin, Qingshun Q. Li ⏱ 90 days 📋 9 phases 🧫 Patient-Derived iPSC (ALS-PDC); Human iPSC (healthy control)

Abstract

This protocol describes the generation of microglia-containing cerebral organoids from human pluripotent stem cells using growth factor supplementation and 10% CO₂ culture conditions. The organoids contain integrated neuron-astrocyte-microglia networks suitable for modeling neurodegenerative diseases such as ALS-PDC, enabling investigation of microglia polarization, phagocytic function, and interferon signaling in disease pathogenesis.

Cell source
Patient-Derived iPSC (ALS-PDC); Human iPSC (healthy control)
Application
Disease modeling; neuroinflammation and neurodegeneration research

Protocol overview

61 steps across 9 phases

Generation of patient-specific iPSCs from lymphoid cell lines 0-32
  1. 1 Culture lymphoid cells to confluence
  2. 2 Transfect lymphoid cells with episomal plasmids
  3. 3 Plate transfected cells on Matrigel-coated plates
  4. 4 Medium transition at day 12
  5. 5 Select and expand iPSC colonies
  6. 6 Assess pluripotency by immunostaining
  7. 7 Perform karyotype analysis
  8. 8 Validate pluripotency by RNA-seq
Differentiation of embryoid bodies and neural rosette formation 0-18
  1. 1 Prepare and dissociate iPSCs or H9 hESCs
  2. 2 Culture dissociated cells in suspension for embryoid body formation
  3. 3 Verify embryoid body morphology
  4. 4 Induce neural fate with dual-SMAD inhibition
  5. 5 Fragment EBs for rosette formation
  6. 6 Plate fragments on Matrigel for rosette formation
  7. 7 Identify and characterize neural rosettes
  8. 8 Note reduced microglial progenitors in ALS-PDC rosettes
Generation of 3D cerebral organoids 18-90
  1. 1 Detach neural rosettes
  2. 2 Transfer rosettes to suspension culture
  3. 3 Culture organoids in differentiation medium
  4. 4 Culture organoids to 3 months
  5. 5 Measure organoid diameter
Generation of 2D monolayer neuronal cultures 18-32
  1. 1 Dissociate neural rosettes
  2. 2 Plate dissociated cells
  3. 3 Culture monolayer in differentiation medium
  4. 4 Confirm neuronal maturation
BMAA neurotoxin exposure 0-14
  1. 1 Perform BMAA dosing optimization (optional)
  2. 2 Expose mature organoids to BMAA
  3. 3 Prepare control organoids
Immunohistochemistry and marker analysis 14-21
  1. 1 Fix organoid samples
  2. 2 Cryoprotect organoids
  3. 3 Embed and freeze organoids
  4. 4 Prepare cryosections
  5. 5 Permeabilize tissue sections
  6. 6 Block non-specific binding
  7. 7 Incubate with primary antibodies
  8. 8 Wash and add secondary antibodies
  9. 9 Final wash and mounting
  10. 10 Image tissue sections
  11. 11 Quantify fluorescence and analyze
Beta-amyloid phagocytosis assay 0-2
  1. 1 Prepare neurons and microglia
  2. 2 Incubate with fluorescently labeled amyloid-beta
  3. 3 Wash and fix cells
  4. 4 Visualize internalized amyloid-beta
  5. 5 Quantify phagocytic capacity
  6. 6 Assess phagocytic phenotype
Interferon-gamma supplementation and rescue experiments 0-7
  1. 1 Establish baseline phenotype
  2. 2 Supplement culture medium with IFN-gamma
  3. 3 Maintain control organoids without IFN-gamma
  4. 4 Assess IFITM expression by immunostaining
  5. 5 Quantify M2 microglia numbers
  6. 6 Assess beta-amyloid uptake recovery
  7. 7 Quantify extracellular amyloid-beta accumulation
RNA sequencing and transcriptome analysis 0-21
  1. 1 Prepare organoid samples for RNA extraction
  2. 2 Extract total RNA
  3. 3 Submit samples for library preparation and sequencing
  4. 4 Align reads to human genome
  5. 5 Quantify gene expression
  6. 6 Identify differentially expressed genes (DEGs)
  7. 7 Perform pathway analysis
  8. 8 Create visualizations
  9. 9 Validate selected genes by RT-qPCR

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Hong et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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