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BRAIN Publication-derived

Cerebral Organoids for Disease Modelling and Neuroinflammation

Full SOP with Academic or a plan

Source Hong et al., 2023 · Western University of Health Sciences, College of Veterinary Medicine, Pomona, CA · 10.1016/j.isci.2023.106267

Authors: Yiling Hong, Xu Dong, Lawrence Chang, Chen Xie, Mariann Chang, Jose S. Aguilar, Jimmy Lin, Juncheng Lin, Qingshun Q. Li Duration: 90 days 9 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Hong et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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