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BRAIN Publication-derived

Generation and Characterization of 3D Cerebral Organoids from PSEN2[N141I] Familial Alzheimer's Disease Patient-Derived iPSCs

Source Yin et al. · Program in Neuroscience and Behavioural Disorders, Duke-NUS Medical School, Singapore · 10.1101/2020.07.07.192781;

👤 Juan Yin, Antonius M. VanDongen ⏱ 63 days 📋 12 phases 🧫 Patient-Derived iPSC (Familial Alzheimer's Disease, PSEN2[N141I] mutation)

Abstract

This protocol describes the generation of three-dimensional cerebral organoids from human pluripotent stem cells (hPSCs) derived from a patient with familial Alzheimer's disease carrying a heterozygous PSEN2[N141I] point mutation, alongside isogenic controls with the corrected mutation. The organoids recapitulate key AD pathological features including elevated Aβ42/Aβ40 ratio, enhanced apoptosis, reduced organoid size, asynchronous calcium transients, and neuronal hyperactivity, providing a functional 3D model for studying AD pathophysiology and testing therapeutics.

Cell source
Patient-Derived iPSC (Familial Alzheimer's Disease, PSEN2[N141I] mutation)
Application
Disease modeling and functional assessment of Alzheimer's disease pathology

Protocol overview

70 steps across 12 phases

hPSC Maintenance and Characterization Ongoing
  1. 1 Culture hPSCs on Matrigel-coated plates
  2. 2 Verify pluripotency by immunostaining
CRISPR/Cas9-Mediated Correction of PSEN2[N141I] Mutation Days 0–21
  1. 1 Design and prepare sgRNA vectors
  2. 2 Transfect hPSCs with CRISPR components
  3. 3 Select corrected clones with puromycin
  4. 4 Isolate and characterize individual clones
  5. 5 Verify cDNA integrity by RT-PCR
  6. 6 Expand successfully corrected clones
Organoid Generation: Embryonic Body Formation Days 0–6
  1. 1 Dissociate hPSC colonies to single cells
  2. 2 Form embryonic bodies (EBs) in ultra-low-attachment plates
  3. 3 Change EB Formation Medium daily without Y-27632
  4. 4 Monitor EB morphology
Neuroectoderm Induction Days 6–8
  1. 1 Transfer EBs to 24-well ultra-low-attachment plates
  2. 2 Culture EBs in Induction Medium
  3. 3 Observe neuroectoderm formation
Neuroepithelial Expansion in 3D Matrix Days 8–11
  1. 1 Embed EBs in Matrigel droplets
  2. 2 Transition to Expansion Medium
  3. 3 Monitor organoid expansion
  4. 4 Observe neural progenitor cell proliferation
Organoid Maturation and Neuronal Differentiation Days 11–45
  1. 1 Transfer organoids to Maturation Medium
  2. 2 Culture organoids on orbital shaker
  3. 3 Replace medium regularly
  4. 4 Monitor neuronal differentiation
  5. 5 Measure organoid size
  6. 6 Prepare organoids for functional assays
Aβ Quantification by Immunostaining Day 45
  1. 1 Fix organoids
  2. 2 Cryoprotect and embed organoids
  3. 3 Section organoids
  4. 4 Permeabilize tissue
  5. 5 Block non-specific binding
  6. 6 Incubate with primary antibodies
  7. 7 Wash and apply secondary antibodies
  8. 8 Stain nuclei and mount slides
  9. 9 Acquire and analyze fluorescence images
Apoptosis and Proliferation Assessment by Immunostaining Days 12, 16, 25, 40, 45
  1. 1 Prepare organoid sections at multiple timepoints
  2. 2 Perform immunostaining for Ki67 (proliferation marker)
  3. 3 Apply secondary antibody and DAPI for Ki67 staining
  4. 4 Perform immunostaining for Cleaved Caspase-3 (apoptosis marker)
  5. 5 Apply secondary antibody and DAPI for Cleaved Caspase-3 staining
  6. 6 Quantify Ki67 and Cleaved Caspase-3 expression
Organoid Dissociation for 2D Monolayer Culture Day 40
  1. 1 Prepare organoids for dissociation
  2. 2 Dissociate organoids enzymatically
  3. 3 Collect and wash dissociated cells
  4. 4 Resuspend cells in neural culture medium
  5. 5 Count and assess cell viability
  6. 6 Plate cells on coated substrates
  7. 7 Change medium regularly
Calcium Imaging of Neuronal Network Activity in 3D Organoids Days 40–63 (3 weeks post-sectioning)
  1. 1 Section organoid and plate on glass-bottom dish
  2. 2 Monitor neuron migration
  3. 3 Load cells with Fluo-4 calcium indicator
  4. 4 Wash excess dye
  5. 5 Acquire time-lapse calcium imaging
  6. 6 Define regions of interest (ROIs) around individual neurons
  7. 7 Calculate ΔF/F (relative fluorescence change)
  8. 8 Identify calcium transients and spikes
  9. 9 Analyze synchronization
  10. 10 Measure amplitude of calcium transients
  11. 11 Statistical comparison between cell lines
Pharmacological Manipulation of Calcium Activity Days 61–63 (during calcium imaging)
  1. 1 Establish baseline calcium activity
  2. 2 Apply 4-AP (potassium channel blocker)
  3. 3 Analyze 4-AP response
  4. 4 Wash out 4-AP and allow recovery
  5. 5 Establish post-washout baseline
  6. 6 Apply Bicuculline methochloride (GABA antagonist)
  7. 7 Analyze Bicuculline response
  8. 8 Final washout and recovery
  9. 9 Compare drug sensitivity between cell lines
Additional Immunostaining for Neuronal and Morphological Markers Days 25, 40, 45
  1. 1 Fix and section organoids at key timepoints
  2. 2 Perform immunostaining for Tuj1 (early neuronal marker)
  3. 3 Apply secondary antibody and DAPI

Full SOP

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Attribution

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

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