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BRAIN Publication-derived

Generation and Electrophysiological Characterization of AD hiPSC-Derived Cerebrocortical Neurons and Cerebral Organoids

Source Ghatak et al., 2019 · The Scripps Research Institute, Department of Molecular Medicine · 10.7554/elife.50333

👤 Swagata Ghatak, Nima Dolatabadi, Dorit Trudler, XiaoTong Zhang, Yin Wu, Madhav Mohata, Rajesh Ambasudhan, Maria Talantova, Stuart A Lipton ⏱ 60 days 📋 13 phases 🧫 Patient-Derived iPSC (Familial Alzheimer's Disease - PS1 ΔE9, PS1 M146V, APP Swedish)

Abstract

This protocol generates cerebrocortical neurons and cerebral organoids from hiPSCs bearing familial Alzheimer's disease (AD) mutations (PS1 ΔE9, PS1 M146V, APP Swedish) and their isogenic wild-type controls. The protocol enables comprehensive characterization of AD pathophysiology including neuronal hyperexcitability, altered ion channel properties, disrupted synaptic transmission, and neurite loss that recapitulate key features of human AD brains.

Cell source
Patient-Derived iPSC (Familial Alzheimer's Disease - PS1 ΔE9, PS1 M146V, APP Swedish)
Application
Disease modeling

Protocol overview

89 steps across 13 phases

hiPSC Maintenance and Neural Induction Days 1-6
  1. 1 Culture feeder-free hiPSCs on Matrigel
  2. 2 Initiate neural induction with small molecule cocktail
  3. 3 Maintain cultures for 6 days in induction medium
Neurosphere Formation and Expansion Days 7-21
  1. 1 Scrape cells to form PAX6+ neurospheres
  2. 2 Culture neurospheres in expansion medium
  3. 3 Plate neurospheres to form rosette structures
  4. 4 Manually pick and expand rosettes
Terminal Neuronal Differentiation (Protocol Option 1: Astrocyte Co-culture) Days 22-49
  1. 1 Prepare poly-ornithine/laminin-coated coverslips
  2. 2 Co-seed hNPCs with neonatal mouse astrocytes
  3. 3 Add neurotrophic factors
  4. 4 Switch to BrainPhys medium at week 3
  5. 5 Continue culture for 5-6 weeks total differentiation
Terminal Neuronal Differentiation (Protocol Option 2: Compound E Treatment) Days 22-49
  1. 1 Treat hNPCs with 100 nM Compound E
  2. 2 Transfer to Compound E-free BrainPhys medium
  3. 3 Continue culture to 5-6 weeks
Cerebral Organoid Generation Days 1-60
  1. 1 Seed hiPSCs in embryoid body (EB) formation medium
  2. 2 Maintain EB formation for 5 days
  3. 3 Transfer EBs to 24-well plates with induction medium
  4. 4 Culture in induction medium for 2 days
  5. 5 Embed EBs in Matrigel
  6. 6 Culture in expansion medium
  7. 7 Transfer to maturation medium at 72 hours post-embedding
  8. 8 Continue culture in maturation medium on orbital shaker
Whole-Cell Patch-Clamp Electrophysiology Variable (at specified timepoints)
  1. 1 Prepare patch pipettes
  2. 2 Fill pipettes with internal solution
  3. 3 Prepare external recording solution
  4. 4 Mount neuron on microscope stage
  5. 5 Establish whole-cell configuration
  6. 6 Record spontaneous action potentials (current-clamp mode)
  7. 7 Record evoked action potentials
  8. 8 Switch to voltage-clamp mode for sodium/potassium currents
  9. 9 Record spontaneous excitatory postsynaptic currents (sEPSCs)
  10. 10 Record spontaneous inhibitory postsynaptic currents (sIPSCs)
  11. 11 Record miniature EPSCs (mEPSCs) with tetrodotoxin
  12. 12 Record miniature IPSCs (mIPSCs) with tetrodotoxin
  13. 13 Measure readily releasable pool (RRP) with hypertonic sucrose
Pharmacological Manipulation (γ-Secretase and BACE1 Inhibitors) Variable (days 2-4 before electrophysiology)
  1. 1 Pre-treat cultures with γ-secretase inhibitor
  2. 2 Pre-treat cultures with BACE1 inhibitor (alternative)
  3. 3 Perform electrophysiology recordings
Immunocytochemistry of 2D Neuronal Cultures Variable (typically at 5-6 weeks)
  1. 1 Fix neurons with paraformaldehyde
  2. 2 Permeabilize and block
  3. 3 Incubate with primary antibodies
  4. 4 Wash and incubate with secondary antibodies
  5. 5 Counterstain with DAPI
  6. 6 Mount and image
  7. 7 Quantify Tuj1-positive neurite area
  8. 8 Quantify synaptic density
  9. 9 Quantify VGLUT1 and VGAT immunofluorescence
Immunocytochemistry and Morphology Analysis of Cerebral Organoids Variable (typically at 2 months)
  1. 1 Fix organoids with paraformaldehyde
  2. 2 Cryoprotect organoids with sucrose
  3. 3 Embed in OCT medium and freeze
  4. 4 Section frozen organoids
  5. 5 Stain for cortical layer markers
  6. 6 Stain for Aβ deposition
  7. 7 Stain for glutamatergic and GABAergic markers
  8. 8 Image with confocal microscopy
  9. 9 Perform Golgi staining for morphology
  10. 10 Trace and analyze neurite length
Enzyme-Linked Immunosorbent Assay (ELISA) for Amyloid-β Variable (at 6 weeks for 2D cultures; 1-2 months for organoids)
  1. 1 Collect culture medium from 2D cultures
  2. 2 Collect organoid culture medium
  3. 3 Prepare ELISA kit
  4. 4 Prepare samples in duplicate
  5. 5 Load samples on ELISA plate
  6. 6 Wash and develop plate
  7. 7 Read ELISA plate
  8. 8 Calculate Aβ ratios and concentrations
Multielectrode Array (MEA) Recording from Cerebral Organoids Variable (typically 2 months organoid age + 2 weeks post-plating)
  1. 1 Prepare MEA plates with coating
  2. 2 Plate mature cerebral organoids
  3. 3 Allow organoid attachment (2 weeks)
  4. 4 Perform MEA recording
  5. 5 Analyze firing rate and burst activity
Western Blot Analysis of VGLUT1 and VGAT Variable (typically at 4-5 weeks)
  1. 1 Harvest neuronal cultures
  2. 2 Lyse cells in RIPA buffer
  3. 3 Collect cell lysate
  4. 4 Quantify total protein
  5. 5 Prepare samples for SDS-PAGE
  6. 6 Separate proteins by SDS-PAGE
  7. 7 Transfer to PVDF membrane
  8. 8 Block and incubate with primary antibodies
  9. 9 Wash and incubate with secondary antibodies
  10. 10 Detect and image
  11. 11 Quantify band intensity
Transfection and Neurite Morphology Analysis Variable (typically at 4-5 weeks)
  1. 1 Prepare neurons for transfection
  2. 2 Transfect with GFP plasmid
  3. 3 Allow GFP expression
  4. 4 Image GFP-labeled neurons
  5. 5 Trace neurites using ImageJ
  6. 6 Quantify total neurite length
  7. 7 Quantify branching

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ghatak et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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