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BRAIN Publication-derived

ABCA7 Deficiency in iPSC-Derived Cortical Organoids and Neurons: Disease Modeling and Mitochondrial Analysis

Source Kawatani et al., 2024 · Mayo Clinic, Jacksonville, FL · 10.1038/s41380-023-02372-w

👤 Keiji Kawatani, Marie-Louise Holm, Skylar C. Starling, Yuka A. Martens, Jing Zhao, Wenyan Lu, Yingxue Ren, Zonghua Li, Peizhou Jiang, Yangying Jiang, Samantha K. Baker, Ni Wang, Bhaskar Roy, Tammee M. Parsons, Ralph B. Perkerson III, Hanmei Bao, Xianlin Han, Guojun Bu, Takahisa Kanekiyo ⏱ 60 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of ABCA7-deficient human iPSC-derived cortical organoids and neurons using CRISPR/Cas9 gene editing to model Alzheimer's disease-related neuronal dysfunction. The method includes differentiation of isogenic iPSC pairs, characterization of mitochondrial morphology and respiration, assessment of synaptic function, and quantification of lipid metabolism alterations to elucidate ABCA7's role in neuronal homeostasis.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

55 steps across 12 phases

ABCA7 Deletion and iPSC Characterization Day 0 to Day 30
  1. 1 Prepare iPSC culture
  2. 2 Perform CRISPR/Cas9-mediated ABCA7 deletion
  3. 3 Perform karyotyping
  4. 4 Validate ABCA7 knockout at protein level
Differentiation into Cortical Organoids Day 0 to Day 60
  1. 1 Initiate embryoid body (EB) formation
  2. 2 Change EB formation media
  3. 3 Transfer EBs to low-attachment plates
  4. 4 Plate EBs on Matrigel and initiate expansion
  5. 5 Switch to maturation medium
  6. 6 Culture on orbital shaker with regular medium changes
Differentiation into Neural Progenitor Cells (NPCs) and Neurons Day 0 to Day 40+
  1. 1 Initiate NPC differentiation with EB formation
  2. 2 Transfer EBs and induce neural rosettes
  3. 3 Isolate and expand neural rosettes
  4. 4 Switch to NPC expansion medium
  5. 5 Expand NPCs with regular passaging
  6. 6 Initiate neuronal differentiation
  7. 7 Dissociate and plate neurons on maturation substrate
  8. 8 Mature neurons with regular medium changes
Characterization—Immunocytochemistry, ROS, and Mitochondrial Imaging Day 60+ (organoids) or Week 6–7 (neurons)
  1. 1 Perform immunocytochemistry on organoids
  2. 2 Quantify ROS with MitoSOX and MitoTracker
  3. 3 Visualize lipid trafficking with NBD-PA
  4. 4 Acquire confocal microscopy images
Characterization—Transmission Electron Microscopy (TEM) Day 60+ (organoids) or Week 6–7 (neurons)
  1. 1 Fix samples for TEM
  2. 2 Dehydrate and stain samples
  3. 3 Infiltrate and embed in Epon 812
  4. 4 Section and post-stain
  5. 5 Image and measure mitochondrial morphology
Functional Assays—ATP Synthase, AOPP, and OCR Week 6–7 (neurons) or Day 60 (organoids)
  1. 1 Measure ATP synthase activity
  2. 2 Quantify advanced oxidation protein products (AOPP)
  3. 3 Measure oxygen consumption rate (OCR) by Seahorse analysis
Functional Assays—Electrophysiology and Synaptic Analysis Week 2–9 (neurons)
  1. 1 Prepare neurons for microelectrode array (MEA) recording
  2. 2 Record spontaneous electrophysiological activity
  3. 3 Detect burst firing
  4. 4 Assess synaptic protein levels
Intervention—Supplementation with NMN, PG, CL, and PA Week 6–7 (neurons); timing dependent on experiment
  1. 1 Prepare stock solutions
  2. 2 Apply treatments to neurons
  3. 3 Assess functional rescue after treatment
Molecular Analysis—qRT-PCR and RNA-seq Day 60 (organoids) or Week 6–7 (neurons)
  1. 1 Isolate total RNA
  2. 2 Perform reverse transcription
  3. 3 Perform qRT-PCR
  4. 4 Perform bulk RNA-seq
Lipidomics and Biochemical Analysis Day 60 (organoids)
  1. 1 Prepare samples for lipidomic analysis
  2. 2 Perform shotgun lipidomic mass spectrometry
  3. 3 Perform weighted gene co-expression network analysis (WGCNA) on lipidomics data
Validation in Neuron-Specific ABCA7 Knockout Mice Mouse ages 4 and 20 months
  1. 1 Generate neuron-specific Abca7 knockout mice
  2. 2 Confirm neuronal ABCA7 deletion by immunostaining
  3. 3 Isolate synaptosomes from mouse brain
  4. 4 Confirm ABCA7 loss in synaptosomes by Western blot
  5. 5 Assess synaptic mitochondrial morphology in synaptosomes by TEM
  6. 6 Measure synaptic protein levels in synaptosomes
  7. 7 Quantify gene expression in mouse cortex
Statistical Analysis Upon completion of all experiments
  1. 1 Organize and validate data
  2. 2 Perform statistical tests
  3. 3 Analyze RNA-seq data
  4. 4 Present results with descriptive statistics

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kawatani et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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