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BRAIN Publication-derived

Generation and Characterization of Human Neural Organoids with MAPT R406W Mutation for Study of GRAMD1B-Mediated Lipid Dysregulation and Tau Pathology

Source Ingram et al., 2025 · Department of Neuroscience, College of Medicine, The Ohio State University · 10.1038/s41467-025-58585-w

👤 Diana Acosta Ingram, Emir Turkes, Tae Yeon Kim, Sheeny Vo, Nicholas Sweeney, Marie-Amandine Bonte, Ryan Rutherford, Dominic L. Julian, Meixia Pan, Jacob Marsh, Andrea R. Argouarch, Min Wu, Douglas W. Scharre, Erica H. Bell, Lawrence S. Honig, Jean Paul Vonsattel, Geidy E. Serrano, Thomas G. Beach, Celeste M. Karch, Aimee W. Kao, Mark E. Hester, Xianlin Han, Hongjun Fu ⏱ 120 days 📋 12 phases 🧫 Patient-Derived iPSC (MAPT R406W mutation)

Abstract

This protocol describes the generation and comprehensive characterization of human neural organoids (HNOs) derived from iPSCs carrying the MAPT R406W mutation associated with familial frontotemporal lobar degeneration. The HNOs are cultured for 120 days to recapitulate tau phosphorylation, lipid dysregulation, and altered autophagy associated with FTLD-tau pathology, enabling investigation of GRAMD1B as a regulator of lipid homeostasis and tau phosphorylation in excitatory neurons.

Cell source
Patient-Derived iPSC (MAPT R406W mutation)
Application
Disease modeling (Frontotemporal Lobar Degeneration with tau pathology)

Protocol overview

63 steps across 12 phases

iPSC Culture and Maintenance Prior to differentiation
  1. 1 Obtain iPSC lines
  2. 2 Culture iPSCs in standard conditions
  3. 3 Passage iPSCs
Embryoid Body (EB) Formation Days 1-13
  1. 1 Initiate EB formation
  2. 2 Remove ROCK inhibitor
  3. 3 Begin neuroectoderm induction
  4. 4 Monitor neuroectoderm formation
  5. 5 Encapsulate EBs in Matrigel
Human Neural Organoid (HNO) Expansion and Growth Days 13-60
  1. 1 Culture HNOs in expansion medium
  2. 2 Perform half-medium changes
  3. 3 Monitor HNO size
HNO Maturation and Characterization Days 60-120
  1. 1 Continue culture on rotating shaker
  2. 2 Collect HNOs for scRNA-seq at Day 72
  3. 3 Collect HNOs for analysis at Day 120
Single-Cell RNA Sequencing (scRNA-seq) Days 72 and 120
  1. 1 Dissociate HNOs to single cells
  2. 2 Generate cDNA libraries
  3. 3 Perform sequencing
Immunofluorescence Staining of HNOs Days 60 and 120
  1. 1 Fix HNOs
  2. 2 Perform sucrose gradient
  3. 3 Store embedded HNOs
  4. 4 Section HNOs
  5. 5 Dry frozen sections
  6. 6 Acetone treatment
  7. 7 Wash with PBS
  8. 8 Antigen retrieval
  9. 9 Cool and wash sections
  10. 10 Block sections
  11. 11 Incubate with primary antibodies
  12. 12 Wash with PBST
  13. 13 Incubate with secondary antibodies
  14. 14 Final wash
  15. 15 Mount and image
Functional Analysis via Microelectrode Array (MEA) Day 75
  1. 1 Prepare MEA probes
  2. 2 Transfer HNOs to MEA chips
  3. 3 Record extracellular field potentials
  4. 4 Analyze electrophysiological metrics
Western Blot Analysis Days 60 and 120
  1. 1 Collect HNO samples
  2. 2 Prepare HNO lysates
  3. 3 Centrifuge samples
  4. 4 Measure protein concentration
  5. 5 Separate proteins by gel electrophoresis
  6. 6 Probe with primary antibodies
  7. 7 Incubate with secondary antibodies
  8. 8 Image and quantify
Lipidomic Analysis of HNO Culture Media Days 60 and 120
  1. 1 Collect conditioned medium
  2. 2 Add internal lipid standards
  3. 3 Perform lipid extraction
  4. 4 Dilute lipid extract
  5. 5 Perform mass spectrometry
  6. 6 Identify and quantify lipid species
GRAMD1B Modulation in HNOs and iPSC-Derived Neurons Days 4-10 (neurons) or 7-17 (HNOs)
  1. 1 Transduce with GRAMD1B lentivirus
  2. 2 Validate GRAMD1B modulation
  3. 3 Assess autophagy markers
Autophagy Flux Monitoring with FUW-mCherry-GFP-LC3 Reporter Days 3-10 (neurons) or 7-17 (HNOs)
  1. 1 Transduce with autophagy reporter virus
  2. 2 Treat with BAFA1 (optional)
  3. 3 Perform live imaging
  4. 4 Quantify puncta
AI-3d Aster Inhibitor Treatment of HNOs 1 week
  1. 1 Prepare HNOs for treatment
  2. 2 Treat HNOs with AI-3d
  3. 3 Fix and embed treated organoids
  4. 4 Stain for free cholesterol and lipid droplets
  5. 5 Assess autophagy and tau phosphorylation
  6. 6 Verify unchanged excitatory neuron populations

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ingram et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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