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BRAIN Publication-derived

Generation and Analysis of Granulin-Loss-of-Function Mature Brain Organoids (mbOrgs) for TDP-43 Proteinopathy Modeling

Source de Majo et al., 2023 · University of California San Francisco · 10.1016/j.stemcr.2023.01.012

👤 Martina de Majo, Mark Koontz, Elise Marsan, Nir Salinas, Arren Ramsey, Yien-Ming Kuo, Kyounghee Seo, Huinan Li, Nina Dräger, Kun Leng, Santiago L. Gonzales, Michael Kurnellas, Yuichiro Miyaoka, Joseph R. Klim, Martin Kampmann, Michael E. Ward, Eric J. Huang, Erik M. Ullian ⏱ 30 days 📋 9 phases 🧫 Human iPSC (isogenic wild-type and GRN knockout lines)

Abstract

This protocol describes the generation of three-dimensional mature brain organoids (mbOrgs) composed of iPSC-derived cortical-like neurons and astrocytes assembled at defined ratios to model GRN-loss-of-function-associated TDP-43 pathology. When composed of GRN-null cells, mbOrgs spontaneously recapitulate key features of human frontotemporal dementia including TDP-43 mis-localization, hyperphosphorylation, and cryptic STMN2 splicing, providing a platform for mechanistic studies and therapeutic screening.

Cell source
Human iPSC (isogenic wild-type and GRN knockout lines)
Application
Disease modeling of frontotemporal dementia and ALS via TDP-43 proteinopathy

Protocol overview

32 steps across 9 phases

iPSC Maintenance and Expansion Ongoing
  1. 1 iPSC Culture and Maintenance
  2. 2 iPSC Passage Using EDTA
Cortical-Like Neuronal Induction (iNeurons) Days 1–3
  1. 1 Prepare Matrigel-Coated Plates
  2. 2 Seed and Induce NGN2-Inducible Neurons
  3. 3 Harvest Induced Neurons (iNeurons)
Mature Cortical-Like Astrocyte Differentiation (iAstrocytes) Days 1–30 (parallel to neuronal induction)
  1. 1 iPSC-to-Astrocyte Differentiation (Method Previously Described)
  2. 2 Characterize Astrocyte Identity and Maturity
Assembly of 3D Mature Brain Organoids (mbOrgs) Day 1 (assembly) to Day 28 (culture endpoint)
  1. 1 Prepare Cells for Organoid Assembly
  2. 2 Mix and Assemble Genotypes
  3. 3 Culture mbOrgs for 2 and 4 Weeks
Phenotypic Analysis of mbOrgs at 2 and 4 Weeks Days 14 (2-week) and 28 (4-week)
  1. 1 Immunostaining for Cell-Type Markers
  2. 2 Assess TDP-43 Pathology by Immunostaining
  3. 3 Quantify Extranuclear TDP-43 Particles (IMARIS 3D Reconstruction)
  4. 4 Western Blot Analysis: pTDP-43 and Total TDP-43
  5. 5 Quantitative PCR for Cryptic STMN2 Splicing (CrSTMN2)
  6. 6 Synaptic Marker Immunostaining and Analysis
  7. 7 Western Blot: Synaptic Proteins
Gene Expression Analysis: RNA Sequencing and qPCR Day 28 (4-week timepoint)
  1. 1 Bulk RNA Sequencing
  2. 2 Analyze RNA-seq Data for Phagocytosis-Related Gene Expression
  3. 3 Confirmation qPCR for Phagocytosis Marker Genes
Functional Assay: Astrocyte Synaptosome Phagocytosis (2D Cultures) Days 1–28 (2D co-culture setup and analysis)
  1. 1 Prepare 2D Astrocyte–Neuron Co-Cultures
  2. 2 Prepare mCherry-Labeled Rat Synaptosome
  3. 3 Phagocytosis Assay: FACS Analysis
  4. 4 Quantify Phagocytosis Results
Heterotypic mbOrg Assembly and Analysis Day 1 (assembly) to Day 28 (analysis)
  1. 1 Generate All Four Genotype Combinations for 3D mbOrgs
  2. 2 Immunostaining for TDP-43 in Heterotypic mbOrgs
  3. 3 Quantify CrSTMN2 Expression in Heterotypic mbOrgs
  4. 4 Confirm Phagocytosis Gene Downregulation in Heterotypic Conditions
Rescue Experiments: Treatment with Recombinant PGRN (2D Co-Cultures) Days 1–28 (parallel 2D culture setup with PGRN treatment)
  1. 1 Determine Optimal PGRN Concentration and Dosing
  2. 2 Confirm PGRN Cellular Uptake by Immunostaining
  3. 3 Assess CrSTMN2 Rescue in PGRN-Treated 2D Co-Cultures
  4. 4 Assess Phagocytosis Gene Rescue (MERTK and MEGF10)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in de Majo et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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