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BRAIN Publication-derived

Human Brain Organoid Model of Maternal Immune Activation: Hyper-IL-6 Treatment of Dorsal Forebrain Organoids

Source Sarieva et al., 2023 · Hertie Institute for Clinical Brain Research, University of Tübingen · 10.1038/s41380-023-01997-1

👤 Kseniia Sarieva, Theresa Kagermeier, Shokoufeh Khakipoor, Ezgi Atay, Zeynep Yentür, Katharina Becker, Simone Mayer ⏱ 90 days 📋 7 phases 🧫 Human iPSC

Abstract

This protocol establishes a three-dimensional in vitro model of maternal immune activation (MIA) using induced pluripotent stem cell-derived dorsal forebrain organoids treated with Hyper-IL-6, a constitutively active form of interleukin-6. The model demonstrates that radial glia cells are selectively vulnerable to IL-6 signaling and exhibit transcriptional changes associated with immune response activation and protein translation downregulation, recapitulating molecular features of autism spectrum disorder risk.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

42 steps across 7 phases

iPSC Culture and Dorsal Forebrain Organoid Generation Days 0-45
  1. 1 iPSC Culture Setup
  2. 2 Dorsal Forebrain Organoid Generation
Hyper-IL-6 and IL-6 Treatment of Dorsal Forebrain Organoids Days 45-55
  1. 1 Preparation of Vehicle Control Solution
  2. 2 Preparation of IL-6 Treatment Solution
  3. 3 Preparation of Hyper-IL-6 Treatment Solution
  4. 4 Treatment Initiation at Day 45
  5. 5 Prolonged Treatment Period
  6. 6 Assessment of STAT3 Phosphorylation (Day 50-55)
Immunohistochemistry Analysis of Cell Type Composition Days 50-55 and Day 90
  1. 1 Organoid Fixation and Cryopreservation
  2. 2 Immunohistochemical Staining for Radial Glia Markers
  3. 3 Immunohistochemistry for Intermediate Progenitor Cells and Excitatory Neurons (Day 50-55)
  4. 4 Immunohistochemistry for Layer-Specific Neurons (Day 90)
  5. 5 Quantification of Cell Counts and Distribution
Western Blotting for STAT3 Phosphorylation and NR2F1 Expression Days 50-55
  1. 1 Organoid Protein Extraction
  2. 2 Protein Quantification
  3. 3 Western Blot Sample Preparation
  4. 4 SDS-PAGE Gel Electrophoresis
  5. 5 Protein Transfer to Membrane
  6. 6 Blocking and Antibody Incubation
  7. 7 Secondary Antibody Incubation and Detection
  8. 8 Detection with Li-COR Odyssey System
  9. 9 Quantification and Statistical Analysis
Bulk mRNA Sequencing Days 50-55
  1. 1 Sample Preparation for RNA Sequencing
  2. 2 mRNA Sequencing Library Preparation
  3. 3 Differential Gene Expression Analysis
  4. 4 Weighted Gene Co-Expression Network Analysis (WGCNA)
Single-Cell RNA Sequencing (scRNAseq) Day 53
  1. 1 Organoid Dissociation into Single Cells
  2. 2 Dead Cell Removal
  3. 3 Sample Multiplexing with CellPlex
  4. 4 10x Chromium Encapsulation
  5. 5 scRNAseq Library Preparation
  6. 6 Library Pooling and Sequencing
  7. 7 scRNAseq Data Processing and Quality Control
  8. 8 Clustering and Cell Type Identification
  9. 9 Regional Identity Assessment with VoxHunt
  10. 10 Trajectory Analysis of Excitatory Neuron Lineage
  11. 11 Cell Type-Specific Differential Gene Expression Analysis
  12. 12 Transcription Factor Network Analysis with SCENIC
  13. 13 ASD Relevance Analysis
Temporal Assessment of Long-Term Effects Day 90
  1. 1 Long-Term Culture to Day 90
  2. 2 Assessment of Long-Term Neuronal Changes
  3. 3 Analysis of Cortical Lamination

Full SOP

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Attribution

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

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