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BRAIN Publication-derived

iPSC-Derived Forebrain Organoid Generation and Drug Treatment for Single-Cell Transcriptomics Analysis

Source Ho et al., 2024 · Mayo Clinic, Rochester, MN, USA · 10.1038/s41380-022-01837-8

👤 Ming-Fen Ho, Cheng Zhang, Irene Moon, Xiujuan Zhu, Brandon J. Coombes, Joanna Biernacka, Michelle Skime, Tyler S. Oesterle, Victor M. Karpyak, Kristen Schmidt, Kate Gliske, Quyen Ngo, Cedric Skillon, Marvin D. Seppala, Hu Li, Richard M. Weinshilboum ⏱ 90 days 📋 11 phases 🧫 Patient-Derived iPSC (Opioid Use Disorder)

Abstract

This protocol describes the generation of iPSC-derived forebrain organoids from opioid use disorder patients and their treatment with oxycodone or buprenorphine for single-cell transcriptomics analysis. The method enables cell-type-specific assessment of transcriptional responses to opioid medications at single-nucleus resolution.

Cell source
Patient-Derived iPSC (Opioid Use Disorder)
Application
Disease modeling and drug response characterization in opioid use disorder

Protocol overview

56 steps across 11 phases

iPSC Generation and Characterization 0-14
  1. 1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
  2. 2 iPSC Reprogramming
  3. 3 iPSC Culture on Matrigel
  4. 4 iPSC Characterization and Quality Control
iPSC-Derived Forebrain Organoid Differentiation (Days 0-14) 14-14
  1. 1 Embryoid Body Formation and Patterning
  2. 2 Seven-Day Culture in Patterning Medium
  3. 3 Mechanical Dissociation from Matrigel
Forebrain Organoid Maturation in Bioreactor (Days 14-70+) 14-70
  1. 1 Transfer to Bioreactor
  2. 2 Culture in Basal Maturation Medium (Days 14-70)
  3. 3 Culture in Maturation Medium with Neurotrophic Factors (Day 70 Onward)
Drug Treatment of Forebrain Organoids 83-90
  1. 1 Preparation of Drug Stock Solutions
  2. 2 Drug Treatment Initiation
  3. 3 Seven-Day Drug Treatment with Daily Medium Changes
  4. 4 Harvest Organoids for Analysis
iPSC-Derived Neuron Differentiation (Parallel to Organoids) 0-34
  1. 1 Initial EB Formation in Non-Treated Plates
  2. 2 Neural Induction (Days 6-7)
  3. 3 Rosette Maintenance (Days 7-22)
  4. 4 Neural Progenitor Sphere Formation (Days 22-24)
  5. 5 Neuronal Differentiation
iPSC-Derived Astrocyte Differentiation (Parallel to Organoids) 22-34
  1. 1 Neural Progenitor Preparation
  2. 2 Astrocyte Differentiation
STAT1 Inhibitor Treatment of iPSC-Derived Neurons 28-34
  1. 1 Preparation of STAT1 Inhibitor (Fludarabine) Stock Solution
  2. 2 STAT1 Inhibitor Treatment of Neurons
  3. 3 RNA Isolation Following STAT1 Inhibition
Immunofluorescence Staining and Confocal Imaging Various
  1. 1 Cell Fixation
  2. 2 Permeabilization
  3. 3 Blocking
  4. 4 Primary Antibody Incubation
  5. 5 Secondary Antibody Incubation
  6. 6 Nuclear Counterstaining and Mounting
  7. 7 Confocal Microscopy Imaging
Single Nuclei RNA Sequencing (snRNA-seq) and Analysis Post-treatment
  1. 1 Nuclei Isolation from Frozen Organoids
  2. 2 Library Preparation
  3. 3 RNA Library Sequencing
  4. 4 Quality Control and Data Filtering
  5. 5 Data Normalization and Scaling
  6. 6 Feature Selection
  7. 7 Principal Component Analysis (PCA)
  8. 8 UMAP Visualization
  9. 9 Graph-Based Clustering
  10. 10 Cluster Annotation
  11. 11 Differential Expression Analysis
  12. 12 Pathway Analysis
  13. 13 Protein-Protein Interaction Network Analysis
Quantitative Real-Time PCR (qPCR) Post-treatment
  1. 1 RNA Isolation
  2. 2 qPCR Reaction Setup
  3. 3 qPCR Amplification
  4. 4 Data Analysis
Western Blot Analysis Post-treatment
  1. 1 Protein Sample Isolation
  2. 2 SDS-PAGE Gel Loading and Electrophoresis
  3. 3 Protein Transfer to PVDF Membrane
  4. 4 Membrane Blocking
  5. 5 Primary Antibody Incubation
  6. 6 Secondary Antibody Incubation
  7. 7 Chemiluminescent Detection
  8. 8 Quantitative Analysis

Full SOP

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Attribution

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

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