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BRAIN Publication-derived

Parechovirus Infection in Human Brain Organoids: Infection and Analysis Protocol

Source Capendale et al., 2024 · Amsterdam UMC, Academic Medical Center, Amsterdam Institute for Infection and Immunity · 10.1038/s41467-024-46634-9

👤 Pamela E. Capendale, Inés García-Rodríguez, Anoop T. Ambikan, Lance A. Mulder, Josse A. Depla, Eline Freeze, Gerrit Koen, Carlemi Calitz, Vikas Sood, Renata Vieira de Sá, Ujjwal Neogi, Dasja Pajkrt, Adithya Sridhar, Katja C. Wolthers ⏱ 77 days 📋 11 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of unguided neural organoids (UNOs) from human iPSCs and their infection with parechovirus A (PeV-A) genotypes to model CNS infection. The protocol enables characterization of viral replication kinetics, cell tropism, and host inflammatory responses to distinguish neuropathogenic from non-neuropathogenic viral genotypes.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

87 steps across 11 phases

iPSC Culture and Maintenance Ongoing, weekly passages
  1. 1 Coat culture plates with laminin 521
  2. 2 Culture hiPSCs in mTeSR+ medium
  3. 3 Passage hiPSCs weekly
  4. 4 Perform routine mycoplasma testing
Unguided Neural Organoid Generation Days 0–10 (induction and initial expansion)
  1. 1 Dissociate hiPSCs into single cell suspension
  2. 2 Form embryoid bodies (EBs)
  3. 3 Induce neuroectoderm differentiation
  4. 4 Embed EBs in ESC-qualified Matrigel
  5. 5 Transfer organoids to orbital shaker
  6. 6 Refresh maturation medium every 3–4 days
Viral Culture and Preparation Prior to infection (timing variable)
  1. 1 Maintain virus-producer cell lines
  2. 2 Amplify lab-adapted virus strains
  3. 3 Maintain clinical isolate stocks
  4. 4 Prepare heat-inactivated virus controls
Organoid Infection and Sampling Days 67–77 (infection through day 10 post-infection)
  1. 1 Prepare 96-well plate with anti-adherence solution
  2. 2 Transfer organoids and inoculate with virus
  3. 3 Allow viral adsorption
  4. 4 Wash organoids three times with PBS
  5. 5 Transfer to coated 48-well plate with maturation medium
  6. 6 Incubate and collect 0 h time-point
  7. 7 Collect supernatant at multiple time points
  8. 8 Perform back-titration of inoculum
Viral Replication Analysis Days 0–10 post-infection (samples collected at each time point)
  1. 1 Extract RNA from supernatant
  2. 2 Perform reverse transcription
  3. 3 Perform quantitative RT-qPCR
  4. 4 Perform TCID50 assay for infectious virus
  5. 5 Calculate TCID50 using Reed and Muench method
Immunofluorescence and Viral Tropism Analysis Days 5 and 10 post-infection (or as designated)
  1. 1 Fix organoids with formaldehyde
  2. 2 Wash fixed organoids with PBS
  3. 3 Cryoprotect organoids with sucrose
  4. 4 Embed organoids in OCT medium
  5. 5 Section organoids on cryostat
  6. 6 Block sections
  7. 7 Apply primary antibodies
  8. 8 Wash sections with PBS
  9. 9 Apply secondary antibody and Hoechst
  10. 10 Quench autofluorescence
  11. 11 Wash sections again with PBS
  12. 12 Mount slides with coverslips
  13. 13 Image organoid sections
Cytokine and Gene Expression Analysis Days 5 and 10 post-infection (sample collection and analysis)
  1. 1 Harvest organoids into lysis buffer
  2. 2 Homogenize and resuspend samples
  3. 3 Extract RNA from organoid lysates
  4. 4 Synthesize cDNA
  5. 5 Perform RT-qPCR for cytokine genes
  6. 6 Normalize gene expression data
  7. 7 Prepare samples for Luminex assay
  8. 8 Perform Luminex cytokine multiplex assay
  9. 9 Process and visualize cytokine data
Proteomic Analysis (LC-MS/MS) Day 10 post-infection (sample collection and processing)
  1. 1 Prepare organoid specimens for proteomic analysis
  2. 2 Add protease inhibitors and sonicate
  3. 3 Adjust pH and sonicate with probe
  4. 4 Clarify lysate by centrifugation
  5. 5 Estimate protein concentration
  6. 6 Prepare protein for digestion
  7. 7 Reduce disulfide bonds
  8. 8 Alkylate free cysteine residues
  9. 9 Quench excess alkylation reagent
  10. 10 Perform trypsin digestion
  11. 11 Stop digestion with formic acid
  12. 12 Purify peptides on C18 plate
  13. 13 Perform TMT labeling and multiplexing
  14. 14 Fractionate by high-pH reversed-phase UPLC
  15. 15 Prepare peptides for LC-MS/MS
  16. 16 Analyze by LC-MS/MS
  17. 17 Process raw MS data with Proteome Discoverer
Proteomic Data Analysis and Bioinformatics Post-MS/MS data acquisition (analysis phase)
  1. 1 Check normality and apply normalization
  2. 2 Perform missing value imputation
  3. 3 Correct for batch effects
  4. 4 Perform differential abundance analysis
  5. 5 Identify differentially abundant proteins (DAPs)
  6. 6 Perform pathway enrichment analysis
  7. 7 Analyze interferon-related proteins
  8. 8 Generate visualization of results
JAK-STAT Pathway Inhibition Experiment Days 67–77 (parallel to main infection protocol)
  1. 1 Pre-treat organoids with Ruxolitinib or DMSO vehicle
  2. 2 Verify JAK/STAT pathway blockade with IFN stimulation
  3. 3 Infect Rux-pre-treated organoids
  4. 4 Continue Rux/DMSO treatment throughout infection
  5. 5 Collect samples for ISG expression analysis
  6. 6 Collect supernatant for viral replication analysis
  7. 7 Analyze effect of Rux on ISG expression
  8. 8 Analyze effect of Rux on viral replication
Statistical Analysis and Data Visualization Post-data collection (analysis phase)
  1. 1 Compile experimental data
  2. 2 Perform statistical analysis for non-proteomics data
  3. 3 Generate standard graphs and plots
  4. 4 Generate proteomic visualizations
  5. 5 Generate comprehensive heatmaps

Full SOP

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Attribution

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

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