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BRAIN Publication-derived

Downregulation of neurodevelopmental gene expression in iPSC-derived cerebral organoids upon infection by human cytomegalovirus

Source O'Brien et al., 2022 · Medical College of Wisconsin · 10.1016/j.isci

👤 Benjamin S. O'Brien, Rebekah L. Mokry, Megan L. Schumacher, Kirthi Pulakanti, Sridhar Rao, Scott S. Terhune, Allison D. Ebert ⏱ 44 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation and infection of human iPSC-derived cerebral organoids with HCMV strain TB40/E-GFP, followed by fluorescence-activated cell sorting and transcriptomic analysis to assess infection-dependent downregulation of neurodevelopmental genes. The method enables investigation of HCMV-mediated disruption of neural differentiation pathways and signaling networks.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

36 steps across 9 phases

Embryoid Body Formation and Neural Induction Day 0–7
  1. 1 Seed iPSCs for embryoid body formation
  2. 2 Initiate neural induction
Organoid Expansion and Maturation Day 7–30
  1. 1 Embed neural tissue in Matrigel and transfer to 6-well plates
  2. 2 Transfer to rocker platform
  3. 3 Change media every 3 days with maturation media
Organoid Infection with HCMV Day 30–44
  1. 1 Weigh organoids and prepare for infection
  2. 2 Infect organoids with HCMV TB40/E-GFP
  3. 3 Monitor GFP signal progression
  4. 4 Change media every 3–4 days post-infection
Organoid Dissociation and FACS Sorting Day 13–16 post-infection
  1. 1 Dissociate infected and mock organoids into single cells
  2. 2 Establish live cell gating
  3. 3 Sort infected organoid cells by GFP fluorescence intensity
  4. 4 Verify sort quality and cell viability
Viral Gene Expression Quantification Concurrent with Phase 4
  1. 1 Extract total RNA from sorted cell populations
  2. 2 Quantify viral gene expression via qRT-PCR
  3. 3 Quantify viral vs. host genomes
RNA Sequencing Library Preparation Concurrent with Phase 5
  1. 1 Assess RNA quantity and quality
  2. 2 Add ERCC spike-in controls
  3. 3 Perform mRNA isolation and cDNA library generation
  4. 4 Quantify and validate cDNA libraries
  5. 5 Pool and re-quantify libraries
RNA Sequencing and Quality Control Day of sequencing
  1. 1 Prepare sequencing cartridge with PhiX control
  2. 2 Run NextSeq sequencing
  3. 3 Assess sequencing quality metrics
RNA-Seq Bioinformatic Analysis Post-sequencing
  1. 1 Map reads to reference genome and quantify transcripts
  2. 2 Perform principal component analysis (PCA)
  3. 3 Identify differentially expressed genes (DEGs)
  4. 4 Perform gene ontology (GO) enrichment analysis
  5. 5 Perform Gene Set Enrichment Analysis (GSEA)
  6. 6 Perform Ingenuity Pathway Analysis (IPA)
  7. 7 Generate and interpret heatmaps
Validation with TB40r mGFP-IE-FKBP Shield-1 System (Optional/Parallel) 3–7 days for NPC infection
  1. 1 Culture and plate neural progenitor cells (NPCs)
  2. 2 Infect NPCs with TB40r mGFP-IE-FKBP
  3. 3 Monitor viral gene expression over infection time course
  4. 4 Assess viral tegument protein expression
  5. 5 Quantify neurodevelopmental gene targets

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in O'Brien et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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