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BRAIN Publication-derived

Congenital Cytomegalovirus Infection Cerebral Organoids

Full SOP with Academic or a plan

Source O'Brien et al., 2022 · Medical College of Wisconsin · 10.1016/j.isci.2022.104098

Authors: Benjamin S. O'Brien, Rebekah L. Mokry, Megan L. Schumacher, Kirthi Pulakanti, Sridhar Rao, Scott S. Terhune, Allison D. Ebert Duration: 44 days 9 phases Cell source: 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

Phase 1 · Embryoid Body Formation and Neural Induction Day 0-7
  1. a Seed iPSCs for embryoid body formation
  2. b Initiate neural induction
Phase 2 · Organoid Expansion and Maturation Day 7-30
  1. a Embed neural tissue in Matrigel and transfer to 6-well plates
  2. b Transfer to rocker platform
  3. c Change media every 3 days with maturation media
Phase 3 · Organoid Infection with HCMV Day 30-44
  1. a Weigh organoids and prepare for infection
  2. b Infect organoids with HCMV TB40/E-GFP
  3. c Monitor GFP signal progression
  4. d Change media every 3-4 days post-infection
Phase 4 · Organoid Dissociation and FACS Sorting Day 13-16 post-infection
  1. a Dissociate infected and mock organoids into single cells
  2. b Establish live cell gating
  3. c Sort infected organoid cells by GFP fluorescence intensity
  4. d Verify sort quality and cell viability
Phase 5 · Viral Gene Expression Quantification Concurrent with Phase 4
  1. a Extract total RNA from sorted cell populations
  2. b Quantify viral gene expression via qRT-PCR
  3. c Quantify viral vs. host genomes
Phase 6 · RNA Sequencing Library Preparation Concurrent with Phase 5
  1. a Assess RNA quantity and quality
  2. b Add ERCC spike-in controls
  3. c Perform mRNA isolation and cDNA library generation
  4. d Quantify and validate cDNA libraries
  5. e Pool and re-quantify libraries
Phase 7 · RNA Sequencing and Quality Control Day of sequencing
  1. a Prepare sequencing cartridge with PhiX control
  2. b Run NextSeq sequencing
  3. c Assess sequencing quality metrics
Phase 8 · RNA-Seq Bioinformatic Analysis Post-sequencing
  1. a Map reads to reference genome and quantify transcripts
  2. b Perform principal component analysis (PCA)
  3. c Identify differentially expressed genes (DEGs)
  4. d Perform gene ontology (GO) enrichment analysis
  5. e Perform Gene Set Enrichment Analysis (GSEA)
  6. f Perform Ingenuity Pathway Analysis (IPA)
  7. g Generate and interpret heatmaps
Phase 9 · Validation with TB40r mGFP-IE-FKBP Shield-1 System (Optional/Parallel) 3-7 days for NPC infection
  1. a Culture and plate neural progenitor cells (NPCs)
  2. b Infect NPCs with TB40r mGFP-IE-FKBP
  3. c Monitor viral gene expression over infection time course
  4. d Assess viral tegument protein expression
  5. e Quantify neurodevelopmental gene targets

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Attribution

This SOP was written by Organthis based on the published method in O'Brien et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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