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BRAIN Publication-derived

Herpes Simplex Virus Infection Brain Organoids for Drug Screening

Full SOP with Academic or a plan

Source D'Aiuto et al., 2019 · University of Pittsburgh School of Medicine · 10.1128/jvi.00111-19

Authors: Leonardo D'Aiuto, David C. Bloom, Jennifer N. Naciri, Adam Smith, Terri G. Edwards, Lora McClain, Jason A. Callio, Morgan Jessup, Joel Wood, Kodavali Chowdari, Matthew Demers, Eric E. Abrahamson, Milos D. Ikonomovic, Luigi Viggiano, Roberta De Zio, Simon Watkins, Paul R. Kinchington, Vishwajit L. Nimgaonkar 11 phases Cell source: Human iPSC

Abstract

This protocol establishes hiPSC-derived two-dimensional (2D) and three-dimensional (3D) brain organoid cultures to model acute, latent, and reactivated HSV-1 infections in human CNS neurons. The 2D cultures recapitulate lytic infection and enable efficient viral reactivation from latency, while 3D organoids model the complex laminar structure of the CNS and demonstrate inefficient HSV-1 reactivation, more closely paralleling in vivo CNS infection dynamics.

Cell source
Human iPSC
Application
Disease modeling; Viral infection studies; Drug screening platform development

Protocol overview

55 steps across 11 phases

Phase 1 · Generation of neural progenitor cells (NPCs) from hiPSCs 0-14 days
  1. a Culture hiPSC lines
  2. b Differentiate hiPSCs into neural rosettes
  3. c Expand neural rosettes as monolayer NPC cultures
Phase 2 · Generation and characterization of 2D hiPSC-derived CNS neuronal cultures 15-42 days
  1. a Seed NPCs for 2D neuronal differentiation
  2. b Maintain 2D neuronal cultures
  3. c Characterize neuronal phenotype by immunofluorescence
Phase 3 · Generation of 3D brain organoids 0-84 days
  1. a Seed NPCs in Millicell transwell plates
  2. b Add differentiation medium and growth factors (days 0-4)
  3. c Withdraw morphogen factors (day 4)
  4. d Continue 3D differentiation (days 4-28)
  5. e Transfer organoids to low-attachment plates
  6. f Culture organoids long-term
Phase 4 · Acute HSV-1 infection of 2D neuronal cultures 0-2 days (after 6-week neuronal differentiation)
  1. a Prepare hiPSC-derived neurons for infection
  2. b Adsorb HSV-1 onto neuronal monolayer
  3. c Remove inoculum and wash
  4. d Culture infected neurons
  5. e Assess viral infection by ICP4 immunostaining
Phase 5 · Establishment of HSV-1 latency in 2D neuronal cultures 0-7 days (after 6-week neuronal differentiation)
  1. a Pretreat neurons with antivirals
  2. b Infect neurons with HSV-1 in presence of antivirals
  3. c Confirm latency by viral titer assay
  4. d Analyze viral gene expression by RT-qPCR
  5. e Visualize viral DNA by 3D-FISH
Phase 6 · Reactivation of HSV-1 latency in 2D neuronal cultures 7-12 days postinfection
  1. a Withdraw antivirals without reactivation stimulus
  2. b Treat with HDAC inhibitor (sodium butyrate)
  3. c Detect reactivation by viral titer assay
  4. d Analyze chromatin changes by 3D-FISH
Phase 7 · Chromatin immunoprecipitation (ChIP) analysis of HSV-1 latency 0-7 days
  1. a Prepare acutely and latently infected chromatin
  2. b Cross-link chromatin
  3. c Sonicate chromatin
  4. d Perform immunoprecipitation with antibodies
  5. e Recover immunoprecipitated and input DNA
  6. f Analyze enrichment of histone marks and proteins
Phase 8 · Chromatin accessibility analysis (ChART-PCR) 0-1 day
  1. a Infect neurons and collect nuclei
  2. b Digest with micrococcal nuclease
  3. c Extract DNA and assess digestion
  4. d Quantify viral DNA accessibility by qPCR
  5. e Statistical analysis
Phase 9 · Acute HSV-1 infection of 3D brain organoids 0-2 days (after 10+ weeks organoid differentiation)
  1. a Prepare organoids for infection
  2. b Prepare viral inoculum
  3. c Infect organoids
  4. d Incubate infected organoids
  5. e Remove inoculum and wash
  6. f Return organoids to 24-well plates
  7. g Assess viral infection by immunohistochemistry
  8. h Detect LAT expression by RNAscope
Phase 10 · Establishment of HSV-1 latency in 3D organoids 0-11 days
  1. a Pretreat organoids with antivirals
  2. b Infect organoids with HSV-1 in presence of antivirals
  3. c Monitor EGFP and RFP expression
  4. d Confirm latency by viral titer assay
Phase 11 · HSV-1 reactivation attempts in latently infected 3D organoids 8-33 days postinfection
  1. a Assess spontaneous reactivation
  2. b Withdraw antivirals for spontaneous reactivation assessment
  3. c Treat with HDAC inhibitor (sodium butyrate)
  4. d Treat with HDAC inhibitor (trichostatin A)
  5. e Treat with phosphatidylinositol 3-kinase inhibitor
  6. f Analyze organoid architecture post-reactivation

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Attribution

This SOP was written by Organthis based on the published method in D'Aiuto et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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