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BRAIN Publication-derived

The Impaired Neurodevelopment of Human Neural Rosettes in HSV-1-Infected Early Brain Organoids

Source D'Aiuto et al., 2022 · Western Psychiatric Institute and Clinic, Department of Psychiatry, School of Medicine, University of Pittsburgh · 10.3390/cells11223539

👤 Leonardo D'Aiuto, Jill K. Caldwell, Callen T. Wallace, Tristan R. Grams, Maribeth A. Wesesky, Joel A. Wood, Simon C. Watkins, Paul R. Kinchington, David C. Bloom, Vishwajit L. Nimgaonkar ⏱ 35 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol models neurodevelopmental impairment caused by HSV-1 infection of early-stage brain organoids derived from human induced pluripotent stem cells (iPSCs). Early-stage organoids composed of neural rosettes are infected with HSV-1 and cultured in the presence or absence of acyclovir to investigate neuropathological effects on neural differentiation and the emergence of antiviral-resistant viral mutants.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

73 steps across 12 phases

Cell Culture Maintenance Ongoing
  1. 1 Maintain Vero cells for plaque assay
  2. 2 Culture human iPSC line SC0000020 (subclone SF)
Virus Preparation Days 1–3
  1. 1 Prepare HSV-1 virus stock
  2. 2 Harvest infected cells
  3. 3 Centrifuge cell suspension
  4. 4 Resuspend cell pellet
  5. 5 Freeze-thaw cycle
  6. 6 Remove debris
  7. 7 Store viral stock
Virus Titration by Plaque Assay Days 1–4
  1. 1 Prepare Vero cell monolayers
  2. 2 Infect with serial dilutions of HSV
  3. 3 Overlay with CMC medium
  4. 4 Incubate plates
  5. 5 Remove overlay and fix cells
  6. 6 Stain and count plaques
Acyclovir Resistance Assay Days 1–3
  1. 1 Prepare rabbit skin cells
  2. 2 Pretreat with acyclovir
  3. 3 Infect with culture supernatant
  4. 4 Replace medium with ACV-containing medium
  5. 5 Incubate and fix cells
  6. 6 Enumerate plaques
Neural Rosette Generation from iPSCs Days 1–7
  1. 1 Prepare hiPSCs for neural induction
  2. 2 Withdraw ROCK inhibitor
  3. 3 Change culture medium daily
  4. 4 Monitor for neural rosette appearance
Isolation and Expansion of Neural Rosettes Days 7–10
  1. 1 Manually dissect neural rosette clusters
  2. 2 Culture rosette clusters in suspension
  3. 3 Transfer to low-attachment 96-well plates
Infection of Early-Stage Organoids from Neural Rosettes Days 10–30
  1. 1 Wash organoids
  2. 2 Prepare infection inoculum
  3. 3 Infect organoids
  4. 4 Incubate organoids
  5. 5 Remove inoculum
  6. 6 Wash organoids twice
  7. 7 Transfer to culture dishes
  8. 8 Monitor organoid development
Generation of Early-Stage Brain Organoids from iPSCs (Alternative Method) Days 1–9
  1. 1 Generate embryoid bodies
  2. 2 Culture embryoid bodies
  3. 3 Switch to neural induction medium
  4. 4 Observe neuroectoderm formation
  5. 5 Verify neural rosette presence
  6. 6 Prepare organoids for infection
Infection of Early-Stage Brain Organoids from Embryoid Bodies Day 9 onwards
  1. 1 Infect organoids individually
  2. 2 Allow viral absorption
  3. 3 Remove inocula
  4. 4 Wash organoids
  5. 5 Culture in neuronal differentiation medium
  6. 6 Change culture medium regularly
  7. 7 Monitor organoid status
Generation and Infection of Adherent 3D (A-3D) Cultures Days 1–35
  1. 1 Generate monolayer NPC cultures
  2. 2 Infect NPC monolayers
  3. 3 Remove inocula and dissociate cells
  4. 4 Seed cells at high density
  5. 5 Culture in NPC maintenance medium
  6. 6 Switch to differentiation medium
  7. 7 Withdraw small molecules
  8. 8 Change medium schedule
  9. 9 Monitor A-3D cultures
Immunofluorescence Analysis Days 4, 8, 15, 20, 35
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Wash organoids
  3. 3 Cryoprotect organoids
  4. 4 Embed organoids
  5. 5 Prepare frozen sections
  6. 6 Prepare slides
  7. 7 Apply primary antibodies
  8. 8 Apply secondary antibodies
  9. 9 Acquire images
Quantitative Confocal Analysis Post-immunofluorescence
  1. 1 Acquire confocal images
  2. 2 Process images with DenoiseAI
  3. 3 Perform segmentation
  4. 4 Count nuclei
  5. 5 Measure and normalize volumes
  6. 6 Generate representative images

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in D'Aiuto 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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