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BRAIN Publication-derived

Congenital Cytomegalovirus Infection Brain Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Sun et al., 2020 · Beckman Research Institute of City of Hope · 10.1016/j.xcrm.2020.100002

Authors: Guoqiang Sun, Flavia Chiuppesi, Xianwei Chen, Cheng Wang, E Tian, Jenny Nguyen, Mindy Kha, Daniel Trinh, Hannah Zhang, Maria C. Marchetto, Hongjun Song, Guo-Li Ming, Fred H. Gage, Don J. Diamond, Felix Wussow, Yanhong Shi Duration: 75 days 7 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of human iPSC-derived brain organoids and their infection with clinical-like HCMV strain TB40/E to model virus-induced microcephaly. The infected organoids exhibit reduced growth, impaired cortical layer formation, abnormal calcium signaling, and disrupted neural network activity. Neutralizing antibodies targeting the HCMV pentamer complex can effectively prevent HCMV-induced brain organoid abnormalities.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

28 steps across 7 phases

Phase 1 · iPSC Generation and Maintenance Variable (before organoid differentiation)
  1. a iPSC Derivation from Fibroblasts
  2. b iPSC Culture and Expansion
Phase 2 · Brain Organoid Generation (Days 0-15) Days 0-15
  1. a Embryoid Body Formation
  2. b Neural Induction
  3. c Matrigel Embedding
  4. d Organoid Transfer
  5. e Transfer to Suspension Culture
Phase 3 · Brain Organoid Maturation and Characterization (Days 15-75) Days 15-75
  1. a Continued Culture in Differentiation Medium
  2. b Characterization by Immunostaining (Days 45-75)
  3. c Calcium Imaging
  4. d Multielectrode Array (MEA) Recording
Phase 4 · HCMV Infection of Brain Organoids (Day 45) Day 45 to Day 65
  1. a Viral Preparation and Titration
  2. b Brain Organoid Infection
  3. c Measurement of GFP Fluorescence Intensity and Organoid Size
  4. d Immunostaining for Infected Organoids
Phase 5 · Neutralizing Antibody (NAb) Treatment Day 45 to Day 75
  1. a Antibody Preparation
  2. b Pre-incubation of Virus with NAbs
  3. c Measurement of NAb-Mediated Protection
  4. d Immunostaining for Cortical Layer Analysis
Phase 6 · Analysis of Cellular Receptors (Parallel Study) Day 45 (4-day pre-treatment + 20 days post-infection)
  1. a siRNA-Mediated Receptor Knockdown
  2. b Viral Infection of siRNA-Treated Organoids
  3. c Western Blot Verification of Receptor Expression
  4. d Receptor Overexpression in hiPSCs
Phase 7 · Analysis of Calcium Signaling and Neural Network Function Day 45 to Day 49 (calcium imaging) and Day 45 to Day 59 (MEA)
  1. a RNA Sequencing to Identify Affected Genes
  2. b qRT-PCR Validation of Gene Expression
  3. c Calcium Imaging of Infected Organoids
  4. d Multielectrode Array (MEA) Analysis of Neural Network Activity
  5. e Pharmacological Validation of MEA Results

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Attribution

This SOP was written by Organthis based on the published method in Sun et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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