Skip to content
← Back to browse
BRAIN Publication-derived

Human Cytomegalovirus Disruption of Calcium Signaling in Neural Progenitor Cells and Organoids

Source Sison et al., 2019 · Medical College of Wisconsin · 10.1128/jvi.00954-19

👤 Samantha L. Sison, Benjamin S. O'Brien, Amanda J. Johnson, Emily R. Seminary, Scott S. Terhune, Allison D. Ebert ⏱ 60 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol investigates HCMV infection of human iPSC-derived neural progenitor cells and cortical organoids to model congenital CMV-induced microcephaly. The protocol measures calcium signaling dysfunction in infected neurons and astrocytes using live-cell imaging and assesses organoid structure and neural differentiation markers under viral infection with and without antiviral treatment.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

22 steps across 8 phases

Viral Stock Preparation and Cell Culture Setup Day -30 to Day 0
  1. 1 Prepare HCMV TB40/E-eGFP Viral Stock
  2. 2 Culture MRC-5 Fibroblasts
  3. 3 Generate iPSC-Derived Neural Progenitor Cells (NPCs)
NPC and Fibroblast Infection with HCMV Day 1–28
  1. 1 Infect NPCs with HCMV
  2. 2 Infect MRC-5 Fibroblasts with HCMV
Live-Cell Calcium Imaging Day 2–14 (relative to infection)
  1. 1 Prepare Cells for Calcium Imaging
  2. 2 Baseline Calcium Measurement
  3. 3 ATP Stimulation for Purinergic Response
  4. 4 KCl Stimulation for Voltage-Gated Calcium Channel Response
  5. 5 Data Analysis and Quantification
Thapsigargin Treatment (ER Calcium Depletion Control) Day 3 (fibroblasts) or Day 14 (NPCs)
  1. 1 Treat Cells with Thapsigargin
Cortical Organoid Generation Day 0–60
  1. 1 Embryoid Body (EB) Formation
  2. 2 Neural Induction (Days 6–10)
  3. 3 Organoid Maturation in Matrigel (Days 11–14)
  4. 4 Organoid Maturation with Vitamin A (Days 15–60)
Cortical Organoid Infection Day 30 or Day 60
  1. 1 Organoid Infection with HCMV
  2. 2 Monitor Viral Infection Progress
Organoid Dissociation and Neural Cell Calcium Imaging Day 14 (postinfection)
  1. 1 Dissociate Organoids
  2. 2 Perform Calcium Imaging on Organoid-Derived Cells
RNA and Immunofluorescence Analysis of Organoids Day 14 (postinfection)
  1. 1 RNA Isolation and RT-PCR
  2. 2 Organoid Fixation and Cryosectioning
  3. 3 Immunofluorescence Staining

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Sison et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence