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BRAIN Publication-derived

Herpes Simplex Virus Infection Choroid Plexus Organoids

Full SOP with Academic or a plan

Source Qiao et al., 2023 · Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California · 10.1002/jmv.28472

Authors: Haowen Qiao, Yuanpu Chiu, Xinyan Liang, Shangzhou Xia, Mariam Ayrapetyan, Siqi Liu, Cuiling He, Ruocen Song, Jianxiong Zeng, Xiangxue Deng, Weiming Yuan, Zhen Zhao Duration: 90 days 6 phases Cell source: Human ESC (H9) and Patient-Derived iPSC (mTagRFP-expressing AICS-0054-091)

Abstract

This protocol describes the generation of microglia-containing choroid plexus organoids (M-ChPO) from human H9 embryonic stem cells and induced pluripotent stem cells for modeling herpes simplex virus type 1 (HSV-1) infection. The protocol details the differentiation of hESCs into microglia and choroid plexus epithelial structures, their coculture integration, and subsequent HSV-1 infection to investigate microglia-mediated antiviral defense and blood-cerebrospinal fluid barrier function.

Cell source
Human ESC (H9) and Patient-Derived iPSC (mTagRFP-expressing AICS-0054-091)
Application
Disease modeling and viral infection studies

Protocol overview

26 steps across 6 phases

Phase 1 · H9 hESC Maintenance and Microglia Differentiation Phase 1 (Mesoderm and Hematopoiesis) Days 0-12
  1. a Maintain H9 hESCs in Pluripotent State
  2. b Initiate Microglia Differentiation with STEMdiff Hematopoietic Kit
  3. c Continue Hematopoiesis and HPC Emergence
  4. d Harvest and Characterize HPCs
Phase 2 · Microglia Maturation Phase (HPC to Functional Microglia) Days 12-46
  1. a Initiate Microglia Differentiation and Maturation
  2. b Monitor Microglia Precursor Generation
  3. c Complete Maturation to Functional Microglia
Phase 3 · Choroid Plexus Organoid (ChPO) Generation Days 0-60 (parallel to microglia differentiation)
  1. a Prepare Single Cell Suspension of H9 hESCs
  2. b Form Embryoid Bodies (EBs)
  3. c Apply Neural Induction Media
  4. d Embed EBs in Matrigel
  5. e Transfer to 6-Well Plate and Expand
Phase 4 · M-ChPO Generation via Microglia-Organoid Coculture Days 60-67 (after ChPO maturation)
  1. a Prepare Mature ChPO and Functional Microglia
  2. b Coculture Microglia with ChPO
  3. c Resume Shaking and Maintain Coculture
  4. d Confirm Microglia Integration
Phase 5 · HSV-1 Infection Protocol Day 87 onward (or as needed)
  1. a Pretreatment with Pathway Inhibitors (Optional)
  2. b Prepare HSV-1 Virus Inoculum
  3. c Inoculate Organoids with HSV-1
  4. d Replace Medium at 24 Hours Post-Infection
  5. e Collect Supernatant for Analysis
Phase 6 · Sample Preparation for Analysis Upon completion of infection (Day 72-90 hpi)
  1. a Fix Organoids for Immunostaining
  2. b Cryoprotect with Sucrose
  3. c Cryosection Organoids
  4. d Prepare for RNA Extraction
  5. e Wash and Lyse Organoids

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Attribution

This SOP was written by Organthis based on the published method in Qiao et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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