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BRAIN Publication-derived

Microglia-Containing Choroid Plexus Organoid (M-ChPO) Generation and HSV-1 Infection Protocol

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

👤 Haowen Qiao, Yuanpu Chiu, Xinyan Liang, Shangzhou Xia, Mariam Ayrapetyan, Siqi Liu, Cuiling He, Ruocen Song, Jianxiong Zeng, Xiangxue Deng, Weiming Yuan, Zhen Zhao ⏱ 90 days 📋 6 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Qiao et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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