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BRAIN Publication-derived

A human three-dimensional neural-perivascular 'assembloid' promotes astrocytic development and enables modeling of SARS-CoV-2 neuropathology

Source Wang et al., 2021 · University of California San Diego · 10.1038/s41591-021-01443-1

👤 Lu Wang, David Sievert, Alex E. Clark, Sangmoon Lee, Hannah Federman, Benjamin D. Gastfriend, Eric V. Shusta, Sean P. Palecek, Aaron F. Carlin, Joseph G. Gleeson ⏱ 74 days 📋 6 phases 🧫 Human iPSC-derived neural crest stem cells (NCSCs), H1-hESC

Abstract

This protocol describes the generation of pericyte-containing cortical organoids (PCCOs), a three-dimensional neural-perivascular assembloid model. GFP-labeled pericyte-like cells (PLCs) derived from human pluripotent stem cell-derived neural crest stem cells are integrated into cortical brain organoids at 60 days in vitro. PCCOs recapitulate key features of the neurovascular unit, including astrocytic maturation, basement membrane formation, and neuronal differentiation. The model supports productive SARS-CoV-2 infection with viral replication primarily in PLCs and secondary infection of astrocytes, enabling study of viral neuropathology and type I interferon responses.

Cell source
Human iPSC-derived neural crest stem cells (NCSCs), H1-hESC
Application
Disease modeling, SARS-CoV-2 neuropathology

Protocol overview

31 steps across 6 phases

iPSC and NCSC Generation Day 0 to Day 15
  1. 1 Reprogram fibroblasts into iPSCs
  2. 2 Validate pluripotency by immunostaining
  3. 3 Generate embryoid bodies (EBs) for differentiation potential
  4. 4 Verify karyotype integrity
  5. 5 Derive NCSCs from hPSCs
  6. 6 Enrich NCSCs using magnetic activated cell sorting (MASC)
Pericyte-Like Cell (PLC) Generation and GFP Labeling Day 0 to Day 9 (PLC differentiation)
  1. 1 Prepare lentiviral particles
  2. 2 Differentiate NCSCs into PLCs
  3. 3 Infect PLCs with GFP lentivirus
  4. 4 Select GFP-positive cells
  5. 5 Maintain selected GFP+ PLCs
Cortical Organoid (CO) Generation Day 0 to Day 69
  1. 1 Prepare and dissociate hPSCs
  2. 2 Plate cells for aggregate formation (Phase 1: Days 0-4)
  3. 3 Continue initial differentiation (Days 4-17)
  4. 4 Transfer to 6-well plates and organoid differentiation phase (Days 17-34)
  5. 5 Maturation phase with Matrigel (Days 35-69)
  6. 6 Transition to long-term maintenance (Day 70 onwards)
PLC-Containing Cortical Organoid (PCCO) Assembly Day 60 to Day 74
  1. 1 Prepare GFP+ PLCs for integration
  2. 2 Integrate PLCs into cortical organoids
  3. 3 Culture PCCOs in Maturation medium (Days 60-73)
  4. 4 Transition to long-term maintenance (Day 74 onwards)
SARS-CoV-2 Infection (Optional) Variable (performed at desired timepoint)
  1. 1 Prepare SARS-CoV-2 inoculum
  2. 2 Infect COs or PCCOs with SARS-CoV-2
  3. 3 Refresh medium post-infection
  4. 4 Harvest organoids for analysis
Quality Control and Analysis Throughout culture and post-harvest
  1. 1 Verify hPSC pluripotency
  2. 2 Confirm NCSC purity by flow cytometry or immunostaining
  3. 3 Validate PLC marker expression
  4. 4 Assess organoid structure and composition
  5. 5 Confirm PLC integration into organoids
  6. 6 Verify viral infection (if applicable)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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