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BRAIN Publication-derived

HIV-1 Infection of Human iPSC-Derived Microglia and Cerebral Organoids

Source Boreland et al., 2024 · Rutgers Robert Wood Johnson Medical School, Child Health Institute of New Jersey · 10.1016/j.isci.2024.109628

👤 Andrew J. Boreland, Alessandro C. Stillitano, Hsin-Ching Lin, Yara Abbo, Ronald P. Hart, Peng Jiang, Zhiping P. Pang, Arnold B. Rabson ⏱ 144 days 📋 11 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human iPSC-derived microglia from three iPSC lines and their infection with replication-competent, macrophage-tropic HIV-1 strains (JRFL and YU2). The infected microglia are integrated into sliced neocortical organoids to model HIV-associated neurocognitive disorder (HAND) and study persistent type I interferon signaling in a human neural context.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

61 steps across 11 phases

Human iPSC Maintenance and Embryoid Body Formation Days 1-6
  1. 1 Culture iPSCs in maintenance medium
  2. 2 Prepare iPSCs for embryoid body formation
  3. 3 Prepare yolk sac induction medium
  4. 4 Culture embryoid bodies in yolk sac induction medium
  5. 5 Transfer yolk sac-embryoid bodies to factory medium
  6. 6 Harvest primitive macrophage precursors (PMPs)
PMP Maturation to Microglia Days 7-21
  1. 1 Transition PMPs to maturation medium
  2. 2 Change maturation medium regularly
  3. 3 Harvest mature microglia
HIV-1 Stock Preparation Days 1-10
  1. 1 Obtain HIV-1 JRFL and YU2 stocks
  2. 2 Propagate HIV-1 JRFL in PHA-stimulated PBMCs
  3. 3 Generate HIV-1 YU2 stocks by transfection
  4. 4 Further propagate YU2 stocks in PHA-PBMCs
  5. 5 Quantify viral p24 antigen
HIV-1 Infection of Microglia Days 1-30
  1. 1 Prepare mature microglia for infection
  2. 2 Incubate microglia with HIV-1
  3. 3 Expand infected culture
  4. 4 Maintain infected culture
  5. 5 Collect samples for analysis
Cerebral Organoid Generation and Slicing Days 1-144
  1. 1 Form uniform embryoid bodies
  2. 2 Initiate neural induction (Day 1)
  3. 3 Add small molecule inhibitors (Days 5-6)
  4. 4 Embed organoids in Matrigel (Day 7)
  5. 5 Transfer to low-adherence plates (Day 14)
  6. 6 Feed organoids with expansion medium (Days 16-30)
  7. 7 Mature organoids (Days 30-144)
  8. 8 Slice organoids using vibratome
  9. 9 Prepare bivalent cation ACSF
  10. 10 Embed and section organoids
  11. 11 Wash sliced organoids
Integration of Infected Microglia into Organoid Slices Days 1-14 (post-coculture)
  1. 1 Prepare HIV-1-infected microglia for organoid integration
  2. 2 Add infected or control microglia to organoid slices
  3. 3 Maintain microglia-containing organoid (mSNO) cultures
  4. 4 Sample co-cultures at day 14 post-coculture
RNA Extraction and RT-qPCR Analysis Post-infection timepoints (Days 9, 16, 23, 30)
  1. 1 Extract total RNA from infected microglia
  2. 2 Generate cDNA
  3. 3 Perform RT-qPCR
  4. 4 Analyze qPCR results
Immunofluorescence and Confocal Microscopy Post-fixation analysis
  1. 1 Fix cells for immunocytochemistry (monolayer cultures)
  2. 2 Fix and prepare organoid sections
  3. 3 Cryosection organoids
  4. 4 Permeabilize fixed cells (monolayer)
  5. 5 Permeabilize organoid sections
  6. 6 Block nonspecific binding (monolayer)
  7. 7 Block nonspecific binding (organoid sections)
  8. 8 Apply primary antibodies
  9. 9 Wash and apply secondary antibodies
  10. 10 Final washing and mounting
  11. 11 Image by confocal microscopy
p24 ELISA Assay Post-infection analysis (Days 1-30)
  1. 1 Collect culture supernatant for p24 detection
  2. 2 Perform HIV-1 Gag p24 DuoSet ELISA
  3. 3 Quantify p24 concentration
HIV-1 2-LTR Circles Detection by PCR Post-infection (typically Day 4)
  1. 1 Extract total DNA from infected cells
  2. 2 Perform endpoint PCR for 2-LTR circles
  3. 3 Analyze PCR products
Bulk RNA Sequencing Post-infection Day 16
  1. 1 Extract and prepare total RNA for sequencing
  2. 2 Library construction
  3. 3 Perform sequencing
  4. 4 Generate FastQ files and quality control
  5. 5 Align reads to reference genome
  6. 6 Extract transcript counts

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Boreland et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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