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BRAIN Publication-derived

HIV-Associated Neurocognitive Disorder Cerebral Organoids

Full SOP with Academic or a plan

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

Authors: Andrew J. Boreland, Alessandro C. Stillitano, Hsin-Ching Lin, Yara Abbo, Ronald P. Hart, Peng Jiang, Zhiping P. Pang, Arnold B. Rabson Duration: 144 days 11 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Boreland et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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