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BRAIN Publication-derived

Glial cell diversity and methamphetamine-induced neuroinflammation in human cerebral organoids

Source Dang et al., 2021 · University of California San Diego · 10.1038/s41380-020-0676-x

👤 Jason Dang, Shashi Kant Tiwari, Tariq M. Rana ⏱ 11 days 📋 8 phases 🧫 Human ESC (H9)

Abstract

This protocol describes the generation of self-organizing human cerebral organoids from embryonic stem cells and their characterization using single-cell RNA sequencing to study methamphetamine-induced neuroinflammation and glial cell diversity. Organoids treated with METH display marked transcriptional changes in astrocytes and neural progenitor cells, including upregulation of neuroinflammatory, immune response, and apoptosis-related genes.

Cell source
Human ESC (H9)
Application
Disease modeling (prenatal methamphetamine exposure, neuroinflammation)

Protocol overview

42 steps across 8 phases

H9 hESC Culture and Embryoid Body Formation Days 0-6
  1. 1 Prepare H9 hESCs from feeder layer
  2. 2 Form embryoid bodies using hanging drop method
  3. 3 Transfer embryoid bodies to petri dishes
  4. 4 Transition to neural induction media
Cerebral Organoid Differentiation in Matrigel and Bioreactor Culture Days 11-METH treatment (variable)
  1. 5 Embed organoids in Matrigel droplets
  2. 6 Transfer organoids to stir flask bioreactors
  3. 7 Perform regular media changes during organoid maturation
METH Treatment of Cerebral Organoids Day treatment varies (10-month-old organoids treated for 1 week)
  1. 8 Prepare METH treatment solution
  2. 9 Administer METH to cerebral organoids
  3. 10 Collect supernatants for ELISA analysis
Organoid Dissociation and Single-Cell RNA Sequencing (scRNA-seq) End of treatment period
  1. 11 Dissociate organoids into single-cell suspension
  2. 12 Filter and assess cell viability
  3. 13 Load cells into 10x Chromium microfluidic system
  4. 14 Perform reverse transcription in GEMs
  5. 15 Recover and amplify cDNA
  6. 16 Construct sequencing libraries
  7. 17 Analyze library quality and quantify
  8. 18 Sequence libraries on HiSeq4000
Immunohistochemistry and Immunofluorescence Analysis End of treatment period or in parallel
  1. 19 Prepare organoids for immunostaining
  2. 20 Prepare organoid tissue sections
  3. 21 Block non-specific antibody binding
  4. 22 Incubate with primary antibodies
  5. 23 Wash away primary antibody
  6. 24 Incubate with secondary antibodies
  7. 25 Final washing and mounting
BrdU Incorporation and Proliferation Analysis During neural stem cell culture
  1. 26 Pulse cells with BrdU
  2. 27 Denature DNA for BrdU detection
  3. 28 Block and incubate with anti-BrdU antibody
  4. 29 Detect BrdU with fluorescent secondary antibody
Protein Analysis by Immunoblotting End of treatment period
  1. 30 Prepare organoid and astrocyte lysates
  2. 31 Quantify total protein
  3. 32 Separate proteins by SDS-PAGE
  4. 33 Block and incubate with primary antibodies
  5. 34 Wash and incubate with secondary antibody
  6. 35 Detect immunoreactive proteins
  7. 36 Analyze and quantify protein bands
ELISA for IL-6 Detection During and after METH treatment
  1. 37 Coat ELISA plate with capture antibody
  2. 38 Block and prepare wells
  3. 39 Add standards and samples
  4. 40 Wash and add detection antibody
  5. 41 Add Avidin-HRP and substrate
  6. 42 Read and analyze ELISA results

Full SOP

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Attribution

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

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