Skip to content
← Back to browse
BRAIN Publication-derived

Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation

Source Thomas et al., 2017 · University of California, San Diego · 10.1016/j.stem.2017.07.009

👤 Charles A. Thomas, Leon Tejwani, Cleber A. Trujillo, Priscilla D. Negraes, Roberto H. Herai, Pinar Mesci, Angela Macia, Yanick J. Crow, Alysson R. Muotri ⏱ 60 days 📋 17 phases 🧫 Human ESC and Patient-Derived iPSC (Aicardi-Goutières syndrome)

Abstract

This protocol generates TREX1-deficient human neural cells and cortical organoids to model Aicardi-Goutières syndrome (AGS), a neuroinflammatory disorder. The protocol demonstrates that TREX1 deficiency leads to accumulation of L1 retrotransposon-derived ssDNA in the cytosol, triggering neuronal toxicity and astrocyte-mediated type I interferon secretion. Reverse-transcriptase inhibitors (3TC and d4T) rescue the neurotoxic phenotype, suggesting therapeutic potential.

Cell source
Human ESC and Patient-Derived iPSC (Aicardi-Goutières syndrome)
Application
Disease modeling

Protocol overview

113 steps across 17 phases

CRISPR/Cas9-Mediated TREX1 Mutagenesis in H9 ESCs 1-21
  1. 1 Prepare single-cell suspension of H9 ESCs
  2. 2 Transfect cells with Cas9 and guide RNA
  3. 3 Recovery and eGFP selection
  4. 4 Post-FACS recovery and clonal expansion
  5. 5 Assess mutagenesis efficiency and validate mutations
  6. 6 Verify genomic stability by exome sequencing
Generation of iPSC Line from AGS Patient Fibroblasts 1-60
  1. 1 Obtain patient fibroblasts with TREX1 V201D mutation
  2. 2 Reprogram fibroblasts to iPSCs using episomal vectors
  3. 3 Isolate and expand iPSC clones
  4. 4 Characterize iPSC pluripotency and mutation status
Differentiation into Neural Precursor Cells (NPCs) 1-32
  1. 1 Prepare pluripotent stem cells for differentiation
  2. 2 Initiate neural induction with small molecules
  3. 3 Form and expand embryoid bodies
  4. 4 Plate embryoid bodies and induce rosette formation
  5. 5 Isolate and expand neuroectodermal rosettes
  6. 6 Dissociate rosettes into neural progenitor cells
  7. 7 Validate NPC identity and expand
Differentiation into Neurons and FACS Purification 1-35
  1. 1 Initiate neuronal differentiation from NPCs
  2. 2 Morphological assessment of neuronal differentiation
  3. 3 Dissociate differentiated neuronal culture
  4. 4 FACS sorting of purified neurons
  5. 5 Plate purified neurons
  6. 6 Validate neuronal identity
Differentiation into Astrocytes 1-35
  1. 1 Form neurospheres from NPCs
  2. 2 Switch to astrocyte-promoting medium
  3. 3 Plate astrocyte-containing neurospheres
  4. 4 Expand and passage astrocytes
  5. 5 Validate astrocytic identity
Treatment with Reverse-Transcriptase Inhibitors (RTi) and Control Drugs Throughout differentiation
  1. 1 Prepare RTi and control drug stocks
  2. 2 Chronic treatment of cell lines with RTi
  3. 3 Treatment with control drug (NVP)
  4. 4 Removal of RTi prior to downstream assays
Extrachromosomal DNA Extraction and Sequencing Variable
  1. 1 Prepare cells for DNA extraction
  2. 2 Lyse cells and precipitate chromosomal DNA
  3. 3 Precipitate and isolate extrachromosomal DNA
  4. 4 Treat extracted DNA with dsDNase for sequencing
  5. 5 Convert ssDNA to dsDNA for library preparation
  6. 6 Submit libraries for deep sequencing
  7. 7 Bioinformatic analysis of sequencing data
ssDNA Immunofluorescence and Quantification 1-3 (per batch)
  1. 1 Prepare cells for immunofluorescence
  2. 2 RNase treatment to eliminate RNA-signal contamination
  3. 3 Prepare controls for antibody validation
  4. 4 Block and stain cells with anti-ssDNA antibody
  5. 5 Incubate with secondary antibodies and DAPI
  6. 6 Acquire images with consistent microscope settings
  7. 7 Manually quantify ssDNA puncta
  8. 8 Statistical analysis and graphing
Cortical Organoid Generation and Culture 1-35
  1. 1 Dissociate PSC colonies into single cells
  2. 2 Initiate neural induction in suspension
  3. 3 Promote NPC proliferation
  4. 4 Initiate neuronal maturation
  5. 5 Measure organoid diameter
Apoptosis Assessment in Organoids (CC3 and Ki67 Staining) 1-2 (per analysis timepoint)
  1. 1 Harvest organoids for immunofluorescence
  2. 2 Fix organoids
  3. 3 Permeabilize and block organoids
  4. 4 Stain for Cleaved Caspase-3 (CC3) and/or Ki67
  5. 5 Secondary antibody incubation and mounting
  6. 6 Acquire Z-stack confocal images
  7. 7 Quantify CC3-positive and Ki67-positive cells
  8. 8 Statistical analysis
Astrocyte-Conditioned Media (ACM) Preparation and Neuronal Toxicity Assay 2-3 days (per ACM batch)
  1. 1 Prepare astrocytes for media conditioning
  2. 2 Remove astrocytes from regular growth medium
  3. 3 Condition media with astrocytes
  4. 4 Harvest astrocyte-conditioned media (ACM)
  5. 5 Prepare purified neurons for toxicity assay
  6. 6 Overlay ACM onto neurons
  7. 7 Assess neuronal toxicity after ACM treatment
  8. 8 Quantify neuronal apoptosis and statistical analysis
Quantitative PCR (qPCR) Analysis of Gene Expression 1-2 days (per batch)
  1. 1 Extract total RNA from cells
  2. 2 Reverse transcription to cDNA
  3. 3 Prepare qPCR master mix and samples
  4. 4 Perform qPCR thermal cycling
  5. 5 Analyze qPCR data and calculate relative expression
  6. 6 Report qPCR results
Quantitative PCR (qPCR) for Extrachromosomal L1 Copies 1-2 days (per batch)
  1. 1 Extract extrachromosomal DNA from NPCs
  2. 2 Prepare qPCR reactions for L1 quantification
  3. 3 Perform qPCR thermal cycling
  4. 4 Calculate L1 copy number
  5. 5 Analyze Alu and HERV as negative controls
  6. 6 Statistical analysis and reporting
Retrotransposition Assay Using L1 Reporter Constructs 1-10
  1. 1 Prepare NPCs for nucleofection
  2. 2 Nucleofect NPCs with L1 reporter construct
  3. 3 Plate nucleofected NPCs
  4. 4 Maintain culture and daily feeding
  5. 5 Optional: Treat subset of samples with histone deacetylase inhibitor (TSA)
  6. 6 Prepare cells for FACS analysis
  7. 7 Perform FACS analysis
  8. 8 Calculate adjusted retrotransposition rate
  9. 9 Statistical analysis
L1 shRNA Knockdown Validation 1-21
  1. 1 Prepare lentiviral vector expressing L1 shRNA
  2. 2 Transduce NPCs with L1 shRNA vector
  3. 3 Expand transduced NPCs
  4. 4 Validate L1 mRNA knockdown by qPCR
  5. 5 Assess ssDNA reduction in shLINE1 NPCs
  6. 6 Differentiate shLINE1 NPCs into neurons and assess toxicity
  7. 7 Statistical analysis and reporting
Western Blot Analysis of TREX1 and L1 Proteins 1-2 days (per batch)
  1. 1 Extract total protein from cells
  2. 2 Prepare SDS-PAGE gel and load samples
  3. 3 Perform electrophoresis
  4. 4 Transfer proteins to nitrocellulose membrane
  5. 5 Block membrane and incubate with primary antibodies
  6. 6 Wash and incubate with secondary antibodies
  7. 7 Image and quantify bands
  8. 8 Statistical analysis and reporting
Quality Control Checkpoints and Troubleshooting Throughout protocol
  1. 1 Verify pluripotency of ESC/iPSC lines
  2. 2 Verify CRISPR-induced mutations by Sanger sequencing
  3. 3 Verify absence of off-target CRISPR mutations
  4. 4 Validate neural cell identity at each differentiation stage
  5. 5 Assess TREX1 protein expression in astrocytes
  6. 6 Monitor mycoplasma contamination
  7. 7 Validate antibody specificity for immunofluorescence
  8. 8 Verify drug effects on L1 reverse transcriptase (not off-target effects)
  9. 9 Assess data quality and statistical power

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Thomas et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence