Skip to content
← Back to browse
BRAIN Publication-derived

KCNJ2 inhibition mitigates mechanical injury in human brain organoid model of traumatic brain injury

Source Lai et al., 2024 · Keck School of Medicine, University of Southern California · 10.1016/j.stem.2024.03.004

👤 Jesse D. Lai, Joshua E. Berlind, Gabriella Fricklas, Cecilia Lie, Jean-Paul Urenda, Kelsey Lam, Naomi Sta Maria, Russell Jacobs, Violeta Yu, Zhen Zhao, Justin K. Ichida ⏱ 62 days 📋 9 phases 🧫 Human iPSC, Patient-derived iPSC (C9ORF72 ALS/FTD)

Abstract

This protocol describes the generation of human iPSC-derived cortical organoids and their mechanical injury via high-intensity focused ultrasound (HIFU) to model traumatic brain injury (TBI) pathophysiology. The model recapitulates key TBI hallmarks including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. Through genome-wide CRISPRi screening, KCNJ2 inhibition is identified as protective against injury-induced neuronal death in both healthy and C9ORF72 ALS/FTD organoids.

Cell source
Human iPSC, Patient-derived iPSC (C9ORF72 ALS/FTD)
Application
Disease modeling, traumatic brain injury and ALS pathology in organoids

Protocol overview

37 steps across 9 phases

iPSC maintenance and cortical organoid generation Day 0-45
  1. 1 Prepare iPSC spheroids
  2. 2 Neural induction (Days 1-5)
  3. 3 Neural expansion phase (Days 6-25)
  4. 4 Transition to bioreactor and maturation (Days 20-43)
  5. 5 Prepare organoids for injury experiments
Lentiviral transduction for cell tracking Day 57-62 (5 days prior to injury)
  1. 6 Prepare lentiviral vector (SYN1::EGFP)
  2. 7 Infect organoids with SYN1::EGFP lentivirus
  3. 8 Verify GFP expression and prepare for immobilization
Organoid immobilization and mechanical injury via HIFU Day 62 (day 0 of injury protocol)
  1. 9 Immobilize organoid in Matrigel
  2. 10 Calibrate HIFU apparatus
  3. 11 Deliver mechanical injury via HIFU at 0.6 MPa
  4. 12 Transfer injured organoid to culture dishes
Live imaging of neuronal survival Day 0-7 post-injury (7 days post-injury; dpi)
  1. 13 Longitudinal imaging of GFP+ neurons
  2. 14 Track single-neuron survival from day 0 to day 7
  3. 15 End-point fixation at day 7 post-injury
Immunofluorescence analysis of TDP-43 and neuronal markers Day 7 post-injury (tissue processing and staining)
  1. 16 Tissue rehydration and permeabilization
  2. 17 Primary antibody staining for TDP-43 and neuronal markers
  3. 18 Secondary antibody labeling and DAPI counterstaining
  4. 19 Confocal imaging of TDP-43 localization
Western blot analysis of neuronal injury markers Day 7 post-injury (protein preparation and blotting)
  1. 20 Organoid lysis and protein extraction
  2. 21 Protein quantification
  3. 22 SDS-PAGE gel preparation and protein separation
  4. 23 Protein transfer and membrane blocking
  5. 24 Primary and secondary antibody incubation
  6. 25 Membrane imaging and quantification
Tau phosphorylation biomarker analysis (supernatant) Day 0-7 post-injury (sample collection and ELISA)
  1. 26 Collect organoid supernatant
  2. 27 Measure phospho-tau (Thr231) and total tau by ELISA
  3. 28 Statistical analysis of tau biomarkers
Optional: Chemical inhibition of KCNJ2 (ML133 treatment) 2 h prior to injury through day 7 post-injury
  1. 29 Prepare ML133 solution
  2. 30 Pre-treat organoids with ML133
  3. 31 Perform HIFU injury (as in Phase 3)
  4. 32 Analyze neuroprotective effect of ML133
Optional: CRISPRi-based genetic screening for mechanosensitive genes NGN2-spheroid generation through day 7 post-injury
  1. 33 Generate NGN2-inducible spheroids
  2. 34 Transduce NGN2-spheroids with CRISPRi library
  3. 35 Divide spheroids into HIFU-injured and sham control groups
  4. 36 Culture injured and sham spheroids for 7 days; harvest for sequencing
  5. 37 Analyze sgRNA enrichment and identify protective hits

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 Lai et al., 2024. 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