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BRAIN Publication-derived

Neurodevelopmental signatures of narcotic and neuropsychiatric risk factors in 3D human-derived forebrain organoids

Source Notaras et al., 2021 · Center for Neurogenetics, Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, Cornell University · 10.1038/s41380-021-01189-9

👤 Michael Notaras, Aiman Lodhi, Estibaliz Barrio-Alonso, Careen Foord, Tori Rodrick, Drew Jones, Haoyun Fang, David Greening, Dilek Colak ⏱ 48 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of 3D human-derived dorsal forebrain organoids from induced pluripotent stem cells and their systematic exposure to enviromimetic compounds modeling narcotic use and neuropsychiatric risk factors (opiates, cannabinoids, alcohol, nicotine, chronic stress, and maternal immune activation). The organoids undergo multi-omic analysis including TMT-LC/MS proteomics, metabolomics, flow cytometry, and neurogenesis assays to assess neurodevelopmental effects within human cortical tissue.

Cell source
Human iPSC
Application
Disease modeling; Neurotoxicity screening; Developmental pathology

Protocol overview

49 steps across 10 phases

iPSC Culture and Maintenance Ongoing (every 24-48 hours)
  1. 1 Expand iPSC colonies on vitronectin-coated plates
  2. 2 Passage iPSCs for maintenance or differentiation
Embryoid Body Formation Days 0–7
  1. 1 Dissociate iPSC colonies into single-cell suspension
  2. 2 Seed cells into Aggrewell V-Bottom Plates
  3. 3 Culture embryoid bodies in suspension
Cortical Differentiation Media (CDM) Cycles Days 7–30
  1. 1 Transfer embryoid bodies to ultra-low adhesion 6 cm dishes
  2. 2 Sequential CDM feeding: Progress through CDM1→CDM2→CDM3→CDM4
  3. 3 Quality control assessment at ~30 DIV
Enviromimetic Treatment Exposure Days 30–37 (7 days of treatment)
  1. 1 Prepare enviromimetic treatment compounds
  2. 2 Replace media with treatment compounds
  3. 3 Maintain chronic treatment for 7 Days In Vitro (7 DIV)
  4. 4 Harvest organoids at 37 DIV (end of 7 DIV treatment)
BrdU Pulse-Chase Neurogenesis Assay Days 30–37 (parallel to treatment)
  1. 1 Pulse with BrdU at treatment commencement (Day 30)
  2. 2 Chase with treatment media for 7 DIV
  3. 3 Harvest and fix organoids at 37 DIV
  4. 4 Cryosection and immunostain for BrdU and MAP2
  5. 5 Quantify newborn and total neurons
Flow Cytometry Assays Days 30–37 (harvest at 37 DIV)
  1. 1 Dissociate organoids to single-cell suspension
  2. 2 Prepare cells for apoptosis and DNA damage detection
  3. 3 Treat with DNAse to reveal DNA damage
  4. 4 Label with anti-cleaved PARP and anti-pH2AX antibodies
  5. 5 Acquire data on flow cytometer
  6. 6 Analyze flow cytometry data
Single-Cell DNA Content Analysis (Cell-Cycle Determination) Days 30–37 (harvest at 37 DIV)
  1. 1 Dissociate organoids to single-cell suspension
  2. 2 Fix cells in 100% ethanol
  3. 3 Rehydrate and treat with Triton-X and DNAse
  4. 4 Stain cells with propidium iodide (PI)
  5. 5 Acquire DNA content data on flow cytometer
  6. 6 Analyze cell-cycle distribution and DNA fragmentation
TMT-LC/MS Proteomics Days 30–37 (harvest at 37 DIV)
  1. 1 Prepare organoid lysates for mass spectrometry
  2. 2 Reduce, alkylate, and digest with trypsin
  3. 3 Desalt peptide mixtures using C18 stage-tips
  4. 4 Label peptides with TMT reagents
  5. 5 Desalt and prepare pooled samples for LC-MS
  6. 6 LC-MS/MS data acquisition
  7. 7 Process raw MS data using MaxQuant
  8. 8 Quantify proteins and perform statistical analysis
  9. 9 Conduct pathway enrichment analysis
Hybrid MS Metabolomics Days 30–37 (harvest at 37 DIV)
  1. 1 Prepare organoid samples for metabolomic analysis
  2. 2 Quantify protein for normalization
  3. 3 Targeted hybrid MS metabolomics analysis
  4. 4 Process and normalize metabolomics data
  5. 5 Perform statistical analysis of metabolite data
  6. 6 Biological interpretation of metabolome changes
Quality Control and Reproducibility Assessment Throughout all phases (ongoing)
  1. 1 Morphological assessment of organoids
  2. 2 Forebrain-specific marker validation
  3. 3 Proteomics reproducibility assessment
  4. 4 Statistical validation of findings
  5. 5 Cross-validation of key findings

Full SOP

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Attribution

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

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