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BRAIN Publication-derived

CYP3A5 Mediates Effects of Cocaine on Human Neocorticogenesis: 3D hPSC-Derived Neocortical Organoid Differentiation Protocol

Source Lee et al., 2016 · National Institute on Drug Abuse, National Institutes of Health · 10.1038/npp.2016.156

👤 Chun-Ting Lee, Jia Chen, Abigail A Kindberg, Raphael M Bendriem, Charles E Spivak, Melanie P Williams, Christopher T Richie, Annelie Handreck, Barbara S Mallon, Carl R Lupica, Da-Ting Lin, Brandon K Harvey, Deborah C Mash, William J Freed ⏱ 66 days 📋 5 phases 🧫 Human ESC (H1, H9, H14, CT2) and Human iPSC (NIH-i5, NIH-i7)

Abstract

This protocol describes the generation of self-organized 3D human neocortical organoids from pluripotent stem cells without extracellular scaffolding, designed to model neurodevelopmental effects of cocaine exposure. The organoids recapitulate key features of human neocortical development including neuroepithelial proliferation, neurogenesis, and neuronal migration, and are used to investigate CYP3A5-mediated mechanisms of cocaine toxicity during development.

Cell source
Human ESC (H1, H9, H14, CT2) and Human iPSC (NIH-i5, NIH-i7)
Application
Disease modeling and drug screening

Protocol overview

10 steps across 5 phases

Embryoid Body Formation and Neuroectodermal Induction Day 0-4
  1. 1 hPSC Colony Dissociation
  2. 2 Embryoid Body Formation
  3. 3 Dual SMAD and FGF Pathway Inhibition
Neuroepithelial Rosette Formation and Specification Day 4-16
  1. 1 Transfer to Neural Differentiation Medium
  2. 2 Adhesion Culture on Laminin-Coated Plates
  3. 3 Dorsal Cortical Rosette Isolation
Neocortical Organoid Priming Day 16-24
  1. 1 Transition to Suspension Culture with FGF Priming
Self-Organized Neocortical Organoid Development Day 24-66
  1. 1 Switch to Neuronal Differentiation Medium
  2. 2 Extended Suspension Culture for Organoid Maturation
Drug Treatment (Optional: For Cocaine Exposure Studies) Day 32-44
  1. 1 Cocaine Exposure

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lee et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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