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BRAIN Publication-derived

Generation of Human Spinal Cord Organoids (hSCOs) Recapitulating Neural Tube Morphogenesis for Drug Screening

Source Lee et al. · Department of Anatomy, Brain Korea 21 Plus Program for Biomedical Science, Korea University College of Medicine, Seoul, Republic of Korea · 10.1101/2020.12.02.409177;

👤 Ju-Hyun Lee, Hyogeun Shin, Mohammed R. Shaker, Hyun Jung Kim, June Hoan Kim, Namwon Lee, Minjin Kang, Subin Cho, Tae Hwan Kwak, Jong Woon Kim, Mi-Ryong Song, Seung-Hae Kwon, Dong Wook Han, Sanghyuk Lee, Se-Young Choi, Im Joo Rhyu, Hyun Kim, Dongho Geum, Il-Joo Cho, Woong Sun ⏱ 140 days 📋 5 phases 🧫 Human ESC (H9-GFP) and Human iPSC (#5-1, #56-2, AICS-0023)

Abstract

This protocol describes the generation of three-dimensional human spinal cord organoids (hSCOs) from pluripotent stem cells that recapitulate neurulation-like morphogenesis. The hSCOs exhibit neural tube formation, differentiation of major spinal cord neuron and glial cell types, and mature synaptic functional networks suitable for quantitative drug screening to assess neural tube defect risk from antiepileptic drugs.

Cell source
Human ESC (H9-GFP) and Human iPSC (#5-1, #56-2, AICS-0023)
Application
Disease modeling, Drug screening, Toxicology testing for neural tube defects

Protocol overview

17 steps across 5 phases

Neural Stem Cell Induction (2D culture) Days 0–3
  1. 1 Prepare hPSC monolayer culture
  2. 2 Replace medium with differentiation medium
  3. 3 Treat with CHIR99021 and SB431542
Neuroepithelial Expansion and 3D Sphere Formation Days 3–7
  1. 1 Detach cNSC colonies
  2. 2 Transfer to uncoated dishes
  3. 3 Culture spheres and establish neuroepithelial layer
Neural Plate Morphogenesis and Neural Tube Formation Days 7–15
  1. 1 Switch to retinoic acid-containing medium without bFGF
  2. 2 Observe neural folding morphogenesis
  3. 3 Complete neural tube closure
Long-term Maturation and Functional Development Days 15–140
  1. 1 Switch to maturation medium
  2. 2 Monitor glial cell emergence
  3. 3 Assess neuronal maturation and connectivity
Optimized 96-Well Plate Protocol for High-Throughput Drug Screening Days 3–15
  1. 1 Prepare dissociated cells
  2. 2 Seed cells into 96-well low-attachment plate
  3. 3 Feed organoids daily for 4 days
  4. 4 Switch to RA-containing medium and apply drug treatments
  5. 5 Image and analyze neural tube morphogenesis

Full SOP

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Attribution

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

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