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BRAIN Publication-derived

OLIG2 Drives Abnormal Neurodevelopmental Phenotypes in Human iPSC-Based Organoid and Chimeric Mouse Models of Down Syndrome

Source Xu et al., 2019 · Rutgers University · 10.1016/j.stem.2019.04.014

👤 Ranjie Xu, Andrew T. Brawner, Shenglan Li, Jing-Jing Liu, Hyosung Kim, Haipeng Xue, Zhiping P. Pang, Woo-Yang Kim, Ronald P. Hart, Ying Liu, Peng Jiang ⏱ 180 days 📋 7 phases 🧫 Patient-Derived iPSC (Down syndrome)

Abstract

This protocol generates ventral forebrain organoids and neuronal chimeric mouse brains from Down syndrome and control human iPSCs to study OLIG2-driven overproduction of GABAergic interneurons. The 3D organoid model demonstrates increased OLIG2 expression in DS cells and excess CR+ and SST+ GABAergic neuron production compared to controls, which can be reversed by OLIG2 knockdown.

Cell source
Patient-Derived iPSC (Down syndrome)
Application
Disease modeling

Protocol overview

20 steps across 7 phases

Neural induction and pNPC generation Days 0-21
  1. 1 Initiate neural differentiation in embryoid bodies
  2. 2 Culture on Matrigel-coated dishes
  3. 3 Isolate and expand rosettes
Ventral forebrain organoid generation and patterning Days 21-35
  1. 4 Dissociate pNPCs and initiate organoid formation
  2. 5 Pattern organoids to ventral forebrain fate
  3. 6 Transition to orbital shaker culture
Neuronal differentiation Days 35-56
  1. 7 Initiate neuronal differentiation
  2. 8 Culture organoids to week 8
Cell transplantation into neonatal mice Day 0 (postnatal)
  1. 9 Prepare cells for transplantation
  2. 10 Anesthetize neonatal mice
  3. 11 Perform stereotaxic injection
Post-transplantation monitoring and analysis Days 1-180
  1. 12 Wean and house mice
  2. 13 Perform behavioral testing at 6 months
  3. 14 Prepare brain tissue for analysis
Molecular and cellular analysis of organoids Weeks 5-8
  1. 15 Perform RNA extraction and qPCR
  2. 16 Perform RNA sequencing
  3. 17 Perform chromatin immunoprecipitation (ChIP)
  4. 18 Perform immunostaining of organoid sections
OLIG2 knockdown experiments (optional) Days 0-35
  1. 19 Generate OLIG2 knockdown hiPSC lines
  2. 20 Verify OLIG2 knockdown

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Xu et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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