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BRAIN Publication-derived

Down Syndrome Brain Organoids with Interneurons

Full SOP with Academic or a plan

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

Authors: 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 Duration: 180 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Xu et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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