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BRAIN Publication-derived

Huntington's Disease Striatal Organoids

Full SOP with Academic or a plan

Source Liu et al., 2022 · Nanjing Medical University · 10.15252/emmm.202215851

Authors: Chunyue Liu, Zixing Fu, Shanshan Wu, Xiaosong Wang, Shengrong Zhang, Chu Chu, Yuan Hong, Wenbo Wu, Shengqi Chen, Yueqing Jiang, Yang Wu, Yongbo Song, Yan Liu, Xing Guo Duration: 60 days 8 phases Cell source: Human iPSC (Patient-Derived iPSC (Huntington's disease with 75 CAG repeats); Control iPSC H9 and IMR90-4)

Abstract

This protocol describes the generation of human striatal organoids from iPSCs to model Huntington's disease and investigate the role of heat shock factor 1 (HSF1) mitochondrial accumulation. The organoids express medium spiny neuron (MSN) and lateral ganglionic eminence (LGE) markers and recapitulate developmental gene expression patterns comparable to fetal striatum. The protocol enables assessment of HSF1 localization to mitochondria, mitochondrial dynamics, mtDNA content, and neuronal viability in an HD-relevant 3D tissue model.

Cell source
Human iPSC (Patient-Derived iPSC (Huntington's disease with 75 CAG repeats); Control iPSC H9 and IMR90-4)
Application
Disease modeling; Huntington's disease pathogenesis investigation

Protocol overview

26 steps across 8 phases

Phase 1 · Embryoid Body Formation and Neural Induction Day 0-7
  1. a iPSC Dissociation and EB Formation
  2. b EB Attachment to Culture Plates
  3. c Neural Rosette Formation
Phase 2 · Striatal Specification and Organoid Formation Day 7-25
  1. a Rosette Isolation and Organoid Formation
  2. b Striatal Induction with Sonic Hedgehog
  3. c Continue Brain Organoid Maturation
Phase 3 · Organoid Maturation and Characterization Day 25-60
  1. a Single-Cell Dissociation and Seeding on Substrate
  2. b Immunohistochemical Characterization
  3. c Single-Cell RNA Sequencing
  4. d Sectioning and Cryopreservation
Phase 4 · HSF1 Mitochondrial Localization Assessment Day 30-60
  1. a Immunofluorescence Staining for HSF1 and Mitochondrial Markers
  2. b Colocalization Analysis
  3. c Immunoelectron Microscopy (Optional)
Phase 5 · AAV-Mediated Gene Expression and mtHSF1 Studies Day 42-60 (organoids); Week 0-3 (mice)
  1. a Viral Preparation
  2. b Organoid Viral Injection
  3. c Post-Injection Culture and Analysis
Phase 6 · Biochemical Analysis of Mitochondrial Function Day 30-60 (ongoing)
  1. a mtDNA Content Quantification
  2. b Mitochondrial Morphology Analysis
  3. c Western Blot Analysis of Mitochondrial Proteins
  4. d Cell Death Assessment
Phase 7 · DH1 Peptide Treatment Day 42-60 (organoids); 3-7 days (cell culture)
  1. a DH1 Peptide Synthesis and Preparation
  2. b DH1 Treatment of HD Striatal Organoids
  3. c DH1 Treatment Assessment in Organoids
Phase 8 · Mitochondrial Isolation and Protein Interaction Studies (Supporting Analyses) Concurrent with other phases
  1. a Mitochondria-Enriched Fraction Isolation
  2. b Western Blot of Mitochondrial HSF1
  3. c Coimmunoprecipitation of HSF1 and Drp1

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Attribution

This SOP was written by Organthis based on the published method in Liu et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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