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BRAIN Publication-derived

Generation and Characterization of HD Striatal Organoids and Assessment of HSF1 Mitochondrial Localization and Function

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

👤 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 ⏱ 60 days 📋 8 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Liu et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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