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BRAIN Publication-derived

Huntington's Disease Striatal Assembloids

Full SOP with Academic or a plan

Source Wu et al., 2024 · Nanjing Medical University · 10.1073/pnas.2316176121

Authors: Shanshan Wu, Yuan Hong, Chu Chu, Yixia Gan, Xinrui Li, Mengdan Tao, Da Wang, Hao Hu, Zhilong Zheng, Qian Zhu, Xiao Han, Wanying Zhu, Min Xu, Yi Dong, Yan Liu, Xing Guo Duration: 80 days 12 phases Cell source: Human iPSC, Patient-Derived iPSC (Huntington's disease)

Abstract

This protocol describes the generation of human striatal and substantia nigra organoids from pluripotent stem cells, which are then fused to create 3D striato-nigral assembloids that recapitulate medium spiny neuron (MSN) projections and synaptic connectivity. The assembloids successfully model Huntington's disease-associated neuronal projection defects in patient-derived iPSCs, providing a human in vitro platform for studying striato-nigral circuit dysfunction and testing therapeutic compounds such as BDNF.

Cell source
Human iPSC, Patient-Derived iPSC (Huntington's disease)
Application
Disease modeling

Protocol overview

52 steps across 12 phases

Phase 1 · iPSC Reprogramming and Maintenance 0-14
  1. a iPSC Generation from Patient PBMCs
  2. b Clonal Selection and Early Expansion
  3. c Feeder-Free hPSC Maintenance
Phase 2 · Striatal Organoid Generation 0-60
  1. a Embryoid Body Formation
  2. b Neural Rosette Formation (Day 7-10)
  3. c Organoid Formation with Striatal Induction
  4. d Optional BDNF Treatment
Phase 3 · Midbrain Substantia Nigra Organoid Generation 0-35
  1. a Initial Neural Induction (Days 0-9)
  2. b Dopaminergic Progenitor Expansion (Days 9-13)
  3. c Dopaminergic Maturation Phase 1 (Days 13-20)
  4. d Final Maturation (Days 20-35)
Phase 4 · Striato-Nigral Assembloid Fusion and Culture 35-65
  1. a Assembloid Fusion Setup
  2. b Assembloid Transfer and Culture Initiation
  3. c Optional BDNF Treatment of Assembloids
  4. d Assembloid Maturation Culture
Phase 5 · Tissue Processing and Immunostaining variable
  1. a Organoid/Assembloid Fixation
  2. b Sucrose Dehydration
  3. c Cryosectioning
  4. d Immunostaining Preparation
  5. e Primary Antibody Incubation
  6. f Secondary Antibody and Counterstain
  7. g Image Acquisition
Phase 6 · In Situ Hybridization variable
  1. a Sample Preparation
  2. b Peroxide and Buffer Treatment
  3. c Dehydration and Protease Treatment
  4. d Probe Hybridization and Signal Detection
Phase 7 · Viral Transduction 35-40
  1. a Virus Preparation and Organoid Setup
  2. b Virus Injection
  3. c Post-Injection Culture
Phase 8 · Electrophysiology - Whole Cell Patch Clamp 45-60
  1. a Recording Solution Preparation and Setup
  2. b Electrode Preparation
  3. c Neuron Visualization and Patching
  4. d Voltage Clamp Recording
  5. e Current Clamp Recording (Action Potential)
  6. f Data Acquisition and Analysis
Phase 9 · Electrophysiology - Local Field Potential Recording 60-80
  1. a Microelectrode Preparation
  2. b Optogenetic Stimulation Setup
  3. c Optogenetic Stimulation Protocol
Phase 10 · Calcium Imaging 70-80
  1. a Sample Preparation for Imaging
  2. b Fluo-4 AM Loading
  3. c Dye Equilibration
  4. d Sample Washing and Solution Exchange
  5. e Optogenetic Stimulation and Calcium Imaging
  6. f Data Analysis
Phase 11 · Biotin Filling and Cell Identification 45-60
  1. a Biotin Injection During Patch Clamp
  2. b Pipette Retraction and Sealing
  3. c Tissue Fixation
  4. d Avidin Detection
  5. e Microscopy and Cell Identification
Phase 12 · DAB Staining variable
  1. a DAB Staining Protocol
  2. b DAB Color Development
  3. c Reaction Termination

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Attribution

This SOP was written by Organthis based on the published method in Wu et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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