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BRAIN Publication-derived

Construction of human 3D striato-nigral assembloids to recapitulate medium spiny neuronal projection defects in Huntington's disease

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

👤 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 ⏱ 80 days 📋 12 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wu et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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