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BRAIN Publication-derived

Huntington's Disease Brain Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Conforti et al., 2018 · Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Diseases, Department of Biosciences, University of Milan · 10.1073/pnas.1715865115

Authors: P. Conforti, D. Besusso, V. D. Bocchi, A. Faedo, E. Cesana, G. Rossetti, V. Ranzani, C. N. Svendsen, L. M. Thompson, M. Toselli, G. Biella, M. Pagani, E. Cattaneo Duration: 105 days 11 phases Cell source: Patient-Derived iPSC (Huntington's Disease)

Abstract

This protocol differentiates HD-derived and control human iPSCs to striatal and cortical neuronal populations using stepwise neural induction, allowing characterization of neurodevelopmental defects caused by mutant huntingtin. The method generates both 2D monolayer cultures and 3D cerebral organoids to model striatal specification, cortical identity, and neuronal maturation in HD.

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

Protocol overview

48 steps across 11 phases

Phase 1 · iPSC Culture Maintenance Ongoing
  1. a iPSC Culture on Matrigel
  2. b Cell Passage
Phase 2 · Striatal Differentiation DIV 0-50
  1. a Initial Plating with ROCK Inhibitor
  2. b Neural Induction with SMAD Inhibitors
  3. c Addition of Ventral Patterning Factors
  4. d Cell Dissociation and Replating
  5. e Terminal Differentiation to Striatal Neurons
Phase 3 · Cortical Differentiation DIV 0-21
  1. a Neural Induction for Cortical Fate
  2. b Progenitor Maturation
Phase 4 · 3D Cerebral Organoid Generation DIV 0-60+
  1. a Embryoid Body Formation
  2. b Spheroid Collection and Culture
  3. c Neuroectodermal Induction
  4. d Matrigel Embedding
  5. e Extended Neuronal Differentiation
Phase 5 · Immunocytochemistry Analysis At specific timepoints (DIV 0, 8, 15, 30, 50, 85)
  1. a Cell Fixation
  2. b Blocking
  3. c Primary Antibody Incubation
  4. d Secondary Antibody and Nuclear Staining
  5. e Image Acquisition
Phase 6 · Immunohistochemistry of Organoid Sections At specific timepoints (DIV 60, 85, 105)
  1. a Organoid Fixation
  2. b Sucrose Infiltration
  3. c OCT Embedding and Freezing
  4. d Cryosectioning
  5. e Blocking and Primary Antibody Incubation
  6. f Secondary Antibody and Mounting
Phase 7 · Electrophysiological Characterization DIV 30
  1. a Whole-Cell Patch-Clamp Recording Setup
  2. b Action Potential and Total Ionic Current Recording
  3. c Voltage-Gated Sodium Current Recording
  4. d Current Density Calculation
Phase 8 · Western Blot Analysis At specific timepoints (DIV 30, 50)
  1. a Cell Lysis
  2. b SDS-PAGE and Protein Transfer
  3. c Blocking and Primary Antibody Incubation
  4. d Secondary Antibody Detection
  5. e Normalization
Phase 9 · Real-Time qPCR Analysis At specific timepoints (DIV 0, 8, 15, 30)
  1. a RNA Extraction
  2. b cDNA Synthesis
  3. c Real-Time PCR
  4. d Data Analysis
Phase 10 · ZFP-Based muHTT Repression (Optional Rescue Experiment) DIV 0-30
  1. a ZFP Construct Cloning
  2. b Constitutive Expression Plasmid Generation
  3. c Lentiviral Transduction (Inducible System)
  4. d Doxycycline Induction
  5. e Continuous Constitutive Expression
  6. f Phenotypic Analysis
Phase 11 · ADAM10 Inhibitor Rescue (Optional Pharmacological Approach) DIV 5-30
  1. a GI254023X Treatment
  2. b N-Cadherin CTF Assessment
  3. c Rosette Morphology Assessment
  4. d Late-Stage Striatal Differentiation Analysis

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Attribution

This SOP was written by Organthis based on the published method in Conforti et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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