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BRAIN Publication-derived

Human iPSC Differentiation to Striatal and Cortical Neurons in Huntington's Disease Modeling

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

👤 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 ⏱ 105 days 📋 11 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Conforti et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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