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BRAIN Publication-derived

HESC-Derived Basal Ganglia / Medial Ganglionic Eminence Organoids

Source Xiang et al. · Yale University / Memorial Sloan Kettering, New York, USA · 10.1016/j.stem.2017.07.014

👤 Xiang Y, Tanaka Y, Patterson B, Kang YJ, Govindaiah G, Roselaar N, Cakir B, Kim KY, Bhatt AP, Bhatt SM, Park IH ⏱ 52 days 📋 9 phases 🧫 Human ESC / Human iPSC

Abstract

Xiang et al. generated medial ganglionic eminence (MGE)-like organoids from human pluripotent stem cells that produce GABAergic interneuron subtypes including parvalbumin+ and somatostatin+ neurons. When fused with cortical organoids, MGE-derived interneurons exhibited saltatory migration, establishing a model for studying interneuron migration and inhibitory circuit assembly.

Cell source
Human ESC / Human iPSC
Application
Interneuron Migration Studies

Protocol overview

52 steps across 9 phases

DNA Construct Preparation Day 1-5
  1. 1 Viral RNA Extraction and cDNA Synthesis
  2. 2 RT-PCR Amplification of Viral ORFs
  3. 3 Gateway Entry Cloning
  4. 4 Yeast Expression Vector Construction
  5. 5 Mammalian Expression Vector Construction
Recombinant Protein Expression and Purification Day 1-4
  1. 1 Yeast Culture Inoculation (Day 1)
  2. 2 Primary Seed Culture Preparation (Day 2)
  3. 3 Protein Expression Induction (Day 3)
  4. 4 Yeast Cell Harvest and Storage
  5. 5 Yeast Cell Lysis and Glutathione Affinity Purification
  6. 6 Washing and Elution
  7. 7 Quality Control of Purified Proteins
Protein Microarray Binding Assay Day 1-2
  1. 1 NS2A Protein Labeling
  2. 2 HuProt Array Blocking
  3. 3 NS2A Protein Incubation on Array
  4. 4 Array Washing and Drying
  5. 5 Array Scanning and Data Acquisition
  6. 6 Data Analysis and Hit Identification
In Utero Electroporation and Neural Progenitor Analysis E14.5 (electroporation) to E17.5-E19.5 (analysis)
  1. 1 Embryo Preparation and Anesthesia
  2. 2 Plasmid DNA Injection into Lateral Ventricles
  3. 3 Electroporation
  4. 4 Embryo Recovery and Wound Closure
  5. 5 EdU Pulse Labeling (for E17.5 analysis)
  6. 6 Brain Tissue Collection and Preparation
  7. 7 Immunohistology and Analysis
Co-Immunoprecipitation and Biochemical Analysis Day 1-2
  1. 1 Cell Transfection
  2. 2 Cell Lysis and Immunoprecipitation
  3. 3 Bead Washing and Elution
  4. 4 Western Blot Analysis
  5. 5 Protein Level Quantification
Mouse Neural Progenitor Culture and Viral Protein Expression Day 1-4
  1. 1 Neural Progenitor Isolation and Culture Setup
  2. 2 Lentiviral Transduction
  3. 3 Cell Harvest for Western Blot Analysis
  4. 4 Co-IP Analysis in Neural Progenitors
ZIKV Direct Infection of Embryonic Mouse Brain E13.5/E14.5 (injection) to E18.5/P3 (analysis)
  1. 1 Embryo Preparation for Viral Injection
  2. 2 ZIKV-SZ Injection into Lateral Ventricles
  3. 3 Embryo Recovery and Monitoring
  4. 4 Brain Tissue Collection at E18.5
  5. 5 Brain Tissue Collection at P3
  6. 6 Immunostaining and Analysis
Human iPSC Culture and Forebrain Organoid Generation Day 1-45
  1. 1 Human iPSC Culture Maintenance
  2. 2 iPSC Dissociation and Organoid Initiation (Day 1)
  3. 3 Neural Induction (Days 5-6)
  4. 4 Matrigel Embedding and Continued Induction (Days 7-13)
  5. 5 Bioreactor Transfer and Differentiation (Days 14-45)
Forebrain Organoid Electroporation and Analysis Day 45-52
  1. 1 Day 45 Organoid Preparation for Electroporation
  2. 2 DNA Injection into Organoid Ventricles
  3. 3 Electroporation Delivery
  4. 4 Post-Electroporation Recovery
  5. 5 EdU Pulse Labeling (Day 48 or 52)
  6. 6 Organoid Fixation and Immunostaining
  7. 7 Quantitative Analysis

Full SOP

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Attribution

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

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