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BRAIN Publication-derived

Striatal Assembloids for Developmental Studies

Full SOP with Academic or a plan

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

Authors: Xiang Y, Tanaka Y, Patterson B, Kang YJ, Govindaiah G, Roselaar N, Cakir B, Kim KY, Bhatt AP, Bhatt SM, Park IH Duration: 52 days 9 phases Cell source: 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

Phase 1 · DNA Construct Preparation Day 1-5
  1. a Viral RNA Extraction and cDNA Synthesis
  2. b RT-PCR Amplification of Viral ORFs
  3. c Gateway Entry Cloning
  4. d Yeast Expression Vector Construction
  5. e Mammalian Expression Vector Construction
Phase 2 · Recombinant Protein Expression and Purification Day 1-4
  1. a Yeast Culture Inoculation (Day 1)
  2. b Primary Seed Culture Preparation (Day 2)
  3. c Protein Expression Induction (Day 3)
  4. d Yeast Cell Harvest and Storage
  5. e Yeast Cell Lysis and Glutathione Affinity Purification
  6. f Washing and Elution
  7. g Quality Control of Purified Proteins
Phase 3 · Protein Microarray Binding Assay Day 1-2
  1. a NS2A Protein Labeling
  2. b HuProt Array Blocking
  3. c NS2A Protein Incubation on Array
  4. d Array Washing and Drying
  5. e Array Scanning and Data Acquisition
  6. f Data Analysis and Hit Identification
Phase 4 · In Utero Electroporation and Neural Progenitor Analysis E14.5 (electroporation) to E17.5-E19.5 (analysis)
  1. a Embryo Preparation and Anesthesia
  2. b Plasmid DNA Injection into Lateral Ventricles
  3. c Electroporation
  4. d Embryo Recovery and Wound Closure
  5. e EdU Pulse Labeling (for E17.5 analysis)
  6. f Brain Tissue Collection and Preparation
  7. g Immunohistology and Analysis
Phase 5 · Co-Immunoprecipitation and Biochemical Analysis Day 1-2
  1. a Cell Transfection
  2. b Cell Lysis and Immunoprecipitation
  3. c Bead Washing and Elution
  4. d Western Blot Analysis
  5. e Protein Level Quantification
Phase 6 · Mouse Neural Progenitor Culture and Viral Protein Expression Day 1-4
  1. a Neural Progenitor Isolation and Culture Setup
  2. b Lentiviral Transduction
  3. c Cell Harvest for Western Blot Analysis
  4. d Co-IP Analysis in Neural Progenitors
Phase 7 · ZIKV Direct Infection of Embryonic Mouse Brain E13.5/E14.5 (injection) to E18.5/P3 (analysis)
  1. a Embryo Preparation for Viral Injection
  2. b ZIKV-SZ Injection into Lateral Ventricles
  3. c Embryo Recovery and Monitoring
  4. d Brain Tissue Collection at E18.5
  5. e Brain Tissue Collection at P3
  6. f Immunostaining and Analysis
Phase 8 · Human iPSC Culture and Forebrain Organoid Generation Day 1-45
  1. a Human iPSC Culture Maintenance
  2. b iPSC Dissociation and Organoid Initiation (Day 1)
  3. c Neural Induction (Days 5-6)
  4. d Matrigel Embedding and Continued Induction (Days 7-13)
  5. e Bioreactor Transfer and Differentiation (Days 14-45)
Phase 9 · Forebrain Organoid Electroporation and Analysis Day 45-52
  1. a Day 45 Organoid Preparation for Electroporation
  2. b DNA Injection into Organoid Ventricles
  3. c Electroporation Delivery
  4. d Post-Electroporation Recovery
  5. e EdU Pulse Labeling (Day 48 or 52)
  6. f Organoid Fixation and Immunostaining
  7. g Quantitative Analysis

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Attribution

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

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