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BRAIN Publication-derived

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration

Source Xiang et al., 2017 · Yale Stem Cell Center, Yale School of Medicine · 10.1016/j.stem.2017.07.007

👤 Yangfei Xiang, Yoshiaki Tanaka, Benjamin Patterson, Young-Jin Kang, Gubbi Govindaiah, Naomi Roselaar, Bilal Cakir, Kun-Yong Kim, Adam P. Lombroso, Sung-Min Hwang, Mei Zhong, Edouard G. Stanley, Andrew G. Elefanty, Janice R. Naegele, Sang-Hun Lee, Sherman M. Weissman, In-Hyun Park ⏱ 105 days 📋 13 phases 🧫 Human ESC (HES-3 NKX2-1[GFP/w], H1), Human iPSC (1090)

Abstract

This protocol describes the generation of human medial ganglionic eminence (MGE)-like organoids (hMGEOs) and cortical-like organoids (hCOs) from human pluripotent stem cells that recapitulate MGE and cortical development respectively. By fusing hMGEOs and hCOs, a 3D model is established to investigate human interneuron migration and functional integration into cortical networks.

Cell source
Human ESC (HES-3 NKX2-1[GFP/w], H1), Human iPSC (1090)
Application
Disease modeling; Brain development study; Interneuron migration modeling

Protocol overview

86 steps across 13 phases

hPSC Preparation and Neural Induction Day 0–10
  1. 1 Prepare hPSC colonies for dissociation
  2. 2 Plate cells for neural induction
  3. 3 Perform neural induction with dual SMAD inhibition
  4. 4 Maintain static culture with appropriate supplements
hMGEO-Specific Patterning (Ventral Specification) Day 10–18
  1. 1 Transfer organoids to 6-well ultra-low-attachment plate
  2. 2 Initiate ventral patterning with SHH signaling
  3. 3 Culture on orbital shaker
hCO-Specific Patterning (Dorsal Specification) Day 10–18
  1. 1 Transfer organoids to 6-well ultra-low-attachment plate
  2. 2 Apply neural differentiation media without ventral patterning
  3. 3 Culture on orbital shaker
Maturation and Extended Culture (Both hMGEO and hCO) Day 18–105
  1. 1 Switch to neural differentiation media with vitamin A
  2. 2 Maintain spinning culture on orbital shaker
  3. 3 Monitor organoid development milestone markers
Fusion of hMGEO and hCO for Interneuron Migration Modeling Day 18 (organoid age)
  1. 1 Select and prepare organoids for fusion
  2. 2 Initiate spontaneous fusion in 96-well plate
  3. 3 Perform partial media change post-fusion
  4. 4 Transfer fused organoids to spinning culture
Characterization: RNA Extraction and qRT-PCR Analysis Days 21, 30, 72
  1. 1 Harvest organoids for RNA extraction
  2. 2 Extract total RNA using RNeasy Mini Kit
  3. 3 Synthesize cDNA
  4. 4 Perform qRT-PCR for gene expression quantification
Characterization: Immunohistochemistry and Cryosectioning Days 21–105 (variable timepoints)
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Wash fixed organoids
  3. 3 Cryoprotect in sucrose solution
  4. 4 Equilibrate in O.C.T. compound
  5. 5 Embed in O.C.T. and freeze
  6. 6 Cryosection organoid
  7. 7 Prepare cryosections for immunostaining
  8. 8 Block non-specific antibody binding
  9. 9 Incubate with primary antibody
  10. 10 Wash and incubate with secondary antibody
  11. 11 Stain nuclei and mount
  12. 12 Image using confocal microscopy
Functional Characterization: Calcium Imaging Days 40–50 (hMGEO), Days 45+ (hCO)
  1. 1 Transduce organoids with calcium indicator
  2. 2 Allow expression of calcium indicator
  3. 3 Set up live-cell imaging chamber
  4. 4 Acquire time-lapse calcium imaging
  5. 5 Assess neuronal activity by TTX blockade
  6. 6 Test GABAergic inhibition with bicuculline
  7. 7 Analyze calcium imaging data
Functional Characterization: Patch-Clamp Electrophysiology Days 40–60
  1. 1 Prepare organoid slices
  2. 2 Cut slices using vibratome
  3. 3 Incubate slices in sucrose solution
  4. 4 Transfer slice to recording chamber
  5. 5 Visualize cells and identify recording targets
  6. 6 Prepare patch pipettes and whole-cell recording setup
  7. 7 Approach cell and achieve whole-cell configuration
  8. 8 Perform current-clamp recording and action potential characterization
  9. 9 Block sodium channels with TTX to confirm neuronal APs
  10. 10 Label recorded cell with biocytin and recover
  11. 11 Visualize recorded cell morphology
Live Imaging of Interneuron Migration in hfMCOs Days 3–21 post-fusion
  1. 1 Prepare hfMCO for live imaging
  2. 2 Mount organoid in heated, controlled imaging chamber
  3. 3 Acquire 4D (x, y, z, t) time-lapse imaging
  4. 4 Identify and track migrating NKX2-1-GFP+ interneurons
  5. 5 Assess growth cone dynamics and soma translocation
  6. 6 Test myosin II inhibition with blebbistatin
  7. 7 Assess calcium activity of migrating interneurons
Characterization: Bulk RNA-Seq and Transcriptome Analysis Days 30, 72
  1. 1 Harvest organoids for RNA extraction
  2. 2 Extract total RNA
  3. 3 Prepare RNA-seq libraries
  4. 4 Perform high-throughput sequencing
  5. 5 Map reads and quantify gene expression
  6. 6 Identify differentially expressed genes
  7. 7 Perform gene ontology and pathway analysis
  8. 8 Compare organoid transcriptomes to in vivo tissues
Characterization: Single-Cell RNA-Seq (scRNA-Seq) Days 30, 72–79
  1. 1 Harvest and dissociate organoids for scRNA-seq
  2. 2 Prepare cells for 10x Chromium loading
  3. 3 Perform 10x Chromium single-cell capture
  4. 4 Construct cDNA libraries and prepare for sequencing
  5. 5 Perform high-throughput sequencing of scRNA-seq libraries
  6. 6 Map reads and generate gene expression matrix
  7. 7 Perform quality control and cell filtering
  8. 8 Normalize, scale, and perform dimensionality reduction
  9. 9 Cluster cells and identify cell types
  10. 10 Perform differential expression and enrichment analyses
  11. 11 Construct co-expression networks of regulatory factors
Characterization: ATAC-Seq (Chromatin Accessibility) Day 72
  1. 1 Harvest and dissociate organoids for ATAC-seq
  2. 2 Prepare nuclei for transposition reaction
  3. 3 Perform Tn5-mediated tagmentation
  4. 4 Recover tagmented DNA and construct libraries
  5. 5 Validate ATAC library quality
  6. 6 Perform high-throughput sequencing of ATAC-seq libraries
  7. 7 Map ATAC-seq reads and identify open chromatin regions
  8. 8 Identify differentially open chromatin regions
  9. 9 Annotate open chromatin regions to genes

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Xiang et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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