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BRAIN Publication-derived

Human iPSC-Derived Cerebral Organoids to Model Leigh Syndrome and Corticogenesis Defects

Source Romero-Morales et al. · Vanderbilt University · 10.1101/2020.04.21.054361;

👤 Alejandra I. Romero-Morales, Anuj Rastogi, Hoor Temuri, Megan L. Rasmussen, Gregory Scott McElroy, Lawrence Hsu, Paula M. Almonacid, Bryan A. Millis, Navdeep S. Chandel, Jean Philippe Cartailler, Vivian Gama ⏱ 365 days 📋 12 phases 🧫 Patient-Derived iPSC (Leigh Syndrome)

Abstract

This protocol describes the generation of human induced pluripotent stem cells (iPSCs) from Leigh syndrome patient fibroblasts and their differentiation into three-dimensional cerebral organoids to model disease phenotypes and corticogenesis defects. The protocol enables comprehensive characterization of neural development abnormalities, mitochondrial dysfunction, and metabolic dysregulation in Leigh syndrome using a patient-derived in vitro platform.

Cell source
Patient-Derived iPSC (Leigh Syndrome)
Application
Disease modeling

Protocol overview

78 steps across 12 phases

Whole Exome and Mitochondrial Sequencing Days 0-1
  1. 1 Harvest Fibroblast Cell Pellets
  2. 2 Perform Whole Exome Sequencing (WES)
  3. 3 Perform Mitochondrial DNA Sequencing
iPSC Generation and Characterization Days 0-30
  1. 1 Obtain Human Fibroblasts
  2. 2 Maintain Fibroblast Cultures
  3. 3 Generate Induced Pluripotent Stem Cells (iPSCs)
  4. 4 Maintain iPSC Cultures
  5. 5 Assess Pluripotency Using PluriTest
  6. 6 Confirm Pluripotency Markers by qPCR
  7. 7 Assess Chromosomal Integrity Using KaryoStat
Trilineage Differentiation and Neural Progenitor Cell Generation Days 31-80
  1. 1 Perform Trilineage Differentiation
  2. 2 Assess Ectoderm Differentiation
  3. 3 Assess Endoderm Differentiation
  4. 4 Assess Mesoderm Differentiation
  5. 5 Generate Neural Progenitor Cells (NPCs) via Dual SMAD Inhibition
  6. 6 Assess NPC Marker Expression
  7. 7 Assess NPC Multipotency
  8. 8 Assess Mitochondrial Network in NPCs
Cell Viability Assessment Under Stress Conditions Days 81-90
  1. 1 Treat NPCs with DNA Damaging Agents
  2. 2 Treat NPCs with Microtubule Depolymerizing Agent
  3. 3 Treat NPCs with Mitochondrial Uncoupler
  4. 4 Perform CellTiter Blue Viability Assay
Neuralized Embryoid Body Generation and Metabolomics Days 91-110
  1. 1 Generate Embryoid Bodies (EBs)
  2. 2 Harvest and Plate EBs
  3. 3 Measure EB Diameter at Day 5
  4. 4 Perform Daily Media Changes
  5. 5 Harvest EBs for Metabolomics at Day 10
  6. 6 Extract Metabolites from EBs
  7. 7 Perform LC-MS-Based Metabolomics Analysis
Neural Rosette Differentiation and Characterization Days 111-135
  1. 1 Generate Embryoid Bodies for Neural Rosettes
  2. 2 Harvest and Plate EBs for Neural Rosette Formation
  3. 3 Maintain Neural Rosette Differentiation
  4. 4 Fix Neural Rosettes
  5. 5 Perform Immunofluorescence Staining for Neural Rosettes
  6. 6 Acquire Confocal Images of Neural Rosettes
  7. 7 Quantify Neural Rosette Numbers and Lumen Area
  8. 8 Statistical Analysis of Neural Rosettes
Cerebral Organoid Generation and Culture Days 136-240
  1. 1 Dissociate iPSCs for Organoid Generation
  2. 2 Generate Homogeneous Embryoid Bodies
  3. 3 Embed EBs in Matrigel (Day 7)
  4. 4 Release Organoids from Matrigel (Day 10)
  5. 5 Culture Organoids on Shaker Platform
  6. 6 Perform Media Changes
  7. 7 Document Organoid Growth
  8. 8 Assess Organoid Formation Efficiency (Day 10)
Organoid Tissue Processing and Immunohistochemistry (Day 30) Days 236-250
  1. 1 Harvest and Fix Organoids at Day 30
  2. 2 Wash Organoids
  3. 3 Incubate in Sucrose Solution
  4. 4 Embed Organoids in Gelatin/Sucrose
  5. 5 Section Organoids with Cryostat
  6. 6 Prepare Slides for Immunostaining
  7. 7 Block Tissue
  8. 8 Incubate with Primary Antibodies (Day 30 Staining)
  9. 9 Wash and Incubate with Secondary Antibodies
  10. 10 Mount Slides and Acquire Images
  11. 11 Assess Day 30 Organoid Architecture
Mitochondrial Morphology Analysis (Day 30) Days 236-250
  1. 1 Perform Immunostaining for Mitochondrial Markers
  2. 2 Acquire Super-Resolution Images of Mitochondria
  3. 3 Analyze Mitochondrial Morphology
Organoid Tissue Processing and Immunohistochemistry (Day 100) Days 236-350
  1. 1 Harvest and Fix Organoids at Day 100
  2. 2 Section and Prepare Organoid Slides
  3. 3 Incubate with Upper Cortical Layer Markers
  4. 4 Incubate with Astrocyte Marker
  5. 5 Incubate with Neural Progenitor Marker
  6. 6 Wash and Incubate with Secondary Antibodies
  7. 7 Mount and Image Day 100 Organoids
  8. 8 Quantify Upper Layer Marker Expression
  9. 9 Quantify CTIP2+ Deep Layer Cells
  10. 10 Quantify S100β+ Astrocytes
Day 40 Organoid Metabolomics Analysis Days 226-260
  1. 1 Harvest Day 40 Organoids for Metabolomics
  2. 2 Extract Metabolites from Organoids
  3. 3 Perform LC-MS-Based Metabolomics
  4. 4 Identify Altered Metabolites
Data Analysis and Interpretation Days 351-365
  1. 1 Perform Statistical Analysis of Organoid Growth
  2. 2 Compare Immunohistochemical Staining Results
  3. 3 Integrate Metabolomic and Phenotypic Data
  4. 4 Document Quality Control (QC) Results
  5. 5 Generate Final Report

Full SOP

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Attribution

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

This wording is awaiting legal review.

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