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BRAIN Publication-derived

Leigh Syndrome Brain Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 365 days 12 phases Cell source: 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

Phase 1 · Whole Exome and Mitochondrial Sequencing Days 0-1
  1. a Harvest Fibroblast Cell Pellets
  2. b Perform Whole Exome Sequencing (WES)
  3. c Perform Mitochondrial DNA Sequencing
Phase 2 · iPSC Generation and Characterization Days 0-30
  1. a Obtain Human Fibroblasts
  2. b Maintain Fibroblast Cultures
  3. c Generate Induced Pluripotent Stem Cells (iPSCs)
  4. d Maintain iPSC Cultures
  5. e Assess Pluripotency Using PluriTest
  6. f Confirm Pluripotency Markers by qPCR
  7. g Assess Chromosomal Integrity Using KaryoStat
Phase 3 · Trilineage Differentiation and Neural Progenitor Cell Generation Days 31-80
  1. a Perform Trilineage Differentiation
  2. b Assess Ectoderm Differentiation
  3. c Assess Endoderm Differentiation
  4. d Assess Mesoderm Differentiation
  5. e Generate Neural Progenitor Cells (NPCs) via Dual SMAD Inhibition
  6. f Assess NPC Marker Expression
  7. g Assess NPC Multipotency
  8. h Assess Mitochondrial Network in NPCs
Phase 4 · Cell Viability Assessment Under Stress Conditions Days 81-90
  1. a Treat NPCs with DNA Damaging Agents
  2. b Treat NPCs with Microtubule Depolymerizing Agent
  3. c Treat NPCs with Mitochondrial Uncoupler
  4. d Perform CellTiter Blue Viability Assay
Phase 5 · Neuralized Embryoid Body Generation and Metabolomics Days 91-110
  1. a Generate Embryoid Bodies (EBs)
  2. b Harvest and Plate EBs
  3. c Measure EB Diameter at Day 5
  4. d Perform Daily Media Changes
  5. e Harvest EBs for Metabolomics at Day 10
  6. f Extract Metabolites from EBs
  7. g Perform LC-MS-Based Metabolomics Analysis
Phase 6 · Neural Rosette Differentiation and Characterization Days 111-135
  1. a Generate Embryoid Bodies for Neural Rosettes
  2. b Harvest and Plate EBs for Neural Rosette Formation
  3. c Maintain Neural Rosette Differentiation
  4. d Fix Neural Rosettes
  5. e Perform Immunofluorescence Staining for Neural Rosettes
  6. f Acquire Confocal Images of Neural Rosettes
  7. g Quantify Neural Rosette Numbers and Lumen Area
  8. h Statistical Analysis of Neural Rosettes
Phase 7 · Cerebral Organoid Generation and Culture Days 136-240
  1. a Dissociate iPSCs for Organoid Generation
  2. b Generate Homogeneous Embryoid Bodies
  3. c Embed EBs in Matrigel (Day 7)
  4. d Release Organoids from Matrigel (Day 10)
  5. e Culture Organoids on Shaker Platform
  6. f Perform Media Changes
  7. g Document Organoid Growth
  8. h Assess Organoid Formation Efficiency (Day 10)
Phase 8 · Organoid Tissue Processing and Immunohistochemistry (Day 30) Days 236-250
  1. a Harvest and Fix Organoids at Day 30
  2. b Wash Organoids
  3. c Incubate in Sucrose Solution
  4. d Embed Organoids in Gelatin/Sucrose
  5. e Section Organoids with Cryostat
  6. f Prepare Slides for Immunostaining
  7. g Block Tissue
  8. h Incubate with Primary Antibodies (Day 30 Staining)
  9. i Wash and Incubate with Secondary Antibodies
  10. j Mount Slides and Acquire Images
  11. k Assess Day 30 Organoid Architecture
Phase 9 · Mitochondrial Morphology Analysis (Day 30) Days 236-250
  1. a Perform Immunostaining for Mitochondrial Markers
  2. b Acquire Super-Resolution Images of Mitochondria
  3. c Analyze Mitochondrial Morphology
Phase 10 · Organoid Tissue Processing and Immunohistochemistry (Day 100) Days 236-350
  1. a Harvest and Fix Organoids at Day 100
  2. b Section and Prepare Organoid Slides
  3. c Incubate with Upper Cortical Layer Markers
  4. d Incubate with Astrocyte Marker
  5. e Incubate with Neural Progenitor Marker
  6. f Wash and Incubate with Secondary Antibodies
  7. g Mount and Image Day 100 Organoids
  8. h Quantify Upper Layer Marker Expression
  9. i Quantify CTIP2+ Deep Layer Cells
  10. j Quantify S100β+ Astrocytes
Phase 11 · Day 40 Organoid Metabolomics Analysis Days 226-260
  1. a Harvest Day 40 Organoids for Metabolomics
  2. b Extract Metabolites from Organoids
  3. c Perform LC-MS-Based Metabolomics
  4. d Identify Altered Metabolites
Phase 12 · Data Analysis and Interpretation Days 351-365
  1. a Perform Statistical Analysis of Organoid Growth
  2. b Compare Immunohistochemical Staining Results
  3. c Integrate Metabolomic and Phenotypic Data
  4. d Document Quality Control (QC) Results
  5. e Generate Final Report

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Attribution

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

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