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BRAIN Publication-derived

Leigh Syndrome Brain 2D Cell Cultures

Full SOP with Academic or a plan

Source Inak et al., 2021 · Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany · 10.1038/s41467-021-22117-z

Authors: Gizem Inak, Agnieszka Rybak-Wolf, Pawel Lisowski, Sebastian Diecke, Alessandro Prigione, Markus Schuelke 9 phases Cell source: Patient-Derived iPSC (Leigh syndrome, SURF1 mutations)

Abstract

This protocol describes the generation of patient-derived induced pluripotent stem cells (iPSCs) carrying SURF1 mutations and their differentiation into neural progenitor cells (NPCs) and mature neurons to model Leigh syndrome. The protocol includes two-dimensional (2D) neural cultures and three-dimensional (3D) cerebral organoids, with assessments of mitochondrial bioenergetics, neuronal morphogenesis, and therapeutic interventions including SURF1 gene augmentation and bezafibrate treatment.

Cell source
Patient-Derived iPSC (Leigh syndrome, SURF1 mutations)
Application
Disease modeling; Mechanistic study of mitochondrial neurodegeneration

Protocol overview

33 steps across 9 phases

Phase 1 · iPSC Generation and Genomic Correction Days 0-60
  1. a Skin fibroblast collection and reprogramming
  2. b iPSC culture and characterization
  3. c CRISPR/eCas9-mediated genome editing for mutation correction
  4. d Generation of isogenic control lines
Phase 2 · Neural Progenitor Cell (NPC) Differentiation Days 0-20
  1. a Formation of embryoid bodies
  2. b Neural induction (Days 2-6)
  3. c Plating on Matrigel and NPC expansion
Phase 3 · Differentiation to Mature Neurons (2D Culture) Days 9-56 (4 weeks to 8 weeks)
  1. a Initiation of neuronal differentiation
  2. b Medium transition (Week 2)
  3. c Final neuronal maturation medium (Day 9 onwards)
Phase 4 · Generation of 3D Cerebral Organoids Days 0-90
  1. a Embryoid body formation (Days 0-6)
  2. b Neural induction and maturation (Days 8-15)
  3. c Organoid maturation on orbital shaker (Days 15-90)
  4. d Tissue preparation for analysis
Phase 5 · Mitochondrial Bioenergetic Analysis Day of measurement
  1. a Oxygen consumption rate (OCR) measurement
  2. b Extracellular acidification rate (ECAR) measurement
  3. c Lactate measurement
Phase 6 · Neuronal Morphogenesis Assessment Day of measurement
  1. a Immunostaining for neuronal markers
  2. b High-content analysis (HCA) for neurite morphology
  3. c Electrophysiological recording (optional)
Phase 7 · Therapeutic Intervention: SURF1 Gene Augmentation Day of transduction + 3-7 days
  1. a Lentiviral vector preparation
  2. b Lentiviral transduction of NPCs or DNs
  3. c Adeno-associated virus (AAV) vector production and transduction
  4. d Assessment of gene augmentation efficacy
Phase 8 · Therapeutic Intervention: Bezafibrate Treatment and PGC1A Induction Overnight treatment or 24-72 hours
  1. a Bezafibrate treatment protocol
  2. b Assessment of mitochondrial biogenesis
  3. c Assessment of proliferation and metabolic reprogramming
  4. d PGC1A overexpression via lentiviral transduction
Phase 9 · Molecular Analysis: Transcriptomics and Metabolomics Day of sample collection
  1. a Bulk RNA-sequencing sample preparation
  2. b Single-cell RNA-sequencing (Drop-seq)
  3. c Proteomics analysis
  4. d Targeted metabolomics analysis
  5. e Multi-omics integration

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Attribution

This SOP was written by Organthis based on the published method in Inak et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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