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BRAIN Publication-derived

Lithium Treatment of iPSC-Derived Human Cortical Spheroids from Bipolar Disorder Patients

Source Osete et al., 2023 · NORMENT, Institute of Clinical Medicine, University of Oslo, and Division of Mental Health and Addiction, Oslo University Hospital · 10.1038/s41380-023-01944-0

👤 Jordi Requena Osete, Ibrahim A. Akkouh, Oleksandr Ievglevskyi, Matthieu Vandenberghe, Denis Reis de Assis, Thor Ueland, Elena Kondratskaya, Børge Holen, Attila Szabo, Timothy Hughes, Olav B. Smeland, Vidar Martin Steen, Ole A. Andreassen, Srdjan Djurovic ⏱ 180 days 📋 10 phases 🧫 Patient-Derived iPSC (Bipolar Disorder)

Abstract

This protocol describes the generation of iPSC-derived human cortical spheroids (hCS) from healthy controls and bipolar disorder patients, and their treatment with lithium (1 mM for 1 month) to investigate transcriptional and functional effects. The study examines neuronal excitability, gene expression, cytokine secretion, and mitochondrial function in 180-day-old hCS to elucidate lithium's mechanism of action in bipolar disorder.

Cell source
Patient-Derived iPSC (Bipolar Disorder)
Application
Disease modeling and drug screening for bipolar disorder

Protocol overview

33 steps across 10 phases

iPSC Derivation and Quality Control Passage 15-20
  1. 1 Fibroblast isolation and expansion
  2. 2 Reprogramming to induced pluripotent stem cells (iPSCs)
  3. 3 Pluripotency marker expression characterization
  4. 4 Karyotyping analysis
Cortical Spheroid Differentiation Day 0-180
  1. 1 Differentiation to cortical spheroids
  2. 2 Organoid size monitoring
Lithium Treatment Day 150-180
  1. 1 Lithium solution preparation
  2. 2 Apply lithium treatment to spheroids
  3. 3 Medium and drug refreshment
RNA Extraction and RT-PCR Analysis Day 180
  1. 1 Cell lysis and total RNA extraction
  2. 2 Reverse transcription and RT-PCR
Immunohistochemistry and Microscopy Day 180
  1. 1 Fixation and cryoprotection
  2. 2 Embedding and sectioning
  3. 3 Immunofluorescence staining
  4. 4 Image acquisition and analysis
Whole-Cell Patch-Clamp Electrophysiology Day 180
  1. 1 Spheroid slicing
  2. 2 ACSF preparation
  3. 3 Whole-cell patch-clamp recordings
Live Calcium Imaging Day 180
  1. 1 Spheroid slicing and recovery
  2. 2 Calcium dye loading
  3. 3 Calcium imaging and analysis
RNA Sequencing and Library Preparation Day 180
  1. 1 Total RNA extraction for sequencing
  2. 2 Library preparation
  3. 3 RNA sequencing
  4. 4 Quality assessment and trimming
  5. 5 Read mapping and quantification
ELISA Cytokine Quantification Day 30-180
  1. 1 Supernatant collection and storage
  2. 2 Cytokine measurement by U-PLEX assay
  3. 3 Cytokine concentration normalization
Mitochondrial Function Assessment (Seahorse Bioenergenics) Day 180
  1. 1 Spheroid slicing and recovery
  2. 2 Sample plating for Seahorse assay
  3. 3 Mitochondrial respiration assessment
  4. 4 Protein normalization

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Osete et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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