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An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids
María José Castellanos-Montiel1, Mathilde Chaineau1, Anna Kristyna Franco-Flores1
1The Neuro's Early Drug Discovery Unit (EDDU), McGill University, 3801 University Street, Montreal, QC H3A 2B4, Canada.
Cells
|February 25, 2023
Summary
Researchers developed a 3D motor neuron (MN) spheroid model using induced pluripotent stem cells (iPSCs) to study motor neuron diseases (MNDs). This model allows for comprehensive analysis of MNs, aiding in understanding disease mechanisms.
Area of Science:
- Neuroscience
- Stem Cell Biology
- Disease Modeling
Background:
- Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) are crucial for modeling motor neuron diseases (MNDs).
- Three-dimensional (3D) spheroid models offer advantages for high-throughput screening and complex biological assays.
- Developing standardized workflows for these 3D models is essential for reproducible research.
Purpose of the Study:
- To generate and characterize 3D motor neuron spheroids derived from iPSCs.
- To establish a comprehensive workflow for analyzing spheroid morphology, gene expression, protein levels, and functional activity.
- To validate the utility of this 3D model for studying MNs and potentially MNDs.
Main Methods:
- Generation of MN spheroids from iPSCs.
- Morphological profiling using a custom image analysis pipeline.
- Quantitative PCR (qPCR) for gene expression analysis.
- Immunocytochemistry on cleared tissue for protein validation.
- Microelectrode array (MEA) recordings for functional assessment of action potentials and bursts.
Main Results:
- Successful generation of iPSC-derived MN spheroids.
- Established a workflow for morphological, transcriptomic, proteomic, and functional characterization.
- Confirmed MN identity and expression of key markers at both transcript and protein levels.
- Demonstrated functional neuronal activity (action potentials, bursts) via MEA.
- Identified the presence of other neural cell types, including interneurons and oligodendrocytes.
Conclusions:
- A robust 3D MN spheroid model and associated analytical workflow have been successfully developed.
- The model provides a platform for comprehensive, time-course profiling of MNs.
- This approach facilitates the investigation of selective MN degeneration in MNDs and other neurological disorders.
