A pipeline for cell migration analysis in live-cell imaging data from human iPSC-derived forebrain assembloids.

Area of Science:

• Neuroscience
• Developmental Biology
• Stem Cell Biology

Background:

• Inhibitory interneurons and oligodendrocyte progenitor cells migrate extensively during forebrain development.
• Human induced pluripotent stem cell-derived forebrain assembloids (FAs) offer an in vitro model to study this migration.
• Tracking these cells in 3D models is crucial for understanding neural development.

Purpose of the Study:

• To develop and validate a processing pipeline for tracking cell migration in human forebrain assembloids (FAs).
• To analyze the migratory behavior of key neural cell types within FAs.
• To provide an open-source resource for cell tracking in 3D stem cell models.

Main Methods:

• Utilized viral labeling (EYFP, tdTomato) in FAs to track cells via EF1α and SOX10 promoters.
• Employed spinning disk confocal microscopy for 4D volumetric imaging over 15-17 hours.
• Developed an end-to-end image processing and manual cell tracking pipeline, including background subtraction and drift correction.

Main Results:

• Image preprocessing significantly enhanced data quality for manual tracking.
• Trajectory analysis of over 670 labeled cells revealed super-diffusive directed motility in most migrating cells.
• The developed pipeline successfully characterized migratory cell behavior in FAs.

Conclusions:

• The established pipeline is a valuable resource for cell tracking in FAs and similar 3D stem cell platforms.
• This work provides the first open tracking resource for iPSC-derived FAs.
• The pipeline can serve as a ground-truth for developing automated cell tracking algorithms in developmental neuroscience.