Multi-scale imaging and informatics pipeline for in situ pluripotent stem cell analysis

Area of Science:

• Stem cell biology
• Developmental biology
• Bioinformatics

Background:

• Human pluripotent stem (hPS) cells are vital for regenerative medicine and developmental studies.
• In vitro culture of hPS cells leads to heterogeneity, hindering clinical applications.
• Analyzing spatial organization within hPS cell colonies is crucial for understanding cell behavior.

Purpose of the Study:

• To develop and validate a novel imaging informatics pipeline for analyzing spatial heterogeneity in hPS cell colonies.
• To quantify single-cell and sub-cellular features within the context of colony structure.
• To investigate the relationship between spatial location, cell cycle, and pluripotency marker expression in hPS cells.

Main Methods:

• Integration of automated fluorescent microscopy with a multi-scale image analysis pipeline.
• High-throughput colony detection at low resolution and single-cell analysis at high resolution.
• Development of image processing techniques for handling high-content, high-resolution single molecular mRNA FISH data.

Main Results:

• The tool generates seamless in situ maps of single-cellular data organized by colony.
• Cells in the G1 phase were more frequent at the colony periphery (less pluripotent), while G2 phase cells were enriched in the first pluripotent layer.
• Colony density classes showed distinct pluripotency marker distributions and differential responses to DNA damage.

Conclusions:

• The developed imaging informatics pipeline enables comprehensive analysis of hPS cell heterogeneity at multiple scales.
• Spatial context within colonies significantly influences hPS cell cycle regulation and pluripotency.
• This tool advances the analysis of stem cell populations for both basic research and clinical translation.