An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells

Area of Science:

• Stem cell biology
• Cell cycle regulation
• Gastrointestinal research

Background:

• The intestinal epithelium dynamically controls cell cycle phase lengths.
• No existing experimental platform allows direct analysis of cell cycle phases in living human intestinal stem cells (ISCs).

Approach:

• Developed primary human ISC lines with two reporter constructs (PIP-FUCCI and PIP-H2A) for fluorescent cell cycle readouts.
• Utilized 3D-printed collagen presses for chamber slides supporting ISC growth and confocal microscopy.
• Employed live imaging, EdU pulsing, and a computational analysis pipeline with CellPose2 and TrackMate.

Key Points:

• PIP-FUCCI ISCs are optimal for snapshot cell cycle phase assignment from single images.
• PIP-H2A ISCs facilitate automated analysis of cell cycle phase lengths through live imaging.
• The developed platforms integrate soft collagen chamber slides, reporter constructs, and computational tools.

Conclusions:

• Presents two comprehensive platforms for analyzing cell cycle phases in living primary human ISCs.
• PIP-FUCCI and PIP-H2A constructs enable distinct methods for cell cycle analysis in ISCs.
• These platforms are valuable for future research on pharmaceutical effects, cell cycle regulation, and ISC biology.