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

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

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

Background and Aims: The intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs. Methods: 3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results. Results: Chamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells. Conclusions: We present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.