Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity

Rivera, Kristina R.; Bliton, R. Jarrett; Burclaff, Joseph; Czerwinski, Michael; Liu, Jintong; Trueblood, Jessica M.; Hinesley, Caroline M.; Breau, Keith A.; Deal, Halston; Joshi, Shlok; Pozdin, Vladimir A.; Yao, Ming; Ziegler, A; Blikslager, Anthony T.; Daniele, Michael A.; Magness, Scott T.

doi:10.1016/j.jcmgh.2023.07.012

Cited by 1 publication

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References 118 publications

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“…While many genes were originally demonstrated to mark ISCs in mice, our recent scRNAseq atlas supports that these markers are conserved in primary human ISCs 23 . IEC monolayers containing stem cells defined by gene markers is consistent with our earlier study demonstrating that IECs plated as monolayers retain functional stem cell activity, with a 2–3% organoid formation efficiency 31 . The presence of both stem and progenitor cells in our IECs might also explain the biphasic cycle lengths shown by both reporters (Figs.…”

Section: Discussionsupporting

confidence: 90%

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

Cotton,

Ariel,

Chen

et al. 2024

Sci Rep

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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.

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Section: Discussionsupporting

confidence: 90%