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Single Cell Transcriptomics Uncovers Stem Cell That Regenerates Intestinal Epithelium After Injury

NEW YORK (GenomeWeb) – Through single-cell transcriptomics, researchers have identified a stem cell that regenerates the major cell types of the intestine after injury.

Multipotent LGR5+ crypt-based columnar cells typically power intestinal epithelium turnover, but these cells are lost after injury like radiation, according to researchers led by Alex Gregorieff from Mount Sinai Hospital in Toronto. Despite their loss, the intestinal epithelium is able to recover, which has suggested that other cells step in to regenerate the epithelium.

Through single-cell RNA sequencing of damaged mouse intestinal cells, Gregorieff and his colleagues uncovered a population of rare, typically quiescent cells they called revival stem cells (revSCs). As they reported today in Nature, these cells can give rise to the major intestinal cell types.

"Damage-induced expansion of revSCs — rather than a resident regenerative stem cell or general cell plasticity — may therefore be a key mechanism that underlies tissue repair in response to injury," the researchers wrote in their paper.

Gregorieff and his colleagues exposed mice to 12 Grays of whole-body radiation and isolated single cells from their intestinal epithelium, including their intestinal crypts, for transcriptional profiling to tease out cells involved in epithelial regeneration.

In particular, they noted control crypts had 2.5-fold more LGR5+ CBC cells, as compared to the epithelium, but after radiation, 90 percent of those cells were lost. Radiation also reduced the numbers of other cell types like Paneth cells and goblet cells, while the number of enterocytes, tuft cells, and crypt-associated immune cells increased.

Overall, they identified 17 epithelial cell and two lymphocyte clusters among these cells.

One cluster of cells the researchers identified — dubbed SSC1 — encompassed most of the proliferating cells within undamaged intestinal crypts and was suppressed by irradiation. The SSC2 cluster, meanwhile, included cells from irradiated crypts that typically expressed genes involved in DNA damage response and cell survival like clusterin (Clu).

SSC2 further harbored three subgroups — SSC2a, SSC2b, and SSC2c — that differed in some of their expressed markers. In particular, SSC2c was a quiescent group that highly expressed Clu, a stress-response gene involved in cell survival.

Using transgenic mice with marked endogenous Clu, the researchers found that Clu-GFP-positive cells were rare in healthy intestines, but increased in number following radiation, especially as Lgr5-GFP-positive cells are lost.

They further found that the YAP1 gene signature — a transcriptional regulator that is a key player in intestinal regeneration — was found only among the SSC2c subgroup. Additionally, inactivation of YAP1 leads to CLU+ cell suppression.

This, they added, indicated that CLU+ SSC2c cells are rare, quiescent crypt cells that are induced by intestinal damage in a YAP1-dependent manner.

These cells, which the researchers dubbed revSC, can reconstitute LGR5+ cells to restore intestinal crypts. By labeling different cell types in another mouse model, the researchers found that CLU+ cells could give rise to LGR5+ CBCs as well as differentiated progeny.

Additionally, when the researchers studied mice lacking revSC, they found that those mice, after irradiation, had decreased proliferation, intestinal crypt numbers and crypt length, while uninjured ones had intestines similar to wild-type mice.

"Collectively, our results identify revSCs as a slow-cycling cell type that arises in damaged intestines following YAP1-dependent signaling to reconstitute LGR5+ CBCs and regenerate the intestine," they wrote.