Resolving the complexity of the intestinal stem cell pool with single cell RNA-seq
Intestinal stem cells contribute to homeostasis and regenerative potential of the intestine
The intestine has regenerative potential with populations of intestinal stem cells (ISCs) that contribute to homeostasis and repopulation after injury. Research has revealed several ISC populations that are known to repopulate the stem cell pool upon injury and a variety of cellular markers associated with these diverse ISC populations. Despite these findings, questions still remain around ISC inter-relatedness, hierarchy, and interconversion and how these relate to the cellular plasticity observed in the intestine. The introduction of single cell technologies has enabled researchers to begin addressing these questions on a cell-by-cell basis, revealing ISC population composition and helping to unravel the complexities of the ISC pool.
Exploring quiescent injury-inducible ISC populations
ISCs are suspected to be in one of two states, a persistent self-renewing population that generate mature intestinal lineages, or a quiescent, but injury-inducible state that can help repopulate the intestine upon injury. Mounting evidence suggests that secretory lineage precursors, or enterocytes, can serve as injury-inducible ISCs; however, questions still remain over the relatedness, hierarchy, and interconversion of the ISC pool. In a new Cell Stem Cell publication, Yan et al., take a closer look at enteroendocrine (EE) lineage cells and their possible role in intestinal regeneration after injury. The researchers performed systematic transcriptome profiling of diverse ISC marker populations to gain insights into EE lineage cell heterogeneity and identify a novel injury-inducible ISC marker.
Using single cell RNA-seq to take a closer look at ISC heterogeneity and lineage
ISCs expressing the marker Bmi1 exhibit properties of quiescent but injury-inducible populations. In this study, researchers used bulk RNA-seq data of Bmi1+ cells to zero in on a putative new injury-inducible ISC marker, Prox1. Lacking the resolution needed to tease out the Bmi1+ and Prox1+ subpopulations using bulk RNA-seq, researchers turned to single cell RNA-seq (scRNA-seq). The scRNA-seq data showed distinct subpopulations of Bmi1+ cells, all expressing EE secretory marker mRNAs, and an overlapping Prox1+ subpopulation, also expressing EE secretory marker mRNAs. This overlap suggests a common EE lineage of Bmi1 and Prox1 cells. Along with in vivo data showing Bmi1+ and Prox1+ intestinal epithelial cell functional similarity, the overlapping gene expression data supports Prox1 as a putative new marker for injury-inducible ISCs.
Increasing our understanding of complex cellular populations with scRNA-seq
Where bulk RNA-seq is unable to resolve the heterogeneity ISC populations, scRNA-seq provides a tool to dissect ISC populations on a cell-by-cell basis, shedding light on the true diversity of the ISC pool. Using scRNA-seq data, researchers were able to compare the gene expression of Bmi1+ and Prox1+ cells and also perform lineage tracing, providing insight into the interrelatedness and hierarchy of intestinal epithelial cells. Notably, the gene expression profiles of Bmi1+ cells indicate that they are mature EE cells, providing evidence that highly differentiated EE cells may also serve as injury-inducible ISCs. Importantly, the 10x Chromium™ Single Cell 3' Solution not only enabled Yan et al. to dissect the heterogeneity of the Bmi1+ cells, its high cell capture efficiency actually made single cell analysis on this system possible, as Bmi1+ and Prox1+ cells are extremely rare.
Additional Resources
- Read more about the authors’ work with ISCs: Recent Study Published in Nature Unravels a Novel Pathway for Stem Cell Self-Renewal
- Learn about the Chromium™ Single Cell 3’ Solution for single cell RNA-seq
- Download scRNA-seq datasets