Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity

Abstract

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5⁺ ISCs, the most well-defined ISC pool, but Bmi1-GFP⁺ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP⁺ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1⁺ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP⁺ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Figure 1. Hierarchical clustering of ISC populations by bulk cell mRNA-seq

A. Unsupervised hierarchical clustering reveals the association of Lgr5^hi, Cd166⁺, Cd24^lo, upper SP and Grp78⁻ cells into Cluster 1. Bmi1-GFP⁺ and mTert-GFP⁺ cells are present in Cluster 2, while Hopx⁺, Dclk1⁺ and lower SP cells exhibit intermediate features between Clusters 1 and 2. B. Supervised hierarchical analysis demonstrating enrichment of an Lgr5^hi/CBC gene signature amongst Cluster 1 populations.

Figure 2. Characterization of the Bmi1/mTert ISC cluster

A. Venn diagram of overlapping gene enrichment between Bmi1-GFP⁺ and mTert-GFP⁺ cells. B. Enteroendocrine gene signature in Bmi1-GFP⁺ cells includes numerous EE secretory factors and EE transcription factors (Neurod1, Arx, Prox1) but notably lacks Neurog3. C. Anti-ChgA (white) immunofluorescence co-localization in intestinal crypts is found in 89.9% of crypt Bmi1-GFP cells (anti-GFP, green). Bar = 25 µm. Error bars indicate mean ± s.e.m. D. Overlap between genes enriched in Bmi1-GFP⁺, LRC and multi-capable EE. E. Clonogenic enteroid culture of FACS-isolated Bmi1-GFP⁺ cells validating their clonogenic potential ex vivo. D = day, P = passage number. Bar = 100 µm.

Figure 3. Functional similarities between Bmi1-GFP⁺ and Prox1⁺ cells in vivo

A. Co-localization of Prox1 expression in 74.4% of Bmi1-GFP⁺ crypt cells by immunofluorescence, Bmi1-GFP (anti-GFP, green) and anti-Prox1 (red). Bar = 25 µm. Error bars indicate mean ± s.e.m. B. Clonogenic enteroid growth ex vivo from Prox1-GFP⁺ cells isolated from proximal jejunum, day 5. 20× magnification. C. Short-term (36h) in vivo tamoxifen-induced labeling of Prox1⁺ cells in jejunum of Prox1-GFP; Prox1-CreER; Rosa26 tdTomato mice marks crypt epithelial cells. Bar = 20 µm. D. Persistent lineage tracing in jejunum from a subset of crypt-based Prox1⁺ cells in tamoxifen-treated Prox1-CreER; Rosa26 tdTomato mice, 9 to 365 days after tamoxifen treatment. Lineage tracing was observed in approximately 1/150 crypts. Labeling was also observed in lymphatic vessels (“lv”, arrow) consistent with the known expression of Prox1 in lacteals and in ~10% of villi. Bar = 50 µm. E. Multi-potency of Prox1-expressing cells. Differentiation markers were detected by IF within Prox1 lineage stripes, with Fabp1 for enterocytes, Muc2 for goblet cells, ChgA for EE cells and Lyz for Paneth cells. Bar = 20 µm. F. Injury-inducibility of Prox1⁺ lineage traces in tamoxifen-treated Prox1-CreER; Rosa26 tdTomato mice. Tamoxifen was administered 2 days prior to delivery of 12 Gy whole body irradiation, followed by tissue harvest after an additional 7 days. Bar = 50 µm.

Figure 4. Single-cell transcriptomics of Prox1-GFP⁺ cells

A. t-SNE projection of 1,051 Prox1-GFP⁺ cells colored by clustering. B. Expression of key genes including Prox1 and markers of stem/progenitor cells. Lgr5, Ascl2 and Olfm4 represent canonical CBC markers whereas Nfat5, Nfatc3 and Cd83 are LRC/secretory progenitor makers. C. Tuft cell marker expression among the Prox1-GFP⁺ clusters. D. EE marker expression among the Prox1-GFP⁺ clusters. E. Immunofluorescence validation of EE (ChgA) and tuft (Dclk1) marker expression in Prox1⁺ cells. Bar = 20 µm.

Figure 5. Direct comparison of Prox1-GFP⁺, Bmi1-GFP⁺ and Lgr5-eGFP⁺ cells at single-cell resolution

A. (Top left) Composite t-SNE projection of 1,607 Lgr5-eGFP⁺, 658 crypt Bmi1-GFP⁺, 246 crypt Prox1-GFP⁺ and 1,010 Lgr5-eGFP(−) cells color-coded by FACS-isolated sample. (Top right) Composite t-SNE plot reveals 13 distinct clusters identified by their gene expression. (Bottom) Deconvolution of the cells into their four individual component samples. All five EE clusters visualized by t-SNE represent Bmi1-GFP⁺ cells (Clusters 3, 6, 7, 10, 12) are globally distinct from Lgr5-eGFP⁺ cells. B. Bmi1-GFP⁺ cells express EE genes at the single-cell level. Gene expression heatmaps overlaid on t-SNE plot of crypt Bmi1-GFP⁺ cells. All five EE clusters representing Bmi1-GFP⁺ cells express various EE secretory products or EE cell fate determination transcription factors. Bmi1-GFP⁺ cells (>92%) express EE hormone and transcription factor mRNAs consistent with EE cell identity but lack Lgr5⁺ ISC signature, secretory progenitor and other lineage marker genes.

Figure 6. SPADE single-cell gene expression lineage hierarchy analysis reveals relationship of Prox1-GFP and Bmi1-GFP cell clusters

A. SPADE lineage hierarchy reconstruction of the intestinal epithelium determined for all cells for the annotated 13 lineages by clustering analysis and t-SNE projection of scRNA-seq data. Cells on the same or neighboring branches are expected to be more hierarchically related compared to cells on different branches in a given tree. A. Hierarchical relationships between the 13 clusters colored by cluster identity. B. Hierarchical relationships colored by sample type. Notably, Bmi1-GFP⁺ cells mark clusters at the distal branches of the EE lineage whereas Prox1-GFP⁺ cells mark the tuft lineage and more proximal branches of the EE lineage.