The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations

Abstract

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

Fig. 1.

Basal proliferation status and response to canonical Wnt pathway modulation of Lgr5⁺ vs. Bmi1⁺ ISCs. (A–F and N) Lgr5-eGFP⁺ ISCs in Lgr5-eGFP-IRES-CreERT2 duodenum are actively cycling and incorporate EdU under homeostasis (A–C). In contrast, Bmi1⁺ ISCs labeled with 1.5-d tamoxifen exposure in Bmi1-CreER; Rosa26-YFP duodenum are slowly cycling and do not incorporate EdU (D–F, **P = 0.0049 vs. Lgr5; DAPI colored in blue). (G–I) Lgr5⁺ ISCs in Lgr5-eGFP-IRES-CreERT2; Rosa26-TdTomato duodenum generate progeny much more efficiently than Bmi1⁺ ISCs in Bmi1-CreER; Rosa26-YFP duodenum by 7 d after tamoxifen-mediated lineage tracing (*P = 0.0001). TdTomato is shown in red (G); phalloidin is colored in red and DAPI in blue (H). (J–N) Consequences of canonical Wnt stimulation with Ad Rspo1-Fc on Lgr5⁺ vs. Bmi1⁺ ISCs. Marked expansion of Lgr5-eGFP⁺ ISCs and the transit-amplifying compartment are seen by direct fluorescence microscopy, whereas Bmi1-YFP⁺ ISCs are less responsive to Ad Rspo1-Fc stimulation. Notably, Ad Rspo1-Fc results in enhanced EdU incorporation in Lgr5-eGFP⁺ ISCs (*P = 0.04) but not in Bmi1-YFP⁺ ISCs (P = 0.13) compared with Ad Fc control. (O and P) Electron microscopy reveals expansion of crypt base columnar (CBC) cells (*) consistent with Lgr5⁺ ISC morphology in between Paneth cells (Pa) after Ad Rspo1-Fc treatment. (Q and R) Canonical Wnt inhibition with Ad Dkk1 results in complete loss of Lgr5-eGFP⁺ ISCs. (S and T) In contrast, Bmi1-YFP⁺ cells persist despite systemic Wnt blockade with Ad Dkk1. (Q–T) Phalloidin is colored in red and DAPI in blue. (U–W) In situ hybridization (ISH) analysis reveals enhanced expression of Olfm4, a surrogate marker for Lgr5, in response to Ad Rspo1-Fc stimulation of canonical Wnt signaling (V), whereas Ad Dkk1 results in complete loss of Olfm4 expression (W) compared with Ad Fc control (U). (Scale bars: H, Q–W, 50 μm; J–M, 20 μm; O and P, 5 μm.)

Fig. 2.

Differential responses of Lgr5⁺ vs. Bmi1⁺ ISCs to acute radiation injury. (A–H) Direct fluorescence detection of Lgr5-eGFP⁺ vs. Bmi1-YFP⁺ ISCs. Phalloidin is depicted in red and DAPI in blue. (A and B) Unirradiated duodenum with Lgr5-eGFP⁺ cells in the crypt bases and rare Bmi1-YFP⁺ ISCs labeled with 1-d tamoxifen exposure in Bmi1-CreER; Rosa-YFP mice. (C–F) Lgr5-eGFP⁺ ISCs are completely lost (C and E), whereas Bmi1-YFP⁺ ISCs (D and F) persist by 2 and 4.5 d after 12 Gy whole-body irradiation. (G and H) Lgr5-eGFP⁺ ISCs return in sporadic (≈1/180) crypt bases (G) and Bmi-YFP⁺ ISCs are quantitatively unchanged by 7 d after 12 Gy whole-body irradiation (H). (I and J) Olfm4 ISH demonstrates loss of Olfm4 expression in crypt bases by 2 d after 12 Gy irradiation confirming loss of Lgr5 expression. (K–N) FACS analysis demonstrates loss of Lgr5-eGFP⁺ epithelial cells by 2 d (K and L), in contrast to expansion of Bmi1-YFP⁺ cells/progeny by 4.5 d after 12 Gy whole-body irradiation (M and N). (O–R) Enhanced proliferation of Bmi1-YFP⁺ ISCs by 2 d after 12 Gy irradiation revealed by EdU incorporation (*P = 0.0006, n = 3 mice, unpaired Student's t test). (Scale bars: 50 μm.)

Fig. 3.

Differential responses of Lgr5⁺ vs. Bmi1⁺ lineages to acute radiation injury. (A and B) Tamoxifen-mediated lineage trace of Lgr5-eGFP⁺ progeny in Lgr5-eGFP-IRES-CreERT2; Rosa-YFP mouse duodenum demonstrates prolific lineage generation under homeostasis by 8 d (A). Lgr5-eGFP⁺ ISCs and their lineage are ablated at 7 d after 12 Gy whole-body irradiation (B). (C and D) Bmi1⁺ lineage in Bmi1-CreER; Rosa-YFP duodenum marked by 9-d tamoxifen treatment exhibits marked proliferative regenerative response with progeny observed in adjacent crypts and villi at 7 d after irradiation. (Scale bars; A–D, 100 μm.) Phalloidin is shown in red and DAPI in blue. (E–G) 3D confocal reconstruction of Bmi1⁺ lineage regenerative response in the jejunum at 7 d after 12 Gy irradiaton highlights confluent patches of Bmi1⁺-derived cells in multiple adjacent crypts/villi. Zoomed view of regenerated jejunum immunostained with lysozyme, illustrating Paneth cells within a budding crypt (F). Phalloidin is colored in red and DAPI in blue. (H–K) Monoclonal repopulation of 7 d postirradiated jejunum from Bmi1-CreER; Rosa-Confetti compound heterozygotes, as suggested by monochromatic patches of regenerated crypts/villi illustrated in RFP (H), GFP (I), CFP (J), and YFP (K). Insets represent serial cross-sections through lineage traces indicating involvement of multiple contiguous crypts and villi (G and K).

Fig. 4.

Clonogenic culture of single FACS-isolated Bmi1-YFP⁺ ISCs. (A–D) Single sorted Bmi1-YFP⁺ cells marked by 1 or 2 d tamoxifen administration in Bmi1-CreER; Rosa-YFP mice form intestinal epithelial spheroids in clonogenic culture that demonstrate continued proliferation and self-renewal. Brightfield images of spheroids by 3 (A), 10 (B), and 14 d (C) in culture with pan-YFP expression visualized by direct fluorescence (D), consistent with clonal-derivation from a genetically marked Bmi1-expressing cell. (E–H) Bmi1-YFP⁺ ISCs are multipotent in vitro by immunofluorescence antibody detection of lineage markers within clonogenic spheroids including sucrase-isomaltase for enterocytes (E), Muc2 for goblet cells (F), lysozyme for Paneth cells (G), and chromogranin A for enteroendocrine cells (H). E-cadherin is colored green and DAPI blue. (I) H&E staining of Bmi1-YFP⁺ spheroid. (J) Proliferation of Bmi1-YFP⁺ spheroid by EdU incorporation. E-cadherin is colored green and DAPI blue. (K) Clonogenically cultured Bmi1-YFP⁺ spheroid from FACS-isolated single cells gives rise to multiple Lgr5⁺ cells in vitro by Lgr5 FISH. Simultaneous Lgr5 FISH was performed with immunodetection of YFP, demonstrating that Lgr5⁺ cells are clonally derived from a cell genetically marked with YFP expression in a Bmi1-CreER; Rosa-YFP mouse. DAPI is shown in blue. (Scale bars: A–D, 100 μm; E–I and K, 50 μm; J, 25 μm.)