Interconversion Between Intestinal Stem Cell Populations in Distinct Niches

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Science  09 Dec 2011:
Vol. 334, Issue 6061, pp. 1420-1424
DOI: 10.1126/science.1213214


Intestinal epithelial stem cell identity and location have been the subject of substantial research. Cells in the +4 niche are slow-cycling and label-retaining, whereas a different stem cell niche located at the crypt base is occupied by crypt base columnar (CBC) cells. CBCs are distinct from +4 cells, and the relationship between them is unknown, though both give rise to all intestinal epithelial lineages. We demonstrate that Hopx, an atypical homeobox protein, is a specific marker of +4 cells. Hopx-expressing cells give rise to CBCs and all mature intestinal epithelial lineages. Conversely, CBCs can give rise to +4 Hopx-positive cells. These findings demonstrate a bidirectional lineage relationship between active and quiescent stem cells in their niches.

The multicellular epithelium of the intestine is replaced every few days, and this renewal process is maintained by multipotent intestinal stem cells (ISCs) (1, 2). The location and identity of ISCs have been a subject of much research and debate, with implications for understanding gastrointestinal cancer, repair after intestinal injury, and normal physiology. Numerous reports have suggested that ISCs are located at the +4 position relative to the crypt base (3, 4), while a separate body of work has identified a distinct stem cell niche at the crypt base where crypt base columnar (CBC) cells are interspersed between Paneth cells (57). The +4 cells correspond to the location of slow-cycling, label-retaining cells (LRCs) (3, 8) and colocalize with Bmi1-expressing cells (4), as well as those expressing an mTert transgene (9, 10). CBC stem cells, by contrast, are marked by Lgr5 (5). Although +4 cells and CBCs are clearly distinct, lineage-tracing studies have shown that both can give rise to all the various cell types comprising the small intestine epithelium: goblet cells, neuroendocrine cells, Paneth cells, and epithelial absorptive cells. However, the relationship between these two distinct stem cell populations remains incompletely understood. A recent report suggests that +4 cells can compensate for the loss of CBCs to maintain homeostasis after experimental ablation of Lgr5-expressing cells (11). However, a bidirectional lineage relationship between active and quiescent populations of stem cells in multiple tissues has also been postulated (2), though experimental evidence to support this proposal has been lacking. Here, we show that quiescent +4 ISCs express the atypical homeobox gene Hopx and give rise to Lgr5-expressing CBCs. Conversely, rapidly cycling CBCs expressing Lgr5 give rise to +4 cells expressing Hopx. These findings reconcile controversies regarding the location and identity of ISCs and demonstrate interconversion between organ-specific stem cell niches.

Hopx encodes an atypical homeodomain-containing protein that has previously been studied in the heart and neural stem cells (1214). Analysis of the intestines of Hopx lacZ knock-in (HopxLacZ/+) mice revealed robust expression of β-galactosidase (β-Gal) in intestinal crypts along the entire length of the intestine (Fig. 1A and fig. S1A). Expression was strongest in the +4 region and included label-retaining cells identified after irradiation and pulse labeling with 5-bromodeoxyuridine (BrdU) (9, 15, 16) (Fig. 1B and fig. S1B). Eighty-six percent (68/79) of non-Paneth BrdU-retaining cells expressed β-Gal, and nearly all were at or near the +4 position (Fig. 1C). Similar results were found in unirradiated animals; 92% (35/38) of non-Paneth BrdU-retaining cells expressed β-Gal (fig. S1C). To track the fate of Hopx cells, we generated a tamoxifen-inducible Cre (ERCre) knock-in targeted to the 3′ untranslated region of the Hopx locus following an internal ribosomal entry sequence (IRES) (HopxERCre/+) (fig. S2) and crossed them with R26RstoplacZ (R26LacZ/+) indicator mice (fig. S3) (17, 18). Two months after tamoxifen induction, localized staining indicative of β-Gal activity in cells derived from Hopx-expressing precursors was evident in intestinal crypts with a proximal-to-distal gradient (Fig. 1D). Under the conditions used, tamoxifen induction was only partially efficient; nevertheless, entire crypt-villus structures were labeled, suggesting clonal origins. Hopx descendants were present at least 13 months after induction (Fig. 1, E and F), the latest time point we examined, even though the entire intestinal epithelium replenishes every ~5 days, suggesting that Hopx cells self-renew and/or give rise to multipotent ISCs. All differentiated intestinal epithelial cell types can derive from Hopx cells, including Paneth, goblet, neuroendocrine, and absorptive cells (fig. S4). Eighteen hours after a single pulse of tamoxifen to HopxERCre/+;R26mT-mG/+ mice, distinct single cells at the +4 position expressed green fluorescent protein (GFP) (Fig. 1G). Serial analysis of Hopx descendants after initial pulse labeling of Hopx clones indicated that Hopx cells at the +4 position gave rise to progeny that populated the crypt base and the villus epithelium (Fig. 1H). The entire crypt base, including regions occupied by CBCs expressing Lgr5, expressed β-Gal.

Fig. 1

Hopx labels ISCs at the +4 position in intestinal epithelial crypts. (A) Whole-mount X-gal–stained small intestine from a HopxLacZ/+ mouse. (B) Double staining of HopxLacZ/+ intestine for LacZ and BrdU using a postirradiation regeneration labeling protocol. (C) Quantification of BrdU-positive cells at each crypt position. (D) Composite image (nine images combined into one) of X-gal–stained HopxERCre/+;R26LacZ/+ small intestine 2 months after a 5-day tamoxifen pulse. (E and F) X-gal staining 13 months after tamoxifen induction; whole-mount image (E) and eosin counterstained section (F). (G) Immunohistochemical GFP staining in crypts of HopxERCre/+;R26mT-mG/+ mice 18 hours after tamoxifen induction. GFP-positive cells are at the +4 position. (H) Time course of Hopx lineage tracing (HopxERCre/+;R26LacZ/+) after a single tamoxifen pulse. (I) X-gal staining of Lgr5EGFP-ERCre/+;R26LacZ/+ intestine 18 hours after tamoxifen induction. Lgr5-positive cells are found between Paneth cells at the crypt base. (J) Comparison of the location of Lgr5- and Hopx-positive cells in the small intestine. Lgr5-expressing cells are predominantly at the 1′ or 2′ position, whereas most Hopx-expressing cells are at the +4 or +5 position (5). (Analysis was performed 18 hours after a single pulse of tamoxifen.) (K and L) The pattern of X-gal staining was analyzed in HopxERCre/+;R26LacZ/+ (K) and Lgr5EGFP-ERCre/+;R26LacZ/+ (L) mice after a 5-day tamoxifen induction (chase periods as indicated; d, days; M, months). Staining patterns were scored according to the key below the graphs. Scale bars: 10 μm (B, H, I), 25 μm (G), 100 μm (E and F), 250 μm (A), and 1000 μm (D).

The location of Hopx-expressing cells in the intestine was distinct from that of Lgr5-positive cells (compare Fig. 1, G and I). The position of β-Gal–positive cells in 334 crypts was recorded 18 hours after tamoxifen induction, confirming a propensity for the +4 position (Fig. 1J). In contrast, a similar analysis performed with Lgr5EGFP-IRES-ERCre/+;R26LacZ/+ mice (Lgr5EGFP-ERCre/+;R26LacZ/+) (5) placed Lgr5-positive cells predominantly between Paneth cells in the +1/+2 position (Fig. 1, I and J). Serial analysis over 13 months indicated that increasing numbers of crypt-villus structures were entirely labeled over time (Fig. 1K, purple). We observed a gradual decrease in the number of crypts that had two or more LacZ-positive cells without ubiquitous labeling (Fig. 1K, green), and an increase in crypts with a single labeled periodic acid–Schiff–positive Paneth cell (Fig. 1K, red), consistent with the relatively long survival of Paneth cells compared with other epithelial cell types. Parallel experiments with Lgr5EGFP-ERCre/+;R26LacZ/+ mice showed more rapid acquisition of ubiquitous crypt-villus labeling (Fig. 1L), consistent with the interpretation that Lgr5-positive cells are more rapidly cycling stem cells (n = 3 mice for each genotype; >190 crypts were scored for each time point). Taken together, these data indicate that Hopx-positive cells at the +4 position are multipotent, slow-cycling, label-retaining ISCs, which are distinct from Lgr5-positive cells at the crypt base.

CBCs have been shown to form crypt-villus structures and generate all intestinal epithelial cell types in organoid culture (19). Cultures of crypt epithelial cells from HopxLacZ/+ mice, which constitutively express β-Gal from the Hopx locus (18), produced two types of organoids: those that express β-Gal and those that do not (Fig. 2A). Over time in culture, the percentage of organoids expressing β-Gal increased from ~70% at day 2 to >95% by day 14 (Fig. 2B). β-Galactosidase produced from the Hopx locus was relatively stable and perdured, as has been reported in other cases [for example, see (20)]. Thus, all cells expressing Hopx and their early descendants express β-Gal. The presence of unlabeled organoids in these experiments indicated the existence of ISCs that do not express, and did not recently express, Hopx. Examination of organoids from HopxLacZ/+;Lgr5EGFP-ERCre/+ mice indicated that β-Gal–negative organoids expressed GFP from the Lgr5 locus (fig. S5, A and B).

Fig. 2

Organoid cultures of Hopx-labeled cells. (A) X-gal–stained organoids from HopxLacZ/+ mice after 9 days of crypt culture. (B) Time course of the percentage of LacZ-positive organoids derived from HopxLacZ/+ mice. Two hundred organoids from three different HopxLacZ/+ mice were analyzed (error bars: ±1 SD). (C) Examples of crypt organoids from HopxLacZ/+ mice stained with X-gal. (D) Examples of crypt organoids from HopxERCre/+;R26LacZ/+ mice (tamoxifen pulse for 12 hours on day 0.5 of culture). X-gal–stained images are shown with a 14-day eosin counterstained image. Scale bars: 50 μm (A) and 20 μm (C and D).

In cultures in which β-Gal expression was identified, labeled cells were initially located immediately above Paneth cells (identified by their dense granules). Labeled cells expanded to generate crypt-villus “buds” in organoid culture (Fig. 2C). Similar experiments in which HopxERCre/+;R26LacZ/+ cultures were exposed to a single pulse of tamoxifen confirmed the clonal origin of developing crypt-villus structures and epithelial derivatives (Fig. 2D). Cultures derived from HopxLacZ/+;Lgr5EGFP-ERCre/+ mice also produced organoids in which β-Gal (perduring in Hopx-derived cells) overlapped with expression of GFP derived from the Lgr5 locus (fig. S5, C and D).

Hopx cells at the +4 position and Hopx descendants located between Paneth cells at the crypt base were isolated by laser capture microdissection (LCM) from HopxERCre/+;R26LacZ/+ mice 18 and 48 hours after tamoxifen induction, respectively, and expression levels of stem cell markers were compared. Lgr5 was robustly expressed in Hopx descendant CBCs, whereas Hopx and Bmi1 were more strongly expressed by +4 cells (figs. S6 and S7). In separate experiments, Hopx-positive cells and their descendants were isolated by fluorescence-activated cell sorting (FACS) from HopxERCre/+;R26mT-mG/+ mice 18 hours, 2 days, and 4 days after tamoxifen treatment (Fig. 3, A and B), and the expression level of stem cell and differentiation markers was analyzed. Expression of Hopx, Bmi1, Msi1 and Tert, markers of +4 cells (4, 9, 10, 21), decreased over time, whereas that of Lgr5, Olfm4, and Ascl2, expressed by CBCs (5, 22, 23), increased (Fig. 3C). Genes expressed by differentiated epithelial derivatives including Alpi, Lyz1, and Muc2, also increased (Fig. 3C). Notably, single Hopx-expressing cells isolated 18 hours after tamoxifen induction in these experiments remained quiescent in culture. For example, in one experiment only 1 of 7500 cells isolated by FACS expanded substantially during 4 days of culture in the presence of Wnt3A (100 ng/ml). By contrast, cells isolated 4 days after tamoxifen treatment proliferated at a rate equivalent to that of Lgr5hi cells [those with the most robust GFP expression (19)] derived from Lgr5EGFP-ERCre/+ mice (Fig. 3D). Taken together, these findings are consistent with the interpretation that Hopx labels a quiescent population of ISCs that can give rise to more rapidly proliferating Lgr5-expressing ISCs. This conversion may take place more prominently in vivo than in vitro, perhaps due to signals present in the niche.

Fig. 3

FACS and gene expression of Hopx descendants. (A and B) FACS-sorted Hopx descendants after a pulse of tamoxifen (HopxERCre/+;R26mT-mG/+). Representative plots from one of three experiments are shown. Analysis of Hopx descendants at the indicated time periods after a single pulse of tamoxifen to HopxERCre/+;R26mT-mG/+ mice demonstrates a gradual increase in the number of cells expressing GFP (orange), representing Hopx derivatives, and a concomitant loss of the tdTomato signal (blue, ubiquitously expressed until inactivated by HopxERCre/+ expression). Percentages of cells expressing GFP are shown in the gated population in (B). (C) Gene expression of GFP-positive cells was determined by quantitative reverse transcription–polymerase chain reaction (normalized to glyceraldehyde-3-phosphate dehydrogenase). Results are expressed relative to the level of gene expression observed 18 hours after tamoxifen induction, n = 3 experiments. (D) Rate of growth per organoid, n = 5 organoids. Hopx derivatives 18 hours (blue) or 4 days (brown) after tamoxifen induction of HopxERCre/+;R26mT-mG/+ mice, and Lgr5hi (green) cells from HopxLacZ/+;Lgr5EGFP-ERCre/+ mice were used (error bars: ±1 SD).

Lgr5-positive cells can also give rise to Hopx-expressing cells. Single Lgr5hi cells derived from HopxLacZ/+;Lgr5EGFP-ERCre/+ mice produced organoids with an efficiency of 13.0 ± 3.0% and 3.08 ± 0.57% with and without Wnt3A (100 ng/ml), respectively (n = 2000 cells in each group from three different mice) (Fig. 4A), similar to results reported previously by others using Lgr5EGFP-ERCre/+ mice (19, 24). After 21 days in culture, robust β-Gal expression was evident in a pattern distinct from that of GFP (Fig. 4B). At 3 days of culture, all organoids were LacZ negative, but by 7 days >20% of the organoids expressed β-Gal (Fig. 4, C and D), and by 21 days 100% (47/47) expressed β-Gal (Fig. 4D), demonstrating that Lgr5-positive cells can give rise to Hopx-expressing cells in vitro. In vivo, fate mapping of Lgr5 cells with HopxLacZ/+;Lgr5EGFP-ERCre/+;R26mT-mG/+ mice, 5 months after a 5-day tamoxifen pulse, revealed entire crypt-villus structures that express membrane-bound GFP, including cells at the +4 position that simultaneously expressed β-Gal, indicating that they were derived from Lgr5-positive precursors (Fig. 4E). We also prepared near single-cell suspensions of crypts from HopxLacZ/+;Lgr5EGFP-ERCre/+;R26tdTomato/+ (HopxLacZ/+;Lgr5EGFP-ERCre/+;R26Tom/+) mice either 18 hours, 5 days, or 10 days after a single pulse of tamoxifen and analyzed the cells for LacZ and tdTomato expression. Eighteen hours after induction, we found no LacZ and tdTomato double-positive cells, consistent with Hopx-expressing cells being distinct from Lgr5-positive cells. However, over the ensuing 10 days, double-positive cells emerged, confirming that Lgr5-positive cells can give rise to Hopx-expressing, +4 cells (Fig. 4F).

Fig. 4

Lgr5-positive cells can give rise to Hopx-positive cells. (A and B) Organoid cultures from single Lgr5hi cells (HopxLacZ/+;Lgr5EGFP-ERCre/+). Days of growth are shown above each panel. (B) Day 21 GFP and β-Gal expression. (C) Day 7 single-Lgr5hi cell organoid cultures. There are both LacZ-negative (top) and -positive organoids (bottom). (D) Percentage of organoids derived from single Lgr5hi cells (HopxLacZ/+;Lgr5EGFP-ERCre/+) that are LacZ-positive. (Number of organoids analyzed is listed above each time point.) (E) (Left) Confocal image of LacZ and GFP double staining 5 months after a 5-day tamoxifen pulse to HopxLacZ/+;Lgr5EGFP-ERCre/+;R26mT-mG/+ mice (black arrowheads point to double-positive cells). (Right) Light microscope image of the same crypt demonstrating LacZ expression; arrowheads point to LacZ-positive, +4 cells. GFP expression (shown as a red membrane-bound signal) demarcates Lgr5 derivatives, and LacZ (blue) indicates Hopx expression. (F) Representative images of double-positive cells isolated after a single pulse of tamoxifen to HopxLacZ/+;Lgr5EGFP-ERCre/+;R26Tom/+ mice. The percentage of double-positive cells as compared to tdTomato cells is shown. At 18 hours after a single pulse, there are zero double-positive cells. White arrowhead points to double-positive cell in representative images. tdTomato (red) indicates Lgr5 derivatives; LacZ (blue) indicates Hopx expression. Scale bars: 25 μm (E and F) and 50 μm (A, B, C).

Our results provide experimental evidence to support a proposed model (2) in which slowly cycling ISCs at the +4 position dynamically interconvert with more rapidly cycling ISCs at the crypt base (CBCs). Both populations display properties of self-renewal and are multipotent, consistent with stem cell identity. These findings help to reconcile prior controversy in the field and suggest that adult organ-specific stem cells in distinct niches can regenerate one another. Further elucidation of the unique properties of each stem cell population and the signals that regulate interconversion will be likely to inform gastrointestinal pathophysiology and stem cell biology in the future.

Supporting Online Material

Materials and Methods

Figs. S1 to S7

References (2529)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank the Epstein laboratory for helpful discussions; C. J. Lengner, A. Padmanabhan, N. Singh, and K. S. Zaret for critical reading of the manuscript; and the Penn Flow Cytometry core and C. Pletcher for assistance with FACS experiments. This work was supported by an American Heart Association Physician-Scientist/Postdoctoral fellowship to R.J. (AHA 0825548D) and funds from the NIH (R01 HL071546, U01 HL100405), the Spain fund for Regenerative Medicine, and W. W. Smith Endowed Chair to J.A.E.

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