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J Anat. Jul 2008; 213(1): 52–58.
PMCID: PMC2475558

Small intestinal stem cell markers

Abstract

Stem cells hold great promise for regenerative medicine but remain elusive in many tissues, including the small intestine, where it is well accepted that the epithelium is maintained by intestinal stem cells located in the crypts. The lack of established markers to prospectively identify intestinal stem cells has necessitated the use of indirect analysis, e.g. long-term label retention, which is based on the hypothesis that intestinal stem cells are slow-cycling. Several intestinal stem cell markers have been proposed, including Musashi-1, BMPR1α, phospho-PTEN, DCAMKL1, Eph receptors and integrins, but their validity, using functional and/or lineage tracing assays, has yet to be confirmed. Recently, Lgr5 has been identified by lineage tracing as an intestinal stem cell marker. In this review we summarize what is known about the currently reported intestinal stem cell markers and provide a rationale for developing model systems whereby intestinal stem cells can be functionally validated.

Keywords: intestines, markers, stem cells

Introduction

Since the work of Cheng and Leblond more than 30 years ago (Cheng & Leblond, 1974) it has been accepted that the small intestinal epithelium is maintained by a population of tissue-specific stem cells. Despite their presumed location within the small intestinal crypt, the identification and isolation of intestinal stem cells (ISCs) has remained a challenge. Long-term label-retaining models have provided indirect evidence that the ISC is located within the lower portion of the small intestinal crypt (Potten, 2004), but definitive phenotypic, functionally validated, markers of the ISC have yet to be established, making study of the ISC challenging.

Remaining to be answered are such fundamental questions as, how many ISCs reside within each crypt and where specifically are they located? In this article we review the various proposed ISC markers and emphasize the need to identify markers that will allow for the prospective isolation and functional validation of ISCs.

The ISC niche

Stem cells are thought to reside in a niche, which regulates the balance between stem cell self-renewal and tissue regeneration (Moore & Lemischka, 2006). Within the ISC niche, a number of signaling pathways are active, which together establish the location of the ISC and presumably serve to regulate ISC proliferation. Genetic studies have provided direct evidence that Wnt/β-catenin signaling is important for ISC maintenance (Korinek et al. 1998), that Notch signaling is involved in intestinal cell fate determination (Fre et al. 2005; Stanger et al. 2005; van Es et al. 2005) and that BMP (bone morphogenetic protein) signaling negatively regulates stem cell proliferation by suppression of Wnt/β-catenin signaling (He et al. 2004). Communication within the niche is complex, with Wnt signaling emanating from both mesenchymal and epithelial cells (Gregorieff et al. 2005), BMP secretion from mesenchymal cells adjacent to the epithelium (Haramis et al. 2004), and additional regulatory factors secreted by myofibroblasts (Powell et al. 2005) and enteric neurons (Bjerknes & Cheng, 2001). In addition, epithelial cell numbers are regulated by paracrine factors, such as Hedgehog and circulating factors binding to the EGF receptor (Sancho et al. 2004). Since activation of these various pathways is through surface receptors one might expect that ISCs would display a unique pattern of surface marker expression, but so far no such pattern has been identified. Without analysis using specific ISC markers, it remains unclear whether these previous studies define mechanisms directly regulating ISC fate and function and/or their downstream progenitors.

Structure and function of small intestinal crypts

Mature intestinal villus epithelium arises continually from rapidly proliferating cells in the intestinal crypts, which form two distinct cell lineages and four mature cell types: the absorptive lineage (absorptive enterocytes) and the secretory lineage (goblet cells, enteroendocrine cells and Paneth cells) (Cheng & Leblond, 1974; Yang et al. 2001). Studies using markers of DNA synthesis, such as 3H-thymidine and BrdU, or antibodies against proliferation markers, such as Ki67, indicate that the lower two thirds of crypt cells are proliferating, and therefore are referred to as the transit amplifying (TA) population (Bach et al. 2000). An average murine small intestinal crypt contains ~250 cells, of which ~150 are actively dividing, with half in S phase at any given time (Bach et al. 2000). The cell cycle time for a TA cell is 12–13 h and each is believed to undergo four to six divisions as it migrates upward in the crypt (Bach et al. 2000).

Evidence for a developmental gradient extending up the crypt has been provided by detailed ultrastructural analyses showing immature cells near the crypt base, more mature cells with small amounts of mucus or endocrine inclusions above these and mature goblet, enteroendocrine and absorptive cells at the crypt–villus junction (Cheng & Leblond, 1974). It is likely that the least differentiated cells located near the base of the crypt, but distinct from Paneth cells, represent ISCs. The continuous supply of crypt cells is presumably maintained by asymmetric division of the ISC, possibly mediated by the orientation of the plane of mitosis within the crypt epithelium, resulting in both ISC self-renewal and differentiated progeny, though direct evidence for asymmetric ISC divisions has yet to be provided (Bjerknes & Cheng, 1989).

Regulation of stem cell proliferation as well as quiescence is emerging as an important area for scientific investigation. Identification of the mechanisms that maintain ISC quiescence and those that induce cell division are key questions in understanding stem cell function. Recently, it has been proposed that stem cell quiescence, previously believed to represent a transcriptionally silent state, may be far more dynamic than has previously been appreciated (Lacorazza et al. 2006; Passegue & Wagers, 2006). Furthermore, recent evidence suggests that ‘emergence from quiescence’ represents a unique developmental state associated with the specification of key outcomes (self-renewal vs. differentiation) (Passegue et al. 2005). Without definitive markers to identify ISCs, however, these and other hypotheses remain untested.

Estimation of ISC number

Studies measuring crypt regeneration following varying doses of irradiation have been used to estimate the number of radiation-sensitive ISCs. Using a dose of 1 Gy, each crypt has been estimated to contain 4–6 ISCs. Exposure to a slightly higher dose of radiation (> 1 Gy) yielded 6 additional so-called ‘clonogenic’ crypt cells with the capacity to regenerate entire lost crypts. Following even higher doses of radiation (8–10 Gy) as many as 30–40 clonogenic cells could be identified per crypt (Bach et al. 2000). Under normal conditions, clonogenic cells are thought to represent early TA cells, which normally differentiate, though the mechanisms underlying clonogenic potential have not been established. Although significant insights have been provided by this work it must be kept in mind that the ISCs and clonogenic cells have been defined in this model using a cytotoxic insult, which may not accurately reflect the normal in vivo situation.

In another model developed by Bjerknes and Cheng, using chemical mutagenesis to genetically mark single crypt cells (clonal analysis), populations of both long-lived and short-lived progenitors have been proposed (Bjerknes & Cheng, 1999). In addition to stem cells, each crypt has been estimated to contain 4–5 long-lived progenitors that may persist for up to 100 days. Many more short-lived progenitors rapidly divide before giving rise to differentiated progeny. Each crypt must contain at least one ISC, but the precise number is unknown in the absence of direct measurements.

Long-term label retention

Based on the hypothesis that ISCs have a significantly longer cell cycle time than do TA cells, Potten and colleagues developed a method to distinguish between the two, which involves pulse labeling with either 3H-thymidine or BrdU immediately following a cytotoxic injury (irradiation). The pulse is then followed by a period of chase sufficient for labeled TA cells to migrate up the villi and be shed and/or undergo sufficient cell divisions to dilute out the signal. In this model the slowly cycling, long-term label-retaining cells (LRCs) are presumed to represent the ISC population, though they may also represent long-lived progenitors. This analysis identified single LRCs along the crypt axis in a distribution that peaks between cell positions 4–9 (Potten et al. 2002). It is important to note, however, that this and other studies (Potten et al. 2002; He et al. 2004, 2007) have employed high doses of radiation, well in excess of that needed to kill the ISCs, to define the distribution of LRCs along the crypt axis, implying that clonogenic cells may confound this analysis. In support of these findings, a similar distribution of LRCs was obtained when juvenile mice received a pulse of 3H-thymidine during a period of active ISC replication and then later were studied as adult animals (Potten et al. 2002). Of note, however, a recent study examining hematopoietic stem cells (HSCs) using the LRC method revealed fewer than 6% of HSCs to be LRCs and fewer than 0.5% of all label-retaining hematopoietic cells to be HSCs. Although this report raises important questions about the utility of this method (Kiel et al. 2007), it remains possible that fundamental differences exist between the hematopoietic and intestinal systems, making the LRC method a valid tool for study of the ISC. Finally, LRCs have yet to be functionally validated, which is required before they can be established as ISCs (see Fig. 1afor schematic representation).

Fig. 1
Schematic Illustration of the location of putative small intestinal stem cell markers. (A) Numbers of long-term label-retaining cells, plotted against cell position in the crypt, indicated by numbers on the left. (B) Expression pattern of cell membrane ...

Putative intestinal stem cell markers

BMPR1a and phospho-PTEN

Mutations in BMPR1α, SMAD4 and PTEN[phosphatase and tensin homolog (mutated in multiple advanced cancers 1)] have been shown to give rise to intestinal polyposis syndromes (Liaw et al. 1997; Howe et al. 1998, 2001), suggesting a role for BMP and PTEN signaling in the intestinal crypt. In addition, BMP signaling has been implicated as a negative regulator of ISC proliferation (Haramis et al. 2004). Using the LRC method to define the ISC, He and colleagues reported BMPR1α to be highly expressed on LRCs and that intestine-specific knockout of BMPR1α resulted in intestinal polyposis, presumably due to an increase in ISC self-renewal (He et al. 2004). They proposed that BMP signaling leads to increases in PTEN activity, which as a negative regulator of PI3 K-Akt suppresses Wnt signaling, leading them to conclude that the inactive phospho-PTEN (which co-localized with LRCs) serves as an ISC marker (He et al. 2004). In a subsequent report, they provided additional evidence for their model using intestine-specific PTEN knockout mice, which demonstrate excessive crypt formation and fission leading again to polyposis (He et al. 2007). Furthermore, they provide mechanistic evidence that the PI3K-Akt pathway specifically phosphorylates β-catenin at Ser552, leading to stabilization and nuclear localization, and resulting in excess proliferation (He et al. 2007). While these data provided important validation that BMPR1α, PTEN-PI3K-Akt and Wnt/β-catenin pathways are essential for the regulation of intestinal homeostasis, the reliance on the LRC method to define the ISC limits the claim that BMPR1α and phospho-PTEN are ISC-specific markers. Definitive proof awaits formal validation, using either lineage-tracing or functional analyses. Furthermore, the claim that phospho-PTEN serves as a specific ISC marker has generated some controversy, in part because its expression within crypt cells does not appear to be solely restricted to ISCs (Bjerknes & Cheng, 2005b) (See Fig. 1d for schematic representation).

DCAMKL1

Doublecortin- and Calmodulin Kinase-Like 1 (DCAMKL1) is a microtubule-associated protein expressed at high levels in the developing brain (Lin et al. 2000). The work of Giannakis and colleagues using gene expression microarray analysis of small intestinal crypt cells identified DCAMKL1 as a potential stem cell marker (Giannakis et al. 2006). To better understand which crypt cells express DCAMKL1, short-term BrdU pulse labeling studies were performed to label the TA population. Co-labeling studies demonstrated DCAMKL1 expression in non-dividing (BrdU) cells in the lower crypt, leading to the conclusion that DCAMKL1 may represent a putative ISC marker. The absence of cell division during short-term labeling, though, does not provide definitive proof of its role as an ISC marker. Furthermore, DCAMKL1 has also been detected within epithelial cells of intestinal villi, raising the question of ISC specificity (Montgomery RK et al., unpublished observations). Confirmation that DCAMKL1 is an ISC-specific marker awaits functional validation (See Fig. 1dfor schematic representation).

Eph receptors

Interactions between Eph receptors and ephrin ligands regulate biological processes such as cell migration and boundary formation during development and tumorigenesis (Dodelet & Pasquale, 2000). EphB2 is strongly expressed on cell membranes at the bottom of the crypt, peaking at cell positions 4–6 (the putative ISC location), with decreased expression on cells higher in the crypt. In addition, EphB3 is strongly expressed on cells in the crypt base. In contrast, ephrin B1 and B2 expression is low on cells at the bottom of the crypt and increased on cells higher in the crypt. In both EphB2−/– and EphB3−/– mice, normal intestinal epithelial cell migration is disrupted, resulting in the intermingling of proliferative and differentiated cells, as well as migration up the villi of Paneth cells, which are normally restricted to the base of the crypt (Batlle et al. 2002). In addition, microarray analysis of crypt cells identified EphA6 as a potential stem cell marker (Giannakis et al. 2006). These receptors may provide useful cell surface markers to study the ISC, but remain to be carefully examined and functionally validated. (See Fig. 1bfor schematic representation).

Integrins

The epithelial cells of the small intestine attach to the underlying basement membrane through interactions between the integrin family of heterodimeric transmembrane glycoproteins and their ligands in the basement membrane, such as collagen, fibronectin and laminin. Integrin-mediated cell signaling pathways have been shown to regulate cell survival, proliferation and differentiation (Gilcrease, 2007). Restricted expression of integrins, such as α2β1, has been described in the stem cell region of the small intestine (Beaulieu, 1992). In addition, intestine-specific deletion of β1 integrin results in hyperplasia of the epithelium due to loss of integrin-mediated Hedgehog signaling (Jones et al. 2006) and elevated β1 integrin levels have been used to purify putative colon stem cells (Fujimoto et al. 2002). Furthermore, mice with β4 integrin null mutations display reduced small intestinal epithelial cell proliferation (Murgia et al. 1998). As cell surface proteins, integrins are attractive candidates for ISC markers given the potential to isolate viable cells using FACS, though a specific expression pattern has not yet been functionally established (See Fig. 1cfor schematic representation).

Musashi

Musashi is an RNA-binding protein that has been shown to be a critical regulator of asymmetric division in neural stem cells of Drosophila (Okano et al. 2005). It is thought to act as a translational repressor and regulate Notch signaling, among whose targets is Hes-1 expression, which is required for differentiation of the secretory lineage (goblet, enteroendocrine and Paneth cells) (Yang et al. 2001). The mammalian homolog Musashi-1 (Msi-1) has been proposed to be a marker for both the ISC as well as early progenitors due to the large number of crypt cells it labels (Potten et al. 2003). Whereas Hes-1 is expressed in most of the crypt cells, Msi-1 is preferentially expressed in cells toward the base of the crypt, including those found between Paneth cells. Although cells co-expressing both Msi-1 and Hes-1 have not been directly demonstrated, it has been postulated that dual expression may define the ISC (Kayahara et al. 2003). Recently, He and colleagues reported single LRCs that co-stained for Msi-1; in addition, other Msi-1-positive cells also co-stained for Ki67, consistent with their identity as proliferating progenitor cells (He et al. 2007). Interestingly, although Msi-1 appears to be expressed in putative ISCs, mice lacking this protein do not exhibit any defect in intestinal growth (Sakakibara et al. 2002). Thus, Msi-1 may not be a specific ISC marker, but is likely expressed in both ISCs and in their early progeny (Fig. 1c).

Lgr5/GPR49

Barker et al. (2007) have recently reported identification of a marker for stem cells in the small intestine and colon. This marker, an orphan receptor known either as Lgr5 or GPR49, was identified in a screen of downstream targets of wnt signaling. In situ hybridization demonstrated that Lgr5 was uniquely expressed in columnar base cells, located among the Paneth cells at the base of the small intestinal crypts. Lineage marking using an inducible Lgr5-Cre knockin and Rosa26-lacZ reporter demonstrated that progeny encompassing absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells of the small intestine were labeled, providing strong evidence that Lgr5 is a marker for intestinal stem cells or long-lived multipotent progenitor cells.

Consistent with this finding, as mentioned above, Kayahara et al. (2003) reported co-staining of crypt base columnar cells for both the putative stem cell markers MSI1 and Hes, and Bjerknes & Cheng (2005a) suggested that the crypt base columnar cells represent stem cells. On the other hand, the findings reported by Barker et al. (2007) contrast with several of the established models for intestinal stem cells. The identified cells are in a location where previous efforts using long-term label retention have demonstrated few cells, the maximum labeling being above the Paneth cell zone. In addition, these cells label very rapidly, in contrast to the widely accepted model of slowly cycling stem cells. It is also of interest that a knockout of the Lgr5 gene is reported to have no observable effect on the development of the intestine (Morita et al. 2004), though there are closely related receptors which may substitute for the inactivated Lgr5. It may be that the Lgr5 receptor is a stem cell marker but is not essential for maintenance of the stem cell. Functional validation of Lgr5 as a stem cell marker for small intestine remains to be carried out (Fig. 1d).

Position of stem cells in the crypt

As with estimates of ISC numbers, the available data identifying the ISC location within the crypt are based largely on long-term label retention studies and clonogenic analysis (He et al. 2004; Potten 2004). Because LRCs were identified at multiple crypt positions, with peak frequencies at positions 4–9, the ISC is often depicted as being located specifically at position 4 in the crypt and nowhere else, but this is an oversimplification of the data. He et al. (2004) have reported Msi-1 and BMPR1α co-expression within LRCs, and other investigators have also demonstrated multiple Msi-1+ cells in lower crypt cells (Kayahara et al. 2003; Potten et al. 2003). Alternatively, Bjerknes & Cheng (2005a) have suggested that cells intercalated among the Paneth cells at the base of the crypt may actually represent ISCs. Lgr5 specifically marks cells at the base of the crypt (Barker et al. 2007). The available evidence indicates that the ISC may be found at multiple locations in the crypt, rather than in a single fixed location, though this remains to be formally established.

Side population

The recent development of cell isolation methods based on particular biochemical properties of stem cells, such as the side-population phenotype (Goodell et al. 1996), have provided additional tools to enrich for stem cells within heterogeneous populations. Dekaney et al. (2005) describe the use of side population (SP) analysis to isolate a putative stem cell population from mouse small intestine. Whereas the SP population had a low percentage of cells positive for Sca-1 and c-kit, markers associated with other stem cell populations, more than 87% of the SP cells were positive for β1 integrin, a proposed ISC marker. In addition, the SP cells were enriched for Msi-1 mRNA and de-enriched for markers of differentiated absorptive cells (sucrase), goblet cells (trefoil factor) and Paneth cells (lysozyme). Interestingly, these cells remained viable in culture for several weeks but failed to proliferate or differentiate. As proliferation and differentiation are key functional properties of stem cells, it remains unclear whether SP cells contain ISCs.

Telomerase

Telomerase activity is essential to prevent telomere shortening and cellular senescence and has been proposed as a stem cell marker in multiple tissues (Allsopp et al. 2003a,b; Greenwood & Lansdorp, 2003; Harrington, 2004; Flores et al. 2005). In addition, mice deficient in telomerase activity demonstrate organ failure in tissues highly dependent on stem cell proliferation, including the intestine (Lee et al. 1998). Booth and Potten have reported telomerase expression in individual cells of the lower small intestinal crypt, consistent with a role for telomerase as an ISC marker (Booth & Potten, 2000). The generation of telomerase reporter mice could serve as a useful tool for the isolation and functional validation of ISCs.

Conclusions

A number of candidate markers for the ISC have been proposed (see Table 1 for summary), based largely on their relationship to LRC in the intestinal crypts. Although identification of LRC has been the best method available to investigate ISCs and progenitors, definitive proof of its role as an ISC marker is lacking, given that it remains to be functionally validated. Musashi-1, the most studied protein marker, may label both ISCs and progenitor cells. Other potential markers, including BMPR1α, P-PTEN, DCAMKL1, β1 integrin, Eph receptors, side population and telomerase expression, remain to be validated using functional assays. Although the cells identified display some characteristics different from accepted stem cell models, the recently reported lineage tracing experiments strongly suggest that Lgr5 is an intestinal stem cell marker. Further experiments using model systems that enable the direct isolation and enrichment of stem cells in combination with lineage-tracing analysis are required for the identification, purification, and functional validation of intestinal stem and progenitor cell populations.

Table 1
List of reported ISC markers

Acknowledgments

The authors would like to acknowledge Dr D. L. Carlone for critical reading of the manuscript. This work was supported by a Seed Grant from the Harvard Stem Cell Institute (DTB) and by a Pilot and Feasibility Grant from the Harvard Digestive Disease Center (RKM, DTB).

Note Added in Proof

Recent work by our group validates mTert as an ISC marker. In addition, Bmi1 has recently been identified and functionally validated as an ISC marker.

Breault DT, Min IM, Carlone DL, et al. (2008) Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells. Proc Natl Acad Sci USA (in press).

Sangiorgi E, Capecchi MR (2008) Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet (Published Online 8 June 2008).

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