Prominin1 (CD133) as an Intestinal Stem Cell Marker: Promise and Nuance

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20. Jalan R, Wright G, Davies NA, et al. L-Ornithine phenylacetate (OP): a novel treatment for hyperammonemia and hepatic encephalopathy. Med Hypotheses 2007;69:1064 –9. 21. Davies NA, Wright G, Ytrebø LM, et al. L-ornithine and phenylacetate synergistically produces sustained reduction in ammonia and brain water in cirrhotic rats. Hepatology 2009; in press. 22. Ytrebø LM, Kristiansen RG, Mæhre H, et al. L-Ornithine phenylacetate attenuates increased arterial and extracellular brain ammonia and prevents intracranial hypertension in pigs with acute liver failure. Hepatology 2009; in press. 23. Lee WM, Hynan LS, Rossaro L, et al; and the ALFSG. Intravenous N-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure. Gastroenterology 2009; in press.

Reprint requests Address requests for reprints to: William M. Lee, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas. e-mail: [email protected]. Conflicts of interest The authors disclose the following: Professor Rajiv Jalan (University College London) has filed for patents surrounding the use of L-ornithine phenylacetate for the treatment of hepatic encephalopathy. The technology has been licensed to Ocera Therapeutics. William M. Lee discloses no conflicts. © 2009 by the 0016-5085/09/$36.00 doi:10.1053/j.gastro.2009.04.016

Prominin1 (CD133) as an Intestinal Stem Cell Marker: Promise and Nuance

See “Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine” by Snippert HJ, Can Es JS, can den Born M, et al, on page 2187.

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he presence of long-lived, self-renewing stem cells in intestinal crypts is well established.1 Although these cells have been studied extensively for their functions and putative hierarchies, molecular markers are as yet unavailable to permit their prospective isolation, their unequivocal identification in tissue sections, or thorough assessment of mechanisms of stem cell replication and differentiation. The self-renewing property of gut epithelium is in many respects reminiscent of cancer, and indeed, cancers are believed to propagate from small subpopulations of stem-like cells that are inherently more tumorigenic than their progeny.2,3 Identification of intestinal tumorinitiating cells holds special interest because it would allow investigators to address fundamental questions about the cell of tumor origin and its biology in relation to defined normal counterparts. We might learn, for example, to what extent tumor-initiating cells resemble the long-lived stem cell or transit-amplifying cells, knowledge that will shape future approaches toward understanding the disease and defining therapeutic opportunities. These ideas gathered recent momentum when 2 groups reported that human colon cancers could be separated into tumor-initiating and noninitiating cell populations based on expression of the neuronal and hematopoietic cell surface marker CD133 (known as Prominin1 in the mouse). Such advances provoke the need for clarity and consensus on surface markers

that reliably distinguish intestinal stem cells from other crypt populations and help to define the relationship between normal and cancerous stem cells. In this issue of GASTROENTEROLOGY, Snippert et al4 clear the air with respect to CD133/Prominin1. Stem cells in most tissues are believed to cycle slowly (although this is not imperative a priori); thus, DNA marked during replication retains the label for extended periods, whereas cycling progeny and migrating cells lose the label with turnover. Applying this principle to the small intestine, Potten et al5 observed that long-term label-retaining cells lie most commonly at crypt position ⫹4, immediately above the Paneth cell zone, with fewer cells present elsewhere in the crypt. By contrast, Bjerknes and Cheng’s analysis6 of migration of labeled cells led to the idea that stem cells may nestle between Paneth cells at the crypt base. Predating identification of candidate stem cell markers, these studies laid important anatomic and conceptual foundations. The RNA-binding protein Musashi-1 is restricted to crypts, but marks a broad population, including transit-amplifying progenitors.7 Using label-retaining cells as a standard, He et al proposed phosphorylated PTEN as a stem cell marker8 and phosphorylation of ␤-catenin on serine 552 as a marker of activated stem cells.9 DCAMKL1 and telomerase also are proposed as products specific to stem cells in gut epithelium.10,11 Each of these markers mainly identifies cells in the predicted “⫹4” position, lying just above the Paneth cell zone (Figure 1), but none has yet been shown directly to mark the operational stem cell. The Clevers group’s characterization of the Wnt target Lgr5 (also called GPR49) provided the first functionally 2051

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validated marker.12,13 These investigators used lineage tracing to show that Lgr5-expressing columnar cells, largely interspersed between Paneth cells at the crypt base, give rise, stably and continually, to all major differentiated cell types in small bowel epithelium. The same group recently demonstrated that a transcription factor expressed selectively in Lgr5⫹ cells, Achaete Scute-Like 2 (Ascl2), is needed to maintain these stem cells.14 Meanwhile, Sangiorgi and Capecchi15 proposed the polycomb factor Bmi1 as an alternative stem cell marker, also by virtue of similar results with lineage tracing, although their data placed a Bmi1⫹ stem cell population at the ⫹4 position.15 The notion of 2 distinct but functionally related stem cell populations and evidence supporting various markers were recently reviewed in detail.16 Bmi1 and Ascl2 are intracellular proteins, but Lgr5, a surface protein and confirmed stem cell marker, is a good candidate for prospective isolation of live stem cells. However, no current antibody recognizes it with sufficient specificity and activity for this purpose. Prospective isolation of stem cells is now an intense focus of attention in many human cancers; in 2007, 2 independent groups reported using monoclonal antibodies against CD133 to enrich colon cancer-initiating cells from bulk tumors by flow cytometry.17,18 The choice of CD133 as an index marker was not based on any known role or particular expression in the colon but on prior success with CD133 antibodies in isolating cancer-initiating cells from brain and other tumors. Indeed, applying the same test of tumor formation in mouse xenografts, a 3rd group showed enrichment of colon cancer-initiating cells in an EPCAMhi/CD44⫹ population.19 Notably, each of these approaches, and indeed similar studies reported for other solid tumors, enriches rather than isolates tu-

mor-initiating cells, which are estimated to comprise well under 1% of CD133⫹ or EPCAMhi/CD44⫹ cell populations. Although the structure of the phylogenetically conserved pentaspan transmembrane protein CD133 suggests receptor properties, its physiologic function is unknown. CD133 was first recognized as a marker for human hematopoietic stem cells, using the AC133 monoclonal antibody,20 and later used to mark neuronal and brain tumor stem cells; its murine homolog Prominin1 appears on embryonic neural cells and other developing epithelia. However, some studies identify wide CD133 expression in differentiated kidney, breast, and other epithelia, questioning its reputed stem cell specificity. Antibody sensitivity toward protein glycosylation, which may change with development and cellular context, and species differences may limit definitive interpretation of CD133 distribution in different sites. Furthermore, in all organs, cell populations likely vary in the level of surface CD133 expression. If colon cancer-initiating cells are present within a CD133⫹ population, it is important to know how this marker is distributed in the normal intestine and if it could be exploited to isolate stem cells from the normal tissue. Three groups have therefore targeted a reporter gene into different locations within the mouse prominin1 locus. In the first such knock-in mouse model, Shmelkov et al21 replaced exons 3– 8 with a LacZ reporter gene, thereby marking all cells where Prominin1 is normally expressed. LacZ staining of these mice marked numerous epithelial cells in the gastrointestinal tract and other organs. Consistent with these data, immunohistochemistry identified Prominin1 expression on many differentiated cells in the colon and elsewhere; these investigators

Figure 1. Cell positions and molecular markers in mouse small intestines crypts. 2052

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did not report on the small intestine. Importantly, analysis of tumor cells from serial xenograft transplants indicated that tumor-forming potential was not restricted to CD133⫹ cells: In contrast with the 2 studies on human colon cancer, tumor-initiating cells in a mouse IL10⫺/⫺ model were about as frequent in CD133⫹ as in CD133depleted populations. These results cast doubt on the use of CD133 as a marker specific to normal or cancer stem cells or as a tool to enrich cancer-initiating cells from bulk tumors. However, a subsequent report by Zhu et al22 built a fresh case for Prominin1 as a marker of central importance. These investigators inserted DNA encoding Cre recombinase and LacZ, separated by an internal ribosome entry site, at the ATG initiation codon in exon 2. Mice carrying this marker Prom1 allele showed widespread reporter expression in many tissues, including colon epithelium. In sharp contrast, LacZ staining in the small intestine was largely confined to a few cells with the crypt base location and columnar morphology characteristic of Lgr5⫹ stem cells. To test the stem-like properties of cells that apparently co-express Lgr5 and Prominin1, Zhu et al crossed their engineered allele into the ROSA26-YFP reporter strain and observed YFP-marked progeny, including all 4 differentiated gut epithelial lineages, emerging in tracks from 25% of small intestine crypts but not in the colon. The authors did not specify how commonly the YFP signal persisted over a prolonged period, but they observed some after 60 days and their very presence served as a de facto standard for gut stem cell function. The authors further used their targeted mouse Cre line to introduce an activating ␤-catenin mutation in cells with endogenous Prominin1 expression. This initially induced proliferation of cells at the crypt base, below the ⫹4 tier, followed over 2 months by gross dysplasia and focal intraepithelial tumors in the small intestine. It is worth noting that no study has argued that CD133 is absent from stem cells, only that it may broadly mark several cell types, possibly including the intestinal stem cell. Thus, the observations of the Zhu et al study can be interpreted to indicate that some fraction of Prominin1⫹ cells in the small intestine corresponds to the long-lived, self-renewing stem cell. However, the reported data are insufficient to determine if the marker is expressed predominantly in long-lived stem cells or their short-lived progeny. Similar considerations apply toward prospective isolation of human CD133⫹ colon cancer stem cells. In the current issue of GASTROENTEROLOGY, Snippert et al4 re-address the precise distribution of mouse Prominin1. In most tissues, their results with in situ hybridization and immunochemistry match those of the other groups; in the small intestine, they find Prominin1 expression not only in Lgr5⫹ stem cells but almost throughout the crypts. They also demonstrate comparable Prominin1

mRNA levels in Lgr5hi stem cells and their presumptive progeny, which were separated by flow cytometry and express less Lgr5. The authors then generated mice in which a reporter construct containing mCherry and tamoxifen-inducible Cre recombinase, separated by an internal ribosome entry site, is inserted in the last exon of the Prom1 locus. Prominin1 expression, tracked by Cherry red fluorescence, again extended beyond the Lgr5⫹ crypt base, and crosses between these mice and the ROSA26LacZ reporter strain permitted lineage tracing. Shortterm Cre induction marked crypt cells higher than the usual position of Lgr5-expressing basal columnar cells. Within 1 or 2 days of Cre induction, the authors observed many villi where uniform LacZ staining pointed to an origin in Prominin1-expressing cells. However, uniformly stained villi were considerably less frequent by day 7, suggesting that their initial appearance reflected Prominin1 expression in short-term progenitors and not in self-renewing stem cells. In comparing 3 papers that used a similar approach, the obvious question is why they arrived at different conclusions about Prominin1 expression in gut stem cells. Different designs for targeting the mouse Prom1 locus could potentially give distinct expression patterns, but all 3 groups reported essentially the same distribution outside the intestine. Reporter expression and staining for the native protein gave similar results, arguing against substantive differences in protein and mRNA stability. Despite using the same Prominin1 antibody, the 3 groups did obtain different immunohistochemistry profiles, which might reflect different experimental conditions or application of different thresholds to distinguish specific signals from background. In any case, the most telling experiments are those that trace the progeny of Prominin1-expressing cells. Here, Snippert et al provide the crucial information that most descendents of prominin1-marked cells disappear rapidly from the gut; only 1 in every 10 villi that carried LacZ-marked cells all along their length 1 day after activation of Prominin1driven Cre remained so marked at 14 or 75 days. Thus, most LacZ-marked cells must have descended from short-lived progenitors, with only a smaller fraction representing the progeny of bona fide stem cells. The aggregate data hence indicate that mouse Prominin1 is expressed broadly in small intestine and colon crypts, including rare long-lived stem cells, and not exclusively in the latter. Prospective isolation of colon cancer stem cells has captured attention at the same time that intestinal stem cell hierarchies and molecular mechanisms are coming into focus. Arguably, no disease positions biologists to probe the links between normal and malignant stem cells better than human colorectal cancer, where the genetic underpinnings are well established and powerful experi2053

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mental tools provide the means to interrogate stem cell properties. Indeed, the Clevers laboratory recently made a persuasive case that tumorigenic mutations in Wnt pathway genes target native intestinal stem cells and not their progeny.23 Further advances depend critically on agreement among investigators about the distribution and functions of putative molecular markers. The study by Snippert et al in this issue of GASTROENTEROLOGY provides welcome resolution on the limits of the role of CD133/Prominin1 as a tool to track normal intestinal stem cells or to isolate their cancer-initiating counterparts.

ROBERT K. MONTGOMERY RAMESH A. SHIVDASANI Dana-Farber Cancer Institute Children’s Hospital and Harvard Medical School Boston, Massachusetts

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Reprint requests Address requests for reprints to: Ramesh A. Shivdasani, MD, PhD, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115. e-mail: [email protected]. Conflicts of Interest The authors disclose no conflicts. © 2009 by the AGA Institute 0016-5085/09/$36.00 doi:10.1053/j.gastro.2009.04.021