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

Abstract

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. 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. The presence of long-lived, self-renewing stem cells in intestinal crypts is well established.1Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine V Unitarian theory of the origin of the four epithelial cell types.Am J Anat. 1974; 141: 537-561Crossref PubMed Scopus (1093) Google Scholar 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.2Polyak K. Hahn W.C. Roots and stems: stem cells in cancer.Nat Med. 2006; 12: 296-300Crossref PubMed Scopus (322) Google Scholar, 3Radtke F. Clevers H. Self-renewal and cancer of the gut: two sides of a coin.Science. 2005; 307: 1904-1909Crossref PubMed Scopus (580) Google Scholar Identification of intestinal tumor-initiating 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 al4Snippert H.J. Can Es J.S. can den Born M. et al.Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine.Gastroenterology. 2009; 136: 2187-2194Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar 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 al5Potten C.S. Owen G. Booth D. Intestinal stem cells protect their genome by selective segregation of template DNA strands.J Cell Sci. 2002; 115: 2381-2388PubMed Google Scholar 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 analysis6Bjerknes M. Cheng H. The stem-cell zone of the small intestinal epithelium III. Evidence from columnar, enteroendocrine, and mucous cells in the adult mouse.Am J Anat. 1981; 160: 77-91Crossref PubMed Scopus (162) Google Scholar 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.7Potten C.S. Booth C. Tudor G.L. et al.Identification of a putative intestinal stem cell and early lineage marker; musashi-1.Differentiation. 2003; 71: 28-41Crossref PubMed Scopus (410) Google Scholar Using label-retaining cells as a standard, He et al proposed phosphorylated PTEN as a stem cell marker8He X.C. Zhang J. Tong W.-G. et al.BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-β-catenin signaling.Nat Genet. 2004; 36: 1117-1121Crossref PubMed Scopus (840) Google Scholar and phosphorylation of β-catenin on serine 552 as a marker of activated stem cells.9He X.C. Yin T. Grindley J.C. et al.PTEN-deficient intestinal stem cells initiate intestinal polyposis.Nat Genet. 2007; 39: 189-198Crossref PubMed Scopus (360) Google Scholar DCAMKL1 and telomerase also are proposed as products specific to stem cells in gut epithelium.10May R. Riehl T.E. Hunt C. et al.Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, Doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice.Stem Cells. 2008; 26: 630-637Crossref PubMed Scopus (231) Google Scholar, 11Breault D.T. Min I.M. Carlone D.L. et al.Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells.Proc Natl Acad Sci U S A. 2008; 105: 10420-10425Crossref PubMed Scopus (102) Google Scholar 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 validated marker.12Barker N. van Es J.H. Kuipers J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (3784) Google Scholar, 13Barker N. Clevers H. Tracking down the stem cells of the intestine: strategies to identify adult stem cells.Gastroenterology. 2007; 133: 1755-1760Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar 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.14van der Flier L.G. van Gijn M.E. Hatzis P. et al.Transcription factor Achaete Scute-Like 2 controls intestinal stem cell fate.Cell. 2009; 136: 903-912Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar Meanwhile, Sangiorgi and Capecchi15Sangiorgi E. Capecchi M.R. Bmi1 is expressed in vivo in intestinal stem cells.Nat Genet. 2008; 40: 915-920Crossref PubMed Scopus (940) Google Scholar 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.15Sangiorgi E. Capecchi M.R. Bmi1 is expressed in vivo in intestinal stem cells.Nat Genet. 2008; 40: 915-920Crossref PubMed Scopus (940) Google Scholar The notion of 2 distinct but functionally related stem cell populations and evidence supporting various markers were recently reviewed in detail.16Scoville D.H. Sato T. He X.C. et al.Current view: intestinal stem cells and signaling.Gastroenterology. 2008; 134: 849-864Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar 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.17O'Brien C.A. Pollett A. Gallinger S. et al.A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.Nature. 2007; 445: 106-110Crossref PubMed Scopus (3396) Google Scholar, 18Ricci-Vitiani L. Lombardi D.G. Pilozzi E. et al.Identification and expansion of human colon-cancer-initiating cells.Nature. 2007; 445: 111-115Crossref PubMed Scopus (3358) Google Scholar 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.19Dalerba P. Dylla S.J. Park I.-K. et al.Phenotypic characterization of human colorectal cancer stem cells.Proc Natl Acad Sci U S A. 2007; 104: 10158-10163Crossref PubMed Scopus (1741) Google Scholar Notably, each of these approaches, and indeed similar studies reported for other solid tumors, enriches rather than isolates tumor-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,20Yin A.H. Miraglia S. Zanjani E.D. et al.AC133, a novel marker for human hematopoietic stem and progenitor cells.Blood. 1997; 90: 5002-5012Crossref PubMed Google Scholar 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 al21Shmelkov S.V. Butler J.M. Hooper A.T. et al.CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors.J Clin Invest. 2008; 118: 2111-2120PubMed Google Scholar 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 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 CD133-depleted 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 al22Zhu L. Gibson P. Currle D.S. et al.Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.Nature. 2009; 457: 603-607Crossref PubMed Scopus (539) Google Scholar 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 β-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 al4Snippert H.J. Can Es J.S. can den Born M. et al.Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine.Gastroenterology. 2009; 136: 2187-2194Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar 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 ROSA26-LacZ reporter strain permitted lineage tracing. Short-term 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 Prominin1-driven 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 experimental 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.23Barker N. Ridgway R.A. van Es J.H. et al.Crypt stem cells as the cells-of-origin of intestinal cancer.Nature. 2009; 457: 608-611Crossref PubMed Scopus (1575) Google Scholar 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. Prominin-1/CD133 Marks Stem Cells and Early Progenitors in Mouse Small IntestineGastroenterologyVol. 136Issue 7PreviewProminin-1(Prom1)/CD133 is used, alone or in combination with other cell surface markers, to identify and isolate stem cells from various adult tissues. We recently identified leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) as a marker of the intestinal stem cells from which all cellular lineages of the gastrointestinal epithelium are derived. To determine whether there is a relationship between these markers, we investigated the intestinal expression pattern of Prom1/CD133 and created knock-in mice to visualize and trace Prom1+ cells. Full-Text PDF DCAMKL-1 Expression Identifies Tuft Cells Rather Than Stem Cells in the Adult Mouse Intestinal EpitheliumGastroenterologyVol. 137Issue 6PreviewIn an editorial of a recent issue of Gastroenterology, Montgomery and Shivdasani comment on the known markers of mammalian intestinal epithelial stem cells.1 We wish to caution that staining for doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL-1), one of the putative stem cell markers mentioned in this editorial, is a highly specific and robust marker of postmitotic, differentiated, tuft cells, a minority cell lineage of the intestinal epithelium, rather than a marker for intestinal epithelial stem cells. Full-Text PDF