OLFM4 Is a Robust Marker for Stem Cells in Human Intestine and Marks a Subset of Colorectal Cancer Cells

The epithelium of the small intestine and colon is the most rapidly self-renewing tissue in mammals. Proliferative cells are confined to crypts, and differentiated cell types predominantly occupy the villi in the small intestine and the surface epithelium in the colon. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression. These cells are intermingled with postmitotic Paneth cells at crypt bottoms in the small intestine. In the colon, they reside at crypt bottoms and are intermingled with goblet cells. Whereas knockin alleles of Lgr5 have been instrumental in defining these cells, Lgr5 mRNA or protein levels are too low to serve as faithful markers. Olfactomedin-4 (Olfm4) has emerged from a gene signature for these Lgr5 stem cells as a robust marker for murine small intestinal stem cells. Herein, we show that OLFM4 is highly expressed in crypt base columnar cells in human small intestine and colon. Moreover, OLFM4 is expressed in cells within adenocarcinomas of the colon. We propose that OLFM4 can serve as a useful marker for Lgr5-type stem cells in human small intestine and colon. Intestinal crypts contain stem cells and their transit-amplifying (TA) daughter cells. Cells exiting the proliferative crypts onto the villi terminally differentiate into enterocytes, goblet cells, and enteroendocrine cells. Paneth cells escape the crypt–villus flow by migrating to crypt bottoms where they live for several weeks. With the exception of stem cells and Paneth cells, the murine small intestinal epithelium is renewed approximately every 5 days.1Barker N. van de Wetering M. Clevers H. The intestinal stem cell.Genes Dev. 2008; 22: 1856-1864Google Scholar Because of the intimate connection between Wnt signaling, crypt biology,2Korinek V. Barker N. Moerer P. et al.Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Google Scholar, 3Kuhnert F. Davis C.R. Wang H.T. et al.Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1.Proc Natl Acad Sci U S A. 2004; 101: 266-271Google Scholar, 4Pinto D. Gregorieff A. Begthel H. et al.Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Google Scholar and colon cancer,5Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Google Scholar, 6Morin P.J. Sparks A.B. Korinek V. et al.Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.Science. 1997; 275: 1787-1790Google Scholar we have previously attempted to unravel the TCF4 target gene program activated by this pathway in crypts and colorectal tumors.7van de Wetering M. Sancho E. Verweij C. et al.The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Google Scholar, 8Van der Flier L.G. Sabates-Bellver J. Oving I. et al.The Intestinal Wnt/TCF Signature.Gastroenterology. 2007; 132: 628-632Abstract Full Text Full Text PDF Scopus (404) Google Scholar, 9van Es J.H. Jay P. Gregorieff A. et al.Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Google Scholar Lgr5 emerged from these studies as a candidate stem cell marker. Using Cre-mediated genetic tracing, we demonstrated that Lgr5 marks long-lived, multipotent stem cells.10Barker 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: 1003-1007Google Scholar, 11Barker 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 Scopus (123) Google Scholar Each crypt bottom harbors about 6 of these small, cycling cells intermingled with Paneth cells. Although Lgr5 stem cells occasionally occupy a position directly above the Paneth cells, they seem to be distinct from another proposed stem cell population located at the so called +4 position12Potten C.S. Extreme sensitivity of some intestinal crypt cells to X and gamma irradiation.Nature. 1977; 269: 518-521Google Scholar, 13Potten C.S. Kovacs L. Hamilton E. Continuous labelling studies on mouse skin and intestine.Cell Tissue Kinet. 1974; 7: 271-283Google Scholar; Lgr5 stem cells are not particularly radiation-sensitive and do not retain DNA labels.1Barker N. van de Wetering M. Clevers H. The intestinal stem cell.Genes Dev. 2008; 22: 1856-1864Google Scholar, 10Barker 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: 1003-1007Google Scholar Recently, it was shown that Bmi1 expression marks cells at position +4 relative to the crypt bottom. Lineage tracing has revealed that Bmi1+ cells mark pluripotent stem cells that replenish the epithelium with similar kinetics to Lgr5 stem cells.14Sangiorgi E. Capecchi M.R. Bmi1 is expressed in vivo in intestinal stem cells.Nat Genet. 2008; 40: 915-920Google Scholar With real-time quantitative polymerase chain reaction analysis, we have found that Bmi1RNA is enriched in Lgr5 stem cells,15van 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: 903-912Google Scholar suggesting that Bmi1 and Lgr5 stem cells represent overlapping populations. We previously determined a differential gene expression profile for Lgr5 stem cells and their immediate daughters, by GFP-based sorting of epithelial cells from isolated crypts of Lgr5-EGFP-ires-CreERT2 mice.15van 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: 903-912Google Scholar When individual genes were tested by in situ hybridization analysis, Olfm4 emerged as a highly specific and robust marker for Lgr5 stem cells. Olfm4 was not expressed in murine colon.15van 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: 903-912Google Scholar Yet, human OLFM4 is enriched in human colon crypts as demonstrated by microarray analysis.16Kosinski C. Li V.S. Chan A.S. et al.Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors.Proc Natl Acad Sci U S A. 2007; 104: 15418-15423Google Scholar The OLFM4 gene encodes a secreted molecule with unknown function originally cloned from human myeloblasts.17Zhang J. Liu W.L. Tang D.C. et al.Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development.Gene. 2002; 283: 83-93Google Scholar Recently, it was shown that Xenopus ONT1, an OLFM4 family member, acts as a BMP antagonist.18Inomata H. Haraguchi T. Sasai Y. Robust stability of the embryonic axial pattern requires a secreted scaffold for chordin degradation.Cell. 2008; 134: 854-865Google Scholar We obtained paraffin sections of normal human small intestine and colon and performed in situ hybridizations. Human OLFM4 is expressed in comparable fashion to murine Olfm4 in the small intestine, that is, in crypt base columnar cells (Figure 1A and B). Although murine OlfM4 is not expressed in the colon, a specific signal was detected in human colonic epithelium (Figure 1C), reminiscent of Lgr5 expression in the colon.10Barker 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: 1003-1007Google ScholarOLFM4 was also highly expressed in subsets of cells within colorectal carcinomas (Figure 1D). OLFM4 expression in these tumor cells was much higher than the expression observed in wild-type crypt base columnar cells at flanking crypt bottoms. β-Catenin staining on serial sections clearly revealed the location of malignant tissue elements within the preparation (Figure 1E). From these observations, we conclude that OLFM4 expression may be a useful tool to study the biology of stem cells in human small intestine and colon. Lgr5 stem cells represent the cells of origin of intestinal malignancies in the mouse.19Barker N. Ridgway R.A. van Es J.H. et al.Crypt stem cells as the cells-of-origin of intestinal cancer.Nature. 2009; 457: 608-611Google Scholar Previous studies in human adenocarcinomas of the colon indicate that these tumors harbor tumor-initiating cells, or so-called cancer stem cells.20Dalerba 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: 10158-10163Google Scholar, 21O'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: 106-110Google Scholar, 22Ricci-Vitiani L. Lombardi D.G. Pilozzi E. et al.Identification and expansion of human colon-cancer-initiating cells.Nature. 2007; 445: 111-115Google Scholar, 23Vermeulen L. Todaro M. de Sousa Mello F. et al.Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.Proc Natl Acad Sci U S A. 2008; 105: 13427-13432Google Scholar Future studies may indicate that OLFM4 marks cells within colon adenocarcinomas that have such cancer stem cell properties. The authors thank Harry Begthel for assistance with histology.