Tumour-wide 'omics' approaches have long held sway as the approach to identifying useful therapeutic targets. This view is changing with the realization that many, if not all, cancers contain a minority population of self-renewing stem cells, the cancer stem cells, which are entirely responsible for sustaining the tumour as well as giving rise to proliferating but progressively differentiating cells that are responsible for much of the cellular heterogeneity that is so familiar to histopathologists. Moreover, although many tumours probably have their origins in normal stem cells, persuasive evidence from the haematopoietic system suggests that genetic alterations in more committed progenitor cells can reactivate the self-renewal machinery, resulting in a further source of cancer stem cells. Thus, the bulk of the tumour is not the problem, and so the identification of cancer stem cells and the factors that regulate their behaviour are likely to have an enormous bearing on the way that we treat neoplastic disease in the future.

The side population (SP) phenotype, defined as the reserpine-blockable ability to efflux the nucleic acid dye Hoechst 33342, has been claimed to be enriched for stem cells in several human normal tissues, cancers and cell lines, and thus may be useful for the identification and isolation of cancer stem cells. We demonstrated the presence of SP fractions in all of seven tested gastrointestinal cancer cell lines. Four cell lines were selected (HT29, HGT101, Caco2 and HRA19a1.1) for detailed phenotypic and behavioural analysis with respect to stem cell characteristics. Cell surface marker analysis showed that, contrary to non-SP cells, the SPs entirely lack the expression of CD34. This difference, however, disappeared when the cells were cultured, rendering both populations CD34-positive. Expression of other putative stem cell markers (CD133, CD44, Hes-1, beta-catenin, Musashi-1, Oct-4 and CD117) was identical on SP and non-SPs before and after culturing. Sorted SP and non-SP cells were similarly clonogenic in vitro, tumourigenic in vivo, and displayed similar multipotential differentiation potential in vitro and in vivo. Additionally, culturing cytometrically-sorted SP and non-SP cells showed that the populations are interconvertible, each giving rise to the other. Expression of ABCG2 and Mdr-1, two membrane transporter proteins that have been suggested to be responsible for the drug-effluxing capacities of SP cells, including Hoechst 33342, was identical in non-SP and SP cells, indicating that there may be additional factors responsible for the Hoechst effluxing property in gastrointestinal cancer SP cells. Here, we show that the SP and non-SP fractions, albeit phenotypically distinct populations, do not differ with respect to stem cell-like cell number or behaviour. We thus conclude that the concept of the SP phenotype as a universal marker for stem cells does not apply to gastrointestinal cancer cells. These findings stand in contrast to the observations made in many other tissues and harbour important implications for the future search for intestinal cancer stem cell markers.

Methods for lineage tracing of stem cell progeny in human tissues are currently not available. We describe a technique for detecting the expansion of a single cell's progeny that contain clonal mitochondrial DNA (mtDNA) mutations affecting the expression of mtDNA-encoded cytochrome c oxidase (COX). Because such mutations take up to 40 years to become phenotypically apparent, we believe these clonal patches originate in stem cells. Dual-color enzyme histochemistry was used to identify COX-deficient cells, and mutations were confirmed by microdissection of single cells with polymerase chain reaction sequencing of the entire mtDNA genome. These techniques have been applied to human intestine, liver, pancreas, and skin. Our results suggest that the stem cell niche is located at the base of colonic crypts and above the Paneth cell region in the small intestine, in accord with dynamic cell kinetic studies in animals. In the pancreas, exocrine tissue progenitors appeared to be located in or close to interlobular ducts, and, in the liver, we propose that stem cells are located in the periportal region. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle. We propose that this is a general method for detecting clonal cell populations from which the location of the niche can be inferred, also affording the generation of cell fate maps, all in human tissues. In addition, the technique allows analysis of the origin of human tumors from specific tissue sites. STEM

Background & Aims:The existence of slowly-cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients.Methods: Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg per m 2 body surface area, maximum dose of 400 mg) over a 30-min period; the IdU had a circulation t 1/2 of 8hs. Tissues were collected at 7, 11, 29 and 67 days following infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. Results:No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly, because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this timepoint. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). Conclusions:LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia is required.4