The worldwide shortage of donor livers to transplant end stage liver disease patients has prompted the search for alternative cell therapies for intractable liver diseases, such as acute liver failure, cirrhosis and hepatocellular carcinoma (HCC). Under normal circumstances the liver undergoes a low rate of hepatocyte 'wear and tear' renewal, but can mount a brisk regenerative response to the acute loss of two-thirds or more of the parenchymal mass. A body of evidence favours placement of a stem cell niche in the periportal regions, although the identity of such stem cells in rodents and man is far from clear. In animal models of liver disease, adopting strategies to provide a selective advantage for transplanted hepatocytes has proved highly effective in repopulating recipient livers, but the poor success of today's hepatocyte transplants can be attributed to the lack of a clinically applicable procedure to force a similar repopulation of the human liver. The activation of bipotential hepatic progenitor cells (HPCs) is clearly vital for survival in many cases of acute liver failure, and the signals that promote such reactions are being elucidated. Bone marrow cells (BMCs) make, at best, a trivial contribution to hepatocyte replacement after damage, but other BMCs contribute to the hepatic collagen-producing cell population, resulting in fibrotic disease; paradoxically, BMC transplantation may help alleviate established fibrotic disease. HCC may have its origins in either hepatocytes or HPCs, and HCCs, like other solid tumours appear to be sustained by a minority population of cancer stem cells.

While cultured embryonic stem (ES) cells can be harvested in abundance and appear to be the most versatile of cells for regenerative medicine, adult stem cells also hold promise, but the identity and subsequent isolation of these comparatively rare cells remains problematic in most tissues, perhaps with the notable exception of the bone marrow. The ability to continuously self-renew and produce the differentiated progeny of the tissue of their location are their defining properties. Identifying surface molecules (markers) that would aid in stem cell isolation is a major goal. Considerable overlap exists between different putative organspecific stem cells in their repertoire of gene expression, often related to self-renewal, cell survival and cell adhesion. More robust tests of 'stemness' are now being employed, using lineage-specific genetic marking and tracking to show production of long-lived clones and multipotentiality in vivo. Moreover, the characterization of normal stem cells in specific tissues may provide a dividend for the treatment of cancer. The successful treatment of neoplastic disease may well require the specific targeting of neoplastic stem cells, cells that may well have many of the characteristics of their normal counterparts.

We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser-capture microdissected and mutations iden- A dult tissue-specific stem cells are thought to reside within a specialized microenvironment, known as the niche, and it is here that stem cell behavior is regulated and maintained. 1 In epithelia with ordered structure and in a state of continual cell renewal, there is often a hierarchical organization with stem cells at the beginning of the flux, and terminally differentiated, reproductively sterile cells at the end of the flux, imminently to be lost from the population. Many studies have attempted to identify stem cells and the location of the niche using histological methods based on the premise that stem cells have inherent properties such as DNA label retention, high integrin expression, and abundant detoxifying enzyme activity. 2 However, many uncertainties remain even in comparatively well-defined instances such as the hematopoietic system. 3 It has been proposed that the gold standard of stem cell identification involves marking putative stem cells to identify the niche, and then performing lineage tracing to demonstrate that the proposed "stem cell" has multipotentiality. 4 This approach commonly uses mice genetically engineered to have a steroid-activated version of Crerecombinase knocked into the putative stem cell marker gene, such that Cre activation mediates excision of a roadblock sequence in the Rosa26-lacZ reporter, thus resulting in an irreversible marker in all the descendants of the putative stem cell. Using this technology, it has been shown, for example, that mouse hair follicle bulge cells expressing K15 generate all the epithelial cells in the lower hair follicle, 5,6 whereas in the murine small intestine, long-lived cell clones containing all the intestinal cell lineages can be generated from both leucine-rich repeat-containing G protein-coupled receptor 5 [Lgr5]-expressing 7 and polycomb ring finger oncogene [Bmi1]-expressing Abbreviations: 3D, mtDNA: mitochondrial DNA; PBS, SDH, succinate dehydrogenase. From the

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

SummaryThere is growing realization that many -if not all -cancer-cell populations contain a subpopulation of self-renewing stem cells known as cancer stem cells (CSCs). Unlike normal adult stem cells that remain constant in number, CSCs can increase in number as tumours grow, and give rise to progeny that can be both locally invasive and colonise distant sites -the two hallmarks of malignancy. Immunodeficient mouse models in which human tumours can be xenografted provide persuasive evidence that CSCs are present in human leukaemias and many types of solid tumour. In addition, many studies have found similar subpopulations in mouse tumours that show enhanced tumorigenic properties when they are transplanted into histocompatible mice. In this Commentary, we refer to CSCs as tumour-propagating cells (TPCs), a term that reflects the assays that are currently employed to identify them. We first discuss evidence that cancer can originate from normal stem cells or closely related descendants. We then outline the attributes of TPCs and review studies in which they have been identified in various cancers. Finally, we discuss the implications of these findings for successful cancer therapies.Key words: Clonogenicity, Immunodeficient mice, Self-renewal, Stem cells, Tumour-propagating cells Journal of Cell Science Small intestineIn the mouse small intestine there is both a slowly cycling stem cell population found ~4-5 cell positions above the base of the crypt (Sangiorgi and Capecchi, 2008), and a rapidly cycling stem cell population composed of slender-shaped cells [so-called crypt base columnar cells (CBCCs)] that are sandwiched between Paneth cells at the base of the crypt (Barker et al., 2007). The stem cell characteristics of the two compartments have been established using elegant genetic-lineage tracking techniques. The former cell population expresses Bmi1, a component of the polycombrepressing complex 1 (PRC1) that prevents stem cell senescence; this Bmi1-expressing population can give rise to clones that contain all intestinal lineages. CBCCs, by contrast, express the Wnt target gene Lgr5, which encodes an orphan G-protein-coupled receptor (Barker and Clevers, 2010); these Lgr5-expressing cells are also multipotential stem cells (Barker et al., 2007). Targeted deletion of the tumour suppressor gene Apc (adenomatous polyposis coli) in Lgr5-expressing CBCCs resulted in rapidly growing adenomas, whereas targeted deletion of Apc in the higher-positioned TACs that are the direct descendants of either stem cell population failed to induce substantial adenoma growth (Barker et al., 2009). These data illustrate that a mutation in a stem cell population is most effective for tumour initiation in the short term. However, small adenomas derived from TACs persisted for nearly a year in this model, so the possibility that cancer later arises from these adenomas cannot be excluded, particularly given the fact that human colorectal cancers grow very slowly (Camplejohn et al., 1973). Intriguingly, 36 days after induction of th...