Locating the stem cell niche and tracing hepatocyte lineages in human liver

Abstract

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

Differentiation of progenitors in the liver: a matter of local choice

“…Alison and colleagues have harnessed the inability of the cell to repair mutations in mitochondrial DNA to ask whether human livers have such progenitors (8,45). By identifying cells in which the mitochondria have a COX mutation, this group described patches of epithelium in the liver in which the mitochondria were mutated, and when random, discrete regions of the patch were analyzed the mitochondrial mutation was consistent across the sample.…”

“…In many liver diseases, such as chronic viral hepatitis (5) and non-alcoholic fatty liver disease (6), hepatocytes become senescent and unable to efficiently regenerate the parenchyma. In this scenario, hepatic progenitor cells (HPCs) become activated and are sufficient to regenerate the biliary and hepatocellular epithelium (7,8). The biology of HPCs is less studied in comparison to analogous progenitor cells in other adult tissues, and markers that delineate the stem versus transit amplifying populations are not clearly defined (9)(10)(11).…”

MECHANISMS LINKING mtDNA DAMAGE AND AGING

“…showed how, in aged individuals, there is an increased number of single randomly distributed myocytes and cardiomyocytes without Cytochrome c oxidase (COX) activity [93,94]. COX-deficient succinate dehydrogenase (SDH)-positive single neurons with high mtDNA deletions increase in aged-linked neurodegenerative diseases such as Alzheimer's and Parkinson's disease [95][96][97]; moreover, focal respiratory chain defects accumulate to significant levels in aging human colon [59], liver, pancreas [98], stomach [99], and small intestine [92]. Through clonal expansion, both de novo and inherited mutations would increase with age ( Figure 1).…”

“…Also, patches of hepatocytes deficient in cytochrome c oxidase activity contain clonal mtDNA mutations, suggesting their origin from a stem cell [98].…”

Stem cell self-renewal: lessons from bone marrow, gut and iPS toward clinical applications

“…[4][5][6][7][8][9][10][11][12] With the recent focus on embryonic SCs (ESCs) and induced pluripotent SCs (iPSCs), interest into in vitro generation of HSCs, including signals for HSC expansion and differentiation from these more primitive SCs, has seen a tremendous boost. [13][14][15] As HSC-based therapy is the only type of SC therapy routinely used, much knowledge obtained from the clinical application of HSCs and from genetic modification of HSCs for gene therapy applications can be used to introduce other types of adult SC therapies, and perhaps in the future extend this to include ESCs and iPSCs.…”