Tracing stem cell lineages in human epithelial tissues

Most people have become familiar with the concept of using stem cells for the healing of tissues ravaged by old age or disease. Most of the headlines have been made by embryonic stem cells, cells that can be extracted from early embryos, and which possess the versatility of being seemingly able to be coaxed into almost any type of tissue. Less well known are adult stem cells; these are thought to be present in almost all tissues and their continued multiplication throughout life is necessary to maintain tissues like the gut lining and skin where millions of cells are exfoliated every day. We know very little about the identity of such stem cells and thus are not able to extract them, perhaps with the notable exception of blood stem cells which have been used for many decades in life-saving transplants. We are going to use some very novel genetic technology to identify adult stem cells and their progeny in a number of human tissues, principally the large intestine, skin, liver and pancreas. In the large intestine and skin we are seeking to discover if cancers arise from these cells, while in the pancreas if we could locate stem cells, we may be able to expand them outside the body and make them into insulin-producing cells for subsequent transplantation for diabetes. Many people are now suffering from liver failure and cannot be treated because a shortage of livers for transplantation. An alternative strategy is cell transplantation into the failing liver using a healthy donor liver?s own cells called hepatocytes. If we could identify stem cells in the liver, these could be greatly expanded in number outside the body, converted to functional hepatocytes, and transplanted. Thus, our investigation centres on the role of stem cells in human diseases, principally bowel cancer, diabetes and liver failure, diseases that all have a dismal outlook and for which there are still no prospects of cure, despite many years of research.

This project proposal sets out a programme of work designed to define stem cell behaviour in several human epithelial tissues namely the colon, liver, pancreas and skin, where our knowledge of their very nature, location and subsequent fate of their progeny is unknown. We aim to locate clonal proliferative units of tissue and infer the location of the stem cell niche. Following this we will deduce the role of stem/long-lived progenitor cells in the colon, pancreas and epidermis, and their role in the development of liver cirrhosis. We will exploit a technique we have been developing that can detect cells that are deficient in mitochondrial DNA (mtDNA) encoded cytochrome c oxidase (CCO), the terminal enzyme (component IV) in the electron transport chain. These are non-pathogenic mutations that mtDNA acquires; over time they dominate a cell that becomes histologically recognised as CCO-deficient. In the colon at least, this process takes many years, and so is believed to occur in stem cells. Initial observations can be made on routine paraffin wax-embedded tissue sections, but definitive analyses will be carried out on fresh frozen sections where CCO deficient cells can be detected by simple enzyme histochemistry. Subsequently, individual cells can be laser-capture microdissected from multiple sites within a ribbon/patch of similarly CCO deficient cells and their complete mtDNA genome sequenced. The finding of the same mutation in all cells of a group establishes monoclonality, since the odds of two cells acquiring the same mutation is 1:2.48 x 109.
We propose to examine normal and diseased human intestine, liver, pancreas and epidermis. Our preliminary results in the colon places the stem cell niche towards at the base of the crypt. We have also discovered clones originating outwith the conventional stem cell niche, which may be long-lived progenitors and we seek to establish their potentiality. We are developing other techniques based on the methylation sequence tags of non-expressed genes not only to identify clones, but as methylation status is a dynamic process, to study the kinetics of niche succession. We seek the identity of the stem cell niche in all tissues and the role of stem/progenitor cells in disease, e.g. cancer and liver regenerative nodules.