A Methodological Approach to Tracing Cell Lineage in Human Epithelial Tissues

Abstract

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

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.…”

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

“…CCO deficiency usually is attributable to a mutation of the mtDNA where the gene is encoded; thus the shared ancestry of a patch of CCO − crypts can be demonstrated by their having a clonal mutation. Furthermore, the presence of multiple cell lineages within a CCO − clone demonstrates that the clone contains a multipotential stem cell (16). A serendipitous means to study dynamics and infer rates of clonal expansion in human tissues is via analysis of methylation patterns of CpG islands associated with nonexpressed genes (17).…”

Clonal expansions in ulcerative colitis identify patients with neoplasia

“…More recently, these investigators have capitalized on the high frequency of epimutations in CpG islands of nontranscribed genes to study the clonal structure of non-MSI tumor development (42). Wright and colleagues have recently used the stainable phenotype of neutral, homoplasmic mutations in a mitochondrially encoded cyclooxygenase gene to study cell lineages in variety of tissues (43). Although the process by which homoplasmy arises is not well understood and 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 takes many years to develop in some tissues (32), the ability to directly visualize the individual cells of a clonal population in an unperturbed tissue context makes this a promising technique with the potential to complement our studies.…”