Introduction Deficiency in the enzyme cytochrome C oxidase (CCO) has proven to be a versatile marker of clonal population in human tissues. CCO is encoded entirely by the mitochondrial DNA (mtDNA) and deficiency in CCO expression is usually attributable to mutation in the mtDNA. CCO-deficient cells are easily detectable by two-colour enzyme histochemistry. This staining provides means to identify clonal patches of CCO-deficient cells. Subsequent sequencing of mtDNA from individual cells within the patch and revealing the same somatic mutation in each cell confirms the clonality of a patch.
In the human intestine, crypts are composed of a population of a contiguous CCO-deficient mtDNA-mutated cells, and also non-mutant CCO-proficient cells are frequently observed: from the small clones occupying only a few cell positions on the crypt circumference (partially-mutated crypts), to crypts composed only CCO-deficient cells (wholly-mutated crypts). Patches of adjacent and clonal CCO-deficient crypts are also observed. Larger patches are more common in older patients indicating that CCO-deficient crypts continue to divide in the ageing colon.
Methods To study stem cells dynamics within intestinal crypts, we aim to characterise the shape and the size of clones in partially CCO-deficient crypts, by combining the two-colour enzyme histochemical staining with image analysis and computer reconstruction.
Multiple serial sections in the transverse plain were taken through frozen samples. Digital images were then taken of each serial section and used to create a ‘crypt map’ using in-house analytical software. A crypt map is a representation of the whole 3D tubular crypt unfurled and laid flat with colour enhancement post-processing.
Results Our results in normal and diseased human colon show that clone size can be approximated by the percentage of the crypt circumference measured from crypt transverse sections and occupied by a CCO-deficient clone.
Conclusion We envisage that analysis of such clonal distributions in the context of a branching process model could be used to determine the patterns of stem cell division within the human colon.
Disclosure of Interest None Declared.
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