ORIGINAL PAPER Journal of Pathology J Pathol 2011; 225: 181–188 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.2965 In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells John K Blackwood, 1† Stuart C Williamson, 2† Laura C Greaves, 1 Laura Wilson, 2 Anastasia C Rigas, 2 Raveen Sandher, 3 Robert S Pickard, 3,4 Craig N Robson, 2 Douglass M Turnbull, 1 Robert W Taylor 1 * and Rakesh Heer 2,4 1 Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK 2 Northern Institute of Cancer Research, Newcastle University, Newcastle upon Tyne, UK 3 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK 4 Department of Urology, Freeman Hospital, Newcastle upon Tyne, UK *Correspondence to: Professor Robert W Taylor, Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. e-mail: r.w.taylor@ncl.ac.uk † These authors contributed equally to this work. Abstract Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology. Copyright  2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: stem cell fate; prostate; mitochondrial DNA mutations; respiratory chain deficiency Received 13 May 2011; Revised 4 July 2011; Accepted 5 July 2011 No conflicts of interest were declared. Introduction Stem cells are maintained for the life of a tissue and are essential for homeostasis in lineage-specific cel- lular compartments. These cells differentiate into a highly proliferative, transiently amplifying population to expand their numbers before committing to termi- nal differentiation, replacing cells undergoing natural wastage. With age, changes in stem cell homeostasis are thought to produce proliferative imbalances in tis- sue repair that are considered central mechanisms in both benign prostatic hyperplasia (BPH) and prostate cancer [1,2]. Although a mouse prostate stem cell molecular signature has been characterized that can identify single stem cells [3], these findings have not been translated into human models, where, at best, enrichment is demonstrated with similar markers in both benign and malignant prostates [4–7]. In vitro culture-based approaches which generate single cells grown in 2D, devoid of appropriate extra- cellular matrix and stromal cells, disrupt normal stem cell maintenance regulated by the niche environment and lead to an altered phenotype likely to include com- mitted cells [8,9]. In human prostate, identifying the stem cells and their niche to study lineage tracing using gold standard in situ labelling has not been possible. Consequently, accurate studies of stem cell biology are not available and debate remains concerning organiza- tion of prostate epithelia. In particular, there is evidence that there are separate and hierarchically independent populations of dedicated luminal and basal stem cells, but alternative models suggest that basal cells give rise to luminal cells [10–14]. This confusion has stalled Copyright  2011 Pathological Society of Great Britain and Ireland. J Pathol 2011; 225: 181–188 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com