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WIREs Dev Biol
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Cell lineage tracing in human epithelial tissues using mitochondrial DNA mutations as clonal markers

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The study of cell lineages through heritable genetic lineage tracing is well established in experimental animals, particularly mice. While such techniques are not feasible in humans, we have taken advantage of the fact that the mitochondrial genome is highly prone to nonpathogenic mutations and such mutations can be used as clonal markers to identify stem cell derived clonal populations in human tissue sections. A mitochondrial DNA (mtDNA) mutation can spread by a stochastic process through the several copies of the circular genome in a single mitochondrion, and then through the many mitochondria in a single cell, a process called ‘genetic drift.’ This process takes many years and so is likely to occur only in stem cells, but once established, the fate of stem cell progeny can be followed. A cell having at least 80% of its mtDNA genomes bearing the mutation results in a demonstrable deficiency in mtDNA‐encoded cytochrome c oxidase (CCO), optimally detected in frozen tissue sections by dual‐color histochemistry, whereby CCO activity stains brown and CCO deficiency is highlighted by subsequent succinate dehydrogenase activity, staining the CCO‐deficient areas blue. Cells with CCO deficiency can be laser captured and subsequent mtDNA sequencing can ascertain the nature of the mutation. If all cells in a CCO‐deficient area have an identical mutation, then a clonal population has been identified; the chances of the same mutation initially arising in separate cells are highly improbable. The technique lends itself to the study of both normal epithelia and can answer several questions in tumor biology. WIREs Dev Biol 2016, 5:103–117. doi: 10.1002/wdev.203 This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Methods and Principles Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease
(a) A single cytochrome c oxidase (CCO)‐deficient patch appearing to emanate from the portal tract (PT). (b) High‐power magnification illustrates that within the patch there are CCO‐positive sinusoid‐lining cells (asterisk), clearly pointing to the fact that, as expected, such cells are of different cell lineages from hepatocytes. Patient was a 61‐year‐old man. (Reprinted with permission from Ref . Copyright). The portal tract to hepatic vein orientation of the patch might indicate a stem cell and lineage system emanating from the periportal area (the so‐called streaming liver) and genetic lineage labeling in mice has supported such a proposal. On the other hand, several studies have not found evidence that rodent liver homeostasis is maintained by a constant input of cells from the portal region, while other studies in rats point to clones arising diffusely throughout the parenchyma expanding centrifugally producing fractal patches. (Reprinted with permission from Ref . Copyright 2009 John Wiley and Sons)
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Tracing lineages through heritable genetic changes in mitochondrial DNA (mtDNA). (a) A mtDNA mutation can occur spontaneously in a single circular genome and (b) through genome duplication and turnover can be present in all copies of the genome within a single mitochondrion and then (c) through the many mitochondria in a single cell, evolving from a heteroplasmic state to a near‐homoplasmic state, a process known as genetic drift. A cell in which at least 80% of its mtDNA is mutated results in a deficiency of cytochrome c oxidase (CCO), which can be detected by dual enzyme histochemistry. CCO activity will stain brown, whereas CCO‐deficient cells will stain blue. (d) Laser‐capture microdissection of blue and brown cells and subsequent sequencing of the mitochondrial genome can identify the mutation. If all CCO‐negative cells in a patch harbor an identical mutation, the population is considered to be clonal.
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Recognizing cytochrome c oxidase (CCO)‐deficient cells through CCO/succinate dehydrogenase (SDH) dual enzyme histochemistry. Left panel shows standard H&E staining of colonic crypts. Right panel shows the dual‐color enzyme histochemistry method performed on frozen sections of colonic crypts to simultaneously detect CCO and SDH activity. Cells expressing CCO stain brown and cells deficient in CCO stain blue. (Prepared by Marnix Jansen)
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Multipotent stem cells reside within clonal cytochrome c oxidase (CCO)‐deficient adenomatous crypts. (a‐i) H&E staining showing a tubular adenoma with low‐grade dysplasia. (a‐ii) CCO‐enzyme histochemistry identifying two patches of multiple, blue, CCO‐deficient crypts. (a‐iii and a‐iv) Laser‐capture microdissection of areas in (a‐ii) outlined in red and (a‐v) mtDNA sequencing of single cells from multiple blue crypts and within the same blue crypts versus adjacent brown, wild‐type crypts demonstrated that all blue crypts shared a common, clonal point mutation in their mtDNA that was not present in adjacent brown crypts. (b) Immunofluorescence staining of serial sections from the adenoma. Clonal CCO‐deficient crypts contained cells positive for markers of neuroendocrine cells (chromogranin A) and secretory cells [mucin2 (MUC2) and mucin5AC (MUC5AC)], indicating these crypts contained a multipotential stem cell that had produced these distinct cell types. Detection of CCO expression was conducted on the same section as chromogranin A after visualization for chromogranin A expression. Detection of MUC2 and MUC5AC expression was conducted on the same section simultaneously. Negative controls were isotype matched at the same concentration as the corresponding primary antibody. Asterisks indicate the crypt enlarged in high‐power images. (Scale bars: ∼50 µm in low‐power images and 25 µm in high‐power images. (Reprinted with permission from Ref . Copyright 2013. Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution, PNAS.)
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Cytochrome c oxidase (CCO)/succinate dehydrogenase (SDH) histochemistry and mtDNA sequencing of a basal cell carcinoma (BCC). (a, b) The entire tumor is CCO‐negative (blue) as is the outer root sheath (ORS) of a neighboring hair follicle, which is clearly contiguous with the BCC. (c): Cells from the CCO‐negative BCC and CCO‐negative ORS were laser captured and microdissected, and polymerase chain reaction (PCR) sequencing identified a clonal T>C transversion at position at 13,740 (d) with CCO‐positive cells (brown) remaining wild type (e). This indicates that the inner root sheath lineages and companion layer are derived from a separate stem cell pool to the ORS, and strongly suggests that the BCC has arisen from the ORS. (Reprinted with permission from Ref . Copyright 2008 John Wiley and Sons)
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Prostatic intraepithelial neoplasia (PIN) and prostate cancer are clonally related entities. (a, b) Within a cytochrome c oxidase (CCO)‐normal cancer area, a deficient blue cancer subclone and an area of PIN with both CCO‐positive and deficient acini can be seen (the area of PIN is circled in red). (c) The whole cancer area is circled in blue. (b) PIN classification was confirmed by positive basal cell immunostaining for CK5/6. (c) PIN comprises both brown CCO‐proficient (4 + 6) and blue CCO‐deficient (7, 9, and 10) acini. Deficient areas in PIN and cancer show an identical 5982 G>A mtDNA mutation, indicating a clonal relationship. Notably, not all of the cancer, or the PIN lesion, was CCO deficient, suggesting that the cancer was formed of two independently initiated clones. CCO/succinate dehydrogenase (SDH) staining. (Reprinted with permission from Ref . Copyright 2011 John Wiley and Sons)
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Mitochondrial DNA genotyping indicates that regenerative nodules can be derived from CK19‐positive ductular cells. (a) An entirely cytochrome c oxidase (CCO)‐deficient nodule (blue). (b) Five groups of cells (1–5) from the same CCO‐deficient nodule, cells (6) from the adjacent CCO‐deficient ductular reaction, confirmed by cytokeratin‐19 IHC on the next serial section (c, brown staining), and cells (7) from the CCO‐positive nodule (brown) were laser‐capture microdissected and the entire mitochondrial genome was sequenced. (d) Cell areas 1–5 all contained four different transition mutations: 2145G3 A, 2269G3 A, 12362C3 T, and 15671A3G (black arrows). (e) Cell area 6 from the abutting CCO‐deficient ductular reaction had exactly the same mutations. Heteroplasmy was detected at base‐pair locations 2145 and 2269 (arrowheads), whereas the mutations at base‐pair locations 12362 and 15671 were homoplasmic (black arrows). (f) Cell area 7 from the CCO‐positive nodule had no mutation (white arrows). (Reprinted with permission from Ref . Copyright 2010 John Wiley and Sons)
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Cytochrome c oxidase (CCO)/succinate dehydrogenase (SDH) histochemistry on sections of intestinal metaplasia in the human stomach. All the major differentiated intestinal epithelial cell lineages are present in the metaplastic crypt, which is flanked by CCO‐positive crypts. By immunohistochemistry, (a) CCO, (b) MUC2‐positive goblet cells, (c) chromogranin A‐positive neuroendocrine cells, (d) IgG‐matched isotype control, (e) CD10‐positive enterocytes, and (f) Alcian blue/diastase stain revealing goblet cells. This is strong evidence that intestinal metaplastic crypts can undergo monoclonal conversion and that they contain multipotential stem cells. (Reprinted with permission from Ref . Copyright 2008 Elsevier)
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Human small bowel crypts are clonal and contain multiple stem cells. (a) A mixed crypt in transverse section (arrow). (b) A patch of cytochrome c oxidase (CCO)‐deficient crypts, suggesting that a CCO‐deficient crypt has divided. All differentiated epithelial cells are derived from a single stem cell. Serial sections showing (c) an entirely CCO‐negative crypt (arrow) contains: (d) PAS/Alcian blue/diastase‐stained goblet cells, (e) CD10‐positive enterocytes, (f) lysozyme‐positive Paneth cells, and (g) chromogranin A‐positive neuroendocrine cells. (h) An isotype‐matched negative control. (a–h: Reprinted with permission from Ref . Copyright 2009 John Wiley and Sons)
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Dual‐color cytochrome c oxidase (CCO)/succinate dehydrogenase (SDH) histochemistry can detect partially mutated colonic crypts. Partially mutated crypts indicate the presence of multiple stem cells within the crypt. Niche succession can lead to monoclonal conversion of the crypt whereby both all stem cells and their progeny have an identical mutation, or the mutated stem cell is lost from the crypt. (Prepared by Marnix Jansen)
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