Paul J. Tadrous

Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per‐operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol‐fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the ‘lifetime’ factor τ. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger τ (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean τ of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy‐associated stroma (p < 0.05). Also, benign collagen had a longer τ than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy‐associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy‐associated stroma were obtained even with small patient numbers, indicating that lifetime‐based instruments can be developed for real‐time diagnostic imaging with microscopic resolution. Copyright

Locating the stem cell niche and tracing hepatocyte lineages in human liver.

We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser‐capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of cytochrome c oxidase–deficient hepatocytes were clonal, suggesting an origin from a long‐lived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase‐deficient hepatocytes were nonpathogenic. Conclusion: these data show, for the first time, that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins. (HEPATOLOGY 2009.)

The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice

After liver injury, parenchymal regeneration occurs through hepatocyte replication. However, during regenerative stress, oval cells (OCs) and small hepatocyte like progenitor cells (SHPCs) contribute to the process. We systematically studied the intra‐hepatic and extra‐hepatic sources of liver cell replacement in the hepatitis B surface antigen (HBsAg‐tg) mouse model of chronic liver injury. Female HBsAg‐tg mice received a bone marrow (BM) transplant from male HBsAg‐negative mice, and half of these animals received retrorsine to block indigenous hepatocyte proliferation. Livers were examined 3 and 6 months post‐BM transplantation for evidence of BM‐derived hepatocytes, OCs, and SHPCs. In animals that did not receive retrorsine, parenchymal regeneration occurred through hepatocyte replication, and the BM very rarely contributed to hepatocyte regeneration. In mice receiving retrorsine, 4.8% of hepatocytes were Y chromosome positive at 3 months, but this was frequently attributable to cell fusion between indigenous hepatocytes and donor BM, and their frequency decreased to 1.6% by 6 months, as florid OC reactions and nodules of SHPCs developed. By analyzing serial sections and reconstructing a 3‐dimensional map, continuous streams of OCs could be seen that surrounded and entered deep into the nodules of SHPCs, connecting directly with SHPCs, suggesting a conversion of OCs into SHPCs. In conclusion, during regenerative stress, the contribution to parenchymal regeneration from the BM is minor and frequently attributable to cell fusion. OCs and SHPCs are of intrinsic hepatic origin, and OCs can form SHPC nodules. (HEPATOLOGY 2006;43:316–324.)

Studying biological tissue with fluorescence lifetime imaging: microscopy, endoscopy, and complex decay profiles

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.

Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging.

This second article in the series shows how fluorescence lifetime imaging allows natural biochemical and physiological properties of tissues to act as contrast agents and so provide a basis for distinguishing normal and diseased tissue components. When combined with methods for imaging through non‐transparent tissues and tomographic reconstruction it shows promise as a new optical biopsy technique. In addition to this, specially designed vital fluorescent probes of specific biochemical, secondary messenger and receptor activity in living cells may be imaged using FLIM. This is the youngest of the techniques covered in these review articles on imaging, the first FLIM images of cells having been produced in 1994. Copyright

Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

Summary Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+). Furthermore, we show that, in adenomatous crypts (APC−/−), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.

A methodological approach to tracing cell lineage in human epithelial tissues.

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 cells 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 CELLS 2009;27:1410–1420

Use of Methylation Patterns to Determine Expansion of Stem Cell Clones in Human Colon Tissue

BACKGROUND & AIMS It is a challenge to determine the dynamics of stem cells within human epithelial tissues such as colonic crypts. By tracking methylation patterns of nonexpressed genes, we have been able to determine how rapidly individual stem cells became dominant within a human colonic crypt. We also analyzed methylation patterns to study clonal expansion of entire crypts via crypt fission. METHODS Colonic mucosa was obtained from 9 patients who received surgery for colorectal cancer. The methylation patterns of Cardiac-specific homeobox, Myoblast determination protein 1, and Biglycan were examined within clonal cell populations, comprising either part of, or multiple adjacent, normal human colonic crypts. Clonality was demonstrated by following cytochrome c oxidase-deficient (CCO⁻) cells that shared an identical somatic point mutation in mitochondrial DNA. RESULTS Methylation pattern diversity among CCO⁻ clones that occupied only part of a crypt was proportional to clone size; this allowed us to determine rates of clonal expansion. Analysis indicated a slow rate of niche succession within the crypt. The 2 arms of bifurcating crypts had distinct methylation patterns, indicating that fission can disrupt epigenetic records of crypt ancestry. Adjacent clonal CCO⁻ crypts usually had methylation patterns as dissimilar to one another as methylation patterns of 2 unrelated crypts. Mathematical models indicated that stem cell dynamics and epigenetic drift could account for observed dissimilarities in methylation patterns. CONCLUSIONS Methylation patterns can be analyzed to determine the rates of recent clonal expansion of stem cells, but determination of clonality over many decades is restricted by epigenetic drift. We developed a technique to follow changes in intestinal stem cell dynamics in human epithelial tissues that might be used to study premalignant disease.

The human urothelium consists of multiple clonal units, each maintained by a stem cell

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual‐colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO‐proficient and ‐deficient areas were individually laser‐capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO‐deficient patches could be observed in normal urothelium of all cystectomy specimens. The two‐dimensional length of these negative patches varied from 2–3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO‐deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell‐derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another. Copyright

Methods for imaging the structure and function of living tissues and cells: 1. Optical coherence tomography.

This is the first in a series of review articles which aim to present a concise and systematic overview of the principles, limitations, advantages, and uses of some of the more important recently developed techniques capable of imaging living histology. Optical coherence tomography (OCT) is now an established optical biopsy method, imaging 2–3 mm into opaque tissue. It is analogous to optical ‘ultrasound’ but has an outstanding resolution, being capable of imaging single cells in the intact animal via a surface, intravascular or endoscopic approach. Both two‐dimensional (2D) and three‐dimensional (3D) image datasets can be acquired and studied over time (4D imaging) in the live animal or human subject without the need to remove tissue or perform any tissue processing or staining. It has been used in ophthalmology, gastrointestinal tract (GI) studies, gynaecological tract investigation, and endovascular imaging, to name but a few areas. A degree of differential tissue contrast information can also be gleaned, since different tissue components give different OCT reflectivity signals such that adipose, muscle, collagen, and elastic components may all be resolved without staining. Continuing developments include faster data acquisition for real‐time recording and Doppler OCT for more functional imaging. Copyright