Stems to GEMs

Since 2005, the American College of Veterinary Pathologists Intersociety Experimental Pathology Committee has organized an annual symposium in conjunction with the American Society for Investigative Pathology (http://www.asip.org/), during its annual meeting at Experimental Biology (http://experimentalbiology.org/content/AboutEB.aspx). The mission of these meetings is to create scientific sessions that further the interactions and foster the collaborations between veterinary and physician scientists and pathologists. The 2011 symposium focused on stem cells and genetically engineered mouse models (GEMs).

The principal goal of this symposium was to provide a broad view of ways that stem cell technology has influenced our understanding of the biology of human and animal disease, as well as our concepts of therapeutic horizons. There have been great strides in the development, use, and interpretation of GEMs, and the collaborative role of the veterinary pathologist has been one key component of the successful advancement of this technology. This year’s symposium highlighted the spectrum of advances in this technology and emphasized many important questions that still remain.

The first talk, presented by Dr Bruce Bunnell (Tulane University), “Stem Cell Therapies in Animal Models of Lysosomal Storage Disease,” began with a perspective of the disease course and the therapeutic options for lysosomal storage diseases in human beings. He focused on the pathology of Krabbe disease, or globoid cell leukodystrophy, which occurs when there is a deficiency of functional galactosylceramidase caused by a mutation in the GALC gene. Enzyme replacement therapies have not worked well, in part because of poor blood-brain barrier penetration. While bone marrow transplantation can result in durable responses, there is no cure for this devastating and ultimately fatal disease. Genetic models of globoid cell leukodystrophy have been identified in multiple animal species, from mice to rhesus monkeys. The twitcher mouse is a well-studied model that is a true genetic homolog to the human disease; however, the enzyme deficiency in mice manifests with more peripheral than central nervous system effects, as compared to humans. ¹² In studies evaluating the potential therapeutic role of stem cells, Dr Bunnell presented evidence that one mechanism contributing to the delay in neurologic dysfunction with syngeneic transplantation of stem cells is a decrease in inflammation in the twitcher mouse. ¹¹ Green fluorescent protein–expressing stem cells derived from mesenchymal and neural compartments persisted in vivo up to approximately 20 days following injection in 10-day-old mice and could be detected by polymerase chain reaction and fluorescence microscopy. Interestingly, while injected stem cells did not undergo neural differentiation and integration into the neuropil, the progression of disease was retarded, as evidenced by reduced proinflammatory cytokines, macrophage infiltration, and microglial activation. These immunomodulatory effects persisted 10 to 15 days beyond the survival of the stem cell population itself. Ongoing development of improved delivery methods, predifferentiation of cells prior to therapeutic delivery, as well as use of stem cells engineered to overexpress cerebroside-β-galactosidase are all resulting in prolonged therapeutic benefits of stem cell transplantation in the model. These data indicate that stem cell therapies continue to show promise in the treatment of lysosomal storage disorders.

In the second presentation, “ES Cells: Little GEMs of Knowledge,” Dr Rani Sellers (Albert Einstein College of Medicine) spoke about the importance of genetic contributions from embryonic stem (ES) cells, many unintended, that can influence the resultant phenotype of the GEM. Dr Sellers began by presenting the history of the origin and evolution of many strains of laboratory mice that are important in developing genetic engineering tools in common use today. She provided an essential history for understanding the use of ES cells in constructing GEMs, and she introduced experimental considerations that are critical for successful phenotypic analysis of the GEM. She highlighted the importance of understanding the wide variety of strain-related features (anatomic, clinical, immune, etc) that are commonly found in GEM phenotypes.^4,7,15,16 The origin of these phenotypes can frequently be traced to the specific mouse strain from which the different ES cells were derived; these phenotypes are notably different from the background strain phenotype into which the ES cells have been introduced. She discussed several strain-dependent pathologies, such as ulcerative dermatitis and age-related hearing deficits typical of C57BL/6 mice, and the potential for teratoma formation and failed corpus callosum development in 129 mice. These strain-related features may be observed in GEMs created with ES cells derived from these strains, and, regrettably, such strain-related effects are sometimes published inadvertently as target gene effects. Other important points that Dr Sellers raised included the benefits of evaluating phenotype in congenic mice, effects of genetic drift among distinct populations of mouse strains, and properties acquired from insertion of DNA flanking a transgene.^3,6

A presentation by Dr Ruth Sullivan (University of Wisconsin), “Advanced Imaging Methods to Characterize GEMs,” provided a cutting-edge view of translational research using advanced imaging technologies to study GEMs. She presented 3 scenarios that integrated the functional biology, anatomy, and histology of the whole animal model through temporal and spatial analysis of gene expression at the cellular level. In the first example, the work directly tested the paradigm that tumors are monoclonal and demonstrated the polyclonality of familial murine adenomas. Initial studies involved mice generated from aggregation chimeras with LacZ-tagged cells using 2-dimensional light microscopy, progressing to current work employing multiphoton laser-scanning microscopy on cell populations tagged with fluorescent protein reporter genes. ¹³ Dr Sullivan demonstrated that both confocal imaging and multiphoton imaging provide optical sectioning capabilities but that greater tissue penetration with improved signal to noise and less tissue damage is possible with multiphoton imaging. In the second portion of the talk, she illustrated advantages of exploiting photon emission duration (fluorescence lifetime) or wavelength (fluorescence spectrum) of endogenous fluorophores (eg, flavin adenine dinucleotide) to detect the redox state of living cells or interpret the biochemistry of protein binding—for example, in differentiating cancer cells within normal tissues. ² This technology is moving into increased use within the living animal (intravital imaging), which will provide interpretation of cellular biochemistry in the context of the same animal over time. Finally, Dr Sullivan introduced the usefulness of studying biology during structural remodeling of development or disease through interpreting second harmonic signals naturally generated via the anisotropy of collagen. She summarized work identifying the progression of mammary and prostate cancer by evaluating stromal remodeling.^1,9,10 She exemplified the value of veterinary pathologists, with their background in histopathology, physiology, and biochemistry, as ideal research team members investigating imaging at the cellular level in the context of the whole animal.

A presentation by Dr Dorothy French (Genentech), “Shedding Light on Ubiquitin Modification in Cancer,” concluded with an elegant dissection of regulatory and physiologically critical processes in ubiquitination, a posttranslational mechanism of controlling intracellular protein levels. After introducing ubiquitin modification of target proteins as a multifunctional molecular process, she provided examples of linkage-specific effects of polyubiquitination, such as K48-linked ubiquitin targeting of p53 or IKKβ for degradation, versus K63-linked ubiquitin modification of NEMO/IKKγ ⁵ functionality. With this background, she focused on how GEMs were used to define contributions of ubiquitination to prostate cancer progression. The protein encoded by COP1 (constitutively photomorphogenic, relating to its discovery in plants) is responsible for regulation of more than 20% of the plant genome as an E3 ligase in the ubiquitination pathway, and it has functional tumor suppressor activity in mammalian cell systems. ⁸ COP1, with one of its interaction partners, DET, leads to the degradation of ETV1, a nuclear transcription factor. ETV1 has been found to be overexpressed in numerous prostate cancer patients. Examination of the translocated gene demonstrated fusions that resulted in loss of the degron (COP1 binding motifs), thereby allowing the ETV1 fusion product to escape ubiquitination and degradation mediated by COP1. Utilizing tissue-specific COP1 deletion mice, Dr French and her colleagues first demonstrated that early neoplastic changes, prostate intraepithelial neoplasia, correlated with elevated ETV1 protein expression in areas of COP1 loss. A subsequent cross of COP1 conditional knockout mice to PTEN conditional knockout mice resulted in high-grade prostate intraepithelial neoplasia that progressed to invasive carcinoma, supporting the hypothesis that loss of COP1-regulated ETV1 degradation contributes to tumorigenesis in the prostate gland. ¹⁴

Summary

Collectively, these presentations introduced the audience to the roles of ES cells in generating phenotypes of transgenic animals, and they provided examples where the GEMs were used to define molecular mechanisms of disease or where ES cells were used as a therapeutic modality.

Points of discussion among audience members reinforced the importance of strain-associated background lesions in animal models, technological advances in imaging functional biology, opportunities for stem cell therapies, and ubiquitination in regulation of cell proliferation.

The 2012 American College of Veterinary Pathologists symposium “Evolutionary Aspects of Animal Models” will focus on the proper selection of a relevant animal model in biomedical research as critical to investigative success.

Recent work characterizing rapid evolutionary changes and differences in physiology between species questions the validity of some comparative models.

Dr Robert Hamlin will be speaking on cardiovascular disease in “Animals as Models of Human Cardiovascular Disease: Or the Search to Overcome Outdated Evolutionary Homeostatic Mechanisms.” Dr Stefan Niewiesk will discuss evolutionary factors that affect modeling the human immune system in “Of Mice and Men: Evolutionarily, What Are the Best Rodent Models of the Human Immune System for Infectious Disease Research?” Dr Steven Austad will consider evolution in “Evolutionary Aspects of Animal Models of Aging.” Finally, Dr Elizabeth Uhl will conclude the session with “Modeling Disease Phenotypes: How an Evolutionary Perspective Enhances the Questions.”

2011 Intersociety Experimental Pathology Committee

• Elizabeth Galbreath, Lilly Research Laboratories, Indianapolis, Indiana

• Mark McArthur, The University of Texas MD Anderson Cancer Center, Bastrop, Texas

• Wendy Halpern, Genentech, South San Francisco, California

• Elizabeth Uhl, University of Georgia, Athens, Georgia

• Michael J. Oglesbee, Ohio State University, Columbus, Ohio

• Arlin B. Rogers, University of North Carolina, Chapel Hill, North Carolina

• Tim LaBranche, Pfizer, Cambridge, Massachusetts

• Wayne Buck, Abbott Laboratories, Abbott Park, Illinois

• Elizabeth M. Whitley, Iowa State University, Ames, Iowa

• R. Mark Simpson, National Cancer Institute, Bethesda, Maryland