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The mouse is the primary genetic model to functionally annotate the mammalian genome and dissect a diverse array of genetic diseases due to the genetic and physiological conservation between mice and humans and, the availability of numerous powerful mutagenic strategies. We are using various strategies, including gene targeting, gene trapping and ENU mutagenesis, to generate and study animal models of hematopoietic, musculoskeletal, and cardiovascular diseases as well as various types of cancer. While gene targeting and gene trapping can be used as a candidate gene approach, ENU mutagenesis is used as a forward genetic strategy in which mutagenized mice are screened for specific disease phenotypes. Genetic mapping is used to clone the ENU mutations. We have developed a sensitized ENU screen to identify mutations affecting hematopoietic development by inducing transient cytopenia and analyzing the recovery. We have identified numerous mutant strains demonstrating clinically-relevant phenotypes, including polycythemia, thrombocythemia, leukocytosis, anemia, and thrombocytopenia. We have recently cloned our first hematopoietic mutant, which presents with thrombocythemia. We are also involved in the generation and analysis of animal models of skeletal diseases using ENU mutagenesis. We and our colleagues have recently generated a mouse model of Oculodentodigital Dysplasia (ODDD), which results from a dominant mutation in Gja1 (Connexin 43) (Flenniken et al., 2005).
One of our primary interests is stem cell biology and stem cell based diseases, including degenerative diseases and cancer. Using a candidate gene approach, we utilize gene targeting and gene trapping (see functional annotation of the mammalian genome and the gene trap web page for more information) to generate mutations in genes we suspect are involved in stem cell function and thus defective expression could lead to disease. One such gene product is Sca-1 (Stem Cell Antigen-1) or Ly6a, which is expressed in many somatic stem cell and early progenitor lineages including those that give rise to the hematopoietic and mesenchymal tissues. Our targeted Sca-1 mutants (Stanford et al., 1997) have a defective hematopoietic (Ito et al., 2003) and mesenchymal stem cell (Bonyadi et al., 2003) self-renewal capacity, resulting in defects in bone marrow transplantation and early onset age-related osteoporosis, respectively. We are using these mice to understand the role of stem cells in tissue maintenance and regeneration.
We have developed strong collaborations within the Toronto cardiovascular scientific and medical community to utilize animal mutants to better understand cardiovascular disease pathogenesis and develop novel cell and pharmacological therapeutics. We have identified two important druggable pathways affecting the initiation and extent of angioplasty-induced restenosis (Wang et al., 2004 and Wang et al., 2005). Current projects include analyzing the role of specific signaling pathways in cardiac remodeling following myocardial infarction and the molecular pathways of endothelial precursor cell function.
In addition to generating models of human disease, the range of molecular lesions generated by ENU leading to subtle amino acid substitutions, splicing errors, or premature termination - a range of mutations similar to those often found in human disease -- can provide a multitude of mutant alleles of a given gene (an allelic series) which can better define the full extent of a gene's function. A complementary approach to ENU mutagenesis and gene targeting for mouse mutant generation is gene trapping, a vector-based, insertional random mutagenesis strategy. Gene trap mutagenesis of mouse embryonic stem (ES) cells efficiently generates random, which can be identified by a sequence tag and can often report the endogenous expression of the mutated gene (reviewed Stanford et al., 2001). Like ENU mutagenesis, gene trapping can create an allelic series, depending on the structure of the gene and where within the gene the vector inserts. As a part of the Centre for Modeling Human Disease (CMHD), we have developed a resource of gene trap insertions screened by in vitro expression assays and sequence tags, organized within relational databases, and freely available to the research community via a user-friendly web interface. The novel vectors that we have developed and implemented in our screen do not require expression of the trapped gene in undifferentiated ES cells for selection, enabling us to mutagenize a unique set of genes, complementing other insertional mutagenesis resource centres of the International Gene Trap Consortium (IGTC), which utilize vectors requiring ES cell expression to select the trapped gene. To date, more than 4000 sequence tags have been deposited in the CMHD searchable database, most of which have been deposited in the NCBI dbGSS and can be found as a ribbon on genome browsers. With our collaborators, we are using computational methods to annotate the tags and predict potential physiological roles of uncharacterized genes, and to generate and phenotypically characterize gene trap mouse mutants in targeted biochemical and developmental pathways. These computational tools to predict gene function are being developed as a new web application for the entire research community. Our lab is primarily interested in using the tools to predict gene function to identify new candidate genes that control stem cell function and are involved in pathogenesis. The CMHD and IGTC gene trap libraries are then used to generate new models of human disease.
Regenerative Medicine is devoted to replacing diseased cells, tissues, or organs, or repairing tissues in vivo by augmenting natural or inducing latent regenerative processes. While the best source of cells to replace a diseased tissue is dependent upon the particular tissue or disease, the limited proliferative capacity of most differentiated cell types, means that in most cases progenitor or stem cells are required to generate new tissue. Thus, central to the goals of regenerative medicine is the manipulation - both expansion and directed differentiation - of stem cells, which are the primary source of de novo tissue regeneration and maintenance of organ homeostasis. Stem cells are unique in their properties to remain undifferentiated or to differentiate. This process must be highly regulated, otherwise the pool of stem cells may prematurely exhaust. In addition to genetic strategies to tease apart the mechanisms driving self-renewal and differentiation described above, we are performing microarray, siRNA, and small molecule screens of embryonic stem cells to identify molecules that influence the balance between self-renewal and differentiation. Working with collaborators, we have developed a high throughput cell-based assay to measure self-renewal versus differentiation using high content screening by the Cellomics ArrayScan automated fluorescent microscope. To mine the data from these screens and develop new hypotheses to test, we are working with collaborators to use support vector machines and other machine learning tools. The results from this work will feed into our cell and molecular therapeutic regenerative medicine research described below.
Through tissue engineering, embryonic stem cells (ESC) have enormous potential to treat cancer and degenerative diseases such as Muscular Dystrophy or Heart Failure. Nearly two decades of differentiating mouse ESC in vitro has demonstrated that an impressively wide variety of developmental programs are accessible in culture. These developmental programs are excellent model systems to study in vivo development, thereby providing proof of principle that human ESC may be used to treat disease using innovative cell replacement therapies. However, there has been limited success in "driving" ESC down specific developmental pathways through cell culture techniques or addition of exogenous molecules such as cytokines. Yet, genetic manipulation of ES cells has allowed researchers to isolate specific cell lineages at high purity, which is critical to prevent teratocarcinoma formation by undifferentiated ESC. One of the reasons for the limited success in driving ESC development along specific developmental pathways has been the lack of sensitive methods to quantify differentiation and the fact that researchers have focused on isolation of mature cells late in the cultures. We have developed and are utilizing novel interrelated genetic and cell culture approaches to instruct mouse and human ESC differentiation to specific lineages beginning with the mesodermal lineages which will enable the development of tissue-engineered constructs for mesoderm-derived tissues including cardiac, vascular, hematopoietic, bone, cartilage, and skeletal muscle.
Concomitant with our embryonic stem cell-based tissue engineering, we are pursuing, where applicable, somatic stem cell-based tissue engineering strategies. Mesenchymal progenitors or stem cells have been best studied from the bone marrow stroma and are relatively accessible, as they can be obtained by a bone marrow aspirate. As a population, these cells have the capacity to differentiate into bone, fat, cartilage, muscle, and support fibroblasts; however, the individual cells capable of this multi-lineage differentiation have not been characterized. Musculoskeletal diseases represent the largest burden to the North American health care system and are major contributors to long-term disability, chronic pain and reduced quality of life. Of this group of diseases, osteoarthritis -- which involves the loss of joint cartilage and is associated with a reduction in joint mobility and increased pain - is the most prevalent. Osteoarthritis is managed with analgesics to reduce inflammation and pain, while replacement of diseased articular joints with prostheses represents the optimal treatment for late-stage joint degeneration. However, synthetic prostheses used to replace joints with end stage arthritis often fail. Working with materials science engineers and pathologists, we are using bone marrow aspirates from sheep, expanding progenitors, inducing differentiation, and growing the chondrocytes on an artificial bone substrate to develop a biphasic cartilage-bone construct for joint replacement. We are currently developing a preclinical model.
Our collaborators have recently identified a novel source of mesenchymal stem cells, derived from the human umbilical cord perivascular cells (HUCPV cells). Together we have demonstrated single HUCPV cells can give rise to at least 5 different mesenchymal cell types in vitro. This is the first demonstration of a true mesenchymal stem cell. We are performing research to understand the biology of HUCPV cells and initiate studies to determine their potential use in regenerative therapies.
Finally, endothelial progenitor cells or EPCs can be derived from peripheral blood or bone marrow. In addition to their potential for use in treatment of vascular diseases such as ischemia or pulmonary hypertension, improved blood flow would expedite recovery from many diseases, suggesting that EPCs may have broad therapeutic potential. We are also interested in manipulating EPCs to form tissue-engineered blood vessels.
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