Office: Science Center room #223
Phone: (415) 482-1910
While heading a local biotechnology enterprise I lead a scientific team investigating the capacity of human cells to spontaneously cell sort to reform tissue layers. Our results lead to the commercialization of 3-D organ models (AccuOrgans), where multiple tissue layers reform correctly by tapping into cell motility and adhesion. The fundamental discoveries made open up many new opportunities to use and characterize these new products of tissue engineering. I list a few areas where Dominican students can learn cell and molecular biology techniques while gaining experience with our novel products, enabling them with unique skills. Students will have the chance to work on research projects, in some cases in collaboration with other scientists located internationally.
Project 1: Active ingredient screening and toxicity testing in in vitro 3-D organ cultures, animal-free testing
Evaluation of the biological properties of drugs and active ingredients is a key step in new product development of pharmaceuticals and personal care products that have greatly improved the quality of life. Functional assays conducted on biological material, both on cultured cells and animals, comprise most of the steps in the development of pharmaceuticals, yet the biological materials currently relied upon, cell monolayers and tests on laboratory animals, just have not provided the sensitive assays needed, are prone to artifacts, and often do not identify off-target effects. Students will gain hands on experience with the new biological material that will replace many of the older assays, human 3-D organ cultures setup in multi-well plates for use in higher throughput plate readers. Using test samples containing known compounds for evaluation the new assays will be validated for measurement of viability using MTT as a measure of toxicity over a range of concentrations. Immunological methods, ELISA assays and immunohistochemistry on known biomarkers will be conducted to evaluate morphological changes in response to the test compounds. Panels of known toxicological standards as well as known active ingredients will be evaluated and the results standardized. The complete replacement of animals through appropriate internationally validated animal-free testing methods is a societal goal. The European Centre for Validation of Alternative Methods (ECVAM), and a similar agency in the U.S. (ICCVAM) established in the U.S. in 1997 as an offshoot of the Food and Drug Administration (FDA), will be contacted concerning our project results.
Project 2: Exploring mechanisms of cell motility and adhesion using in vitro 3-D organ cultures
The fact that cells expanded in tissue culture retain the ability to reorganize into tissues and organs in vitro creates a unique opportunity to explore the timed sequence of events responsible, and uncover the mechanisms involved. Cell motility is influenced by cell-matrix interactions and responds to cytokines and chemokines. Integrins, cadherins, and selectins are major classes of adhesion molecules that are investigated to determine their roles. Specific molecules can be blocked using specific antibody reagents and RNAi can be used to knock down expression levels, offering tools to dissect the role of each component. Likewise, the formation of cell junctions can be explored, including adherens junctions, desmosomes, focal contacts, gap junctions, hemidesmosomes, and tight junctions. The 3-D organ cultures offer a unique opportunity to detect the timed sequence of events occurring during tissue remodeling because all the cells respond en masse, simplifying detection and characterization.
Project 3: Recapitulation of disease phenotypes in vitro by the use of 3-D organ models
Modeling a disease in vitro can provide a valuable tool for a more complete understanding of the disease and also can provide convenient assays for screening compounds that can be used in treatment. Two types of disease will be pursued, genetic diseases, where a genetic defect is the underlying cause, and autoimmune diseases, where the patient makes an antibody against one of their own proteins, resulting in some loss of function. Several diseases can be modeled by simply incorporating cells from a patient into the appropriate 3-D organ model. For example, epidermolysis bullosa (EB) is a skin blistering disease that is caused by a genetic defect in collagen VII (dystrophic EB), laminin-5 or other hemidesmosome components (junctional), or keratins 5 or 14 (simplex). By incorporating cells from patients into our 3-D organ models, a similar phenotype will be recapitulated. An example of an autoimmune disease, pemphigus vulgaris causes blistering and sores and occurs when antibodies attack desmoglein (dsg) 3, a protein that keeps cells bound together. By adding blocking antibodies to the 3-D organ models we will characterize the extent of recapitulation of the disease. A shortage of good in vitro disease models has slowed the progress in treatments for a variety of diseases, so our contribution of providing models that recapitulate the pathophysiology of disease has the potential of advancing many fields. We will collaborate with outside medical research institutions to provide a variety of disease models to assist in the advancement of treatments and cures.
Project 4: Adult human stem cells derived from skin, their role in skin morphology, and their differentiation into other cell types
Adult human stem cells reside in both the bulge region of the hair follicle and interspersed in the interfollicular epidermis. Adult stem cells are present throughout the body to serve as a source of potent replicative capacity for supplying replacement cells. For example, in the epithelia of the skin, the mouth, and the gut the outer layers of cells are constantly being sloughed off. The successful engineering of replacement tissues and organs requires that stem cells are incorporated into the newly made replacements. This requires routine successful isolation and propagation of stem cells, for which we established a dependable procedure using skin stem cells. We are researching the best ways to reestablish the stem cell niche within reconstructed epithelial tissues, and whether stem cells can be added into in vitro cell sorting systems and will migrate to appropriate positions within the constructs, which so far seems to be the case based on observed positioning of fluorescently tagged stem cells (by CFDA or quantum dot). Because the skin is a rich source of stem cells, we are interested in exploring potential applications for these cells and to what extent do our skin stem cells retain their ability to differentiate into other cell types.
Project 5: Wound healing and the role of epithelial cells in the process of revascularization
Our unique in vitro 3-D model of a skin wound (AccuWound) has resulted in surprising observations that will be further investigated. Although epithelial cells were known to migrate into the wound bed others data has not been convincing about what their role is, and they are typically described as ‘sealing off the wound’. Our data presented at the Society of Dermatology Conference 2007 indicates that the migrating epithelial cells form a spongy scaffold of apoptotic cells that we believe serves as a provisional matrix for holding endothelial cells and other blood cell types required to achieve revascularization of the wound. Students working with AccuWound will determine if the epithelial cell provisional matrix contains the necessary adhesion molecules to allow endothelial cells to migrate in and bind, and using in vitro cell migration assays will determine if this occurs in vitro using boyden chamber assays. The role of cytokines, such as VEGF and PDGF will be evaluated, and the possible role for inflammatory cytokines in preparing the endothelial cells for binding the matrix. If our modeled provisional matrix has the characteristics needed to localize the revascularization of wounds it would serve as a medically useful graftable material for use in acceleration of wound healing.
Musical training in organ and piano, enjoy composition. Hiking, running, swimming, skiing