"The Bioengineering-Clinical Collaboration: Considerations for Biotechnology Developent"
Raymond M. Dunn, MD
Professor and Chief
UMass Memorial Plastic Surgery
“Biotechnology” is at the nexus of interdisciplinary development, demographic need and scientific advance where the current moment and proceeding years will display extraordinary potential “clinical” applications for newly established knowledge and technology.
The realization or success of “clinical translation” of these applications will depend upon the breakdown of many traditional disciplinary “silos” and the establishment of new paradigms of collaboration.
The purpose of my comments today will be to present some provocative comments on our existing “development” paths and their strengths and deficiencies. I will discuss some thoughts on areas of bottleneck and deficiency that will need to be addressed if we are to fully realize the ultimate potential of our science and engineering in timely and effective clinical application.
“Remodelling the Genome for Animal Cloning and Cell Therapies: Somatic Cell Nuclear Transfer other Novel Approaches”
Keith Campbell. BSc, DPhil.
Professor of Animal Development
Head, Division of Animal Physiology
The University of Nottingham, School of Biosciences
The production of live offspring by somatic cell nuclear transfer demonstrated that the nuclear genome of somatic cells was not fixed and could be reprogrammed resulting in de-differentiation and re-differentiation for the production of all cell types required for the production of a conceptus. Originally reported in sheep, SCNT has successfully produced offspring in a range of species and has also been used for the production of embryonic stem (ES) cells in the mouse. Although the mechanisms which control this process are only now being unravelled, it is known that reprogramming does not involve changes in DNA sequence but rather epigenetic modification. The ability of the oocyte to reprogram gene expression following SCNT suggested a number of other routes to achieve de-differentiation or trans-differentiation and produce autologous cells for cell transplantation. The lack of human oocytes for production of embryos and isolation of ES cells, prompted trans-species SCNT, some studies have reported the development of blastocyst stage embryos and also the isolation human ES like cells. The production of human embryos for isolation of ES cells is the subject of significant moral and ethical debates and alternatives which did not produce embryos would be a significant advance. As oocyte proteins can reprogram the genome the use of oocyte extracts would provide such an alternative, however the availability and size of human oocytes precludes this approach. Oocytes and eggs of amphibian species are large and readily available and experiments have demonstrated that they are able to reprogram the epigenome of somatic cells. Similarly, extracts from differentiated cells or fusion to ES cells can alter somtic cell gene expression. All of these studies have provided information into the mechanisms underlying reprogramming. More recently this has culminated in the production of ES like cells following transfection of specific genes into somatic cells.
This paper will review the approaches towards nuclear reprogramming; in particular results from my laboratory using SCNT and amphibian extract approaches and discuss some of the factors which may facilitate the reprogramming process.
“Mechanisms and consequences of asymmetric events during cytokinesis”
Stephen Doxsey, Ph.D.
Professor, Cell Biology Department
University of Massachusetts Medical School
Cell division is the fundamental process by which a cell replicates to create two genetically identical daughter cells. Our recent work in cultured cells demonstrates that daughter cells generated during cell division are dramatically different. Through a series of asymmetric events during the final stage of cell division, one daughter inherits the singular midbody and the older copy of the centrosome, two organelles involved in cell division. The cell with the so-called midbody derivative continued to inherit and accumulate these structures in subsequent divisions. Inspection of human tissues unexpectedly demonstrated that cells with accumulated midbodies were primarily, if not exclusively, found instem cells. They were also found in vitro in human embryonic and adult stem cell lines and in most cancer cells, but not in dividing non-stem cells or non-cancer cells. This suggested that several asymmetric event produce long-lived cells and cells destined to die. These observations have important implications for human life span, stem cell self-renewal, stem cell markers, cancer cell immortality, aging disorders and neurodegenerative dementias.
“Cell-Based Approaches to Vascular Tissue Engineering”
Marsha Rolle, Ph.D.
Assistant Professor, Department of Biomedical Engineering
Worcester Polytechnic Institute
Elastin is the predominant extracellular matrix component of muscular arteries. One of the primary unmet challenges in vascular graft tissue engineering is to generate sufficient elastin and elastic fibers to mimic the structure and function of normal blood vessels. Overexpression of V3, an isoform of the proteoglycan versican, upregulates elastin synthesis in adult smooth muscle cells, but also inhibits proliferation, which may limit the utility of this strategy for tissue engineering applications. A biphasic approach to vascular graft tissue engineering, in which elastogenic conditions are introduced following a period of cell expansion, may be necessary to achieve optimal cell growth and elastin deposition.
To generate smooth muscle microvessels, cells overexpressing V3 or vector-transduced control cells were seeded onto nylon mandrels. Microvessels were cultured for four weeks in a growth factor enriched medium (GF medium) to suppress elastin synthesis, then cultured for an additional two weeks in GF medium or switched to standard culture medium to allow elastin synthesis. After six weeks in culture, V3 microvessels were composed of flattened cell layers packed tightly together, with elastin deposits concentrated at the mandrel surface. Control microvessels exhibited a loose architecture with increased matrix deposition but very little elastin. V3 microvessels that were switched to standard growth medium for the last 2 weeks of culture exhibited elastin deposits between the cell layers and not just concentrated at the mandrel surface, suggesting that both matrix gene expression and exogenous factors influenced elastin deposition. Ongoing studies include optimization of cell aggregation around mandrels to rapidly and reproducibly generate tissue engineered vessels from smooth muscle cells without exogenous scaffolds, as well as further characterization of the functional and mechanical properties of vascular grafts composed entirely from cells and cell-derived extracellular matrix.
Current research in the Rolle lab is focused on developing genetic and cellular engineering approaches to control extracellular matrix (ECM) synthesis in tissue engineered constructs, and to quantify the effects of ECM manipulation on the mechanical and physiological properties of engineered tissues.
“Stem Cell-Based Repair of the Infarcted Heart”
Charles E. Murry, MD, Ph.D.
Professor, Department of Pathology; Director, Center for Cardiovascular Biology &
University of Washington
The heart is one of the least regenerative organs in the body. As such, it stands to benefit greatly from stem cell-based approaches to repopulate the myocardium or coronary circulation. Studies in transplanted human hearts using gender-mismatched donors and recipients indicate that there is little repopulation of cardiomyocytes from circulating cells (0.04%), even over a 10-year period. In contrast, there is extensive repopulation of endothelial cells from circulating sources, averaging 25% of the coronary microvasculature. These data suggest that circulating progenitors, e.g. marrow-derived, are promising for revascularization but are not intrinsically suited for remuscularizing the heart.
Because of difficulties in obtaining definitive cardiomyocytes from adult stem cells, our group has focused on embryonic stem cells (ESCs). Transplantation of undifferentiated ESCs into the normal or infarcted heart leads to teratoma formation, indicating that cells must first be induced toward a cardiac phenotype before transplantation. In collaboration with colleagues at Geron Corporation, we have established a protocol to induce cardiac differentiation from human ESCs. This protocol uses sequential application of activin A and BMP-4 and yields ~50% cardiomyocytes. Cardiogenesis requires endogenous Wnt/b-catenin signaling, as extracellular Wnt inhibitors prevent cardiac differentiation in the presence of these inducers.
Transplantation studies with directly differentiated human cardiomyocytes into the athymic (nude) rat are underway. Initial studies failed to form new myocardium in the infarcted heart due to extensive cell death. A large number of interventions targeting candidate death pathways were unsuccessful when applied individually, suggesting multiple pathways were operative. We therefore prepared a pro-survival cocktail that targeted multiple points in both apoptotic and oncotic death pathways, and this resulted in human myocardial grafts forming in >90% of recipient animals. Using echocardiography and MRI to assess ventricular function, we found that formation of human myocardium prevented the decline in global systolic function post-infarction, attenuated ventricular remodeling, and markedly enhanced regional wall thickening. Studies are currently underway to determine the mechanism for these beneficial effects.
“Biologic Scaffold for Tissue Reconstruction: Mechanisms of Remodeling”
Stephen F. Badylak, DVM, PhD, MD
Research Professor, Department of Surgery; Director, Center for Pre-Clinical Tissue Engineering, McGowan Institute for Regenerative Medicine
University of Pittsburgh
Biologic scaffolds, particularly those composed of naturally occurring extracellular matrix, have shown great utility in both pre-clinical and clinical studies for the reconstruction of numerous tissue types. These scaffolds promote the deposition and organization of site specific tissues. The mechanisms by which host cells are attracted to these scaffolds, migrate, proliferate, and differentiate into site specific structures is largely unknown. It is clear however, that a complex system of cell signaling processes occurs and that the extent to which wound healing can be modulated to mimic developmental biology dictates the degree of success in the clinical setting.
“Human Limb Regeneration: Understanding the Challenge”
Ken Muneoka, Ph.D.
Professor, Department of Cell and Molecular Biology
In response to minor injury, most tissues of the human body have the capacity to undergo self-repair and restore physiological function. In contrast, tissues present at larger wound sites, such as limb amputations, display no repair response and undergo wound healing that results in scar tissue deposition. There are exceptions to this rule. For example, fingertips of children are known to undergo a regenerative response if allowed to heal conservatively, and the limbs of adult salamanders are able to regenerate a perfect functional replica of the amputated limb. In addition, embryonic tissues have enhanced regenerative capabilities that gradually decline with maturation. As we explore deeper into the mechanisms that control injury responses in these regeneration models, we gain insight into the prospects of induced regenerative abilities humans. For human limb regeneration the challenges are many, but a roadmap is emerging.
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Last modified: September 17, 2007 13:32:38