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Burns Service Division
CENTER FOR ENGINEERING IN MEDICINE

Principal Investigators:
Ronald G. Tompkins, M.D., Sc.D.
Martin L. Yarmush, M.D., Ph.D.
Mehmet Toner, Ph.D.
Francois Berthiaume, Ph.D.
Laurence G. Rahme, Ph.D.
Arul Jayaraman, Ph.D.
Arno Tilles, M.D.
John Levine, M.D., Ph.D.
Zak Megeed, Ph.D.
Koby Nahmias, Ph.D.

INTRODUCTION

The CEM's research power comes from its ability to define the frontiers of healthcare technologies and organize concerted efforts that push the envelope. Examples include creating integrated microsystems as diagnostic and therapeutic tools and the development of computer models which describe biological processes using sophisticated mathematical techniques. These unique capabilities, when merged with expertise in viewing, constructing, and analyzing biological molecules and in cultivating and manipulating living cells, provide a most powerful platform for designing the molecular and cellular machines of tomorrow.

The CEM’s focus is neither on a single disease nor on a single group of technologies. Instead, vitality springs from creating novel applications using the tools of disparate disciplines ranging from molecular biology and biochemistry to engineering design and analysis. These tools are currently being applied to “thrust areas” in artificial organ development, applied immunology and virology, biopreservation, metabolic engineering, stem cell bioengineering, genomics and proteomics, microfabrication and nanotechnology, drug delivery and novel therapeutics, and tissue engineering and repair.

Current representative research projects include:

DEVELOPMENT OF BIOARTIFICIAL LIVER SUPPORT SYSTEMS:
Each year over 17,000 individuals develop severe enough liver failure to require serious clinical support. Of these patients, less than 7,000 will undergo liver transplantation, which is currently the only available method for effectively treating severe liver failure. We have been working on several critical technologies needed for the development and deployment of bioartificial liver devices which would help those patients who still have regenerative potential and as well serve as a bridge to transplant. The first is developing a cell source for such a device, and this work is described below in the section on Stem Cells. The second technology that we have focused on is bioreactor development, where we are using microfabrication techniques to build reactors that are hepatocyte-friendly with the least dead volume possible. Finally, we are also developing several liver failure animal models with which to evaluate our devices, their proper usage (e.g. frequency, duration, cell number, etc.), and ways to minimize any immune sensitization.

STEM CELL ENGINEERING: APPLICATION TO EFFICIENT HEPATOCYTE GENERATION:
Stem cells are undifferentiated cells capable of proliferation and self-renewal, with the added ability to give rise to multiple differentiated cell types. They offer the possibility of a renewable source of replacement cells and tissues to treat various diseases. Our laboratory is currently involved in developing a number of novel approaches aimed at differentiation of embryonic stem cells into mature and functional hepatocytes. To date, we have showed through extensive microarray analysis and modeling, that the preferred conventional technique (the Hamazaki technique which relies on embryoid body formation and serial addition of various growth factors), generates a low yield of a mixed, very primitive, hepatocyte lineage cell population. To drive this challenging field forward, we are focused on several scalable manipulations to differentiate and separate pure populations of hepatocyte–like cells. These approaches, some of which have already yielded dramatic results, include the use of: 1) microencapsulation, 2) microfabrication with controlled growth factor delivery, 3) techniques for inducing the metabolic machinery that accompanies hepatocyte differentiation, and 4) co-culture with adult hepatocytes and mesenchymal cells.

WIDENING THE SOURCE OF LIVERS FOR TRANSPLANTATION:
Steatosis or lipid accumulation in nonadipocytes occurs in about 25% of cadaveric livers used for transplantation. Although usually asymptomatic, hepatic steatosis is a significant risk factor for postoperative liver failure, as fatty livers are much more sensitive to ischemia-reperfusion injury than normal “lean” livers. Thus fatty livers are often considered to be “marginally acceptable” for transplantation. Our long-term goal is to develop a metabolic pre-conditioning regimen, which reduces hepatic lipid storage and increases the liver’s ability to withstand ischemia-reperfusion injury. We hypothesize that metabolic pre-conditioning will reduce the risk of postoperative liver dysfunction to a level similar to that observed in nonsteatotic livers. The proposed studies should (1) provide the rationale basis for increasing the liver donor pool size, as severely steatotic livers are usually discarded, (2) improve the outcome of patients which receive liver transplants with mild to moderate steatosis, (3) provide new ways to prevent or limit hepatic fibrosis, as hepatic steatosis often precedes fibrosis in degenerative liver diseases.

A NOVEL REAL-TIME FUNCTIONAL GENOMICS PLATFORM: THE LIVING CELL MICROARRAY:
The tools of modern biology have revolutionized biomedical research, enabling an exponential growth in the acquisition of data regarding the expression and function of genes and proteins in normal and diseased states. However, it is often not easy to correlate the trends and relationships observed in normal or abnormal states to the phenotype resulting from the gene expression profile. We are developing a new functional genomics approach for studying gene expression, in which we use microfluidics and dynamic arrays of intact cells for the simultaneous temporal expression profiles of multiple genes. The technique relies on GFP fluorescence of different proteins in a massively parallel, high throughput format. Our goals are: 1) to identify the genes whose expression is altered by molecular mediators of the stress response and to generate green fluorescence protein (GFP)-tagged expression constructs of these genes; 2) to use microfabricated and microfluidic techniques where cells can be cultivated and exposed to multiple inputs; and 3) to obtain temporal gene expression profiles of cells exposed to a variety of physiologic and pathologic environments. We are using these tools and the information they provide in studies of liver fibrosis, liver regeneration, and wound healing.

BLOOD-ON-A-CHIP:
Blood cells represent a wealth of information pertaining to diseases, infections, malignancies or allergic conditions. However, extracting unaltered and accurate information depends not only on sophisticated genomic and proteomic instrumentation, but can be seriously biased by improper blood handling and preparation procedures. We are building an integrated platform to automatically and systematically handle blood while avoiding artifacts, and extract scientific or clinically relevant information from target populations of cells in blood. Individual modules are being developed for depleting red blood cells, sorting leukocytes into homogenous phenotypes and interrogating cells based on phenotypic or genotypic characteristics. Integrated microsystems for rapid and comprehensive blood analysis are poised to become single use, disposable, point-of-care diagnostic tools for clinical applications or science research tools for blood analysis from small laboratory animals.

DYNAMIC TISSUE MICROSYSTEMS:
In this project, we are developing a new tool which will hopefully fill the gap between cell culture and perfused organs studies. A dynamic tissue microsystem is an array of tissue units which can be used for studying tissue processes in vitro. The tissue that we are currently working with is liver tissue and the process that we are investigating is ischemia and reperfusion. In these systems, we assemble liver sinusoidal units in which the hepatocytes are modified to express a GFP construct of inducible nitric oxide synthetase and the endothelial cells are modified to express a YFP construct of ICAM. By varying the stimuli through a microfluidic network, we are able to visualize the relative dynamics of gene expression and the adhesion of neutrophils and other inflammatory cells to the sinusoidal units. The dynamic tissue microsystem is adaptable to any tissue and can be used for study of many tissue processes (e.g. wound healing, immune attack, tumor invasion, etc.).

NOVEL APPROACHES TO THE STORAGE OF CELLS AND TISSUES:
The lack of reliable and economical methods for long-term preservation of biomaterials presents a significant obstacle for many applications in the fields of medicine, biotechnology, and basic biological research. We are currently exploring new strategies for the preservation of cells, tissues, and biomaterials. One possible alternative to traditional cryoprotective agents is mono- and disaccharides, such as trehalose and sucrose, which possess superior physiochemical properties as cryprotectants and are minimally toxic to cells. Unfortunately, mammalian cell membranes are naturally impermeable to disaccharides, and as a result, cryobiologists cannot directly exploit these molecules for use as intracellular cryoprotectants. In order to use disaccharides as intracellular cryoprotectants we have employed a genetically engineered mutant of the pore-forming S. aureus alpha-toxin, equipped with a metal actuated switch to controllably and reversibly change cell membrane permeability to small molecules, while maintaining cell viability. More recently we have developed another approach to cryopreservation of cells by using laser annealing to obtain ultra-rapid cooling rates to achieve a glassy state in the presence of minimal (or no) cryoprotectant. First, we energize a small region of the solution with a Q-switched YAG laser in order to keep it in a liquid state while cooling the surrounding area. Thereafter, the thermal energy is rapidly quenched away by turning the laser off. We can achieve rates of cooling that are in excess of 105 K/s. This approach has the potential to be used for biological materials that are extremely sensitive to cryoprotectant treatments, such as human oocytes for in vitro fertilization purposes.

METABOLIC ENGINEERING AND HUMAN DISEASE:
Metabolic anomalies exist in most severe injuries and chronic diseases like cancer and AIDS. Drastic alterations in substrate turnover, including increased gluconeogenesis and urea synthesis, lead to malnutrition and loss of lean body mass. Despite the seriousness of these complications, few effective therapies have been developed because the underlying mechanisms are not well understood. Our overall objective is to quantitatively account for the metabolic alterations that are produced by the inflammatory response to major injury or disease. In our studies, the flow of substrates through an organ is experimentally determined (e.g. liver or muscle) under both normal and pathologic conditions. Flux balance equations are formulated, which enable simultaneous calculations of the rates of overall substrate utilization and production and intracellular reactions. Model reaction schemes that describe the major characteristics of the metabolic reaction network are used to determine the distribution of flows of material within the model systems. These experimental and theoretical analyses of metabolic alterations produced by injury and/or disease help identify the key differences between normal and pathologic conditions, give insight into the evolution of diseases, and suggest better nutritional therapies for patients with severe injuries and/or chronic diseases.

INFLAMMATION AND THE HOST RESPONSE:
Our goal, to integrate proteomics and genomics biology of inflammation with care of the injured patient, can only be realized through the integration and simultaneous accomplishment of multiple tasks. These tasks include: enrollment of sufficient numbers of patients with stringent entry criteria conducting large-scale cellular and protein assessments necessary to delineate the human immuno-inflammatory phenotype determination of the gene expression profiles using state-of-the-art platforms design and implementation of complex web-enabled databases of clinical, physiological, outcomes, proteomic, and genomic expression and genotype data analysis of these complex data using multiple independent methodologies To fill the gaps in trauma research, a large-scale collaborative program has been organized into several components, each distinctly different, yet highly interrelated to provide a networked approach of interactions. There are several clinical studies in which trauma and burn patients, and LPS-challenged and normal volunteers, are studied to generate data and samples for physiological, proteomic, and genomic analyses. Multiple institutions collaborate as analytical sites for performance of high-throughout genomics and proteomics analyses on the samples. Clinical, demographic, outcomes, cellular, and gene expression data from the patients and samples are highly integrated into useful information for analysis and informatics. There is also an administration infrastructure in place that provides the foundation to support the administration, project management, and finances of the research program.

GENE THERAPY AND CELLULAR ENGINEERING:
Recent advances in molecular genetics have resulted in the development of new technologies for the introduction and expression of genes in human somatic cells. Although there has been a large cohort of clinical trials using several different viral vectors, a firm bioprocessing technological base has yet to be established. Our laboratory has been involved with identifying the rate limiting steps in the retroviral transfection process with an eye toward large-scale vector manufacturing which is efficient and cost-effective. We are currently focused on understanding the mechanism of retrovirus decay. It appears that retroviral activity decays because of RNA degradation, and so we are currently developing several strategies to efficiently block or overcome this degradation. In a second collaborative project, we are using genetic transfer techniques to overexpress various growth factors in skin cells with an eye toward: 1) developing more robust and biologically active skin grafts, and 2) understanding the role of these growth factors in the wound healing process.

WOUND REPAIR:
Wound healing research in the Department of Surgery at MGH and Shriners Hospital focuses both on understanding basic cellular mechanisms and on the application of tissue engineering principles to skin. Work is directed toward understanding the fundamental roles of epidermal keratinocytes, inflammatory cells, endothelial cells and fibroblasts in the healing process. Experimental approaches include animal models of excisional and incisional wounding, the transplantation of either engineered skin using purified cells, of normal human skin, or of hyperptrophic scars onto athymic animals. These surgical techniques are complemented by application of retroviral technologies to overexpress growth factors, the use of blocking antibodies, recombinant proteins and peptides to modulate and study the wound healing process. The work is supported by state-of-the-art morphological facilities.

NEUTROPHIL FUNCTION IN INJURY:
Major improvements in clinical care have dramatically increased the chance of surviving major injuries. Non-survivors no longer die as a direct result of injury, but rather due to complications following the injury. A frequent occurrence in trauma patients is infections arising several days after the initial injury leading to pneumonia and progressive multiple organ dysfunction syndrome. Trauma patients exhibit a severe suppression of both the antigen specific (T-cell-dependent) and nonspecific (neutrophil and macrophage-dependent) arms of the immune system, which results in an increased susceptibility to infection. Our overall goal is to devise therapeutic strategies which enhance the immune function of the patient. Circulating neutrophils in trauma patients often exhibit decreased functions and an immature phenotype reminiscent of that found in the bone marrow. We hypothesize that impaired neutrophil function is due to the premature release of neutrophils from the bone marrow before they have completed their maturation process. In this investigation, we are interested in the role played by "redox-sensitive" factors which alter the growth and development of hematopoietic progenitors and the production of neutrophils. Two of these factors, the pentapeptide pEECDK and transforming growth factor-b (TGF-b), can modulate the early stages of hematopoietic stem cell development and lead to the observed alterations in immune function in burned patients. In our studies, we mouse and rat models of burns and burns with infection to characterize the dynamics of hematopoietic cell differentiation and long-term bone marrow cultures to investigate the specific roles of hematopoietic growth regulators on neutrophil production. These studies involve both biochemical characterization of cells and mathematical models to track the rates of differentiation, division, and death of various bone marrow-derived cell populations.

INTRACELLULAR PROCESSING IN SKELETAL MUSCLE AND CELLS OF THE IMMUNE SYSTEM:
Burn patients experience prolonged negative nitrogen balance with severe muscle wasting and debilitation; this is largely attributable to increased protein catabolism. The mechanisms underlying this process have not been fully characterized, in part due to the lack of available stable long-term differentiated culture systems for skeletal myocytes, as well as the difficulties associated with using whole muscle tissue preparations. We hypothesize that normal intracellular protein processing mechanisms, and specifically, cytosolic ubiquitin-proteasomal degradation pathways are responsible for the majority of the increased protein turnover. Moreover, we propose that the same hypercatabolic pathways are active to varying extents in cells and tissues other than skeletal muscle, and in particular, occur in peripheral blood lymphocytes (PBL). Use of PBL facilitates analysis of the mechanisms of increased protein catabolism, since a large library of well-characterized immunologic reagents relevant to these cells already exists, and these cells are easily cultured while maintaining assayable functional phenotypes. In addition, PBL may be readily sampled from patients to assess the ongoing level of catabolism and efficacy of treatment. The specific aims of this project are to examine 1) the effects of burn injury on the intracellular catabolic pathways of murine PBL and other lymphocyte populations in comparison to those effects seen in skeletal muscle, myoblast cell lines, and stable hepatocyte cultures, 2) the specific effects of a hypercatabolic state on normal lymphocyte function, and 3) the molecular mechanisms mediating the increased intracellular catabolism in lymphocytes.

BACTERIAL PATHOGENS AND BURN INJURIES:
A common occurrence with compensatory anti-inflammatory response syndrome (CARS) is the development of secondary infections, which augment the burden on the immune system. Pseudomonas aeruginosa is the most common causative organism of sepsis in burn patients. Our group focuses on the elucidation of the molecular basis of P. aeruginosa pathogenesis and the dissection of host responses during infection. One of the two major fields of interest in our group is the study of the mechanisms a bacterial pathogen employs to attack its host. To facilitate our studies, we have developed pathogenesis model systems in which plants, nematodes and insects are utilized to model mammalian bacterial pathogenesis. Utilizing these alternative non-vertebrate model systems, we demonstrated the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts. We are employing molecular and biochemical approaches to determine the fundamental mechanisms that underlie bacterial pathogenesis. The second field of interest lies in understanding of the role of host responses in limiting disease development. We are utilizing both D. melanogaster and mice to identify and characterize the signal transduction pathways involved in the immune responses during infection with P. aeruginosa. Currently, we are expanding our efforts to perform genome-wide expression studies utilizing both D. melanogaster and mice to identify genes expressed during host-pathogen interactions.

Rev. 8/10/06

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