NFP 46 Tissue Engineering 4046-58630

Bioengineering of a Composite Nerve Graft for Peripheral Nerve Reconstruction

Background
Loss of peripheral nerve tissue is common after severe injuries or surgery for malignant tumors. Nerve autograft is presently considered the "gold standard" for the surgical reconstruction of peripheral nerves.

Problem
A major hindrance is the lack of donor tissue for extensive nerve reconstructions. To this end, efforts are made to find unlimited supplies of natural or synthetic material to replace these autografts.

Objective
The specific goals of the project are:
(i) the engineering of nerve grafts made of extracellular matrix protein scaffolds seeded with cultures of isogenic Schwann cells.
(ii) the investigation of the influence of the scaffold structure on the extent of nerve regeneration and reinnervation specificity.
(iii) the development of an artificial nerve substitute for clinical use, envisaged in collaboration with an industrial partner, the Geistlich Company.

Approach
Part of the project is the development of nonimmunogenic nerve implants to achieve reinnervation with adequate target specificity and to provide surgeons with large amounts of graft material to reconstruct extensive nerve defects with strong enhancement of the functional outcome.

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NFP 46 Tissue Engineering 4046-58623

Engineering Human Cartilaginous Implants in Bioreactors Using Mature Chondrocytes and Mesenchymal Progenitor Cells from Donors of Different Age Groups: A Collaborative Study by Two Institutions

Intermediate Results May 2002

Background
Today, new molecular techniques can be used to monitor the differentiation of chondrogenic cells and to identify differential patterns of gene expression among cell populations. Furthermore, use of recently available human recombinant growth factors may induce a more effective generation of implants. Recently, animal experiments have shown that cartilaginous tissues generated in vitro and grafted into cartilage defects can lead to histological and functional consolidation of articular surfaces.

Problem
However, engineering equivalents of human cartilage tissue from culture-expanded autologous cells has not yet been achieved. Human chondrocytes harvested form normal articular cartilage dedifferentiate into a fibroblastic stage during in vitro expansion in monolayers, and - particularly if cells are from old donors - they fail to reproducibly redifferentiate and to generate a functional matrix after expansion. Human mesenchymal progenitor cells (MPC) harvested from the bone marrow, although able to display a chondrogenic potential after expansion, have yet to be shown to reach and maintain a stable articular chondrocyte phenotype.

Objective
The project pursues the following objectives: (i) to identify donor age related differences in the pattern of gene expression of normal human articular chondrocytes and MPC; (ii) to define markers of chondrocytes in native cartilage; (iii) to enhance the chondrogenic potential of chondrocytes and MPC; and (iv) to improve production and accumulation of cartilage-specific extracellular matrix by enpanded chondrocytes and MPC.

Approach
The hypotheses of our poposed collaborative research are (i) that differences in the patterns of gene expression of primary chondrocytes from donors of different age groups may be used to identify markers correlating with the cell's chondrogenic potential; (ii) that exogenously applied molecules and bioreactor conditions can modulate the differentiation and extracellular matrix production of culture-expanded human chondrocytes and MPC.

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NFP 46 Tissue Engineering 4046-101116

Characterization and Evaluation of Cell Phenotype and Extracellular Matrix in Tissue Engineered Heart Valves Based on Human Cardiovascular Cells

Background
Based on a novel "biomimetic" (pulse-duplicator) in vitro-environment we have been able to demonstrate the principal feasibility of tissue engineered living, completely autologous heart valves. These tissue engineered heart valves showed excellent functional in vivo-performance in long-term animal experiments. The tissue engineered heart valves were based on ovine vascular derived myofibroblasts and endothelial cells and gradually evolved to resemble native valves as to extracellular matrix and cell composition.

Objective
The aim of this study is to define "quality criteria" for a near future clinical application of tissue engineered heart valves as to human cellular and matrix characteristic to provide maximum safety for the cardiac surgery patients. Accordingly, the objective of this project is to investigate cell phenotype, immunology and matrix maturation processes in tissue engineered heart valves based on human cells and grown in biomimetic in vitro-conditions. A meticulous understanding of the developmental biology during the multi-step engineering procedure is a prerequisite to safe transferral of the tissue engineering concept to human applications. The following specific aims will be addressed:

Aim 1: Characterization of human myofibroblast cell phenotype during the in vitro-tissue evolution of human tissue engineered heart valves. Study of the influence of biomimetic in vitro-conditions and cell donor age.

Aim 2: Investigation of extracellular matrix composition, maturation, and biomechanical properties in tissue engineered heart valves based on human myofibroblasts with specific focus on the influence of physiological in vitro-conditions and cell donor age.

Aim 3: Determine the phenotype, immunological and functional potential of human endothelial cells used for tissue engineered heart valves evaluated in pulsatile and non-pulsatile in vitro-conditions.

Approach
Prior to clinical application it is crucial for researchers and clinicians to have reliable "quality criteria" of tissue engineered heart valves regarding adequate cell differentiation, sufficient extracellular matrix maturation, and the absence of detrimental immunogenic characteristics. "Benchmark" for the definition of these criteria is human native heart valve tissue. Therefore, the results of this research plan may represent a concrete step towards the first successful clinical application of a human autologous tissue engineered heart valve.

Budget: 254'948
Time Frame: 1/6/2003-31/5/2005

NFP 46 Tissue Engineering 4046-101114

Bone 35: Fetal bone cells for tissue engineering (abstract / full text [EPFL])

Bone Tissue Engineering Using Foetal Cell Therapy

Background
This proposed study is to be included in the vast schematic project of bone tissue engineering which is composed of several laboratories having competence in cell and molecular biology, scaffold development, biomechanics, biocompatibility, and clinical applications. In addition to its interdisciplinary shown by the combination of biological (gene and protein analysis) and engineering (biomechanics and biocompatibility) aspects, this study is developed in very close collaboration with orthopedic surgeons allowing a continuity of the project development with the physicians whom will ultimately used the engineered tissue. This approach allows us to solve practical clinical problems at each development level of the project.

Objective
The project will make use of foetal cell therapy in the development of bone tissue engineering. This approach is motivated by several factors. These cells have been shown to resist strenuous conditions which are present in a bone tissue engineering application. Moreover, foetal cells do not induce an immunological reaction from the host tissue and based on our previous results on skin tissue engineering, one single donor will be enough to treat thousands of patients. This renders the cell therapy approach very efficient, as only one cell line will have to be characterized.

Approach
We will establish a foetal cell bank obtained from a piece of femur. The isolated cells will be extensively characterized at the gene and protein levels and compared to osteoblasts obtained from femur of young and old adults. The foetal cells will then be mechanically stimulated in situations mimicking the clinical applications. The two majors goals of this work are:

1) to compare bone gene/protein expression in foetal and adult cell bone and to verify that foetal cells can produce adult bone of good quality;

2) to verify that foetal cells seeded in a scaffold keep a long-term maintenance of the osteoblast differentiated phenotype in a mechanical situation close to the clinical application and do not induce tissue fibrous formation or osteolysis.

Budget: CHF 231'824
Time Frame: 1/3/2003-28/2/2005

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NFP 46 Tissue Engineering 4046-101119

Trachea Reconstruction Using Novel Tissue Engineered Constructs

Background
The problem of reconstructing long segmental tracheal defects remains unsolved, despite the recent progress in the field of in vitro-cartilage tissue engineering. Yet, employing chondrocyte-seeded polymer constructs, may serve as an emerging concept to overcome the encountered difficulties. Developing applicable and novel tissue engineering techniques may lead to the creation of functional neocartilage constructs for bridging long segmental tracheal defects.

Objective
We propose the technical and biological feasibility of successfully creating tissue engineered neocartilage. Using an improved dynamic pressure bioreactor for cultivation, in combination with a novel polyestheruretane scaffold, our research will focus on three questions that are central in establishing the functional and structural integration of tissue engineered neocartilage into the host environment:
1) finding the optimal extracellular matrix (ECM) profiles and ex vivo-cultivation sequence.
2) assessing the issue of construct nutrition and neovascularization.
3) determining the interaction of the multilayer design with the corresponding host structures.

Approach
Design and construction of a new generation of dynamic pressure bioreactor. Conventional and dynamic fabrication of chondrocyte-polymer constructs. In vitro- and in vivo-assessment of the fabricated neocartilage, including histological evaluation, SEM, biomechanical testing, MTT, GAG, RT-PCR, etc. Orthotopic transplantation of in vitro-generated constructs into tracheal defects of rats will be performed microsurgically.

Budget: CHF 238'800
Time Frame: 1/4/2003-31/3/2005

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NFP 46 Generating Matrices 4046-58681

Engineered Cell Ingrowth Matrices for Growth Factor Presentation

Background
The growth factors vascular endothelial cell growth factor (VEGF), keratinocyte growth factor (KGF), and activin A each have unique properties that may be useful in promoting cutaneous healing. VEGF is highly specific for endothelial cells and is inductive of angiogenesis, KGF is highly specific for epithelial cells, and activin A is a potent stimulator of matrix protein production and a modulator of fibroblast and keratinocyte differentiation.

Problem
In healing, each of these factors is presented in the context of interaction with a matrix, as each possesses heparin-binding character, yet when evaluated therapeutically, none have been explored in the direct context of a matrix.

Objective
Our collaborative team will employ engineered fibrin as a cell ingrowth and growth factor presentation matrix, with the dual objective to better understand the physiological function of these factors and to develop a more efficacious therapeutic system of matrix plus growth factor.

Approach
Two concepts will be investigated toward the objective of growth factor presentation: (i) Heparin affinity. For VEGF, KGF and activin A, the heparin-binding character of the growth factors will be exploited. Heparin-binding peptides will be enzymatically incorporated into a fibrin matrix (via the transglutaminase activity of factor XIIIa), along with heparin; the heparin that is thereby bound into the fibrin will serve as an affinity-binding site, which will retain the growth factor within the matrix until the matrix is degraded during cell infiltration, thus releasing the growth factor to become bioavailable. (ii) Enzymatic incorporation. For the example of VEGF, fusion proteins will be expressed that include a factor XIIIa substrate domain, thus directly incorporating the growth factor into the fibrin matrix during coagulation. This will provide for unequivocal retention within the cell ingrowth matrix until the matrix is degraded during cell infiltration. Mouse models of impaired wound healing will be employed, supplemented by in vitro systems. Comparison of these two approaches, one employing the native therapeutic protein and one utilizing a fusion protein, will permit greater insight into the function of the growth factor and into approaches for optimal therapeutic presentation of cutaneous repair.

Budget: CHF 591'090
Time frame: 1/7/2000-30/6/2003

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NFP 46 Tissue Engineering 4046-101113

In vitro-Engineering of Implantable Human Urinary Tract Tissue Matrices

Background
To increase bladder volume or to replace diseased urological tissues extensive surgical procedures may become necessary in the treatment of congenital or acquired diseases of the urinary tract. The current techniques of bladder augmentation using intestinal or gastric tissues for replacing diseased tissues or substituting missing tissues of the urinary tract are prone to severe and in particular metabolic complications. Tissue engineering of urological tissue offers an alternative solution to overcome these problems.

Problem
The engineered tissue should be primarily composed of the components of native urological tissues, the smooth muscle and urothelial cells, seeded on a cell carrier matrix. The success of the method depends on the capacity of in vitro-cell expansion providing enough cells for seeding in adequate time, and a suitable matrix allowing cell adherence, proliferation and promoting vascularization. Natural and synthetic degradable or non-degradable polymers can act as cell-carrier matrices. Synthetic polymers have the advantage to allow manufacturing in a reproducible way and offering adequate mechanical behaviour. However, these matrices are not optimal for cell adherence and growth. To overcome this problem, extracellular matrix proteins, permitting cell adhesion, proliferation and differentiation can be adsorbed onto polymer surfaces. For surface tailoring these polyesters, a new technology (patent in process) has been developed for non-degradable polymers and will be extended to degradable supports.

Objective
The aim of this study is to further develop non-degradable and degradable surface-functionalized polymer matrices and knittings for extracellular matrix proteins adsorption, favoring cell adherence and growth and acting as scaffolds for tissue engineering. Furthermore special constructions of knitted textile supports will be featured and optimized. The textile parameters, including area weight, loop geometry and water permeability, will be determined. Particular emphasis is put on the relation between structure and material as well as mechanical properties.

Approach
Human and porcine primary urothelial and smooth muscle cell cultures were previously established arising either from tissue biopsies obtained from children undergoing open ureteral or bladder surgery or from animals. Furthermore, stratified urothelial growth was induced and functional non-permeable urothelial sheets were engineered. For the characterization of urothelial cells specific cytokeratin markers and for smooth muscle cells anti-a smooth muscle actin, -desmin and -myosin were used. Surfaces of non-degradable and degradable polymers are modified by graft polymerization of acrylic acid for subsequent extracellular protein (collagen type I and III, laminin, fibronectin etc.) adsorption. Smooth muscle and urothelial cell adhesion and growth are analyzed on these modified polymer films.

In addition the in-vitro engineered structures will be implanted into in-vivo systems using the nude mice and mini-pig models to study the tissue integration behavior of the constructed bladder wall patch. Differentiation and function of the in-vivo transferred cultured cells will be studied.

The mechanical properties of the reconstructed bladder wall will be determined.

Budget: CHF 213'556
Time Frame: 1/3/2003-28/2/2005

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NFP 46 Allogenous Cellular Transplants 4046-58663

Immunology of Encapsulated Cell Transplantation Using Erythropoietin-Secreting Cells

Artikel | Article

Background
Recent trials have demonstrated the rationality of cell transplantation for either replacing damaged tissues or releasing bioactive proteins. Cell transplantation provides a promising alternative to the treatment of numerous diseases, such as diabetes or anemia, relying on the repetitive administration of therapeutic proteins. Chronic injections necessitate patient compliance and do not allow a tight regulation of the protein delivery leading, in the case of type I diabetes, to long-term complications due to lack of minute glucose regulation. Cell transplantation may offer an opportunity to circumvent these drawbacks by an in vivo regulated delivery of bioactive proteins. Furthermore, the in situ secretion of bioactive proteins permits their localized administration in poorly accessible sites such as the eye or the central nervous system.

Problem
Cell transplantation, however, faces two main problems, i.e. host immune rejection and safety issues. Cell encapsulation within a permeable membrane has been intended to avoid immune rejection by limiting molecular exchanges and preventing cell-to-cell contacts between graft and host tissues. Recent observations have evidenced important aspects influencing acceptance of encapsulated cells by the host immune system. Encapsulated xenogeneic cells survive long-term in the central nervous system whereas they do not in the subcutaneous tissue. On the other hand, encapsulated allogeneic cells survive in all tested sites including the subcutaneous tissue. These observations suggest that encapsulation only partially prevents affector and effector mechanism of the host immune response.

Objective
The present proposal aims at clarifying the immune protection provided by encapsulation, as compared to the transplantation of naked allogeneic and xenogeneic cells.

Approach
The project will evaluate strategies of immunosuppression and immunomodulation for their ability to improve the survival and induce host tolerance of encapsulated xenogeneic cells. This work will lead to a clinical trial based on the subcutaneous transplantation of encapsulated allogeneic cells deliverging the erythropoietin hormone in kidney failure patients.

Budget: CHF 426'243
Time frame: 1/7/2000-30/6/2003

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NFP 46 Tissue Engineering 4046-58682

Diabetologia 46: Destruction of conditional insulinoma cell lines in NOD mice: A role for autoimmunity (abstract)
Diabetes 51: cFLIP Protein Prevents Tumor Necrosis (abstract)

Development and Preclinical Assessment of a Bioartificial Pancreas

Background
A bioartificial pancreas consists of properly functioning glucose-regulated insulin secreting cells placed in an encapsulation device. The possibility to transplant such an bioartificial organ in type I diabetic patients is widely thought to be the solution for the treatment of this disease, to avoid in particular the development of long-term micro- and macrovascular complications.

Problem
The development of a bioartificial pancreas, however, is progressing slowly.
because of the use of pancreatic islets of human or pig origin. The complexity of these cellular preparations and the variability of their purity and functional state make it very difficult to interpret results of any transplantation experiment. In fact, failure of graft acceptance may be influenced by each of the cellular parameters, in addition to those associated with the host response.

Objective
The project proposes to rigorously evaluate the parameters that may allow the successful development and use of a bioartificial pancreas.

Approach
In thew first place, a well defined insulin secreting cell line, the ßTC-Tet cells, will be used. These insulin secreting cells are perfectly glucose regulated and their proliferation can be controlled by an easily controllable genetic switch. These cells can correct diabetic hyperglycemia, when transplanted under the kidney capsule of syngeneic or allogeneic mice. Furthermore, they can be genetically modified without losing their unique functional properties and will thus be used to evaluate the effects of genetic modifications to enable them to survive in an autoimmune (NOD mice) or xenogeneic (rat) environment. The requirement for additional protection, in particular against direct cellular contact with inflammatory or immune cells, will be tested by means of cellular encapsulation. The encapsulation techniques to be used for these studies will include alginate-poly-L-lysine microbeads, thermoplastic-based flat sheet and vascular devices.

As the ultimate insulin secreting cell lines to be used in a bioartificial pancreas should be of human origin, the project will, in parallel, attempt the conditional immortalization of human ßcells.

Project Leader: Prof. Dr. Bernhard Thorens
Project Team:Prof. Dr. Patrick Aebischer
Dr. William Pralong
Dr. Markus Borkenhagen

Budget: CHF 415'972
Time frame: 1/1/2001-31/12/2003

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NFP 46 Allogenous Cellular Transplants 4046-58685

Islet Transplantation for the Treatment of Diabetes - Microencapsulation for Immunoprotection

News Item

Background
The wide-spread application of human islet transplantation is hampered by the limited islet supply as well as the systemic immunosuppression required to prevent rejection and recurrence of auto-immune diabetes. The transplantation of encapsulated porcine islets, or genetically reproduced human b-cells, has been proposed to overcome the limited supply of human tissue. Microcapsules would not only offer protection against allo- or xeno-geneic immunoresponse, but also increase the biosafety of the graft with regard to transmissible diseases.

Problem
Immunoprotection by microencapsulation has been confronted with several obstacles such as irreproducible, impure and endotoxin rich sources of polymers, the lack of adequate long term in vivo-mechanical properties, inadequate material elasticity and permeability, as well as uncertainties as the to the optimal site for transplantation.

Objective
The study will determine whether the concept of encapsulated islet transplantation, as allotransplantations of genetically modified and reproducible b-cell-grafts or as porcine islet xenografts, will cure type I diabetes.

Approach
The project brings togethertwo teams with biomedical (Geneva) resp. bio-technological (EPFL) expertise in the field of islet transplantation. The EPFL team has developed and patented a new microencabsulation system ensuring the survival of rat islets for more than 6 months in the NOD-mouse model. For future clinical application, the EPFL team intends to develop a large-scale quality controlled process for materials and islet encapsulation. Such a device has been successfully tested on large animal models. The Geneva team will evaluate the biological characteristics of the newly developed and select the most appropriate capsule system. The EPFL's bioartificial pancreas has the advantage of a degree of freedom which permits modification of its design and properties according to clinical feedback. To determine the optimal site for transplantation, ensuring both optimal oxygen and nutrient supply as will as maximal sefety for the recipient, will be an important part of the project.

Budget: CHF 299'730
Time frame: 1/1/2001-31/12/2003

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NFP 46 Xenogenous Cellular Transplants 4046-58668

ABO-Incompatibility and Hyperacute Xenograft Rejection: Minor ABO-incompatible Bone Marrow Transplantation as a Model for B-Cell Tolerance and the Prevention of Natural Antibody-mediated Graft Rejection

Background
The current organ shortage in transplantation medicine stimulates the exploration of new strategies to expand the donor pool including the utilization of living donors, ABO-incompatible grafts and xenotransplantation.

Problem
Preformed natural antibodies (NAb) such as anti-A/B histo-blood group isoagglutinins and xenoreactive anti-Gala1,3Gal antibodies (Abs) mediate hyperacute graft rejecton and thus represent a major hurdle to the employment of such strategies.

Objective
The immediate goal is to analyze the immunological mechanism leading to the spontaneous disappearance of anti-A/B isoagglutinins. Furthermore, the expression of ABH antigens (Ag) and the association with the clinical outcome of minor ABO-incompatible BMT will be examined. The long-rang goal is to expand the donor pool for cellular and solid organ transplantation by developing therapeutic approaches to prevent hyperacute rejection in ABO-incompatible allogeneic and xenogenic transplantation.

Approach and Specific Aims
This research proposal will study the resistance against NAb as observed in recipients of minor ABO-incompatible bone marrow transplants (BMT), e.g. the transplantation of BM from a type O donor into a type A recipient.
(i) Analyze the time course and level of anti-A/B Abs and ABH Ag expression in recipients of minor-ABO-incompatible BMT. Study the association of ABO-incompatbility with the rate of hemolysis, GvHD, transplant-related mortality and overall survival. (ii) Investigate the molecular mechanisms responsible for the loss of agglutinating anti-A/B Abs and the occurrence of B cell tolerance to A/B carbohydrate Ag. The role of absorption, shift of anti-A/B Ab effector functions, glycosyltransferase activity, anti-idiotypic antibodies, and presence of A/B Ag-specific b-cells will be addressed. (iii) Study the potential of immunoabsorbtion, anti-idiotypic antibodies, donor type RBC transfusion and glycosyltransferases in combination with complement-inhibition to prevent NAb-mediated rejection in ABO-incompatible allo- as well as xenotransplantation.

NFP 46 Stem Cell Transplants 4046-58662

Nabelschnurblut-Stammzellen: Banking und Transplantation (Daniel Surbek)

In-Utero Transplantation of Haematopoietic Stem Cells for Definitive Treatment or Tolerance Induction
in Non-immunodeficient Recipients Affected by Genetic Diseases

Background/Problem
Most genetic diseases of the lymphohaematopoietic system and inborn errors of metabolism can now be diagnosed early in gestation. However, no prenatal treatment is available to date. Postnatal therpy by hematopoietic stem cell (HSC) transplantation from bone marrow or cord blood is possible for some of these diseases, but is often limited because of lack of histocompatible donor, severe treatment-associated morbidity, and preexisting organ damage developing before birth. In-utero allogeneic HSC transplantation has several potential advantages over postnatal treatment and has been successfully performed in several animal models and recently in humans. However, clinical success of this novel treatment is limited to diseases leading to severe immunodeficiency of the foetus. The lack of donor cell engraftment in non-immunocompromised hosts is thought to be due to competitive foetal marrow population by host HSC's, and probably immunologic barriers.

Objective
The objective of this proposal is to assess several potential approaches to provide competitive advantage to donor cells: Modification of the graft by addition of donor-specific stromal cells or growth factors, or modification of the host hematopoietic microenvironment by minimal myeloablation or blocking antibodies. Additionally, another potential way to overcome engraftment barriers which includes induction of microchimerism and donor-specific tolerance in the host for postnatal transplantation form the same donor without immunosuppression or myeloablation will be assessed.

Approach
Transplantation experiments will initially make use of the immunodeficient NOD/SCID mouse in utero transplantation model; the next step will include non-immunideficent NOD&SCID mice in utero transplantation model; the next step will include non-imunodeficient mice. With the present project, important information on conditions leading to improved engraftment after in utero HSC transplantation will be obtained. Provided that this novel therapy would be successful in non-immunodeficient humans, the quality of life of children affected by these diseases would improve dramatically, and it would have major implications on the reduction of health costs.

Budget: CHF 320'397
Time frame: 1/7/2000-30/6/2003

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NFP 46 Stem Cell Transplants 4046-58689

Mit Mäusen Menschen helfen - Nachahmung des krebszellenzerstörenden Mechanismus der natürlichen Killerzellen im Tier (Bio World, 22. März 2006)

Stem Cell Transplantation in Leukemia:
The Potential of Natural Killer and Dendritic Cells for Immunotherapy

Background/Problem
Natural killer (NK) cells and dendritic cells (DC) are important for immune surveillance and may play a significant role in preventing recurrence of leukemia after stem cell transplantation. The proposed studies may provide experimental approaches to new immunotherapy with DC and genetically-modified NK cells.

Objective
The aim of the project is to characterize the development and functional properties of DC and NK cells during post-transplant immune reconstitution, and to understand how the function of these cells can be manipulated to increase specific cytotoxicity against tumor cells. In addition, the anti-leukemic role of flt3 ligand (FL) will be explored.

Approach
The first part comprises the 3 steps: (i) DC differentiating from grafted cells will be characterized; these studies aim at understanding the functional phenotypes of DC capable to promote priming and expansion of leukemia-specific cytotoxic T cells; (ii) NK cells appearing after transplantation will be analyzed; the study will determine the repertoire of NK cell inhibitory and activatory receptors, and will investigate whether graft-versus-leukemia responses in allogeneic transplants are associated with the presence of mismatches between NK receptors and host HLA class 1 specificity; (iii) Level and duration of chemotherapy-induced overexpression of FL will be monitored; these studies aim at undestanding the role of endogenous FL in reconstitution of DC and NK cell compartments, and whether high FL levels in vivo correlate with a decrease of the relapse rate.
The second part will focus on the role of natural cytotoxicity receptors (NCR) in NK cell functions: (i) to upregulate expression of NCR in order to increase the probabitity of tumor recognition by NK cells. These studies will use lentiviral vectors to transfer NCR genes into NK and hematopoietic progenitor cells. (ii) to examine the effect of FL on proliferation and differentiation of defined DC and NK cell populations, (iii) to establish a human leukemia transplantation model in NOD/SCID mice, with the goal to analyze in vivo the anti-leukemic responses to DC and NK cells, in particular those maintained by NCR-genetically modified haematopoietic progenitors.

Budget: CHF 406'450
Time frame: 1/1/2001-31/12/2003

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NFP 46 Stem Cell Transplants 4046-58610

Haematopoietic Stem Cell Quantitation, Gene Transduction and
Study of In Vitro-Expansion with Immortalized Stromal Cells

Background
Haematopoietic stem cell (HSC) transplantation, autologous or allogeneic, with HSC collected from mobilized peripheral blood or from cord blood, is increasingly utilized in therapies for cancer/leukemia and primary or acquired diseases of blood and immune system. HSC are also the target cells for somatic gene therapy aimed at such conditions.

Problem
Quantitation of HSC is difficult; a practicable in vitro assay would be helpful for research on HSC (mobillization, in vitro expansion, etc.).

Objective/Approach
The project has three specific aims:
(i) Quantitation of HSC: It is planned to develop a clonal assay which utilizes quantitative reverse transcription-polymerase chain reaction (RT-PCR) for enumeration of cells capable of generating progeny with myeloid and lymphoid markers (Ec, Mega, Mono, Dendritic, Granulo, NK, pro-B and possibly early pro-T cells) in series of single-cell (CD34+/lin- cell) - derived short-term cultures (2 weeks), i.e. an assay which detects cells with multilineage potential as is the hallmark of HSC. The assay will then be applied to research in HSC expansion and to the assessment of clinical HSC mobilization protocols.
(ii) Optimization of gene transduction into HSC: Ongoing collaborative work will be continued, in which the group's laboratory tests novel HIV-1-derived lentiviral vectors developed in Prof. Didier Trono's laboratory in Geneva, regarding optimal gene transduction into quiescent HSC with safer vectors. Lineage-specific gene expression, particularly in the B lymphoid-lineage, will be studied.
(iii) Generation of human stromal cell lines: In collaboration with Prof. Trono it is planned to immortalize stromal cells with a combination of oncogenes transduced with the above vectors. Stromal cell lines will be tested in the laboratory. The aim is to find a line which permits efficient expansion of HSC, which then could be used to identify molecules relevant for HSC regulation and applicable in the future to clinical HSC expansion.

Budget: CHF 280'772
Time frame: 1/7/2000-30/6/2003

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NFP 46 Stem Cell Transplants 4046-58671

Human Retinal Stem Cells for Transplantation in Patients Suffering from Retinal Degeneration: Preclinical Studies

Background
Retinal degenerations such as age-related macular degeneration and retinitis pigmentosa are incurable diseases, affecting thousands of people in Switzerland and millions in the world. These diseases lead to the loss of photoreceptors and consequently of visual function. Patients suffering from these diseases need the replacement of photoreceptors to recover their eyesight. Several studies in rodent models of retinal degeneration revealed that such an approach, using retinal cell transplantation, starts to be effective.

Problem
The efficacy of grafting has to be improved before introducing this technique into clinical practice. Moreover, human fetal primary cells are not available for thousands of patients.

Objective
As an alternative, the project proposes to isolate human retinal stem cells, since stem cells are known to copiously expand in vitro and to generate all the cell phenotypes composing an organ. It will investigate the potency of retinal stem cells versus photoreceptors (derived from stem cells) to re-establish the photoreceptor layers in an animal model of retinal degeneration (high light damage). It also intends to study whether adult stem cells conserve the biological properties of fetal stem cells. Finally, the project heads towards evaluating the biological potency of transplantable cells taking into account contextual ethical problems.

Approach
The gene expression "identity" of cells before transplantation will be investigated by microarray technology. This approach should reveal which genes could be required for cell integration before transplantation. The genes that will be investigated belong to the families of receptor (notch?), extracellular-matrix molecules (integrins?), transcription factors (Pax6?), and diffusible factors (bFGF?). The roles of the potentially most interesting genes will be investigated (n=5) by gene overexpression. The functionality of the graft will be recorded by electroretinogramme and by behaviour depending on simple visual functions.

Budget: CHF 383'835
Time frame: 1/9/2000-31/8/2003

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NFP 46 Stem Cell Transplants 4046-58638

Hepatocyte Transplantation for End Stage Liver Disease:
Potential of Hepatocyte Progenitor Cells and Bone Marrow Stem Cells

Background
To date, liver transplantation is the only therapeutic option for treatment of end stage cirrhotic liver disease. If this research project is successful, it will ultimately lead to a therapy for cirrhotic end stage liver disease which does not depend upon liver transplantation.

Problem
The option of liver transplants is compromised by a severe shortage of donor organs and by high costs.

Objective
The ultimate goal of this research proposal is the development and evaluation of autologous stem cell transplantation as an alternative to whole liver transplantation for therapy of end stage liver cirrhosis.

Approach
To achieve this goal, the project is divided into three parts:
(i) Development of the methodology for autologous transplantation of Mrp2 transfected liver stem cells in GY/TR- rats to correct their inherited hyperbilirubinemia.
(ii) Development of the methodology for autologous transplantation of Mrp2 transfected bone marrow derived stem cells in GY/TR- rats to correct their inherited hyperbilirubinemia.
(iii) Development of a therapy of experimental liver cirrhosis in rat models by transplantation of human HGF transfected bone marrow stem cells.

(ii) and (iii) are based on the experimental experiences gained in (i). These studies will pave the way for the eventual replacement of whole liver transplantation for the treatment of end stage liver disease by autologous cell transplantation. Since it is based on autologous bone marrow derived stem cell transplantation, this therapy will not be limited by a shortage of organs and will be cheaper than whole liver transplantation.

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NFP 46 Stem Cell Transplants 4046-58639

Clinical Use of Human and Myoblast and Myogenic Stem-Cell Transplants for In vivo-Regeneration of Muscles

Background
It is most probable that, in a near future, myoblast transplants will have clinical applications in domains as different as orthopaedics, endocrinology, management of heart infarct, and therapies of muscle diseases. Two Swiss teams have developed methods for collection, rapid purification and culture of myogenic stem cells and human myoblasts, and have also set up a transfection technique that allows for high transfection rates of myoblasts. Depending on the clinical problem, myogenic stem cells or myoblasts collected from a patient will be back-transplanted into the same patient with or without previous genetic engineering.

Problem
Muscle damages are frequent complications of traumas and sports accidents with serious consequences both in terms of permanent disabilities and of health economics. Often these lesions heal very poorly. A number of growth factors show promise as healing agents, but are difficult to deliver clinically.

Objective
The project aims at combining the know-how and the unique expertise of two Swiss teams in the preparation and genetic manipulation of pure human myoblast cultures on the one hand, and in the surgical transplantation of these cells in animal models on the other hand. The initial effort will be centred on orthopaedics. The objectives of the clinical application of myoblast auto-transplantations will be (i) to accelerate healing and recovery of normal function after severe muscle injuries, (ii) to prevent the complications of compartment syndromes and those induced by muscle contractures in situations requiring limb-lenghtening , (iii) to restore muscle mass and function after localised massive muscle loss .

The project proposes to introduce the use of myoblast transplantation in patients, after preliminary tests in an animal model, the pig.The goal is to use ex vivo somatic gene therapy with myoblasts modified to secrete growth factors, with the aim of improving muscle healing in patients and of demonstrating the potential of this technology.

Budget: CHF 400'000
Time frame: 1/10/2000-30/9/2003

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NFP 46 Stem Cell Transplants 4046-58712

La pénurie d'embryons guette la Suisse, l'Hebdo, 16.02.2006

Marisa Jaconi: "Nur wenn wir auch in der Schweiz das Know-how haben, können wir im internationalen Netz der Stammzellforscher mitreden und auch von deren Wissen profitieren."

Marisa Jaconi: Keine Kompromisse in Herzensangelegenheiten
Marisa Jaconi: Wir müssen die Forschung entmystifizieren

Cell Therapy of Congestive Heart Failure Using In vitro-generated Cardiomyocytes

Background/Problem
Chronic congestive heart failure (CHF) represent a major cause of cardiovascular morbidity and mortality in developed countries. Besides heart transplantation, no causal therapy is available.

Objective
As the use of human embryonic stem cells is ethically problematic, we will use an embvrxonic stem cell clone from mouse origin. In general, in vitro ESC are characterized by their ability to differentiate spontaneously into various cell lines, amongst them CCCs. Stable transfection of ESC with green fluorescent protein under the control of a cardiac-specific promoter will allow easy identiication of CCCs as well as their rapid purification by fluoresent cell sorting.

Approach
Consequently, in vitro obtained CCC, will bed delivered to the myocardium of mice by either direct intramyocasrdial injection or indirectly via application into laser-generated transmyocardial channels. In initial eperiments, mouse-derived CCCs will be delivered to healthy isogenic mice in order to test cell aurvival and integration of implanted cells in the caardiac tissue. Once this technology is optimized, subsequent experiments will finally consist in the engraftmnt of CCCs into transgenic animals with CHF. Short a long-term effects of cell therapy in, first, healthy and, utimately, in animals with CHF will bechaacterized by measuring hemodynamic, cardiac and motphologic parameters.

In the future, cell therapy might help to treat CHF effectively, avoiding either long-standing and costly drug treatment or to treat patients on a waiting list for a transplant. Of importace for the clinical impact of the proposed project is its nature as platform for collaboraion between a cell biologist, a geriatric physician, a heart surgeon and a pahologist. The important participation of clinicians in this project will ensure rapid transfer of positive results into clinical studies.

Budget: CHF 314'333
Time frame: 1/1/2001-31/12/2003

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NFP 46 Stem Cell Transplants 4046-101103

Rebuilding Thymic Function: Replacement Therapy with Thymic Epithelial Stem Cells

Background
The thymus provides the physiological microenvironment for the development of the majority of T-lymphocytes, and its function is critical for the successful establishment and maintenance of the immune system's capacity to distinguish between vital self and injurious non-self. There are numerous conditions in the course of which the integrity of thymic structure and function is severely altered, leading to life-threatening immunodeficiency secondary to defects in T-cell repertoire and number. These pathologies include (i) the thymic epithelial cell (TEC) injury caused by preconditioning with radiation/chemotherapy prior to lymphohaematopoietic stem cell transplantation (LHSCT) and (ii) the structural alterations concurrent with the emergence of certain autoimmune diseases. TEC replacement may, thus, constitute an important therapeutic strategy to accelerate T-cell reconstitution and to regain a suitable T-cell receptor repertoire.

Problem
A major impediment in the attempts to reconstitute the thymic stromal compartment has been the lack of detailed knowledge as to the existence, precise nature and phenotype of the thymic epithelial stem cell. We have recently identified a population of thymic epithelial progenitor cells in the mouse by cell surface phenotyping. These cells are able to give rise to both cortical and medullary epithelial lineages. These progenitor cells are present within the population of TECs identified by cell surface expression of the glycoprotein MTS24. Heterotopic transplantation of purified MTS24+ TECs derived from E15.5 embryos demonstrated that cells of this phenotype are sufficient to form a phenotypically and functionally normal thymic microenvironment able to initiate and sustain regular thymopoiesis, the expected selection of immature thymocytes to single positive T cells and their regular export to the periphery. Hence, these results indicate that MTS24+ TECs include a population of intrathymic progenitor/stem cells capable of giving rise to fully functional cortical and medullary thymic epithelium. Here we propose to perform an in-depth study of the phenotypic characteristics of the murine thymic epithelial precursor/stem cells and to use these (i) to efficiently rebuild the immune system in conditioned recipients of LHSCT and (ii) to generate a regular T-cell repertoire selection in mice prone to develop spontaneous autoimmune disease.

Objective
We propose that the engraftment of MTS24+ thymic epithelial precursor cells will be an effective immunotherapy for thymic injury and dysfunction. We hypothesize that the establishment of an ectopic thymus in recipients exposed to radio/chemotherapy will enhance the restoration of T-cell repertoire complexity and T-cell function after allogeneic LHSCT, and that the provision of normal TECs in certain mouse strains prone for autoimmunity will prevent the occurrence of organ-specific disease secondary to the generation of regulatory T cells.

Aim 1: To determine the precise phenotypes, gene expression patterns and in vivo functions of MTS24+ thymic epithelial progenitor/stem cells. Aim 2.: To determine the role and efficiency of thymic epithelial progenitor/stem cells (i) to correct T-cell immunodeficiency in the context pre-LHSCT conditioning.

Approach
To investigate the cellular, molecular and functional characteristics of thymic epithelial progenitor/stem cells in mice, we will take advantage of a combination of state-of-the-art techniques including flow cytometry, microsurgery, molecular biology, immunohistology, T-cell functional analysis, and in vivo models of human LHSCT and autoimmunity, respectively.

T-cell deficiencies constitute a life-threatening condition to the affected individual and an economically extremely taxing burden to the health care system as a whole. Therapeutic strategies are therefore urgently needed to rebuild the immune system in a highly efficient and accelerated way. We are convinced that the provision of epithelial progenitor/stem cells that form a ectopic thymus will be paramount for this important task. Our results will provide the critical basis for the development of a novel therapeutic strategy beneficial ti the treatment of side effects affecting T-cell repertoire selection and output. Taken together, the cell replacement strategy proposed in this grant is attuned to the overall goal of NRP 46 Implants and Transplants

Budget: CHF 350'000
Time Frame: 1/1/2003-31/12/2004

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NFP 46 Stem Cell Transplants 4046-101098

Cell Replacement Therapy in Mouse Models with an Inherited Degeneration of Motorneurons

Background/Problem
The human CNS has a limited capacity to undergo regeneration and repair; therefore the discovery of stem cells which can re-populate regions of the degenerating brain and spinal cord has incited great interest.

Objective
The goal of our work is to examine mechanisms for replacing motoneurons in the context of human diseases in which these neurons specifically degenerate (amyotrophic lateral sclerosis and spinal muscular atrophy).

Approach
We are proposing a series of experiments to examine the capability of three different stem cell lines to replace degenerated motoneurons. For these studies, we plan to inject stem cells into two mouse mutants (pmn/progressive motor neuronopathy and wobbler) that have an inherited degeneration of motoneurons in the spinal cord. Due to the presence of a reporter gene (lacZ and GFP), it will be possible to examine the survival and migration of the grafted cells at different times following transplantation. One of the fundamental questions we plan to address concerns the hypothesis that the degenerated zone may release "factors" or "signals" that would guide the stem cells to re-populate this region. In our work, we plan to determine whether the grafted stem cells will migrate into a zone of the spinal cord of the mutant mice in which degeneration has occurred. These experiments would have important implications to suggest that degeneration may reactivate developmental mechanisms that are necessary for neuronal migration and survival.

Budget: CHF 90'948
Time Frame: 1/2/2003-31/1/2005

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NFP 46 Stem Cell Transplants 4046-101109

Stem Cell Engineering as a Tool for Cell Therapy of Neurodegenerative Diseases

Konferenz zur Stammzellforschung

Background/Problem
Cell therapy is an attractive concept for the treatment of diseases that are characterized by cell loss. This holds particularly true for neurodegenerative diseases, for which presently hardly any therapeutic options exist. Until recently, it was very difficult to obtain neurons for cell therapy. Recent progress in stem cell research, both adult and embryonic stems cells, now provide very promising solutions in this respect.

Objective
However, in order to advance towards a clinical use of cell therapy, several additional obstacles have to be overcome. Among them figure prominently the optimization of i) neuronal differentiation of stem cells and ii) survival and integration of implanted stem cell-derived neurons in the resident neuronal network.

Approach
In this grant application we propose to address these points through a cellular engineering approach:
i) generate cells that express transcription factors from early neurogenesis to control neuronal phenotypic differentiation,
ii) generate cells that express 61 integrins and/or a dominant negative Nogo receptor to optimize axon outgrowth and neuronal integration in the nervous system. We propose to use embryonic stem cells as well as bone-marrow-derived pluripotent adult stem cells in our studies. All cells will express GFP to allow the distinction of implanted cells from resident cells.

Cells will initially be investigated in vitro through immunohistological and morphological analysis for neuronal differentiation. If the in vitro phenotype of the cells is promising, we will test their in-vivo behavior by implantation in mouse brain. Engineered cells will be implanted in normal mice as well as in mice subjected to a cerebral stroke. Implanted cells will be recognized by GFP fluorescence and analyzed for their phenotypical and neurochemical properties, with a particular focus on the integration into the resident neuronal network.

Budget: CHF 252'563
Time Frame: 1/2/2003-31/1/2005

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NFP 46 Stem Cell Transplants 4046-101111

Comprehending the Cells Involved in Skin Morphogenesis and Renewal: a Step Toward the Reconstruction of Hair Follicles and Sweat Glands

Background/Problem
The skin is a self-renewing organ the main function of which is to protect the body against environmental hazards. When the skin is extensively wounded the reconstruction (regeneration) of a functional epidermal barrier becomes life saving. For that purpose, cultured keratinocytes that have an extended capacity for proliferation and that can sustain epidermal renewal for years when transplanted, have been used for decades to treat deep extensive burn wounds. Thus far only epidermis, but not the epidermal appendages (hair follicles, sebaceous glands and sweat glands), is generated from transplanted autologous cultured stem keratinocytes. Patients and surgeons demand that the functionality and the aesthetic of the transplanted skin improve, meaning reconstruction of sweat glands and hair follicles, a thick dermis, and a homogenous pigmentation.

Objective/Approach
The vibrissal follicle, the largest and the most evolved of all hair follicles is an excellent model system to study the development and renewal of a mammalian epidermal appendage. In previous work, we have demonstrated that the upper region of the outer root sheath of vibrissal follicles of rats contains hundred of clonogenic keratinocytes (Kobayashi et al., Proc. Natl. Acad. Sci. 1993). We also demonstrated that human hair follicles also contain hundreds of clonogenic keratinocytes with extensive growth potential, a characteristic of stem cells (Rochat et al., Cell 1994). We have now demonstrated that the upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands and epidermis, i.e. all the lineages of the hairy skin (Oshima*, Rochat* et al., Cell 2001 co first authors*). This result is an important step toward the reconstruction of epidermal appendages. However, it remains to demonstrate that human skin contains multipotent keratinocytes and to identify the factors that affect stem cell trafficking, stem cell fate and appendage renewal. Most importantly, it is crucial to demonstrate that multipotent stem cells can be
cultivated before one can consider to reconstruct appendages ex vivo. Only then the demand for better skin will cease to be a dream.

Budget: CHF 240'000
Time Frame: 1/3/2003-28/2/2005

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NFP 46 Stem Cell Transplants 4046-101112

In vitro-Requirements for Maintenance and Expansion of Transplantable Haematopoietic Stem and Progenitor Cells

Background
In the past decade, much progress has been made in the phenotypic characterization of the rare pluripotent haematopoietic human stem cell (HSC), though for more than 40 years transplantation of HSC-activity has been used in a variety of clinical settings. However, at both the molecular and the cellular level the functional characteristics of HSCs are poorly understood, and it is still not possible to expand true pluripotent HSCs in sufficient numbers in vitro. The projects described herewith aim at a better understanding of the molecular and cellular biology of HSCs, and at defining their requirements for ex vivo maintenance and expansion. They may be of great clinical relevance in areas such as stem cell transplantations and gene therapy of hematological disorders.

Objective
To shed more light on the requirements and the cellular biology of hematopoietic stem cells, we plan
first to systematically address the question of the optimal requirements that allow the long-term maintenance and expansion of murine HSCs in tissue culture. Second, we will address the question, whether cells with similar features are present in normal mice. Third, we plan to refine methods allowing the isolation of human haematopoietic progenitor populations highly enriched for HSC activity. These populations will then be used to systematically determine the optimal growth-factor and stromal cell requirements allowing their ex vivo expansion.

Approach
Detailed characterization of the phenotypic and functional characteristics of haematopoietic stem cells by serial transplantation and in vitro- and invivo-analysis HSC in sublethally irradiated immunodeficient mice will be used to understand the requirements and the biology of HSC. Based on our previous findings that in vitro cultured pre BI cells from Pax-5 deficient mice have HSC properties, pre BI cells in which Pax-5 expression is temporarily or permanently suppressed by in vitro manipulation will be generated and tested for HSC activity. Because there are currently no reliable assays, in vitro assays for human HSC activity will be combined with serial in vivo transplantation experiments using appropriate murine models for human stem cell engraftment to measure the long-term in vivo self-renewal capacity of human HSCs.

Budget: CHF 250'000
Time Frame: 1/2/2003-31/1/2005

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NFP 46 Stem Cell Transplants 4046-101115

Transplantation of Human Stem Cells into Mouse Blastocysts

Background/Problem
The factors controlling self-renewal and expansion of stem cells or their development into different mature tissue types are among the most intensely researched areas. Recently, a promising alternative approach has been developed for human haematopoietic stem cells (HSC). HSC injected into mouse blastocysts survived during mouse embryogenesis and remained detectable in adult mice derived from injected embryos. This system offers the advantage that a few purified stem cells or even single cells can be analyzed. However, at present the efficiency of engraftment of human HSC is too low to make this system generally applicable.

Objective
Our project aims at finding conditions for improving the efficiency of engraftment of human HSC injected into mouse blastocysts. A key advantage of the proposed system is that a small number of stem cells or even single stem cells can be studied. This is of particular relevance for all studies attempting to define the phenotype of the most pluripotent stem cell populations and for protocols with the goal to expand stem cells in vitro or to assess the influence of exogenous growth factors and cytokines on stem cell commitment and differentiation.

Approach
We will pursue the following approaches:
- We will test the hypothesis that poor engraftment is due to competition with endogenous embryonic cells. To create a selective advantage for engraftment of human HSC, we will use as recipients mutant mouse blastocysts that develop into embryos incapable of forming their own HSC. The efficiency of engraftment of human HSC from different ontogenetic stages, i.e. adult, cord blood and fetal liver, will be compared.
- We will then examine engraftment of non-haematopoietic stem cells, such as human mesenchymal, muscle, pancreatic islet and hepatic stem cells.
- We will finally use mouse blastocysts as recipients for human stem cells with an inherent growth advantage, i.e. leukemic stem cells.

Budget: CHF 274'742
Time Frame: 1/1/2003-31/12/2004

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NFP 46 Stem Cell Transplants 4046-101297

Quest for "Adult Multipotent Progenitor Cell Descendants" in Recipients of Allogeneic Haematopoietic Stem Cell Transplants

Background
The almost unlimited potential of stem cells generates hopes for new forms of medical treatment. In theory, any failing organ should be accessible to corrective therapy. In practice, best access to such cells and clinical use is still unknown.

There are indications that multipotent stem cells reside in the bone marrow, in the circulation and in other tissues. They could be accessible for harvest and transplantation. Proof that they could expand after transplantation and repair tissue is lacking.

Objective
Aim of this project is to find evidence for the presence of non-haematopoietic progenitor cells within haematopoietic stem cell transplant products and for their expansion and proliferation in vivo after transplantation. We will search for donor derived cells within non-haematopoetic tissue in long term survivors of haematopoietic stem cell transplants.

Approach
We will focus on tissues with high need of organ repair following conditioning for haematopoietic stem cell transplants, e.g. hair follicles and, in men, sperm. This material is easy and repetitively acccessible by non-invasive techniques and can be collected without contaminating blood cells. Using standard donor-recipient chimerism analyses by short tandem repeat polymorphisms we should be able to detect relevant macrochimerism of at least two percent donor cells within such tissues.

The finding of donor type cells within hair or sperm samples of haematopoietic stem cell transplant recipients could be proof of principle. It would confirm the concept of adult multipotent stem cells in normal persons, document their transplantability, clarify their capacity for in vivo expansion and challenge our current concepts of paternity.

Furthermore, evidence of multipotent stem cells in clinical haematopoietic stem cell transplant products would provide a rational basis for autologous cord blood banking.

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NFP 46 Stem Cell Transplants 4046-101232

Stammzellentherapie bei Diabetes mellitus?

Ex vivo-Generation of Insulin Secreting Cells from Nestin Expressing Adult Stem Cells

Background
Our recent discovery of heretofore unknown, multipotential stem cells within the embryonic and adult islets of Langerhans suggests new therapeutic options for the treatment of diabetes mellitus type 1. These cells are characterized by the expression of the neural stem cell marker nestin and by their ability to differentiate ex vivo into pancreatic endocrine, exocrine and hepatic phenotypes with the expression of insulin, glucagon, amylase, cytokeratin-19 as well as liver specific proteins such as alpha-fetoprotein. It was also shown, that the insulinotropic hormone glucagon-like peptide-1 enhances the differentiation of these nestin positive islet derived progenitor (NIP) cells into insulin secreting cells.

Problem
The isolation of a pure NIP cell population for evaluation of their developmental properties is hampered by the lack of cell surface markers. Another problem is due to the difficulty to follow the fete of nestin positive cells and only of these cells during differentiation into insulin secreting cells and thus to give the unequivocal proof that NIP cells really are progenitors of insulin secreting p-cells. And third, it is not known whether nestin expressing cells of haematopoietic origin also have the developmental potential to differentiate into insulin secreting cells.

Objective and approach
The major goal of the proposed research project is the ex vivo-generation of insulin-secreting cells for transplantation purposes using adult human nestin expressing stem cells. In order to achieve this goal we define three projects.
Project 1: We will perform a promotor-targeted isolation of NIPs using a nestin promoter expression cassette that drives the green fluorescence protein (GFP). Cultured human islet cells will be transduced by the newest generation of lentiviral vectors. In addition, we will isolate the side population (SP) phenotype cells from cultured human islets by targeting the ABC transporter ABCG2 with a monoclonal antibody. ABCG2 is a transport protein that confers the SP phenotype. The pure nestin expressing cells as well as SP cells will be then studied for their stem cell properties; their capacity to proliferate, to form stable colonies and to differentiate into insulin secreting cells. Single cell culture studies will also be performed in order to give the unequivocal proof that NIPs are progenitors for insulin secreting cells in vitro.
Project 2: The second project will analyse the differentiation of stem cells into insulin secreting cells using the approach of promoter-targeted monitoring of ex vivo-cell differentiation. The promoters of two key genes of P-cells, IDX-1 and insulin will drive the expression of red fluorescence protein (RFP for IDX-1) or cyan fluorescence protein (CFP for insulin), respectively. These two living colours will enable the monitoring of cell differentiation by FACS analysis. This method will allow us to identify the best progenitor cell population as well as the best culture conditions for differentiation into pancreatic p-cells.
With the third project we want to test our hypothesis that human nestin expressing cells of haematopoietic origin also harbour the potential to differentiate into insulin secreting cells ex vivo, given the appropriate stimuli. Our first preliminary results indicate, that cultured human umbilical cord blood cells express the mRNA for nestin.

Significance and feasibility
A stem cell based treatment for diabetes mellitus type 1 can be envisioned with islet derived stem/progenitor cells. The proposed strategies for isolation and differentiation of islet derived stem cells comprise of very powerful tools, like the promoter targeted isolation of NIPs and promotor-targeted monitoring of cell differentiation. With the development of highly effective and well standardized isolation and expansion protocols for NIPs it will be possible to produce insulin secreting cells on a large scale. In addition, the proposed research project will give us an essential gain in knowledge on the very nature of islet derived stem cells. This will be an important step toward therapeutic application of these cells, and finally a step toward a stem cell based treatment of diabetes mellitus type 1.

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NFP 46 Mechanism of Transplant Tolerance and Rejection 4046-58609

Mechanism of Transplantation Tolerance

Background
For several years immunologist have been attracted by the idea of identifying immunoregulatory factors for use as potential therapeutic agents. A recurrent theme of immunological tolerance to islet allografts is the presence of a dense T-cell infiltrate, which surrounds, but does not invade the transplanted islets. Similar to rejection, the infiltrate of the tolerant host is composed of both CD4+ and CD8+ T-cells. Moreover, studies demonstrate the capacity of these cells to proliferate and to possess cytotoxic T lymphocyte (CTL) activity in vitro, when stimulated with an alloantigen.

Problem
The hypothesis is favoured that immunoregulatory factors exist in the microenvironment of the graft, which preclude alloreactive T-cells from effecting allograft destruction. The problem has been the difficulty in detecting these factors and the cells producing them. In supplementing cells with a soup containing multiple growth factors, there is a risk of modifying the initial phenotype or of selecting a minor subpopulation with no relevance to tolerance. Subtractive hybridisationhas been hampered by its technical difficulties. A further problem with all these techniques is the amount of total RNA required for analysis.

Objective
The principal goal of this proposal is to test the hypothesis, that true tolerance is associated with the release of immunoregulatory molecules into the microenvironement of the graft, and to determine, if the graft associated infiltrate in tolerant mice is producing unknown molecules with anti-inflammatory activity.

Approach
Using representational difference analysis (RDA) technique technique, it is possible to study the graft infiltrate in situ without introducing the pitfalls associated with cloning or the technical difficulties associated with subtractive hybridisation. Two genes, which are sociated with tolerance in the model presented, have so far been isolated. In the proposed study these genes will be expressed as proteins and tested in vitro and in vivo for immunregulatory potential. It is further palnned to perform RDA in islet graft recipients challenged with donor Ag after they acquired tolerance.

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NFP 46 Mechanism of Transplant Tolerance and Rejection 4046-58715

Topical CTLA4 fragments for tolerance induction in corneal transplantation

Background
Corneal transplantation is one of the most frequently performed surgical procedures in transplantation medicine. In spite of standard immunosuppression with topical corticosteroids eye drops, the overall 10-year graft survival is only 62 %.Irreversible graft rejection is the major cause of corneal graft failure.

Problem
Soluble CTLA4-IG that blocks CD80 and CD86 mediated co-stimulatory signals on antigen presenting cells (APC) can prolong graft survival in many experimental models including corneal transplantation when given systemically. However, the molecule is too large (100-200kDA) to penetrate through the ocular surface when given topically as eye drops. The assumption is that engineered CTLA4 fragments with a molecular weight of 14 kDa are small enough to penetrate through the conjuctiva and cornea to block CD80 and CD86 receptors on APC. A short-term (several weeks) blockade of co-stimulation by topical treatment with CTLA4 fragments is expected to result in prolonged or indefinite corneal graft survival.

Objective
The aim of this project is to engineer small recominant CTLA4 fragments with improved binding affinity to CD80/CD86 and to evaluate the immuno-modulatory effect of such fragments in vitro and in vivo when applied topically as eye drops in a rat orthotopic corneal transplantation model.

Approach
Soluble CTLA4 fragments with a size of 14 kDa will be engineered by directed molecular evolution, using the techniques of DNA shuffling in conjunction with ribosomal and phage display. This will also allow for the selection of fragments with improved binding affinity, molecular stability and protein folding. In vitro testing of the improved CTLA4 fragments will include: 1) measurement of binding affinity using a Biacore optical biosenor, 2) evaluation of in vitro immunosuppressive activity in an APC-dependent mitogenesis assay (concanavalin A) and 3) conreal penetration studies in a perfusion chamber. Subsequently, topically applied CTLA4 fragments will be tested in vivo for their tolerogenic potential in a rat model of orthotopic corneal transplantation.

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NFP 46 Mechanism of Transplant Tolerance and Rejection 4046-101082

Matrix Metalloproteinases (MMP) as Proinflammatory Mediators ant Therapeutic Targets to Sustain Long-term Graft Survival

Background
Chronic rejection remains one of the leading causes of late allograft loss. Thus, if MMP inhibition attenuates tissue damage, MMP represent novel targets for the prevention or even therapy of chronic allograft rejection in order to promote long-term graft survival. Associated with improved allograft half-lives are considerable social and economical benefits

Accumulation of extracellular matrix (ECM) proteins and increased proliferation/migration of certain cell types, particularly vascular smooth muscle cells leading to intimal hyperplasia and fibroblasts causing fibrosis and sclerosis, are characteristic histological findings in chronic rejection processes. Both features may be regulated by matrix metalloproteinases (MMP), which are generally increased in inflammatory processes. The precise role, however, of MMP as regulators of tissue remodeling in chronic allograft rejection remains to be defined.

Objective
Our studies will describe the role of MMP as novel, pro-inflammatory mediators in chronic rejection processes. Importantly, the clinical relevance of these proteases will be assessed by the intervention aimed at inhibiting excess MMP activity.

Approach
In accordance with previously published studies and our own preliminary results, we hypothesize the following:
- MMP are up-regulated during the earlier decisive stages of chronic rejection, prior to the occurrence of end-stage tissue sclerosis. Thereby, MMP act as pro-inflammatory mediators and actively participate in tissue damage associated with chronic allograft rejection. Possible mechanisms are direct tissue injury, augmentation of excess cell proliferation/migration (e.g.vascular smooth muscle cells and fibroblasts), and facilitation of tissue invasion by extrinsic inflammatory cells (e.g. lymphocytes).
- MMP inhibition, for instance by the use of synthetic MMP inhibitors, ameliorates tissue damage during chronic allograft rejection
MMP expression and activity may vary between different organs. Thus, several types of allografts need to be analyzed to define the clinical significance of these proteases.

Part 1: We propose to investigate MMP activity and expression in three different types of rat allografts, such as kidney (F344-to-Lewis), heart (F344-to-Lewis), and trachea (Brown-Norway-to-Lewis) transplantation

Part 2: We propose to analyze the functional role of up-regulated MMP by systemic application of synthetic MMP inhibitors.

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NFP 46 Mechanism of Transplant Tolerance and Rejection 4046-101255

T Cell Biology and Tolerance

Background/Problem
One of the major barriers to successful organ transplantation is the aggressive T cell response to non-self MHC (allo) antigens. As much as 1% (representing 1010 T cells) of the human T cell repertoire, can recognize a single alloantigen. Since most transplanted organs contain multiple MHC mismatches, the strength of the allo-response is enormous. Most transplant patients receive pharmacological immunosuppression, which acts non-specifically, leaving patients susceptible to infection and other side effects. For transplantation to progress beyond its current limitations, the allo-MHC response must be specifically controlled, allowing the recipients' immune System to carry out its normal functions.

Objective
The grant has 3 aims.
Aim 1: Characterize the parametetres of T cell expansion and apoptosis during graft rejection
Aim 2: Characterize the generation and function of regulatory T cells, which inhibit graft rejection.
Aim 3: Characterize the parameters of graft rejection mediated by T cells deficient in activation induced cell death (AICD).
Two issues seem to be at the core of regulating the T cell response to alloantigens.
1) How does the T cell receptor (TCR) control the balance between proliferation and activation induced cell death (AICD). This is a critical variable, which determines the extent of T cell expansion.
2) How are regulatory T cells generated and how do they carry out their regulatory functions?

Approach
Ad 1) We plan to continue to develop our model of transplantation using transgenic mice expressing a TCR specific for the class II MHC alloantigen, I-Abml2. In particular, we want to characterize the expansion and apoptosis of these alloreactive T cells in the context of skin graft rejection.
Ad 2) We would like to generate regulatory T cells (Tregs) in transgenic mice expressing an anti-I-Abm12 TCR. We are optimistic about our prospects, since this has been done in a variety of TCR transgenic mice. We are interested to know some of the parameters, which determine whether a T cell will develop into regulatory T cell. We also want to determine if Tregs can be generated under conditions of pharmacological immunosuppression and whether the transfer of Tregs to an immunosuppressed, skin grafted animal would allow the cessation of pharmacological immunosuppressive therapy.
Ad 3) We have generated a mutant TCR that is defective in generating AICD Signals. Transgenic mice expressing the mutant receptor exhibit a hyper-proliferative phenotype, which is characterized by a decreased expression of FasL, p73 and E2F-1. The decrease in FasL expression leads to a significant accumulation of T cells during an anti-I-Abm12 response. The experiments in this aim are designed to study these AICD deficient T cells in the context of graft rejection.

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NFP 46 Psychological Aspects 4046-58661

Psychosocial Risk Factors Before and After Organ Transplantation and Psychosocial Risk Factors

Report (February 2002) - Abstract Berlin 2004 - Report 2004 - University research database

Background/Problem
So far, researchers have not achieved a good understanding of psychosocial factors and their potentially beneficial or detrimental effects on transplant patients' quality of life, life satisfaction, psychological and physical well-being before and after transplantation. Yet, about one third of organ recipients show relevant psychological problems. Factors thought to increase the patient's risk for developing psychological problems include, e.g. substance abuse, lack of social support, and psychiatric symptoms.

Objective
The study investigates the associations between risk factors and quality of life, physical and psychological well-being, spiritual belief systems and life satisfaction before and after transplantation. The research project is furthermor aimed at developing a standardised screening instrument and guidelines for the identification of patients who are at high risk to develop psychological problems that affect transplant outcome.

Approach
A consecutive sample of 200 heart, liver, and lung, kidney and stem cell transplant patients (around 40 from each organ group) will be recruited at the University Hospital Zurich during the routine interdisciplinary screening procedure before waiting list placement. Participants will be assessed by means of interview and standardised questionnaires every three months during time on the waiting list and at 6, 12, and 24 months post-operation. Single case studies will be conducted with twenty patients who will be assessed at frequent, regular intervals.

Budget: CHF 351'160
Time frame: 1/9/2000-31/8/2003

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NFP 46 Psychological Aspects 4056-58619

The Experience of Relatives Who Consented or Not to Organ Donation:
A Prospective Study Six Months after Their Decision

Background/Problem
Little is known about the experience of relatives who consented or not to organ donation.

Objective
In the proposed study their experience will be investigated six months after their decision by questionnaire and interview.

Approach
It is intended that all Swiss transplant centres and all major donor hospitals in Switzerland participate in the study. Specific areas of interest are:
(i) The perception of the quality of care given by the staff of the intensive care unit,
(ii) The perception of the information process concerning brain death,
(iii) Knowledge of the wishes of the deceased regarding organ donation,
(iv) Perception of the request of organ donation,
(v) The stability of the decision to donate or not to donate over time,
(vi) Influence of the donation request upon the mourning process,
(vii) Current attitudes and beliefs about brain death and organ donation,
(viii) Suggestions to improve the performance of the staff, based upon personal experience.

The results of the study will deepen our understanding of relatives' experience with regard to the request for organ donation and the impact of this event on the following six months. The results will be integrated into the ongoing training workshops for doctors and nurses in intensive care units, with a view to improve their communication skills in dealing with grieving relatives and the donation request. The data of the study could be used by the participating intensive care units in the quality control of their care for relatives and could contribute to improve the quality of care, where necessary.
Project Leader: Prof. Dr. Alexander

Budget: CHF173'313
Start: 1/7/2000-30/6/2003

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NFP 46 Psychological Aspects 4046-058697

Improving Long-Term Successful Outcome after Renal Transplantation: A Randomized Controlled Trial to Decrease Subclinical Noncompliance

Background/Problem
Noncompliance with the immunosuppressive regimen is recongnized as an important cause of late graft loss in renal transplant recipients.

Objective
The purpose of this study is twofold: (1) to study the revalence, determinants and consequences of subclinical noncompliance in a sample of renal transplant recipients with a fonctioning graft more than 1 year posttransplant using electronic event monitoring methodology (EEM). (2) to randomize the effectiveness of an individudalized combined educational/behavioral intervention to improve compliance with immunosuppressive therapy (Randomized Controlled Trial (RCT). The study will be guided by following research questions:

Approach
Part I:What is the prevalence of subclinical noncompliance with immunosuppressive therapy in RTX recipients (>1 year posttransplant) as measured by EEM? What are selected derterminants of subclinical noncompliance with immunosuppressive therapy in RTX patients? What is the association between subclinical noncompliance and the prevalence of negative clinical outcome [(i.e. acute rejection(s) in RTX recipients beyond 1 year post-RTX until inclusion in the study (retrospective analysis)}?
Part II: What is the effect of a 3-month indiviualized combined educational/behavioral intervention on compliance with immunosuppressive regimen in subclinical noncompliers? How long does the hypothesized beneficial impact of the compliance intervention last (after completion of the 3-month intervention in the intervention cohort)?
Findings of this study will be disseminated to transplant clinicians at international and national scientific conferences and will be published in the transplant literature. Moreover, if the compliance intervention would prove to be successful, guidelines for clinical transplant practice can be developed and training sessions for transplant clinicians could be organized.

Budget: CHF 310’903
Time frame: 1/3/2001-29/2/2004

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NFP 46 Legal and Ethical Aspects 4046-58664

Legal and Ethical Issues Raised by Collection, Banking and Use of Stem Cells from Cord Blood

Background
Although collecting, processing and banking of stem cells from cord blood has been rapidly accepted worldwide as an alternative to bone marrow tranplantation, a number of legal and ethical questions pertaining to the use of stem cells from cordblood. These questions are have to be looked at in context of basic fundamental principles of legal ethics. The two main complexes are consent to collecting and legal aspects of banking. These complexes cannot be wholly separated, since consent may depend on the kind of banking envisaged.

Problem
There are similarities between the medical novelty of collection and banking of cord blood with the well-known practice of blood donation on the one hand, since blood is involved, but alo with spinal bone marrow donation on the other, since stem cells are involved. From the point of view of the law and of legal ethics, cord blood donation falls between the two, as different prerequisites are involved. Newborn children are involved, cord blood cannot be regenerated, there is no direct manipulation of the body.

Objective
Private cord blood banks, which offer collection and banking of cord blood for private use only, are stepping up pressure worldwide to be allowed to start cord blood banking on these lines. There are 5 firms active in the USA, six in Germany, and one - recently granted a licence - in Switzerland. The project aims at dealing with aspects of property - who is the legal owner of cord blood? - and of physical integrity - protection of the individual also for severed part of the body?: Consent raises another issue: who may consent to collection and what limits (welfare of the child) exist?

Approach
In particular the questions of consent, property rights, data protection and physical integrity are to be examined systematically. Interdisciplinary activities will involve the project of Alberto Bondolfi, which looks at general legal and ethical issues raised by transplant technologies.

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NFP 46 Legal and Ethical Aspects 4046-58691

Alberto Bondolfi zum Verhältnis von Information derBevölkerung und Akzeptanz umstrittener Forschung

Legal and Ethical Issues of Transplantation Technologies: Fundamental Questions

Background
In the last thirty years, organ transplantation has become medical routine. Since this technology involves serious infringmnt of physical integrity, it has been accompanied right from the beginning by intenive and controversial normative discussions among specialists about fundamental ethical issues as well as statutory regulation.

Problem
Criteria for evaluation and decision making with a view to the social dimensions of medical progress in this field have been proposed and discussed, consensus has not been reached yet. The general public are yet largely unaware of this discussion and its implications.

Objective
The projet aims at scientifically claryfying the elments of this complexe controversy and, thus, at contibuting to a more differentiated and better accepted consensus

Approach
The main aspects of research will be the citeria for consent to donation, for allocation in times of a shortage of organs, for cross-over donation and specific aspects in the context of xenotransplants.

All the projects in the field of humanities and social sciences submitted for the NRP 46 will be pursued in close contat and be co-ordinated. This projects also encompasses the creation of an informaion network and the promotion of interaction with international experts (workshops, discussion groups, scientific meetings, public conferences).

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NFP 46 Ethical and Historical Aspects 4046-58674

Brain Death in Switzerland 1960 - 2000: The Making of a Medical Innovation

Background/Problem
The definition and determination of death have recently become an issue in public discussion of euthanasia and transplantation medicine. Various concepts of human life and death are being debated. To some people the concept of brain death signifies a rupture with tradition, the medicalization of the 'formerly' natural death with the sole aim of facilitating the 'harvesting' of organs for transplantation. Others claim that the concept of brain death is not new at all. What is true for Switzerland? What are the explanations for the fact that readiness to donate organs is three times higher in the Canton Ticino than in the rest of Switzerland?

Objective
The general aim of this study is to provide some guidance for public policy making as well as for individual judgement of the still ambiguous concept of brain death.

Approach
The cultual history of brain death in Switzerand will be studied comparatively in the Canton Ticino and the German-speaking part of Swtzerland. Results will help to differentiate between medico-scientific and other cultural factors in the making and the public reception of the concept of brain death. The study will thereby make transparent ethical values constitutive for medicine since the 1960s, which may not necessarily be shared by all strata of Swiss society. The results may, thus, have considerable relevance for present and future morality.

Budget: CHF 200'595
Time frame: 1/11/2000-31/10/2003

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NFP 46 Ethical Aspects of Xenotransplantation 4046-58607

Xenotransplantation:
An Ethical Evaluation Giving Special Consideration to Animal Ethical Aspects

Background/Problem
The systematic investigation of xenotransplantation from the point of view of animal, social and medical ethics has so far not got beyond an outline of some relevant aspects. Thus, the two fundamental aspects, whether it is acceptable and feasible to make up for the shortage of human organs by providing animal organs, and, secondly, whether the use of animal organs can be justified from the point of view of animal ethics. Existing guidelines, positions and recommendations concentrate on the specific problem of transmission of zoonoses and rarely deal with other fundamental issues.

Objective
The project aims at broadening the ongoing discussion about the concept of dignity of the ceature with respect of a revision of the scope of this concept in connection with breeding and using transgenic animals, in particular as organ donors.

Approach
This project will consider ethical - especially animal ethical - problems in the context of xenotransplantation.
(i) The study will address the question of whether a different ethical evaluation for production and for housing conditions of transgenic animals used as organ donors and those used for experimentation in biomedical research is admissible. Instructive answers shall be found through "field research", qualitative interviews, questinnaires and a close collaboration with ethicists and laboratory animal specialists.
(ii) A content analysis of the contributions concering the use of primates as organ donors and laboratory animals in pre-clinical research will allow a systematization and evaluation of the arguments and will make a contribution to the clarification of controversial questions.
(iii) A third complex of problems concerns the topic of cellular xenotransplants (as opposed to organ transplants)with special emphasis on questions of different evaluation of neuronal and non-neuronal tissue transplantation and on the ethical evaluation of chimeric tissue use. A systematic evaluation of cellular xenotransplantation from an ethical perspective - taking into account current practice and possible future developments - shall be presented.

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NFP 46 Organizational Aspects of Transplantation 4046-58646

The Administrative Organization of Organ Transplantation in Switzerland

Background/Problem
Until today, there have been no legal rules dealing with the organizational aspects of transplantation activities in Swiss hospitals. When an organ is harvested, there are, again, no laws governing the attribution of that organ. Swisstransplant, a private foundation that serves as a national co-ordination centre, is based on a consensus among professionals, i.e. it works without any legal basis. Swisstransplant allocates the organs to a transplantation centre, not to a specific patient. Each transplantation centre then decides which patient it will attribute the organ to, following internal guidelines developed over the years. It is therefore submitted that in Switzerland, even though transplantation seems to be performed efficiently and satisfactorily, it is performed without any real democratic control.

Objective
This research project aims at gathering and analyzing relevant information from different systems to establish a catalogue of possible solutions regarding the most appropriate administrative framework for organ transplantation. That should prove especially valuable in the context of pending Federal legislation on organ transplantation.

Approach
The study will explore legal and administrative solutions found abroad for three different problems related to organ transplantation :
- the accreditation of transplantation centres ;
- the legal status and activities of a national co-ordination centre ;
- the rules applicable to organ allocation.

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NFP 46 Raising Public Awareness 4046-58627

Raising Public Awareness of Organ Donation

Background
Improvements in transplant medicine have transformed transplantation from an experimental stage tothe therapy of choice for patients with organ failure. Consequently, there is a growing gap between the numbers of patients needing a transplant and those receiving one. Partly this development is due to the lack of awareness of the population of organ transplantation. Publicity campaigns have an important role to play in raising aswareness of the need for more donors. Such campaigns, hoewever, require a better insight into the informational needs of people faced with the option of organ donation.

Objective
The goals of this project are

to assess the factors of people's awareness of and attitudes to organ donation
to evaluate the correlation between these factors and the consent rate
to outline and des ign efficient communicatife strategies for efective public and proessional education programmes

Approach
Starting from a theoretical model of factors which influence the intention to donate organs, we shall identify the principal influence factors by a large-scale survey. Their impact (positive or negative) on the willingness to donate together with the analysis of the ethical and legal requirements for correct information will then be used to develop and test communication strategies with a group of citizens.

The project's results will then be diffused among professionals involved in this issue through the publication of a handbook on communication strategies for organ donation and through a series of workshops with prsactitioners, officers ind the public sector and journalists.

Budget: CHF 345'435
Time Frame: 1/1/2003-31/12/2005

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