Darwin J. Prockop - US grants

The purpose of this program is to bring together several independent investigator so that they can take advantage of an unusual opportunity both to advance the basic information we have about the biology of connective tissues and to explore the possible implications that this information may have for our understanding of diseases which affect these tissues. A major part of the work on diseases will focus on the relatively well-defined genetic diseases osteogenesis imperfecta and Ehlers-Danlos syndrome. These diseases will be studied both because of their own intrinsic importance and because of the implications that several recent discoveries about these diseases have for more common conditions such as osteoporosis, osteoarthritis, and related disorders. The research will include collaborative studies in the following specific areas: Studies on the enzymes which leave the N-terminal propeptide in the conversion of procollagen to collagen and studies on the enzyme which cleaves the C- terminal propeptide in the conversion of procollagen to collagen; formation of collagen fibers in vitro by the enzymatic cleavage of procollagen; structure and function of altered procollagens synthesized by fibroblasts from patients with osteogenesis imperfecta and related disorders; and mutation in genes for type I procollagen in patients with osteogenesis imperfecta and related disorders. Human subjects are used in this work only in the sense that we receive cultured fibroblasts or skin biopsy samples either from repositories (especially the Medical Research Institute in Camden and the ATCC in Bethesda) and from physicians at a large number of hospitals in this country and abroad. Also, one of the enzymes we are studying is prepared from normal, full-term placentas. We use warm-blooded animals in the sense that we prepare antibodies by injecting antigens we have purified into rabbits. Also, we inject isolated collagen genes into mouse embryos to generate transgenic mice. These experiments will be carried out in collaboration with Dr. Robert Brent and in the Stein Laboratory of the Department of Pediatrics. (We understand Dr. Brent has filed a separate application to this committee for the experiments on transgenic mice.)

The general aim of this proposal is to carry out DNA linkage studies to identify the gene defects that can cause osteoporosis. The proposal is based on the assumption that the necessary technology is now available to definitively identify and characterize these gene defects with the same experimental strategies that have recently been successful in defining mutated genes causing a number of other diseases. The first part of the project consists of completing DNA linkage studies on seven large families with osteopenia and osteoporosis that we and our collaborators have recently identified and shown to inherit low bone mineral density (BMD) in an apparently monogenic manner. A second part will consist of searching for mutations in one candidate gene (lysyl hydroxylase) for which we have suggestive data for linkage to low BMD in one of the seven families, and a second candidate gene (BMP-2) for which we have suggestive data for linkage to low BMD in another of the seven families. A third part of the project will consist of recruiting additional large families for DNA linkage analyses through our continuing collaboration with Dr. Alan Tenenhouse who sees about 400 new patients per year at his McGilI Bone Centre, and who is currently establishing a network of nine collaborating bone centres through a grant from the Canadian government that will assay BMD and define the bone status in about 9,000 randomly selected adults. A fourth part of the study will extend our collaboration with Dr. Tenenhouse and his colleagues to recruit smaller families that can be used for the newer strategies of affected sib-pair analysis and the affected-pedigree- member method. The data from linkage studies on large families and from the sib-pair and pedigree analyses on smaller families will be used to identify candidate intervals in the genome for genes causing osteopenia and osteoporosis. When the candidate interval contains a gene that is expressed in bone and produces a gene product that is likely to be essential for the normal structure and function of bone, we will analyze the gene for mutations that cause osteopenia and osteoporosis. When the candidate interval is convincingly identified by linkage analyses, but is not known to contain a reasonable candidate gene, we will carry out detailed analyses of the interval to identify the gene or genes at fault.

This application is based on a series of new technologies that now make it possible --- for the first time --- to begin true protein engineering of type I collagen, the most abundant structural protein of higher organisms. Therefore, it is possible to resolve a series of fundamental questions about the role of collagen in normal biology and its role in a series of human diseases caused by mutations in the structure of the protein. The new technologies that now make this proposal feasible are: (a) A highly reproducible system whereby we can examine assembly of type I collagen into fibrils by enzymic cleavage of partially processed procollagen in a physiological buffer and over a physiological range of temperatures. (b) An efficient host-vector system in which milligram quantities of recombinant procollagen can be generated. (c) An experimental design whereby we overcome the technical problems posed by the large and repetitive coding sequences of type I procollagen by preparing gene constructs that consist of exchangeable DNA cassettes coding for the 4 D- periods, the telopeptides, and the propeptides of the protein. (d) Synthesis of most of the DNA cassettes required and successful expression of a parent construct that can readily be modified to obtain expression of a series of highly informative hybrid, chimeric, and mutated recombinant procollagens. We will employ these experimental tools and strategies to pursue the following Specific Aims: A. Define the structural domains and then the amino acid sequences of the type I collagen molecule that are critical for normal assembly of the protein into fibrils. B. Define the high- and low-melting domains in the triple helix of type I collagen and then define the amino acid sequences that characterize the domains. C. Define the structural domains and then the amino acid sequences of type I collagen that are binding sites for cells and cell receptors. D. Define the structural domains and then the amino acid sequences of type I collagen that contain binding sites for fibromodulin and decorin. E. Define how the binding of fibromodulin or decorin alters the assembly of type I collagen into fibrils in terms of the kinetics and thermodynamics of the assembly process and the morphology of the fibrils formed.

DESCRIPTION (Adapted from the Applicant's Abstract): The overall aim of this proposal is to answer two related questions about marrow stromal cells (MSCs): (1) do MSCs normally serve as a continuing source of precursor cells for renewal of many of the mesenchymal tissues of the body?; and (2) will further experiments in transgenic mice support preliminary observations, suggesting that systemic administration of MSCs can be used for cell replacement therapy and ex vivo gene therapy of osteogenesis imperfecta, and perhaps several other genetic diseases? The experimental protocols used to attempt to answer these two questions will be designed so that, if they produce positive results in transgenic mice, they can be subsequently used to design protocols for testing similar therapies, first in larger animals, and then in patients with osteogenesis imperfecta (OI) and related genetic diseases. The Specific Aims are: (1) to further define the tissue fate of MSCs that are infused systemically into mice, by preparing MSCs expressing marker genes driven by tissue-specific promoters, and infusing these cells into irradiated and non-irradiated mice; and (2) to determine the degree to which the OI phenotype can be rescued in transgenic mice expressing a mutated COL1A1 gene, by administration of MSCs from normal mice, and MSCs over-expressing a normal COL1A1 allele.

The overall aim of the present proposal is to carry out experiments in transgenic mice on the possible genetic causes of OA that cannot be carried out with human subjects. The experiments will address two questions: (a) Is there a close correlation between the dystrophic changes in cartilage of young mice and the severity of OA in adult mice with mutations in collagen genes expressed in cartilage? (B) Do mutations in collagen genes that cause cartilage disorders modify each other so that the severity of the defects caused by a mutation in one collagen gene are increased by coinheritance of mutations in a second collagen gene? If we find there is a correlation between late onset OA and early dystrophic changes, the results will support the evidence we have obtained from studies on one family (see Preliminary Results) that suggests X-ray examinations of young individuals can be used to predict the development of early onset OA in some families with heritable forms of the disease. If we find that mutations in two collagen genes can modify each other, they will help explain the difficulty that we and others have encountered in establishing Mendelian inheritance in many families with OA. We propose to use lines of transgenic mice already prepared by us and our collaborations as well as several additional lines we will prepare to pursue the following Specific Aims: A. Determine whether there is a correlation between (1) qualitative and quantitative assays for dystrophic changes in cartilage of young mice and (2) the severity of OA in adult mice in transgenic lines of mice with knock-out mutations in three collagen genes expressed in cartilage (Col2a1, Col9a1 and Col11a2). B. Determine whether the mild cartilage phenotypes produced by inactive alleles for each of the four collagens become more severe phenotypes when coinherited with an inactive allele for a second collagen gene expressed in cartilage. C. Determine whether the cartilage phenotypes produced by dominant negative alleles of the COL2A1 gene become more severe when coinherited with an inactive allele or a second dominant negative allele.

The cells have been variously referred to as colony-forming fibroblasts, mesenchymal stem cells, or marrow stromal cells (MSCs), have attracted increasing interest both for their biological properties as multi-potential stem-like cells and their potential use for cell and gene therapy. And their potential use for cell and gene therapy. However, a major problem in the field has been that reproducible conditions for isolation and expansion of the cells in culture have not been defined. The overall aim of the present proposal is to develop effective conditions for the isolation and expansion of human hMSCs (hMSCs) in cultured based on three recent observations made in our laboratory: (a) We have developed culture conditions whereby hMSCs can be expanded over 10/8-fold without significant loss in replicative capacity. (b) We have found that replication of hMSCs in culture is regulated autocrine/paracrine factors that can be recovered from the culture medium. (c) We have identified a sub-population of small granular cells in cultures of hMSCs that are highly replicative and appear to be the earliest progenitors in the cultures. The Specific Aims of this proposal are: (1) Determine whether the highly replicative cells we have obtained after 10/8-fold expansion of hMSCs retain their multi- potentiality to differentiate into osteoblasts, chrondrocytes and adipocytes. (2) Isolate and characterize the secreted factors that regulate replication of hMSCs in culture. (3) Isolate the small, granular, and highly replicative cells we have identified in cultures of hMSCs and define their surface epitopes.

DESCRIPTION (provided by applicant): This proposal will explore the possibility that patients with osteogenesis imperfecta (OI) can potentially be treated with their own osteoprogenitors by gene-engineening the cells from their bone marrow that are referred to as mesenchymal stem cells or marrow stromal cells (MSCs). We will use antibodies to surface epitopes we have recently identified to prepare clonal and homogeneous preparations of the earliest progenitors in cultures of MSCs that have an enhanced ability to undergo multilineage differentiation and that are rapidly self-renewing cells (RS cells). The RS cells will be used to test the hypothesis that they are similar to CD34 positive stem cells used for bone marrow transplants and that therefore they will be the most effective cells to provide long-term engraftment of osteoprogenitors into bone. We will also test the hypothesis that RS cells from patients with OI can be gene-engineered to correct the deleterious effect, of mutations in type I Collagen that produce the disease. The Specific Aims are (1) Use a series of antibodies to surface epitopes we have recently identified to prepare clonal and homogeneous preparation, of RS cells from cultures of human MSCs. In the process, test the hypothesis that RS cells can be further fractionated to obtain homogeneous preparations of stem cells that are even more effective as osteoprogenitors for engrafment into bone. (2) Define the osteogenic potential in vitro of the RS cell preparations by assays of the rates of mineralization and assays of expressed genes by mRNA microarrays and proteomics. (3) Determine the osteogenic potential in vivo of the RS cell preparations by assays of differentiation into bone after subcutaneous implantation in vehicles or after systemic infusion into immunodeficient mice. (4) Determine the feasibility of correcting the gene defect in a patients own RS cells, (a) by overexpression of a cDNA for the wildtype COL I A I gene, or (b) by replacing a mutated COLlAl gene by homologous recombination.

DESCRIPTION (provided by applicant): This proposal is for basic research on the adult stem cells from bone marrow stroma referred to as mesenchymal stem cells or marrow stromal cells (MSCs). The overall aims of the proposal are: (a) to improve our understanding of the basic biology of human MSCs (hMSCs); (b) to develop new protocols for the ex vivo expansion of hMSCs and (c) to develop ex vivo and in vivo assays for the cells that will be of value for both further basic research with the cells and for their potential clinical applications to diseases of lung. The Specific Aims are: (1) Elucidate the molecular events that permit and enhance ex vivo expansion of hMSCs. (2) Develop an ex vivo assay for the potential of hMSCs to repair injured lung by co-culturing hMSCs with lung cells that have been damaged by heat-shock and hypoxia. (3) Develop an in vivo assay for engraftment and differentiation of hMSCs to sites of tissue injury in lung. (4) Identify the properties of hMSCs that enhance their engraftment to specific sites of tissue injury in lung.

DESCRIPTION (provided by applicant): Recent reports have raised the possibility that use of adult stem cells from bone marrow may provide dramatic new therapies for both acute and chronic cardiac diseases. However, to develop a safe and effective therapy with adult stem cells, a number of problems and issues must be resolved. We will focus our attention on the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). The cells have several advantages over other stem cells in that they can readily be isolated from a patient to be treated, they are not tumorogenic, they are multipotential for differentiation into many cell phenotypes, and they have shown a remarkable ability to home to sites of tissue injury and repair the injury. We will first thoroughly characterize distinct subclasses of MSCs that we have recently identified and that apparently include the earliest progenitors and the most multipotential cells in standard cultures of MSCs. We will then compare standardized preparations of the cells for engraftment and differentiation first in a simple ex vivo co-culture system with cardiac cells, and then in rat in vivo models for acute and chronic ischemic damage to heart. The Specific Aims are: (1) Further characterize the special subclass of stem-like cells that we have recently isolated from cultures of MSCs and that have unusually long telomeres and propagate more rapidly than parallel samples of MSCs. We will test the hypothesis that the cells are pre-RS cells, i.e. precursors of the rapidly self-renewing cells (RS cells) that we previously identified as a sub-population of early progenitors in standard cultures of MSCs. (2) Use an ex vivo co-culture system to compare the ability of the putative pre-RS cells, RS cells, mMSCs, and marrow mononuclear cells, to repair hypoxic damage to cardiac cells either through direct differentiation of the cells or through cell fusion. (3) Isolate and characterize putative pre-RS ceils, RS cells, mMSCs, and mononuclear cells from rat bone marrow for testing in in vivo models of cardiac ischemia in Specific Aim 4. (4) Determine the ability of rMSCs, mMSCs, pre-RS, RS, and unfractionated mononuclear cells from rat marrow to engraft into and produce functional improvement and reduce damage in rat models of acute myocardial infarction (ischemia-reperfusion) and coronary arteriogenesis (intermittent repetitive ischemia).

[unreadable] DESCRIPTION (provided by applicant): The overall aim of this proposal is to establish a Center for preparation, quality testing, and distribution to multiple investigators of the adult stem cells referred to as MSCs. The cells have attracted increasing attention because they are readily isolated from the patient, expanded in culture, and genetically engineered. They can differentiate into multiple cell lineages and have recently reported to have some efficacy in animal models for diseases such as osteogenesis imperfecta, spinal cord injury, stroke, and cardiomyopathies. The Center will address a critical problem that has delayed research with MSCs; the difficulties that many investigators have encountered in obtaining standardized preparations of the cells. The urgent need for a Center of the kind proposed here is reflected by the fact that 84 other investigators have provided Letters of Support for the present application. The list includes essentially all the leading investigators in this rapidly growing field. The Specific Aims are to establish a Center that will: 1) prepare a continuous supply of human MSCs that are thoroughly quality tested and distribute them on request to other investigators at multiple institutions for research on the cells; 2) prepare a similar continuous supply of rat MSCs for distribution to investigators at multiple institutions; 3) prepare MSCs from human bone marrow aspirates sent by investigators from other institutions and return the quality-tested MSCs to same investigators. Also, carry out quality testing of MSCs prepared by investigators at other institutions; and 4) develop improved methods of isolating and characterizing human MSCs using the antibodies to a series of surface epitopes that have recently been identified on the cells.

DESCRIPTION (provided by applicant): Recent reports have raised the possibility that use of adult stem cells from bone marrow may provide dramatic new therapies for both acute and chronic cardiac diseases. However, to develop a safe and effective therapy with adult stem cells, a number of problems and issues must be resolved. We will focus our attention on the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). The cells have several advantages over other stem cells in that they can readily be isolated from a patient to be treated, they are not tumorogenic, they are multipotential for differentiation into many cell phenotypes, and they have shown a remarkable ability to home to sites of tissue injury and repair the injury. We will first thoroughly characterize distinct subclasses of MSCs that we have recently identified and that apparently include the earliest progenitors and the most multipotential cells in standard cultures of MSCs. We will then compare standardized preparations of the cells for engraftment and differentiation first in a simple ex vivo co-culture system with cardiac cells, and then in rat in vivo models for acute and chronic ischemic damage to heart. The Specific Aims are: (1) Further characterize the special subclass of stem-like cells that we have recently isolated from cultures of MSCs and that have unusually long telomeres and propagate more rapidly than parallel samples of MSCs. We will test the hypothesis that the cells are pre-RS cells, i.e. precursors of the rapidly self-renewing cells (RS cells) that we previously identified as a sub-population of early progenitors in standard cultures of MSCs. (2) Use an ex vivo co-culture system to compare the ability of the putative pre-RS cells, RS cells, mMSCs, and marrow mononuclear cells, to repair hypoxic damage to cardiac cells either through direct differentiation of the cells or through cell fusion. (3) Isolate and characterize putative pre-RS ceils, RS cells, mMSCs, and mononuclear cells from rat bone marrow for testing in in vivo models of cardiac ischemia in Specific Aim 4. (4) Determine the ability of rMSCs, mMSCs, pre-RS, RS, and unfractionated mononuclear cells from rat marrow to engraft into and produce functional improvement and reduce damage in rat models of acute myocardial infarction (ischemia-reperfusion) and coronary arteriogenesis (intermittent repetitive ischemia).

stem cells; technology /technique development; tissue /cell culture; stromal cells; chondrocytes; cell differentiation; adipocytes; osteoblasts; clinical research;

stem cells; tissue /cell preparation; biomedical facility; animal colony; clinical research;

stem cells; sample collection; technology /technique development; stromal cells; biomedical facility; blood; bone marrow; tissue /cell culture; chondrocytes; adipocytes; cell differentiation; mesenchyme; osteoblasts; clinical research; tissue resource /registry;

0514242
O'Connor

The objective of the proposed research is to identify factors within the microenvironment of injured tissue that may enhance stem-cell plasticity across germ layers, using an in vitro co-culture of human bone marrow stromal cells (MSC) and human bronchial epithelial cells (BEC) as a model system. The overall hypothesis is that MSC plasticity is influenced by the extent of BEC differentiation, severity of injury, and delay time between injury and repair with MSC. In testing this hypothesis the investigators propose 2 specific aims: aim 1 tests the hypothesis that MSC will acquire an epithelial phenotype, as assessed by polarity, tight cell contacts, gene expression profile, morphology and mucosa production, when co-cultured in vitro with heat-shocked BEC in a regenerated bronchial epithelium more profoundly than in a poorly differentiated monolayer, and aim 2 tests the hypothesis that (1) epithelial differentiation of MSC can be induced in vitro by an alternative mode of BEC injury - - bleomycin exposure, and (2) the extent of MSC differentiation is dependent on the severity of BEC injury and delay time between injury and addition of MSC to culture.

DESCRIPTION (provided by applicant): This application is to foster collaborative research among four groups of investigators at two neighboring institutions that will develop definitive data about the potential usefulness of adult stem cells to treat important pulmonary diseases. The staff includes one group of investigators with expertise in the preparation and characterization of adult stem cells, two groups of investigators with expertise in animal models for diseases such as fibrosis and emphysema, and a fourth group with expertise in the development of viral vectors for correction of the genetic disease cystic fibrosis (CF). The research will focus on the special class of adult stem cells that can be isolated from a patient's own bone marrow and that are referred to as mesenchymal stem cells or marrow stromal cells (MSCs). The program project format will allow the investigators to advance the field by performing collaborative work that cannot be carried out with individual research grants. Three sub-populations of MSCs that are well characterized will be tested in an innovative co-culture system that assays quantitatively their potential for repairing damaged pulmonary cells. We will also explore the possibility that cell fusion may play a role in the repair of pulmonary cells by MSCs. In addition, it will use a new assay for competitive engraftment to determine which sub-population of MSCs engrafts most efficiently in lungs of immunodeficient mice. The three sub-populations of cells will be assayed for engraftment and differentiation in a model for lung damage by asbestos and in a tracheal explant model. The three sub-populations of cells will be tested for their effectiveness in repairing lung damage in an elastase model for emphysema. In addition, we will attempt to define the mechanisms by which MSCs engraft in lung. MSCs from patients with CF will be engineered to correct the gene defect and then tested to determine whether they can become functional ciliated epithelial cells. Of necessity, the investigators will have to quickly share data as their experiment progresses. For example, data will suggest which sub-populations of MSC should be tested in the other three projects. As another example, the experience developed with lentiviruses will make it possible to use viruses for tracking MSCs.

We will use a recently developed co-culture system and an assay for competitive engraftment to identify the best candidatesfor use. The Specific Aims are: (1.) Use standard and unfractionated preparations of human and rat MSCs in the recently developed co-culture system with small airway epithelial cells (SAECs) damaged by heat shock or hyperoxia to generate quantitative assays for (a) differentiation of MSCs; (b) fusion of MSCs with SAECs; and (c) the fate of MSCs that fuse with SAECs. (2.) Use the co-culture assay developed in Specific Aim 1 to determine which of three sub-populations of MSCs that have recently been characterized in our laboratory are the most effective in differentiating into SAECs: (a) small and rapidly self-renewing cells (RS cells); (b) large and slowly replicating mature MSCs (mMSCs); and (c) cells that we have recently isolated and tentatively referred to as pre-RS cells because they are more similar to embryonic cells than RS cells. To define molecular mechanisms that explain any observed differences among cells in the assay, we will carry out microarray assays on the cells before and after addition to the co-cultures. (3.) Use the co-culture assay from Specific Aim 1 to test the same populations of MSCs in analogous co-culture systems in which the target cells are large bronchial epithelial cells, alveolar epithelial cells, or pulmonary vascular endothelial cells. (4.) Determine which of the subpopulations of MSCs from Specific Aim 2 can most efficiently engraft in lung by using a competitive engraftment assay in immunodeficient mice.

2-Deoxy-2-((methylnitrosoamino)carbonyl)amino-D-glucose; 2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose; 2-deoxy-2-[[(methylnitrosamino)-carbonyl]amino]-D-glucopyranose; 21+ years old; Adult; Animal Model; Animal Models and Related Studies; Animals; Applications Grants; Autologous; Blood Glucose; Blood Sugar; Body Tissues; Bone Marrow; Bone marrow biopsy; CRISP; Cell Function; Cell Process; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Clinical Trials; Clinical Trials, Unspecified; Colony-forming units; Computer Retrieval of Information on Scientific Projects Database; Data; Diabetes Mellitus; Diabetes Mellitus, Brittle; Diabetes Mellitus, Insulin-Dependent; Diabetes Mellitus, Juvenile-Onset; Diabetes Mellitus, Ketosis-Prone; Diabetes Mellitus, Sudden-Onset; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type I; Funding; Future; Gastrointestinal Tract, Pancreas; Grant; Grant Proposals; Grants, Applications; Home; Home environment; Human; Human, Adult; Human, General; Humulin R; IDD; IDDM; Immunodeficient Mouse; Injection of therapeutic agent; Injections; Institution; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin, Regular; Insulin-Dependent Diabetes Mellitus; Investigational New Drug Application; Investigators; Kidney; Kidney Glomerulus; Malpighian Tuft; Mammals, Mice; Man (Taxonomy); Man, Modern; Mesenchymal; Mesenchymal Progenitor Cell; Mesenchymal Stem Cells; Mice; Modeling; Mother Cells; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Novolin R; Numbers; Pancreas; Pancreatic; Patients; Progenitor Cells; Property; Property, LOINC Axis 2; Renal function; Research; Research Personnel; Research Resources; Researchers; Resources; Reticuloendothelial System, Bone Marrow; STZ; Source; Stem cells; Streptozocin; Streptozotocin; Stromal Cells; Subcellular Process; T1 diabetes; T1D; T1DM; Testing; Therapeutic; Tissues; Translations; Transplantation; Type 1 diabetes; United States National Institutes of Health; Urinary System, Kidney; Wound Healing; Wound Repair; Zanosar; adult human (21+); clinical investigation; design; designing; diabetes; experiment; experimental research; experimental study; glucose tolerance; improved; injured; insulin dependent diabetes; juvenile diabetes; juvenile diabetes mellitus; ketosis prone diabetes; kidney function; model organism; mouse model; non-human primate; nonhuman primate; renal; renal glomerulus; repair; repaired; research study; stem; stem cell therapy; tissue repair; transplant; type I diabetes

[unreadable] DESCRIPTION (provided by applicant): This is a competing renewal application for a Center to prepare adult stem/progenitor cells from the bone marrow of human volunteers and rodents, define the quality of the cells, and then distribute them to multiple investigators. The adult stem/progenitor cells we prepare were originally referred to as fibroblastic colony forming - units, then as marrow stromal cells in the hematological literature, subsequently as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells or MSCs. Since its establishment in June of 2003, the Center has made 451 shipments of 1,013 vials of frozen MSCs to 220 individual investigators, 129 of which are NIH funded (Appendix A). An additional 41 investigators have submitted MTAs to receive MSCs; of which 14 are NIH funded. A total of 114 investigators have provided letters of support for this competing renewal application. The Specific Aims are: 1. To continue to produce standardized preparations of human MSCs (hMSCs) for distribution to other investigators. 2. To continue to provide similarly characterized preparations of rat MSCs (rMSCs) for distribution to other investigators. 3. To continue to provide similar preparations of mouse MSCs (moMSCs) for distribution to other investigators. 4. To develop improved methods for isolating and characterizing hMSCs. The need for experiments with fully characterized MSCs has become critical with the current efforts by us and others to initiate Phase I and Phase II clinical trials with MSCs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a R21 application to test the hypothesis that effective new therapies can be developed for noninfectious inflammatory diseases of the cornea with either readily available adult stem/progenitor cells or the therapeutic proteins the cells produce in response to signals from injured tissues. The hypothesis will be tested through a collaboration between (a) the PI, a member of the National Academy of Sciences, who has been a pioneer in research with adult stem/progenitor cells known as MSCs, and (b) a fully-trained ophthalmologist and scientist (Joo Youn Oh, Co-Investigator). The hypothesis is based on the dramatic discovery by Dr.Oh that after a chemical burn to the cornea of a rat, application of MSCs or conditioned medium from MSCs reduced inflammation and neovascularization (Oh et al., 2008;Oh et al., 2009). Aim 1. Test the hypothesis that MSCs pre-activated in culture to express therapeutic proteins will be more effective in reducing inflammation and neovascularization following chemically-induced injury to the cornea than the standard cultures of MSCs used previously (Oh et al., 2008). Aim 2. Test the hypothesis that inflammation and neovascularization of the cornea can be reduced by application of two of the therapeutic proteins produced by activated MSCs: the anti- inflammatory protein TSG-6 and/or the anti-apoptotic protein STC-1. Aim 3. Use a previously successful strategy employed by the PI to search for additional therapeutic factors produced by MSCs in response to corneal injury. SIGNIFICANCE: If successful, the application will provide a basis for more effective therapies for the 750,000 Americans who each year suffer from acute and severe injuries of the cornea and for the 9 million Americans who suffer from dry eye syndrome. PUBLIC HEALTH RELEVANCE: There are no efficient and safe therapeutic strategies for corneal surface diseases ranging from quality-of-life deteriorating conditions (e.g. dry eye) to vision-threatening conditions (e.g. chemical burn). Most of these conditions are accompanied by noninfectious corneal inflammation and wound healing defects. This is a R21 application to test the hypothesis that effective new therapies can be developed for noninfectious inflammatory diseases of the cornea with either readily available adult stem/progenitor cells or the therapeutic proteins the cells produce in response to signals from injured tissues. If successful, the application will provide a basis for more effective therapies for the 750,000 Americans who each year suffer from acute and severe injuries of the cornea and for the 9 million Americans who suffer from dry eye syndrome.

Project Summary/Abstract ?Mesenchymal Stem Cell Derived A1-Exosomes for Traumatic Brain Injury? This proposal will develop a novel therapy for severe traumatic brain injury (TBI), a condition that has devastating effects on the victims and creates a large burden over $55 billion per year on the US healthcare system. One of the distressing features of TBI is that after the acute phase, the patients gradually develop behavioral deficits. As a result, the disease is now considered as a chronic syndrome that involves a viscous cycle in which the initial inflammatory response is excessive so that it causes considerable injury to the brain and thereby triggers another round of lasting inflammation. Numerous anti-inflammatory agents have been tested for treating TBI, but none have yet been accepted as part of standard medical care. This proposal is based on earlier publications that demonstrated administration of mesenchymal stem/stromal cells produced beneficial effects in animal models of TBI (by MSCs), primarily through reducing inflammation, and the more recent observation by us and another lab that the beneficial effects of MSCs can be reproduced with small vesicles (exosomes) produced by MSCs. The proposed studies will establish the efficacy, safety, and mode of action of A1-exosomes in a mouse model of TBI. A1-exosomes are prepared from MSCs with a scalable protocol, which can be used as ?off-the-shelf? reagents. We will compare the administration of A1-exosomes either intravenously or intranasally since we have recently found that intranasal administration of A1-exosomes dramatically reduces induced-neuroinflammation. If successful, the proposal will provide the basis for a novel clinical therapy for TBI and perhaps other diseases involving neuroinflammation such as Parkinsonism and Alzheimer?s disease.