Bone marrow contains a class of progenitor cells, termed Mesenchymal Stem Cells, which can differentiate into osteoblasts and chondrocytes: these stem cells are distinct from hematopoietic stem cells. Previous studies have shown that Mesenchymal Stem Cells can be isolated from the marrow of a variety of animals and mitotically expanded many-fold in culture. Culture-expanded cells have been implanted into immunocompatible host animals, in diffusion chambers or ceramic cubes where they differentiate into cartilage and bone, depending on the assay conditions. In addition, culture-expanded Mesenchymal Stem Cells from rabbit marrow have recently been used to regenerate cartilage in full-thickness articular cartilage defects in the rabbit knee. Methods have also been developed in our laboratory to culture expand Mesenchymal Stem Cells from human marrow. Until recently, there were no known phenotypic markers (i.e. morphology, enzyme staining or extracellular matrix or cell surface proteins) unique to Mesenchymal Stem Cells. As a first step to identifying such markers, we generated three monoclonal antibodies, SH2, SH3, and SH4 that selectively stain cell surface antigens on culture-expanded human marrow-derived Mesenchymal Stem Cells. In this study, we propose to identify the cell surface antigens recognized by the SH2, SH3, and SH4 monoclonal antibodies by liberating, purifying and biochemically characterizing, including partial amino acid sequencing, the SH2, SH3, and SH4 antigens. The partial amino acid sequences will be compared to known amino acid sequence databases to determine if the SH-antigens are known or previously uncharacterized proteins. Experimentation will then be developed to identify and sequence the genes encoding these proteins. In addition, binding assays will be used to investigate possible functional roles of the SH-antigens on the surface of Mesenchymal Stem Cells. The data generated from these studies should add to our ability to manipulate and monitor Mesenchymal Stem Cells and learn more about the role these cells have in cartilage tissue development and repair.
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