Introduction Mesenchymal stem cells (MSCs) can be derived from adult bone marrow and have been shown to differentiate into cells of connective tissues such as ligament, tendon, and bone. Characterization of mesenchymal stem cells (MSCs) has proven difficult because there is little consensus regarding MSC phenotype. In addition, current MSCs are derived from methods that result in heterogeneous MSC populations, making the study of their basic biology difficult. Furthermore, it is unclear whether currently isolated MSCs can provide sustained regeneration and remodeling in vivo. We aim to address these issues in an effort to isolate the ideal MSC for potential clinical use. Specifically, we propose the following: (1) to optimize methods for MSC harvest and expansion and (2) to develop a clonal assay to determine the multipotentiality and expandability of MSC populations in vitro. We aim to develop a high-throughput screen using small volumes in 96- or 384-well plates that will allow direct comparison between various differentiating cells. This project constitutes an important component of an interdisciplinary collaboration that aims to produce a MSC-driven, biomaterials-based, regenerative therapy for revision total hip replacement surgery with severe osteolysis.
Methods We will develop protocols to enrich for MSCs from primary tissue culture and will assess the clonogenicity of each cell population. Single cells will be isolated, and those that form colonies will be further assessed for mesenchymal multipotentiality by culture in various differentiation media. Cell differentiation will be analyzed by histology and RT-PCR and Western blot analysis of lineage-specific gene products.
Preliminary Results We have isolated and characterized bulk MSC cultures and have assessed their multipotentiality using an RT-PCR-based differentiation assay. We are currently developing a method for MSC enrichment using cell surface markers and are in the midst of assessing single-cell derived colonies for self-renewal, proliferation, and differentiation along the mesenchymal lineage.
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