Mesenchymal stem cells (MSCs) of the bone marrow replicate to produce identical daughter stem cells and can differentiate into many different cell types inculding osteoblasts. However, MSCs lose their unique properties when cultured on tissue culture plastic. This indicates that a critical feature of the marrow microenvironment that facilitates retention of stem cell properties is missing in such culture systems - a situation that impairs the study of MSCs and limits their therapeutic potential. In other tissues, the extracellular matrix (ECM) plays an important role in controlling stem cell behavior by governing their exposure to growth factors. It is also known that a proteoglycan component of the ECM, biglycan, plays an important role in the maintenance of MSC number and function. Preliminary studies indicated that culture of MSCs on a cell-free ECM made by murine marrow-derived stromal cells preserved their stem cell properties, including the capacity to self-renew, the maintenance of an undifferentiated state, and responsiveness to the pro-osteoblastogenic effect of exogenous BMP-2. This finding may be explained by evidence that the ECM sequestered endogenous BMP-2 secreted by the stromal cells. The above observations form the basis of the hypothesis that the ECM made by marrow stromal cells specifically governs the ability of MSCs to both self-renew and differentiate into a particular lineage, at least in part, by regulating exposure of cells to factors that control their replication and differentiation. Experiments are proposed to identify the principal collagen, adhesion protein and proteoglycan components of murine marrow stromal cell-derived ECM using immunostaining and confocal microscopy; and to determine whether the stromal cell-derived ECM is unique in its ability to preserve MSC properties. In addition, the amount of pro-osteoblastogenic factors, including IGF-I, TGF-beta, PDGF, VEGF, and BMP-2, in the stromal cell-derived ECM will be determined; and the impact of biglycan deficiency on the level of these growth factors in this ECM will be investigated. Finally, the question of whether MSCs grown on this ECM retain their stem cell properties will be tested in vivo following transplantation into immunodeficient mice. These studies will set the stage for investigating whether age- related bone loss is due in part to aberrant MSC behavior caused by alteration in the ECM of the bone marrow. ? ? ?
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