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The difficulty in long-term expansion of mesenchymal stem cells (MSCs) using standard culture systems without the loss of their stem cell properties suggests that a critical feature of their microenvironment necessary for retention of stem cell properties is absent in these culture systems. We report here the reconstitution of a native extracellular matrix (ECM) made by human marrow cells ex vivo, which consists of at least collagen types I and III, fibronectin, small leucine-rich proteoglycans such as biglycan and decorin, and major components of basement membrane such as the large molecular weight proteoglycan perlecan and laminin. Expansion of human MSCs on this ECM strongly promoted their proliferation, retained their stem cell properties with a low level of reactive oxygen species (ROS), and substantially increased their response to BMP-2. The quality of the expanded cells following each passage was further tested by an in vivo transplantation assay. The results showed that MSCs expanded on the ECM for multiple passages still retained the same capacity for skeletogenesis. In contrast, the bone formation capacity of cells expanded on plastic was dramatically diminished after 6-7 passages. These findings suggest that the marrow stromal cell-derived ECM is a promising matrix for expanding largescale highly functional MSCs for eventualuse in stem cell-based therapy. Moreover, this system should also be invaluable for establishment of a unique tissue-specific ECM, which will facilitate control of the fate of MSCs for therapeutic applications.

stem cells and development

Reconstitution of Marrow-Derived Extracellular Matrix Ex Vivo: A Robust Culture System for Expanding Large-Scale Highly Functional Human Mesenchymal Stem Cells