CHONDROGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS WITHIN AN ALGINATE LAYER CULTURE SYSTEM

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Abstract

Human mesenchymal stem cells (hMSCs) derived from bone marrow have the capacity to differentiate along a number of connective tissue pathways and are an attractive source of chondrocyte precursor cells. When these cells are cultured in a three-dimensional format in the presence of transforming growth factor-beta, they undergo characteristic morphological changes concurrent with deposition of cartilaginous extracellular matrix (ECM). In this study, factors influencing hMSC chondrogenesis were investigated using an alginate layer culture system. Application of this system resulted in a more homogeneous and rapid synthesis of cartilaginous ECM than did micromass cultures and presented a more functional format than did alginate bead cultures. Differentiation was found to be dependent on initial cell seeding density and was interrelated to cellular proliferation. Maximal glycosaminoglycan (GAG) synthesis defined an optimal hMSC seeding density for chondrogenesis at 25 x 10(6) cells/ml. Inclusion of hyaluronan in the alginate layer at the initiation of cultures enhanced chondrogenic differentiation in a dose-dependent manner, with maximal effect seen at 100 microg/ml. Hyaluronan increased GAG synthesis at early time points, with greater effect seen at lower cell densities, signifying cell-cell contact involvement. This culture system offers additional opportunities for elucidating conditions influencing chondrogenesis and for modeling cartilage homeostasis or osteoarthritic changes.

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Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components.

F Barry, R E Boynton, B. Liu … (2001)

Transforming growth factor (TGF)-beta-induced chondrogenesis of mesenchymal stem cells derived from bone marrow involves the rapid deposition of a cartilage-specific extracellular matrix. The sequential events in this pathway leading from the undifferentiated stem cell to a mature chondrocyte were investigated by analysis of key matrix elements. Differentiation was rapidly induced in cells cultured in the presence of TGF-beta 3 or -beta 2 and was accompanied by the early expression of fibromodulin and cartilage oligomeric matrix protein. An increase in aggrecan and versican core protein synthesis defined an intermediate stage, which also involved the small leucine-rich proteoglycans decorin and biglycan. This was followed by the appearance of type II collagen and chondroadherin. The pathway was also characterized by the appearance of type X collagen, usually associated with hypertrophic cartilage. There was also a change in the pattern of sulfation of chondroitin sulfate, with a progressive increase in the proportion of 6-sulfated species. The major proportion of newly synthesized glycosaminoglycan was part of an aggregating proteoglycan network. These data allow us to define the phenotype of the differentiated cell and to understand in greater detail the sequential process of matrix assembly. Copyright 2001 Academic Press.

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Chondrogenic Differentiation of Cultured Human Mesenchymal Stem Cells from Marrow

Frank Barry, J Mary Murphy, Clinton Chichester … (1998)

In the adult human, mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. In this study, culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days, cells secreted an extracellular matrix incorporating type II collagen, aggrecan, and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine, the withdrawal of TGF-beta(3), and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues, their maintenance, and their regeneration.