Objective. Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo.Methods. During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology.Results. The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content.Conclusion. An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.

Objective. To compare the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (BMSC) and adipose tissue-derived stromal cells (ATSC), because the availability of an unlimited cell source replacing human chondrocytes could be strongly beneficial for cell therapy, tissue engineering, in vitro drug screening, and development of new therapeutic options to enhance the regenerative capacity of human cartilage.Methods. Quantitative gene expression of common cartilage and cell interaction molecules was analyzed using complementary DNA array technology and reverse transcription-polymerase chain reaction during optimization of cell differentiation, in order to achieve a molecular phenotype similar to that of chondrocytes in cartilage.Results. The multilineage potential of BMSC and ATSC was similar according to cell morphology and histology, but minor differences in marker gene expression occurred in diverse differentiation pathways. Although chondrogenic differentiation of BMSC and ATSC was indistinguishable in monolayer and remained partial, only BMSC responded (with improved chondrogenesis) to a shift to high-density 3-dimensional cell culture, and reached a gene expression profile highly homologous to that of osteoarthritic (OA) cartilage.Conclusion. Hypertrophy of chondrocytes and high matrix-remodeling activity in differentiated BMSC spheroids and in OA cartilage may be the basis for the strong similarities in gene expression profiles between these samples. Differentiated stem cell spheroids represent an attractive tool for use in drug development and identification of drug targets in OA cartilage-like tissue outside the human body. However, optimization of differentiation protocols to achieve the phenotype of healthy chondrocytes is desired for cell therapy and tissue engineering approaches.

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor β (TGFβ)-mediated induction in vitro.Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGFβ 3 , dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles.After TGFβ-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture.In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage. 2005;23:403-411 Stem Cells