Regular articleRedifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis
Introduction
Chondrocytes differentiate from mesenchymal cells during embryonic development [1], and the phenotype of the differentiated chondrocyte is characterized by the synthesis, deposition, and maintenance of cartilage-specific extracellular matrix (ECM) molecules, including type II collagen and aggrecan [2], [3], [4]. It is rapidly lost during serial monolayer culture since the phenotype of differentiated chondrocytes is unstable in culture [5], [6], [7], [8]. This process is referred to as “dedifferentiation” and is a major impediment to use of in mass cell populations for cell therapy or tissue engineering of damaged cartilage. When isolated chondrocytes are cultured in a monolayer at low density, the typical round chondrocytes morphologically transform into flattened fibroblast-like cells, with profound changes in biochemical and genetic characteristics, including reduced synthesis of type II collagen and cartilage proteins [9]. When cultured three-dimensionally in a scaffold such as agarose, collagen, and alginate, redifferentiated chondrocytes reexpressed the chondrocytic differentiation phenotype. We have successfully induced differentiation of chondrocytes and marrow stromal cells by chondrosphere formation.
Mesenchymal stem cells persist in adult bone marrow as a population of cells [10] with the capacity to differentiate to osteogenic [11], chondrogenic [12], [13], [14], or adipogenic [15] lineages both in vitro and when implanted subcutaneously in SCID mice [14]. These cells can be isolated from the marrow by standardized techniques and expanded in culture through many generations, while retaining their capacity to differentiate along these pathways when exposed to appropriate culture conditions. This property of mesenchymal stem cells opens up therapeutic opportunities for the treatment of lesions in mesenchymal tissues, and protocols have been devised for the treatment of defects in articular cartilage [16], bone [17], tendon [18], and meniscus [19] and for bone marrow stromal recovery [20] and osteogenesis imperfecta [21]. It also provides an opportunity to study the differentiation of mesenchymal cells in chondrocytes in the developing limb in detail.
We have generated cDNA libraries and carried out large-scale sequencing of cDNA constructed with the oligocapping methods [22] with the objective of conducting a global survey of gene expression and differentiation of genes that are specific for redifferentiated chondrocytes and marrow stromal-derived chondrogenic progenitor. We have successfully established human diploid chondrocyte strains and marrow stromal cell strains, and although these strains are mortal, they serve a useful tool as reproducible systems of human chondrogenesis with cell-type-specific profiling patterns.
Section snippets
Isolation and cell culture of human bone marrow stromal cells (BMSCs) and dedifferentiated chondrocytes (DECs)
Bone marrow samples and normal human articular chondrocytes were obtained from normal human donors with the approval of the internal Keio Ethics Committee (#13-11). Cells were resuspended in BMSC culture medium [10% fetal bovine serum in Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (DMEM-HG)] with antibiotics/antimycotics supplements (Gibco BRL, Gaithersburg, MD, USA). Chondrocytes were maintained in chondrocyte growth medium (CGM; Bio Whittacker/Clonetics, Walkersville, MD,
Redifferentiation of DECs by chondrosphere culture
Human DECs were isolated from femoral head cartilage and designated as DECs. We used DECs beyond the 5th passage and induced rechondrogenesis by chondrosphere culture. In monolayer culture, the chondrocytes were flat with a fibroblast-like morphology (Fig. 1A and B), but they became round and in close contact with each other after chondrosphere culture at a high density cell condensation of 2 × 107 cells/ml and 2-h incubation (Fig. 1C and D). On day 5, the cells produced an ECM that exhibited
Discussion
BMSCs were successfully cultured for more than 25 passages. H4-1 cells proliferated and retained differentiation capability even after 48 population doublings (PDs), while most marrow-derived mesenchymal cells reached senescence before 30 PDs. Human marrow progenitor cells have been successfully cultured for more than 60 cell doublings with multipotency under specific culture conditions [31]. H4-1 cells had a surface marker pattern similar to that of mesodermal progenitor cells, and thus it is
Acknowledgements
We express our sincere thanks to K. Sakurada and Y. Yamada (Kyowa Hakko Kogyo Co., Ltd.) for their support throughout the work, to A. Wakamatsu for construction of the cDNA libraries and sequencing analysis, to S. Ishii, J. Yamamoto, K. Saito, and Y. Kawai for sequencing analysis, and to T. Inomata, Y. Setoyama, and N. Onoda for providing expert technical assistance. The study was supported in part by Health and Labour Sciences Research Grants, Translational Research from Ministry of Health
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Present address: National Research Institute for Child Health and Development, Tokyo, 157-8535, Japan.