title: Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells.
abstract: High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on "hanging drop" cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies.
authors.affiliation: Institute of Pathology, University of Regensburg Medical School Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Ruedel
authors.forename: Simone
authors.initials: S
authors.lastname: Hofmeister
authors.forename: Anja-Katrin
authors.initials: AK
authors.lastname: Bosserhoff
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D020219:Chondrogenesis; D005786:Gene Expression Regulation; D005819:Genetic Markers; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D005819:Genetic Markers; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human
keywords: Chondrogenic differentiation; collagen; hanging drop; human mesenchymal stem cells