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Int J Clin Exp Pathol 2013;6(12):3042-3048

Original Article
Development of a model system to analyze chondrogenic differentiation of
mesenchymal stem cells

Anke Ruedel, Simone Hofmeister, Anja-Katrin Bosserhoff

Institute of Pathology, University of Regensburg Medical School, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. Equal
contributors.

Received September 17, 2013; Accepted October 2, 2013; Epub November 15, 2013; Published December 1, 2013

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. (IJCEP1309047).

Keywords: Chondrogenic differentiation, hanging drop, human mesenchymal stem cells, collagen

Address correspondence to: Dr. Anja-Katrin Bosserhoff, Institute of Pathology, University of Regensburg Medical School, Franz-Josef-Strauss-
Allee 11, D-93053 Regensburg, Germany. Tel: +49 941 944 6705; Fax: +49 941 944 6602; E-mail: anja.bosserhoff@klinik.uni-regensburg.de