Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage Sandy A. van Gool1., Joyce A. M. Emons1., Jeroen C. H. Leijten2, Eva Decker3, Carsten Sticht4, Johannes C. van Houwelingen5, Jelle J. Goeman 5 , Carin Kleijburg6, Sicco A. Scherjon 6 , Norbert Gretz4, Jan Maarten Wit1, Gudrun Rappold3, Janine N. Post2, Marcel Karperien1,2* 1 Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands, 2 Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands, 3 Department of Human Molecular Genetics, University of Heidelberg, Heidelberg, Germany, 4 Medical Research Center, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany, 5 Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands, 6 Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands Abstract We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFb3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. Citation: van Gool SA, Emons JAM, Leijten JCH, Decker E, Sticht C, et al. (2012) Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage. PLoS ONE 7(11): e44561. doi:10.1371/journal.pone.0044561 Editor: Dmitry I. Nurminsky, University of Maryland School of Medicine, United States of America Received March 15, 2012; Accepted August 6, 2012; Published November 5, 2012 Copyright: 2012 van Gool et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the European Society for Paediatric Endocrinology Research Unit and by grants from ZonMW, the Netherlands Organisation for Health Research and Development, to S.A. van Gool (grant number 920-03-392) and J.A.M. Emons (grant number 920-03-358) and a grant from the Deutsche Forschungsgemeinschaft to G. Rappold (Ra380/12-1). The authors gratefully acknowledge the TeRM Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: h.b.j.karperien@utwente.nl . These authors contributed equally to this work. Introduction Longitudinal bone growth is the result of a tightly orchestrated proliferation and differentiation program called endochondral ossification. In the epiphyseal growth plate, chondrocytes origi- nating from mesenchymal stromal cells subsequently undergo proliferation, hypertrophic differentiation, and programmed cell death before being replaced by bone. At the time of puberty, growth first increases but at the end of puberty epiphyseal fusion and termination of growth occurs. Although estrogen has been identified as a key regulator of growth plate maturation and fusion [1], our knowledge of the molecular mechanisms underlying human growth regulation during puberty is limited. Gaining a detailed understanding of growth regulatory processes is essential to facilitate the development of novel strategies for the treatment of various growth disorders. Commonly used animal models for studying growth plate regulation represent the human epiphyseal growth plate poorly. One of the most obvious differences is that rodent growth plates do not fuse at the end of sexual maturation [2], an important hallmark of human growth plate development. The shortcoming of the mouse model is furthermore demonstrated by the contrast between the marginally affected growth phenotype of the estrogen receptor alpha (ERa) knock out mouse (aERKO) [3] and the prominent growth phenotype of a male patient lacking functional ERa [4], which is characterized by the absence of epiphyseal fusion and continuation of growth into adulthood. In order to elucidate the mechanisms involved in growth plate regulation and fusion, we need to employ different strategies. The most optimal strategy would be to investigate these processes in human growth plate specimens at different times during develop- PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e44561