hfMSCs Differentiate toward Growth Plate Cartilage Figure 1. hfMSCs micromasses undergo chondrogenic differentiation. Expression of (A) glycosaminoglycans visualized by toluidine blue staining, nuclei are counterstained with haematoxylin, (B) collagen type II immunofluorescence, and (C) collagen type X immunohistochemistry (red- brown) during 5 weeks of chondrogenic differentiation of hfMSCs to chondrocytes. The top panel shows a magnification of the pellet cultures at week 1 and week 5 stained by toluidine blue demonstrating the change in cell morphology and the deposition of the extracellular matrix. The insets in panel B show higher magnifications of collagen type II positive chondrocytes. doi:10.1371/journal.pone.0044561.g001 thereafter and profile 4 (n = 15) displaying a short-term expression dip between t1-t2 (week 1- week 2). A total of 83 out of 315 probes could not be annotated and was discarded from further analysis. The remaining 232 probes that could be matched to genes (table S1) were used to identify 9 KEGG pathways that were significantly associated with chondro- genic differentiation and contained 39 genes (figure 4). Some genes were present solely in one pathway (n = 23), but others were found in 2 (n = 6) or 3 (n = 10) pathways (Figure 4). Three functional groups of genes were recognized: 1) growth factor (GF) and GF- related genes; 2) genes associated with the extracellular matrix; and 3) genes associated with signal transduction, cell cycle, and cell survival. In table S2, we have listed the top hits of upregulated and down-regulated genes (with a cut-off of at least 3.29-fold change) as identified by the microarray analysis at week 5 compared to undifferentiated hfMSCs. 2.1.3 Network analysis of significantly changed genes identifies many chondrogenesis associated clusters. In order to identify protein-protein interactions between proteins encoded by genes shown in table S2 STRING 9.0 (http://string- db.org) was used (figure 5). The clusters identified include many proteins involved in skeletal development and regulation of ECM homeostasis (figure 5 A, E), such as ACAN, COL2A1, ADAMTS5, PTH1R and GDF5, transcription regulators (figure 5C), such as JUN and FOS, as well as a node containing TGFb superfamily proteins (figure 5B), such as GREM1. In addition, WNT11 and FZD2 seem to play a role in the chondrogenic differentiation of hfMSCs. We propose that if proteins in a related network mode change simultaneously, this increases the likelihood that these proteins play an important role in the differentiation of the hfMSCs toward the chondrogenic lineage. 2.2 hfMSCs acquire a partial GP phenotype after 5 weeks of differentiation Histological and gene expression analyses showed that the differentiating hfMSCs acquired a hyaline cartilage phenotype. In order to investigate how the expression profile changes from a MSC toward a GP profile we compared the gene profiles of the primary hfMSCs with prepubertal GP samples and identified many genes that are differentially expressed (table S3). In order to visualize the protein-protein interactions of the proteins encoded by these genes, we used STRING9.0. STRING (Search Tool for the retrieval of Interaction Genes/Proteins) is a freely available tool to visualize known and predicted protein interactions [12]. As such, it visualizes underlying protein networks that that are significantly changed within our data set. Interestingly, there is a large overlap of clusters identified in the profiles of MSC week 0 vs. MSC week 5 (figure 5) and the profile of MSC week 0 vs prepubertal GP (figure 6). Note that these clusters are based on protein-protein interactions, and not functional similarities. Protein clusters include ECM related proteins (figure 6A, E), TGFb superfamily related proteins and their interactants (figure 6B), transcription regulatory proteins (figure 6C), and cytoskeleton related proteins (figure 6D. In addition, a large cluster containing proteins that are important in chondrocyte hypertro- phy is visible in the MSC vs. GP data. PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e44561