hfMSCs Differentiate toward Growth Plate Cartilage Figure 2. Increase in growth plate enriched genes and decrease of articular cartilage enriched genes in time. Expression of growth plate enriched genes PANX3, EPYC, LEF1 and articular cartilage enriched genes DKK1, FRZB, GREM1 during 4 weeks of chondrogenic differentiation of hfMSCs. The y-axis (left) indicates the qPCR results as normalized fold expression on a log-scale. The x-axis (right) indicates the time in weeks. Fold changes are calculated from qPCR data expressed as delta delta CT values corrected for the housekeeping gene GAPDH. doi:10.1371/journal.pone.0044561.g002 Figure 5A depicts a large cluster of ECM related proteins that change over 3.29 fold between week 0 vs. week 5. The expression of genes encoding cartilaginous ECM proteins, such as collagens (COL1A1, COL1A2, COL2A1, COL5A2, COL11A1), chondroad- herin (CHAD), cartilage oligomeric matrix protein (COMP), secreted phosphoprotein 1 (osteopontin, SPP1), and fibronectin type III (FNDC1), was up regulated. Growth and differentiation factor 5 (GDF5), previously reported as stimulator of chondrocyte proliferation [13], was highly expressed at the earliest time point observed and down regulated thereafter. Cluster 5B depicts proteins involved in TGFb signaling. BMP2 and its downstream effector ID3 are up regulated early in differentiation consistent with previous reports on the importance of BMP signaling in chondrogenesis [14,15]. The BMP inhibitor follistatin (FST) was highly expressed at the earliest time point, and down regulated at later times and was previously shown to be expressed by proliferative, but not by hypertrophic chondrocytes [16]. This is in concordance with the observation of Kearns et al, who described the activation of a feedback loop in which the expression of follistatin is induced by BMP2 [17]. Cluster 5C depicts proteins interactions with a variety of functions, such as regulation of transcription, coagulation, matrix mineralization and early hypertrophic differentiation. Members of the complement and coagulation family of proteins were expressed during in vitro chondrogenic differentiation. Coagulation factor XIII (F13A1) expression was up regulated, while other genes like complement component I (C1R), urokinase-type plasminogen activator (PLAU) and bradykinin receptor B2 (BDKRB2) were downregulated during differentiation in our model. These proteins are associated with matrix mineralization [18–20] or matrix degradation in the growth plate [21–23], indicating early hypertrophic differentiation in the model, but not late hypertro- phy. Proto-oncogene KIT (cluster F, hypertrophy related genes), transcription factor JUN and FOS are all associated with the regulation of cell proliferation [24,25]. Cluster 5C depicts several genes involved in the regulation of cell survival and proliferation and signal transduction. Caveolin 1 (CAV1), a multifunctional scaffolding protein located at cell surface caveolae, regulates TGF, Wnt, cytokine and VEGF signaling by modulating their down- stream signaling cascades such as the JAK/STAT, b-catenin/ LEF1, MAPK/ERK and PI3K/AKT signaling pathways [26–29]. In cluster D we find proteins involved in cell cycle regulation. Expression of the nuclear envelope protein lamin B2 (LMNB2, interacts with proteins in cluster D) was first up regulated and later in differentiation down regulated in our model. Constantinescu et al describe that B-type lamins are differentially expressed during different stages of ESC differentiation and suggest a role of these nuclear envelope proteins in the regulation of of the differentia- tion. [30]. The role for LMNB2 in chondrogenic differentiation is not known, but the changes in gene expression during chondro- genic differentiation may imply a role for regulation of the timing of hypertrophic differentiation in our in vitro cultures. In figure 6 we identify the same clusters of interacting protein as in figure 5. The comparison between these images gives us insight into the route that MSCs take toward the differentiation into growth plate cartilage. The most obvious difference between the clusters identified in figure 5 and the ones in figure 6 is cluster 6E, which contains many proteins involved in late hypertrophic differentiation. Other genes showing significant differences in expression are clustered in the same groups, but the changes in gene expression profile between growth plate chondrocytes and undifferentiated MSCs is larger than that between week 0 and PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e44561