Figure 3. Analysis of changes in gene expression by micro- array. Gene selection based on principal component analysis. A) variance explained by components 1–6 from principal component analysis. B) principal components 1, 2, and 3 as expression profiles. C) selection of probes based on their factor 2 and 3 scores. D) scatterplot view of gene data in respect to their correlation (factor score) to principal components 2 and 3. Subgroups 1, 2, 3, and 4 are represented by blue, green, yellow, and pink dots, respectively. Side-placed graphs depict the gene expression profiles for genes found in the four subgroups. doi:10.1371/journal.pone.0044561.g003 week 5 differentiated MSCs. The presence of the most important nodes in larger protein interaction clusters, emphasize that this cannot be a casualty. In the differentiating MSCs we identified a steep increase in expression of the early hypertrophic marker COL10A1, however, the addition of many more hypertrophic markers in the GP samples indicate that in order to fully form GP hfMSCs Differentiate toward Growth Plate Cartilage cartilage, MSCs will have to activate genes that induce later stages of hypertrophy. Discussion 3.1 hfMSCs can serve as a model for human growth plate development In this study we identified many protein clusters involved in the differentiation of hfMSCs toward chondrocytes. In order for hfMSCs to serve as a model for the development of the epiphyseal growth plate, the cells need to obtain gene expression profiles that contain protein clusters that have been shown to play functional roles in the different stages of growth plate development. Fetal human bone marrow-derived MSCs seem to display better chondrogenic differentiation capacity than adult MSCs [10]. Consequently, it seems appropriate to use fetal bone marrow- derived MSCs as a model for chondrogenesis. However, fetal MSCs are not easily obtained, due to ethical and legal considerations, and as a consequence, adult bone marrow-derived MSCs have been used in most previous studies [31–34]. Previously, we have demonstrated that the hfMSCs differenti- ating towards chondrocytes acquired a GP cartilage-like pheno- type [11]. Chondrogenic differentiation occurred in our in vitro model, as illustrated by the progressive increase in expression of the chondrocyte markers collagen type II and X and the cartilaginous matrix constituent glycosaminoglycan over time. Interestingly, hypertrophic differentiation as evidenced by an increase in cell size and positive staining for collagen X was only observed in a discrete zone of the pellet, which was surrounded at both sites by chondrocytes positive for collagen type II. Further confirmation of chondrogenesis was obtained by analysis of mRNA expression of cartilage markers, which, in addition, also validated our microarray results. Similar gene expression patterns were obtained during chondrogenic differentiation of fetal MSCs derived from other donors. This suggested that the selected 22 weeks old fetal MSC-donor was representative for fetal bone marrow in general. Most studies in which MSCs are used to produce cartilage are aimed at obtaining articular cartilage. Cartilage matrix analysis is often limited to examining collagen type 2 and aggrecan expression. These markers are characteristic for hyaline cartilage and cannot distinguish growth plate cartilage from articular cartilage. Indeed, Huang et al. performed global microarray analysis of adult bovine MSCs at time 0 and after 28 days of differentiation in agarose constructs and compared the gene expression profile to that of chondrocytes isolated from articular cartilage. They showed that chondrogenically differentiating MSC do not form articular cartilage at 28 days in the presence of TGFb3 [35]. This is in line with our present and previous study [11]. Although many pathways are involved in chondrocyte differ- entiation, all the studies leading to the identification of these pathways have been performed in animal chondrocytes and the number of studies using human chondrocytes is limited. A recent publication of our group identified 3 soluble antagonists to the Wnt and BMP pathway as highly expressed in articular cartilage and not growth plate cartilage [36]. In this article, we showed that when MSCs differentiate towards chondrocytes, they adopt a GP- like phenotype, whereas the addition of recombinant DKK1, GREM1 and FRZB during this differentiation results in inhibition of hypertrophic differentiation and mineralization. The data presented in the current manuscript shed light on the route taken for mesenchymal stromal cells toward the chondrogenic pheno- type and may be useful in determining strategies for selective PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e44561