ment. However, human growth plate specimens are difficult to obtain [5]. In vitro models such as chondrosarcoma cell lines or articular cartilage-derived chondrocyte cultures have limited differentiation capacity, are often difficult to maintain under laboratory conditions or tend to dedifferentiate [6,7]. Further- more, articular cartilage and growth plate cartilage have distinct functions and it is therefore debatable whether articular cartilage- derived chondrocytes are representative for epiphyseal growth plate chondrocytes. Multipotent human mesenchymal stromal cells (hMSCs) are promising for studying chondrogenesis in vitro. hMSCs are an alternative cell source for articular cartilage reconstruction and for studying endochondral ossification as it occurs in the epiphyseal growth plate [8]. In addition, we found that MSCs in co-culture with chondrocytes contribute greatly to better cartilage generation in vitro [9]. In this study, we explored the cartilage forming capacity of human fetal (hf)MSCs in time. In addition, we explored whether differentiating hfMSCs may provide an in vitro model for the human growth plate. We have chosen human fetal bone marrow-derived MSC for their superior differentiation characteristics compared to adult bone marrow-derived MSCs [10]. Efficient cartilage formation was demonstrated by immuno- histochemical analysis and gene expression profiling was applied to identify genetic pathways involved in the differentiation process. In addition, gene expression profiles of the differentiating hfMSCs were compared with global gene expression patterns of human articular and growth plate cartilage to assess whether differenti- ating hfMSCs represent either articular or growth plate chondro- cytes. Results 2.1 Chondrogenically differentiating hfMSCs express typical markers of hyaline cartilage 2.1.1. Evaluation of protein and mRNA expression. Two major types of hyaline cartilage can be distinguished, namely articular and epiphyseal cartilage. In order to serve as a model for the epiphyseal growth plate, differentiating hfMSCs should obtain a growth plate signature. Pellet cultures were used to induce chondrogenic differentiation of hfMSCs and samples were collected at 1, 2, 3, 4 and 5 weeks of culture. Immunohistological evaluation showed an increasing expression of cartilage markers with time and a gradual morphological change from stromal cells to mature and hyper- trophic chondrocytes (Figure 1). The mean diameter of the pellets increased with time, as well as the amount of glycosaminoglycans, a major constituent of the cartilaginous extracellular matrix. Immunofluorescent staining for collagen type II demonstrated the presence of chondrocytes after 1 week of pellet culture. The expression of collagen type II increased over time. Hypertrophic chondrocytes were first detected after 3 weeks, as evidenced by immunohistochemical staining for collagen type X. These collagen type X positive cells were located in a discrete ring-like zone surrounded by collagen type II positive chondrocytes. In all stages of differentiation, the chondrogenic core of the pellets was surrounded by a thin layer of two to three undifferentiated cells (Figure 1). Matrix mineralization was not observed after 5 weeks of differentiation (data not shown), suggesting that the matrix was not ready for mineralization or that environmental stimuli necessary to induce this process were absent. From each time point RNA was isolated and subjected to microarray analysis. Changes in gene expression of a subset of genes consisting of both established marker genes for chondro- genesis and differentially expressed genes identified by microarray hfMSCs Differentiate toward Growth Plate Cartilage analysis were validated using qPCR (Figure S1). In concordance with the observations of immunohistological markers of chondro- genesis, microarray data and qPCR showed time-dependent increases in the expression of the cartilage markers COL2A1, ‘COL10A1, SOX9, and ACAN mRNA. Previously, we have shown that chondrogenic differentiation of MSCs results in a growth plate (GP-) like rather than an articular phenotype [11]. In this study, we compared the gene expression profiles of human articular cartilage to human growth plate cartilage of the same donors. Global gene expression microarray analysis showed that the Wnt antagonist DKK1 and FRZB and the BMP antagonist GREM1 are highly expressed in articular cartilage as compared to growth plate cartilage. In contrast, PANX3, EPYC, WNT11 and LEF1 are highly expressed in growth plate cartilage as compared to articular cartilage. To investigate whether the differentiation of the hfMSCs in the chondrogenic lineage results in expression of markers specifically found in the GP vs AC, we selected genes that are significantly differentially expressed in these two types of hyaline cartilage; for GP: PANX3, EPYC, WNT11, LEF1, and for AC: GREM1, FRZB, DKK1. We observed a sharp increase in the expression of PANX3, EPYC and LEF1 (GP enriched genes) in the differentiating MSCs while the AC enriched genes FRZB, and DKK1 showed a marked decrease in their expression over time (figure 2). The gene expression of GREM1 hardly changed over time. This indicates that the cells acquire a GP-like expression profile early during the differentiation. 2.1.2. Principal component analysis and KEGG pathway analysis identifies 9 pathways associated with chondrogenic differentiation. The sequential changes that occur during chondrogenic differentiation in the hfMSC model were studied with bioinformatics analysis of the microarray data. Using principal component analysis, three components were found to explain 99.6% of the variance within our dataset (figure 3A). The factor loadings in figure 3B show that component 1 describes a general level of gene expression, as expected. Component 2 shows to what extent gene expression changed with time during chondrogenic differentiation and component 3 signifies there was an additional, short-term elevation or dip in expression around 2 to 3 weeks of differentiation. Since components 2 and 3 were most likely to contain genes associated with the loss of stromal cell characteristics or the gain of a chondrocyte phenotype, we focused on those components. Using the $3.29 cut-off in combination with a 5% significance test, we distinguished four subgroups of probes. The precise definitions and the resulting numbers of these subgroups are given in figure 3C. The scatter plot in figure 3D illustrates that the numbers of probes in subgroups 1 and 2 are much larger than the 9 probes (0.05%) that would have been expected under purely random selection. Moreover, in these two subgroups nearly all probes in the first selection are significant at the 5% level, suggesting that the number of false discoveries in these two groups is quite small. More noise is presumably present in the smaller subgroups 3 and 4 based on factor 3 scores. The profiles of the selected probes demonstrate that subgroup 1 containing the largest number of probes (n = 146) describes a peak of expression on t0 (week 0) followed by a decrease in expression thereafter. In contrast, the second largest subgroup of probes (n = 105) in profile 2 demonstrates increasing expression levels from t0 onward. The smaller subgroups 3 and 4 demonstrate lower levels of expression with profile 3 (n = 49) showing a short-term increase in expression at t1 (week 1) followed by decreases PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e44561