hfMSCs Differentiate toward Growth Plate Cartilage Figure 4. Gene expression of genes by KEGG signaling pathway. KEGG signaling pathways significantly associated with chondrogenic differentiation of hfMSCs. For each pathway, genes showing the same distinct expression profile during 5 weeks of chondrogenic differentiation are depicted as groups. doi:10.1371/journal.pone.0044561.g004 generation of either functional GP cartilage for research into growth plate (GP) maturation and fusion or generation of functional articular cartilage for tissue engineering strategies. 3.2 Differentiating hfMSCs show signs of early chondrogenesis, not endochondral ossification and matrix mineralization In order to get insight into the requirement for a growth plate gene expression profile, we generated a map of the protein-protein interactions of the proteins of which the gene expression changed more than 3.29 fold. We compared that map to a map of protein- protein interactions of genes that change between undifferentiated MSCs and growth plate chondrocytes. We observed the same clusters of interacting proteins in both maps, and obtained clear evidence that there is early hypertrophic differentiation, as witnessed by the increased expression of collagen X. We obtained less evidence for late hypertrophic differentiation, although markers such as VEGF, LIF, PLAU, C1R and F13A1 are upregulated during week 4+5 of chondrogenic differentiation. We have found no indication of mineralization in our cultures. This indicates that the chondrogenic differentiation of hfMSCs is a good model for the early phases of growth plate development, but under these conditions, not for the late phase. Other reports, more specific in the field of bone tissue engineering have indicated Figure 5. Protein interactions of significantly changed genes after 5 weeks of chondrogenic differentiation. Analysis of protein interactions of all genes that were $3.29-fold changed after 5 weeks of chondrogenic differentiation as compared to undifferentiated hfMSC (week 0) using STRING9.0. Different clusters of interacting proteins can be identified, clusters A–F. Many other genes are identified that have no known protein interactions with other genes in the list. doi:10.1371/journal.pone.0044561.g005 PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e44561