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. 2012;7(11):e44561.
doi: 10.1371/journal.pone.0044561. Epub 2012 Nov 5.

Fetal Mesenchymal Stromal Cells Differentiating Towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

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Free PMC article

Fetal Mesenchymal Stromal Cells Differentiating Towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

Sandy A van Gool et al. PLoS One. .
Free PMC article

Abstract

We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
Figure 3. Analysis of changes in gene expression by microarray.
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.
Figure 4
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.
Figure 5
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.
Figure 6
Figure 6. Protein interactions of differentially expressed genes between undifferentiated hfMSC and prepubertal growth plate cartilage.
Analysis of protein interactions of genes that are differentially expressed in undifferentiated hfMSC (week 0) compared to average expression profiles of growth plate cartilage of 3 prepubertal donors using STRING 9.0. Large clusters of interacting proteins can be identified (A–F). Many other genes are identified that have no known protein interactions with other genes in the list.

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Grant support

This work was supported by a grant from the European Society for Paediatric Endocrinology Research Unit and by grants from ZonMW, the Netherlands Organisation for Health Research and Development, to S.A. van Gool (grant number 920-03-392) and J.A.M. Emons (grant number 920-03-358) and a grant from the Deutsche Forschungsgemeinschaft to G. Rappold (Ra380/12-1). The authors gratefully acknowledge the TeRM Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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