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. 2011 Apr;17(7-8):1003-13.
doi: 10.1089/ten.TEA.2010.0499. Epub 2011 Jan 10.

Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells in Self-Gelling Alginate Discs Reveals Novel Chondrogenic Signature Gene Clusters

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Free PMC article

Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells in Self-Gelling Alginate Discs Reveals Novel Chondrogenic Signature Gene Clusters

Sarah Roxana Herlofsen et al. Tissue Eng Part A. .
Free PMC article

Abstract

We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants.

Figures

FIG. 1.
FIG. 1.
Quantitative real-time reverse transcription–polymerase chain reaction analysis of genes expressed in human bone marrow (hBM)–mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation in self-gelling alginate scaffold. Samples from three different donors were gathered at days 0, 7, 14, and 21. Shown values are the mean and SD of triplicate determination of each single donor, normalized to the expression of the housekeeping gene GAPDH. The mRNA expression profiles shown are for (A) type II, I, and X collagen; (B) the chondrogenesis-associated transcription factors SOX9, SOX5, and SOX6; (C) the noncollagenous matrix molecules aggrecan (ACAN), cartilage oligomeric matrix protein (COMP), and versican (VCAN); and (D) the hypertrophic marker genes matrix metalloproteinase 13 (MMP13), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL).
FIG. 2.
FIG. 2.
Fluorescence immunohistochemical analysis of protein expression in chondrogenically differentiated hBM-MSCs. (A) Co-staining of SOX9 (green) and COL2 (red) in samples derived after 7, 14, and 21 days of chondrogenic differentiation of hBM-MSCs in alginate. (B) Expression on day 21 of chondrogenic differentiation of the COL1 and COL10, SOX5, and SOX6 transcription factors and the noncollagenous matrix molecules ACAN and VCAN at day 21 of differentiation. Nuclei are counterstained with DAPI (blue). Scale bars = 50 μm.
FIG. 3.
FIG. 3.
Synthesis of sulfated glycosaminoglycans (sGAG) during chondrogenic differentiation of hBM-MSC in alginate discs. sGAG concentrations were measured in supernatants from days 7, 14, and 21 in cultures of hBM-MSC in alginate discs maintained in the culture medium (hatched bars) and in the chondrogenic differentiation medium (dark bars). The concentration of synthesized sGAG in the supernatant was measured after 3 days without changing the medium. Data presented are mean values for the three donors, each measured in triplicate. *p < 0.05.
FIG. 4.
FIG. 4.
(A) Expression profile of genes obtained from COL2A1 similarity search. Data were obtained from three donors under five different conditions: undifferentiated monolayer cells (MSC), 7 days of chondrogenic differentiation (DIF7), 14 days of chondrogenic differentiation (DIF14), 21 days of chondrogenic differentiation (DIF21), and undifferentiated three-dimensional control cells, which were cultured in alginate for 3 weeks using 10% fetal bovine serum (FBS; FBS21). The 98 shown genes (shown in gray) are the 5% most similar differentially expressed genes (>2-fold change, p < 0.05) compared to expression of COL2A1 (shown in black). Relative expression levels (log2 ratios) after mean normalization are shown. (B) The 20 most similar genes (left column) and genes within the 5% most similar differentially expressed genes belonging to three selected gene ontology (GO) terms are shown together with their similarity rank number relative to COL2A1 expression. All of these GO terms were significantly upregulated in the similarity cluster (p < 0.001).
FIG. 5.
FIG. 5.
(A) Upregulation of SOX8 mRNA during chondrogenesis was validated by quantitative real-time reverse transcription–polymerase chain reaction. Data shown were obtained from three different donors, each measured in triplicate, at different time points of chondrogenesis. Day 0 corresponds to undifferentiated monolayer MSCs. Expression levels are relative to GAPDH expression. (B) To characterize the protein expression of SOX8, we performed fluorescent immunohistochemical staining at day 21 of differentiation. Nuclei are counterstained with DAPI (blue). Scale bar = 50 μm.
FIG. 6.
FIG. 6.
(A) Expression profile of genes obtained from CXCL12 similarity search. Data were obtained from three donors under five different conditions: undifferentiated monolayer cells (MSC), 7 days of chondrogenic differentiation (DIF7), 14 days of chondrogenic differentiation (DIF14), 21 days of chondrogenic differentiation (DIF21), and undifferentiated three-dimensional control cells, which were cultured in alginate for 3 weeks using 10% FBS (FBS21). The 98 shown genes (shown in gray) are the 5% most similar differentially expressed genes (>2-fold change, p < 0.05) compared to expression of CXCL12 (shown in black). Relative expression levels (log2 ratios) after mean normalization are shown. (B) The 20 most similar genes (left column) and genes within the 5% most similar differentially expressed genes belonging to three selected GO terms are shown together with their similarity rank number relative to CXCL12 expression. All of these GO terms were significantly upregulated in the similarity cluster (p < 0.001).

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