J Korean Med Sci.  2011 Jul;26(7):851-858. 10.3346/jkms.2011.26.7.851.

Gene Expression Profile during Chondrogenesis in Human Bone Marrow derived Mesenchymal Stem Cells using a cDNA Microarray

• Yoo HJ
• Yoon SS
• Park SY
• Lee EY
• Lee EB
• Kim JH
• Song YW

• KMID: 1094253
• DOI: http://doi.org/10.3346/jkms.2011.26.7.851

Abstract

Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. Cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. This study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns. These findings provide novel information concerning genes involved in the chondrogenesis of human BM-MSCs.

Keyword

Chondrogenesis; Mesenchymal Stem Cells; cDNA Microarray

MeSH Terms

Bone Marrow Cells/*cytology
Cell Differentiation
Chondrocytes/metabolism
Chondrogenesis/*genetics
*Gene Expression Profiling
Humans
Mesenchymal Stem Cells/cytology/*metabolism
Oligonucleotide Array Sequence Analysis
Reverse Transcriptase Polymerase Chain Reaction
Time Factors

Figure

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  Fig. 1 Schematic representation of the experimental procedure (A) and growth curve of bone marrow derived mesenchymal stem cells (BM-MSCs) in culture (B). Increases in the numbers of viable adherent cells during incubation were measured by counting trypan blue negative cells. Cultures were started at 5,000 cells per well, harvested daily for 10 days, and numbers of adherent cells were counted. Data represent mean cell numbers ± SD of three experiments performed in duplicate. BM, bone marrow; RBC, red blood cell; QC, quality control.

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  Fig. 2 Human bone marrow derived mesenchymal stem cells (BM-MSCs) confirmation by morphology, immunofluorescence staining, and flow cytometry. (A) BM-MSCs show a spindle-shaped fibroblastic morphology after culture expansion under the phase contrast microscope. Original magnification × 100. (B) Cultured BM-MSCs are positive for SH2 by immunofluorescence staining. (C) Flow cytometric analysis of BM-MSCs for the expression of SH2.

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  Fig. 3 Histochemical analyses of undifferentiated and differentiated human mesenchymal stem cells (MSCs). (A) Time course of MSC chondrogenesis. Macro picture of a differentiating MSC pellet on days 0, 3, 7, 14, and 21. A 1-mm rule is included. (B) Safranin O staining of undifferentiated (Con) and differentiated MSCs (after 3 and 7 days of differentiation).

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  Fig. 4 Gene expressions of microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). (A) Two-way hierarchical clustering of 1,486 genes expressed by undifferentiated (con) and differentiating (at 3, 7, 14, and 21 days) bone marrow derived mesenchymal stem cells. Expression profiles were clustered by average linkage hierarchical clustering. The color scale of standardized signal intensities in the microarray extends from bright green (down-regulation) to bright red (up-regulation). (B) RT-PCR analysis of 10 selected genes in undifferentiated (con) and differentiating stem cells (at 3, 7, 14, and 21 days). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

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  Fig. 5 Comparisons of Axl, synaptotagmin IV, Hrad 6B, peroxiredoxin-1, BMP-7, heat shock transcription factor-2, annexin A2, contactin-1, IL-15 and serotonin receptor-7 gene expression patterns as determined by microarray and RT-PCR analysis. The x-axis indicates the five time points (differentiation days 0, 3, 7, 14 and 21), and the y-axis represents fold changes determined by microarray and RT-PCR analysis.

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