Elucidating mechanisms of osteogenesis in human adipose-derived stromal cells via microarray analysis.
BACKGROUND
The osteogenic potential of human adipose-derived stromal cells (hASCs), the ease of cell procurement, and the shortcomings of conventional skeletal reconstruction call for further analysis of the molecular mechanisms governing hASC osteogenic differentiation. We have examined the expression profile of the human transcriptome during osteogenic differentiation of ASCs using microarray. Subsequently, we analyzed those genes related to osteogenesis that have not been previously studied about hASCs. We have preliminarily assessed the role of IGFBP3, TGF-B3, TNC, CTGF, DKK-1, and PDGFRB in hASC osteogenic differentiation.


METHODS
We compared the expression profile of undifferentiated hASCs to that of hASCs treated with osteogenic differentiation medium for 1, 3, or 7 days using the Human Exonic Evidence-Based Oligonucleotide chip. Genes significantly overexpress or underexpressed were validated with quantitative reverse transcription-polymerase chain reaction. The osteogenic capability of ASCs was verified by Alizarin Red staining.


RESULTS
IGFBP3, TGF-B3, TNC, CTGF, and PDGFRB were all upregulated in early osteogenesis, and TGF-B3, TNC, and PDGFRB were upregulated in late osteogenesis by microarray and quantitative reverse transcription analysis. In contrast, DKK-1 was downregulated in early and late osteogenesis. Alizarin Red staining showed a significant increase in mineralization in hASCs, even after 1 day in osteogenic differentiation medium.


CONCLUSIONS
Factors that commit hASCs to an osteogenic pathway remain largely unknown. We have described 6 genes that play key roles in hASC osteogenic differentiation. We plan to further exploit these data via in vitro treatment of hASCs with these soluble cytokines and in vivo translation using a nude mouse calvarial defect model.
Lee
Jackie
J
Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, USA.
Gupta
Deepak
D
Panetta
Nicholas J
NJ
Levi
Benjamin
B
James
Aaron W
AW
Wan
Derrick
D
Commons
George W
GW
Longaker
Michael T
MT
D000273:Adipose Tissue / Q000166:cytology* / Q000502:physiology*; D000704:Analysis of Variance; D000818:Animals; D000880:Anthraquinones; D002454:Cell Differentiation; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D020411:Oligonucleotide Array Sequence Analysis; D010012:Osteogenesis / Q000235:genetics*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology* / Q000502:physiology*; D019063:Tenascin / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D000880:Anthraquinones; C526317:CCN2 protein, human; C501126:DKK1 protein, human; C515497:IGFBP3 protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D019063:Tenascin; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; C010078:alizarin; D020797:Receptor, Platelet-Derived Growth Factor beta