J Korean Neurosurg Soc.  2013 Apr;53(4):207-212. 10.3340/jkns.2013.53.4.207.

Analysis of Molecular Expression in Adipose Tissue-Derived Mesenchymal Stem Cells : Prospects for Use in the Treatment of Intervertebral Disc Degeneration

• Jin ES
• Min J
• Jeon SR
• Choi KH
• Jeong JH

• KMID: 1426205
• DOI: http://doi.org/10.3340/jkns.2013.53.4.207

Abstract

OBJECTIVE
Recent studies have shown encouraging progress toward the use of autogenic and allogenic mesenchymal stem cells (MSCs) to arrest, or even lead to partial regeneration in, intervertebral disc (IVD) degeneration. However, this technology is still in its infancy, and further development is required. The aim of this study was to analyze whether rat adipose-derived mesenchymal stem cells (ADMSC) can differentiate towards IVD-like cells after treatment with transforming growth factor beta3 (TGF-beta3) in vitro. We also performed quantitative analysis of gene expression for ADMSC only, ADMSCs treated with TGF-beta3, and co-cultured ADMSCs treated with TGF-beta3.
METHODS
ADMSCs were sub-cultured to homogeneity and used in fluorocytometry assays for CD11, CD45, and CD90/Thy1. ADMSCs were differentiated in spheroid culture towards the chondrogenic lineage by the presence of TGF-beta3, dexamethasone, and ascorbate. We also co-cultured pure ADMSCs and nucleus pulposus cells in 24-well plates, and performed immunohistochemical staining, western blotting, and RT-PCR for quantitativeanalysis of gene expression.
RESULTS
Results of fluorocytometry were positive for CD90/Thy1 and negative for CD11 and CD45. TGF-beta3-mediated induction of ADMSCs led to the expression of the differentiation markers of intervertebral disc-like cells, such as aggrecan, collagen II, and sox-9. Co-cultured ADMSCs treated with TGF-beta3 showed higher expression of differentiation markers and greater extracellular matrix production compared with ADMSCs treated with TGF-beta3 alone.
CONCLUSION
ADMSC treated with TGF-beta3 may be an attractive source for regeneration therapy in degenerative IVD. These findings may also help elucidate the pathologic mechanism of MSC therapy in the degeneration of IVD in vivo.

Keyword

Adipose tissue; Mesenchymal stem cells; Intervertebral disc; Cell and tissue engineering

MeSH Terms

Adipose Tissue
Aggrecans
Animals
Antigens, Differentiation
Blotting, Western
Collagen
Dexamethasone
Extracellular Matrix
Gene Expression
Intervertebral Disc
Intervertebral Disc Degeneration
Mesenchymal Stromal Cells
Rats
Regeneration
Transforming Growth Factor beta3
Aggrecans
Antigens, Differentiation
Collagen
Dexamethasone
Transforming Growth Factor beta3

Figure

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  Fig. 1 Schematic diagram shows co-culture system. Insert has low-protein binding property of PET membranes, wide contact area and numerous micro pore. ADMSC : adipose-derived mesenchymal stem cells, NP : nucleus pulposus.

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  Fig. 2 FACS analysis shows that rat ADMSCs express high levels of CD90/thy1 and low levels of CD45 and CD11b. FACS : fluorescence-activated cell sorting, ADMSCs : adipose-derived stem cells.

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  Fig. 3 Immunohistochemical analysis shows positive staining for aggrecan and collagen II (scale bar=100 µm). ADMSCs : adipose-derived stem cells, TGF-β3 : transforming growth factor β3.

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  Fig. 4 Results of RT-PCR show that ADMSCs treated with TGF-β3 displayed a significant increase in the gene expression of aggrecan, collagen II, and Sox-9 over untreated ADMSCs. Co-cultured ADMSCs treated with TGF-β3 was further elevated compared with the ADMSCs treated only with TGF-β3.

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  Fig. 5 Western blotting showed that co-cultured ADMSCs treated with TGF-β3 had greater expression of extracellular matrix compared with ADMSCs alone or ADMSCs treated only with TGF-β3.

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