In vitro osteogenic potential of human mesenchymal stem cells is predicted by Runx2/Sox9 ratio - PubMed (original) (raw)

In vitro osteogenic potential of human mesenchymal stem cells is predicted by Runx2/Sox9 ratio

Claudia Loebel et al. Tissue Eng Part A. 2015 Jan.

Abstract

Introduction: Runx2 is one of the most studied transcription factors expressed in mesenchymal stem cells (MSCs) upon their commitment toward an osteogenic differentiation. During endochondral bone formation in vivo, Sox9 directly interacts with Runx2 and represses its activity; however, the role of Sox9 in direct osteogenesis in vitro has been largely overlooked.

Methods: Bone marrow-derived human MSCs (hMSCs) were cultured in vitro either in the control or osteogenic medium supplemented with dexamethasone (DEX). To further investigate the role of Sox9 in direct osteogenesis in vitro, hMSCs were treated with Sox9 siRNA.

Results: We show here that Sox9 is the key early indicator during in vitro osteogenic differentiation of hMSCs. Osteogenic induction leads to a significant decrease of Sox9 gene and protein expression by day 7. Treatment of hMSCs with Sox9 siRNA enhanced mineralization in vitro, suggesting that downregulation of Sox9 is involved in direct osteogenesis. siRNA knockdown of Sox9 did not in itself induce osteogenesis in the absence of DEX, indicating that other factors are still required.

Conclusion: Screening of not preselected donors of different ages and gender (n=12) has shown that the Runx2/Sox9 ratio on day 7 is correlated to the (45)Ca incorporation on day 28. The impact of Sox9 downregulation in the mineralization of human MSCs in vitro indicates a so far unprecedented role of Sox9 as a major regulator of direct osteogenesis. We propose that the Runx2/Sox9 ratio is a promising, early, in vitro screening method for osteogenicity of human MSCs.

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Figures

<b>FIG. 1.</b>

**FIG. 1.

Sox9 gene expression of (A) on day 2 and 7 (_n_=12); (B) on day 14 and 21 (_n_=4), based on expression fold change to day 0 (mean±SEM *p<0.05); (C) Sox9 protein analysis: western blot and image analysis on day 7 either in control or dexamethasone (DEX) medium. Laminin B 1 served as internal control; one representative donor (_n_=3).

<b>FIG. 2.</b>

**FIG. 2.

Runx2 gene expression (A) on day 2 and 7 (_n_=12); (B) on day 14 and 21 (_n_=4); Runx2/Sox9 ratio (C) on day 2 and 7 (_n_=12), (D) on day 14 and 21 (_n_=4), based on expression fold change to day 0 (mean±SEM *p<0.05).

<b>FIG. 3.</b>

**FIG. 3.

(A) Runx2/Sox9 ratio on day 7 (mean±SEM *p<0.05); (B) 45Ca incorporation on day 28 (mean±SEM *p<0.05), (C) alkaline phosphatase (ALP) activity on day 14, for 8 representative donors with low (_n_=4) and high (_n_=4) osteogenic potential (high osteogenic potential defined as above 100,000 CPMI/μg DNA on day 28; low osteogenic potential as below 80,000 CPMI/μg DNA on day 28); (D) ALP activity for donors with both low and high osteogenic potential (_n_=8); (E) correlation of 45Ca incorporation on day 28 with Runx2/Sox9 ratio on day 7; for unselected population of donors (_n_=12).

<b>FIG. 4.</b>

**FIG. 4.

(A) Electroporation of hMSCs with Sox9 siRNA, gene expression after 48 h based on expression fold change to day 0; *****indicates significant difference between Sox9 siRNA and controls (_n_=6, mean±SEM, *p<0.05), (B) protein analysis: western blot and image analysis of Sox9 protein after Sox9 siRNA electroporation (48 h) in control medium. Laminin B 1 served as internal control; one representative donor (_n_=3); (C) Runx2/Sox9 ratio after 48 h, one representative donor; (D) 45Ca incorporation on day 28 either in control or DEX medium; *indicates significant difference between Sox9 siRNA treatment and scrambled control and control, respectively (_n_=6, mean±SEM, *p<0.05); (E) staining of alizarin red S on day 28 after Sox9 siRNA treatment, either in control or DEX medium; representative images (scale bar: 200 μm); (F) optical density graphs in DEX medium; *****indicates significant difference between Sox9 siRNA and control groups (_n_=6, mean±SEM *p<0.05). Color images available online at

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References

    1. Lefebvre V., and Smits P.Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res C Embryo Today 75**,**200, 2005 -PubMed
    1. Zhao Q., Eberspaecher H., Lefebvre V., and de Crombrugghe B.Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev Dyn 209**,**377, 1997 -PubMed
    1. Hattori T., Muller C., Gebhard S., Bauer E., Pausch F., Schlund B., Bosl M.R., Hess A., Surmann-Schmitt C., von der Mark H., de Crombrugghe B., and von der Mark K.SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development 137**,**901, 2010 -PubMed
    1. Ducy P., Zhang R., Geoffroy V., Ridall A.L., and Karsenty G.Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89**,**747, 1997 -PubMed
    1. Yamaguchi A., Komori T., and Suda T.Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 21**,**393, 2000 -PubMed

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