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Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells.

Kwong FN, Richardson SM, Evans CH - Arthritis Res. Ther. (2008)

Bottom Line: The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Molecular Orthopaedics, Harvard Medical School, Longwood Avenue, Boston, Massachusetts 02115, USA. fcng@yahoo.com

ABSTRACT

Introduction: Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods: Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results: We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion: We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.

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Effect of chordin siRNA on cellular proliferation. Cellular proliferation was measured with the metabolic indicator WST-1 at the indicated periods after the start of culture. The cells were either not exposed to anything (Blank), to the transfection reagent Lipofectamine only (Lipo), or to the transfection reagent and a control siRNA (small interfering RNA) or chordin siRNA. The cells were then grown in osteogenic media for different periods. Each value is the mean ± standard deviation of three independent experiments. *P < 0.005 versus control siRNA.
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Figure 4: Effect of chordin siRNA on cellular proliferation. Cellular proliferation was measured with the metabolic indicator WST-1 at the indicated periods after the start of culture. The cells were either not exposed to anything (Blank), to the transfection reagent Lipofectamine only (Lipo), or to the transfection reagent and a control siRNA (small interfering RNA) or chordin siRNA. The cells were then grown in osteogenic media for different periods. Each value is the mean ± standard deviation of three independent experiments. *P < 0.005 versus control siRNA.

Mentions: There were no significant calcium deposits in cells cultured with BM (Figure 1). There was no difference in morphology between the control siRNA and chordin siRNA treated cells. Human MSC proliferation in the presence of Lipofectamine control, control siRNA and chordin siRNA was monitored for 7 days (as described in Materials and methods [above]). The relative number of cells was determined spectrophotometrically using WST-1 assay at 3, 5 and 7 days of culture. After 5 and 7 days, cells treated with chordin siRNA had decreased proliferation, as compared with untreated cells, and cells treated with transfection reagent only or control siRNA (P < 0.005; Figure 4).


Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells.

Kwong FN, Richardson SM, Evans CH - Arthritis Res. Ther. (2008)

Effect of chordin siRNA on cellular proliferation. Cellular proliferation was measured with the metabolic indicator WST-1 at the indicated periods after the start of culture. The cells were either not exposed to anything (Blank), to the transfection reagent Lipofectamine only (Lipo), or to the transfection reagent and a control siRNA (small interfering RNA) or chordin siRNA. The cells were then grown in osteogenic media for different periods. Each value is the mean ± standard deviation of three independent experiments. *P < 0.005 versus control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483456&req=5

Figure 4: Effect of chordin siRNA on cellular proliferation. Cellular proliferation was measured with the metabolic indicator WST-1 at the indicated periods after the start of culture. The cells were either not exposed to anything (Blank), to the transfection reagent Lipofectamine only (Lipo), or to the transfection reagent and a control siRNA (small interfering RNA) or chordin siRNA. The cells were then grown in osteogenic media for different periods. Each value is the mean ± standard deviation of three independent experiments. *P < 0.005 versus control siRNA.
Mentions: There were no significant calcium deposits in cells cultured with BM (Figure 1). There was no difference in morphology between the control siRNA and chordin siRNA treated cells. Human MSC proliferation in the presence of Lipofectamine control, control siRNA and chordin siRNA was monitored for 7 days (as described in Materials and methods [above]). The relative number of cells was determined spectrophotometrically using WST-1 assay at 3, 5 and 7 days of culture. After 5 and 7 days, cells treated with chordin siRNA had decreased proliferation, as compared with untreated cells, and cells treated with transfection reagent only or control siRNA (P < 0.005; Figure 4).

Bottom Line: The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Molecular Orthopaedics, Harvard Medical School, Longwood Avenue, Boston, Massachusetts 02115, USA. fcng@yahoo.com

ABSTRACT

Introduction: Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods: Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results: We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion: We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.

Show MeSH
Related in: MedlinePlus