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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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Transfection efficiency of siRNA in relation to concentration. Final concentration of small interfering RNA (siRNA) in culture media in nanomoles/litre (nM). Histograph analysis of flow cytometry data shows the transfection efficiency of siRNA. The darker peak represents the fluorescence-positive cell population, which is clearly shifted from the position of the gated untransfected cells (lighter peaks). Upper univariate range indicates proportion of fluorescent cells. These data are representative of two independent experiments.
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Figure 3: Transfection efficiency of siRNA in relation to concentration. Final concentration of small interfering RNA (siRNA) in culture media in nanomoles/litre (nM). Histograph analysis of flow cytometry data shows the transfection efficiency of siRNA. The darker peak represents the fluorescence-positive cell population, which is clearly shifted from the position of the gated untransfected cells (lighter peaks). Upper univariate range indicates proportion of fluorescent cells. These data are representative of two independent experiments.

Mentions: To assess the effect of chordin knockdown on osteogenic differentiation, we first determined whether we could transfect siRNA into human MSCs, by using BLOCK-iT Oligo fluorescent siRNA and detecting its presence using a Cytomics FC500 MPL flow cytometer. As measured by fluorescence-labelled siRNA using a flow cytometer, transfection with 100, 200 and 300 nmol/l oligomucleotides respectively resulted in 51%, 91% and 99% efficiencies of transfection (Figure 3). Based on these data, the dose of 300 nmol/l siRNA was used in further experiments to screen the designed candidate siRNAs.


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

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

Transfection efficiency of siRNA in relation to concentration. Final concentration of small interfering RNA (siRNA) in culture media in nanomoles/litre (nM). Histograph analysis of flow cytometry data shows the transfection efficiency of siRNA. The darker peak represents the fluorescence-positive cell population, which is clearly shifted from the position of the gated untransfected cells (lighter peaks). Upper univariate range indicates proportion of fluorescent cells. These data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Transfection efficiency of siRNA in relation to concentration. Final concentration of small interfering RNA (siRNA) in culture media in nanomoles/litre (nM). Histograph analysis of flow cytometry data shows the transfection efficiency of siRNA. The darker peak represents the fluorescence-positive cell population, which is clearly shifted from the position of the gated untransfected cells (lighter peaks). Upper univariate range indicates proportion of fluorescent cells. These data are representative of two independent experiments.
Mentions: To assess the effect of chordin knockdown on osteogenic differentiation, we first determined whether we could transfect siRNA into human MSCs, by using BLOCK-iT Oligo fluorescent siRNA and detecting its presence using a Cytomics FC500 MPL flow cytometer. As measured by fluorescence-labelled siRNA using a flow cytometer, transfection with 100, 200 and 300 nmol/l oligomucleotides respectively resulted in 51%, 91% and 99% efficiencies of transfection (Figure 3). Based on these data, the dose of 300 nmol/l siRNA was used in further experiments to screen the designed candidate siRNAs.

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