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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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Related in: MedlinePlus

Expression of BMP-2 and chordin transcripts during osteogenic differentiation of human mesenchymal stem cells. (a) Temporal progression of bone morphogenetic protein (BMP)-2 and chordin expression during osteogenic differentiation of human MSCs over a 10-day period. Human MSCs were cultured in basal media (BM) or osteogenic media (OM) for 5 or 10 days. (b) Effect of small interfering RNA (siRNA) on chordin expression. Mesenchymal stem cells (MSCs) were transfected with either control or chordin siRNA and then grown in OM for the 3 or 10 days. RNA was extracted and reverse transcribed, and PCR amplification of chordin, BMP-2 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) sequences was performed.
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Figure 2: Expression of BMP-2 and chordin transcripts during osteogenic differentiation of human mesenchymal stem cells. (a) Temporal progression of bone morphogenetic protein (BMP)-2 and chordin expression during osteogenic differentiation of human MSCs over a 10-day period. Human MSCs were cultured in basal media (BM) or osteogenic media (OM) for 5 or 10 days. (b) Effect of small interfering RNA (siRNA) on chordin expression. Mesenchymal stem cells (MSCs) were transfected with either control or chordin siRNA and then grown in OM for the 3 or 10 days. RNA was extracted and reverse transcribed, and PCR amplification of chordin, BMP-2 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) sequences was performed.

Mentions: BMP-2 expression was weakly detectable in MSCs exposed to 5 and 10 days of BM (Figure 2a). Osteogenic differentiation induced an increase in the expression of BMP-2 (Figure 2a). Chordin mRNA expression was not detectable by RT-PCR when the MSCs were cultured in BM. However, there was detectable expression of chordin transcripts in MSCs cultured in OM, with an apparent increased expression at day 10 compared with day 5 (Figure 2a).


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

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

Expression of BMP-2 and chordin transcripts during osteogenic differentiation of human mesenchymal stem cells. (a) Temporal progression of bone morphogenetic protein (BMP)-2 and chordin expression during osteogenic differentiation of human MSCs over a 10-day period. Human MSCs were cultured in basal media (BM) or osteogenic media (OM) for 5 or 10 days. (b) Effect of small interfering RNA (siRNA) on chordin expression. Mesenchymal stem cells (MSCs) were transfected with either control or chordin siRNA and then grown in OM for the 3 or 10 days. RNA was extracted and reverse transcribed, and PCR amplification of chordin, BMP-2 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) sequences was performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Expression of BMP-2 and chordin transcripts during osteogenic differentiation of human mesenchymal stem cells. (a) Temporal progression of bone morphogenetic protein (BMP)-2 and chordin expression during osteogenic differentiation of human MSCs over a 10-day period. Human MSCs were cultured in basal media (BM) or osteogenic media (OM) for 5 or 10 days. (b) Effect of small interfering RNA (siRNA) on chordin expression. Mesenchymal stem cells (MSCs) were transfected with either control or chordin siRNA and then grown in OM for the 3 or 10 days. RNA was extracted and reverse transcribed, and PCR amplification of chordin, BMP-2 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) sequences was performed.
Mentions: BMP-2 expression was weakly detectable in MSCs exposed to 5 and 10 days of BM (Figure 2a). Osteogenic differentiation induced an increase in the expression of BMP-2 (Figure 2a). Chordin mRNA expression was not detectable by RT-PCR when the MSCs were cultured in BM. However, there was detectable expression of chordin transcripts in MSCs cultured in OM, with an apparent increased expression at day 10 compared with day 5 (Figure 2a).

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