Effect of transforming growth factor β1 on chondrogenic differentiation of cultured equine mesenchymal stem cells

Allison A. Worster Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Present address is Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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 DVM, MS
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Alan J. Nixon Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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 BVSc, MS
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Brent D. Brower-Toland Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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 MS
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Janice Williams Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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 BS

Abstract

Objective—To determine the morphologic and phenotypic effects of transforming growth factor β1 (TGF-β1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes.

Sample Population—Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8- month-old horses.

Procedure—After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-β1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen.

Results—MSC cultures exposed to TGF-β1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-β1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF- β1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-β1 led to dose-related increases in area and intensity of type-II collagen immunoreaction.

Conclusion—Results suggest that TGF-β1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner. (Am J Vet Res 2000;61:1003–1010)

Abstract

Objective—To determine the morphologic and phenotypic effects of transforming growth factor β1 (TGF-β1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes.

Sample Population—Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8- month-old horses.

Procedure—After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-β1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen.

Results—MSC cultures exposed to TGF-β1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-β1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF- β1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-β1 led to dose-related increases in area and intensity of type-II collagen immunoreaction.

Conclusion—Results suggest that TGF-β1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner. (Am J Vet Res 2000;61:1003–1010)

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