Induction of chondrogenesis from human embryonic stem cells without embryoid body formation by bone morphogenetic protein 7 and transforming growth factor β1

Toshiyuki Nakagawa, Sang Yang Lee, A Hari Reddi

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Objective. Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor β1 (TGFβ1)-induced human ESC differentiation toward the articular cartilage phenotype. Methods. Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFβ1, CM with BMP-7, or CM with both TGFβ1 and BMP-7. Results. The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFβ1 treatment, CM plus BMP-7 treatment, and CM plus TGFβ1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFβ1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions. Conclusion. These results demonstrate an efficient and reproducible model system of human ESC-induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.

Original languageEnglish (US)
Pages (from-to)3686-3692
Number of pages7
JournalArthritis and Rheumatism
Volume60
Issue number12
DOIs
StatePublished - Dec 2009

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Bone Morphogenetic Protein 7
Embryoid Bodies
Chondrogenesis
Transforming Growth Factors
Articular Cartilage
Chondrocytes
Cartilage
Joints
Phenotype
Aggrecans
Weights and Measures
Regenerative Medicine
Collagen Type II
Gelatin
Tissue Engineering
Human Embryonic Stem Cells
Glycosaminoglycans
Cell Differentiation
Proteins
Gene Expression

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Rheumatology
  • Pharmacology (medical)

Cite this

Induction of chondrogenesis from human embryonic stem cells without embryoid body formation by bone morphogenetic protein 7 and transforming growth factor β1. / Nakagawa, Toshiyuki; Lee, Sang Yang; Reddi, A Hari.

In: Arthritis and Rheumatism, Vol. 60, No. 12, 12.2009, p. 3686-3692.

Research output: Contribution to journalArticle

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abstract = "Objective. Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor β1 (TGFβ1)-induced human ESC differentiation toward the articular cartilage phenotype. Methods. Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFβ1, CM with BMP-7, or CM with both TGFβ1 and BMP-7. Results. The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFβ1 treatment, CM plus BMP-7 treatment, and CM plus TGFβ1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFβ1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions. Conclusion. These results demonstrate an efficient and reproducible model system of human ESC-induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.",
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N2 - Objective. Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor β1 (TGFβ1)-induced human ESC differentiation toward the articular cartilage phenotype. Methods. Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFβ1, CM with BMP-7, or CM with both TGFβ1 and BMP-7. Results. The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFβ1 treatment, CM plus BMP-7 treatment, and CM plus TGFβ1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFβ1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions. Conclusion. These results demonstrate an efficient and reproducible model system of human ESC-induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.

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