title: Primary murine limb bud mesenchymal cells in long-term culture complete chondrocyte differentiation: TGF-beta delays hypertrophy and PGE2 inhibits terminal differentiation.
abstract: In vitro models of endochondral bone formation using both primary and immortalized cells have provided insight regarding factors and signaling pathways involved in chondrocyte maturation and endochondral bone formation. However, primary murine cell culture models of chondrocyte differentiation have not been established but have enormous potential due to the possible use of cells from transgenic and knockout animals. Here, we show that stage E11.5 embryonic murine limb bud mesenchymal stem cells in micromass cell culture progress through the stages of chondrogenesis, chondrocyte hypertrophy, terminal differentiation, and matrix calcification. This cell culture system recapitulated the sequential expression of genes that characterize chondrocyte differentiation, including Sox9, col2, colX, MMP13, VEGF, and osteocalcin. TGF-beta treatment for up to 21 days markedly delayed the rate of chondrocyte maturation and inhibited matrix calcification and its related gene expression. In TGF-beta-treated cultures, the hypertrophic and terminal differentiation markers colX, VEGF, MMP13, and osteocalcin were reduced or absent. PGE2 had minimal effects on chondrocyte hypertrophy but delayed terminal differentiation and matrix calcification. Thus, primary murine mesenchymal cells sequentially differentiate through the various stages of chondrocyte maturation and establish a model whereby the role of specific signaling molecules can be examined in cells derived from transgenic or knockout mice.
authors.affiliation: Department of Orthopaedics, Center for Musculoskeletal Research, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Xinping
authors.initials: X
authors.lastname: Zhang
authors.forename: Navid
authors.initials: N
authors.lastname: Ziran
authors.forename: J Jeffery
authors.initials: JJ
authors.lastname: Goater
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: J Edward
authors.initials: JE
authors.lastname: Puzas
authors.forename: Randy N
authors.initials: RN
authors.lastname: Rosier
authors.forename: Michael
authors.initials: M
authors.lastname: Zuscik
authors.forename: Hicham
authors.initials: H
authors.lastname: Drissi
authors.forename: Regis J
authors.initials: RJ
authors.lastname: O'Keefe
mesh_terms: D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects*; D017931:DNA Primers; D015232:Dinoprostone / Q000494:pharmacology*; D005260:Female; D017403:In Situ Hybridization; D018878:Limb Buds / Q000166:cytology*; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D016212:Transforming Growth Factor beta; D015232:Dinoprostone