Searchable abstracts of presentations at key conferences on calcified tissues
Bone Abstracts (2013) 1 PP266 | DOI: 10.1530/boneabs.1.PP266

ECTS2013 Poster Presentations Chondrocytes and cartilage (20 abstracts)

The Rho/ROCK GTPase pathway differentially modulates chondrocyte and osteoblast differentiation from pluripotent stem cells

Dalea M Bukhary , Fraser McDonald & Agamemnon E Grigoriadis


King’s College London, London, UK.


It is well-established that in vitro differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can recapitulate embryonic development through germ layer induction, enrichment and expansion of specific lineages. We have used PSC technology and developed a novel, mESC differentiation system for investigating the mechanisms of chondrocyte and osteoblast lineage commitment and differentiation. This step-wise, serum-free protocol uses specific recombinant factors to investigate i) the mechanisms of PSC commitment to mesoderm and bone/cartilage cell lineages and ii) the role of Rho GTPase signaling in ESC-derived chondrocyte/osteoblast differentiation. Activation of the Nodal/Activin and canonical Wnt pathways together with inhibition of BMP signaling (Noggin) directed ESCs to form a primitive streak-like population expressing Brachyury, which was further enriched to mesodermal subpopulations expressing both lateral plate and paraxial mesoderm markers, which subsequently differentiated efficiently in monolayer culture to chondrocyte and osteoblast lineages. Inhibition of Rho/ROCK signaling using the ROCK inhibitor, Y-27632, at different stages of mesoderm enrichment and differentiation phases modulated chondrogenesis and osteogenesis, showing up to a two- to threefold increase in cartilage and bone nodule formation. This was confirmed by qPCR analysis of osteoblast (Runx2, ALP, and BSP) and chondrocyte (Sox9 and Coll2)-specific genes, as well as by Alcian Blue Staining and Coll2 antibody staining of differentiated chondrocyte monolayers. Preliminary data also suggest that differential exposure to bFGF and BMP4 together with stage-specific addition of Y-27632 enhanced differentiation and/or expansion of hypertrophic chondrocytes and mineralizing osteoblasts. Finally, renal capsule grafting studies showed that the ESC-derived mesodermal populations gave rise to both cartilage and bone in vivo, mimicking endochondral ossification. The ESC model system provides defined, manipulatable and expandable chondro-osteoprogenitor populations that will provide insights into the molecular basis of bone/cartilage development and disease, as well as for generating specific populations for bone and cartilage tissue repair and replacement.

Volume 1

European Calcified Tissue Society Congress 2013

Lisbon, Portugal
18 May 2013 - 22 May 2013

European Calcified Tissue Society 

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