AbstractIt is well-established that in vitro differentiation of Embryonic Stem Cells (ESCs) can recapitulate embryonic development. In this project a novel, step-wise, serum-free differentiation system was developed using specific recombinant growth factors for investigating the differentiation of mESCs, through formation of a primitive streak-like population expressing Brachyury and specification of a subpopulation of mesodermal progenitors expressing both paraxial and lateral plate mesoderm markers. These cells subsequently differentiated efficiently in monolayer cultures to chondrocyte and osteoblast lineages marked by cell morphology, Alcian blue/ALP staining as well as by chondrocyte and osteoblastspecific gene expression.
The role of the Rho kinase (ROCK) pathway in cartilage and bone cell differentiation is controversial. Exposure of ESC cultures to the ROCK inhibitor, Y-27632, at mesoderm enrichment and/or monolayer differentiation phases revealed that continuous exposure to Y27632 modulated differentiation in a developmental phase-dependent manner, with up to a 7-fold and 2-fold increase in chondrogenesis and osteogenesis, respectively. In contrast, temporal exposure of Y27632 favoured chondrogenic over osteogenic differentiation. This was confirmed by qPCR analysis of chondrocyte (Sox9, Sox5, Acan, Coll II, Col X) and osteoblast (Runx2, Osx, ALP, BSP, OC)-specific gene expression. Furthermore, temporal exposure to FGF2 and BMP4 together
with phase-specific addition of Y-27632 enhanced differentiation/expansion of hypertrophic chondrocytes and mineralising osteoblasts. Finally, renal capsule grafting studies showed that the mesoderm-derived ESCs mimicked endochondral ossification, which was enhanced by Y27632 treatment.
This study established a novel ESC model system, which generated defined, manipulatable and expandable chondro-osteoprogenitor populations that will provide insights into the molecular basis of bone/cartilage development. Moreover, a phase-dependent inhibition of ROCK signalling modulated early chondro-osteoprogenitor lineage commitment and enhanced cartilage and bone
formation. These studies provide a novel targetable pathway for generating specific populations for potential bone and cartilage tissue repair and replacement.
|Date of Award
|Agamemnon Grigoriadis (Supervisor) & Fraser McDonald (Supervisor)