Chemical and Mechanical regulation of osteoblast differentiation from pluripotent stem cells through the Rho/ROCK pathway

Abstract

It is well-established that the commitment and differentiation of stem cells can be influenced by both chemical as well as mechanical signals provided by the niche and the extracellular environment that they interact with. It has been established recently that matrix stiffness plays a key role in regulating osteoblast differentiation from mesenchymal stem cells. However, the regulation of osteogenic differentiation from pluripotent stem cells by defined growth factors and matrices of different stiffnesses requires further investigation. In this study, I have used the in vitro differentiation of pluripotent mouse Embryonic Stem Cells (mESCs) to osteoblasts and chondrocytes, to investigate the interplay between BMP signalling, matrix stiffness and Rho/ROCK signalling on chondro-osteo differentiation. Specifically, differentiation of mESCs through a step-wise protocol involving primitive streak/mesoderm specification and FGF-2-dependent mesoderm enrichment, yields a population of precursors that have both chondrogenic and osteogenic potentials when cultured in specific media. Our current studies now show that addition of BMP-4 during a small interval, designated the mesoderm-enrichment phase of differentiation, induces the osteoblast lineage over the chondrocyte lineage, as determined by histochemical staining and molecular marker gene expression for lineage-specific markers. The addition of a ROCK inhibitor enhanced the BMP-4 effect, suggesting an important additional role of cell spreading and cell-matrix interactions. To test this, mESC differentiation was carried out on polyacrylamide gel substrates of low (2KPa) and high (50KPa) stiffness. It appears that matrix stiffness regulates both osteoblast and chondrocyte lineages, however, BMP-4 dominates over the effects of matrix stiffness by inducing bone and reducing cartilage differentiation. ROCK inhibition enhances the BMP-4 effect on osteogenesis and has no effect on chondrogesis as BMP-4 suppresses cartilage in the presence or absence of ROCK inhibitor. The findings also suggest that inhibition of ROCK reverses the cell rounding phenotype induced by seeding on 2KPa substrates, therefore subsequent effects on differentiation are related to cell spreading, as ROCK inhibition increases cell spreading on soft substrates as well as induces osteogenic differentiation. These results suggest that although mESC-derived chondro-osteo lineage commitment is regulated by both chemical and mechanical signals, there is a greater dependence for differentiation on chemical/biological It is well-established that the commitment and differentiation of stem cells can be influenced by both chemical as well as mechanical signals provided by the niche and the extracellular environment that they interact with. It has been established recently that matrix stiffness plays a key role in regulating osteoblast differentiation from mesenchymal stem cells. However, the regulation of osteogenic differentiation from pluripotent stem cells by defined growth factors and matrices of different stiffnesses requires further investigation. In this study, I have used the in vitro differentiation of pluripotent mouse Embryonic Stem Cells (mESCs) to osteoblasts and chondrocytes, to investigate the interplay between BMP signalling, matrix stiffness and Rho/ROCK signalling on chondro-osteo differentiation. Specifically, differentiation of mESCs through a step-wise protocol involving primitive streak/mesoderm specification and FGF-2-dependent mesoderm enrichment, yields a population of precursors that have both chondrogenic and osteogenic potentials when cultured in specific media. Our current studies now show that addition of BMP-4 during a small interval, designated the mesoderm-enrichment phase of differentiation, induces the osteoblast lineage over the chondrocyte lineage, as determined by histochemica