Embryonic stem cells (ESCs) are an ideal source for chondrogenic progenitors for the repair of damaged cartilage tissue. It is currently difficult to induce uniform and scalable ESC differentiation in vitro, a process required for stem cell therapy. This is partly because stem cell fate is determined by complex interactions with the native microenvironment and mechanical properties of the extracellular matrix. Mechanical signaling is considered to be one of the major factors regulating the proliferation and differentiation of chondrogenic cells both in vitro and in vivo. We used biocompatible and elastic polydimethylsiloxane (PDMS) scaffolds, capable of transducing mechanical signals, including compressive stress in vitro. ESCs seeded into the PDMS scaffolds and subjected to mechanical loading resulted in induction of differentiation. Differentiated ESC derivatives in three-dimensional (3-D) PDMS scaffolds exhibited elongated single cell rather than round clonal ESC morphology. They expressed chondrogenic marker, Col2, with concomitant reduction in the expression of pluripotent marker, Oct4. Immunocytochemical analysis also showed that the expression of COL2 protein was significantly higher in ESCs in 3-D scaffolds subjected to compressive stress. Further analysis showed that compressive stress also resulted in expression of early chondrogenic makers, Sox9 and Acan, but not hypertrophic chondrogenic markers, Runx2, Col10, and Mmp13. Compressive stress induced differentiation caused a reduction in the expression of β-Catenin and an increase in the expression of genes, Rhoa, Yap, and Taz, which are known to be affected by mechanosignaling. The chondroinductive role of RhoA was confirmed by its downregulation with simultaneous decrease in the transcriptional and translational expression of early chondrogenic markers, SOX9, COL2, and ACAN, when ESCs in PDMS scaffolds were subjected to compressive stress and treated with RhoA inhibitor, CCG-1432. Based on these observations, a model for compression induced chondrogenic differentiation of ESCs in 3-D scaffolds was proposed.
Keywords: 3-D cell culture; PDMS; chondrogenic differentiation; embryonic stem cells; mechanosignaling; scaffold.