Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment.

Heterogeneous nanomechanical properties of superficial and zonal regions of articular cartilage of the rabbit proximal radius condyle by atomic force microscopy.

Articular chondrocytes have been thought to reside in a homogenous matrix. The physical characteristics of the intercellular matrix of articular cartilage are not well characterized, especially at a nanoscopic scale. The present work tested the hypothesis that the nanomechanical properties of the intercellular matrices of articular cartilage in both the articulating surface and various cellular zones are non-homogeneous. Nanoindentation by atomic force microscopy was applied to the geometric center of the medial, lateral and groove regions of the superficial zone of the rabbit proximal radius cartilage, and then the intercellular matrices of chondrocytes from the superficial to calcifying zones in 40 microm increments. The elastic modulus of the articular surface of the medial condyle (1.46+/-0.11 MPa) was significantly higher than the lateral condyle (1.18+/-0.10 MPa), and the groove (0.96+/-0.07 MPa). There is a significant gradient increase in Young's moduli from the superficial zone (0.52+/-0.05 MPa) to calcifying zone (1.69+/-0.12 MPa). Thus, the nanomechanical properties of the intercellular matrices of the articulating surface are region-specific and likely related to articular function. Heterogeneous biophysical properties of intercellular matrices along the depth from the superficial to calcifying zones suggest that chondrocytes likely reside in a heterogeneous matrix.

Microstructural and elastic properties of the extracellular matrices of the superficial zone of neonatal articular cartilage by atomic force microscopy.

The structural and mechanical properties of the superficial zone of articular cartilage are not well understood. Most previous studies have focused on the overall properties of articular cartilage in the adult. In the present work, the extracellular matrices of the superficial zone of the jaw-joint condyle in the 7-day-old rabbit were subjected to dynamic indentation with atomic force microscopy (AFM). The surface topography of four equally divided regions of the entire articular surface lacked substantial variations, with mean roughness from 95.4 nm (+/- 28.0) to 130.1 nm (+/- 13.8). Indentations of the articular surface and the microdissected, orthogonal transverse surface revealed a narrow distribution of Young's moduli ranging from 0.92 MPa (+/- 0.12) to 1.02 MPa (+/- 0.22). These rather uniform structural and mechanical properties of the superficial zone of the neonatal articular cartilage are in contrast to our previous finding of a gradient distribution of Young's moduli of the superficial zone of adult articular cartilage from 0.95 (+/- 0.06 MPa) to 2.34 (+/- 0.26 MPa) (Hu et al.: J Struct Biol 2001:136:46-52), indicating that the mechanical properties of the articular surface are modified during development. Thus, articular cartilage's anisotropic mechanical properties may be specific to the adult, rather than the neonatal. It is further postulated that the structural and mechanical properties of the superficial zone of articular cartilage are regulated by chondrocytes in addition to their unidirectional development pathway toward subchondral bone formation.