Intrinsic Mechanical Cues and Their Impact on Stem Cells and Embryogenesis.

Although understanding how soluble cues direct cellular processes revolutionised the study of cell biology in the second half of the 20th century, over the last two decades, new insights into how mechanical cues similarly impact cell fate decisions has gained momentum. During development, extrinsic cues such as fluid flow, shear stress and compressive forces are essential for normal embryogenesis to proceed. Indeed, both adult and embryonic stem cells can respond to applied forces, but they can also detect intrinsic mechanical cues from their surrounding environment, such as the stiffness of the extracellular matrix, which impacts differentiation and morphogenesis. Cells can detect changes in their mechanical environment using cell surface receptors such as integrins and focal adhesions. Moreover, dynamic rearrangements of the cytoskeleton have been identified as a key means by which forces are transmitted from the extracellular matrix to the cell and vice versa. Although we have some understanding of the downstream mechanisms whereby mechanical cues are translated into changes in cell behaviour, many of the signalling pathways remain to be defined. This review discusses the importance of intrinsic mechanical cues on adult cell fate decisions, the emerging roles of cell surface mechano-sensors and the cytoskeleton in enabling cells to sense its microenvironment, and the role of intracellular signalling in translating mechanical cues into transcriptional outputs. In addition, the contribution of mechanical cues to fundamental processes during embryogenesis such as apical constriction and convergent extension is discussed. The continued development of tools to measure the biomechanical properties of soft tissues in vivo is likely to uncover currently underestimated contributions of these cues to adult stem cell fate decisions and embryogenesis, and may inform on regenerative strategies for tissue repair.

Dchs1-Fat4 regulation of osteogenic differentiation in mouse.

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of Yap-Runx2 and Taz-Runx2 complexes is altered in Dchs1/Fat4 mutant osteoblasts. In conclusion, these data identify Dchs1-Fat4 as a signalling pathway in osteoblast differentiation, reveal its crucial role within the early Runx2 progenitors, and identify distinct requirements for Yap and Taz during osteoblast differentiation.

Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation.

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-ß1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-ß may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-ß3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.