Sachi Watanabe

RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hMSCs) have the potential to differentiate into tendon/ligament‐like lineages when they are subjected to mechanical stretching. However, the means through which mechanical stretch regulates the tenogenic differentiation of hMSCs remains unclear. This study examined the role of RhoA/ROCK, cytoskeletal organization, and focal adhesion kinase (FAK) in mechanical stretch‐induced tenogenic differentiation characterized by the up‐regulation of tendon‐related marker gene expression. Our findings showed that RhoA/ROCK and FAK regulated mechanical stretch‐induced realignment of hMSCs by regulating cytoskeletal organization and that RhoA/ROCK and cytoskeletal organization were essential to mechanical stretch‐activated FAK phosphorylation at Tyr397. We also demonstrated that this process can be blocked by Y‐27632 (a specific inhibitor of RhoA/ROCK), cytochalasin D (an inhibitor of cytoskeletal organization) or PF 573228 (a specific inhibitor of FAK). The results of this study suggest that RhoA/ROCK, cytoskeletal organization, and FAK compose a “signaling network” that senses mechanical stretching and drives mechanical stretch‐induced tenogenic differentiation of hMSCs. This work provides novel insights regarding the mechanisms of tenogenesis in a stretch‐induced environment and supports the therapeutic potential of hMSCs. J. Cell. Physiol. 227: 2722–2729, 2012.

Evaluation of Precise Optimal Cyclic Strain for Tenogenic Differentiation of MSCs

Although there are a number of papers relating to tenogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) using uniaxial cyclic stretching stimulation with homogeneous strain field, it has been pretty hard to figure out the optimal normal strain in the stretch direction for the differentiation. In the present study, our group has developed a non-uniform strain field system to elucidate the optimal normal strain in one-time experiment in principle. A relationship between the normal strain of membrane and expression levels of the differentiation marker proteins, type I collagen (Col I) and tenascin-C (Tnc), derived from stretched cells was obtained. Finally, the rigorous optimal normal strains were clarified 7.9 and 8.5 % for Col I and Tnc, respectively. Additionally, we found that a dependence of protein expression levels with the normal strain of membrane was different in each protein, which would be crucial in the field of embryology and regenerative medicine.

In Vitro Experimental Study for the Determination of Cellular Characteristics of Mesenchymal Stem Cells Using a Non-uniform Deformation Field

In the present study, relationship between cell orientation angle and strain value of membrane was comprehensively investigated using inhomogeneous strain field. And an axial strain threshold of cell, which corresponds to launch of cell orientation migration, was elucidated. One of the advantages in this study was that the inhomogeneous strain distribution was easily created by making a little improvement in a commonly-used uniaxial stretching device. The strains of two-dimensional stretched membrane were quantified position by position using digital image correlation (DIC) method. A 3D histogram of the cell frequency, which correlated with the cell orientation angle and normal strain of the membrane, made it possible to determine the axial strain threshold accurately. The value was 4.4 ± 0.3 %, which was reasonable compared with past study conducted by other researcher, although the past experiments were based on cyclic uniaxial stretch stimulation (homogeneous strain field). In addition, a preferential axial strain of the cell was achieved using the same technique of the determination of the axial strain threshold. This work has novel values at three points: (i) Determining axial strain threshold of the cells precisely. (ii) First suggestion of preferential axial strain of the cells. (iii) Investigating methodically cell behavior in inhomogeneous strain field.