Li Huaqiong, Wen Feng, Wang Xincai, Tan Lay Poh
School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798; School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou 325027, China; and Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences, Wenzhou 325035, China.
School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798.
Biointerphases. 2015 Jun 1;10(2):021003. doi: 10.1116/1.4916624.
In our previous report, the authors have demonstrated that direct laser machined microchannels would trigger upregulation of myogenic markers in human mesenchymal stem cells (hMSCs) through promotion of cell elongation. However, the molecular basis signaling pathways behind this observation remains unclear. In this work, three types of microchannels generated by femtosecond laser were utilized to investigate possible mechanisms behind the induction of hMSCs myogenesis by microchannels. The authors hypothesized that small G-proteins RhoA and Rac1 play a vital role on myogenesis of hMSCs through regulating cytoskeleton rearrangement, via cell tension signaling cascades. The RhoA and Rac1 activities were evaluated for cells cultured on the micropatterned substrates, using a flat unpatterned substrate as control. It was found that significant activation of RhoA GTPase was exhibited for cells cultured on narrow microchannels (20-20-20 and 30-30-20), while no obvious differences were obtained on wide ones (80-30-20). Meanwhile, no significant difference was found for Rac1 activities on all tested groups. To further deduce the role of RhoA signaling pathway in microchannel directed stem cell myogenesis, the effectors of Rho, Rho kinase (ROCK) was chosen to explore how cell shape regulate myogenesis of hMSCs cultured on laser micropatterned substrate. A pharmacological ROCK inhibitor, Y-27632, was used to treat the cells and the effect on RhoA activation was investigated. Our data on the role of RhoA/ROCK in regulating cell myogenic differentiation on lasered microchannels substrates may provide a mechanistic insight on hMSCs fate directed by substrate topography.
在我们之前的报告中，作者已经证明，直接激光加工的微通道会通过促进细胞伸长来触发人间充质干细胞（hMSCs）中肌源性标志物的上调。然而，这一观察结果背后的分子基础信号通路仍不清楚。在这项工作中，利用飞秒激光产生的三种类型的微通道来研究微通道诱导hMSCs成肌的可能机制。作者假设小G蛋白RhoA和Rac1通过细胞张力信号级联调节细胞骨架重排，在hMSCs的成肌过程中起关键作用。使用平坦无图案的底物作为对照，评估在微图案化底物上培养的细胞的RhoA和Rac1活性。结果发现，在狭窄微通道（20-20-20和30-30-20）上培养的细胞表现出RhoA GTP酶的显著激活，而在宽微通道（80-30-20）上没有明显差异。同时，所有测试组的Rac1活性没有发现显著差异。为了进一步推断RhoA信号通路在微通道定向干细胞成肌中的作用，选择Rho的效应器Rho激酶（ROCK）来探索细胞形状如何调节在激光微图案化底物上培养的hMSCs的成肌。使用一种药理学ROCK抑制剂Y-27632处理细胞，并研究其对RhoA激活的影响。我们关于RhoA/ROCK在调节激光微通道底物上细胞成肌分化中的作用的数据，可能为底物拓扑结构指导hMSCs命运提供一个机制性的见解。
