State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai 200433, China.
Biomaterials. 2013 Jan;34(4):930-9. doi: 10.1016/j.biomaterials.2012.10.052. Epub 2012 Nov 9.
The present study is aimed to examine the shape effect on lineage commitment of stem cells in growth medium free of external chemical induction factors. Aspect ratios (ARs) of cells were controlled by micropatterns with cell-adhesive microislands of AR 1, 2 and 8 on the potent nonfouling background of poly(ethylene glycol) hydrogels, and the single stem cells were well shaped for 19 days. Mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured in osteogenic medium, adipogenic medium, mixed coinduction medium, and also growth medium; alkaline phosphatase (ALP) and oil droplets were employed as indicators of osteoblasts and adipocytes, respectively. Those indicators were well observed in all of three induction media as early as day 7, and also in growth medium at a longer culture time till day 13. While a significant monotonic decrease of adipogenesis was observed with the increase of AR, a non-monotonic change of osteogenesis was found with optimal AR about 2. The relative gene expressions further verified the above findings. As a result, cell shape itself is an inherent cue to regulate stem cell differentiation, let alone with or without external chemical induction factors. Such a shape effect disappeared upon addition of a microfilament inhibitor cytochalasin D or a Rho-associated protein kinase (ROCK) inhibitor Y-27632. So, formation of cytoskeleton is necessary for the shape effect, and the ROCK-pathway-related cell tension is responsible for the shape effect on the lineage commitment of stem cells even in growth medium.
本研究旨在探讨在无外部化学诱导因子的生长培养基中，细胞形状对干细胞谱系分化的影响。通过具有 AR1、2 和 8 的细胞粘附微岛的微图案来控制细胞的纵横比（AR），并且单个干细胞在强大的非粘性聚乙二醇水凝胶背景下能够很好地保持形状长达 19 天。从大鼠骨髓中分离得到的间充质干细胞（MSCs）在成骨培养基、成脂培养基、混合共诱导培养基以及生长培养基中进行培养；碱性磷酸酶（ALP）和油滴分别用作成骨细胞和脂肪细胞的标志物。这些标志物在所有三种诱导培养基中早在第 7 天就得到了很好的观察，并且在更长的培养时间（直到第 13 天）的生长培养基中也得到了观察。虽然随着 AR 的增加，脂肪生成呈显著的单调下降趋势，但成骨作用呈非单调变化，最佳 AR 约为 2。相对基因表达进一步验证了上述发现。因此，细胞形状本身就是调节干细胞分化的固有线索，更不用说是否存在外部化学诱导因子了。这种形状效应在添加微丝抑制剂细胞松弛素 D 或 Rho 相关蛋白激酶（ROCK）抑制剂 Y-27632 后消失。因此，细胞骨架的形成对于形状效应是必要的，并且 ROCK 途径相关的细胞张力对于即使在生长培养基中干细胞谱系分化的形状效应也是负责的。
