Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
FASEB J. 2011 Apr;25(4):1208-17. doi: 10.1096/fj.10-168971. Epub 2010 Dec 23.
Shear stress is a ubiquitous environmental cue experienced by stem cells when they are being differentiated or expanded in perfusion cultures. However, its role in modulating self-renewing stem cell phenotypes is unclear, since shear is usually only studied in the context of cardiovascular differentiation. We used a multiplex microfluidic array, which overcomes the limitations of macroperfusion systems in shear application throughput and precision, to initiate a comprehensive, quantitative study of shear effects on self-renewing mouse embryonic stem cells (mESCs), where shear stresses varying by >1000 times (0.016-16 dyn/cm(2)) are applied simultaneously. When compared with static controls in the presence or absence of a saturated soluble environment (i.e., mESC-conditioned medium), we ascertained that flow-induced shear stress specifically up-regulates the epiblast marker Fgf5. Epiblast-state transition in mESCs involves heparan sulfate proteoglycans (HSPGs), which have also been shown to transduce shear stress in endothelial cells. By disrupting (with sulfation inhibitors and heparinase) and partially reconstituting (with heparin) HSPG function, we show that mESCs also mechanically sense shear stress via HSPGs to modulate Fgf5 expression. This study demonstrates that self-renewing mESCs possess the molecular machinery to sense shear stress and provides quantitative shear application benchmarks for future scalable stem cell culture systems.
切应力是干细胞在灌注培养中分化或扩增时普遍存在的环境信号。然而，其在调节自我更新的干细胞表型中的作用尚不清楚，因为通常仅在心血管分化的背景下研究切应力。我们使用了一种多重微流控阵列，该阵列克服了宏观灌注系统在切应力应用通量和精度方面的局限性，从而对切应力对自我更新的小鼠胚胎干细胞（mESC）的影响进行了全面的定量研究，其中应用的切应力变化超过 1000 倍（0.016-16 dyn/cm2）。与存在或不存在饱和可溶性环境（即 mESC 条件培养基）的静态对照相比，我们确定流动引起的切应力特异性地上调了胚外层标记物 Fgf5。mESC 中的胚外层状态转变涉及硫酸乙酰肝素蛋白聚糖（HSPG），已知内皮细胞中的 HSPG 也能传递切应力。通过破坏（用硫酸化抑制剂和肝素酶）和部分重建（用肝素）HSPG 功能，我们表明 mESC 还通过 HSPG 机械感知切应力来调节 Fgf5 的表达。这项研究表明，自我更新的 mESC 具有感知切应力的分子机制，并为未来可扩展的干细胞培养系统提供了定量切应力应用基准。
