Dynamic blebbing: A bottleneck to human embryonic stem cell culture that can be overcome by Laminin-Integrin signaling.
Dynamic blebbing: A bottleneck to human embryonic stem cell culture that can be overcome by Laminin-Integrin signaling.
复制标题
动态起泡：人类胚胎干细胞培养的瓶颈，可以通过层粘连蛋白-整合素信号传导来克服。
DOI：
10.1016/j.scr.2018.10.022
复制
发表时间：
2018
影响因子：
1.2
通讯作者：
Talbot,Prue
中科院分区：
文献类型：
--
作者：
Weng,NikkiJo-Hao;Cheung,Cindy;Talbot,Prue
这项研究描述了人胚胎干细胞 (hESC) 中动态和凋亡出泡的特征，确定动态出泡是传代过程中细胞成功附着的瓶颈，并证明通过将细胞铺板在重组人层粘连蛋白上可以快速停止动态出泡。在新鲜铺板的 hESC 中，动态和细胞凋亡在发生时间、泡回缩率、线粒体膜电位以及 caspase 3 和 7 激活方面存在差异。虽然动态起泡可以通过抑制肌球蛋白 II 的药物来控制，但这些方法具有脱靶效应，不适合临床应用。重组人层粘连蛋白 521 或在基质胶中添加层粘连蛋白 111 提供了一种安全的方法，可大幅减少动态起泡并改善细胞与内细胞团中通常存在的蛋白质的附着。抑制粘着斑激酶（通过整合素与层粘连蛋白结合而激活），延长动态起泡并抑制附着。这些数据表明，hESC 通过整合素快速与层粘连蛋白结合，从而激活粘着斑激酶，进而下调动态起泡。层粘连蛋白使 hESC 在传代过程中快速附着，提高铺板效率，实现单个多能干细胞的传代，并避免使用具有非特异性脱靶效应的抑制剂。这些数据提供了使用生物安全的重组人蛋白改进 hESC 培养的策略。
This study characterizes dynamic and apoptotic blebbing in human embryonic stem cells (hESC), identifies dynamic blebbing as a bottleneck to successful cell attachment during passaging, and demonstrates that dynamic blebbing can be rapidly stopped by plating cells on recombinant human laminin. In freshly plated hESC, dynamic and apoptotic blebbing differed in time of occurrence, bleb retraction rate, mitochondrial membrane potential, and caspase 3&7 activation. While dynamic blebbing can be controlled with drugs that inhibit myosin II, these methods have off-target effects and are not suitable for clinical applications. Recombinant human laminin-521 or addition of laminin-111 to Matrigel provided a safe method to drastically decrease dynamic blebbing and improve cell attachment with proteins normally found in the inner cell mass. Inhibition of focal adhesion kinase, which is activated by binding of integrins to laminin, prolonged dynamic blebbing and inhibited attachment. These data show that hESC bind rapidly to laminins through an integrin, which activates focal adhesion kinase that in turn downregulates dynamic blebbing. Laminins enabled hESC to rapidly attach during passaging, improved plating efficiency, enabled passaging of single pluripotent stem cells, and avoided use of inhibitors that have non-specific off-target effects. These data provide a strategy for improving hESC culture using biologically safe recombinant human proteins.