Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Laboratory for Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Sci Rep. 2017 Mar 7;7(1):93. doi: 10.1038/s41598-017-00083-1.
The undifferentiated state of human induced pluripotent stem cells (hiPSCs) depends on their cell-cell and cell-substrate adhesions. In this study, we report that exposure to botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, selectively removed cells that deviated from the undifferentiated state in hiPSC colonies. After HA treatment, cell-cell adhesion was disrupted, deviated cells detached from colony centers, and dividing cells filled these spaces. Because E-cadherin-mediated adhesion was disrupted in undifferentiated cells, stress-fiber formation and focal adhesions were diminished; however, these were subsequently restored, and the cells retained expression of undifferentiated stem cell markers and their differentiation potential. In contrast, actin structures and focal adhesions were lost from deviated cells, and they subsequently died. In undifferentiated and deviated cells, the cadherin/integrin-regulator Rap1 was localized at cell-cell adhesions and in the cytoplasm, respectively. Concurrent HA and Rap1-inhibitor treatment accelerated the deviated-cell detachment and delayed the recovery of hiPSC morphology, but this effect was significantly attenuated by co-treatment with Rap1 activator. Thus, Rap1 regulated E-cadherin-integrin interplay in hiPSC colonies exhibiting deviation, while HA-mediated selective removal of these deviated cells helped maintain the undifferentiated state in the remaining hiPSCs.
人类诱导多能干细胞（hiPSC）的未分化状态取决于其细胞-细胞和细胞-基质的黏附。在这项研究中，我们报告称，暴露于肉毒杆菌血凝素（HA），一种 E-钙黏蛋白功能阻断剂，可选择性地去除 hiPSC 集落中偏离未分化状态的细胞。HA 处理后，细胞-细胞黏附被破坏，偏离的细胞从集落中心脱落，分裂细胞填充这些空间。由于未分化细胞中 E-钙黏蛋白介导的黏附被破坏，应力纤维形成和焦点黏附减少；然而，随后这些被恢复，细胞保留未分化干细胞标志物的表达及其分化潜能。相比之下，偏离细胞的肌动蛋白结构和焦点黏附消失，随后死亡。在未分化和偏离的细胞中，钙黏蛋白/整合素调节剂 Rap1 分别位于细胞-细胞黏附处和细胞质中。同时使用 HA 和 Rap1 抑制剂处理加速了偏离细胞的脱落，并延迟了 hiPSC 形态的恢复，但 Rap1 激活剂的共同处理显著减弱了这种效果。因此，Rap1 调节 hiPSC 集落中偏离细胞的 E-钙黏蛋白-整合素相互作用，而 HA 介导的这些偏离细胞的选择性去除有助于维持剩余 hiPSC 的未分化状态。
