Maintenance of an undifferentiated state of human-induced pluripotent stem cells through botulinum hemagglutinin-mediated regulation of cell behavior
2019
Applications of human
induced pluripotent stem cell(hiPSC) culture are impaired by problems with long term maintenance of pluripotency. In this study, we report that exposure to botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, suppressed deviation from an undifferentiated state in hiPSC colonies. Time-lapse imaging of live cells revealed that cells in central regions of colonies moved slowly and underwent a morphological change to a cobblestone-like shape via interaction between contacting cells, forming dense, multiple layers. Staining and migration analysis showed that actin
stress fibersand
paxillinspots were diminished in colony central regions, and this was associated with alteration of cellular morphology and migratory behavior. However, in culture with HA exposure, cells in the central and peripheral regions of hiPSC colonies were migratory and arranged in loose monolayers, resulting in relatively uniform dispersion of cells in colonies. We also found that a well-organized network of actin
stress fiberswas of significance in the central and peripheral regions of a colony, resulting in activation of
paxillinand E-cadherin expression in hiPSCs. After routine application of HA for
serial passages, hiPSCs remained pluripotent and capable of differentiating into all three
germ layers. These observations indicate that relaxation of cell–
cell junctionsby HA induced rearrangements of the cytoskeleton and cell adhesion in hiPSC colonies by promoting migratory behaviors. These results suggest that this simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures.
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