4 years ago

A simple and robust method for culturing human induced pluripotent stem cells in an undifferentiated state using botulinum haemagglutinin

Mee-Hae Kim, Yoshifumi Matsubara, Masahiro Kino-oka, Yukako Fujinaga
Clinical and industrial applications of human induced pluripotent stem cells (hiPSCs) have been hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, we developed a simple and robust hiPSC-culture-propagation strategy incorporating botulinum haemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state. After HA treatment, cell-cell adhesion was disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. We found that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA were confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintained expression of pluripotent markers and readily formed embryoid bodies expressing markers for all three germ-cell layers. This method enabled highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/biot.201700384

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