title: Botulinum hemagglutinin-mediated selective removal of cells deviating from the undifferentiated state in hiPSC colonies.
abstract: 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.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Mee-Hae
authors.initials: MH
authors.lastname: Kim
authors.affiliation: 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.
authors.forename: Yo
authors.initials: Y
authors.lastname: Sugawara
authors.affiliation: 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.|Department of Bacteriology, Graduate School of Medical Sciences, Kanazawa University, 13-1 Takara, Kanazawa, Ishikawa, 920-8640, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Fujinaga
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. kino-oka@bio.eng.osaka-u.ac.jp.
authors.forename: Masahiro
authors.identifier: 0000-0002-4912-5811
authors.initials: M
authors.lastname: Kino-Oka
mesh_terms: D000818:Animals; D015703:Antigens, CD; D001905:Botulinum Toxins / Q000633:toxicity*; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006388:Hemagglutinins / Q000633:toxicity*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D020690:GTPase-Activating Proteins; D006388:Hemagglutinins; C484658:RAP1GAP protein, human; D001905:Botulinum Toxins