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.
Highlights
Human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells hold great promise for clinical and industrial applications because they can self-renew and differentiate into all cell types[1, 2]
Cells in the deviated region invade and occupy the colony, thereby accelerating loss of their self-renewal ability and pluripotency during subculturing. Human pluripotent stem cells (hPSCs) cultures invariably exhibit a small amount of differentiation, and the cultures must be routinely cleaned by manually removing differentiated cells to prevent the differentiated areas from initiating morphological changes that can trigger colony-wide differentiation
In some colonies cultured with SNL feeder cells, human induced pluripotent stem cells (hiPSCs) deviated from the undifferentiated state and transformed into large flattened cells at the colony center (Fig. 1a, arrowheads)
Summary
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) hold great promise for clinical and industrial applications because they can self-renew and differentiate into all cell types[1, 2]. HESC and hiPSC culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium From these cultures, hPSCs spontaneously deviate from the undifferentiated state, a widely recognized phenomenon, whereby their morphology changes drastically into large flattened cells[8]. In hPSC cultures, E-cadherin-mediated cell–cell adhesion induces changes in both cell and colony morphologies, which potentially activates signaling pathways involved in either maintaining the undifferentiated state or committing to a lineage[21,22,23,24,25,26,27,28,29,30]. In hESC cultures, Rap[1] affects the endocytic recycling pathway involved in the formation and maintenance of E-cadherin-mediated cell–cell adhesion, which is essential for the colony formation and self-renewal[29] These findings provide insight into successful strategies for the regulating the hPSC self-renewal and differentiation[23]. Based on the observed differences in morphological behaviors between undifferentiated and deviated regions in single hiPSC colonies, we discuss the fundamental mechanisms of cell–cell and cell–substrate adhesion in relation to hiPSC survival, self-renewal, and death
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