Abstract
New methods are needed to reduce dynamic blebbing which inhibits cell attachment and survival during passaging of pluripotent stem cells. We tested the hypothesis that activation of the P2X7 receptor by extracellular ATP during passaging initiates dynamic blebbing. The P2X7 receptor was present in human embryonic stem cells (hESC), but not in differentiating cells. Extracellular ATP concentrations were 14× higher in medium during passaging. Addition of ATP to culture medium prolonged dynamic blebbing and inhibited attachment. Inhibition of P2X7 by specific drugs or by siRNA significantly reduced dynamic blebbing and improved cell attachment. When cells were incubated in calcium chelators (EGTA or BAPTA), blebbing decreased and attachment improved. Calcium influx was observed using Fura-4 when ATP was added to culture medium and inhibited in the presence of the P2X7 inhibitor. Over-expressing activated Rac in hESC reduced blebbing and promoted cell attachment, while a Rac inhibitor prolonged blebbing and reduced attachment. These data identify a pathway involving P2X7 that initiates and prolongs dynamic blebbing during hESC passaging. This pathway provides new insight into factors that increase dynamic blebbing and identifies new targets, such as P2X7, that can be used to improve the culture of cells with therapeutic potential.
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