Characterizing the Pathways That Initiate and Stop Dynamic Blebbing in Human Embryonic Stem Cells
Dynamic blebs are membrane protrusions on the surface of healthy cells that function in cell division and migration. The purpose of this dissertation was to discover better methods to control dynamic blebbing by identifying pathways that initiate and inhibit blebbing in human embryonic stem cells (hESC). Live cell imaging experiments demonstrated that dynamic and apoptotic blebbing were morphologically and temporally distinct during passaging of hESC. Dynamic blebs retracted faster than apoptotic blebs and had an intact cytoskeleton. Dynamic blebbing was prolonged by depolymerization of microtubules and stopped by drugs that disrupted actin microfilaments or inhibited myosin II. Plating on laminin-521 or Matrigel overcoated with laminin-111 efficiently stopped blebbing by activating an integrin-focal adhesion kinase pathway.
To identify the pathway that initiates dynamic blebbing, we tested the hypothesis that the P2X7 calcium channel is activated by ATP released during passaging. Immunocytochemistry and PCR showed that the P2X7 receptor is expressed in hESC, but not in cells starting differentiation. P2X7 inhibitors and siRNA decreased dynamic blebbing. Extracellular ATP concentration increased during passaging. While apyrase, which degrades ATP, reduced the percentage of blebbing cells, addition of ATP to the culture medium prolonged blebbing. When Ca2+ was chelated by either EGTA or BAPTA, dynamic blebbing was inhibited. Rac activation was associated with decreased blebbing and cell attachment, while blebbing was activated though the ROCK pathway. These data support the idea that dynamic blebbing in hESC is initiated by extracellular ATP binding to P2X7, allowing Ca2+ influx which in turn initiates dynamic blebbing via the ROCK- myosin II pathway. Decreased extracellular ATP is accompanied by activation of Rac, cessation of blebbing, and attachment.
These results introduce better ways to control dynamic blebbing and improve cell survival during passaging. The use of P2X7 inhibitors and laminin substrates enables attachment and improves single hESC plating efficiency, which will facilitate quantitative work in drug discovery and toxicology. These results also introduce: (1) P2X7 as a cell surface marker for pluripotency; (2) hESC as an excellent model for studying dynamic blebbing and (3) the possibility of using hESC, which are similar to epiblast cells, to study cell movements during gastrulation.