br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion During passaging, hESC undergo vigorous dynamic blebbing that prevents attachment and spreading of cells. If dynamic blebbing does not stop, oxidative phosphorylation fail to attach, and eventually undergo apoptosis (Weng, 2016). Dynamic blebbing, which stresses cells and causes loss of cytoplasm, can be stopped rapidly when laminins are used as the substrate for attachment. Binding of laminin to hESC integrins activates a FAK, and signaling through the FAK leads to cessation of blebbing. In this study, we showed that the key upstream event that initiates dynamic blebbing in hESC is the activation of the P2X7 receptor which responds to extracellular ATP released during cell passaging (Figs. 3 and 4). P2X7 activation leads to calcium influx, which in turn correlated with the initiation of blebbing through activation of the Rho pathway. P2X7, along with Oct4, dissapeared during differentiation and identified a new cell surface marker for pluripotency. Because dynamic blebbing stresses cells and decreases cell survival, it is important to identify new ways to prevent or stop dynamic blebbing during passaging of hESC. Reagents that inhibit myosin II, such as ROCK inhibitors and blebbistatin, have been widely used to accelerate attachment of hESC during passaging and for plating single hESC (Watanabe et al., 2007; Harb et al., 2008; Li et al., 2009; Ohgushi et al., 2010). However, hESC treated with ROCK inhibitor appear stressed (Supplemental Fig. 1; Ikenouchi & Aoki, 2016), suggesting ROCK inhibitor may have off target effects that would preclude using ROCK inhibitor treated pluripotent stem cells in drug/toxicant testing and in clinical applications. Plating hESC on laminin-521 or on Matrigel coated with laminin-111 significantly reduces the time for attachment by stopping dynamic blebbing (Weng, 2016) and offers a safer method for passaging hESC. Little is known about the factors that initiate dynamic blebbing in hESC. In 2011, RNA interference was used to demonstrate that Active BCR-Related protein (Abr), a unique Rho-GEF family factor containing a functional GAP domain for Rho-class GTPase, is an upstream regulator of ROCK-myosin hyperactivation and apoptosis in hESC (Watanabe et al., 2007). In other cell types, multiple pathways may converge on Rho (Panupinthu et al., 2007; Ratz et al., 2009), suggesting additional regulators may be involved in initiating dynamic blebbing. For example, lysophosphatidic acid (LPA) is a phospholipid derivative that can act as a signaling molecule to activate the small GTPase Rho, which in turn activates ROCK. This pathway can lead to inhibition of myosin light-chain phosphatase (Frisca et al., 2013). However, there is currently no evidence that LPA is increased in hESC during culture. Our study demonstrates that the P2X7 receptor is involved in initiating dynamic blebbing in hESC during passaging. The P2X7 receptor is an ion channel that opens in response to extracellular ATP allowing cations to enter the cell, thereby triggering diverse responses such as secretion, apoptosis, survival, and proliferation (Bulanova et al., 2009; Adinolfi et al., 2005; Giannuzzo et al., 2005). P2X7 is expressed in a variety of cell types including monocytes/macrophages, dendritic cells, mast cells, mesangial, and microglial cells (Coutinho-Silva et al., 1999; Hickman et al., 1994; Brough et al., 2002). Its widespread expression in immune cells suggests that the P2X7 receptor has multiple proinflammatory actions (Cotrina & Nedergaard, 2009; Coutinho-Silva et al., 2009). In addition, P2X7 is found in many non-immune cells such as human cervical epithelium, retinal pigmented epithelium, and corneal epithelium, where it has been proposed to function in apoptosis, lysosomal alkalization, cell migration, cell proliferation and cell death (Wakx et al., 2016). The normal role of P2X7 in hESC may be to facilitate proliferation, a function ascribed to P2X7 in mouse ESC (Glaser et al., 2014). However, the P2X7 inhibitor did not alter hESC proliferation during 24h of incubation. In both human and mouse ESC, P2X7 was not present when cells began differentiating, indicating that it is an excellent surface marker for pluripotent cells.