Various cytokines and downstream signaling

Various cytokines and downstream signaling pathways, including FGF, BMP4 (Qi et al., 2004; Xu et al., 2005), TGFβ (Watabe and Miyazono, 2009; Peiffer et al., 2008), p38 MAPK (Binetruy et al., 2007), JNK pathway (Brill et al., 2009), and ERK pathway (Armstrong et al., 2006; Li et al., 2007) regulate hESC self-renewal. Growth factors also influence apoptosis via PKC, PI3K, and Akt pathways (Thompson and Thompson, 2004). Our study using inhibitors for specific signaling pathways indicated that Bcl-xL promoted single-cell survival of hESCs independent of those signaling pathways (Supplementary Fig. 7).
Materials and methods
Author contributions The following are the supplementary materials related to this article.
Acknowledgments This work was supported by NIHNCRR 2P20RR018789, NIH/RC1 Challenge GrantRC1 HL100117, and American Society of Hematology Faculty Award (ZW). Additional technical core support was provided by NIH Center of Excellence in Stem Cell Biology and Regenerative Medicine core facilities (2P20RR18789) and Center of Excellence in Vascular Biology core facilities (2P20RR15555).
Introduction Directional migration of endothelial progenitor thymidylate synthase (EPCs) is critical in vasculogenesis, angiogenesis, and vasculature repair. In embryos, EPCs migrate, differentiate, and coalesce into clusters of hemangioblasts to form new blood vessels (vasculogenesis). In adults, EPCs may move to sites of injury or a site of neovascularization and then differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization (angiogenesis) (Asahara et al., 1997; Akeson et al., 2000; Iwatsuki et al., 2005; Shi et al., 1998). Injury to vascular endothelium leads to the loss of the anti-thrombotic properties of the vessel wall, which could result in thrombosis, intimal hyperplasia and stenosis (Asahara et al., 1997; Zhao et al., 2008). Minor damage may be repaired by migration and proliferation of surrounding mature endothelial cells. Larger damage requires migration and differentiation of EPCs that are marked in general by CD133+/CD34+/vascular endothelial growth factor receptor 2+ (VEGFR-2)/VE-cadherin− (Akeson et al., 2000, 2001, 2010; Yamashita et al., 2000; Hristov and Weber, 2004, 2008). Directional migration of EPCs is meditated by varied chemokines, extracellular matrix, growth factors and membrane receptors (Hristov and Weber, 2004, 2008). Vascular endothelial growth factor (VEGF) is the most important growth factor in angiogenesis, controlling and increasing blood vessel formation (Yamashita et al., 2000; Yancopoulos et al., 2000). Endogenous electric fields (EFs) occur naturally at wounds and around vasculature, which may be an important signal in guiding cell migration (McCaig et al., 2005; Zhao et al., 2006; Nuccitelli, 2003; Barker et al., 1982). Applied EFs induced important pre-angiogenic responses in mature endothelial cells in culture. Human umbilical vein endothelial cells, bovine aortic endothelia cells, human dermal microvascular endothelial cells respond to applied EFs by directional migration, elongation and alignment (Li and Kolega, 2002; Bai et al., 2004; Zhao et al., 2004). Importantly, electrical stimulation has been demonstrated as an effective approach to induce angiogenesis in ischemia and wound healing (Zhao et al., 2004; Patterson and Runge, 1999; Kanno et al., 1999; Linderman et al., 2000; Sheikh et al., 2005; Kloth, 2005; Nagasaka et al., 2006). Electrical stimulation significantly enhances angiogenesis in ischemic and non-ischemic rat limbs, possibly mediated by VEGF and hepatocyte growth factor (HGF) expressed in muscle cells (Kanno et al., 1999; Sheikh et al., 2005; Nagasaka et al., 2006; Hang et al., 1995). Small EFs directly stimulate VEGF production by endothelial cells and direct reorientation, elongation and migration of endothelial cells in culture, through phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB or Akt), Rho-ROCK signaling pathways (Bai et al., 2004; Zhao et al., 2004).