Most published protocols for the endothelial differentiation

Most published protocols for the endothelial differentiation of hESCs employ stromal co-culture (Boyd et al., 2007; Vodyanik et al., 2005) or utilise media with undefined or animal derived components such as serum, albumin fractionated from bovine serum, conditioned media or extracellular matrices (Chen et al., 2007; Ferreira et al., 2007; Kane et al., 2010; Lagarkova et al., 2008; Levenberg et al., 2010; Li et al., 2008; Prado-Lopez et al., 2010; Sun et al., 2012; Wang et al., 2007). Albumin, a common constituent in many formulations, is an avid transporter of lipids and hormones, whose composition may vary based on the source. In contrast, our studies used a fully defined buy flumazenil culture medium, denoted APEL (Ng et al., 2008), that contained only recombinant human proteins (albumin, transferrin and insulin). In addition, EB, rather than monolayer differentiation, was highly efficient and generally yielded ~40% CD34+KDR+ progenitor cells after 6days of differentiation. In HUVECs that are cultured in serum-containing medium, eNOS is thought to be associated with caveolae, invaginated plasma membrane micro-domains rich in cholesterol and sphingolipids (Govers and Rabelink, 2001), where it is found in association with caveolin-1 (Govers et al., 2002). The intracellular localisation of eNOS in HUVECs is reportedly also influenced by cell density. In sub-confluent cultures, eNOS is predominantly confined to the golgi whereas in dense cultures, eNOS is found in the caveolae (Govers et al., 2002; Liu et al., 1997; Sessa et al., 1995). However, eNOS in hESC-ECs cultured in APEL medium was similarly concentrated in a perinuclear compartment, consistent with golgi localisation. The highly defined nature of our culture system enabled us to identify LPA and PAF as factors that induced redistribution of eNOS such that it subcellular localisation resembled that seen in HUVECs. LPA is associated with cellular proliferation (van Corven et al., 1989), wound healing, adhesion and cell migration (Moolenaar, 1995) whilst PAF is a mediator of leukocyte adhesion to the endothelium and also regulates vessel tone. In the systemic vasculature, PAF elevates NO production and vascular permeability, whilst mediating bronchoconstriction in lung endothelial cells (Kuebler et al., 2010). In the circulation, both LPA (Eichholtz et al., 1993) and PAF (Clay et al., 1990) are present in albumin-containing complexes. Our data are consistent with the hypothesis that exogenous factors known to be present in serum can influence the phenotypic characteristics of ECs.
Conclusion The following are the supplementary data related to this article.
Acknowledgments We thank Dr. S. Hawes and Dr. A. Pebay for their advice. This work was supported by the Australian Stem Cell Centre, Stem Cells Australia, The National Health and Medical Research council of Australia, The Juvenile Diabetes Research Foundation and The Australian Research Council.