Introduction The vascular endothelium the single cell layer

Introduction The vascular endothelium, the single-cell layer lining blood vessels, is a multifunctional interface that displays a striking phenotypic plasticity necessary for maintaining vascular homeostasis. In this context, the vascular endothelium is critical to initiate an inflammatory response, trigger thrombosis, regulate vasomotor tone, and control vascular permeability. Dysfunction of the endothelium plays a significant pathogenic role in cardiovascular diseases, namely, atherosclerosis and its consequences: heart attacks and strokes (Gimbrone et al., 2000; Hansson, 2005). Notably, studies at the genetic and molecular level of human endothelium have been limited by the availability of relevant tissue derived from cadaveric, discarded surgical, or umbilical vasculature sources. Recent developments in stem cell biology promise new resources for modeling genetic diseases. In particular, induced pluripotent stem IOX2 (iPSCs) offer the ability to study the effects of genetic alterations and mechanisms of genetic diseases in currently inaccessible cell types (Takahashi and Yamanaka, 2006). Although iPSCs have been differentiated into many cell types including endothelium (Choi et al., 2009; Homma et al., 2010; Li et al., 2011; Park et al., 2010; Rufaihah et al., 2011, 2013; Taura et al., 2009; White et al., 2013), the fidelity and functional mimicry of stem cell-derived tissues and their relevance to human disease remain poorly characterized. This functionality must be carefully assessed before their scientific and therapeutic potential can be realized (Soldner and Jaenisch, 2012).
Results
Discussion In this study, we have generated vascular ECs from human iPSCs and characterized their humoral-, pharmacological-, and biomechanical-induced functional phenotypes, advancing iPSC-ECs as an experimental platform to study endothelial biology. The methodology to differentiate iPSCs into vascular endothelium was reproducible and robust across several iPSC lines created with different technologies. Previous studies have differentiated iPSCs to isolate ECs based on expression of CD31 or KDR (Choi et al., 2009; Homma et al., 2010; Li et al., 2011; Park et al., 2010; Rufaihah et al., 2011, 2013; Taura et al., 2009; White et al., 2013). In contrast, we isolated populations of ECs from the EBs based on VE-cadherin expression, a strong marker of endothelial identity, as we observed CD31+, and KDR+ cells subpopulations lacking VE-cadherin, and a CD31+ subpopulation expressing CD45. After isolation and culture, iPSC-ECs displayed molecular markers of vascular endothelium and contained vWF-positive WPBs that can be rapidly exocytosed, a critical function for hemostasis. It is possible that the perinuclear localization observed in many of the WPBs may suggest an immature stage in WPB biogenesis (Valentijn et al., 2011; Zenner et al., 2007). iPSC-ECs are competent for the functional repertoire displayed by vascular endothelium critical in pathophysiological settings. Specifically, we demonstrated that proinflammatory stimuli induce an activated proinflammatory phenotype that included expression of leukocyte adhesion molecules, secretion of proinflammatory cytokines, and support for human leukocyte transmigration. We also observed that iPSC-EC monolayers display a dynamic permeability in response to a panel of physiological stimuli. Human iPSC-ECs are a promising platform for studying endothelial contributions to cardiovascular diseases in the context of specific patients’ genetic backgrounds. We demonstrated that iPSC-ECs have the plasticity to acquire distinct flow-dependent phenotypes, namely atheroprotective and atheroprone phenotypes critical for atherosclerosis resistance and susceptibility. We observed that iPSC-ECs respond to atheroprotective shear stress by activating an atheroprotective gene expression program transcriptionally mediated by KLF2 and KLF4 expression, shown to integrate the flow-mediated endothelial atheroprotective functional phenotype by regulating leukocyte adhesion, redox state, and thrombotic function in cultured human ECs (Dekker et al., 2006; Lin et al., 2005; Parmar et al., 2006; SenBanerjee et al., 2004; Villarreal et al., 2010). The ability to direct the acquisition of flow-dependent functional and dysfunctional phenotypes using patient-specific endothelium should allow the study of specific genetic contributions to endothelial function and dysfunction in the context of human cardiovascular disease. Furthermore, we demonstrated that simvastatin activated an atheroprotective gene expression program and downregulated genes associated with an atheroprone phenotype in iPSC-ECs. This suggests that iPSC-ECs may be a suitable surrogate to assess the effects of drugs on the endothelia of specific patients.