Functional human endothelial cells differentiated

Functional human endothelial cells differentiated from hPSCs could be beneficial for many potential clinical applications (Burridge et al., 2012; Kaupisch et al., 2012; Levenberg et al., 2002; van der Meer et al., 2013), including engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration (Robey et al., 2008), and induction of angiogenesis for treatment of regional ischemia (Liu et al., 2014). Endothelial cell dysfunction is also associated with many diseases, including Alzheimer’s disease, stroke, multiple sclerosis, and atherosclerosis (Boyle et al., 1997; Weiss et al., 2009). hPSC-derived endothelial progenitors and endothelial cells may provide building blocks for the establishment of in vitro disease models for screening and development of drugs to treat these diseases. Functionality of hPSC-derived endothelial cells has been shown using in vitro cell culture platforms and in vivo animal models (Adams et al., 2013; Kusuma et al., 2013; Orlova et al., 2014; Samuel et al., 2013; Wang et al., 2007). Similar to other somatic cells derived from hPSCs, differentiated CD31+ endothelial cells exhibited functional heterogeneity (Rufaihah et al., 2013). Previously reported studies of hPSC differentiation to endothelial cells have demonstrated that Activin/Nodal/transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF), and microRNA-21 signaling promote this differentiation (Di Bernardini et al., 2013; James et al., 2010; Kane et al., 2010; Lu et al., 2007; Marchand et al., 2014; Rufaihah et al., 2011; Wang et al., 2004; Zambidis et al., 2005). In addition, mechanical sheer stress also promoted embryonic stem cell-derived endothelial phenotypes (Wolfe and Ahsan, 2013). During murine embryogenesis, hemangioblasts, which can differentiate into multipotent hematopoietic stem cells and endothelial progenitors, are derived from a subpopulation of mesoderm that coexpresses brachyury and KDR (Huber et al., 2004). Similar blast colony-forming cells were also isolated from mouse embryonic stem cell moexipril Supplier in the presence of cytokines (Kennedy et al., 1997). When cocultured with OP9 stromal cells, hPSCs differentiated to mesodermal progenitors with the capacity to form blast or hemangioblast colonies in response to fibroblast growth factor 2 (FGF2) (Vodyanik et al., 2010). As another approach, hPSCs cultured as embryoid bodies were exposed to a growth factor cocktail containing activin A, BMP4, FGF2, and VEGF to induce differentiation to CD34+CD31+ endothelial progenitors (Costa et al., 2013; Levenberg et al., 2002; Song et al., 2013). The CD34+CD31+ vascular progenitor population generated endothelial cells and smooth muscle cells in the proper culture environments (Bai et al., 2010). TGF-β signaling enhanced smooth muscle cell differentiation from these endothelial progenitors, whereas the TGF-β signaling inhibitor SB431542 promoted endothelial cell generation and expansion (James et al., 2010). Global gene transcription analysis demonstrated low variability between endothelial cells (ECs) differentiated from multiple lines of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in the presence of these cytokines (White et al., 2013). Although prior studies have demonstrated differentiation of hPSCs to endothelial progenitors, and subsequently to ECs and smooth muscle cells, by applying growth factors from different signaling pathways, it is largely unknown whether these distinct differentiation protocols produce identical endothelial cells and their progenitors, and which developmental signaling mechanisms are necessary and sufficient to specify these differentiation fates. Here, we describe a simple and efficient method for the conversion of hPSCs to CD34+CD31+ endothelial progenitors. Appropriate temporal activation of regulators of WNT signaling alone, in the absence of exogenous FGF2 and VEGF signaling, was sufficient to drive multiple hPSC lines to differentiate to greater than 50% CD34+CD31+ endothelial progenitors. However, endogenous MEK signaling was required for hPSC differentiation to endothelial progenitors because MEK inhibitor treatment substantially diminished the yield of CD34+CD31+ cells. These hPSC-derived endothelial progenitors were further enriched to 99% purity with a single step of CD34-based magnetic separation. Single-cell clonal differentiation assays revealed that CD34+CD31+ endothelial progenitors generated by WNT pathway activation were bipotent and could differentiate to functional endothelial cells and smooth muscle cells.