Abstract
The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.
Highlights
A marker of functional endothelium, Von Willebrand Factor (VWF) was upregulated along with markers associated with endothelial progenitor cells, suggesting that Insulin-like growth factor (IGF) promote development of a mature, functional endothelial cells. (Figure S1A) Because IGF promoted differentiation appears to be specific for the mesoderm lineage, we looked at the mRNA expression of a panel of mesoderm markers at 7 days following daily IGF1 and IGF2 treatment in differentiating embryoid bodies (EB) including those for cardiomyocyte, skeletal myocyte and hematopoietic lineages
Our results demonstrate that treatment with IGF1 and IGF2 increase the generation of endothelial cells in a spontaneously differentiating EB model
These results suggest that insulin-like growth factors play a role in promoting vasculogenesis at both of these critical stages
Summary
Stem cell differentiation into endothelial cells is the first step of vasculogenesis. [1,2] This process occurs spontaneously in vitro in embryonic stem cell derived embryoid bodies (EB). [3] The formation of vascular channels in EB closely mimics vasculogenesis in vivo. [4] Many groups have attempted to preferentially drive endothelial differentiation from embryonic stem cells but the process remains inefficient or labor intensive. [5,6] Several critical signaling factors have been identified, including , TGF-b, BMP4, and VEGF, that drive the differentiation of pluripotent stem cells first into mesoderm, endothelial progenitor cells and into mature endothelium. [7,8] In a recent study, we demonstrated that the cell surface glycome plays a critical role in this process. [9] a better understanding of the signaling pathways that control vasculogenesis is needed for efficient endothelial differentiation.Insulin-like growth factor (IGF) signaling mediates many critical cell responses including mitogenesis, proliferation, growth, differentiation, and angiogenesis. [10,11,12] IGFs circulate in the bloodstream at nanomolar concentrations and are generally bound to one of six IGF binding proteins (IGFBPs), which regulate their availability to cell surface receptors. [13] Glycosaminoglycans play a critical role in liberating the ligands from their binding proteins and making them available to bind receptors. [14] Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) share about 50% of amino acid sequence with insulin and have some affinity for the insulin receptor, but have distinct physiological functions from insulin mediated by signaling through the IGF-1 receptor. [15] Binding to this tyrosine kinase receptor activates downstream PI3K and MAPK cascades that stimulate growth and survival of particular cell types. [5,6] Several critical signaling factors have been identified, including , TGF-b, BMP4, and VEGF, that drive the differentiation of pluripotent stem cells first into mesoderm, endothelial progenitor cells and into mature endothelium. Insulin-like growth factor (IGF) signaling mediates many critical cell responses including mitogenesis, proliferation, growth, differentiation, and angiogenesis. While several studies have shown a connection between IGFs and neovascularization, their specific role in vasculogenesis is largely unknown. [27] Interestingly, studies have established a role for the insulin-like growth factor pathway in maintaining stem cell pluripotency. [28,29] In this study, we investigated the role of insulin-like growth factors 1 and 2 in embryonic stem cell vasculogenesis by focusing on endothelial differentiation in embryoid bodies. Our results establish that both IGF1 and IGF2 promote embryonic stem cell differentiation into endothelial cells acting through the IGFR1 pathway
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