Insulin-Like Growth Factors Promote Vasculogenesis in Embryonic Stem Cells

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

Enhancing taxol delivery with novel taxol conjugated liposomal nanoparticles

Cancer has been a leading cause of death in many developed nations. Although there exists a variety of anti-cancer drugs, standard chemotherapy has a severe limitation in which all dividing cells are been targeted as well. In order to enhance the efficiency of anti-cancer drug delivery, we adopt a nano-particle mediated approach whereby targeted anti-cancer therapeutics will be conjugated to biodegradable nano-particles. Due to the extra-permeability and retention (EPR) effect, the nano-particles will be entrapped in the solid tumors, but not in healthy tissues. The conjugated anti-cancer therapeutics will then be released and destroy the cancer cells. Possible improvements will include the use of selective signaling pathway inhibitors to inhibit cancer cell division, and the conjugation of selective ligands to improve the targeting ability of the nano-particles.Bachelor of Engineering (Chemical and Biomolecular Engineering

IGF1 increases HIF1α protein levels in differentiating embryoid bodies and inhibition of HIF1α with rapamycin decreases VEGF and VEGFR2 expression in embryoid bodies treated with IGF1.

<p>A) HIF1αlevels increase significantly following treatment with IGF1. Rapamycin reduces protein levels when added to cell cultures prior to IGF1 stimulation. * denotes P<0.05 compared to conrol, ** denotes P<0.05 compared to +IGF B) Day 3 EB treated with IGF1 express significantly higher levels of VEGF and VEGFR2 compared with control (# denotes P<0.05 compared with control), which are significantly inhibited by treatment with HIF1α inhibitor rapamycin. (* denotes P<0.05 compared to IGF1 treatment). C) Proposed mechanism of IGF1 and IGF2 stimulated endothelial differentiation.</p

IGF1 and IGF2 promote mesoderm and endothelial differentiation.

<p>A–B) Day 3 EB treated with IGF1 and IGF2 display increased mRNA levels of Brachyury and no significant change in Oct4, Nanog, and Sox2. C–D) Levels of AFP and PAX6 are not affected. # denotes P<0.05, Rn denotes normalized reporter. Day 7 EB treated with IGF1 (E,F) and IGF2 (G,H) possess increased levels of endothelial specific markers.</p

Expression of Insulin Like Growth Factors, Receptors, and Binding Proteins with Embryonic Stem Cell Differentiation.

<p>A) mRNA levels of IGFR-1, IGFR-2, IGF1, and IGF2 increase with time in differentiating embryoid bodies measured from days 1–7, although IGF1 appears to remain steady, pointing to its role in both pluripotency and differentiation. * denotes P<0.05 to day 1 control, Rn denotes normalized reporter.</p

IGFs induce endothelial differentiation at early and late stages of vasculogenesis and increase expression of VEGF.

<p>A–B) VEGFR2 and Tie-2 levels at day 7 are significantly increased in EB treated on days 1 and 2 only or on days 4–7 only. C) Treatment with IGF1 or IGF2 post-mesoderm development results in an increase in the percentage of VWF-positive cells compared to control differentiation EB. D–E) PPP treatment on days 1 and 2 only or on days 4–7 only reduced levels of endothelial markers at day 7. Levels of VEGF increased with IGF1 and IGF2 treatment, and decreased with PPP treatment (F–H), at day 3. * denotes P<0.05 compared to control ** P<0.01, Rn denotes normalized reporter.</p

Picropodophyllin treatment inhibits mesoderm and endothelial formation in differentiating embryoid bodies.

<p>Day 3 EB treated with PPP have unchanged or increased levels of pluripotency markers significantly decreased levels of Brachyury. B) PPP treatment decreases expression of vascular markers at day 7. C) Differentiated EB cultures treated with PPP and stained for CD31 (TRITC) had strikingly less vessels compared to control on day 10. *denotes P<0.05, Rn denotes normalized reporter.</p