FROM EMBRYONIC STEM CELLS TO ARTERIES AND VEINS

A fundamental question in developmental biology is how a single fertilized egg cell and its progeny are programmed or instructed to form thousands of different cell types and how these are organized into a complex and functional organism. In this thesis I have explored how a specific subset of cells is formed the vascular endothelial cells lining our blood vessels. As the embryo grows in size, passive diffusion is not sufficient to support the developing tissues with oxygen and nutrients. The cardiovascular system is therefore the first functional organ to be established during embryonic development. Understanding the mechanism driving vascular development could have great implications to our understanding and treatment of multiple pathologies and diseases such as cancer, diabetes and ischemic heart disease. My studies have been focused on the initial de novo generation of endothelial lineage and further specification into arterial and venous endothelial subtypes. These processes have been explored using in vitro differentiation of embryonic stem cells (ESC) to mimic the sequence of events in the developing embryo. In the first paper we addressed the function of vascular endothelial growth factor recptor-2 (VEGFR2) in the generation of endothelial cells and their integration into vascular structures. We showed that ESC lacking VEGFR2 fail to form and incorporate into vascular structures. By reintroducing VEGFR2 using lentiviral transduction we could rescue these defects. Interestingly, neither loss of or over expression of VEGFR2 seemed to influence the de novo generation of, at least immature, endothelial cells. In paper II I present data showing that it is possible to generate endothelial cells with transcriptional and functional arterial and venous characteristics through in vitro differentiation of ESC. VEGF played a critical role where high concentration promoted arterial fate in contrast to intermediate or low VEGF concentration which supported venous differentiation. I could further show that the VEGF signal driving arterial fate was dependent on functional Notch signaling. Having established the model system detailed above, I explored whether the hypoxic state of the embryo prior to functional circulation may influence the arterial venous lineage. Hypoxia did indeed strongly potentiate arterial over venous transcription in a Notch dependent manner. The hypoxia inducible factor (HIF) 1 and more potently 2 activated Adrenomedullin gene transcription. I showed that Adrenomedullin drives Notch signaling through transcriptional induction of the arterially expressed Notch ligand Dll4. In the final paper I aimed to dissect the individual role of two VEGF co-receptors, Neuropillin1 and heparan sulfate (HS) proteoglycans in arterial venous specification. Surprisingly we found NDST1/2 -/ESC which produce HS lacking a critical modification, N-sulfation, to be resistant to in vitro differentiation. These cells maintained transcriptional, morphological and functional characteristics of undifferentiated ESC following four days of embryoid body differentiation in the absence of leukemia inhibiting factor (LIF). We show that N-sulfation is critical specifically for endogenous FGF4 which is responsible for down-regulation of Nanog and thereby initiation of ESC differentiation. Finally, N-sulfation of HS has a functional importance as the efficiency of de novo ESC line derivation from mouse blastocysts greatly increased by the addition of a general sulfation inhibitor. These studies suggest that in vitro differentiation of ESC can be an excellent model system to compliment studies performed in the actual embryo, especially when addressing lineage specifications. LIST OF PUBLICATIONS I. Xiujuan Li, Dan Edholm, Fredrik Lanner, Georg Breier, Filip Farnebo, Anna Dimberg, Lena Claesson-Welsh. Lentiviral rescue of vascular endothelial growth factor receptor-2 expression in Flk1-/embryonic stem cells show early priming of endothelial precursors. Stem Cells (2007) 25, 2987-2995. II. Fredrik Lanner, Marcus Sohl, Filip Farnebo. Functional arterial and venous fate is determined by graded VEGF signaling and Notch status during embryonic stem cell differentiation. Arterioscler Thromb Vasc Biol. (2007) 27, 487-493. III. Fredrik Lanner, Marcus Sohl, Emil Hansson, Peter Carmeliet, Lorenz Poellinger, Urban Lendahl, Filip Farnebo. Hypoxic induction of Adrenomedullin and Notch signaling promotes arterial differentiation of embryonic stem cells. Manuscript (2008). IV. Fredrik Lanner, Marcus Sohl, Katarina Holmborn, Johannes Wilbertz, Filip Farnebo. N-sulfation of heparan sulfate is critical for embryonic stem cell differentiation. Manuscript (2008).