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The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study,we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM),Flk-1,tie-1,tie-2,vascular endothelial (VE) cadherin,MECA-32,and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4,whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1,PECAM,VE-cadherin,MECA-32,and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin,interleukin-6,fibroblast growth factor 2,and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis. View Publication

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival,self-renewal,and differentiation. Thus,hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel,i.e. on standard substrates,without affecting hPSC morphology,growth rate or the ability to differentiate into multiple lineages both invitro and invivo. Importantly,QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically,the interaction of QHREDGS with ??1-integrins increased expression of integrin-linked kinase (ILK),increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2),and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance. ?? 2014 Elsevier Ltd. View Publication

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture,PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next,we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2),a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise,platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine,interleukin (IL)-1-β,via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration,viability,and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis,which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease. View Publication

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture,they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands,a process which we call vasculogenesis,was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum,fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response,which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis,the development of blood vessels from in situ differentiating endothelial cells,and angiogenesis,the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated. View Publication

BACKGROUND: Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).backslashnbackslashnMETHODS AND RESULTS: To induce endothelial cell differentiation,undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days,CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation,these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%),CD31 (97.2+/-2.5%),and VE-cadherin (93.7+/-2.8%),form vascular-like channels,and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward,5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly,bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally,in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).backslashnbackslashnCONCLUSIONS: This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs,and enhance their angiogenic effects in vivo. Furthermore,activation of nAChRs has anti-apoptotic,angiogenic,and proliferative effects through MAPK and Akt signaling pathways. View Publication

Definitive mesoderm arises from a bipotent mesendodermal population,and to study processes controlling its development at this stage,embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context,we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively,EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation,EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic,vascular,and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition,the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. View Publication

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells,the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially,and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture,and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels,although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro,and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation. View Publication

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation,reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study,we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter,tie-1. Using EGFP as a reporter gene,we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently,tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected,puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers,including CD31,CD34,VEGFR-1,VEGFR-2,Tie-1,VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1,two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally,we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together,these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies. View Publication

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells,whereas mural cells are believed to derive from mesoderm,neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation,whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system. View Publication