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Purpose: Endothelial derivatives of human pluripotent stem cells hold out hope for therapeutic angiogenesis. Our aim was to investigate the effect of differentiation protocols on the development of arterial and venous endothelial cells and to study the arterial and venous gene expression pattern after in vivo engraftment of endothelial cells.Methods: H7 human embryonic stem cells (hESC, from Wicell) were differentiated via embryoid body (EB) formation method in normoxygenic (20%) and hypoxic (5%) conditions as well as via monolayer method in the presence of vascular-endothelial growth factor (VEGF, 1ng/ml). Human induced pluripotent stem cells (hIPSC, from ReproCell) were differentiated under normoxygenic condition via EB formation. CD31-positive endothelial cells (EC) were sorted by FACS. For engineering 3D constructs, human aortic wall samples were decellularised with detergent solution (2% sodium dodecyl sulfate) and hESC-EC and hIPSC-EC were seeded onto this extracellular matrix. Human ESC-EC and hIPSC-EC were transplanted into three months old athymic nude rats using Matrigel as an extracellular matrix carrier.Results: The mRNA levels of angiopoietin2 showed an increase in hESC-EC when differentiated with EB method (EB normoxia 353.17±86.29; EB hypoxia 323.89±86.63, p<0.001, n=3, mRNA levels normalized to those in undifferentiated hESC). As shown by immunocytochemistry, differentiated hESC-EC and hIPSC-EC were stained positive for anti-CD31, von Willebrand factor and ve-cadherin; cells formed capillary-like tubules on Matrigel and took up acetylated-LDL. Quantitative PCR showed abundant expressions of both arterial (EphrinB2, Notch1-2) and venous (EphB4) endothelial markers; however, lymphatic endothelial cells were not detectable. As assessed by proteome profiling, a wide range of angiogenesis-related proteins were detected in both in hESC-EC and hIPSC-EC. Human ESC-EC and hIPSC-EC seeded onto decellularised human extracellular matrix remained viable as shown by calcein AM staining. Cells remained viable also upon in vivo engraftment; marked increase was found in mRNA levels of all arterial and venous marker genes in re-isolated cells (EphrinB2, EphB4, Notch 1-2, CD31, n=3). Conclusions: EB-based differentiation protocol of endothelial cells from human pluripotent stem cells has the highest angiogenic potency in vitro. After in vivo conditioning, expression of endothelial markers increased, suggesting the functional role of engrafted endothelial cells and possibly supporting future therapeutic purposes with specific angiogenic cells. [ABSTRACT FROM PUBLISHER]

Purpose: Endothelial cells have an essential role in the maintenance of cardiac function. Cardiac endothelial cells in response of different cardiovascular risk factors become activated and this activation is often followed by endothelial dysfunction. The change of endothelial phenotype may influence interactions with surrounding cardiac fibroblasts, myocytes, pericytes.The aim of our work is to produce in vitro culture system with high cell purity to investigate endothelial cells in different heart diseases. Poor quality of these tissue samples indicates the optimalization of isolation and culture conditions for both cardiac microvascular and endocardial endothelial cells (CMVE and EE cells).Methods: Human heart samples were obtained from valve surgery or explanted hearts during transplantation. CMVE cells were isolated from left ventricle with collagenase and trypsin digestion. EE cells were digested with collagenase from mitral valve. After separation the pellets were seeded to culture dishes and grown in a medium containing endothelial cell growth supplement. These basic methods would be changed as follows. The time of enzymatic digestion was changed. The collected cell suspensions were preincubated to allow fibroblasts to attach to the bottom of culture dish. The decanting medium was centrifuged repeatedly and collected cells were cultured in specific endothelial medium completed with sera from different origins. CMVE cells were treated with puromycin to eliminate pericytes. Cells were seeded on different artificial extracellular surface components (collagen, laminin, gelatin, fibronectin). The CMVE and EE cells were characterized by uptake of acetylated LDL and immunostaining of vonWillebrand factor, VE-cadherin and CD31. Fibroblasts were identified by vimentin staining.Results: Primary cells isolated with basic methods exhibited different cell morfologies, they were multilayered and only the 4% and 10% of the cells were endothelial cells, CMVE and EE respectively, separated by flourescent-activated cell sorting (FACS). The attachment of fibroblasts and the puromycin treatment were effective in the primary cell cultures. Numerous cell colonies were observed within 2-3 days on gelatin coated surface and confluence was achieved within 10-14 days.Conclusion: With use of these culture conditions the endothelial cell rate is increased in primery cultures and further separation by FACS or immunomagnetic methods will be more effective. These optimalized cell culture systems provide possibility to investigate endothelial functions in different heart diseases. [ABSTRACT FROM PUBLISHER]

Purpose: Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet.Methods: We studied PI3K-FOXO1A pathways during differentiation of H7 hESC towards endothelial lineage and on proliferation, maturation and cell death of hESC-derived endothelial cells (hESC-EC). We differentiated hESC towards endothelial lineage and used FACS to isolate CD31+ cells at day 13 after initiation of differentiation.Results: During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodelling-related genes. PI3K inhibitor LY294002 (10 μM) activated FOXO1A as shown by real-time PCR and nuclear translocation assay, and induced formation of CD31+ hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by siRNA showed lower mRNA levels of CD31 and angiopoietin2. Similarly, overexpression of FOXO1A-eGFP construct in hESC-EC resulted in an increased angiopoietin2 expression (1.5-fold, p<0.5, n=3) as well as higher percentage of Topro3-positive necrotic cells (p<0.001). LY294002 decreased proliferative activity of purified hESC-EC, whilst FOXO1A siRNA increased their proliferation. LY294002 inhibited migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. Using a small-animal PET/MRI system along with gallium-labelled NOTA-based conjugates for in vivo multimodality imaging showed an active angiogenic activity of hESC-EC in athymic nude rats. After in vivo conditioning of cells for three weeks, cells retain their low FOXO1A expression levels.Conclusions: PI3K/FOXO1A pathway is important for function and survival of hESC-EC as well as in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs. [ABSTRACT FROM PUBLISHER]