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.