Introduction
Vascular patches made of synthetic polymers [e.g. expanded polytetrafluoroethylene (ePTFE), polyethylene terephthalate (Dacron)] are used to reconstruct and repair damaged blood vessels. Unfortunately, these patches have limitations related to thrombus formation and calcification due to blood and tissue incompatibility[1][2]. Tissue-engineered vascular patches could overcome the problems of synthetic vascular patches. Here, we developed a clinical relevant methodology to derive arterial and venous endothelial cells from induced pluripotent stem cells (iPSCs) in chemically defined and serum-free conditions. We generated vascular patches by seeding the iPSC-derived endothelial cells in nanofilms composed of poly(caprolactone) or poly(dimethylsiloxane) (PDMS). We have studied the pro-inflammatory properties of the vascular patches and compared to the ones commercially available.
Results and Discussion
Approximately 90% of the isolated cells were positive for CD31 and vascular endothelial receptor II (VEGFR2/KDR). At this stage cells expressed low levels of arterial, venous and lymphatic endothelial markers, suggesting that their sub-phenotype is not yet defined. After four passages in serum-free medium supplemented with vascular endothelial growth factor (VEGF165, 50 ng/mL), the cells, characterized by the expression of endothelial markers (CD31: 88.3±4.2%; KDR: 89.3±5.2%; VE-cadherin: 54±6,7%), preferentially expressed arterial markers, such as EphrB2: 26.5±2%(a percentage similar to the 35±7,9% expressed by human umbilical artery endothelial cells) and other arterial related genes such as JAG1, EFNB1, EFNB2, HEY-2, together with low levels of venous markers EFNB4 and COUP-TFII. In contrast, vascular progenitor cells cultured in serum-free medium supplemented with a lower concentration of VEGF165 (10 ng/mL) differentiated preferentially into venous endothelial cells, characterized by increased expression of the venous marker COUP-TFII, and low expression levels of arterial markers in comparison with cells expanded with a higher VEGF concentration. Both iPSC-derived arterial and venous endothelial cells have the capacity to uptake acetylated low-density-lipoprotein (Ac-LDL), to form a capillary like-network and respond to pro-inflammatory stimuli. These cells are biocompatible with PCL and PDMS NFs and, have a similar adhesion and proliferation profile that when cultured in commercially available vascular patches, used as a control. Additionally, culture in NFs did not induce the expression of pro-inflammatory markers E-Selectin, ICAM-1 as evaluated by qPCR and flow citometry. We observed that the inflammatory profile of iPSC-derived ECs in response to TNF-α is similar when cells are cultured in NFs and Vascular patches.
The authors would like to thank the funding support of FCT (SFRH/BPD/79323/2011; MITP-TB/ECE/0013/2013) and COMPETE funding (Project “Stem cell based platforms for Regenerative and Therapeutic Medicine”, Centro-07-ST24-FEDER-002008).
References:
[1] Cho, SW., et al. Vascular patches tissue-engineered with autologous bone marrow-derived cells and decellularized tissue matrices. Biomaterials, 2004.
[2] Muto, A., et al. Patches for Carotid Artery Endarterectomy: Current Materials and Prospects. J Vasc Surg., 2009.