**5. Exploring iPSC-ECs Features in 3D Environments**

In a recent study, Campisi et al. used iPSC-ECs and primary cultures to develop an innovative 3D model of the microvascular network of the Blood Brain Barrier (BBB), able to replicate the physiologic neurovascular organization of BBB [94]. Indeed, this model displayed a selective microvasculature, with a degree of permeability lower than the one showed by the conventional in vitro models and more similar to the levels measured in rat brain.

The authors based their study on a microfluidic model comparing BBB derived from culturing human iPSC-EC alone and with a co-culture of human pericytes (PCs) and astrocytes (ACs). BBB from triple culture iPSC-ECs together with PCs and ACs showed the best performance in terms of stability and permeability.

These results indicate that iPSC-ECs co-cultured BBB functionally responds to physiological stimuli and that iPSC-ECs are a reliable model to investigate blood vessel properties in vitro [94]. Kurokawa et al. in 2017 developed a variety of methods to create a functional 3D vasculature in vitro. In particular, they compared iPSC-EC derived from CDH5-mCherry iPSCs to primary ECs [95].

After the phenotypic and functional characterization of iPSC-ECs, the authors created a microfluidic device loaded with HUVECs, Endothelial Colony Forming Cell Derived-Endothelial Cells (ECFC-ECs) and fluorescent iPSC-ECs in order to test their proliferative and vasculogenic potential.

Fluorescent iPSC-ECs displayed the physiological functions of endothelial cells when tested in standard culture condition, and a predominant venous phenotype as well as a response to shear stress when cultured into a 3D microfluidic device where they formed a perfusable vascular network. Furthermore, the cell loaded device when used for drug screening purposes showed a different behavior of the newly formed vasculature according to the different molecules used to modulate angiogenesis.

Within the microfluidic device, the authors investigated the performance of a co-culture composed of human lung fibroblasts and iPSC-ECs in comparison to the one displayed when these fibroblasts were cultured with primary ECs. More in details, they featured the characteristics of the 3D vascular network, the parameters related to the vascular barrier function, and the ability of the cells to aggregate into tube-like structures [95]. Additionally, Belair et al. described a model of engineered blood vessel developed using iPSC-EC cultured into a microfluidic device and then investigated iPSC-EC barrier function in response to a wound healing stimulus [96]. They demonstrate that human iPSC-EC reproduce functional properties of primary ECs in two in vitro platforms. After a physiological and phenotypic characterization, the iPSC-ECs and primary endothelial cells (HUVECs) were used for a comparison between Matrigel culture in standard conditions and a 3D culture in capillary-like structures embedded in fibrin. Afterwards, the authors investigated the iPSC-barrier function in response to a wound healing stimulus measured by means of impedance-based platform that records the electrical resistance as a direct indicator of cell junction damage. They generated a barrier by seeding an iPSC-EC monolayer and then assessed the expression of Zona Occludent-1 after a treatment with a low concentration of thrombin to damage the tight junctions. The authors investigated the capacity of iPSC-ECs to express cell adhesion molecules (CAMs) in response to Tumor Necrosis Factor-α stimulation, thus recapitulating EC properties necessary for cell recruitment during wound healing and inflammation. Flow cytometry analysis demonstrated that TNF-α treatment induced upregulation of Intercellular Adhesio Molecule-1 and Melanoma Cell Adhesion Molecule, which are expressed by ECs to promote attachment of immune and progenitor cells to blood vessels. The results indicate that the barrier formed with iPSC-ECs responded to a wound healing stimulus in a way that resembles mature ECs.

In the same study, the iPSC-ECs were seeded in fibrin gels into a microfluidic device and co-cultured with normal human lung fibroblasts. The co-cultured cells assembled into the three dimensional pattern developed inter-connected capillary networks and remarkably formed cord-like structures containing visibly hollow lumens [96].
