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Derivation of putative brain endothelial cells from porcine induced pluripotent stem cells
The use of pigs in neuroscience has increased over the past years because the pigs are closely related to humans in terms of anatomy and physiology. Especially, the blood-brain barrier (BBB) maintains the homeostatic microenvironment in the central nervous system (CNS) and they can provide a valuable tool for studying the neurobiology. However, only a few putative blood-brain barrier (BBB) models have been generated by co-culture of porcine primary cells. The fundamental problem is that they lose some of their phenotypes when maintained in vitro for long-term culture. To establish improved in vitro porcine BBB models, we differentiated novel brain microvascular endothelial cells (BMECs) from porcine induced pluripotent stem cells (iPSCs) using a modified human-based protocol. Briefly, the dissociated single cells from iPSCs were seeded in Geltrex. For differentiation, cells were maintained for 3 days of expansion and then switched to unconditioned medium (UM) lacking bFGF for 6-7 days. Then, we subcultured cells onto collagen/fibronectin coated plates and changed BMEC medium for 2-3 weeks. About two weeks later, we observed a cluster of round cells surrounded by spindle shaped adherent cells termed as colony-forming units (CFU) of putative BMECs. Over time, the cluster of cells disappears and remained adherent spindle-shaped cells showed properties of endothelial cells. Although further studies will be needed, this study would be a great comparative analysis of the porcine and human in vitro BBB model.
