Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells

Abstract

The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.

Paving the Way Toward Complex Blood-Brain Barrier Models Using Pluripotent Stem Cells

“…This was further verified by detecting claudin, occludin, and ZO-1 expression, as well as by observing vascular-like structures in Matrigel Ò and uptake of acetylated low-density lipoprotein [52]. However, these cells alone yielded a TEER of only 55 Ocm 2 , indicating a barrier tightness that is far from representative of the in vivo BMEC [52]. This indicated that induction by co-culturing with neural cultures is not sufficient for inducing BMEC properties, possibly due to the importance of factors secreted and/or mechanical interactions that pericytes and astrocytes have on BMECs.…”

“…The study by Minami et al on co-culturing with a glioma cell line used CD34 and CD144 (VE-cadherin) as markers for ECs, which were sorted from the population and analyzed for CD31 (PECAM) and Von Willebrand factor to identify BMECs. This was further verified by detecting claudin, occludin, and ZO-1 expression, as well as by observing vascular-like structures in Matrigel Ò and uptake of acetylated low-density lipoprotein [52]. However, these cells alone yielded a TEER of only 55 Ocm 2 , indicating a barrier tightness that is far from representative of the in vivo BMEC [52].…”

“…Also, co-culturing of hPSCs [50] or hiPSCs [51] with the mouse bone marrow stromal cell line OP9 has yielded ECs. Improved protocols that lead to the derivation of BBBspecific BMECs include co-culturing with glioma cell lines [52], NPCs [53], or astrocytes [54].…”

Establishment of a Human iPSC- and Nanofiber-Based Microphysiological Blood–Brain Barrier System

“…19,57 Current in vitro BBB models are usually based on commercially available plastic and porous transwell membranes. In this study, we designed and 3D-printed a holder that can support an electrospun PLGA mesh and can perfectly fit into the transwell insert.…”

“…16 In addition, hiPSC-ECs cocultured with primary rat astrocytes or C6 rat glioma cells also showed brain EC phenotypes. 20,57 A hallmark of the BBB is a high TEER because of the tight junctions between ECs to maintain barrier integrity. In the current study, although the TEER value was lower than the native BBB (~1000–2000 Ω·cm 2 ), 10,12 it is still within the reported range for in vitro BBB models.…”