In vitro BBB Models

One longstanding goal of BBB research has been the development of cell-based in vitro experimental models that accurately mimic the BBB characteristics observed in vivo. Such models would be amenable to drug permeability screening and a priori prediction of brain uptake. Typically, such in vitro BBB models are much Figure 1morepermeable than the situation found in vivo and are thusnot very predictive. Our laboratory has made contributions towards the improvement of blood-brain barriermodels. One of the major difficulties hampering in vitro BBB modeling is endothelial culture impurity after primary isolation. In particular, pericytes often contaminate primary brain microvascular endothelial cell (BMEC) cultures in an uncontrollable manner and therefore hinder the study of these cells. We developed an approach for culture purification that takes advantage of a unique BMEC property, a chemical efflux transport system. Employing the translation inhibitor puromycin resulted in selective toxicity that leaves BMECs unscathed. Rat BMEC cultures with purities routinely as high as 99.8% were generated, and with the subsequent addition of BBB-inducing agent, hydrocortisone, the barrier properties of the puromycin-treated layers were far superior (Figure 1).

The co-cultured cells that are essential to BBB models, such as pericytes, neurons, and astrocytes, can also be difficult to obtain in sufficient quantities from primary sources. To this end, we have established design principles for utilizing neural progenitor cells (NPCs) in place of neurons and astrocytes. NPCs are multipotent progenitors that can form astrocytes, neurons, and oligodendrocytes, and because of their stem-like properties, they are ideal for generating large numbers of cells for modeling purposes. We determined that rat NPCs could be differentiated for 12 days under specific conditions to generate a mixture of neurons and astrocytes that could induce a number of BBB properties in cultured rat BMECs, including high trans-endothelial electrical resistance (TEER; Figure 2a). Importantly, human NPCs isolated from fetal tissue could be differentiated under similar conditions to form astrocytes and neurons which generated a TEER response indistinguishable from the one generated by differentiated rat NPCs and primary rat astrocytes (Figure 2b). These results indicate that human NPCs could be utilized as a source of co-cultured cells for eventual translation to a human BBB model.

Figure 3aFigure 2b

While we have made substantial progress in BBB modeling using primary rodent cultures, it is well-accepted that to truly harness the power of in vitro models for translational medicine, a robust human BBB model must be developed. Perhaps the most daunting hurdle in constructing a human BBB model is the lack of high-fidelity human brain endothelium. Only a select few research groups possess the necessary expertise to acquire these cells from primary sources such as biopsied tissue, and even in these situations, human BMECs cannot be obtained in large enough quantities for significant modeling studies and drug screening. For these reasons, we Figure 3recently focused our efforts on utilizing human pluripotent stem cells (hPSCs) for BBB modeling purposes. hPSCs are an attractive source of cells for human models because they have unlimited self-renewal capabilities, can form any lineage in the human body, and are practical for conducting human cellular studies that would otherwise be intractable in vivo. As such, we have developed a novel method for differentiating hPSCs, including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into human endothelial cells with specific properties of the BBB. These hPSC-derived BMECs express transcription and protein markers of the BBB, exhibit extremely high TEER (up to 1450±140 Ωxcm2), and possess efflux transport proteins such as p-glycoprotein, breast cancer resistance protein (BCRP), and members of the multidrug resistance protein (MRP) family, which allows them to discriminate between small molecule drugs with varying efflux transporter recognition properties in an in vitro permeability screen (Figure 3). Given the rapid progression of iPSC technology and growing usage of stem cells by many research facilities, we anticipate these hPSC-derived BMECs will be readily accessible for broad neuroscience applications.