The blood-brain barrier (BBB) plays an important role in brain health and is often compromised in disease. Moreover, as a result of its significant barrier properties, this endothelial interface restricts neurotherapeutic uptake. Thus, a renewable source of human BBB endothelium could prove enabling for brain research and pharmaceutical development. Herein, we demonstrate that endothelial cells generated from human pluripotent stem cells (hPSCs) can be specified to possess many BBB attributes, including well-organized tight junctions, expression of nutrient transporters, and polarized efflux transporter activity. Importantly, hPSC-derived BBB endothelial cells respond to astrocytic cues yielding impressive barrier properties as measured by transendothelial electrical resistance (1450±140 Ωxcm2) and molecular permeability that correlates well with in vivo brain uptake. In addition, specification of hPSC-derived BBB endothelial cells occurs in concert with neural cell co-differentiation via Wnt/β-catenin signaling, consistent with previous transgenic studies. This study represents the first example of organ-specific endothelial differentiation from hPSCs.

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications.

BackgroundBrain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood–brain barrier (BBB) phenotype.MethodsA range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay.ResultsThe initial hPSC seeding density of approximately 30,000 cells/cm2 served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm2) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype.ConclusionsGiven the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-015-0007-9) contains supplementary material, which is available to authorized users.

Accumulating evidence suggests that endothelial cells (ECs) display significant heterogeneity across tissue types, playing an important role in tissue regeneration and homeostasis. Recent work demonstrating the derivation of tissue-specific microvascular endothelial cells (TS-MVECs) from human pluripotent stem cells (hPSCs) has ignited the potential to generate tissue-specific models which may be applied to regenerative medicine and in vitro modeling applications. Here we review techniques by which hPSC-derived TS-MVECs have been made to date and discuss how current hPSC-EC differentiation protocols may be directed towards tissue-specific fates. We begin by discussing the nature of EC tissue specificity in vivo and review general hPSC-EC differentiation protocols generated over the last decade. Finally, we describe how specificity can be integrated into hPSC-EC protocols to generate hPSC-derived TS-MVECs in vitro, including EC and parenchymal cell co-culture, directed differentiation, and direct reprogramming strategies.

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm, equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.