Scientists have developed a process for generating large quantities of functional blood-brain barrier (BBB)-like endothelial cells from human pluripotent stem cells (hPSCs), including both induced and embryonic stem cells. The pure populations of differentiated endothelial cells express BBB markers, respond to astrocyte cues, and exhibit both barrier and transport properties that mimic those of primary brain microvascular endothelial cells (BMECs) that make up the BBB in vivo.
The University of Wisconsin–Madison (UWM) and University of Iowa (UI) investigators say their technology could provide a ready source of differentiated BMECs for developing models of diseases that compromise BBB function, screening drug candidates against specific neurological disorders, and evaluating drug neurotoxicity.
UWM’s Eric V Shusta, Ph.D., Sean P. Palacek, Ph.D., and colleagues report their development in Nature Biotechnology. “The nice thing about deriving endothelial cells from iPSCs is that you can make disease-specific models of brain tissue that incorporate the BBB,” Dr. Palacek states. “The cells you create will carry the genetic information of the condition you want to study.” The authors describe their achievement in a paper titled “Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.”
The BBB comprises specialized brain BMECs that feature intercellular tight junctions to prevent the passive diffusion of molecules into the brain, and active transporters that shuttle small lipophilic molecules that diffuse into BMECs back into the bloodstream. The BBB finely regulates the flow of substances including nutrients and metabolites into and out of the brain via specific transporter systems, and represents an impenetrable barrier to intravenously delivered biological drugs. BBB dysfunction and breakdown is also associated with a range of neurological disorders, including Alzheimer disease, stroke, multiple sclerosis, and brain tumors.
Unfortunately, the ability to screen drugs for brain permeability/neurotoxicity or carry out research on BBB dysfunction is hampered by a lack of easily generated in vitro models, the investigators note. Most models use brain microvessels isolated from animal sources, and even though human models have been created by culturing primary human BMECs from autopsy tissue or freshly resected brain specimens, such tissue isn’t widely available.
The UWM and UI researchers set out to develop a stem cell-based technology that could feasibly provide a scalable platform for generating potentially limitless supplies of BMECs for research and drug screening. The basic approach hinges on the simultaneous codifferentation of either hIPSCs or hESCs into both endothelial and neural cells, followed by purification of the BBB-like endothelial population on a selective matrix. This two-dimensional hPSC differentiation approach effectively provides a microenvironment resembling the embryonic brain in vitro, to promote differentiation into BBB-like cells, the authors explain.
iPSCs or hESCs were first on expanded on Matrigel-coated plates in defined mTeSR1 medium for 2–3 days. To initiate neural and endothelial codifferentiation, the colonies were then cultured in unconditioned medium over the course of a week, during which cells differentiated into populations of both neural cells and endothelial cells that expressed the BBB glucose transporter GLUT-1, tight junction proteins and p-glycoprotein. The hPSC-derived BMEC population was then further expanded for two days in a custom endothelial cell medium that includes factors that facilitate primary BMEC growth, and subsequently subcultured as a monolayer onto plates coated with a collagen-fibronectin extracellular matrix commonly used for primary BMEC culture. The resulting cell population expressed PECAM-1 together with requisite BBB markers, and endothelial transcripts encoding von Willebrand factor (vWF) and VE-cadherin.
A series of assays confirmed that monolayers of the hPSC-derived BMECs responded to astrocytic cues, expressed functional transport systems, and exhibited tight barrier properties. BMECs were first seeded onto Transwell filters coated with collagen-fibronectin matrix and cultured in endothelial cell medium. The cells grew to confluence and generated monolayers on the filter surface that maintained continuous cell-cell contacts, and demonstrated raised transendothelial electrical resistance (TEER), a feature of the BBB and a consequence of tight junction protein interactions between adjacent cells.
Freeze-fracture electron microscopy confirmed the presence of complex networks of tight junction strands that mirrored that of the BBB endothelium in vivo. Importantly, when the monolayers were co-cultured with rat astrocytes the TEER increased dramatically, and remained elevated for up to 96 hours, “indicating a specific response to astrocytic cues, as we expected of BMECs,” the team notes.
Purified iPSC-derived BMECs co-cultured with astrocytes also expressed transcripts encoding receptors and transporters found at the BBB in vivo, including amino acid and peptide transporters. Notably, the iPSC-derived BMEC monolayers co-cultured with astrocytes demonstrated levels of permeability to a range of molecules including sucrose, glucose, inulin, diazepam, colchicines, and vincristine that correlated with those of the BBB in vivo.
“To our knowledge no previous methods for differentiating hPSCs have generated organ-specific endothelial cells,” the authors conclude. “BBB specification occurred in the presence of co-differentiating neural cells, which probably supplied many of the necessary cues normally provided by the embryonic brain microenvironment in vivo.”
The investigators say that in comparison with primary animal and human BMEC cultures, their hPSC approach represents a relatively easy and scalable method: endothelial differentiation is efficient, and purification of the differentiated BMECs is a simple process. “Given that BMEC populations were obtained from both hESC and iPSC lines derived via different reprogramming strategies, our model can be readily adopted by the research community for studies of brain development, disease mechanisms, and drug delivery ... As hPSC-derived BMECs have good barrier characteristics with appropriate molecular exclusion and functional transport systems, this cellular platform should be useful in drug screens to develop pharmaceuticals with desired brain permeability.”