Wnt signaling mediates acquisition of blood-brain barrier properties in naïve endothelium derived from human pluripotent stem cells.

ABSTRACT

Endothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood-brain barrier (BBB) properties in response to extrinsic signals, with Wnt/ß-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here, we show that activation of Wnt signaling in hPSC-derived naïve endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, decreased PLVAP, and decreased permeability. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.

The cells that line the inside of blood vessels are called endothelial cells. In the blood vessels of the brain, these cells form a structure called the 'blood-brain barrier', which allows nutrients to pass from the blood into the brain, while at the same time preventing harmful substances like toxins from crossing. Faults in the blood-brain barrier can contribute to neurological diseases, but the blood-brain barrier can also restrict drugs from accessing the brain, making it difficult to treat certain conditions. Understanding how the endothelial cells that form the blood-brain barrier develop may offer insight into new treatments for neurological diseases. During the development of the embryo, endothelial cells develop from stem cells. They can also be generated in the laboratory from human pluripotent stem cells or 'hPSCs', which are cells that can produce more cells like themselves, or differentiate into any cell type in the body. Scientists can treat hPSCs with specific molecules to make them differentiate into endothelial cells, or to modify their properties. This allows researchers to monitor how different types of endothelial cells form. Endothelial cells at the blood-brain barrier are one of these types. During their development, these cells gain distinct features, including the production of proteins called GLUT-1, claudin-5 and LSR. GLUT-1 transports glucose across endothelial cells' membranes, while claudin-5 and LSR tightly join adjacent cells together, preventing molecules from leaking into the brain through the space between cells. In mouse endothelial cells, a signaling protein called Wnt is responsible for turning on the genes that code for these proteins. But how does Wnt signaling impact human endothelial cells? Gastfriend et al. probed the effects of Wnt signaling on human endothelial cells grown in the lab as they differentiate from hPSCs. They found that human endothelial cells developed distinct blood-brain barrier features when Wnt signaling was activated, producing GLUT-1, claudin-5 and LSR. Gastfriend et al. also found that human endothelial cells were more responsive to Wnt signaling earlier in their development. Additionally, they identified the genes that became activated in human endothelial cells when Wnt signaling was triggered. These findings provide insight into the development and features of the endothelial cells that form the human blood-brain barrier. The results are a first step towards a better understanding of how this structure works in humans. This information may also allow researchers to develop new ways to deliver drugs into the brain.