We report a defined method for differentiating human pluripotent stem cells to brain endothelial cells.

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.

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy.

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.

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Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's Modified Eagle Medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3β inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+CD31+ endothelial progenitors that can be purified to>95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications.

A linear frequency−thickness (F−T) relation was established for surface-initiated atom transfer radical polymerization (SI-ATRP) in a quartz crystal microbalance (QCM). This quantitative F−T relation is monomer dependent but independent of polymerization rate, initiator and polymer density. With this F−T relation and the online monitoring capacity, QCM was successfully applied to study the kinetics of SI-ATRP mechanisms. QCM was also demonstrated to be useful in controlling film thickness at the angstrom level, which is critical in nanofabrication.

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation stage-specific application of Gsk3 inhibitor, Wnt inhibitor, then Gsk3 inhibitor is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 days. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation to fibroblasts and smooth muscle cells is also described. In addition, culture in the presence of TGFβ inhibitors allows long-term expansion of hPSC-derived epicardial cells for at least 25 population doublings. Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.