Correction to: An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons

Abstract Following publication of the original article [1], the author has reported that in Figure 1 (b and c) the y-axis TEER (© x cm2) should be replaced with TEER (Ω x cm2). Erratum for An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons. [Fluids Barriers CNS. 2019

Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Progenitors via Small-Molecule Activation of WNT Signaling

SummaryHuman pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34+CD31+ endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was β-catenin dependent. Furthermore, by clonal analysis, we showed that CD34+CD31+CD117+TIE-2+ endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31+CD144+vWF+I-CAM1+ endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs

Antibody Screening Using a Human iPSC-based Blood-Brain Barrier Model Identifies Antibodies that Accumulate in the CNS

Drug delivery across the blood-brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood-borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor-mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off-target effects, driving the search for new and potentially improved brain targeting reagent-RMT pairs. To this end, we deployed human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells as a model BBB substrate on which to mine for new RMT-targeting antibody pairs. A nonimmune, human single-chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC-derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1-scFv, exhibiting a 26-fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1-scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor-mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain

Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection

Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs

Blood–Brain Barrier Modulation to Improve Glioma Drug Delivery

The blood–brain barrier (BBB) is formed by brain microvascular endothelial cells that are sealed by tight junctions, making it a significant obstacle for most brain therapeutics. The poor BBB penetration of newly developed therapeutics has therefore played a major role in limiting their clinical success. A particularly challenging therapeutic target is glioma, which is the most frequently occurring malignant brain tumor. Thus, to enhance therapeutic uptake in tumors, researchers have been developing strategies to modulate BBB permeability. However, most conventional BBB opening strategies are difficult to apply in the clinical setting due to their broad, non-specific modulation of the BBB, which can result in damage to normal brain tissue. In this review, we have summarized strategies that could potentially be used to selectively and efficiently modulate the tumor BBB for more effective glioma treatment

Past and current perspectives in modeling bacteria and blood–brain barrier interactions

The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future

Expert Review Blood-Brain Barrier Transport of Therapeutics via Receptor-Mediation

Abstract. Drug delivery to the brain is hindered by the presence of the blood-brain barrier (BBB). Although the BBB restricts the passage of many substances, it is actually selectively permeable to nutrients necessary for healthy brain function. To accomplish the task of nutrient transport, the brain endothelium is endowed with a diverse collection of molecular transport systems. One such class of transport system, known as a receptor-mediated transcytosis (RMT), employs the vesicular trafficking machinery of the endothelium to transport substrates between blood and brain. If appropriately targeted, RMT systems can also be used to shuttle a wide range of therapeutics into the brain in a noninvasive manner. Over the last decade, there have been significant developments in the arena of RMT-based brain drug transport, and this review will focus on those approaches that have been validated in an in vivo setting

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

The yeast Saccharomyces cerevisiae is an attractive host for the production of heterologous proteins. However, low-yield production of many proteins (from micrograms to milligrams/liter) leaves considerable room for optimization. By engineering the yeast cell via traceable genome-wide libraries, genes that can enhance protein expression level because of their roles in protein transcription, translation, folding, and trafficking processes can be readily identified. This report details a novel approach that combines yeast cDNA overexpression libraries with yeast surface display to allow the rapid flow cytometric screening of engineered yeast for gene products that improve the display of heterologous proteins. After optimization of the screening conditions, a genome-wide scan yielded five yeast gene products that promoted increased display levels of a single-chain T-cell receptor (scTCR). The display-enhancing genes included those coding for cell wall proteins (CCW12, CWP2, and SED1), a ribosomal subunit protein (RPP0), and an endoplasmic reticulum-resident protein (ERO1). Under the premise that yeast surface display levels could be used as a predictor of secretion efficiency, each display-enhancing gene product was tested for its ability to affect secretion levels of multiple scTCR and single-chain antibodies (scFv). All of the selected yeast gene products were shown to promote increased secretion of active protein (1.5-fold to 7.9-fold), with CCW12 and ERO1 being the most generalizable enhancers of scFv/scTCR secretion

Blood-brain barrier transport of therapeutics via receptormediation

Abstract Drug delivery to the brain is hindered by the presence of the blood-brain barrier (BBB). Although the BBB restricts the passage of many substances, it is actually selectively permeable to nutrients necessary for healthy brain function. To accomplish the task of nutrient transport, the brain endothelium is endowed with a diverse collection of molecular transport systems. One such class of transport system, known as a receptor-mediated transcytosis (RMT), employs the vesicular trafficking machinery of the endothelium to transport substrates between blood and brain. If appropriately targeted, RMT systems can also be used to shuttle a wide range of therapeutics into the brain in a noninvasive manner. Over the last decade, there have been significant developments in the arena of RMT-based brain drug transport, and this review will focus on those approaches that have been validated in an in vivo setting