Defined Differentiation of Human Pluripotent Stem Cells to Brain Microvascular Endothelial-Like Cells for Modeling the Blood-Brain Barrier.

The blood-brain barrier (BBB) comprises brain microvascular endothelial cells (BMECs) that form a high-resistance cellular interface that separates the blood compartment from the brain parenchyma. An intact BBB is pivotal to maintaining brain homeostasis but also impedes the entry of neurotherapeutics. There are limited options for human-specific BBB permeability testing, however. Human pluripotent stem cell models offer a powerful tool for dissecting components of this barrier in vitro, including understanding mechanisms of BBB function, and developing strategies to improve the permeability of molecular and cellular therapeutics targeting the brain. Here, we provide a detailed, step-by-step protocol for differentiation of human pluripotent stem cells (hPSCs) to cells exhibiting key characteristics of BMECs, including paracellular and transcellular transport resistance and transporter function that enable modeling the human BBB.

Evidence that the catalytic mechanism of heme a synthase involves the formation of a carbocation stabilized by a conserved glutamate.

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa3-type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site. Previous reports indicate that the C-terminal site is occupied by a heme b cofactor. The N-terminal site may function as the substrate (heme o) binding site, although this has not been confirmed experimentally. Here, we assess the role of conserved residues from the N- and C-terminal heme-binding sites in HAS from prokaryotic (Shewanella oneidensis) and eukaryotic (Saccharomyces cerevisiae) species - SoHAS/CtaA and ScHAS/Cox15, respectively. A glutamate within the N-terminal site is found to be critical for activity in both types of HAS, consistent with the hypothesis that a carbocation forms transiently during catalysis. In contrast, the residue occupying the analogous C-terminal position is dispensable for enzyme activity. In SoHAS, the C-terminal heme ligands are critical for stability, while in ScHAS, substitutions in either heme-binding site have little effect on global structure. In both species, in vivo accumulation of heme o requires the presence of an inactive HAS variant, highlighting a potential regulatory role for HAS in heme o biosynthesis.

Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability.

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1-2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

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

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.

Integrative analysis of the human brain mural cell transcriptome.

Brain mural cells, including pericytes and vascular smooth muscle cells, are important for vascular development, blood-brain barrier function, and neurovascular coupling, but the molecular characteristics of human brain mural cells are incompletely characterized. Single cell RNA-sequencing (scRNA-seq) is increasingly being applied to assess cellular diversity in the human brain, but the scarcity of mural cells in whole brain samples has limited their molecular profiling. Here, we leverage the combined power of multiple independent human brain scRNA-seq datasets to build a transcriptomic database of human brain mural cells. We use this combined dataset to determine human-mouse species differences in mural cell transcriptomes, culture-induced dedifferentiation of human brain pericytes, and human mural cell organotypicity, with several key findings validated by RNA fluorescence in situ hybridization. Together, this work improves knowledge regarding the molecular constituents of human brain mural cells, serves as a resource for hypothesis generation in understanding brain mural cell function, and will facilitate comparative evaluation of animal and in vitro models.

Neuronal Activity Regulates Blood-Brain Barrier Efflux Transport through Endothelial Circadian Genes.

The blood vessels in the central nervous system (CNS) have a series of unique properties, termed the blood-brain barrier (BBB), which stringently regulate the entry of molecules into the brain, thus maintaining proper brain homeostasis. We sought to understand whether neuronal activity could regulate BBB properties. Using both chemogenetics and a volitional behavior paradigm, we identified a core set of brain endothelial genes whose expression is regulated by neuronal activity. In particular, neuronal activity regulates BBB efflux transporter expression and function, which is critical for excluding many small lipophilic molecules from the brain parenchyma. Furthermore, we found that neuronal activity regulates the expression of circadian clock genes within brain endothelial cells, which in turn mediate the activity-dependent control of BBB efflux transport. These results have important clinical implications for CNS drug delivery and clearance of CNS waste products, including Aß, and for understanding how neuronal activity can modulate diurnal processes.

Transcriptomic comparison of human and mouse brain microvessels.

The brain vasculature maintains brain homeostasis by tightly regulating ionic, molecular, and cellular transport between the blood and the brain parenchyma. These blood-brain barrier (BBB) properties are impediments to brain drug delivery, and brain vascular dysfunction accompanies many neurological disorders. The molecular constituents of brain microvascular endothelial cells (BMECs) and pericytes, which share a basement membrane and comprise the microvessel structure, remain incompletely characterized, particularly in humans. To improve the molecular database of these cell types, we performed RNA sequencing on brain microvessel preparations isolated from snap-frozen human and mouse tissues by laser capture microdissection (LCM). The resulting transcriptome datasets from LCM microvessels were enriched in known brain endothelial and pericyte markers, and global comparison identified previously unknown microvessel-enriched genes. We used these datasets to identify mouse-human species differences in microvessel-associated gene expression that may have relevance to BBB regulation and drug delivery. Further, by comparison of human LCM microvessel data with existing human BMEC transcriptomic datasets, we identified novel putative markers of human brain pericytes. Together, these data improve the molecular definition of BMECs and brain pericytes, and are a resource for rational development of new brain-penetrant therapeutics and for advancing understanding of brain vascular function and dysfunction.

Human pluripotent stem cell-derived cardiac stromal cells and their applications in regenerative medicine.

Coronary heart disease is one of the leading causes of death in the United States. Recent advances in stem cell biology have led to the development and engineering of human pluripotent stem cell (hPSC)-derived cardiac cells and tissues for application in cellular therapy and cardiotoxicity studies. Initial studies in this area have largely focused on improving differentiation efficiency and maturation states of cardiomyocytes. However, other cell types in the heart, including endothelial and stromal cells, play crucial roles in cardiac development, injury response, and cardiomyocyte function. This review discusses recent advances in differentiation of hPSCs to cardiac stromal cells, identification and classification of cardiac stromal cell types, and application of hPSC-derived cardiac stromal cells and tissues containing these cells in regenerative and drug development applications.

Bioremediation of Hexanoic Acid and Phenanthrene in Oil Sands Tailings by the Microbial Consortium BioTiger™.

Polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are toxic contaminants of environmental concern found in process water and mature fine tailings, or tailings, from the oil sands industry. BioTiger™, a patented microbial consortium of twelve natural environmental isolates, was found to cometabolically biodegrade the NA hexanoic acid and the PAH phenanthrene in the presence of tailings. Hexanoamide was found to be produced and consumed during cometabolism of hexanoic acid. Mechanistic analysis demonstrated three of the BioTiger™ strains generated biosurfactants with the bacterial adhesion to hydrocarbons assay, seven with the methylene blue active substances assay, and nine with a hemolysis assay. Serial transfers of the BioTiger™ consortium demonstrated the stability of hexanoic acid degradation over several generations. The results demonstrate that BioTiger™ cometabolically biodegrades combinations of phenanthrene and hexanoic acid in tailings. This work reveals the potential for in situ bioremediation of tailings with this natural microbial consortium.