An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843.

Differentiation and characterization of human pluripotent stem cell-derived brain microvascular 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 human 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.

The damaging effects of short chain fatty acids on Escherichia coli membranes.

Carboxylic acids are an attractive biorenewable chemical. However, like many other fermentatively produced compounds, they are inhibitory to the biocatalyst. An understanding of the mechanism of toxicity can aid in mitigating this problem. Here, we show that hexanoic and octanoic acids are completely inhibitory to Escherichia coli MG1655 in minimal medium at a concentration of 40 mM, while decanoic acid was inhibitory at 20 mM. This growth inhibition is pH-dependent and is accompanied by a significant change in the fluorescence polarization (fluidity) and integrity. This inhibition and sensitivity to membrane fluidization, but not to damage of membrane integrity, can be at least partially mitigated during short-term adaptation to octanoic acid. This short-term adaptation was accompanied by a change in membrane lipid composition and a decrease in cell surface hydrophobicity. Specifically, the saturated/unsaturated lipid ratio decreased and the average lipid length increased. A fatty acid-producing strain exhibited an increase in membrane leakage as the product titer increased, but no change in membrane fluidity. These results highlight the importance of the cell membrane as a target for future metabolic engineering efforts for enabling resistance and tolerance of desirable biorenewable compounds, such as carboxylic acids. Knowledge of these effects can help in the engineering of robust biocatalysts for biorenewable chemicals production.

Differentiation of Brain Pericyte-Like Cells from Human Pluripotent Stem Cell-Derived Neural Crest.

Brain pericytes regulate diverse aspects of neurovascular development and function, including blood-brain barrier (BBB) induction and maintenance. Primary brain pericytes have been widely employed in coculture-based in vitro models of the BBB, and a method to generate brain pericytes from human pluripotent stem cells (hPSCs) could provide a renewable, genetically tractable source of cells for BBB modeling and studying pericyte roles in development and disease. Here, we describe a protocol to differentiate hPSCs to NG2+ PDGFRß+ αSMAlow brain pericyte-like cells in 22-25 days through a p75-NGFR+ HNK-1+ neural crest intermediate, which mimics the developmental origin of forebrain pericytes. The resulting brain pericyte-like cells have molecular and functional attributes of brain pericytes. We also provide protocols for maintenance, cryopreservation, and recovery of the neural crest intermediate, and for molecular and functional characterization of the resulting cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of hPSCs to neural crest Basic Protocol 2: Differentiation of neural crest to brain pericyte-like cells Support Protocol 1: Flow cytometry analysis of neural crest cells Support Protocol 2: Maintenance, cryopreservation, and recovery of neural crest cells Support Protocol 3: Molecular characterization of brain pericyte-like cells Support Protocol 4: Cord formation assay with endothelial cells and brain pericyte-like cells.

Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration.

Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.

Sonic Hedgehog Signaling in Cranial Neural Crest Cells Regulates Microvascular Morphogenesis in Facial Development.

Sonic hedgehog (Shh) pathway disruption causes craniofacial malformations including orofacial clefts (OFCs) of the lip and palate. In normal craniofacial morphogenesis, Shh signals to multipotent cranial neural crest cells (cNCCs) and was recently discovered to regulate the angiogenic transcriptome, including expression markers of perivascular cells and pericytes. The mural cells of microvasculature, pericytes in the brain and face differentiate from cNCCs, but their role in facial development is not known. Here, we examined microvascular morphogenesis in a mouse model of Shh pathway antagonist-induced cleft lip and the impact of cNCC-specific Shh pathway activation in a cNCC-endothelial cell co-culture system. During cleft pathogenesis in vivo, disrupted microvascular morphogenesis localized with attenuated tissue outgrowth in the medial nasal processes that form the upper lip. In vitro, we found that human umbilical vein endothelial cell (HUVEC) cord formation was not affected by direct Shh pathway perturbation. However, in a co-culture system in which cNCCs directly interact with endothelial cells, cNCC-autonomous Shh pathway activity significantly prolonged endothelial cord network stability. Taken together, these findings support the premise that Shh pathway activation in cNCCs promotes pericyte-like function and microvascular stability. In addition to suggesting a previously unrecognized role for Shh signaling in facial development, these studies also identify perivascular differentiation and microvascular morphogenesis as new focuses for understanding normal and abnormal craniofacial development.

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

BACKGROUND: Brain microvascular endothelial cells (BMECs) astrocytes, neurons, and pericytes form the neurovascular unit (NVU). Interactions with NVU cells endow BMECs with extremely tight barriers via the expression of tight junction proteins, a host of active efflux and nutrient transporters, and reduced transcellular transport. To recreate the BMEC-enhancing functions of NVU cells, we combined BMECs, astrocytes, neurons, and brain pericyte-like cells. METHODS: BMECs, neurons, astrocytes, and brain like pericytes were differentiated from human induced pluripotent stem cells (iPSCs) and placed in a Transwell-type NVU model. BMECs were placed in co-culture with neurons, astrocytes, and/or pericytes alone or in varying combinations and critical barrier properties were monitored. RESULTS: Co-culture with pericytes followed by a mixture of neurons and astrocytes (1:3) induced the greatest barrier tightening in BMECs, supported by a significant increase in junctional localization of occludin. BMECs also expressed active P-glycoprotein (PGP) efflux transporters under baseline BMEC monoculture conditions and continued to express baseline active PGP efflux transporters regardless of co-culture conditions. Finally, brain-like pericyte co-culture significantly reduced the rate of non-specific transcytosis across BMECs. CONCLUSIONS: Importantly, each cell type in the NVU model was differentiated from the same donor iPSC source, yielding an isogenic model that could prove enabling for enhanced personalized modeling of the NVU in human health and disease.

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

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).

Human pluripotent stem cell-derived brain pericyte-like cells induce blood-brain barrier properties.

Brain pericytes play important roles in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system disorders. While human pluripotent stem cells (hPSCs) have been used to model other NVU cell types, including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, hPSC-derived brain pericyte-like cells have not been integrated into these models. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from hPSCs and subsequently differentiated NCSCs to brain pericyte-like cells. These cells closely resembled primary human brain pericytes and self-assembled with endothelial cells. The brain pericyte-like cells induced blood-brain barrier properties in BMECs, including barrier enhancement and reduced transcytosis. Last, brain pericyte-like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human model that should prove useful for the study of the NVU.

Activation of RARα, RARγ, or RXRα Increases Barrier Tightness in Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells.

The blood-brain barrier (BBB) is critical to central nervous system (CNS) health. Brain microvascular endothelial cells (BMECs) are often used as in vitro BBB models for studying BBB dysfunction and therapeutic screening applications. Human pluripotent stem cells (hPSCs) can be differentiated to cells having key BMEC barrier and transporter properties, offering a renewable, scalable source of human BMECs. hPSC-derived BMECs have previously been shown to respond to all-trans retinoic acid (RA), and the goal of this study was to identify the stages at which differentiating human induced pluripotent stem cells (iPSCs) respond to activation of RA receptors (RARs) to impart BBB phenotypes. Here the authors identified that RA application to iPSC-derived BMECs at days 6-8 of differentiation led to a substantial elevation in transendothelial electrical resistance and induction of VE-cadherin expression. Specific RAR agonists identified RARα, RARγ, and RXRα as receptors capable of inducing barrier phenotypes. Moreover, RAR/RXRα costimulation elevated VE-cadherin expression and improved barrier fidelity to levels that recapitulated the effects of RA. This study elucidates the roles of RA signaling in iPSC-derived BMEC differentiation, and identifies directed agonist approaches that can improve BMEC fidelity for drug screening studies while also distinguishing potential nuclear receptor targets to explore in BBB dysfunction and therapy.

Modeling Group B Streptococcus and Blood-Brain Barrier Interaction by Using Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells.

Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs after bacteria interact with and penetrate the blood-brain barrier (BBB). The BBB is comprised of highly specialized brain microvascular endothelial cells (BMECs) that function to separate the circulation from the CNS and act as a formidable barrier for toxins and pathogens. Certain bacteria, such as Streptococcus agalactiae (group B Streptococcus [GBS]), possess the ability to interact with and penetrate the BBB to cause meningitis. Modeling bacterial interaction with the BBB in vitro has been limited to primary and immortalized BMEC culture. While useful, these cells often do not retain BBB-like properties, and human primary cells have limited availability. Recently, a human induced pluripotent stem cell (iPSC)-derived BMEC model has been established that is readily renewable and retains key BBB phenotypes. Here, we sought to evaluate whether the iPSC-derived BMECs were appropriate for modeling bacterial interaction with the BBB. Using GBS as a model meningeal pathogen, we demonstrate that wild-type GBS adhered to, invaded, and activated the iPSC-derived BMECs, while GBS mutants known to have diminished BBB interaction were attenuated in the iPSC-derived model. Furthermore, bacterial infection resulted in the disruption of tight junction components ZO-1, occludin, and claudin-5. Thus, we show for the first time that the iPSC-derived BBB model can be utilized to study BBB interaction with a bacterial CNS pathogen. IMPORTANCE Here for the first time, human iPSC-derived BMECs were used to model bacterial interaction with the BBB. Unlike models previously used to study these interactions, iPSC-derived BMECs possess robust BBB properties, such as the expression of complex tight junctions that are key components for the investigation of bacterial effects on the BBB. Here, we demonstrated that GBS interacts with the iPSC-derived BMECs and specifically disrupts these tight junctions. Thus, using this BBB model may allow researchers to uncover novel mechanisms of BBB disruption during meningitis that are inaccessible to immortalized or primary cell models that lack substantial tight junctions.

In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use.

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.