RESUMEN
Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3. Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRß, TBX2, FOXS1, KCNJ8, SLC6A12, and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional KATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.
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Células Endoteliales , Células Madre Pluripotentes , Humanos , Células Endoteliales/metabolismo , Cresta Neural/metabolismo , Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismo , Encéfalo/metabolismo , Factores de Transcripción Forkhead/metabolismoRESUMEN
Mural cells directly contact macrophages in the dural layer of the meninges to suppress pro-inflammatory phenotypes, including antigen presentation and lymphocyte differentiation. These mechanisms represent new targets for modulating CNS immune surveillance and pathological inflammation (Min et al. 2024. J. Exp. Med.https://doi.org/10.1084/jem.20230326).
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Antiinflamatorios , Pintura , Humanos , Inflamación , Presentación de Antígeno , Vigilancia InmunológicaRESUMEN
The blood-brain barrier (BBB) hinders therapeutic delivery to the central nervous system (CNS), thereby impeding the development of therapies for brain injury and disease. Receptor-mediated transcytosis (RMT) systems are a promising way to shuttle a targeted therapeutic into the brain. Here, we developed and evaluated an RMT antibody-targeted liposomal system. A previously identified antibody, scFv46.1, that binds to the human and murine BBB and can pass through the murine BBB by transcytosis after intravenous injection was used to decorate the surface of liposomes. Using an in vitro BBB model, we demonstrated the cellular uptake of scFv46.1-modified liposomes (46.1-Lipo). Next, the biodistribution and brain uptake capacity of 46.1-targeted liposomes were assessed after intravenous administration. Our results showed that 46.1-Lipo can lead to increased brain accumulation through targeting of the brain vasculature. Initial rate pharmacokinetic experiments and biodistribution analyses indicated that 46.1-Lipo loaded with pralidoxime exhibited a 10-fold increase in brain accumulation compared with a mock-targeted liposomal group, and this increased accumulation was brain-specific. These studies indicate the potential of this 46.1-Lipo system as a synthetic vehicle for the targeted transport of therapeutic molecules into the CNS.
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Barrera Hematoencefálica , Liposomas , Animales , Anticuerpos , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Humanos , Ratones , Distribución TisularRESUMEN
Blood-brain barrier (BBB) breakdown and immune cell infiltration into the CNS are early hallmarks of multiple sclerosis (MS). The mechanisms leading to BBB dysfunction are incompletely understood and generally thought to be a consequence of neuroinflammation. Here, we have challenged this view and asked if intrinsic alterations in the BBB of MS patients contribute to MS pathogenesis. To this end, we made use of human induced pluripotent stem cells derived from healthy controls and MS patients and differentiated them into brain microvascular endothelial cell (BMEC)-like cells as in vitro model of the BBB. MS-derived BMEC-like cells showed impaired junctional integrity, barrier properties and efflux pump activity when compared to healthy controls. Also, MS-derived BMEC-like cells displayed an inflammatory phenotype with increased adhesion molecule expression and immune cell interactions. Activation of Wnt/ß-catenin signalling in MS-derived endothelial progenitor cells enhanced barrier characteristics and reduced the inflammatory phenotype. Our study provides evidence for an intrinsic impairment of BBB function in MS patients that can be modelled in vitro. Human iPSC-derived BMEC-like cells are thus suitable to explore the molecular underpinnings of BBB dysfunction in MS and will assist in the identification of potential novel therapeutic targets for BBB stabilization.
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Células Madre Pluripotentes Inducidas , Esclerosis Múltiple , Humanos , Barrera Hematoencefálica/patología , Esclerosis Múltiple/patología , Células Cultivadas , Células Madre Pluripotentes Inducidas/metabolismo , Encéfalo/fisiologíaRESUMEN
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.
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Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Células Madre Pluripotentes/metabolismo , Vía de Señalización Wnt/genética , Línea Celular , HumanosRESUMEN
Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.
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Astrocitos/virología , Encéfalo/virología , COVID-19/patología , Pericitos/virología , Tropismo Viral/fisiología , Astrocitos/citología , Encéfalo/patología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Interferón Tipo I/inmunología , SARS-CoV-2 , Replicación Viral/fisiologíaRESUMEN
We describe the extended endothelial cell culture method (EECM) for the differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cell (BMEC)-like cells. EECM-BMEC-like cells resemble primary human BMECs in morphology, molecular junctional architecture, and diffusion barrier characteristics. A mature immune phenotype with proper endothelial adhesion molecule expression makes this model distinct from any other hPSC-derived in vitro blood-brain barrier (BBB) model and suitable to study immune cell migration across the BBB in a disease relevant and personalized fashion. For complete details on the use and execution of this protocol, please refer to Lian et al. (2014), Nishihara et al. (2020a).
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Encéfalo/irrigación sanguínea , Diferenciación Celular , Endotelio Vascular/citología , Microvasos/citología , Células Madre Pluripotentes/citología , Células Cultivadas , Endotelio Vascular/inmunología , Humanos , Microvasos/inmunología , Células Madre Pluripotentes/inmunologíaRESUMEN
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.
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Encéfalo/irrigación sanguínea , Encéfalo/citología , Miocitos del Músculo Liso/metabolismo , Pericitos/metabolismo , Transcriptoma/genética , Animales , Barrera Hematoencefálica/fisiología , Humanos , Hibridación Fluorescente in Situ/métodos , Medicina Integrativa/métodos , Ratones , Acoplamiento Neurovascular/fisiología , ARN Citoplasmático Pequeño/genética , RNA-Seq/métodosRESUMEN
Clinical evidence suggests the central nervous system (CNS) is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although mechanisms remain unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points 1 . Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Prior to infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model 2 that supports SARS-CoV-2 entry and replication in neural tissue, and PCCOs serve as an experimental model for neural infection.
RESUMEN
Clinical evidence suggests the central nervous system (CNS) is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although mechanisms remain unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points 1 . Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Prior to infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model 2 that supports SARS-CoV-2 entry and replication in neural tissue, and PCCOs serve as an experimental model for neural infection.
RESUMEN
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.
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Pericitos , Células Madre Pluripotentes , Diferenciación Celular , Células Endoteliales , Humanos , Cresta NeuralRESUMEN
Endothelial cells (EC) in vivo are continuously exposed to a mechanical microenvironment from blood flow, and fluidic shear stress plays an important role in EC behavior. New approaches to generate physiologically and pathologically relevant pulsatile flows are needed to understand EC behavior under different shear stress regimes. Here, we demonstrate an adaptable pump (Adapt-Pump) platform for generating pulsatile flows from human pluripotent stem cell-derived cardiac spheroids (CS) via quantitative imaging-based signal transduction. Pulsatile flows generated from the Adapt-Pump system can recapitulate unique CS contraction characteristics, accurately model responses to clinically relevant drugs, and simulate CS contraction changes in response to fluidic mechanical stimulation. We discovered that ECs differentiated under a long QT syndrome derived pathological pulsatile flow exhibit abnormal EC monolayer organization. This Adapt-Pump platform provides a powerful tool for modeling the cardiovascular system and improving our understanding of EC behavior under different mechanical microenvironments.
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Células Endoteliales , Miocitos Cardíacos , Humanos , Flujo Pulsátil , Transducción de Señal , Células Madre , Estrés MecánicoRESUMEN
Human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC-derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)-like cells with good barrier properties and mature tight junctions. Importantly, EECM-BMEC-like cells exhibited constitutive cell surface expression of ICAM-1, ICAM-2, and E-selectin. Pro-inflammatory cytokine stimulation increased the cell surface expression of ICAM-1 and induced cell surface expression of P-selectin and VCAM-1. Co-culture of EECM-BMEC-like cells with hiPSC-derived smooth muscle-like cells or their conditioned medium further increased the induction of VCAM-1. Functional expression of endothelial ICAM-1 and VCAM-1 was confirmed by T-cell interaction with EECM-BMEC-like cells. Taken together, we introduce the first hiPSC-derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.
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Barrera Hematoencefálica/diagnóstico por imagen , Comunicación Celular/fisiología , Células Madre Pluripotentes Inducidas/citología , Adulto , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Moléculas de Adhesión Celular/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
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.
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Barrera Hematoencefálica/metabolismo , Células Endoteliales , Células Madre Pluripotentes Inducidas , Anticuerpos de Cadena Única/farmacocinética , Transcitosis , Animales , Barrera Hematoencefálica/citología , Células Cultivadas , Portadores de Fármacos/farmacocinética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Biblioteca de PéptidosRESUMEN
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.
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Barrera Hematoencefálica/metabolismo , Bases de Datos de Ácidos Nucleicos , Células Endoteliales/metabolismo , Microvasos/metabolismo , Pericitos/metabolismo , RNA-Seq , Transcriptoma , Animales , Femenino , Humanos , Masculino , Ratones , Especificidad de la EspecieRESUMEN
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).
RESUMEN
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.
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Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Técnicas de Cocultivo/métodos , Células Endoteliales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Neuronas/fisiología , Pericitos/fisiología , Células 3T3 , Animales , Diferenciación Celular , Humanos , Ratones , Microvasos/fisiología , Ocludina/metabolismo , Uniones Estrechas/fisiologíaRESUMEN
Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood-borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood-brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/microbiología , Infecciones Estreptocócicas/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Streptococcus agalactiaeRESUMEN
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.