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.
- Award ID(s):
- Publication Date:
- NSF-PAR ID:
- Journal Name:
- Scientific Reports
- Nature Publishing Group
- Sponsoring Org:
- National Science Foundation
More Like this
Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier DysfunctionThe blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics ofmore »
Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-Culture From Human Stem CellsBackground: Recently, the in vitro blood–brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications they have with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB, and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry, and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF, and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood–brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for iPCs and iECs in the transwell system and 3D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, and brain-specific genes FOXF2 , ABCC9 , KCNJ8 , and ZIC1 . The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, and BBB-associated genesmore »
Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues
Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier genemore »
Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC)
in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a−/−mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assay in vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.
Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn
The blood-brain barrier (BBB) hinders the brain delivery of therapeutic immunoglobulin γ (IgG) antibodies. Evidence suggests that IgG-specific processing occurs within the endothelium of the BBB, but any influence on transcytosis remains unclear. Here, involvement of the neonatal Fc receptor (FcRn), which mediates IgG recycling and transcytosis in peripheral endothelium, was investigated by evaluating the transcytosis of IgGs with native or reduced FcRn engagement across human induced pluripotent stem cell-derived brain endothelial-like cells. Despite differential trafficking, the permeability of all tested IgGs were comparable and remained constant irrespective of concentration or competition with excess IgG, suggesting IgG transcytosis occurs nonspecifically and originates from fluid-phase endocytosis. Comparison with the receptor-enhanced permeability of transferrin indicates that the phenomena observed for IgG is ubiquitous for most macromolecules. However, increased permeability was observed for macromolecules with biophysical properties known to engage alternative endocytosis mechanisms, highlighting the importance of biophysical characterizations in assessing transcytosis mechanisms.