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1. Automated patterning of human brain endothelial cells on microstructures using a microfluidic manufacturing approach: An in vitro study

   https://doi.org/10.1002/amp2.10169  

   Aykar, Saurabh S. ; Ouedraogo, Lionel J. ; Petersen, Isaac S. ; Trznadel, Mychal J. ; Alimoradi, Nima ; Montazami, Reza ; Brockman, Amanda L. ; Hashemi, Nicole N. ( August 2023 , Journal of Advanced Manufacturing and Processing)

   Barrier functionality of the blood–brain barrier (BBB) is provided by the tight junctions formed by a monolayer of the human brain endothelial cells (HBECs) internally around the blood capillaries. To mimic such barrier functionality in vitro, replicating the hollow tubular structure of the BBB along with the HBECs monolayer on its inner surface is crucial. Here, we developed a microfluidic manufacturing technique to pattern the HBECs on the surface of alginate‐based microstructures. The HBECs were seeded on the inner surface of these hollow microfibers using a custom‐built microfluidic device. The seeded HBECs were monitored for 9 days after manufacturing and cultured to form a monolayer on the inner surface of the alginate hollow microfibers in the maintenance media. A higher cell seeding density of 217 cells/mm length of the hollow microfiber was obtained using our microfluidic technique. Moreover, high accuracy of around 96% was obtained in seeding cells on the inner surface of alginate hollow microfibers. The microfluidic method illustrated in this study could be extrapolated to obtain a monolayer of different cell types on the inner surface of alginate hollow microfibers with cell‐compatible ECM matrix proteins. Furthermore, it will enable us to manufacture a range of microvascular systems in vitro by closely replicating the structural attributes of the native structure.

    

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2. Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-Culture From Human Stem Cells

   https://doi.org/10.3389/fceng.2022.927188  

   Marzano, Mark ; Chen, Xingchi ; Russell, Teal A. ; Medina, Angelica ; Wang, Zizheng ; Hua, Timothy ; Zeng, Changchun ; Wang, Xueju ; Sang, Qing-Xiang ; Tang, Hengli ; et al ( July 2022 , Frontiers in Chemical Engineering)

   Background: 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 genes BRCP , GLUT-1 , PGP , ABCC1 , OCLN , and SLC2A1 . The co-culture of the two cell types responded to the stimulation of amyloid β42 oligomers by the upregulation of the expression of TNFa , IL6 , NFKB , Casp3 , SOD2 , and TP53 . The co-culture also showed the property of trans-endothelial electrical resistance. The proof of concept vascularization strategy was demonstrated in a 3D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties, and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening. 

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3. MiR-34a Interacts with Cytochrome c and Shapes Stroke Outcomes

   https://doi.org/10.1038/s41598-020-59997-y  

   Hu, Heng ; Hone, Emily A. ; Provencher, Edward A. P. ; Sprowls, Samuel A. ; Farooqi, Imran ; Corbin, Deborah R. ; Sarkar, Saumyendra N. ; Hollander, John M. ; Lockman, Paul R. ; Simpkins, James W. ; et al ( February 2020 , Scientific Reports)

   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 assayin 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.

    

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4. Differentiation of Brain Pericyte‐Like Cells from Human Pluripotent Stem Cell−Derived Neural Crest

   https://doi.org/10.1002/cpz1.21  

   Gastfriend, Benjamin D. ; Stebbins, Matthew J. ; Du, Feifan ; Shusta, Eric V. ; Palecek, Sean P. ( January 2021 , Current Protocols)

   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⁺PDGFRβ⁺αSMA^lowbrain 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.

   This article was corrected on 18 July 2022. See the end of the full text for details.

   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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5. Imaging effects of hyperosmolality on individual tricellular junctions

   https://doi.org/10.1039/c9sc05114g  

   Huang, Kaixiang ; Zhou, Lushan ; Alanis, Kristen ; Hou, Jianghui ; Baker, Lane A. ( February 2020 , Chemical Science)

   The use of hyperosmolar agents (osmotherapy) has been a major treatment for intracranial hypertension, which occurs frequently in brain diseases or trauma. However, side-effects of osmotherapy on the brain, especially on the blood–brain barrier (BBB) are still not fully understood. Hyperosmolar conditions, termed hyperosmolality here, are known to transiently disrupt the tight junctions (TJs) at the endothelium of the BBB resulting in loss of BBB function. Present techniques for evaluation of BBB transport typically reveal aggregated responses from the entirety of BBB transport components, with little or no opportunity to evaluate heterogeneity present in the system. In this study, we utilized potentiometric-scanning ion conductance microscopy (P-SICM) to acquire nanometer-scale conductance maps of Madin–Darby Canine Kidney strain II (MDCKII) cells under hyperosmolality, from which two types of TJs, bicellular tight junctions (bTJs) and tricellular tight junctions (tTJs), can be visualized and differentiated. We discovered that hyperosmolality leads to increased conductance at tTJs without significant alteration in conductance at bTJs. To quantify this effect, an automated computer vision algorithm was designed to extract and calculate conductance components at both tTJs and bTJs. Additionally, lowering Ca 2+ concentration in the bath facilitates tTJ disruption under hyperosmolality. Strengthening tTJ structure by overexpressing immunoglobulin-like domain-containing receptor 1 (ILDR1) protein abrogates the effect of hyperosmolality. We posit that osmotic stress physically disrupts tTJ structure, as evidenced by super-resolution microscopy. Findings from this study not only provide a high-resolution view of TJ structure and function, but also can inform current osmotherapy and drug delivery strategies for brain diseases. 

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