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1. Inflammatory Response and Exosome Biogenesis of Choroid Plexus Organoids Derived from Human Pluripotent Stem Cells

   https://doi.org/10.3390/ijms24087660  

   Muok, Laureana ; Liu, Chang ; Chen, Xingchi ; Esmonde, Colin ; Arthur, Peggy ; Wang, Xueju ; Singh, Mandip ; Driscoll, Tristan ; Li, Yan ( April 2023 , International Journal of Molecular Sciences)

   The choroid plexus (ChP) is a complex structure in the human brain that is responsible for the secretion of cerebrospinal fluid (CSF) and forming the blood–CSF barrier (B-CSF-B). Human-induced pluripotent stem cells (hiPSCs) have shown promising results in the formation of brain organoids in vitro; however, very few studies to date have generated ChP organoids. In particular, no study has assessed the inflammatory response and the extracellular vesicle (EV) biogenesis of hiPSC-derived ChP organoids. In this study, the impacts of Wnt signaling on the inflammatory response and EV biogenesis of ChP organoids derived from hiPSCs was investigated. During days 10–15, bone morphogenetic protein 4 was added along with (+/−) CHIR99021 (CHIR, a small molecule GSK-3β inhibitor that acts as a Wnt agonist). At day 30, the ChP organoids were characterized by immunocytochemistry and flow cytometry for TTR (~72%) and CLIC6 (~20%) expression. Compared to the −CHIR group, the +CHIR group showed an upregulation of 6 out of 10 tested ChP genes, including CLIC6 (2-fold), PLEC (4-fold), PLTP (2–4-fold), DCN (~7-fold), DLK1 (2–4-fold), and AQP1 (1.4-fold), and a downregulation of TTR (0.1-fold), IGFBP7 (0.8-fold), MSX1 (0.4-fold), and LUM (0.2–0.4-fold). When exposed to amyloid beta 42 oligomers, the +CHIR group had a more sensitive response as evidenced by the upregulation of inflammation-related genes such as TNFα, IL-6, and MMP2/9 when compared to the −CHIR group. Developmentally, the EV biogenesis markers of ChP organoids showed an increase over time from day 19 to day 38. This study is significant in that it provides a model of the human B-CSF-B and ChP tissue for the purpose of drug screening and designing drug delivery systems to treat neurological disorders such as Alzheimer’s disease and ischemic stroke. 

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2. A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood‐Brain Barrier Mouse Model

   https://doi.org/10.1002/cpim.101  

   Walter, Fruzsina R. ; Gilpin, Trey E. ; Herbath, Melinda ; Deli, Maria A. ; Sandor, Matyas ; Fabry, Zsuzsanna ( July 2020 , Current Protocols in Immunology)

   In vitro culture models of the blood‐brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to testMycobacterium tuberculosis(Mtb) dissemination across brain endothelial cells. One‐third of the global population is infected with Mtb, and in 1%‐2% of cases bacteria invade the CNS through a largely unknown process. The “Trojan horse” theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB‐granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells

   Basic Protocol 2: Isolation of primary mouse bone marrow–derived dendritic cells

   Support Protocol 1: Validation of dendritic cell purity by flow cytometry

   Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells

   Support Protocol 2: Isolation of primary mouse spleen cells

   Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells

   Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load

   Support Protocol 4: In vivo and in vitro infection with mycobacteria

   Basic Protocol 5: Assembly of the BBB co‐culture model

   Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model

    

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3. Transcriptomic comparison of human and mouse brain microvessels

   https://doi.org/10.1038/s41598-020-69096-7  

   Song, Hannah W. ; Foreman, Koji L. ; Gastfriend, Benjamin D. ; Kuo, John S. ; Palecek, Sean P. ; Shusta, Eric V. ( July 2020 , Scientific Reports)

   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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4. Influence of basal media composition on barrier fidelity within human pluripotent stem cell‐derived blood‐brain barrier models

   https://doi.org/10.1111/jnc.15532  

   Neal, Emma H. ; Katdare, Ketaki A. ; Shi, Yajuan ; Marinelli, Nicholas A. ; Hagerla, Kameron A. ; Lippmann, Ethan S. ( November 2021 , Journal of Neurochemistry)

   It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principal component of the blood‐brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human‐induced pluripotent stem cell (iPSC)‐derived BMEC‐like cells cultured under serum‐free conditions in DMEM/F12 and Neurobasal media, which have fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays.

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5. Simple design for membrane-free microphysiological systems to model the blood-tissue barriers

   https://doi.org/10.1016/j.ooc.2023.100032  

   Young, By Ashlyn ; Deal, Halston ; Rusch, Gabrielle ; Pozdin, Vladimir A. ; Brown, Ashley C. ; Daniele, Michael ( December 2023 , Organs-on-a-Chip)

   Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ/cm2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations. 

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