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Abstract Extracellular matrix (ECM) in the human tissue contains vesicles, which are defined as matrix‐bound nanovesicles (MBVs). MBVs serve as one of the functional components in ECM, recapitulating part of the regulatory roles and in vivo microenvironment. In this study, extracellular vesicles from culture supernatants (SuEVs) and MBVs are isolated from the conditioned medium or ECM, respectively, of 3D human mesenchymal stem cells. Nanoparticle tracking analysis shows that MBVs are smaller than SuEVs (100–150 nm). Transmission electron microscopy captures the typical cup shape morphology for both SuEVs and MBVs. Western blot reveals that MBVs have low detection of some SuEV markers such as syntenin‐1. miRNA analysis of MBVs shows that 3D microenvironment enhances the expression of miRNAs such as miR‐19a and miR‐21. In vitro functional analysis shows that MBVs can facilitate human pluripotent stem cell‐derived forebrain organoid recovery after starvation and promote high passage fibroblast proliferation. In macrophage polarization, 2D MBVs tend to suppress the pro‐inflammatory cytokine IL‐12 β , while 3D MBVs tend to enhance the anti‐inflammatory cytokine IL‐10. This study has the significance in advancing the understanding of the bio‐interface of nanovesicles with human tissue and the design of cell‐free therapy for treating neurological disorders such as ischemic stroke. 

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. 

Kaposi's sarcoma–associated herpesvirus (KSHV) inhibitor of cyclic GMP–AMP synthase (cGAS) (KicGAS) encoded by ORF52 is a conserved major tegument protein of KSHV and the first reported viral inhibitor of cGAS. In our previous study, we found that KicGAS is highly oligomerized in solution and that oligomerization is required for its cooperative DNA binding and for inhibiting DNA-induced phase separation and activation of cGAS. However, how KicGAS oligomerizes remained unclear. Here, we present the crystal structure of KicGAS at 2.5 Å resolution, which reveals an “L”-shaped molecule with each arm of the L essentially formed by a single α helix (α1 and α2). Antiparallel dimerization of α2 helices from two KicGAS molecules leads to a unique “Z”-shaped dimer. Surprisingly, α1 is also a dimerization domain. It forms a parallel dimeric leucine zipper with the α1 from a neighboring dimer, leading to the formation of an infinite chain of KicGAS dimers. Residues involved in leucine zipper dimer formation are among the most conserved residues across ORF52 homologs of gammaherpesviruses. The self-oligomerization increases the valence and cooperativity of interaction with DNA. The resultant multivalent interaction is critical for the formation of liquid condensates with DNA and consequent sequestration of DNA from being sensed by cGAS, explaining its role in restricting cGAS activation. The structure presented here not only provides a mechanistic understanding of the function of KicGAS but also informs a molecular target for rational design of antivirals against KSHV and related viruses.