Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10-5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10-3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10-5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.

Application of blood brain barrier models in pre-clinical assessment of glioblastoma-targeting CAR-T based immunotherapies.

Human blood brain barrier (BBB) models derived from induced pluripotent stem cells (iPSCs) have become an important tool for the discovery and preclinical evaluation of central nervous system (CNS) targeting cell and gene-based therapies. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary form of gene-modified cell-based immunotherapy with potential for targeting solid tumors, such as glioblastomas. Crossing the BBB is an important step in the systemic application of CAR-T therapy for the treatment of glioblastomas and other CNS malignancies. In addition, even CAR-T therapies targeting non-CNS antigens, such as the well-known CD19-CAR-T therapies, are known to trigger CNS side-effects including brain swelling due to BBB disruption. In this study, we used iPSC-derived brain endothelial-like cell (iBEC) transwell co-culture model to assess BBB extravasation of CAR-T based immunotherapies targeting U87MG human glioblastoma (GBM) cells overexpressing the tumor-specific mutated protein EGFRvIII (U87vIII). Two types of anti-EGFRvIII targeting CAR-T cells, with varying tonic signaling profiles (CAR-F263 and CAR-F269), and control Mock T cells were applied on the luminal side of BBB model in vitro. CAR-F263 and CAR-F269 T cells triggered a decrease in transendothelial electrical resistance (TEER) and an increase in BBB permeability. CAR-T cell extravasation and U87vIII cytotoxicity were assessed from the abluminal compartment using flow cytometry and Incucyte real-time viability imaging, respectively. A significant decrease in U87vIII cell viability was observed over 48 h, with the most robust cytotoxicity response observed for the constitutively activated CAR-F263. CAR-F269 T cells showed a similar cytotoxic profile but were approximately four fold less efficient at killing the U87vIII cells compared to CAR-F263, despite similar transmigration rates. Visualization of CAR-T cell extravasation across the BBB was further confirmed using BBTB-on-CHIP models. The described BBB assay was able to discriminate the cytotoxic efficacies of different EGFRvIII-CARs and provide a measure of potential alterations to BBB integrity. Collectively, we illustrate how BBB models in vitro can be a valuable tool in deciphering the mechanisms of CAR-T-induced BBB disruption, accompanying toxicity and effector function on post-barrier target cells.

Development of a Blood-Brain Barrier Permeability Assay Using Human Induced Pluripotent Stem Cell Derived Brain Endothelial Cells.

The development of translational and predictive models in vitro for assessing blood-brain barrier (BBB) delivery has become an important requirement in preclinical testing of CNS-targeting therapeutics. Here we describe a directed monolayer differentiation strategy to generate a population of brain endothelial-like cells (BECs) from human induced pluripotent stem cell (iPSC) with robust BBB properties. To generate BBB permeability assays, the BECs are seeded as a monolayer on a semipermeable Transwell insert placed inside a companion plate to generate a two-compartment Transwell model. The BECs provide a BBB-like separation between the luminal (blood) and abluminal (brain) compartments to assess BBB permeability of CNS-targeting therapeutics.

Differentiation of Adipose-Derived Stem Cells into Vascular Smooth Muscle Cells for Tissue Engineering Applications.

Synthetic grafts have been developed for vascular bypass surgery, however, the risks of thrombosis and neointimal hyperplasia still limit their use. Tissue engineering with the use of adipose-derived stem cells (ASCs) has shown promise in addressing these limitations. Here we further characterized and optimized the ASC differentiation into smooth muscle cells (VSMCs) induced by TGF-ß and BMP-4. TGF-ß and BMP-4 induced a time-dependent expression of SMC markers in ASC. Shortening the differentiation period from 7 to 4 days did not impair the functional property of contraction in these cells. Stability of the process was demonstrated by switching cells to regular growth media for up to 14 days. The role of IGFBP7, a downstream effector of TGF-ß, was also examined. Finally, topographic and surface patterning of a substrate is recognized as a powerful tool for regulating cell differentiation. Here we provide evidence that a non-woven PET structure does not affect the differentiation of ASC. Taken together, our results indicate that VSMCs differentiated from ASCs are a suitable candidate to populate a PET-based vascular scaffolds. By employing an autologous source of cells we provide a novel alternative to address major issues that reduces long-term patency of currently vascular grafts.

Analysis and Simulation of Glioblastoma Cell Lines-Derived Extracellular Vesicles Metabolome.

Glioblastoma (GBM) is one of the most aggressive cancers of the central nervous system. Despite current advances in non-invasive imaging and the advent of novel therapeutic modalities, patient survival remains very low. There is a critical need for the development of effective biomarkers for GBM diagnosis and therapeutic monitoring. Extracellular vesicles (EVs) produced by GBM tumors have been shown to play an important role in cellular communication and modulation of the tumor microenvironment. As GBM-derived EVs contain specific "molecular signatures" of their parental cells and are able to transmigrate across the blood-brain barrier into biofluids such as the blood and cerebrospinal fluid (CSF), they are considered as a valuable source of potential diagnostic biomarkers. Given the relatively harsh extracellular environment of blood and CSF, EVs have to endure and adapt to different conditions. The ability of EVs to adjust and function depends on their lipid bilayer, metabolic content and enzymes and transport proteins. The knowledge of EVs metabolic characteristics and adaptability is essential for their utilization as diagnostic and therapeutic tools. The main aim of this study was to determine the metabolome of small EVs or exosomes derived from different GBM cells and compare to the metabolic profile of their parental cells using NMR spectroscopy. In addition, a possible flux of metabolic processes in GBM-derived EVs was simulated using constraint-based modeling from published proteomics information. Our results showed a clear difference between the metabolic profiles of GBM cells, EVs and media. Machine learning analysis of EV metabolomics, as well as flux simulation, supports the notion of active metabolism within EVs, including enzymatic reactions and the transfer of metabolites through the EV membrane. These results are discussed in the context of novel GBM diagnostics and therapeutic monitoring.

Histological assessment of angiogenesis in the hypoxic central nervous system.

Angiogenesis, the sprouting of new capillaries from preexisting vessels, is an integral part of both normal development and numerous pathological conditions such as tumor growth, inflammation, and stroke. The development of angiogenesis assays has been critical in understanding this process in both the context of disease and normal physiology. With the growing availability of antibodies against angiogenic markers as well as advances in microscopy and imaging analysis software, a more comprehensive assessment of the angiogenesis process is beginning to take form (Milner et al., Stroke 39:191-197, 2008; Freitas-Andrade et al., J Cereb Blood Flow Metab 32:663-675, 2012; Li et al., Glia 58:1157-1167, 2010; Dore-Duffy and LaManna, Antioxid Redox Signal 9:1363-1371, 2007). This chapter describes an in vivo method of inducing brain angiogenesis in mice by chronic exposure to mild hypoxia. In addition, a detailed procedure of quantifying angiogenesis using multiple immunofluorescent labeling of mouse brain tissue sections is also presented.

PlGF knockout delays brain vessel growth and maturation upon systemic hypoxic challenge.

In this study, we have investigated the potential role of placental growth factor (PlGF) in hypoxia-induced brain angiogenesis. To this end, PlGF wild-type (PlGF(+/+)) and PlGF knockout (PlGF(-/-)) mice were exposed to whole body hypoxia (10% oxygen) for 7, 14, and 21 days. PlGF(+/+) animals exhibited a significant ~40% increase in angiogenesis after 7 days of hypoxia compared with controls, while in PlGF(-/-) this effect only occurred after 14 days of hypoxia. No differences in pericyte/smooth muscle cell (SMC) coverage between the two genotypes were observed. After 14 days of hypoxia, PlGF(-/-) microvessels had a significant increase in fibrinogen accumulation and extravasation compared with those of PlGF(+/+), which correlated with endothelial cell disruption of the tight junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked both pericyte/SMC coverage and endothelial vascular endothelial growth factor expression, and regressed after 21 days of hypoxia. Vascular endothelial growth factor expression levels were found to be significantly lower in the frontal cortex of PlGF(-/-) compared with those in PlGF(+/+) animals during the first 5 days of hypoxia, which in combination with the lack of PlGF may have contributed to the delayed angiogenic response and the prothrombotic phenotype observed in the PlGF(-/-)animals.

Insulin-like growth factor binding protein 7 exhibits tumor suppressive and vessel stabilization properties in U87MG and T98G glioblastoma cell lines.

Insulin-like growth factor binding protein 7 (IGFBP7) is downregulated in several solid cancers. IGFBP7 has been proposed to act as a tumor suppressor gene through mechanisms involving senescence and apoptotic pathways. The tumor suppressor effect of IGFBP7 in glioblastoma multiforme (GBM) was examined in this study using two human GBM cell lines, U87MG and T98G. Exogenously applied IGFBP7 (20 and 100 nM) significantly reduced U87MG (~70 and ~75%, respectively) and T98G (~37 and ~50%, respectively) cell growth in soft agar. IGFBP7 stimulated senescence-associated ß-galactosidase in both U87MG and T98G cells without stimulating apoptosis (annexin V and propidium iodide staining, expression of SMARCB1 or BNIP3L and caspase cleavage) or affecting phosphorylation of p44/42 MAPK. The inhibitory effect of IGFBP7 on U87MG cell growth was further assessed in vivo using U87MG cells grafted on the chick chorioallantoic membrane. In this model, U87MG cells formed solid and highly vascularized tumors that were reduced in size (~40%) when treated with 500 nM IGFBP7 compared with control tumors. Vessels in IGFBP7-treated tumors were clustered, unevenly distributed and associated with higher number of α-SMA positive cells compared with those in untreated tumors. IGFBP7 induced both aortic smooth muscle cell (AoSMC) chemoattraction and endothelial cell (EC) transdifferentiation into a SM-like cell phenotype. U87MG conditioned media-induced IGFBP7 expression in ECs was significantly inhibited by the cross-talk/interaction with SMCs. This study indicates that IGFBP7 suppresses U87MG tumor cell growth, induces cell senescence and participates in tumor vessel stabilization by promoting SMC/pericyte recruitment and differentiation.

Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation.

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell-type-specific epigenomes and suggest mechanisms by which they may be created and maintained.