B cells in the pneumococcus-infected lung are heterogeneous and require CD4+ T cell help including CD40L to become resident memory B cells.

Recovery from respiratory pneumococcal infections generates lung-localized protection against heterotypic bacteria, mediated by resident memory lymphocytes. Optimal protection in mice requires re-exposure to pneumococcus within days of initial infection. Serial surface marker phenotyping of B cell populations in a model of pneumococcal heterotypic immunity revealed that bacterial re-exposure stimulates the immediate accumulation of dynamic and heterogeneous populations of B cells in the lung, and is essential for the establishment of lung resident memory B (BRM) cells. The B cells in the early wave were activated, proliferating locally, and associated with both CD4+ T cells and CXCL13. Antagonist- and antibody-mediated interventions were implemented during this early timeframe to demonstrate that lymphocyte recirculation, CD4+ cells, and CD40 ligand (CD40L) signaling were all needed for lung BRM cell establishment, whereas CXCL13 signaling was not. While most prominent as aggregates in the loose connective tissue of bronchovascular bundles, morphometry and live lung imaging analyses showed that lung BRM cells were equally numerous as single cells dispersed throughout the alveolar septae. We propose that CD40L signaling from antigen-stimulated CD4+ T cells in the infected lung is critical to establishment of local BRM cells, which subsequently protect the airways and parenchyma against future potential infections.

Crystal ribcage: a platform for probing real-time lung function at cellular resolution.

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real time. Despite advances in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that allows multiscale optical imaging of the functioning lung from whole-organ to single-cell level. It enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal and optogenetic interventions, and it preserves the three-dimensional architecture, air-liquid interface, cellular diversity and respiratory-circulatory functions of the lung. Utilizing these capabilities on murine models of pulmonary pathologies we probed remodeling of respiratory-circulatory functions at the single-alveolus and capillary levels during disease progression. The crystal ribcage and its broad applications presented here will facilitate further studies of nearly any pulmonary disease as well as lead to the identification of new targets for treatment strategies.

Three-dimensional microenvironment regulates gene expression, function, and tight junction dynamics of iPSC-derived blood-brain barrier microvessels.

The blood-brain barrier (BBB) plays a pivotal role in brain health and disease. In the BBB, brain microvascular endothelial cells (BMECs) are connected by tight junctions which regulate paracellular transport, and express specialized transporter systems which regulate transcellular transport. However, existing in vitro models of the BBB display variable accuracy across a wide range of characteristics including gene/protein expression and barrier function. Here, we use an isogenic family of fluorescently-labeled iPSC-derived BMEC-like cells (iBMECs) and brain pericyte-like cells (iPCs) within two-dimensional confluent monolayers (2D) and three-dimensional (3D) tissue-engineered microvessels to explore how 3D microenvironment regulates gene expression and function of the in vitro BBB. We show that 3D microenvironment (shear stress, cell-ECM interactions, and cylindrical geometry) increases BBB phenotype and endothelial identity, and alters angiogenic and cytokine responses in synergy with pericyte co-culture. Tissue-engineered microvessels incorporating junction-labeled iBMECs enable study of the real-time dynamics of tight junctions during homeostasis and in response to physical and chemical perturbations.

Effects of acute and chronic oxidative stress on the blood-brain barrier in 2D and 3D in vitro models.

Oxidative stress is a shared pathology of neurodegenerative disease and brain injuries, and is derived from perturbations to normal cell processes by aging or environmental factors such as UV exposure and air pollution. As oxidative cues are often present in systemic circulation, the blood-brain barrier (BBB) plays a key role in mediating the effect of these cues on brain dysfunction. Therefore, oxidative damage and disruption of the BBB is an emergent focus of neurodegenerative disease etiology and progression. We assessed barrier dysfunction in response to chronic and acute oxidative stress in 2D and 3D in vitro models of the BBB with human iPSC-derived brain microvascular endothelial-like cells (iBMECs). We first established doses of hydrogen peroxide to induce chronic damage (modeling aging and neurodegenerative disease) and acute damage (modeling the response to traumatic brain injury) by assessing barrier function via transendothelial electrical resistance in 2D iBMEC monolayers and permeability and monolayer integrity in 3D tissue-engineered iBMEC microvessels. Following application of these chronic and acute doses in our in vitro models, we found local, discrete structural changes were the most prevalent responses (rather than global barrier loss). Additionally, we validated unique functional changes in response to oxidative stress, including dysfunctional cell turnover dynamics and immune cell adhesion that were consistent with changes in gene expression.

Long-Term Cryopreservation Preserves Blood-Brain Barrier Phenotype of iPSC-Derived Brain Microvascular Endothelial Cells and Three-Dimensional Microvessels.

Brain microvascular endothelial cells derived from induced pluripotent stem cells (dhBMECs) are a scalable and reproducible resource for studies of the human blood-brain barrier, including mechanisms and strategies for drug delivery. Confluent monolayers of dhBMECs recapitulate key in vivo functions including tight junctions to limit paracellular permeability and efflux and nutrient transport to regulate transcellular permeability. Techniques for cryopreservation of dhBMECs have been reported; however, functional validation studies after long-term cryopreservation have not been extensively performed. Here, we characterize dhBMECs after 1 year of cryopreservation using selective purification on extracellular matrix-treated surfaces and ROCK inhibition. One-year cryopreserved dhBMECs maintain functionality of tight junctions, efflux pumps, and nutrient transporters with stable protein localization and gene expression. Cryopreservation is associated with a decrease in the yield of adherent cells and unique responses to cell stress, resulting in altered paracellular permeability of Lucifer yellow. Additionally, cryopreserved dhBMECs reliably form functional three-dimensional microvessels independent of cryopreservation length, with permeabilities lower than non-cryopreserved two-dimensional models. Long-term cryopreservation of dhBMECs offers key advantages including increased scalability, reduced batch-to-batch effects, the ability to conduct well-controlled follow up studies, and support of multisite collaboration from the same cell stock, all while maintaining phenotype for screening pharmaceutical agents.

Tissue-engineered blood-brain barrier models via directed differentiation of human induced pluripotent stem cells.

Three-dimensional (3D) tissue-engineered models of the blood-brain barrier (BBB) recapitulate in vivo shear stress, cylindrical geometry, and cell-ECM interactions. Here we address four issues associated with BBB models: cell source, barrier function, cryopreservation, and matrix stiffness. We reproduce a directed differentiation of brain microvascular endothelial cells (dhBMECs) from two fluorescently labeled human induced pluripotent stem cell lines (hiPSCs) and demonstrate physiological permeability of Lucifer yellow over six days. Microvessels formed from cryopreserved dhBMECs show expression of BBB markers and maintain physiological barrier function comparable to non-cryopreserved cells. Microvessels displaying physiological barrier function are formed in collagen I hydrogels with stiffness matching that of human brain. The dilation response of microvessels was linear with increasing transmural pressure and was dependent on matrix stiffness. Together these results advance capabilities for tissue-engineered BBB models.