BackgroundDue to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood–brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods.MethodsInduced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines.ResultsThe use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm2 and fluorescein permeability below 1.95 × 10−7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated with Parkinson’s Disease. Finally, BMECs differentiated using E6 medium responded to inductive cues from astrocytes and pericytes and achieved a maximum TEER value of 6635 ± 315 Ω × cm2, which to our knowledge is the highest reported in vitro TEER value to date.ConclusionsGiven the accelerated differentiation, equivalent performance, and reduced cost to produce BMECs, our updated methods should make iPSC-derived in vitro BBB models more accessible for a wide variety of applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-017-0059-0) contains supplementary material, which is available to authorized users.

Intimal stiffening has been linked with increased vascular permeability and leukocyte transmigration, hallmarks of atherosclerosis. However, recent evidence indicates age-related intimal stiffening is not uniform but rather characterized by increased point-to-point heterogeneity in subendothelial matrix stiffness, the impact of which is much less understood. To investigate the impact of spatially heterogeneous matrix rigidity on endothelial monolayer integrity, we develop a micropillar model to introduce closely-spaced, step-changes in substrate rigidity and compare endothelial monolayer phenotype to rigidity-matched, uniformly stiff and compliant substrates. We found equivalent disruption of adherens junctions within monolayers on step-rigidity and uniformly stiff substrates relative to uniformly compliant substrates. Similarly, monolayers cultured on step-rigidity substrates exhibited equivalent percentages of leukocyte transmigration to monolayers on rigidity-matched, uniformly stiff substrates. Adherens junction tension and focal adhesion density, but not size, increased within monolayers on step-rigidity and uniformly stiff substrates compared to more compliant substrates suggesting that elevated tension is disrupting adherens junction integrity. Leukocyte transmigration frequency and time, focal adhesion size, and focal adhesion density did not differ between stiff and compliant sub-regions of step-rigidity substrates. Overall, our results suggest that endothelial monolayers exposed to mechanically heterogeneous substrates adopt the phenotype associated with the stiffer matrix, indicating that spatial heterogeneities in intimal stiffness observed with age could disrupt endothelial barrier integrity and contribute to atherogenesis.

Background: Indocyanine green fluorescence (ICG-F) stains hepatic tumours and delineates vascular and biliary structures in real-time. We detail the efficacy of ICG-F in robotic hepatobiliary surgery.Methods: PubMed, EMBASE, Web of Science, and Cochrane Central were searched for original articles and meta-analyses detailing the outcomes of ICG-F in robotic hepatobiliary surgery.Results: 214 abstracts were reviewed; 16 studies are presented. One singleinstitution study reported ICG-F in robotic right hepatectomy reduced postoperative bile leakage (0% vs. 12%, p = 0.023), R1 resection (0% vs. 16%, p = 0.019), and readmission (p = 0.023) without prolonging operative time (288 vs. 272 min, p = 0.778). Improved visualisation aided in attainment of R0 resection in partial hepatectomies and radical gallbladder adenocarcinoma resections. Fewer ICG-Faided robotic cholecystectomies were converted to open procedure compared to laparoscopic cholecystectomies (2.1% vs. 8.9%, p = 0.03; 0.15% vs. 2.6%, p < 0.001).Conclusions: ICG-F improves clinical outcomes in robotic hepatobiliary surgery without prolonging operative time. There is an opportunity to standardise ICG administration protocols, especially for hepatectomies.

Intimal stiffening upregulates endothelial cell contractility disrupting barrier integrity; however, intimal stiffening is non-uniform. The impact of local changes in intimal stiffness on proximal and distal cell-cell interactions is unknown. To investigate the range at which matrix stiffness heterogeneities impact neighboring endothelial cells within a monolayer, we built a micropillar system with adjacent regions of stiff and compliant matrix. The stiffness interface results in an oscillatory pattern of neutrophil transendothelial migration, symmetrical about the interface and well-fit by a sinusoid function. ‘Peaks’ of the sinusoid were found to have increased cellular contractility and decreased barrier function relative to ‘troughs’ of the sinusoid. Pharmacological modulation of contractility was observed to break symmetry, altering the amplitude and wavelength of the sinusoid, indicating that contractility may regulate this effect. This work illuminates a novel biophysical phenomenon of the role of stiffness-mediated cell-matrix interactions on cell-cell interactions at a distance. Additionally, it provides insight into the range at which intimal matrix stiffness heterogeneities will impact endothelial barrier function and potentially contribute to atherogenesis.

Diabetic retinopathy (DR) is a leading cause of vision loss in the United States and throughout the world. With early detection and treatment, sight-threatening sequelae from DR can be prevented. Although artificial intelligence (AI) based DR screening programs have been proven to be effective in identifying patients at high risk of vision loss, adoption of AI in clinical practice has been slow. We adapted the United Kingdom Design Council’s Double-Diamond model to design a strategy for care delivery which integrates an AI-based screening program for DR into a primary care setting. Methods from human-centered design were used to develop a strategy for implementation informed by context-specific barriers and facilitators. The purpose of this community case study is to present findings from this work in progress, including a system of protocols, educational documents and workflows created using key stakeholder input.