Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

Abstract

BackgroundThe endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood–brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells.MethodsTo assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm−2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood–brain barrier were performed.ResultsHuman brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood–brain barrier markers.ConclusionsThe characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood–brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-017-0068-z) contains supplementary material, which is available to authorized users.

In vitro modeling of blood–brain barrier and interface functions in neuroimmune communication

“…Regarding leukocyte trafficking and other BBB properties, it has been found that inducing shear stress, which is normally encountered as a result of blood flow across brain endothelial cells in vivo, can cause some important changes in BEC phenotype. For example, sheer stress induced higher TEER, mRNA upregulation of TJPs and BBB transporters, as well as increased expression of mRNA levels of cell adhesion molecules such as VCAM-1, ICAM-1, and PECAM [98]. In contrast, P-and E-selectin mRNAs were decreased, which may further indicate a BBB phenotype since BECs lack P-and E-selectins in their Weibel-Palade bodies at baseline [99].…”

Recent advances in human iPSC-derived models of the blood–brain barrier

“…Utilizing this capability, several groups have lined microfluidic devices with iBMECs to study the cellular response to flow-induced shear stress. Using a PDMS-based platform with shear rates up to 12 dyne/cm 2 , investigators determined that iBMECs do not elongate or align with the direction of fluid flow in response to shear stress, a phenotype unique to endothelial cells of the brain [60]. Expanding on these observations, subsequent studies showed that while iBMECs do not change their morphology in response to shear stress, they do respond at the transcriptional level in a force-dependent manner [31].…”

“…Several groups have made progress in this area by exposing iBMECs to hypoxia [19], modifying extracellular matrix composition and stiffness [62], or by optimizing hydrogel scaffolds and fluid flow parameters that allow for maintenance of barrier properties for up to 3 weeks [63]. Many of the BBB-chip platforms developed and optimized recently are now being used to investigate drug permeabilities [19,31,64,65], neurodegenerative disease [31,43,66], and other functional aspects of the BBB [33,60,61,67].…”

Lab-On-A-Chip for the Development of Pro-/Anti-Angiogenic Nanomedicines to Treat Brain Diseases

“…In addition, the cellular uptake, transport via the transcellular/paracellular routes and drug-release kinetics, needs to be evaluated. Various device geometries have been reported for brain vasculature models (Figures 2 and 3); parallel channels (PCs), with interconnecting micro-gaps, [42][43][44][45]51,56,90], hollow-microtubes (HTs) [2,47,49,50,52], and others, concentric-ring (CR) channels [54], multi-layered channels [53], hybrid-microchambers [57], and microwells [58]. Each of the above designs has its own advantage in terms of angiogenesis drug screening application.…”

“…Unlike in the static petri dish culture, dynamic flow and shear stress can be generated on microfluidic platforms. LOC provides precise control over the flow rate and shear stress when integrated with a syringe pump [90]. In an LOC, it is possible to dynamically flow culture media and other fluids at a physiological flow rate and create shear stress on the BMVECs (6 dyne cm −2 at 100 µL h −1 ).…”