Chemically defined human vascular laminins for biologically relevant culture of hiPSC-derived brain microvascular endothelial cells

Abstract

Background In recent years, differentiation of human induced pluripotent stem cells (hiPSCs) into brain-specific microvascular endothelial cells (iBMECs) has frequently been used to model the blood–brain barrier (BBB). However, there are limitations in the use of iBMECs for in vitro studies, such as transendothelial electrical resistance (TEER) instability, weak junctional expression of VE-cadherin, and lack of proper fluid shear stress response. In vivo, the basement membrane (BM) composition of the BBB evolves throughout development, and laminins become the dominant component of the mature vascular BM. However, laminin isoforms of the endothelial BM have not been used for culture of differentiated iBMECs. The main goal of this study is to investigate the effect of different laminin isoforms of the endothelial BM on iBMEC functionality and to determine whether better recapitulation of the physiological BM in vitro can address the aforementioned limitations of iBMECs. Methods Using a previously reported method, hiPSCs were differentiated into iBMECs. The influence of main laminins of the endothelial BM, LN 411 and LN 511, on iBMEC functionality was studied and compared to a collagen IV and fibronectin mixture (CN IV-FN). Quantitative RT-PCR, immunocytochemistry, and TEER measurement were utilized to assess gene and protein expression and barrier properties of iBMECs on different extracellular matrices. Single-channel microfluidic devices were used to study the effect of shear stress on iBMECs. Results LN 511, but not LN 411, improved iBMEC barrier properties and resulted in more sustained TEER stability. Immunocytochemistry showed improved junctional protein expression compared to iBMECs cultured on CN IV-FN. iBMECs cultured on LN 511 showed a reduction of stress fibers, indicating resting endothelial phenotype, whereas gene expression analysis revealed upregulation of multiple genes involved in endothelial activation in iBMECs on CN IV-FN. Finally, culturing iBMECs on LN 511 enhanced physiological responses to shear stress, including morphological changes and enhanced junctional protein association. Conclusion LN 511 improves the functionality and long-term barrier stability of iBMECs. Our findings suggest that incorporation of physiologically relevant LN 511 in iBMEC culture would be beneficial for disease modeling applications and BBB-on-a-chip platforms that accommodate fluid flow.

Cell-specific expression and function of laminin at the neurovascular unit

“…28 Similarly, laminin-511 but not laminin-411 improves the barrier properties of pluripotent stem cell-derived BMECs in vitro. 29 Together, these results strongly suggest that laminin-α4 and -α5 differentially regulate BBB permeability under pathological conditions, with laminin-α4 promoting and laminin-α5 inhibiting inflammatory cell infiltration into brain parenchyma.…”

Study of BBB Dysregulation in Neuropathogenicity Using Integrative Human Model of Blood–Brain Barrier

“…Several other factors, including temperature, pore density, and cell insert properties, have been shown to influence TEER values (Vigh et al, 2021). Culturing hiPSC-derived brain endothelial cells on laminin-511 increases and stabilizes TEER over 15 days (Motallebnejad and Azarin, 2020).…”

Induced pluripotent stem cell-based organ-on-a-chip as personalized drug screening tools: A focus on neurodegenerative disorders

“…Another winning strategy to improve barrier properties in a iBMEC-based microfluidic system consists in using a laminin-511 coating. 112 A great breakthrough in barrier integrity studies consists in the integration of sensors that enable a real-time monitoring. Matthiesen et al 113 adopted this strategy in an iPSC-based BBB, in which they were able to observe barrier disruption following the exposure to the nitrosative stressor linsidomine and the protective effects of the antioxidant N-acetylcysteine amide.…”