(I-358) Brain Pericyte-Like Cells are Responsible for Basement Membrane Production in a Human Stem Cell-Derived Blood Brain Barrier Model

Introduction:: The blood-brain barrier (BBB) prevents molecules with the potential to disrupt brain chemistry from crossing from the vasculature to the central nervous system. Inflammation causes breakdown of the BBB and can promote development of neurological disorders. One route that may link inflammation to BBB breakdown is the upregulation of enzymatic activity leading to digestion of the basement membrane (BM). This BM is composed of networks of extracellular matrix proteins, including collagen IV and laminin, that wrap around blood vessels and provide support for the endothelium. Since the presence of BM strengthens endothelial barrier properties, losing the BM would logically compromise those properties and cause BBB disruption [1]. To study the relationship between inflammation, BM, and breakdown of the BBB, we developed an in vitro model consisting of a microfluidic device featuring an ultrathin and continuously nanoporous silicon nitride membrane. We sought to validate that human iPSC-derived cells could form a physiological BM on this membrane, which we evaluated by confirming that major BM protein components were produced and that these proteins were concentrated on the abluminal side of the endothelium.

Materials and Methods:: The top of the nanoporous membrane was coated with the matrix proteins collagen IV and fibronectin to promote endothelial cell adhesion. Human iPSC-derived brain pericyte-like cells (BPLCs) [2] were seeded on the underside of the membrane. One day later, extended endothelial culture method brain microvascular endothelial cell-like (EECM-BMEC-like) cells [3] were seeded on top of the membrane. This cell seeding orientation was designed to mimic the relative cell positions of the physiological vascular wall. After 6 days of co-culture, the cells were fixed with formaldehyde and immunofluorescently labeled with antibodies for collagen IV, fibronectin, and the adherens junction protein VE-cadherin. To validate that the detected collagen IV and fibronectin were cell-secreted and not part of the membrane coating, cell-free membranes were also immunostained. Images were captured at 40x magnification on an Andor Dragonfly Spinning Disc Confocal Microscope with a 0.2 μm step interval.

Results, Conclusions, and Discussions:: Immunostaining for collagen IV and the endothelial marker VE-cadherin revealed that the BM was located on the abluminal side of the EECM-BMEC-like cell monolayer in the BBB model, thus mimicking the relative physiological orientation of the cells and the BM (Fig. 1A,B). Furthermore, there was a subpopulation of cells embedded within the collagen IV layer that were likely the BPLCs since they did not express VE-cadherin. Pericytes have also been observed embedded within the BM in vivo [4]. Interestingly, EECM-BMEC-like cell monocultures did not express as much collagen IV as the co-cultures with both EECM-BMEC-like cells and BPLCs. This indicated that the BPLCs were necessary for the majority of collagen IV production in the model. Similarly, the majority of fibronectin in the BBB model was located below the endothelium and appeared to be contributed by the BPLCs (Fig. 1C,D). These results suggest that pericytes may be essential to include in BBB models that will be used for investigations of BM formation and digestion. Immunostaining of cell-free membranes verified that the coating was not contributing to the collagen IV or fibronectin fluorescence signals. In conclusion, our in vitro model recapitulates the physiological orientation of cell types and BM components of the BBB. The BPLCs are responsible for contributing BM proteins including collagen IV and fibronectin and we will continue to seed these cells in co-culture with EECM-BMEC-like cells so that we can study how various proinflammatory signals impact the BM.

Acknowledgements (Optional): :

References (Optional): : [1] T. Osada, et al., "Interendothelial claudin-5 expression depends on cerebral endothelial cell-matrix

adhesion by β(1)-integrins," Journal of cerebral blood flow and metabolism : official journal of the

International Society of Cerebral Blood Flow and Metabolism, vol. 31, pp. 1972-1985, 2011.

[2] B. D. Gastfriend, et al., "Differentiation of Brain Pericyte-Like Cells from Human Pluripotent Stem Cell-Derived Neural Crest," Curr Protoc, vol. 1, p. e21, Jan 2021.

[3] H. Nishihara, et al., "Differentiation of human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable to study immune cell interactions," STAR protocols, vol. 2, pp. 100563-100563, 2021.

[4] M. S. Thomsen, et al., "Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood–brain barrier," Journal of Neurochemistry, vol. 140, pp. 741-754, 2017/03/01 2017.