Evaluating Microglial Contributions to the Neurovascular Unit in Health and Neurodegeneration Using Human In Vitro Models

Background Microglia are emerging as critical regulators of neurovascular function in health and Alzheimer’s disease (AD), yet their interactions with the human neurovascular unit (NVU), particularly brain endothelial cells, remain incompletely understood. Current in vitro NVU platforms typically exclude microglia and lack perfusable vascular networks with physiologically relevant architecture. Here, we established complementary two-dimensional (2D) and three-dimensional (3D) NVU models to investigate microglia-endothelial and microglia-neurovascular interactions.

Human induced pluripotent stem cell derived-neurons (iNs), astrocytes (iAs), and microglia-like cells (iMGLs) were incorporated into a soft-lithography based engineered microvessel system to establish a multicellular neuroimmune-vascular model. To specifically evaluate iMGL-endothelial cell (EC) interactions, iMGL were co-cultured with primary human brain microvascular endothelial cells (HBMECs) and junctional protein localization was evaluated using immunofluorescence. The barrier integrity of engineered microvessels containing iMGL was evaluated using dextran permeability. Our 2D and 3D systems were stimulated with tumor necrosis factor-α (TNFα) to evaluate whether iMGL would promote or attenuate EC inflammation and barrier breakdown.

Incorporation of iNs, iAs, and iMGLs into a perfusable vascular model enabled a more complete representation of NVU cellular diversity and promoted neuronal health. In monolayer co-culture with iMGL, HBMECs enhanced the junctional localization of tight and adherens junction proteins through both contact-dependent and paracrine mechanisms. Following an inflammatory challenge, iMGLs reduced endothelial inflammatory activation, suggesting a protective role in response to AD-relevant inflammatory conditions. Finally, when embedded in 3D collagen matrices surrounding perfusable endothelialized lumen networks, iMGLs reduced dextran permeability and preserved endothelial barrier integrity following TNFα challenge.

Together, these findings establish a 3D perfusable neuroimmune-vascular model that enables the dissection of microglial contributions to neurovascular function in health and disease. Competing Interest Statement The authors have declared no competing interest. Footnotes Permanent address: 850 Republican St, Building S, Room S570 Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Conflict of interest: The authors declare that they have no competing interest. Availability of data and materials: All data needed to evaluate the conclusions in this paper are present in the main paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the corresponding author. Funding: We acknowledge the financial support of National Institute of Health grants R21AG074373 and R33HL154250-04S1 (to Y.Z. and J.E.Y), R01AG062148 (to J.E.Y) and T32AG066574 (to K.M.E). Authors’ contributions: KME: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing. ET: Investigation, Data curation, Formal analysis, Writing – review and editing. CAW: Investigation, Data curation, Formal analysis, Writing – review and editing. CK: Investigation, Data curation, Writing – review and editing. AR: Investigation, Writing – review and editing. WB: Investigation, Writing – review and editing. KH: Investigation, Writing – review and editing. IB: Investigation, Writing – review and editing. AR: Methodology, Writing – review and editing. YZ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing. JEY: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing. Funding sources had no involvement in publication preparation or research. Acknowledgments: We would like to acknowledge the Lynn and Mike Garvey Imaging Laboratory in the Institute for Stem Cells and Regenerative Medicine (ISCRM). We would also like to acknowledge that schematics were made using BioRender. List of Abbreviations - AD - Alzheimer’s disease - 2D - Two-dimensional - 3D - Three-dimensional - NVU - Neurovascular unit - iNs - Human induced pluripotent stem cell derived-neurons - iAs - Human induced pluripotent stem cell derived-astrocytes - iMGLs - Human induced pluripotent stem cell derived-microglia-like cells - HBMECs - Human brain microvascular endothelial cells - EC - Endothelial cell - TNFα - Tumor necrosis factor α - BBB - Blood brain barrier - CNS - Central nervous system - IL - Interleukin - GWAS - Genome wide association studies - TEER - Transendothelial electrical resistance - iPSC - human induced pluripotent stem cell - LPS - lipopolysaccharide - CSF-1R - Colony-Stimulating Factor 1 Receptor - ZO-1 - zonula occludens-1 - B3TUB - βIII-tubulin - ICC - Immunocytochemistry - MAP2 - microtubule associated protein 2 - MAPT - microtubule associated protein tau - GFAP - glial fibrillary acidic protein - IBA-1 - ionized calcium-binding adaptor molecule 1 - MEA - multielectrode array - MFI - mean fluorescence intensity - VECAD - VE-Cadherin - CM - conditioned media CSF: cerebrospinal fluid - ICAM1 - intercellular adhesion molecule 1 - MSD - Mesoscale Discovery