Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease

Fluids and Barriers of the CNS · 17 authors, 8 centres

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POPULATIONHuman induced pluripotent stem cell-derived brain capillary endothelial-like cells (BCECs) from a LOAD patient (APOE4/4, age 76) and a healthy elderly control (APOE3/3, age 64)

INTERVENTIONCo-culture of BCECs with isogenic astrocytes, microglia-like cells, neural stem cells, or pericytes

COMPARISONBCEC mono-culture vs. co-culture; LOAD vs. control conditions

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TL;DR

This study developed a multicellular isogenic model of the neurovascular unit using human induced pluripotent stem cells from a late-onset Alzheimer disease (LOAD) patient and a healthy control. Co-cultures with neural stem cells tightened the blood-brain barrier in healthy but not LOAD conditions, and transcriptomic analysis revealed cell type-specific dysregulation of tight junctions, transporters, and mucins in LOAD brain capillary endothelial cells. These findings highlight the distinct roles of different brain cell types in maintaining barrier integrity and provide insights into LOAD-associated blood-brain barrier dysfunction.

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**Background:** The blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the molecular mechanisms, particularly related to the APOE4/4 genotype, are poorly understood. The neurovascular unit (NVU) comprises brain capillary endothelial cells (BCECs), astrocytes, microglia, neural stem cells (NSCs), and pericytes, which collectively maintain BBB integrity. This study aimed to develop a multicellular isogenic model of the NVU using human induced pluripotent stem cells (hiPSCs) from a LOAD patient and a healthy control to investigate cell type-specific effects on BCEC function and transcriptome.

**Methods:** HiPSCs were derived from a LOAD patient (age 76, APOE4/4) and a healthy elderly control (age 64, APOE3/3). Isogenic astrocytes, BCECs, microglia-like cells (MGCs), NSCs, and pericytes were differentiated. BCECs were cultured alone or in indirect co-culture with each brain cell type. Barrier integrity was assessed by transendothelial electrical resistance (TEER) and sodium fluorescein permeability (PC_NaF). High-throughput multiplex qPCR analyzed 90 mRNA transcripts relevant to BBB integrity and functionality. Telomere length was measured by monochromatic multiplex qPCR. Cell purity was confirmed by flow cytometry and immunofluorescence.

**Key Results:** Co-cultures with NSCs significantly increased TEER in control BCECs (2674 ± 670 Ω*cm² vs. 2299 ± 123 Ω*cm² in mono-culture, p=0.02), but this effect was absent in LOAD BCECs. MGCs reduced NaF permeability in control BCECs (PC_NaF = 0.840 ± 0.111 μm/min vs. 1.080 ± 0.088 μm/min, p=0.05). Transcriptomic analysis revealed that NSCs had the strongest effect on BCEC gene regulation, with 26 significantly altered transcripts in control and 25 in LOAD BCECs. In LOAD BCECs, co-culture with NSCs led to downregulation of multiple claudins (CLDN3, -4, -7, -8, -10, -11, -12 tv2, -17, -19, -21, -22, -24), tight junction proteins (F11R, JAM2, JAM3, TJP1, TJP3, OCLN, MARVELD2), and transporters (ABCA1, ABCA7, ABCB1, ABCC4, ABCG2, SLC2A1, SLC7A1, SLC7A3, SLC7A5, SLC16A1, SLC16A2). The mucin MUC1 transcript variant a was significantly downregulated in all LOAD co-cultures (PCs p=0.014; ACs p=0.0004; MGCs p=0.00004; NSCs p=0.00002). Telomere length was reduced in LOAD astrocytes, BCECs, and NSCs compared to control.

**Clinical Implications:** This isogenic NVU model demonstrates that different brain cell types exert unique, cell type-specific effects on BCEC transcriptome and barrier function under healthy and LOAD conditions. The loss of NSC-mediated barrier tightening and widespread dysregulation of tight junctions, transporters, and mucins in LOAD BCECs may contribute to BBB breakdown in Alzheimer disease. These findings identify potential therapeutic targets, such as restoring NSC support or modulating specific transporters and mucins, to preserve BBB integrity in LOAD. The model provides a platform for studying LOAD pathophysiology and testing drug candidates, though further studies with additional donors and protein-level validation are needed.

PICO

PPOPULATION

Human induced pluripotent stem cell-derived brain capillary endothelial-like cells (BCECs) from a LOAD patient (APOE4/4, age 76) and a healthy elderly control (APOE3/3, age 64)

IINTERVENTION

Co-culture of BCECs with isogenic astrocytes, microglia-like cells, neural stem cells, or pericytes

OOUTCOME

Transendothelial electrical resistance (TEER), sodium fluorescein permeability (PC_NaF), and mRNA expression of 90 blood-brain barrier markers via high-throughput qPCR

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

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ORIGINAL PAPER

PMC10617216

Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease

Fluids and Barriers of the CNS

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ABSTRACT

Background The function of the blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the associated molecular mechanisms, particularly with respect to the high-risk APOE4/4 genotype, are not well understood. For this purpose, we developed a multicellular isogenic model of the neurovascular unit (NVU) based on human induced pluripotent stem cells. Methods The human NVU was modeled in vitro using isogenic co-cultures of astrocytes, brain capillary endothelial-like cells (BCECs), microglia-like cells, neural stem cells (NSCs), and pericytes. Physiological and pathophysiological properties were investigated as well as the influence of each single cell type on the characteristics and function of BCECs. The barriers established by BCECs were analyzed for specific gene transcription using high-throughput quantitative PCR. Results Co-cultures were found to tighten the barrier of BCECs and alter its transcriptomic profile under both healthy and disease conditions. In vitro differentiation of brain cell types that constitute the NVU was not affected by the LOAD background. The supportive effect of NSCs on the barrier established by BCECs was diminished under LOAD conditions. Transcriptomes of LOAD BCECs were modulated by different brain cell types. NSCs were found to have the strongest effect on BCEC gene regulation and maintenance of the BBB. Co-cultures showed cell type-specific functional contributions to BBB integrity under healthy and LOAD conditions. Conclusions Cell type-dependent transcriptional effects on LOAD BCECs were identified. Our study suggests that different brain cell types of the NVU have unique roles in maintaining barrier integrity that vary under healthy and LOAD conditions. Graphical abstract . Supplementary Information The online version contains supplementary material available at 10.1186/s12987-023-00471-y.

CITATION EXPORT

Undine Haferkamp, Carla Hartmann, Chaudhry Luqman Abid et al.. Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease. Fluids and Barriers of the CNS. (2023). https://doi.org/10.1186/s12987-023-00471-y

AUTHORS

17 · 8 CENTRES

Undine Haferkamp, Carla Hartmann, Chaudhry Luqman Abid, Andreas Brachner, Alevtina Höchner, Anna Gerhartl, Bernadette Harwardt, Selin Leckzik, Jennifer Leu, Marco Metzger, Marina Nastainczyk-Wulf, Winfried Neuhaus, Sabrina Oerter, Ole Pless, Dan Rujescu, Matthias Jung, Antje Appelt-Menzel

1https://ror.org/01s1h3j07grid.510864.eFraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, 22525 Hamburg, Germany
2https://ror.org/05gqaka33grid.9018.00000 0001 0679 2801Institute for Physiological Chemistry, Medical Faculty of the Martin, Luther University Halle-Wittenberg, Hollystrasse 1, 06114 Halle (Saale), Germany
3grid.4332.60000 0000 9799 7097Center Health and Bioresources, Competence Unit Molecular Diagnostics, AIT Austrian Institute of Technology GmbH, Vienna, 1210 Austria
4https://ror.org/05gnv4a66grid.424644.40000 0004 0495 360XFraunhofer Institute for Silicate Research ISC, Translational Center Regenerative Therapies (TLC-RT), 97070 Würzburg, Germany
5https://ror.org/03pvr2g57grid.411760.50000 0001 1378 7891Chair Tissue Engineering and Regenerative Medicine (TERM), University Hospital Würzburg, 97070 Würzburg, Germany
6Institute of Legal Medicine, University Hospital, 06112 Halle (Saale), Germany
7https://ror.org/054ebrh70grid.465811.f0000 0004 4904 7440Department of Medicine, Faculty of Medicine and Dentistry, Danube Private University, Krems, 3500 Austria
8https://ror.org/05n3x4p02grid.22937.3d0000 0000 9259 8492Department of Psychiatry and Psychotherapy, Division of General Psychiatry, Medical University of Vienna, Vienna, 1090 Austria