Undergraduate Researcher University of Rochester Rochester, New York, United States
Introduction: Alzheimer’s Disease (AD) is the most prevalent form of dementia significantly affecting millions worldwide. A critical aspect of AD pathology is the degradation of the blood-brain barrier (BBB), an essential structure for maintaining neuronal function. Emerging evidence suggests that the ε4-allele of apolipoprotein E gene (ApoE4) fails to suppress proinflammatory pathways, leading to BBB instability. This research aims to examine ApoE associated pathology using in vitro models of BBB to investigate the mechanisms by which ApoE4 degrades BBB stability. Here, human induced pluripotent stem cells (iPSCs) were differentiated into brain pericyte-like cells (BPLCs). Those BPLCS were seeded into the microdevices used in this research. Two donors were used: an ApoE3 homozygous healthy control donor and an ApoE4 homozygous sporadic AD donor. These BPLCs were evaluated by immunostaining some of the basement membrane proteins present in the microdevices.
All experiments were conducted in vitro using microchip devices featuring ultrathin, nanoporous silicon nitride membranes known as the µSiM. The advantages of this model are its ultrathin membrane enabling unhindered diffusion between the compartments and glass-like imaging. Utilizing this tissue-on-a-chip model, this research will compare ECM production in brain pericyte-like cells derived from ApoE4 and ApoE3 homozygous donors. The methodology includes culturing BPLCs, staining for ECM proteins, and assessing ECM production through image analysis. Further, differentiation of endothelial cells from the same donors will complement the pericyte studies.
Materials and
Methods: The experiments were conducted using nanoporous membranes with pores approximately 60 nm in diameter. Once the chip containing the nanoporous membrane is assembled into the device, it creates a bottom channel space and a top well space separated through the chip and the membrane. This design allows for two different cell types to grow in different spaces but communicate through the pores on the ultra-thin membrane.
Healthy control and sporadic AD BPLCs were thawed and cultured in 6-well plates and passaged at 90-100% confluence. BPLCs were seeded into the bottom channel of the μSiM device. After BPLCs adhere to the nanoporous membrane, the media is switched to a brain endothelial cell media and changed daily until 7 days after seeding day.
To quantify the basement membrane proteins, μSiM devices are fixed and immunostained for collagen IV, fibronectin, and laminin. Devices are then stained with Hoechst to mark the nuclei and imaged on a dragonfly spinning disk confocal microscope at 10x and 40x, using a z-stack to capture the depth of the basement membrane. FIJI/ImageJ is used to determine the mean fluorescence intensity per z-step of each channel, which is then graphed against micron to study the basement membrane protein expression at different layers.
Results, Conclusions, and Discussions: After aligning the membrane in different devices in each channel, Collagen IV, Fibronectin, and Laminin were on average more intense on ApoE4 sporadic AD BPLCs as compared to ApoE3 healthy control BPLCs. At first glance, this appears contradictory to the prediction, as ApoE4 is the greatest risk factor leading to BBB vulnerability. On the other hand, the confocal images under the 10x microscope show that the production of basement membrane is lower than anticipated in both ApoE3 and ApoE4 devices, suggesting the lack of environmental cues for the basement membrane proteins to develop normally in this experiment. As mentioned earlier, the µSiM device has a top well and bottom channel that allows for coculture of different cells, such as coculturing endothelial cells and pericytes. Since pericytes surround and support the endothelial cells in capillaries in vivo, the experiment could be repeated in the future with endothelial cell and pericyte co-cultured devices to examine the difference in the production of basement membrane proteins in ApoE4 devices and ApoE3 devices.
