Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells
July 14th, 2020
The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.
This protocol demonstrates the usage of iPSC-derived brain-like endothelial cells to examine host pathogen interactions at the blood-brain barriers, such as the meningeal blood-CSF barriers, in a model that can better mimic brain endothelial cells in vivo. In vitro examination of brain endothelial cell barrier properties and interactions with human-specific pathogens can be challenging due to limitations in modeling. iPSC-derived brain endothelial cells are particularly useful for these applications as they possess superior barrier phenotypes and are of human origin.
Maintain the induced pluripotent stem cells, or iPSCs, at 37 degrees Celsius and 5%carbon dioxide in six-well plates with two milliliters of stem cell maintenance medium. To passage the cells, aspirate the medium from a well that is not confluent and add one milliliter of non-enzymatic cell dissociation reagent. Then incubate the plate at 37 degrees Celsius for seven minutes.
Meanwhile, replace the matrix gel solution on a new six-well plate with two milliliters of fresh stem cell maintenance medium per well. Aspirate the cell dissociation reagent, taking care not to aspirate cells that are still attached to the plate. Rinse the bottom of the well with six milliliters of stem cell maintenance medium until all cells are completely detached.
Seed the new six-well plate with varying densities, typically one to six, or one to 12 for normal maintenance. Shake the plate back and forth and left to right, pausing in between alternating shaking motions until the medium has settled. Then put it in the incubator.
Add one milliliter of enzymatic cell dissociation reagent into each well and incubate the plate at 37 degrees Celsius for seven minutes. To deactivate the cell dissociation reagent, transfer one milliliter of the cell suspension into a 15-milliliter tube with at least two milliliters of fresh stem cell maintenance medium. Spin down the cell suspension at 1500 G for five minutes.
Then resuspend the cell pellet in one milliliter of stem cell maintenance medium and count the cells with a hemocytometer. Resuspend 7.5 times 10 to the fifth cells in 12 milliliters of stem cell maintenance medium and 10 micromolar rock inhibitor. Aspirate matrix from a T75 flask and transfer the cell suspension to the flask.
Shake the flask to distribute the cells and place it in the incubator. Refresh the media every day for the next two days to remove rock inhibitor and support the growth of stem cell colonies. On the third day, begin differentiation by changing the media to an unconditioned medium.
Change the medium daily for the next five days. Six days after starting differentiation, selectively expand the endothelial cell population by switching to endothelial cell or EC medium with 20 nanograms per milliliter bFGF and 10 micromolar retinoic acid. Incubate the cells for two days.
Prepare collagen IV and fibronectin solution according to manuscript directions, and use it to coat cell culture plates and membrane inserts. Aspirate the EC medium from the cells and add 12 milliliters of cell dissociation reagent. Incubate the flask at 37 degrees Celsius until 90%of the cells have detached.
During the incubation time, remove the coating solution from the previously prepared plates and inserts, and let them dry in a sterile hood. Once the cells have detached, use a 10-milliliter pipette to create a single cell suspension. Transfer the cells to a 50-milliliter tube and dilute them with an equal volume of human endothelial serum-free medium.
Then count the cells with a hemocytometer. Pellet the cells at 1500 G for 10 minutes. Then resuspend them in freshly prepared EC medium with bFGF and retinoic acid for a concentration of 2 million cells per milliliter.
Add 500 microliters of the cell suspension to the top of a 12-well insert and 1.5 milliliters of medium to the bottom. Distribute the cells evenly across the insert and incubate the plate at 37 degrees Celsius and 5%carbon dioxide. On the next day, change the media to EC without bFGF or retinoic acid.
Place the epithelial volt/ohm meter in the biosafety hood and connect the electrodes. Disinfect the electrodes by submerging them in 70%ethanol for at least five minutes and let them dry completely. Remove the cells from the incubator and immediately measure the transendothelial electrical resistance, or TEER, by placing the shorter electrode on top of the insert and the longer electrode into the medium surrounding the insert.
On the day before the infection experiment, start an overnight culture of the bacteria from frozen stock. Streak Neisseria meningitidis onto Columbia Agar with 5%sheep blood and incubate at 37 degrees Celsius and 5%carbon dioxide overnight. Pathogenic bacteria, such as Neisseria meningitidis, have the potential to cause disease in humans.
It's important to properly train individuals working with such microorganisms and always use best practices and wear proper PPE. On the next day, prepare fresh PPM plus media according to manuscript directions and inoculate 10 milliliters of the media with the bacteria and incubate the culture. While the bacteria are incubating, replace the medium in the iPSC-derived brain endothelial cells, then centrifuge the bacterial culture at 4, 000 G for 10 minutes.
Aspirate the media and resuspend the pellet in 250 microliters of PBS. Adjust to an OD600 of approximately 0.4, which is roughly one times 10 to the eighth colony-forming units per milliliter. Dilute the bacteria to the desired multiplicity of infection with EC medium and infect the cells with 100 microliters of the prepared bacterial suspension per well.
Then incubate the cells for the desired time of infection. When differentiated properly, the iPSC-derived brain endothelial cells exhibit tight barrier properties that are often greater than 2000 ohm centimeter squared and endothelial markers, such as VE-Cadherin and CD-31. Additionally, they express and localize the tight junction markers Claudin-5, Occludin, and ZO-1, as well as transporters, such as Glut-1.
Upon infection with Neisseria meningitidis, the cells upregulate neutrophilic, pro-inflammatory cytokines, such as CXCL8, CXCL1, CXCL2, CCL20, and IL6. When performing cell differentiation, daily maintenance and achieving the initial optimal seeding density when splitting the cells is critical. Following the steps outlined here, researchers can collect a variety of samples to investigate many aspects of post-pathogen interaction from inflammatory responses to brain endothelial cell dysfunction.
The advent of brain endothelial cells derived from iPSCs have opened the doors for the brain barriers field to investigate a wide range of diseases where endothelial cells are compromised. As certain CNS pathogens are human-specific, we demonstrate the use of a human-based system with the human-specific pathogen Neisseria meningitidis
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Overview
This protocol demonstrates the usage of iPSC-derived brain-like endothelial cells to examine host pathogen interactions at the blood-brain barriers. It highlights the preparation of Neisseria meningitidis for infection and sample collection for molecular analyses.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Infectious Diseases
Background
- Brain endothelial cells form a critical barrier in the central nervous system.
- Modeling human-specific pathogen interactions is challenging in vitro.
- Induced pluripotent stem cells (iPSCs) provide a human-derived model.
- iPSC-derived brain endothelial cells exhibit superior barrier properties.
Purpose of Study
- To create a model that mimics brain endothelial cells in vivo.
- To investigate host-pathogen interactions at the blood-brain barrier.
- To facilitate molecular analyses of infection processes.
Methods Used
- Maintain iPSCs at 37 degrees Celsius and 5% carbon dioxide.
- Use non-enzymatic cell dissociation for passaging.
- Prepare Neisseria meningitidis for infection assays.
- Collect samples for further molecular analysis.
Main Results
- iPSC-derived brain-like endothelial cells exhibit enhanced barrier properties.
- Successful modeling of pathogen interactions at the blood-brain barrier.
- Insights into the mechanisms of infection by Neisseria meningitidis.
- Potential applications in studying neurological infections.
Conclusions
- iPSC-derived cells are valuable for studying brain endothelial functions.
- This model can improve understanding of CNS infections.
- Future research can leverage this approach for therapeutic insights.
Frequently Asked Questions
Executive Industry Relevance
Modeling human-specific pathogen interactions at the blood-brain barrier remains a critical challenge in CNS infectious disease research due to limitations of existing in vitro systems. iPSC-derived brain endothelial cells provide a human-relevant platform with enhanced barrier properties that better phenocopy the in vivo microenvironment, enabling mechanistic de-risking of therapeutic candidates targeting meningitis and related neuroinflammatory conditions. This approach supports predictive confidence in target validation by allowing direct observation of host-pathogen dynamics in a scalable, renewable human cell system.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses regarding blood-brain barrier penetration and immune evasion mechanisms of human-specific pathogens like Neisseria meningitidis.
- Operational Value: Provides a renewable source of human brain endothelial cells with measurable barrier integrity via TEER and tight junction marker expression for consistent target validation assays.
- Predictive Value: Supports assessment of biomarker candidates such as CXCL8, IL6, and CCL20 that are upregulated upon infection, aiding in early pathophysiological de-risking.
Screening & Assay Development
- Scientific Value: Generates standardized, barrier-competent endothelial monolayers suitable for high-fidelity screening of compounds or biologics designed to inhibit pathogen translocation or modulate host inflammatory responses.
- Operational Value: Enables reproducible quantification of infection-dependent readouts including cytokine release and barrier dysfunction, facilitating assay optimization and cross-lab comparability.
- Scalability: iPSC-derived cells can be expanded and differentiated in bulk, supporting assay readiness for downstream screening campaigns.
Translational & Preclinical Research
- Scientific Value: Bridges discovery and preclinical stages by modeling key events in meningococcal meningitis pathogenesis, including barrier disruption and innate immune activation in a human cellular context.
- Translational Continuity: Expresses clinically relevant transporters (e.g., Glut-1) and endothelial markers (VE-Cadherin, CD-31, Claudin-5), enhancing relevance for biomarker alignment and PK/PD modeling.
- Risk-Adjusted Decision-Making: Enables evaluation of lead compounds for their ability to preserve barrier function or suppress pro-inflammatory signaling, informing go/no-go criteria in preclinical development.
Pipeline & Workflow Integration
The method fits within the early discovery continuum, supporting target validation through mechanistic insight into blood-brain barrier interactions, and enabling assay development for screening interventions that mitigate pathogen-induced endothelial dysfunction.
- Discovery Biology: Facilitates hypothesis testing on pathogen adherence, invasion, and immune activation mechanisms in a human-relevant endothelial model.
- Screening: Delivers quantitative, barrier-associated outputs such as TEER dynamics and cytokine profiles that allow comparison of experimental conditions.
- Analytics: Generates measurable dependent variables including OD600-adjusted bacterial titers, TEER values (exceeding 2000 Ω·cm² when intact), and multiplex cytokine levels for statistical comparison.
- Translational Research: Models human-specific host-pathogen interactions that inform preclinical continuity for CNS-targeted therapeutics.
- Enterprise Reuse: Establishes a scalable, human-based platform applicable across multiple neurotropic pathogens beyond Neisseria meningitidis, supporting broad target portfolio applications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing reliance on non-human models that fail to recapitulate human-specific pathogen tropism.
- Operational Value: Delivers standardized, barrier-characterized endothelial cells with defined differentiation protocols, enhancing reproducibility across teams and sites.
- Strategic Value: Improves capital efficiency by enabling early de-risking of CNS-active candidates through mechanistic insight into barrier penetration and immune modulation.
- Portfolio Impact: Supports risk-adjusted prioritization of targets based on their ability to preserve endothelial function or attenuate pathogen-induced inflammation in a human-relevant system.
Implementation Considerations
- Requires expertise in stem cell culture, endothelial differentiation, and barrier assay techniques including TEER measurement.
- Dependent on controlled incubation conditions (37°C, 5% CO₂) and access to specialized reagents such as ROCK inhibitor, bFGF, and retinoic acid for differentiation.
- Necessitates standardization of infection parameters including MOI, incubation time, and sample collection timing for consistent inflammatory readouts.
- Involves biosafety level 2 practices for handling Neisseria meningitidis, including PPE use and proper waste decontamination.
- Limited by the time-intensive differentiation protocol (approximately 12 days) and need for routine monitoring of barrier integrity and marker expression.
Biopharma FAQs
Chapters in this video
0:05
Introduction
0:42
IMR90-4 Cell Culture Maintenance
2:15
Differentiation of Brain Endothelial Cells from Human iPSCs
5:57
Transendothelial Electrical Resistance (TEER) as a Measure of Barrier Tightness
6:35
Preparation of Bacteria and Infection of iPSC-BECs
8:22
Results: Upregulation of Cytokines by iPSC-BECs upon Infection with Neisseria meningitidis
9:16
Conclusion
