Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

NOTE: All media / reagent preparation, stem-cell maintenance, and differentiation steps are adapted from Stebbins et al.²².

1. Preparation of materials required for iPSC culture and BEC differentiation.

1. Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
   1. Aliquot basement membrane matrix gel (e.g., Matrigel) into 2.5 mg aliquots and store at -20 °C.
      NOTE: Work quickly when handling the matrix gel and aliquot on ice, as it forms a gel above 4 °C and cannot be aliquoted once it has solidified.
   2. For coating TC plastic, quickly add one aliquot of matrix gel to 30 mL of Dulbecco's Modified Eagle Medium (DMEM)/F12 medium in a 50 mL conical tube. Add 1 mL of the medium onto the frozen matrix gel, pipette up and down until it is thawed, and transfer immediately to the 50 mL conical tube with the remaining medium.
   3. Use 1 mL of this matrix gel-coating solution per well of a 6-well plate and 12 mL per T75 flask.
      NOTE: Matrix gel-coated TC plastic can be prepared up to two weeks before it is used. It is, however, critical to avoid that the matrix gel solution dries out, which may require occasional addition of more DMEM/F12 on top of the wells.
2. Prepare stem-cell maintenance medium by adding 50 mL of 50x supplement to 450 mL of stem cell maintenance basal medium in the sterile environment of a biosafety cabinet.
   NOTE: Other stem-cell maintenance media (mTeSR and E8) have been used in other studies^{13,14,15, 22,23,24,25}.
3. To prepare 500 mL of unconditioned medium (UM), combine 392.5 mL of DMEM/F12 with 100 mL of knock out serum replacement (KOSR), 5 mL of non-essential amino acids, L-glutamine at a final concentration of 1 mM, and 3.5 µL of β-mercaptoethanol. Filter-sterilize and store at 4 °C for up to 2 weeks.
4. To prepare 200 mL of endothelial cell (EC) medium plus retinoic acid (RA) plus bFGF, combine 198 mL of human endothelial serum free medium (hESFM), 2 mL of filter-sterilized platelet-poor derived serum (PDS), and 20 ng/mL bFGF. Filter sterilize and store for up to 2 weeks at 4 °C. Just before addition to cells, add 10 μM of RA to EC medium.
   NOTE: As PDS has been discontinued and may therefore be limited, this protocol has been successfully conducted using B27 in place of PDS^15,23,26.
5. To prepare 200 mL of EC medium without RA or bFGF, combine 198 mL of hESFM and 2 mL of filter sterilized PDS and filter sterilize. Store for up to 4 weeks at 4 °C.

2. Maintenance IMR90-4 cell culture

NOTE: Here we use the IMR90-4 cell line as an example, however other induced pluripotent stem-cell lines such as CC3, CD10, CD12, DF19-9-11T, 83iCTR, 00iCTR, and CS03iCTRn2 have been successfully employed for differentiation into BECs^{13,14,15,16,17,23,27,28}.

1. Culture iPSCs at 37 °C in 5% CO₂ and typically maintain on 6-well plates at various densities with 2 mL of stem-cell maintenance medium per well.
2. For maintenance of the iPSC culture, select a single well for passage that is not confluent and has open spaces between colonies.
   1. Aspirate the culture medium, add 1 mL of non-enzymatic cell dissociation reagent, and incubate at 37 °C for 7 min. While incubation is ongoing, replace the matrix gel solution, on a new 6-well plate, with 2 mL of fresh stem-cell maintenance medium per well.
   2. Carefully aspirate the non-enzymatic cell dissociation reagent taking care to not aspirate cells still attached to the plate.
   3. Add 6 mL of stem-cell maintenance medium and rinse the well bottom a few times until all cells are completely detached. Then, seed the new 6-well plate with varying densities, typically 1:6 or 1:12 for normal maintenance.
      NOTE: Using the aforementioned ratios, the cells are split approximately twice a week.
   4. Move the plate to the incubator and distribute the seeded cells equally across the wells by shaking the plate back and forth and left to right, pausing in between alternating shaking motions until the medium has settled.

3. Differentiation of brain endothelial cells from human iPSCs

1. Plating of iPSCs for differentiation (day -3 of the differentiation protocol)
   1. Per IMR90-4 maintenance culture well used to plate for differentiation, aspirate the culture medium, add 1 mL of enzymatic cell dissociation reagent per well, and incubate at 37 °C for 7 min.
   2. Inactivate the enzymatic cell dissociation reagent by transferring the 1 mL of dissociated cell suspension into a 15 mL conical tube with at least 2 mL of fresh stem-cell maintenance medium per 1 mL of cells. Spin down the cell suspension at 1,500 x g for 5 min.
   3. Resuspend the cell pellet in 1 mL of stem-cell maintenance medium per well of IMR90-4 cells used, and count the cells using a hemocytometer.
      NOTE: It may be helpful to dilute 1:1 with 0.4% trypan blue to distinguish between live and dead cells when counting. Depending on the density of iPSCs, one well of a 6-well plate usually yields 1‒2 x 10⁶ cells.
   4. For differentiation in a T75 flask, add 7.5 x 10⁵ cells to 12 mL of stem-cell maintenance medium and ROCK inhibitor (Y27632 dihydrochloride) at a final concentration of 10 µM. Aspirate matrix gel-coating solution from a T75 flask and transfer the cell suspension to the flask. Distribute the cells equally by shaking the flask back and forth and left to right (see step 2.2.4) and incubate at 37 °C and 5% CO₂.
      NOTE: It is critical to add ROCK inhibitor at this step to enhance survival of the dissociated single stem cells^25,29. Cells should be evenly distributed across the flask and in singlets exhibiting a spread, mesenchymal-like morphology due to the ROCK inhibitor treatment²².
2. On day -2 and day -1, change media to fresh stem-cell maintenance medium; 12 mL per T75.
3. On day 0, start differentiation by changing media to UM; 12 mL per T75.
4. Change UM daily (day 1 – day 5).
   NOTE: The cells typically reach confluence after 2 to 3 days in UM, which can be observed with the naked eye or through an inverted bright field microscope. As the differentiation progresses, nestin⁺ “neural tracts” become visible with PECAM-1⁺ cells in between as previously described^13,14.
5. Selectively expand the endothelial cell population by switching to EC medium with 20 ng/mL bFGF and 10 µM retinoic acid (RA) (day 6) and incubating for two days. EC medium with bFGF can be prepared up to two weeks prior. RA is added from frozen stock on the day of use (e.g., 1 µL of 10 mM RA stock per 1 mL of EC + bFGF).
   NOTE: Successful differentiation can also be achieved without supplementation of RA at days 6 and 8. Omission of RA will, however, yield BECs with reduced TEER^12,13,14.
6. Coat cell culture plates and membrane inserts (e.g., Transwell) with collagen IV and fibronectin (day 7), for purification of the BECs and following experiments.
   1. For coating of membrane inserts, combine 4 parts collagen IV (1 mg/mL in 0.5 mg/mL acetic acid), 1 part fibronectin (1 mg/mL) and 5 parts sterile tissue-grade water. ECM solution can be diluted 1:5 for coating cell culture plates (i.e., 4 parts collagen IV, 1 part fibronectin, 45 parts water). Incubate with coating solution at 37 °C overnight.
      NOTE: Plates and membrane inserts can also be coated for at least 4 h prior to subculturing on the same day of the purification step.
7. Purify BECs by subculturing the differentiated cells on collagen IV and fibronectin-coated plates or membrane inserts (day 8).
   1. Aspirate EC medium and add enzymatic cell dissociation reagent (12 mL per T75). Incubate at 37 °C until 90% of cells have detached from the flask.
      NOTE: Cell dissociation can take up to 1 h.
   2. During the incubation time, remove the collagen IV/fibronectin coating solution from previously prepared plates/inserts and let them dry in a sterile hood. It takes approximately 20 min for the inserts to dry.
   3. Once the cells have detached, wash them off the flask using a 10 mL pipette. Pipette up and down to achieve a single cell suspension.
      NOTE: Single cell suspension is important for reliable cell counting and to achieve solid monolayers.
   4. Dilute with at least equal volume of fresh hESFM in a 50 mL conical tube and count cells using a hemocytometer.
   5. Pellet the cells at 1,500 x g for 10 min.
   6. Resuspend cells in appropriate volume of freshly prepared EC + bFGF + RA to achieve a suspension of 2 x 10⁶ cells/mL for seeding on membrane inserts. Add 500 µL (1 x 10⁶ cells) on top of a 12-well insert and 1.5 mL of EC + bFGF + RA medium on the bottom. For seeding on 24 and 48-well plates, dilute cell suspension 1:2 and add 500 µL (5 x 10⁵ cells) and 250 µL (2.5 x 10⁵ cells) per well, respectively. Distribute cells evenly across the well/insert (see step 2.2.4) and incubate at 37 °C under 5% CO₂.
8. Change media on plates/transwells to EC without bFGF or RA (day 9).
9. Conduct infection experiments, TEER measurement, and immunofluorescence staining as described in the following sections (day 10).
   NOTE: Successfully differentiated and purified BECs typically reach peak TEER on day 10 and express characteristic markers of brain endothelial cells such as PECAM-1 (CD31) and VE-cadherin, the glucose transporter GLUT-1, efflux transporters such as p-glycoprotein, and tight junction components ZO-1, Occludin, and Claudin-5 ^{13,14,16,17,19,22}. Refer to Lippmann et al., Stebbins et al. and others for further details and images of the cell types, morphologies, and expression of cell type specific markers during the differentiation process^{13,14,15,16,17,19,22}. Representative images of the IMR90-4 cells at different stages in the BEC differentiation process can be found in Supplementary Figure 1.

4. Transendothelial electrical resistance (TEER) as a measure of barrier tightness

NOTE: TEER is usually read on membrane inserts on days 9 and 10 of differentiation to confirm successful generation of barrier forming iPSC-BECs (Figure 1A).

1. Place the epithelial volt-ohm meter (EVOM) in the sterile environment of a biosafety hood and connect the electrode to the EVOM.
2. Disinfect the electrode by submerging it in 70% EtOH for at least 5 min and let it dry completely.
   NOTE: Longer incubation in 70% EtOH or decontamination of the electrode using 5% hypochlorite solution is possible if needed.
3. Retrieve the iPSC-BECs on membrane inserts from the incubator and measure TEER.
   NOTE: It is important to read TEER rapidly after removal from the incubator as temperature change may impact TEER measurement.
4. Read TEER by dipping the electrode into the medium so that the shorter electrode is placed on top of the insert and the longer electrode reaches into the medium surrounding the insert.
   NOTE: Make sure the electrodes at the tips of the “chopsticks” are completely covered by liquid. If needed, tilt the well to achieve this before setting the plate down again for measuring.

5. Immunofluorescence (IF) staining to validate BEC phenotype

NOTE: To validate the quality of the fully differentiated and purified cells, iPSC-BEC monolayers are stained for the characteristic markers of brain endothelial cells on day 10 of the differentiation process as previously described (Figure 1B‒G) ^{13,14,15,16,17,19,22}.

1. Aspirate the medium and wash 1x with PBS.
2. Fix cells with ice cold methanol at room temperature (RT) for 15 min.
3. Wash 3x with phosphate buffered saline (PBS) and block with 10% fetal bovine serum (FBS) in PBS at RT for 1 h.
4. Aspirate and add primary antibodies diluted in blocking solution. Incubate at 4 °C overnight.
   NOTE: Refer to the Table of Materials and Stebbins et al. for information related to source and dilution of the antibodies²².
5. Wash 3x with PBS before adding secondary antibody diluted in blocking solution. Incubate at RT for 1 h. Protect samples from light from this point on.
6. Wash 2x with PBS. Then, add DAPI at a 1:5,000 dilution in PBS and stain at RT for 15 min.
7. Wash 1x leaving the PBS on and take images using a fluorescence microscope.

6. Preparation of bacteria and infection of iPSC-BECs

1. On the day before the infection experiment (day 9 of differentiation), start an overnight culture of the bacteria from frozen stock. For infection with Nm, streak bacteria onto Columbia agar with 5% sheep blood (blood-agar). Incubate bacterial cultures at 37 °C and 5% CO₂ overnight.
2. The next day (day 10), prepare fresh PPM+ by supplementing Protease peptone media (PPM) with 50 µL of 2 M MgCl₂, 50 µL of 2 M NaHCO₃, and 100 µL Kellogg’s supplement per 10 mL. Inoculate 10 mL of PPM+ medium in a 50 mL conical tube with Nm from the overnight culture plate using a sterile cotton swab. Incubate shaking at 200 rpm at 37 °C for 1.5 h (i.e., until bacteria are in logarithmic growth phase).
3. During bacterial incubation, prepare iPSC-BECs in a 24-well plate or on membrane inserts for infection by replacing the old medium with 400 µL of fresh EC medium (without bFGF or RA) per well/ top of membrane insert.
4. Centrifuge bacterial culture at 4000 x g for 10 min and aspirate the media. Resuspend the bacterial pellet in 250 µL of PBS.
5. In 4 mL of fresh PBS, use a portion of the bacterial cell suspension and adjust to an OD₆₀₀ of 0.4 (approximately 1 x 10⁸ CFU/mL).
6. Then, dilute the bacteria in cell culture medium (EC medium) according to the desired multiplicity of infection (MOI).
   NOTE: For example, for an MOI of 10, dilute 1:10 or 1:5 when infecting iPSC-BECs in a 24-well plate or on membrane inserts, respectively (1 x 10⁵ cells per monolayer in a 24-well plate). Addition of human serum is not included in the preparation of Nm for infection as was described in other manuscripts as it was observed to have a deleterious impact on the iPSC-BEC barrier phenotype as measured by TEER (data not shown)^30,31. However, the interaction of Nm and iPSC-BECs is unaffected with or without human serum (data not shown).
7. Infect iPSC-BECs in each well with 100 µL of the prepared bacteria suspension and incubate for the desired time of infection.
   NOTE: Expression of a number of proinflammatory cytokines and chemokines is elevated in iPSC-BECs when infected with Nm, most prominently after 8 h of infection as previously described by Martins-Gomes et al (Figure 2)¹⁹.

7. Innate immune activation by quantitative PCR

NOTE: Using a preferred RNA isolation, cDNA synthesis, and qPCR protocol, collect samples and run qPCR on selected cytokines.

1. 7.1. Collect the RNA from BEC samples after infection on day 10 of the differentiation protocol, using reagents form a commercially available RNA isolation kit (see the Table of Materials).
   ​NOTE: Avoid nuclease contamination of samples by working carefully in a cleaned and sterilized environment.
   1. Prepare lysis buffer and add 350 µL to each well/monolayer of iPSC-BECs.
   2. Collect the samples by pipetting up and down numerous times (e.g., 20x) and transferring the suspension to a sterile microcentrifuge tube.
      NOTE: Samples can be stored at -80 °C until ready for RNA isolation.
   3. Follow the protocol provided with the RNA isolation kit for RNA purification from cultured cells and tissue.
   4. After elution in nuclease-free water, keep RNA samples on ice to minimize any potential RNase activity.
   5. Estimate RNA concentrations on a spectrophotometer (e.g., Nanodrop).
2. Generate a cDNA library from the collected RNA using a cDNA synthesis kit (see Table of Materials).
   1. Set up reactions consisting of cDNA synthesis master mix, at least 200 ng (ideally 500 ng) of sample RNA, and nuclease-free water in a defined total reaction volume as described in the protocol of the cDNA synthesis kit.
   2. On a standard thermo cycler, run a program that is appropriate for the reagents of the cDNA synthesis kit used. For instance: 25 °C for 2 min, 55 °C for 10 min, 95 °C for 1 min.
   3. After synthesis, dilute the cDNA up to 1:10 in nuclease-free water and move samples to 4 °C for short term or -20 °C for long term storage.
      NOTE: Lower dilution may be necessary if RNA concentration was low (e.g., below 50 ng). The diluted cDNA can be stored at -20 °C for up to a year.
3. Perform qPCR on the cDNA samples targeting transcripts of innate immune response genes such as cytokines and chemokines with carefully designed and validated primers.
   NOTE: As primer design is very important for the quality of qPCR results, primer efficiency should be tested conducting a dilution series and the absence of multiple products should be confirmed on a DNA gel.
   1. Per 25 µL reaction, use 0.5 µL forward and 0.5 µL reverse primer (10 mM in nuclease-free water), 1 µL cDNA, 10.5 µL nuclease free H₂O, and 12.5 µL qPCR master mix.
   2. Perform the reaction on a qPCR machine using the following cycler protocol: (a) 4 °C for 2 min; (b) 95 °C for 15 min; (c) 95 °C for 15 s; (d) 60 °C for 1 min; cycle through (c) and (d) 45 times; (e) optional melt curve: 30‒99 °C in 1 °C increments; 25 °C for 5 min.
   3. For data analysis, use the ΔΔCT calculation to compare cytokine and chemokine expression levels to a reference housekeeping gene such as 18S or GAPDH.