Differentiating Human-Induced Pluripotent Stem Cells into Brain Microvascular Endothelial Cells

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

NOTE: Briefly, fibroblasts were reprogrammed to iPSC via mRNA-based genetic reprogramming. The iPSCs were maintained in stem cell medium (SCM) (see material list) and stored at a density of ~1.2 x 10² cells/mL with 1 mL of SCM, 10 μM with rho-associated protein kinase inhibitor (ROCKi) Y-27632, and 10% (v/v) dimethyl sulfide (DMSO), in cryopreserved vials in liquid nitrogen at -160 °C. All of the following procedures below are carried out in a biosafety cabinet unless stated otherwise.

1. Basement membrane matrix dilution and plate coating

1. Dilute (1:50) growth factor reduced basement membrane matrix purified from Engelbreth-Holm-Swarm tumor in Dulbecco's Modified Eagle Medium (DMEM) without phenol red.
2. Coat cell culture plates with the appropriate amount of diluted basement membrane matrix (i.e., 6-well plate = 1mL, 12-well plate = 0.5 mL) and incubate these plates at 37 °C for at least 1 hour.

2. Induced pluripotent stem cells (iPSC) maintenance

NOTE: The maximum confluency per well in a 6-well flat-bottom plate is ~1.2 x 10⁶ cells.

1. Thaw cryopreserved iPSCs into SCM with 10 μM Y-27632 and plate onto a 6-well plate coated with diluted growth factor reduced basement membrane matrix.
2. Maintain iPSCs in SCM with 10 μM Y-27632 for the first 24 hours after thawing. Switch to fresh medium after 24 hours.
3. Maintain iPSCs in SCM until cells reach 80-90% confluency before passaging.
   1. Calculate how many iPSCs will be needed for differentiation by multiplying desired density for differentiation (15,600 cells/cm2) by the surface area of the well. For a 6-well flat-bottom plate, multiply 15,600 cells/cm² by 9.6 cm² for a total of 149,760 cells/well.
4. To passage, wash the cells with Hanks' Balanced Salt Solution (HBBS). Then, incubate the cells with non-enzymatic ethylenediaminetetraacetic acid (EDTA) (see material list) for 5 minutes at 37 °C.
   1. Use a cell scraper to gently lift off the cells. Collect cells in fresh SCM.
   2. Plate cells onto cell culture plates coated with diluted SCM and maintain cells as described in step 2.3 or store them at ~1.2 x 10⁶ cells/mL in 1 mL of SCM, 10 μM Y-27632, and 10% DMSO (v/v) in cryopreserved vials in liquid nitrogen at temperature of -160 °C.

3. Differentiation of iPSCs to BMECs

NOTE: Non-enzymatic EDTA separates cells into clumps. Enzymatic EDTA (see Table of Materials) separates cells into single cell suspension. Retinoic acid (RA) should be protected from light.

1. Wash iPSCs once with Dulbecco's Phosphate Buffer Saline (DPBS). Incubate with enzymatic EDTA (1 mL for 6-well plate, 0.5 mL for 12-well plate, and 0.25 mL for 24-well plate) for approximately 5 minutes at 37 °C to yield a single cell suspension.
2. Collect cells and centrifuge at 300 x g (relative centrifugal force) for 5 minutes at room temperature. Resuspend cell pellets in SCM containing 10 μM Y-27632.
3. Determine cell density using Trypan Blue and automated cell counter or a hemocytometer device. Plate cells at a density of 15,600 cells/cm² or 149,760 cells/well of a 6-well flat-bottom plate (with a surface area of 9.6 cm2/well) in SCM containing 10 μM Y-27632 for 24 hours.
4. Initiate differentiation after 24 hours by changing SCM to E6 medium. Change E6 medium daily for the next 4 days.
5. On day 4 of differentiation, replace E6 medium with human endothelial serum-free medium (hESFM) supplemented with diluted (1:200) B27 supplement, 20 ng/mL basic fibroblast growth factor (bFGF), and 10 μM RA. Do not change this medium for the next 48 hours.
6. Prepare 200 mL of hESFM with diluted (1:200) B27, mix 1 mL of 50x concentrated B27 supplement to 199 mL of hESFM.
7. Prepare 20 ng/mL of bFGF by reconstituting 50 μg of bFGF in 250 μL of Tris buffer (5 mM Tris, pH 7.6, 150 mMsodium chloride, NaCl) to make 200 μg/mL stock solution. Prepare 200 mL of hESFM containing 20 ng/mL bFGF by mixing 20 μL of 200 μg/mL bFGF with 200 mL of hESFM.
8. Prepare 10 μM RA by first making a 40 mg/mL of RA stock solution by adding 2.5 mL of DMSO to 100 mg of RA powder. Dilute this concentration to 3 mg/mL to make a 10 mM stock solution. Prepare 200 mL of hESFM containing 10 μM RA by mixing 200 μL of 10μM RA in 200 mL hESFM.

4. Coating collagen IV (COL4) and fibronectin (FN) Matrix for Purification of iPSC-Derived BMEC

1. Add 2 mL of sterile water to 2 mg of FN to make 1 mg/mL FN stock solution. Add 5 mL of sterile water to 5 mg of COL4 to make a 1 mg/mL COL4 stock solution.
   1. Allow FN to dissolve for at least 30 minutes at 37 °C and the COL4 to dissolve at room temperature.
2. Dilute FN stock solution in sterile water to a final concentration of 100 μg/mL and COL4 stock solution to a final concentration of 400 μg/mL.
3. Coat the desired plates (6-well plate = 1 mL of COL4/FN solution, 12-well plate = 0.5 mL, 24-well plate= 0.25 mL, and 12-transwell filtered plate = 0.25 mL) with the mixture of 400 μg/mL COL4 and 100 μg/mL FN.
4. Incubate plates for a minimum of 2 hours or overnight at 37°C; for Transwell filtered plates, a minimum of 4 hours is recommended.

5. Sub-culture and purification of iPSC-Derived BMECs

NOTE: Incubation with enzymatic EDTA may take longer than 15 minutes depending on the confluency of the cells on day 6 of differentiation.

1. On day 6 of differentiation, wash cells twice with DPBS. Incubate with 1 mL of enzymatic EDTA for at least 15 minutes at 37°C until a single cell suspension is obtained.
2. Collect cells via centrifugation at 300 x g for 5 minutes at room temperature. Resuspend cell pellets with fresh hESFM with diluted (1:200) B27 supplement, 20 ng/mL bFGF, and 10 μM RA.
3. Seed cells onto plates coated with a mixture of 400 μg/mL COL4 and 100 μg/mL FN. Seed cells using a ratio of 1 well of a 6-well plate to 3 wells of a 12-well plate, 3 wells of a 12-transwell filtered plate, or 6 wells of a 24-well plate.