Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

Protokoll

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 the 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.
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.
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. 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 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. Cells should be evenly distributed across the flask and in singlets exhibiting a spread, mesenchymal-like morphology due to the ROCK inhibitor treatment.
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.
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.
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 the 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 and incubate at 37 °C under 5% CO₂.
8. Change media on plates/transwells to EC without bFGF or RA (day 9).

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Materialien

Name	Company	Catalog Number	Comments
Accutase (1x)	Sigma	A6964
All-trans retinoic acid (RA)	Sigma	R2625
b-Mercaptoethanol	Merck (Sigma-Aldrich)	805740
Cell culture plates and flasks	Sarstedt
Centrifuge (Heraeus Megafuge 1.0R)	Thermo Scientific
Class II biosafety cabinet	Nuaire	NU-437-400E
Carbon dioxide incubator (DHD Autoflow Carbon dioxide Air-Jacketed Incubator)	Nuaire
Collagen IV	Sigma	C5533
Costar Transwell polyester filters (12- or 24-well)	Corning	3460, 3470
Fibronectin	Sigma	F1141
Hemacytometer (Neubauer)	A. Hartenstein	ZK06
Human basic fibroblast growth factor (bFGF)	PeproTech	100-18B
Human Endothelial Serum Free Medium (hESFM)	Gibco	11111-044
Inverted microscope (Wilovert)	Hund (Will Wetzlar)
iPS(IMR90)-4 cells	WiCell
Knockout serum replacement (KOSR)	Gibco	10828-028
L-glutamine (GlutaMAX)	Invitrogen	35050-038
Matrigel Matrix	Corning	354230
Nonessential amino acids (NEAA)	Gibco	11140-035
StemFlex basal medium + 50x StemFlex supplement	Gibco	A3349401	Stem-cell maintenance medium
Swinging Bucket Rotor (Heraeus #2704)	Thermo Scientific