Mar 24, 2020
Version 2
Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells V.2
- Ethan Lippmann1,
- Hannah Wilson2,
- Emma Neal1
- 1Department of Chemical Engineering, Vanderbilt University, Nashville, TN, USA;
- 2Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bd6ei9be
Manuscript citation:
A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs. Neal EH, Marinelli NA, Shi Y, McClatchey PM, Balotin KM, Gullett DR, Hagerla KA, Bowman AB, Ess KC, Wikswo JP, Lippmann S. Stem Cell Reports. 2019 Jun 11;12(6):1380-1388.doi: 10.1016/j.stemcr.2019.05.008
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working.
Created: March 24, 2020
Last Modified: March 24, 2020
Protocol Integer ID: 34726
Keywords: human pluripotent stem cells, hPSCs, BBB, endothelial cells, blood-brain barrier, defined differentiation, human induced pluripotent stem cell, iPSC, in vitro model
Abstract
Human induced pluripotent stem cell (iPSC)-derived developmental lineages are key tools for in vitro mechanistic interrogations, drug discovery, and disease modeling. iPSCs have previously been differentiated to endothelial cells with blood-brain barrier (BBB) properties, as defined by high transendothelial electrical resistance (TEER), low passive permeability, and active transporter functions. Typical protocols use undefined components, which impart unacceptable variability on the differentiation process. We demonstrate that replacement of serum with fully defined components, from common medium supplements to a simple mixture of insulin, transferrin, and selenium, yields BBB endothelium with TEER in the range of 2,000-8,000 Ω × cm2across multiple iPSC lines, with appropriate marker expression and active transporters. The use of a fully defined medium vastly improves the consistency of differentiation, and co-culture of BBB endothelium with iPSC-derived astrocytes produces a robust in vitro neurovascular model. This defined differentiation scheme should broadly enable the use of human BBB endothelium for diverse applications.
Schematic of E6 method for BBB differentiation
Attachments
Guidelines
We recommend the following antibodies to monitor BMEC differentiation:
Lippmann, E. S.; Al-Ahmad, A.; Azarin, S. M.; Palecek, S. P.; Shusta, E. V. A retinoic acid-enhanced, multicellular human blood- brain barrier model derived from stem cell sources. Sci. Rep. 2014, 4, 4160.
Note
The VE-Cadherin antibody listed above is no longer appropriate.
Materials
MATERIALS
B-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
Essential 8™ MediumGibco - Thermo Fisher ScientificCatalog #A1517001
Insulin solution humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #I9278
Recombinant Human FGF-basic (154 a.a.)peprotechCatalog #100-18B
Human Endothelial-SFMThermo FisherCatalog #11111044
Versene SolutionThermo FisherCatalog #15040066
Essential 6™ MediumThermo Fisher ScientificCatalog #A1516401
Human Holo-Transferrin Protein CFR&D SystemsCatalog #2914-HT-001G
Retinoic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2625-50MG
If desired, E8 and E6 may be purchased commercially rather than prepared in-house.
If purchasing E8 and E6 commercially, human holo-transferrin and human insulin solution are not needed.
Plasticware:
FISHER
Corning Tissue Culture Plates (6- or 12-well, 3513 or 3516)
500 ml filter-top bottles (S2GPT05RE)
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
REAGENT/MEDIUM PREPARATION:
E4 (prepared according to E4 large scale basal media production protocol)
Large batch previously prepared and stored at -80 °C . If the preparation is not urgent, remove a bottle of E4 from the -80°C and place it in the fridge overnight, which will allow the bottle slowly thaw. However, if preparation is desired for the same day, remove a bottle of E4 from the -80°C, place it on the countertop for ~00:20:00 , then place it in the 37 °C water bath until it thaws completely (~01:00:00 – 02:00:00 ). Make sure to follow these steps precisely, as premature addition of a frozen bottle of E4 to a water bath can result in rupture of the plastic bottle.
Insulin
Pre-made solution provided by Sigma (catalog #I9278) that needs no additional preparation. Bottles are stored at 4 °C .
Transferrin
Comes as a powder from R&D Systems (Human Holo-Transferrin, CF; catalog #2914-HT-001G). Add 50 mg of transferrin to 5 mL of phosphate-buffered saline (PBS), aliquot at 500 μl/vial, and store at -80 °C . This mixture does not need to be sterile-filtered.
E6 media (prepared according to E6 and E8 media preparation protocol)
Dispense the thawed bottle of E4 into a bottletop filter attached to a 500 ml glass bottle. Add 100 µL of insulin solution and 500 µL of transferrin solution. Vacuum filter and store at 4 °C . E6 media is stable indefinitely.
B27 Supplement
Thaw 10 ml bottle and mix thoroughly. Aliquot into sterile microcentrifuge tubes at 280 μl/tube and store at -20 °C . Upon thawing, unused portions of an aliquot may be stored at 4 °C for up to 1 week for further media preparation.
bFGF, 100 μg/ml (prepared according to E8 media protocol)
Thaw a 500 μl aliquot of bFGF and dilute 1:5000 in EC medium for a final concentration of 20 Mass Percent as described below. Divide the remaining bFGF in 100 μl aliquots and re-freeze at -80 °C . These remaining aliquots can be thawed and used for EC medium but cannot be refrozen a second time.
Retinoic acid (RA)
Dilute 50 mg RA in 16.6 mL DMSO to create a stock solution of 10 Mass Percent and store 1 mL aliquots at -80 °C . To prepare working stocks, divide a 1 ml stock tube into 50 μl aliquots and store at -20 °C . Dilute working stocks 1:1000 in EC medium for a final concentration of 10 micromolar (µM) .
EC medium w/ 200X B27 + 20 ng/ml bFGF
For 50 ml: add 250 µL of B27 and 10 µL bFGF to 50 mL of hESFM.
Good for up to two weeks at 4 °C .
EC medium w/ 200X B27
For 50 ml: add 250 µL of B27 to 50 mL of hESFM.
BBB differentiation (Day 0–4)
BBB differentiation (Day 0–4)
Note
Note: Cells are seeded for differentiation in E8 medium according to the standardized single cell seeding protocol
On day 0, aspirate E8 medium and add 2 mL of E6 per well.
Change medium every day using 2 mL of E6 per well.
BBB expansion (Day 4–6)
BBB expansion (Day 4–6)
At day 4 of E6 treatment, aspirate and add 2 mL of EC medium with bFGF (basic fibroblast growth factor) and 10 micromolar (µM) RA to each well.
Note
Medium is NOT changed during expansion phase.
BBB subculturing:
On day 6, subculture BBB onto plates and Transwell filters according to the following protocol:
Protocol
NAME
Protocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters
CREATED BY
Emma Neal
.
Please select between subculturing onto plates or filters.
Plates15 steps
Subculturing onto Plates using Accutase.
Coat plates with ECM plate solution for at least 01:00:00 at 37 °C . Volume depends on plate type (see Table):
| Plate type for subculture phase | Volume of ECM solution for coating | Working volume of EC media for cell culture | |
| 6-well | 800 μl | 2 ml | |
| 12-well | 250 μl | 1 ml | |
| 24-well | 200 μl | 500 μl | |
| 48-well | 100 μl | 400 μl | |
| 96-well | 50 μl | 200 μl |
Note
If desired, plates may be coated Overnight . If coating overnight, add necessary volume of ECM and an equal volume of ddH2O to each well to prevent excessive evaporation. If using glass plates, overnight incubation is needed to achieve adequate protein adsorption.
Aspirate plates and allow to dry in sterile hood (place the plate in the back of the hood and leave the lid slightly ajar).
Note
Plates only need to dry for 00:05:00 (can be aspirated during accutase incubation). Do not over dry!
Retrieve cells from incubator and transfer equal volume of spent media to 15 ml conical corresponding to the number of wells being accutased.
Note
For example, if accutasing 4 wells, save 4 ml of spent media and discard the rest.
Wash each well once with 2 mL PBS.
Add 1 mL accutase (warmed to Room temperature ) to each well.
Incubate at 37 °C , length of time depends on cell treatment:
If cells have not been treated with RA9 steps
If cells have not been treated with RA, incubate at 37 °C for 00:20:00 , or until cells are dissociated from plate (whichever comes first).
Using p1000, collect cells, and spray gently over surface 2–3x to dislodge any remaining cells. Triturate briefly to break up cell clumps.
Add cells to 15 ml conical containing spent media.
Spin down cells at 1000 rpm, 00:04:00 .
Aspirate media, and resuspend cells in appropriate volume of EC media. For 6- and 12-wells, cells are seeded based on a split ratio:
- 1 well of a 6-well plate is split to 1 well of a 6-well plate [1:1]
- 1 well of a 6-well plate is split to 3 wells of a 12-well plate [1:3]
- For smaller plates (24-, 48-, or 96-wells), seed 1 million cells/cm2.
- Multiply split ratio by the working volume found in the table to arrive at total volume of EC media in which to resuspend cells.
Thoroughly triturate 3 – 4 times to yield single cell suspension.
Add appropriate volume of cells to each well.
Place plate in incubator, shaking plate back and forth to distribute cells evenly (do not swirl).
24 hours later (i.e., day 7), aspirate spent media and add appropriate volume of EC medium (without bFGF or RA).