Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Summary

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Abstract

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 Ω x cm². BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

Introduction

Blood-Brain Barrier Function
The blood-brain barrier (BBB) forms a boundary that limits movement of substances from the blood to the brain. The BBB is comprised of brain microvascular endothelial cells (BMECs) that form a monolayer lining the vasculature. BMECs, together with astrocytes, neurons, pericytes, microglia, and extracellular matrix, form the neurovascular unit. BMECs have a tightly regulated paracellular structure that allows the BBB to maintain high trans-endothelial electrical resistance (TEER), which limits passive diffusion and serves as an indicator of barrier integrity^1,2

Protocol

Human iPSCs were reprogrammed from the fibroblasts of healthy donors using a protocol approved by the Institutional Review Boards of Massachusetts General Hospital and McLean Hospital, and characterized as described in previous studies^44,45,46.

NOTE: Briefly, fibroblasts were reprogrammed to iPSC via mRNA-based genetic reprogramming⁴⁷. 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 kinas....

Results

BMEC Differentiation
A few critical steps in this protocol should be followed precisely (Figure 1). E6 medium use on day 1 is important, since it is often used for deriving neuroectoderm lineage from iPSCs within a relatively short period of time yielding reproducible results across multiple cell lines³⁶. Another important step is on day 4 of differentiation, where E6 medium should be switched to hESFM with diluted (1:200) B27, 20 ng/mL bFGF and 1.......

Discussion

Modifications and Troubleshooting

In this protocol, we made some modifications in using a commonly used extracellular matrix and cell culture media during iPSC culturing for derivation of BMECs (Figure 1). These changes did not impact the ability to derive BMECS from human iPSCs as described in the Lippmann protocol¹. An iPSC line from a different healthy donor was used to demonstrate that this modified protocol shows resul.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2′,7′-dichlorodihydrofluorescein diacetate	Sigma Aldrich	D6883-50MG
Accutase	Sigma Aldrich	A6964-100mL
Alexa Fluor 488 Donkey anti-Mouse IgG	Life Technologies	A-21202
Alexa Fluor 555 Donkey anti-Rabbit IgG	Life Technologies	A-31572
B27 Supplement	Thermo Fisher Scientific	17504044
CD31 (PECAM-1) (89C2) Mouse mAb	Cell Signaling	3528S
CLDN5 (Claudin-5)	Thermo Fisher Scientific	35-2500
Collagen IV from human placenta	Sigma Aldrich	C5533-5mg
Corning 2 mL Internal Threaded Polypropylene Cryogenic Vial	Corning	8670
Corning Costar Flat Bottom Cell Culture Plates (6-wells)	Corning	353046
Corning Falcon Flat Bottom Cell Culture Plates (24-wells)	Corning	353047
Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts (12-wells)	Corning	3460
Countess slides	Thermo Fisher Scientific	C10228
DMEM/F12 (without phenol red)	Thermo Fisher Scientific	A1413202
DMSO	Sigma Aldrich	D2438-50mL
Donkey serum	Sigma Aldrich	D9663-10ML
DPBS (+/+)	Gibco/Thermo Fisher Scientific	14040-117
Epithelial Volt/Ohm (TEER) Meter (EVOM2) STX2	World Precision Instruments	N/A
Essential 6 Medium (Thermo Fisher)	Thermo Fisher Scientific	A1516401
Fetal Bovine Serum (FBS)	Sigma Aldrich	F2442
Fibronectin	Sigma Aldrich	F2006-2mg
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix	Thermo Fisher Scientific	A1413202
Hanks' Balance Salt Solution with calcium and magnesium	Thermo Fisher Scientific	24020-117
Hoechst 33342, Trihydrochloride, Trihydrate	Thermo Fisher Scientific	H3570
Human endothelial serum-free medium	Thermo Fisher Scientific	11111044
InCell Analyzer 6000	General Electric	N/A
Invitrogen Countess Automated Cell Counter	Thermo Fisher Scientific	N/A
MK-571	Sigma Aldrich	M7571-5MG
NutriStem	Stemgent	01-0005
Occludin	Thermo Fisher Scientific	33-1500
Paraformaldehyde 16%	Electron Microscopy Services	15710
Perkin Elmer Envision 2103 multi-plate Reader	Perkin Elmer	N/A
Recombinant Human VEGF 165	Peprotech	100-20
Recombinant Human FGF-basic (154 a.a.)	Peprotech	100-18B
Retinoic acid	Sigma Aldrich	R2625-100MG
Rhodamine 123	Sigma Aldrich	83702-10MG
SLC2A1 (GLUT-1)	ThermoFisher	PA1-21041
SOX17	Cell Signaling	81778S
TJP-1 (ZO-1)	ThermoFisher	PA5-28869
Triton X-100	Sigma Aldrich	T8787-50ML
Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter	Thermo Fisher Scientific	T10282
Valspodar (Sigma) (cyclosporin A)	Sigma Aldrich	SML0572-5MG
Versene solution	Thermo Fisher Scientific	15040066
Y-27632 dihydrochloride (ROCK inhibitor)	Tocris/Thermo Fisher Scientific	1254