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 integrity1,2. BMECs also have proteins that assist with transcellular movement such as endocytosis, transcytosis, and transmigration, as well as extravasation of leukocytes during an immune response3. BMECs rely on influx and efflux transporters for nourishment and removal of waste products, in order to maintain a homeostatic balance in the brain3. For example, solute carrier family 2 member 1 (SLC2A1) is an influx transporter responsible for the movement of glucose across the BBB4, while efflux transporters such as the ATP binding cassette subfamily B member 1 (ABCB1) and the ATP binding cassette subfamily C member 1 (ABCC1) are responsible for returning substrates back into the blood stream3,5,6,7. ABCB1 substrates include morphine, verapamil4, and antipsychotics such as olanzapine and risperidone8, while the ABCC1 transporter has a variety of substrates including sulfate conjugates, vincristine, and glucuronide conjugates4.
Application of BBB Models in Psychiatric Disorders
BBB dysfunction has been implicated in a number of neurological and psychiatric disorders, including schizophrenia and bipolar disorder9,10. Recently, iPSC-derived ex vivo cellular models are being utilized to interrogate the cellular and molecular underpinnings of psychiatric disorders, but these models currently do not take into account the potential role played by the neurovasculature11,12,13. It is hypothesized that peripheral inflammatory cytokines circulating in the blood can adversely impact the BBB14,15,16,17, but there is also evidence for paracellular18,19,20,21,22, transcellular23,24,25,26,27,28,29, and extracellular matrix20,29,30,31,32 abnormalities contributing to BBB dysfunction. Disruption of the BBB can result in the contents of the blood entering the brain parenchyma and activating astrocytes and/or microglia to release proinflammatory cytokines, which in turn initiate an inflammatory response33 that can have detrimental effects on the brain34. BMECs are the primary component of the BBB and examining the structure and function of these cells can enhance the understanding of BBB dysfunction in neurological and psychiatric disorders.
Alternative BMEC Models
Prior to the development of efficient protocols for deriving BMECs from iPSCs1,6,35,36, researchers had employed immortalized BMECs37 to study BBB function. However, many of these models failed to attain desirable BBB phenotypes, such a physiological range of TEER values38,39. Utilizing iPSCs has the advantage of retaining the genetic background of the individual from which the cells are derived. Scientists are actively working on establishing iPSC-derived ex vivo microenvironment models that recapitulate the structure and function of the human brain. Researchers have developed methods to derive BMECs that are structurally and physiologically similar to BMECs found in vivo. Methods for obtaining purified populations of iPSC-derived BMECs require a number of different steps with protocols being optimized in the last few years1,6,35,36. Generally, iPSC-derived BMECs are cultured in Essential 6 (E6) medium for 4 days, followed by 2 days in human endothelial serum-free medium (hESFM) supplemented with basic fibroblast growth factor (bFGF), retinoic acid (RA), and B27 supplement. The cells are then cultured on a collagen IV (COL4) and fibronectin (FN) matrix to obtain >90% homogeneous BMECs1.
The identity of BMECs are confirmed by immunofluorescence showing the co-expression of BMEC proteins including platelet-endothelial cell adhesion molecule-1 (PECAM1), SLC2A1, and tight junction proteins such as tight junction protein 1 (TJP1), occludin (OCLN), and claudin-5 (CLDN5)6. Sprouting assays have been used to confirm the angiogenic potential of iPSC-derived BMECs.6 The BBB integrity of BMECs is evaluated by the presence of physiologic in vitro TEER values (~2000Ω x cm2)37 and measurable activity for efflux transporters such as ABCB1 and ABCC11,6,36. Recent methodological advances by the Lippmann group have led to iPSC-derived BMEC protocols with reduced experimental variability and enhanced reproducibility1. However, it is not known whether they can be expanded and passaged beyond the sub-culturing stage. Our modified protocol aims to address this issue by passaging iPSC-derived BMECs beyond day 8 and assessing whether they can be further expanded to retain BBB properties after cryopreservation. While no studies have described passaging of iPSC-derived BMECs, a protocol exists for BMEC cryopreservation that retains physiologic BBB properties after undergoing a freeze-thaw cycle40. However, it is not known post-cryopreservation BMECs can be passaged and retain BBB properties.
BMECs derived from iPSCs using the Lippmann protocol have been utilized to model BBB disruption in neurological disorders such as Huntington’s disease7. Such iPSC-derived BMECs have also been used to investigate the effects of bacterial infection such as Neisseria meningitidis or Group B Streptococcus on disruption of blood-CSF barrier and BBB respectively41,42. Also, using iPSC-derived BMECs from 22q deletion syndrome patients with schizophrenia, researchers observed an increase in intercellular adhesion molecule-1 (ICAM-1), a major adhesion molecule in BMECs that assist with recruitment and extravasation of leukocytes into the brain43. Taken together, these studies demonstrate the utility of iPSC-derived BMECs for studying BBB disruption in complex neuropsychiatric disorders.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by a National Institute of Mental Health Biobehavioral Research Awards for Innovative New Scientists (BRAINS) Award R01MH113858 (to R.K.), a National Institutes of Health Award KL2 TR002542 (PL). a National Institute of Mental Health Clinical Scientist Development Award K08MH086846 (to R.K.), a Sydney R Baer Jr Foundation Grant (to P.L.) the Doris Duke Charitable Foundation Clinical Scientist Development Award (to R.K.), the Ryan Licht Sang Bipolar Foundation (to R.K.), the Phyllis & Jerome Lyle Rappaport Foundation (to R.K.), the Harvard Stem Cell Institute (to R.K.) and by Steve Willis and Elissa Freud (to R.K.). We thank Dr. Annie Kathuria for her critical reading and feedback on the manuscript.
Materials
| 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 |
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