Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on sleep in brain health

LIVE: Thursday 2nd May 2024, 18:00-19:30 (CEST)

Quality sleep is essential for health. But what happens to our brains when sleep patterns are disturbed? Join our experts to explore the interplay between sleep disruption and neurological diseases, and the questions that you need to be asking your patients to help you prevent the harmful effects of sleep deprivation.
ADVANCED SEARCH
Log in
Top
Fluids and Barriers of the CNS
Published in: Fluids and Barriers of the CNS 1/2019

Open Access 01-12-2019 | Research

An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons

Authors: Scott G. Canfield, Matthew J. Stebbins, Madeline G. Faubion, Benjamin D. Gastfriend, Sean P. Palecek, Eric V. Shusta

Published in: Fluids and Barriers of the CNS | Issue 1/2019

Login to get access

Abstract

Background

Brain microvascular endothelial cells (BMECs) astrocytes, neurons, and pericytes form the neurovascular unit (NVU). Interactions with NVU cells endow BMECs with extremely tight barriers via the expression of tight junction proteins, a host of active efflux and nutrient transporters, and reduced transcellular transport. To recreate the BMEC-enhancing functions of NVU cells, we combined BMECs, astrocytes, neurons, and brain pericyte-like cells.

Methods

BMECs, neurons, astrocytes, and brain like pericytes were differentiated from human induced pluripotent stem cells (iPSCs) and placed in a Transwell-type NVU model. BMECs were placed in co-culture with neurons, astrocytes, and/or pericytes alone or in varying combinations and critical barrier properties were monitored.

Results

Co-culture with pericytes followed by a mixture of neurons and astrocytes (1:3) induced the greatest barrier tightening in BMECs, supported by a significant increase in junctional localization of occludin. BMECs also expressed active P-glycoprotein (PGP) efflux transporters under baseline BMEC monoculture conditions and continued to express baseline active PGP efflux transporters regardless of co-culture conditions. Finally, brain-like pericyte co-culture significantly reduced the rate of non-specific transcytosis across BMECs.

Conclusions

Importantly, each cell type in the NVU model was differentiated from the same donor iPSC source, yielding an isogenic model that could prove enabling for enhanced personalized modeling of the NVU in human health and disease.
Literature
1.
2.
Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood–brain barrier. Neurobiol Dis. 2010;37(1):13–25.PubMedCrossRef
3.
Abbott NJ, Rönnbäck L, Hansson E. Astrocyte-endothelial interactions at the blood–brain barrier. Nat Rev Neurosci. 2006;7(1):41–53.PubMedCrossRef
4.
Ballabh P, Braun A, Nedergaard M. The blood–brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis. 2004;16(1):1–13.PubMedCrossRef
5.
Daneman R, Agalliu D, Zhou L, Kuhnert F, Kuo CJ, Barres BA. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci USA. 2009;106(2):641–6.PubMedCrossRefPubMedCentral
6.
Daneman R, Zhou L, Kebede AA, Barres BA. Pericytes are required for blood–brain barrier integrity during embryogenesis. Nature. 2010;468(7323):562–6.PubMedPubMedCentralCrossRef
7.
Daneman R, Zhou L, Agalliu D, Cahoy JD, Kaushal A, Barres BA. The mouse blood–brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS ONE. 2010;5(10):e13741.PubMedPubMedCentralCrossRef
8.
Dohgu S, Takata F, Yamauchi A, et al. Brain pericytes contribute to the induction and up-regulation of blood–brain barrier functions through transforming growth factor-beta production. Brain Res. 2005;1038(2):208–15.PubMedCrossRef
9.
Weiss N, Miller F, Cazaubon S, Couraud PO. The blood–brain barrier in brain homeostasis and neurological diseases. Biochim Biophys Acta. 2009;1788(4):842–57.PubMedCrossRef
10.
Sweeney MD, Sagare AP, Zlokovic BV. blood–brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders. Nat Rev Neurol. 2018;14(3):133–50.PubMedPubMedCentralCrossRef
11.
Vatine GD, Al-Ahmad A, Barriga BK, et al. Modeling psychomotor retardation using iPSCs from MCT8-deficient patients indicates a prominent role for the blood–brain barrier. Cell Stem Cell. 2017;20(6):831–43.PubMedPubMedCentralCrossRef
12.
Zlokovic BV. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron. 2008;57(2):178–201.PubMedCrossRef
13.
Deli MA, Abrahám CS, Kataoka Y, Niwa M. Permeability studies on in vitro blood–brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol. 2005;25(1):59–127.PubMedCrossRef
14.
Syvänen S, Lindhe O, Palner M, et al. Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport. Drug Metab Dispos. 2009;37(3):635–43.PubMedCrossRef
15.
Warren MS, Zerangue N, Woodford K, et al. Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human. Pharmacol Res. 2009;59(6):404–13.PubMedCrossRef
16.
Weksler BB, Subileau EA, Perrière N, et al. blood–brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 2005;19(13):1872–4.PubMedCrossRef
17.
Cecchelli R, Berezowski V, Lundquist S, et al. Modelling of the blood–brain barrier in drug discovery and development. Nat Rev Drug Discov. 2007;6(8):650–61.PubMedCrossRef
18.
Calabria AR, Shusta EV. A genomic comparison of in vivo and in vitro brain microvascular endothelial cells. J Cereb Blood Flow Metab. 2008;28(1):135–48.PubMedCrossRef
19.
Förster C, Burek M, Romero IA, Weksler B, Couraud PO, Drenckhahn D. Differential effects of hydrocortisone and TNFalpha on tight junction proteins in an in vitro model of the human blood–brain barrier. J Physiol. 2008;586(Pt 7):1937–49.PubMedPubMedCentralCrossRef
20.
Man S, Ubogu EE, Williams KA, Tucky B, Callahan MK, Ransohoff RM. Human brain microvascular endothelial cells and umbilical vein endothelial cells differentially facilitate leukocyte recruitment and utilize chemokines for T cell migration. Clin Dev Immunol. 2008;2008:384982.PubMedPubMedCentralCrossRef
21.
Hollmann EK, Bailey AK, Potharazu AV, Neely MD, Bowman AB, Lippmann ES. Accelerated differentiation of human induced pluripotent stem cells to blood–brain barrier endothelial cells. Fluids Barriers CNS. 2017;14(1):9.PubMedPubMedCentralCrossRef
22.
Lippmann ES, Azarin SM, Kay JE, et al. Derivation of blood–brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol. 2012;30(8):783–91.PubMedPubMedCentralCrossRef
23.
Lippmann ES, Al-Ahmad A, Azarin SM, Palecek SP, Shusta EV. A retinoic acid-enhanced, multicellular human blood–brain barrier model derived from stem cell sources. Sci Rep. 2014;4:4160.PubMedPubMedCentralCrossRef
24.
25.
Canfield SG, Stebbins MJ, Morales BS, et al. An isogenic blood–brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells. J Neurochem. 2017;140(6):874–88.PubMedPubMedCentralCrossRef
26.
Ribecco-Lutkiewicz M, Sodja C, Haukenfrers J, et al. A novel human induced pluripotent stem cell blood–brain barrier model: applicability to study antibody-triggered receptor-mediated transcytosis. Sci Rep. 2018;8(1):1873.PubMedPubMedCentralCrossRef
27.
Lim RG, Quan C, Reyes-Ortiz AM, et al. Huntington’s disease iPSC-derived brain microvascular endothelial cells reveal WNT-mediated angiogenic and blood–brain barrier deficits. Cell Rep. 2017;19(7):1365–77.PubMedPubMedCentralCrossRef
28.
DeStefano JG, Xu ZS, Williams AJ, Yimam N, Searson PC. Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs). Fluids Barriers CNS. 2017;14(1):20.PubMedPubMedCentralCrossRef
29.
Katt ME, Linville RM, Mayo LN, Xu ZS, Searson PC. Functional brain-specific microvessels from iPSC-derived human brain microvascular endothelial cells: the role of matrix composition on monolayer formation. Fluids Barriers CNS. 2018;15(1):7.PubMedPubMedCentralCrossRef
30.
Helms HC, Abbott NJ, Burek M, et al. In vitro models of the blood–brain barrier: an overview of commonly used brain endothelial cell culture models and guidelines for their use. J Cereb Blood Flow Metab. 2016;36(5):862–90.PubMedPubMedCentralCrossRef
31.
Brown JA, Pensabene V, Markov DA, et al. Recreating blood–brain barrier physiology and structure on chip: a novel neurovascular microfluidic bioreactor. Biomicrofluidics. 2015;9(5):054124.PubMedPubMedCentralCrossRef
32.
Lippmann ES, Weidenfeller C, Svendsen CN, Shusta EV. Blood–brain barrier modeling with co-cultured neural progenitor cell-derived astrocytes and neurons. J Neurochem. 2011;119(3):507–20.PubMedPubMedCentralCrossRef
33.
Qian T, Maguire SE, Canfield SG, et al. Directed differentiation of human pluripotent stem cells to blood–brain barrier endothelial cells. Sci Adv. 2017;3(11):e1701679.PubMedPubMedCentralCrossRef
34.
Appelt-Menzel A, Cubukova A, Günther K, et al. Establishment of a human blood–brain Barrier co-culture model mimicking the neurovascular unit using induced pluri-and multipotent stem cells. Stem Cell Reports. 2017;8(4):894–906.PubMedPubMedCentralCrossRef
35.
Xiang J, Andjelkovic AV, Wang MM, Keep RF. Blood–brain barrier models derived from individual patients: a new frontier: an Editorial Highlight on ‘An isogenic blood–brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells’. J Neurochem. 2017;140(6):843–4.PubMedCrossRef
36.
Stebbins MJ, Gastfriend BD, Canfield SG, et al. Human pluripotent stem cell-derived brain pericyte-like cells induce blood–brain barrier properties. Sci Adv. 2019;5(3):eaau7375.PubMedPubMedCentralCrossRef
37.
Ebert AD, Shelley BC, Hurley AM, et al. EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs. Stem Cell Res. 2013;10(3):417–27.PubMedPubMedCentralCrossRef
38.
Armulik A, Genové G, Mäe M, et al. Pericytes regulate the blood–brain barrier. Nature. 2010;468(7323):557–61.PubMedCrossRef
39.
Armulik A, Genové G, Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev Cell. 2011;21(2):193–215.PubMedCrossRef
40.
Schiera G, Sala S, Gallo A, et al. Permeability properties of a three-cell type in vitro model of blood–brain barrier. J Cell Mol Med. 2005;9(2):373–9.PubMedPubMedCentralCrossRef
41.
Nakagawa S, Deli MA, Kawaguchi H, et al. A new blood–brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int. 2009;54(3–4):253–63.PubMedCrossRef
42.
43.
Wilson HK, Canfield SG, Hjortness MK, Palecek SP, Shusta EV. Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells. Fluids Barriers CNS. 2015;12:13.PubMedPubMedCentralCrossRef
44.
Sareen D, Gowing G, Sahabian A, et al. Human induced pluripotent stem cells are a novel source of neural progenitor cells (iNPCs) that migrate and integrate in the rodent spinal cord. J Comp Neurol. 2014;522(12):2707–28.PubMedPubMedCentralCrossRef
45.
Azevedo FA, Carvalho LR, Grinberg LT, et al. Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J Comp Neurol. 2009;513(5):532–41.PubMedCrossRef
46.
Herculano-Houzel S, Lent R. Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain. J Neurosci. 2005;25(10):2518–21.PubMedPubMedCentralCrossRef
47.
van der Meer AD, Orlova VV, ten Dijke P, van den Berg A, Mummery CL. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device. Lab Chip. 2013;13(18):3562–8.PubMedCrossRef
48.
Kusuma S, Facklam A, Gerecht S. Characterizing human pluripotent-stem-cell-derived vascular cells for tissue engineering applications. Stem Cells Dev. 2015;24(4):451–8.PubMedCrossRef
49.
Nordström U, Maier E, Jessell TM, Edlund T. An early role for WNT signaling in specifying neural patterns of Cdx and Hox gene expression and motor neuron subtype identity. PLoS Biol. 2006;4(8):e252.PubMedPubMedCentralCrossRef
50.
Maden M, Gale E, Kostetskii I, Zile M. Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr Biol. 1996;6(4):417–26.PubMedCrossRef
51.
Liu JP, Laufer E, Jessell TM. Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron. 2001;32(6):997–1012.PubMedCrossRef
52.
53.
Stebbins MJ, Lippmann ES, Faubion MG, Daneman R, Palecek SP, Shusta EV. Activation of RARα, RARγ, or RXRα increases barrier tightness in human induced pluripotent stem cell-derived brain endothelial cells. Biotechnol J. 2018;13(2):1700093.CrossRef
54.
Butt AM, Jones HC, Abbott NJ. Electrical resistance across the blood–brain barrier in anaesthetized rats: a developmental study. J Physiol. 1990;429:47–62.PubMedPubMedCentralCrossRef
55.
Weidenfeller C, Svendsen CN, Shusta EV. Differentiating embryonic neural progenitor cells induce blood–brain barrier properties. J Neurochem. 2007;101(2):555–65.PubMedPubMedCentralCrossRef
56.
Weidenfeller C, Schrot S, Zozulya A, Galla HJ. Murine brain capillary endothelial cells exhibit improved barrier properties under the influence of hydrocortisone. Brain Res. 2005;1053(1–2):162–74.PubMedCrossRef
57.
Calabria AR, Weidenfeller C, Jones AR, de Vries HE, Shusta EV. Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction. J Neurochem. 2006;97(4):922–33.PubMedCrossRef
58.
Berezowski V, Landry C, Dehouck MP, Cecchelli R, Fenart L. Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood–brain barrier. Brain Res. 2004;1018(1):1–9.PubMedCrossRef
59.
Perrière N, Yousif S, Cazaubon S, et al. A functional in vitro model of rat blood–brain barrier for molecular analysis of efflux transporters. Brain Res. 2007;1150:1–13.PubMedCrossRef
60.
Lim JC, Wolpaw AJ, Caldwell MA, Hladky SB, Barrand MA. Neural precursor cell influences on blood–brain barrier characteristics in rat brain endothelial cells. Brain Res. 2007;1159:67–76.PubMedCrossRef
61.
Freese C, Reinhardt S, Hefner G, Unger RE, Kirkpatrick CJ, Endres K. A novel blood–brain barrier co-culture system for drug targeting of Alzheimer’s disease: establishment by using acitretin as a model drug. PLoS ONE. 2014;9(3):e91003.PubMedPubMedCentralCrossRef
62.
Appelt-Menzel A, Cubukova A, Metzger M. Establishment of a human blood–brain barrier co-culture model mimicking the neurovascular unit using induced pluripotent stem cells. Curr Protoc Stem Cell Biol. 2018;47(1):e62.PubMedCrossRef
63.
Alvarez JI, Dodelet-Devillers A, Kebir H, et al. The Hedgehog pathway promotes blood–brain barrier integrity and CNS immune quiescence. Science. 2011;334(6063):1727–31.PubMedCrossRef
64.
Wosik K, Cayrol R, Dodelet-Devillers A, et al. Angiotensin II controls occludin function and is required for blood brain barrier maintenance: relevance to multiple sclerosis. J Neurosci. 2007;27(34):9032–42.PubMedPubMedCentralCrossRef
65.
Nishitsuji K, Hosono T, Nakamura T, Bu G, Michikawa M. Apolipoprotein E regulates the integrity of tight junctions in an isoform-dependent manner in an in vitro blood–brain barrier model. J Biol Chem. 2011;286(20):17536–42.PubMedPubMedCentralCrossRef
66.
Wang YI, Abaci HE, Shuler ML. Microfluidic blood–brain barrier model provides in vivo-like barrier properties for drug permeability screening. Biotechnol Bioeng. 2017;114(1):184–94.PubMedCrossRef
67.
Metadata
Title
An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons
Authors
Scott G. Canfield
Matthew J. Stebbins
Madeline G. Faubion
Benjamin D. Gastfriend
Sean P. Palecek
Eric V. Shusta
Publication date
01-12-2019
Publisher
BioMed Central
Published in
Fluids and Barriers of the CNS / Issue 1/2019
Electronic ISSN: 2045-8118
DOI
https://doi.org/10.1186/s12987-019-0145-6

Other articles of this Issue 1/2019

Fluids and Barriers of the CNS 1/2019 Go to the issue

Research

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells

Short paper

Tau-protein concentrations are not elevated in cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy

Research

Enhanced in vitro model of the CSF dynamics

Research

Claudin-12 is not required for blood–brain barrier tight junction function

Research

The Kuopio idiopathic normal pressure hydrocephalus protocol: initial outcome of 175 patients

Research

Can pulsatile CSF flow across the cerebral aqueduct cause ventriculomegaly? A prospective study of patients with communicating hydrocephalus