Dataset Information

IPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.

ABSTRACT: There is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10-100 times less permeable to sodium fluorescein, 3 kDa dextran, and albumin relative to human umbilical vein endothelial cell and human dermal microvascular endothelial cell controls, and the BMECs maintained barrier function for up to 21 days. Analysis of cell-cell junctions revealed expression patterns supporting barrier formation. Finally, efflux transporter activity was maintained over 3 weeks of perfused culture. Taken together, this work lays the foundation for development of a representative 3D in vitro model of the human NVU constructed from iPSCs.

SUBMITTER: Faley SL

PROVIDER: S-EPMC6409430 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

iPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.

Faley Shannon L SL Neal Emma H EH Wang Jason X JX Bosworth Allison M AM Weber Callie M CM Balotin Kylie M KM Lippmann Ethan S ES Bellan Leon M LM

Stem cell reports 20190214 3

There is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10-100 times less per ...[more]

PMID: 30773484

Similar Datasets

Project description:BACKGROUND:Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. METHODS:We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. RESULTS:We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm2). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. CONCLUSIONS:We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.

Project description:Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events during angiogenesis in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by undefined substrates and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). We rapidly encapsulated iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres using thiol-ene chemistry and subsequently encapsulated cell-dense hydrogel spheres in a cell-free hydrogel layer. The hydrogel sprouting array supported pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. iPSC-ECs in the sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors of vascular endothelial growth factor, NF-?B, matrix metalloproteinase-2/9, protein kinase activity, and ?-tubulin, which confirms their functional role in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds from the US Environmental Protection Agency's ToxCast library identified six compounds that inhibited iPSC-EC sprouting and five compounds that were overtly cytotoxic to iPSC-ECs at a single concentration. The chemically-defined iPSC-EC sprouting model (iSM) is thus amenable to enhanced-throughput screening of small molecular libraries for effects on angiogenic sprouting and iPSC-EC toxicity assessment.Angiogenesis assays that are commonly used for drug screening and toxicity assessment applications typically utilize natural substrates like Matrigel(TM) that are difficult to spatially pattern, costly, ill-defined, and may exhibit lot-to-lot variability. Herein, we describe a novel angiogenic sprouting assay using chemically-defined, bioinert poly(ethylene glycol) hydrogels functionalized with biomimetic peptides to promote cell attachment and degradation in a reproducible format that may mitigate the need for natural substrates. The quantitative assay of angiogenic sprouting here enables precise control over the initial conditions and can be formulated into arrays for screening. The sprouting assay here was dependent on key angiogenic signaling axes in a screen of angiogenesis inhibitors and a blinded screen of putative vascular disrupting compounds from the US-EPA.

Dataset Information

IPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.

Publications

iPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure