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

Faley, Shannon; Neal, Emma H.; Wang, Jason X.; Bosworth, Allison M.; Weber, Callie M.; Balotin, Kylie M.; Lippmann, Ethan S.; Bellan, Leon M.

doi:10.1016/j.stemcr.2019.01.009

Cited by 78 publications

(73 citation statements)

References 59 publications

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“…Finally, physiological fluid flow, which is capable of inducing BBB phenotype, suppressing inflammatory signaling, and improving the tightness of the BMEC barrier [11], was added to the system to determine whether culture on LN 511-E8 could improve the ability of iBMECs to demonstrate physiological responsiveness to shear stress. Other studies have reported iBMECs do not show morphological changes or alterations in junctional protein expression due to shear stress on CN IV-FN [15,92,93]. In contrast, we showed that applying shear stress to iBMECs cultured on LN 511-E8, which strongly interacts with β1 integrins, can result in morphological changes and enhanced junctional association of VE-cadherin, claudin-5, and ZO-1.…”

Section: Discussioncontrasting

confidence: 68%

Chemically defined human vascular laminins for biologically relevant culture of hiPSC-derived brain microvascular endothelial cells

Motallebnejad

Azarin

2020

Fluids Barriers CNS

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Background In recent years, differentiation of human induced pluripotent stem cells (hiPSCs) into brain-specific microvascular endothelial cells (iBMECs) has frequently been used to model the blood–brain barrier (BBB). However, there are limitations in the use of iBMECs for in vitro studies, such as transendothelial electrical resistance (TEER) instability, weak junctional expression of VE-cadherin, and lack of proper fluid shear stress response. In vivo, the basement membrane (BM) composition of the BBB evolves throughout development, and laminins become the dominant component of the mature vascular BM. However, laminin isoforms of the endothelial BM have not been used for culture of differentiated iBMECs. The main goal of this study is to investigate the effect of different laminin isoforms of the endothelial BM on iBMEC functionality and to determine whether better recapitulation of the physiological BM in vitro can address the aforementioned limitations of iBMECs. Methods Using a previously reported method, hiPSCs were differentiated into iBMECs. The influence of main laminins of the endothelial BM, LN 411 and LN 511, on iBMEC functionality was studied and compared to a collagen IV and fibronectin mixture (CN IV-FN). Quantitative RT-PCR, immunocytochemistry, and TEER measurement were utilized to assess gene and protein expression and barrier properties of iBMECs on different extracellular matrices. Single-channel microfluidic devices were used to study the effect of shear stress on iBMECs. Results LN 511, but not LN 411, improved iBMEC barrier properties and resulted in more sustained TEER stability. Immunocytochemistry showed improved junctional protein expression compared to iBMECs cultured on CN IV-FN. iBMECs cultured on LN 511 showed a reduction of stress fibers, indicating resting endothelial phenotype, whereas gene expression analysis revealed upregulation of multiple genes involved in endothelial activation in iBMECs on CN IV-FN. Finally, culturing iBMECs on LN 511 enhanced physiological responses to shear stress, including morphological changes and enhanced junctional protein association. Conclusion LN 511 improves the functionality and long-term barrier stability of iBMECs. Our findings suggest that incorporation of physiologically relevant LN 511 in iBMEC culture would be beneficial for disease modeling applications and BBB-on-a-chip platforms that accommodate fluid flow.

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Section: Discussioncontrasting

confidence: 68%

Chemically defined human vascular laminins for biologically relevant culture of hiPSC-derived brain microvascular endothelial cells

Motallebnejad

Azarin

2020

Fluids Barriers CNS

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“…Most relevant to this commentary, induction and maintenance of endothelial character with a corresponding reduction of epithelial character continues to be a goal. Factors such as hypoxia [19], shear stress [9,19], and three dimensional architecture [10,28,29] have been suggested to increase vascular character, and other models based on induction of BBB character in generic hiPSC-derived ECs are beginning to emerge [32]. Despite these advances, we believe it is prudent to exercise caution when utilizing hPSC-derived BMEC-like cells for studies where the endothelial phenotype is crucial.…”

Section: Main Textmentioning

confidence: 99%

“…We have observed VEGF-dependent network formation in hPSC-derived BMECs using in vitro Matrigel assays [ 1 ], and VEGF has been shown to regulate PLVAP expression in a three-dimensional BBB model that contains hPSC-derived BMECs [ 10 ]. hPSC-derived BMECs also respond to shear; we have observed sprouting-like behavior after culturing these cells in engineered hydrogel matrices under constant perfusion [ 28 ], and others have observed similar phenotypes in hydrogels in response to growth factors and other stimuli such as oxidative stress [ 29 ]. In addition, application of shear after differentiation also led to activation of cholesterol metabolism, proliferation, and angiogenesis transcriptional pathways compared to static controls [ 9 ].…”

Section: Main Textmentioning

confidence: 99%

Commentary on human pluripotent stem cell-based blood–brain barrier models

Lippmann

Azarin

Palecek

et al. 2020

Fluids Barriers CNS

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In 2012, we provided the first published evidence that human pluripotent stem cells could be differentiated to cells exhibiting markers and phenotypes characteristic of the blood–brain barrier (BBB). In the ensuing years, the initial protocols have been refined, and the research community has identified both positive and negative attributes of this stem cell-based BBB model system. Here, we give our perspective on the current status of these models and their use in the BBB community, as well as highlight key attributes that would benefit from improvement moving forward.

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“…The iPSC-derived BMECs, have well-developed tight junctions, express key BBB markers and, most importantly for the application described here, are a reasonable facsimile of both primary human BMECs and acutely isolated human BMECs in terms of their transporter expression profiles. [17][18][19][20] While several iPSC-derived BBB models with sufficient barrier function are available, [21][22][23][24][25] they have not been employed for identification of new BBB penetrating antibodies. Thus, we used iPSC-derived BMECs to perform a transcytosis screen with a phage display scFv library and identified a cohort of antibodies able to react with human BBB antigens and target the murine brain vasculature in vivo.…”

Section: Introductionmentioning

confidence: 99%

Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

et al. 2020

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Drug delivery across the blood‐brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood‐borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor‐mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off‐target effects, driving the search for new and potentially improved brain targeting reagent‐RMT pairs. To this end, we deployed human‐induced pluripotent stem cell (iPSC)‐derived BMEC‐like cells as a model BBB substrate on which to mine for new RMT‐targeting antibody pairs. A nonimmune, human single‐chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC‐derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1‐scFv, exhibiting a 26‐fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1‐scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor‐mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain.

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

Cited by 78 publications

References 59 publications

Chemically defined human vascular laminins for biologically relevant culture of hiPSC-derived brain microvascular endothelial cells

Chemically defined human vascular laminins for biologically relevant culture of hiPSC-derived brain microvascular endothelial cells

Commentary on human pluripotent stem cell-based blood–brain barrier models

Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

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