Browsing by Author "Gastfriend, Benjamin D."

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    Antibody Screening Using a Human iPSC-based Blood-Brain Barrier Model Identifies Antibodies that Accumulate in the CNS
    (Wiley, 2020-09) Georgieva, Julia V.; Goulatis, Loukas I.; Stutz, Charles C.; Canfield, Scott G.; Song, Hannah W.; Gastfriend, Benjamin D.; Shusta, Eric V.; Cellular and Integrative Physiology, School of Medicine
    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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    Correction to: An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons
    (BioMed Central, 2019-09-10) Canfield, Scott G.; Stebbins, Matthew J.; Faubion, Madeline G.; Gastfriend, Benjamin D.; Palecek, Sean P.; Shusta, Eric V.; Cellular and Integrative Physiology, School of Medicine
    Abstract Following publication of the original article [1], the author has reported that in Figure 1 (b and c) the y-axis TEER (© x cm2) should be replaced with TEER (Ω x cm2). Erratum for An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons. [Fluids Barriers CNS. 2019]
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    An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons
    (BioMed Central, 2019-08-07) Canfield, Scott G.; Stebbins, Matthew J.; Faubion, Madeline G.; Gastfriend, Benjamin D.; Palecek, Sean P.; Shusta, Eric V.; Cellular and Integrative Physiology, School of Medicine
    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.