Eric Shusta · University of Wisconsin-Madison · PhdFit
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Eric Shusta
· Howard Curler Distinguished ProfessorVerified
University of Wisconsin-Madison · Biomedical Engineering
Eric Shusta is the Howard Curler Distinguished Professor in the Department of Chemical & Biological Engineering at the University of Wisconsin-Madison. His research focuses on overcoming the blood-brain barrier (BBB) to facilitate non-invasive delivery of pharmaceuticals to the brain for the treatment of neurological diseases. He investigates the properties of brain endothelial cells that form the BBB, as well as the neurovascular unit, which includes various cell types such as pericytes, astrocytes, and nerve terminals that contribute to BBB function.
Shusta employs molecular engineering techniques to utilize endogenous transport mechanisms at the BBB, aiming to develop targeted drug delivery systems using antibodies linked to therapeutic cargo. His work also involves optimizing the production of therapeutic antibodies and proteins, as well as developing in vitro models of the BBB that mimic in vivo characteristics. These models are used for screening drug candidates and understanding BBB permeability, leveraging genomics, proteomics, and pluripotent stem cell technologies, including patient-derived induced pluripotent stem cells, to study BBB function in health and disease.
Abstract Brain microvascular endothelial cells (BMECs) forming the blood-brain barrier (BBB) maintain brain homeostasis through specialized properties such as tight junctions, efflux transporters, and low levels of transcytosis. However, mechanisms governing induction of BBB properties during development remain poorly understood. We mined single-cell RNA sequencing datasets to identify transcription factors (TFs) critical for BBB development. Forty-four TFs were overexpressed in human pluripotent stem cell-derived endothelial cells cultured in the presence of the Wnt pathway agonist CHIR99021 to identify TFs capable of directing acquisition of BBB properties via forward programming. Individual TFs, including KLF2 , KLF4 , FOXF1 , FOXF2 , ZIC2 , ZIC3 , NR4A1 , NR4A2 , FOXC1 , and FOXQ1 , induced distinct BBB-like gene expression patterns. Combinations of these TFs induced many canonical BBB genes, yielding ECs with reduced endocytosis, increased efflux activity, and improved barrier function. The resultant forward programmed CNS-like ECs (fpCECs) offer promising tools for modeling human BBB development and neurovascular disease and for drug screening.
Abstract Background Mechanisms guiding the induction of blood-brain barrier (BBB) properties in central nervous system (CNS) endothelial cells during human development are incompletely understood. For example, there is a limited understanding of signaling pathways that influence the unique property of low vesicular endocytosis and transcytosis in brain microvascular endothelial cells (BMECs) relative to peripheral endothelial cells. Mouse studies suggest the importance of BBB-relevant developmental pathways, including Wnt and Notch signaling, for the induction of this BBB feature in developing BMECs. Methods To explore induction of reduced vesicular endocytosis and transcytosis in human in vitro model of the BBB, we used human pluripotent stem cell (hPSC)-derived endothelial progenitor cells (EPCs) in which Wnt/β-catenin signaling was activated to generate hPSC-derived CNS-like ECs (hPSC-CECs). We assessed the effects of Notch signaling through overexpression of the Notch1 receptor intracellular domain (N1ICD). Results N1ICD overexpression in hPSC-CECs resulted in upregulation of GLUT-1, a BBB-enriched glucose transporter, and decreased expression of both PLVAP and caveolin-1, two vesicular endocytosis-associated proteins. The combination of Wnt/β-catenin activation and N1ICD overexpression resulted in fewer vesicles and reduced albumin uptake. Conclusion These findings indicate that Notch signaling reduces vesicular endocytosis and transcytosis in a human model of the developing BBB and contribute to our understanding of how Notch signaling induces these specific BBB properties in this model of human CNS EC development.
Abstract Background Mechanisms guiding the induction of blood-brain barrier (BBB) properties in central nervous system (CNS) endothelial cells during human development are incompletely understood. For example, there is a limited understanding of signaling pathways that influence the unique property of low vesicular endocytosis and transcytosis in brain microvascular endothelial cells (BMECs) relative to peripheral endothelial cells. Mouse studies suggest the importance of BBB-relevant developmental pathways, including Wnt and Notch signaling, for the induction of this BBB feature in developing BMECs. Methods To explore induction of reduced vesicular endocytosis and transcytosis in human in vitro model of the BBB, we used human pluripotent stem cell (hPSC)-derived endothelial progenitor cells (EPCs) in which Wnt/β-catenin signaling was activated to generate hPSC-derived CNS-like ECs (hPSC-CECs). We assessed the effects of Notch signaling through overexpression of the Notch1 receptor intracellular domain (N1ICD). Results N1ICD overexpression in hPSC-CECs resulted in upregulation of GLUT-1, a BBB-enriched glucose transporter, and decreased expression of both PLVAP and caveolin-1, two vesicular endocytosis-associated proteins. The combination of Wnt/β-catenin activation and N1ICD overexpression resulted in fewer vesicles and reduced albumin uptake. Conclusion These findings indicate that Notch signaling reduces vesicular endocytosis and transcytosis in a human model of the developing BBB and contribute to our understanding of how Notch signaling induces these specific BBB properties in this model of human CNS EC development.
Mechanisms guiding the induction of blood-brain barrier (BBB) properties in central nervous system (CNS) endothelial cells during human development are incompletely understood. For example, there is a limited understanding of signaling pathways that influence the unique property of low vesicular endocytosis and transcytosis in brain microvascular endothelial cells (BMECs) relative to peripheral endothelial cells. Mouse studies suggest the importance of BBB-relevant developmental pathways, including Wnt and Notch signaling, for the induction of this BBB feature in developing BMECs. To explore induction of reduced vesicular endocytosis and transcytosis in human in vitro model of the BBB, we used human pluripotent stem cell (hPSC)-derived endothelial progenitor cells (EPCs) in which Wnt/β-catenin signaling was activated to generate hPSC-derived CNS-like ECs (hPSC-CECs). We assessed the effects of Notch signaling through overexpression of the Notch1 receptor intracellular domain (N1ICD). N1ICD overexpression in hPSC-CECs resulted in upregulation of GLUT-1, a BBB-enriched glucose transporter, and decreased expression of both PLVAP and caveolin-1, two vesicular endocytosis-associated proteins. The combination of Wnt/β-catenin activation and N1ICD overexpression resulted in fewer vesicles and reduced albumin uptake. These findings indicate that Notch signaling reduces vesicular endocytosis and transcytosis in a human model of the developing BBB and contribute to our understanding of how Notch signaling induces these specific BBB properties in this model of human CNS EC development.
BACKGROUND: Antibodies that cross the blood-brain barrier (BBB) by targeting receptor-mediated transport (RMT) systems can allow efficient drug delivery to the central nervous system (CNS). In order to improve brain uptake of antibodies, their binding properties have been engineered, but it is not always clear what antibody properties dictate BBB transport efficiency. In this study, we therefore developed and employed an in vitro phenotypic screen and a quantitative transcytosis assay in an attempt to identify improved variants of a previously identified BBB transcytosing antibody known as 46.1. METHODS: First, a random mutagenic 46.1 antibody phage display library was screened for improved transcytosis through a human induced pluripotent stem cell (iPSC)-derived BBB model. These screens yielded antibody variants that enriched over multiple screening rounds; however, when produced as soluble antibodies, the variants did not display improved in vitro transcytosis over the wild-type (WT) 46.1 antibody. As a second strategy, we performed a targeted histidine point mutation of a solvent-exposed residue in each complementarity-determining region (CDR) and evaluated the in vitro transcytosis capacity of the variants. RESULTS AND CONCLUSIONS: In this way, we identified a 46.1 variant, R162H, with modestly improved in vitro transcytosis properties. These results show that the iPSC-derived BBB screening insights and evaluation strategies presented here could facilitate the engineering and optimization of lead antibodies for CNS delivery.
Variable lymphocyte receptors (VLRs) are the antigen receptors of jawless vertebrates such as lamprey. VLRs are of growing biotechnological interest for their ability to bind certain antigenic targets with higher affinity than traditional immunoglobulins. However, VLRs are disulfide-bonded proteins that are often challenging to produce requiring genetic modifications, fusion partners, non-scalable host cell lines or inclusion body formation and refolding. As a potential VLR expression platform option, the SHuffle Escherichia coli strain has been genetically altered to allow cytoplasmic disulfide bond formation by mutations to thioredoxin reductase (trxB) and glutathione reductase (gor) to create an oxidative cytoplasm. Furthermore, the SHuffle strain expresses disulfide bond isomerase DsbC in the cytoplasm to promote correct disulfide bond pairing. Here, we demonstrate that the SHuffle strain can produce high yield VLRs with titers ranging from 2 to 32 mg of VLR per liter of SHuffle culture. Three VLRs (P1C10, RBC36, VLRA.R2.1) were expressed in SHuffle E. coli and the products were compared directly to those generated using the Rosetta E. coli strain. All VLRs were validated for correct sequence, purity, and activity. For all VLRs, SHuffle E. coli produced 2-9 times more soluble VLRs than Rosetta E. coli. Furthermore, the soluble protein fraction was 2-6 times greater in SHuffle E. coli than Rosetta E. coli for all VLRs. Overall, these results suggest that the E. coli SHuffle strain is a convenient and effective expression system for producing large amounts of VLRs.
Biotechnology and Bioengineering · 2025-03-25 · 6 citations
articleOpen accessCorresponding
Differentiating endothelial cells (ECs) from human pluripotent stem cells (hPSCs) typically takes 2 weeks and requires parameter optimization. Overexpression of cell type-specific transcription factors in hPSCs has shown efficient differentiation into various cell types. ETV2, a crucial transcription factor for endothelial fate, can be overexpressed in hPSCs to induce rapid and facile EC differentiation (iETV2-ECs). We developed a two-stage strategy which involves differentiating inducible ETV2-overexpressing hPSCs in a basal induction medium during stage I and expanding them in an endothelial medium during stage II. By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in 5 days. iETV2-ECs demonstrated in vitro angiogenesis potential, LDL uptake, and cytokine response. Transcriptomic comparisons revealed similar gene expression profiles between iETV2-ECs and traditionally differentiated ECs. Additionally, iETV2-ECs responded to Wnt signaling agonist and TGFβ inhibitor to acquire brain EC phenotypes, making them a scalable EC source for applications including blood-brain barrier modeling.
