72 research outputs found

    Activity Assay of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Triple-Negative Breast Cancer Cells Using Peptide-Conjugated Magnetic Beads

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    Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with limited treatment options. Epidermal growth factor receptor I (EGFR) has emerged as a promising target in TNBC. Limited success of the EGFR kinase inhibiting small molecules in clinical trials may be attributed in part to inaccuracy in identifying EGFR signatures in patient tumors. In light of the absence of a simple correlation between EGFR expression and its degree of activation, a simple and reliable tool that can quantify EGFR kinase activity in tumor samples may be of therapeutic value in predicting patient-specific EGFR targeted therapies. This study reports the development of an assay that can quantitatively profile EGFR kinase activities and inhibitor sensitivities in TNBC cell lysates by using peptide reporters covalently tethered to magnetic beads in a controlled orientation. The use of magnetic beads provides rapid sample handling and easy product isolation. The potential of this approach was demonstrated by screening a set of five clinically relevant EGFR tyrosine kinase inhibitors. Formatted for microwell plates, this magnetic bead-based kinase assay may be used as a complementary approach for direct high-throughput screening of small molecule inhibitors.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140099/1/adt.2012.454.pd

    Epidermal Growth Factor Mediated Healing in Stem Cell-derived Vocal Fold Mucosa

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    Background: The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. Materials and methods: A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation after injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, gefitinib. Results: Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 μm/h, ±2.63) compared with absence of exogenous EGF (7.1 μm/h ±2.84), but inhibited with concurrent addition of Gefitinib (5.2 μm/h, ±2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. Although not statistically significant, increased density of Ki67 staining in the epithelium adjacent to the scratch wound was observed after treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. Conclusions: Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair after injury

    Human pluripotent stem cell-derived epicardial progenitors can differentiate to endocardial-like endothelial cells.

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    During heart development, epicardial progenitors contribute various cardiac lineages including smooth muscle cells, cardiac fibroblasts, and endothelial cells. However, their specific contribution to the human endothelium has not yet been resolved, at least in part due to the inability to expand and maintain human primary or pluripotent stem cell (hPSC)-derived epicardial cells. Here we first generated CDH5-2A-eGFP knock-in hPSC lines and differentiated them into self-renewing WT1+ epicardial cells, which gave rise to endothelial cells upon VEGF treatment in vitro. In addition, we found that the percentage of endothelial cells correlated with WT1 expression in a WT1-2A-eGFP reporter line. The resulting endothelial cells displayed many endocardium-like endothelial cell properties, including high expression levels of endocardial-specific markers, nutrient transporters and well-organized tight junctions. These findings suggest that human epicardial progenitors may have the capacity to form endocardial endothelium during development and have implications for heart regeneration and cardiac tissue engineering

    Fabrication and Selective Functionalization of Amine-Reactive Polymer Multilayers on Topographically Patterned Microwell Cell Culture Arrays

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    We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. Reactive layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone- functionalized polymer poly(2-vinyl-4,4′-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts. © 2011 American Chemical Society

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

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    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

    Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Progenitors via Small-Molecule Activation of WNT Signaling

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    SummaryHuman pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34+CD31+ endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was β-catenin dependent. Furthermore, by clonal analysis, we showed that CD34+CD31+CD117+TIE-2+ endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31+CD144+vWF+I-CAM1+ endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs

    Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells.

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    Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3β inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications
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