Human Vascular Tissue Models Formed from Human Induced Pluripotent Stem Cell Derived Endothelial Cells

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.National Institutes of Health (U.S.) (NIH 1UH2 TR000506-01)National Institutes of Health (U.S.) (3UH2 TR000506-02S1)National Institutes of Health (U.S.) (T32 HL007936-12)National Institutes of Health (U.S.) (RO1 HL093282)National Institutes of Health (U.S.) (R21 EB016381-01

Effect of vessel wettability on the foamability of "ideal" surfactants and "real-world" beer heads

The ability to tailor the foaming properties of a solution by controlling its chemical composition is highly desirable and has been the subject of extensive research driven by a range of applications. However, the control of foams by varying the wettability of the foaming vessel has been less widely reported. This work investigates the effect of the wettability of the side walls of vessels used for the in situ generation of foam by shaking aqueous solutions of three different types of model surfactant systems (non-ionic, anionic and cationic surfactants) along with four different beers (Guinness Original, Banks’s Bitter, Bass No 1 and Harvest Pale). We found that hydrophilic vials increased the foamability only for the three model systems but increased foam stability for all foams except the model cationic system. We then compared stability of beer foams produced by shaking and pouring and demonstrated weak qualitative agreement between both foam methods. We also showed how wettability of the glass controls bubble nucleation for beers and champagne and used this effect to control exactly where bubbles form using simple wettability patterns

Serum-Dependence of Affinity-Mediated VEGF Release from Biomimetic Microspheres

Vascular endothelial growth factor (VEGF) activity is highly regulated via sequestering within the ECM and cell-demanded proteolysis to release the sequestered VEGF. Numerous studies have demonstrated that VEGF activity mediates cellular events leading to angiogenesis and capillary formation in vivo. This has motivated the study of biomaterials to sustain VEGF release, and in many cases, the materials are inspired by the structure and function of the native ECM. However, there remains a need for materials that can bind to VEGF with high specificity, as the in vivo environment is rich in a variety of growth factors (GFs) and GF-binding moieties. Here we describe a strategy to control VEGF release using hydrogel microspheres with tethered peptides derived from VEGF receptor 2 (VEGFR2). Using biomaterials covalently modified with varying concentrations of two distinct VEGFR2-derived peptides with varying serum stability, we analyzed both biomaterial and environmental variables that influence VEGF release and activity. The presence of tethered VEGF-binding peptides (VBPs) resulted in significantly extended VEGF release relative to control conditions, and the resulting released VEGF significantly increased the expansion of human umbilical vein endothelial cells in culture. VEGF release rates were also strongly influenced by the concentration of serum. The presence of Feline McDonough Sarcoma-like tyrosine kinase 1 (sFlt-1), a serum-borne receptor fragment derived from VEGF receptor 1, increased VEGF release rates, although sFlt-1 was not sufficient to recapitulate the release profile of VEGF in serum. Further, the influence of serum on VEGF release was not due to protease activity or nonspecific VEGF interactions in the presence of serum-borne heparin. VEGF release kinetics correlated well with a generalizable mathematical model describing affinity-mediated release of VEGF from hydrogel microspheres in defined conditions. Modeling results suggest a potential mechanism whereby competition between VEGF and multiple VEGF-binding serum proteins including sFlt-1, soluble kinase insert domain receptor (sKDR), and α2-macroglobulin (α2-M) likely influenced VEGF release from microspheres. The materials and mathematical model described in this approach may be useful in a range of applications in which sustained, biologically active GF release of a specific GF is desirable

Engineering human cell spheroids to model embryonic tissue fusion <i>in vitro</i>

<div><p>Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human <i>in vitro</i> assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior <i>in vitro</i> using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.</p></div

Characterization of HWJSC spheroids and time-course of osteogenic differentiation.

<p>A: Schematic of HWJSC spheroid generation in agarose microwells, generated using AggreWell 800 as a template. B: Data representing the mean spheroid diameter of HWJSC spheroids at three different initial seeding densities (1k, 2k, 3k HWJSCs per spheroid) and in either osteogenic differentiation medium (OM) or growth medium (GM). HWJSCs were seeded in GM on day 0 and were subsequently cultured at day 1 and beyond in either OM or GM for the remainder of the culture (with medium changes every 2–3 days). HWJSC spheroid mean diameter ± 95% confidence interval was calculated for 40 spheroids per condition per time point from one representative experiment. C: Quantified mean intensity of 2k HWJSC spheroids cultured in OM and stained using an alkaline phosphatase substrate kit. Data are presented as the mean fluorescence intensity ± SEM for three independent experiments, normalized to the day 1 time point. Asterisks denote statistical significance calculated on raw mean intensity values at α = 0.05 of each condition relative to the day 1 and day 4 time points (*) or relative to the day 7 and day 14 time points (**) using two-way ANOVA and Tukey’s post-hoc test. D: Fluorescent micrographs representing maximum intensity projections of HWJSC spheroids that were stained for alkaline phosphatase activity at the indicated time point after the initiation of spheroid culture. Scale bar represents 200 μm. E-J: Fold change in gene expression over time of 2k HWJSC spheroids cultured in OM. Data represent fold change relative to GAPDH housekeeping gene and the day 1time point for MYC (E), PPARG (F), CD44 (G), VIM (H), BGLAP (I), and RUNX2 (J). Asterisks denote statistical significance relative to a mean value of ‘1’ using a one-tailed Student’s t-test (α = 0.05) for three independent experiments.</p