Biologically Functionalized Expanded Polytetrafluoroethylene Blood Vessel Grafts.

Cardiovascular diseases plague human health because of the lack of transplantable small-diameter blood vessel (SDBV) grafts. Although expanded polytetrafluoroethylene (ePTFE) has the potential to be used as a biocompatible material for SDBV grafts, long-term patency is still the biggest challenge. As discussed in this paper, by virtue of a novel material formulation and a new and benign alcohol/water lubricating agent, biofunctionalized ePTFE blood vessel grafts aimed at providing long-term patency were fabricated. Compared to the most prevalent modification of PTFE, namely surface treatment, this method realized bulk treatment, which could guarantee homogeneous and long-lasting performance throughout PTFE products. These blood vessel grafts included embedded functional biomolecules, such as arginylglycylaspartic acid, heparin, and selenocystamine, using water as a solvent in paste extrusion and in the expansion of ePTFE. Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscope results confirmed the existence of these targeting biomolecules in the as-fabricated ePTFE blood vessel grafts. Meanwhile, the greatly improved biological functions of the grafts were demonstrated via live and dead assays, cell morphology, CD31 staining, nitric oxide (NO) release, and anticoagulation tests. This novel and benign material formulation and fabrication method provides an opportunity to produce multibiofunctional ePTFE blood vessel grafts in a single step, thus yielding a potent product with significant commercial and clinical potential.

Synthetic genome readers target clustered binding sites across diverse chromatin states.

Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to "read" specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.

Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.

Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.

Chemically defined conditions for human iPSC derivation and culture.

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.

Down-regulation of argininosuccinate synthetase is associated with cisplatin resistance in hepatocellular carcinoma cell lines: implications for PEGylated arginine deiminase combination therapy.

BACKGROUND: Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20. METHODS: ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR. RESULTS: A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied. CONCLUSION: ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC.

Surface modification of polytetrafluoroethylene (PTFE) with a heparin-immobilized extracellular matrix (ECM) coating for small-diameter vascular grafts applications.

Intimal hyperplasia, thrombosis formation, and delayed endothelium regeneration are the main causes that restrict the clinical applications of PTFE small-diameter vascular grafts (inner diameter < 6 mm). An ideal strategy to solve such problems is to facilitate in situ endothelialization. Since the natural vascular endothelium adheres onto the basement membrane, which is a specialized form of extracellular matrix (ECM) secreted by endothelial cells (ECs) and smooth muscle cells (SMCs), functionalizing PTFE with an ECM coating was proposed. However, besides ECs, the ECM-modified PTFE improved SMC growth as well, thereby increasing the risk of intimal hyperplasia. In the present study, heparin was immobilized on the ECM coating at different densities (4.89 ± 1.02 µg/cm2, 7.24 ± 1.56 µg/cm2, 15.63 ± 2.45 µg/cm2, and 26.59 ± 3.48 µg/cm2), aiming to develop a bio-favorable environment that possessed excellent hemocompatibility and selectively inhibited SMC growth while promoting endothelialization. The results indicated that a low heparin density (4.89 ± 1.02 µg/cm2) was not enough to restrict platelet adhesion, whereas a high heparin density (26.59 ± 3.48 µg/cm2) resulted in decreased EC growth and enhanced SMC proliferation. Therefore, a heparin density at 7.24 ± 1.56 µg/cm2 was the optimal level in terms of antithrombogenicity, endothelialization, and SMC inhibition. Collectively, this study proposed a heparin-immobilized ECM coating to modify PTFE, offering a promising means to functionalize biomaterials for developing small-diameter vascular grafts.

Expanded Poly(tetrafluoroethylene) Blood Vessel Grafts with Embedded Reactive Oxygen Species (ROS)-Responsive Antithrombogenic Drug for Elimination of Thrombosis.

Treatment of cardiovascular diseases suffers from the lack of transplantable small-diameter blood vessel (SDBV) grafts that can prohibit/eliminate thrombosis. Although expanded poly(tetrafluoroethylene) (ePTFE) has the potential to be used for SDBV grafts, recurrence of thrombus remains the biggest challenge. In this study, a reactive oxygen species (ROS)-responsive antithrombogenic drug synthesis and a bulk coating process were employed to fabricate functional ePTFE grafts capable of prohibiting/eliminating blood clots. The synthesized drug that would release antiplatelet ethyl salicylate (ESA), in responding to ROS, was dissolved in a polycaprolactone (PCL) solution, followed by a bulk coating of the as-fabricated ePTFE grafts with the PCL/drug solution. Nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) were employed to investigate and confirm the synthesis and presence of the ROS-responsive drug in the ePTFE grafts. The ESA release functions were demonstrated via the drug-release profile and dynamic anticoagulation tests. The biocompatibility of the ROS-responsive ePTFE grafts was demonstrated via lactate dehydrogenase (LDH) cytotoxicity assays, live and dead cell assays, cell morphology, and cell-graft interactions. The ROS-responsive, antithrombogenic ePTFE grafts provide a feasible way for maintaining long-term patency, potentially solving a critical challenge in SDBV applications.

Fabrication and modification of wavy multicomponent vascular grafts with biomimetic mechanical properties, antithrombogenicity, and enhanced endothelial cell affinity.

A mismatch of mechanical properties and a high rate of thromboses are two critical challenges of creating viable artificial small-diameter vascular grafts (SDVGs). Herein, we propose a method to fabricate wavy multicomponent vascular grafts (WMVGs) via electrospinning using an assembled rotating collector. The WMVGs consisted of a wavy silk/poly(lactic acid) (PLA) inner layer and a thermoplastic polyurethane (TPU) outer layer, which mimic the structures and properties of collagen and elastin in native blood vessels, respectively. Attributed to the wavy structure and the combination of rigid silk/PLA and elastic TPU biomaterials, WMVGs are capable of mimicking the nonlinear tensile stress-strain relationship and "toe region" of native blood vessels. In addition, they have sufficient mechanical strength to meet implantation requirements in terms of tensile strength, suture retention, and burst pressure. Further modification of silk/PLA fibers with dopamine and heparin gave the grafts antithrombogenic properties and greatly enhanced endothelial cell affinities. Human umbilical vein endothelial cells (HUVECs) cultured on modified silk/PLA showed high viability, high proliferation rate, and favorable cell-substrate interactions. Moreover, HUVECs were able to fully cover and freely migrate upward on the lumen of the modified WMVGs without needing a special circulation bioreactor. Therefore, the modified WMVGs possessed biomimetic properties, antithrombogenicity, and enhanced endothelialization, making them a promising candidate for SDVGs. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2397-2408, 2019.

Quantitative Label-Free Imaging of 3D Vascular Networks Self-Assembled in Synthetic Hydrogels.

Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell-derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues.

Development of biomimetic thermoplastic polyurethane/fibroin small-diameter vascular grafts via a novel electrospinning approach.

A new electrospinning approach for fabricating vascular grafts with a layered, circumferentially aligned, and micro-wavy fibrous structure similar to natural elastic tissues has been developed. The customized electrospinning collector was able to generate wavy fibers using the dynamic "jump rope" collecting process, which also solved the sample removal problem for mandrel-type collectors. In this study, natural silk fibroin and synthetic thermoplastic polyurethane (TPU) were combined at different weight ratios to produce hybrid small-diameter vascular grafts. The purpose of combining these two materials was to leverage the bioactivity and tunable mechanical properties of these natural and synthetic materials. Results showed that the electrospun fiber morphology was highly influenced by the material compositions and solvents employed. All of the TPU/fibroin hybrid grafts had mechanical properties comparable to natural blood vessels. The circumferentially aligned and wavy biomimetic configuration provided the grafts with a sufficient toe region and the capacity for long-term usage under repeated dilatation and contraction. Cell culture tests with human endothelial cells (EC) also revealed high cell viability and good biocompatibility for these grafts. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 985-996, 2018.

A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture.

A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for "3D-like" vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function.

Uniform neural tissue models produced on synthetic hydrogels using standard culture techniques.

The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to in vivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.

Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin.

Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by "thiol-ene" photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol.

Phase I Trial of Arginine Deprivation Therapy with ADI-PEG 20 Plus Docetaxel in Patients with Advanced Malignant Solid Tumors.

PURPOSE: This phase I study examined the toxicity and tolerability of pegylated arginine deiminase (ADI-PEG 20) in combination with docetaxel in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: Eligible patients had histologically proven advanced solid malignancies, with any number of prior therapies, Zubrod performance status 0-2, and adequate organ function. Patients received ADI-PEG 20 weekly intramuscular injection ranging from 4.5 to 36 mg/m(2) and up to 10 doses of docetaxel (75 mg/m(2)) every 3 weeks. Primary endpoints were safety, toxicity, and a recommended phase II dose. Circulating arginine levels were measured before each cycle. Tumor response was measured as a secondary endpoint every 6 weeks on study. RESULTS: Eighteen patients received a total of 116 cycles of therapy through four dose levels of ADI-PEG 20. A single dose-limiting toxicity (grade 3 urticarial rash) was observed at the 1st dose level, with no additional dose-limiting toxicities observed. Hematologic toxicities were common with 14 patients experiencing at least one grade 3 to 4 leukopenia. Fatigue was the most prevalent toxicity reported by 16 patients. Arginine was variably suppressed with 10 patients achieving at least a 50% reduction in baseline values. In 14 patients with evaluable disease, four partial responses (including 2 patients with PSA response) were documented, and 7 patients had stable disease. CONCLUSIONS: ADI-PEG 20 demonstrated reasonable toxicity in combination with docetaxel. Promising clinical activity was noted, and expansion cohorts are now accruing for both castrate-resistant prostate cancer and non-small cell lung cancer at a recommended phase II dose of 36 mg/m(2).

Trophoblast differentiation in embryoid bodies derived from human embryonic stem cells.

Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

Arrays for the combinatorial exploration of cell adhesion.

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.

Functional endothelial cells derived from rhesus monkey embryonic stem cells.

We have used rhesus monkey embryonic stem (ES) cells to study endothelial cell development. Rhesus ES cells (R366.4 cell line) exposed to medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF) assumed a relatively uniform endothelial cell morphology and could be propagated and expanded with a consistent phenotype and normal karyotype. When placed in Matrigel, these rhesus ES cell-derived endothelial cells (RESDECs) formed capillary-like structures characteristic of endothelial cells. Immunohistochemical and flow cytometric analysis of RESDECs showed that they take up acetylated low-density lipoprotein (LDL), express CD146, von Willebrand factor, and the integrin alpha v beta 3, and bind the lectin ulex europaeus agglutinin-1. These cells also express the VEGF receptor Flk-1 and secrete VEGF. When introduced in a Matrigel plug implanted subcutaneously in mice, RESDECs formed intact vessels and recruited new endothelial cell growth. In vivo function was demonstrated by coinjection of RESDECs with murine tumor cells subcutaneously into immunocompromised adult mice. RESDECs injected alone did not form measurable tumors. Tumor cells grew more rapidly and had increased vascularization when coinjected with the RESDECs. Immunohistochemical staining demonstrated that the RESDECs participated in forming the tumor neovasculature. RESDECs provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.