Hydrogels to Recapture Extracellular Matrix Cues That Regulate Vascularization.

The ECM (extracellular matrix) is a 3-dimensional network that supports cellular responses and maintains structural tissue integrity in healthy and pathological conditions. The interactions between ECM and cells trigger signaling cascades that lead to phenotypic changes and structural and compositional turnover of the ECM, which in turn regulates vascular cell behavior. Hydrogel biomaterials are a powerful platform for basic and translational studies and clinical applications due to their high swelling capacity and exceptional versatility in compositions and properties. This review highlights recent developments and uses of engineered natural hydrogel platforms that mimic the ECM and present defined biochemical and mechanical cues for vascularization. Specifically, we focus on modulating vascular cell stimulation and cell-ECM/cell-cell interactions in the microvasculature that are the established biomimetic microenvironment.

Hypoxia-induced blood-brain barrier dysfunction is prevented by pericyte-conditioned media via attenuated actomyosin contractility and claudin-5 stabilization.

The blood-brain barrier (BBB) regulates molecular and cellular entry from the cerebrovasculature into the surrounding brain parenchyma. Many diseases of the brain are associated with dysfunction of the BBB, where hypoxia is a common stressor. However, the contribution of hypoxia to BBB dysfunction is challenging to study due to the complexity of the brain microenvironment. In this study, we used a BBB model with brain microvascular endothelial cells and pericytes differentiated from iPSCs to investigate the effect of hypoxia on barrier function. We found that hypoxia-induced barrier dysfunction is dependent upon increased actomyosin contractility and is associated with increased fibronectin fibrillogenesis. We propose a role for actomyosin contractility in mediating hypoxia-induced barrier dysfunction through modulation of junctional claudin-5. Our findings suggest pericytes may protect brain microvascular endothelial cells from hypoxic stresses and that pericyte-derived factors could be candidates for treatment of pathological barrier-forming tissues.

Regenerative and durable small-diameter graft as an arterial conduit.

Despite significant research efforts, clinical practice for arterial bypass surgery has been stagnant, and engineered grafts continue to face postimplantation challenges. Here, we describe the development and application of a durable small-diameter vascular graft with tailored regenerative capacity. We fabricated small-diameter vascular grafts by electrospinning fibrin tubes and poly(ε-caprolactone) fibrous sheaths, which improved suture retention strength and enabled long-term survival. Using surface topography in a hollow fibrin microfiber tube, we enable immediate, controlled perfusion and formation of a confluent endothelium within 3-4 days in vitro with human endothelial colony-forming cells, but a stable endothelium is noticeable at 4 weeks in vivo. Implantation of acellular or endothelialized fibrin grafts with an external ultrathin poly(ε-caprolactone) sheath as an interposition graft in the abdominal aorta of a severe combined immunodeficient Beige mouse model supports normal blood flow and vessel patency for 24 weeks. Mechanical properties of the implanted grafts closely approximate the native abdominal aorta properties after just 1 week in vivo. Fibrin mediated cellular remodeling, stable tunica intima and media formation, and abundant matrix deposition with organized collagen layers and wavy elastin lamellae. Endothelialized grafts evidenced controlled healthy remodeling with delayed and reduced macrophage infiltration alongside neo vasa vasorum-like structure formation, reduced calcification, and accelerated tunica media formation. Our studies establish a small-diameter graft that is fabricated in less than 1 week, mediates neotissue formation and incorporation into the native tissue, and matches the native vessel size and mechanical properties, overcoming main challenges in arterial bypass surgery.

Tissue Engineering Approaches to Uncover Therapeutic Targets for Endothelial Dysfunction in Pathological Microenvironments.

Endothelial cell dysfunction plays a central role in many pathologies, rendering it crucial to understand the underlying mechanism for potential therapeutics. Tissue engineering offers opportunities for in vitro studies of endothelial dysfunction in pathological mimicry environments. Here, we begin by analyzing hydrogel biomaterials as a platform for understanding the roles of the extracellular matrix and hypoxia in vascular formation. We next examine how three-dimensional bioprinting has been applied to recapitulate healthy and diseased tissue constructs in a highly controllable and patient-specific manner. Similarly, studies have utilized organs-on-a-chip technology to understand endothelial dysfunction's contribution to pathologies in tissue-specific cellular components under well-controlled physicochemical cues. Finally, we consider studies using the in vitro construction of multicellular blood vessels, termed tissue-engineered blood vessels, and the spontaneous assembly of microvascular networks in organoids to delineate pathological endothelial dysfunction.

Cytoskeletal tension regulates mesodermal spatial organization and subsequent vascular fate.

Morphogenesis during human development relies on the interplay between physiochemical cues that are mediated in part by cellular density and cytoskeletal tension. Here, we interrogated these factors on vascular lineage specification during human-induced pluripotent stem-cell (hiPSC) fate decision. We found that independent of chemical cues, spatially presented physical cues induce the self-organization of Brachyury-positive mesodermal cells, in a RhoA/Rho-associated kinase (ROCK)-dependent manner. Using unbiased support vector machine (SVM) learning, we found that density alone is sufficient to predict mesodermal fate. Furthermore, the long-withstanding presentation of spatial confinement during hiPSC differentiation led to an organized vascular tissue, reminiscent of native blood vessels, a process dependent on cell density as found by SVM analysis. Collectively, these results show how tension and density relate to vascular identity mirroring early morphogenesis. We propose that such a system can be applied to study other aspects of the stem-cell niche and its role in embryonic patterning.

Intratumoral oxygen gradients mediate sarcoma cell invasion.

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm(3)) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

Bioinspired Hydrogels to Engineer Cancer Microenvironments.

Recent research has demonstrated that tumor microenvironments play pivotal roles in tumor development and metastasis through various physical, chemical, and biological factors, including extracellular matrix (ECM) composition, matrix remodeling, oxygen tension, pH, cytokines, and matrix stiffness. An emerging trend in cancer research involves the creation of engineered three-dimensional tumor models using bioinspired hydrogels that accurately recapitulate the native tumor microenvironment. With recent advances in materials engineering, many researchers are developing engineered tumor models, which are promising platforms for the study of cancer biology and for screening of therapeutic agents for better clinical outcomes. In this review, we discuss the development and use of polymeric hydrogel materials to engineer native tumor ECMs for cancer research, focusing on emerging technologies in cancer engineering that aim to accelerate clinical outcomes.

Pericytes Derived from Human Pluripotent Stem Cells.

Pericytes wrap blood microvessels and are believed to play important roles in vascular morphogenesis, maturation, and stability. In addition, pericytes have emerged as candidates for targeting cancer growth and for wound healing. In order to model these processes and test new therapies, it is desirable to have a reliable, scalable source of pericytes. Human pluripotent stem cells (hPSCs), which possess the ability to differentiate into any cell type in the body, have been used to generate pericytes in vitro quickly, consistently, and with high yields. In this chapter, we consider the differentiation of pericytes from hPSCs. We compare the approaches taken by multiple groups and discuss characterization of hPSC-pericytes. Studying pericyte differentiation in vitro provides the opportunity to identify factors influencing pericyte development and to establish the ontogenic relationships between pericytes and similar cells. The development of highly specific, defined pericyte populations from hPSCs will enable downstream applications requiring large quantities of cells, including tissue engineered models and cell therapies.

Deregulation of the Hippo pathway in soft-tissue sarcoma promotes FOXM1 expression and tumorigenesis.

Genetic aberrations responsible for soft-tissue sarcoma formation in adults are largely unknown, with targeted therapies sorely needed for this complex and heterogeneous family of diseases. Here we report that that the Hippo pathway is deregulated in many soft-tissue sarcomas, resulting in elevated expression of the effector molecule Yes-Associated Protein (YAP). Based on data gathered from human sarcoma patients, a novel autochthonous mouse model, and mechanistic analyses, we determined that YAP-dependent expression of the transcription factor forkhead box M1 (FOXM1) is necessary for cell proliferation/tumorigenesis in a subset of soft-tissue sarcomas. Notably, FOXM1 directly interacts with the YAP transcriptional complex via TEAD1, resulting in coregulation of numerous critical pro-proliferation targets that enhance sarcoma progression. Finally, pharmacologic inhibition of FOXM1 decreases tumor size in vivo, making FOXM1 an attractive therapeutic target for the treatment of some sarcoma subtypes.

Harnessing developmental processes for vascular engineering and regeneration.

The formation of vasculature is essential for tissue maintenance and regeneration. During development, the vasculature forms via the dual processes of vasculogenesis and angiogenesis, and is regulated at multiple levels: from transcriptional hierarchies and protein interactions to inputs from the extracellular environment. Understanding how vascular formation is coordinated in vivo can offer valuable insights into engineering approaches for therapeutic vascularization and angiogenesis, whether by creating new vasculature in vitro or by stimulating neovascularization in vivo. In this Review, we will discuss how the process of vascular development can be used to guide approaches to engineering vasculature. Specifically, we will focus on some of the recently reported approaches to stimulate therapeutic angiogenesis by recreating the embryonic vascular microenvironment using biomaterials for vascular engineering and regeneration.

Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering.

Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine - including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering - have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies.

Fabrication of 3-dimensional multicellular microvascular structures.

Despite current advances in engineering blood vessels over 1 mm in diameter and the existing wealth of knowledge regarding capillary bed formation, studies for the development of microvasculature, the connecting bridge between them, have been extremely limited so far. Here, we evaluate the use of 3-dimensional (3D) microfibers fabricated by hydrogel electrospinning as templates for microvascular structure formation. We hypothesize that 3D microfibers improve extracellular matrix (ECM) deposition from vascular cells, enabling the formation of freestanding luminal multicellular microvasculature. Compared to 2-dimensional cultures, we demonstrate with confocal microscopy and RT-PCR that fibrin microfibers induce an increased ECM protein deposition by vascular cells, specifically endothelial colony-forming cells, pericytes, and vascular smooth muscle cells. These ECM proteins comprise different layers of the vascular wall including collagen types I, III, and IV, as well as elastin, fibronectin, and laminin. We further demonstrate the achievement of multicellular microvascular structures with an organized endothelium and a robust multicellular perivascular tunica media. This, along with the increased ECM deposition, allowed for the creation of self-supporting multilayered microvasculature with a distinct circular lumen following fibrin microfiber core removal. This approach presents an advancement toward the development of human microvasculature for basic and translational studies.

Diffusing Colloidal Probes of kT-Scale Biomaterial-Cell Interactions.

In the optimization of applied biomaterials, measurements of their interactions with cell surfaces are important to understand their influence on specific and nonspecific cell surface adhesion, internalization pathways, and toxicity. In this study, a novel approach using dark field video microscopy with combined real-time particle and cell tracking allows the trajectories of biomaterial-coated colloids to be monitored in relation to their distance from cell perimeters. Dynamic and statistical mechanical analyses enable direct measurement of colloid-cell surface association lifetimes and interaction potentials mediated by biomaterials. Our analyses of colloidal transport showed polyethylene glycol (PEG) and bovine serum albumin (BSA) lead to net repulsive interactions with cell surfaces, while dextran and hyaluronic acid (HA) lead to reversible and irreversible association to the cell surface, respectively. Our results demonstrate how diffusing colloidal probes can be used for nonobtrusive, sensitive measurements of biomaterial-cell surface interactions important to therapeutics, diagnostics, and tissue engineering.

Three-Dimensional Vascular Network Assembly From Diabetic Patient-Derived Induced Pluripotent Stem Cells.

OBJECTIVE: In diabetics, hyperglycemia results in deficient endothelial progenitors and cells, leading to cardiovascular complications. We aim to engineer 3-dimensional (3D) vascular networks in synthetic hydrogels from type 1 diabetes mellitus (T1D) patient-derived human-induced pluripotent stem cells (hiPSCs), to serve as a transformative autologous vascular therapy for diabetic patients. APPROACH AND RESULTS: We validated and optimized an adherent, feeder-free differentiation procedure to derive early vascular cells (EVCs) with high portions of vascular endothelial cadherin-positive cells from hiPSCs. We demonstrate similar differentiation efficiency from hiPSCs derived from healthy donor and patients with T1D. T1D-hiPSC-derived vascular endothelial cadherin-positive cells can mature to functional endothelial cells-expressing mature markers: von Willebrand factor and endothelial nitric oxide synthase are capable of lectin binding and acetylated low-density lipoprotein uptake, form cords in Matrigel and respond to tumor necrosis factor-α. When embedded in engineered hyaluronic acid hydrogels, T1D-EVCs undergo morphogenesis and assemble into 3D networks. When encapsulated in a novel hypoxia-inducible hydrogel, T1D-EVCs respond to low oxygen and form 3D networks. As xenografts, T1D-EVCs incorporate into developing zebrafish vasculature. CONCLUSIONS: Using our robust protocol, we can direct efficient differentiation of T1D-hiPSC to EVCs. Early endothelial cells derived from T1D-hiPSC are functional when mature. T1D-EVCs self-assembled into 3D networks when embedded in hyaluronic acid and hypoxia-inducible hydrogels. The capability of T1D-EVCs to assemble into 3D networks in engineered matrices and to respond to a hypoxic microenvironment is a significant advancement for autologous vascular therapy in diabetic patients and has broad importance for tissue engineering.

Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix.

The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.

Biomechanical strain induces elastin and collagen production in human pluripotent stem cell-derived vascular smooth muscle cells.

Blood vessels are subjected to numerous biomechanical forces that work harmoniously but, when unbalanced because of vascular smooth muscle cell (vSMC) dysfunction, can trigger a wide range of ailments such as cerebrovascular, peripheral artery, and coronary artery diseases. Human pluripotent stem cells (hPSCs) serve as useful therapeutic tools that may help provide insight on the effect that such biomechanical stimuli have on vSMC function and differentiation. In this study, we aimed to examine the effect of biomechanical strain on vSMCs derived from hPSCs. The effects of two types of tensile strain on hPSC-vSMC derivatives at different stages of differentiation were examined. The derivatives included smooth muscle-like cells (SMLCs), mature SMLCs, and contractile vSMCs. All vSMC derivatives aligned perpendicularly to the direction of cyclic uniaxial strain. Serum deprivation and short-term uniaxial strain had a synergistic effect in enhancing collagen type I, fibronectin, and elastin gene expression. Furthermore, long-term uniaxial strain deterred collagen type III gene expression, whereas long-term circumferential strain upregulated both collagen type III and elastin gene expression. Finally, long-term uniaxial strain downregulated extracellular matrix (ECM) expression in more mature vSMC derivatives while upregulating elastin in less mature vSMC derivatives. Overall, our findings suggest that in vitro application of both cyclic uniaxial and circumferential tensile strain on hPSC-vSMC derivatives induces cell alignment and affects ECM gene expression. Therefore, mechanical stimulation of hPSC-vSMC derivatives using tensile strain may be important in modulating the phenotype and thus the function of vSMCs in tissue-engineered vessels.

Low oxygen tension enhances endothelial fate of human pluripotent stem cells.

OBJECTIVE: A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. APPROACH AND RESULTS: We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor ß+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. CONCLUSIONS: Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.

The design of dextran-based hypoxia-inducible hydrogels via in situ oxygen-consuming reaction.

Hypoxia plays a critical role in the development and wound healing process, as well as a number of pathological conditions. Here, dextran-based hypoxia-inducible (Dex-HI) hydrogels formed with in situ oxygen consumption via a laccase-medicated reaction are reported. Oxygen levels and gradients were accurately predicted by mathematical simulation. It is demonstrated that Dex-HI hydrogels provide prolonged hypoxic conditions up to 12 h. The Dex-HI hydrogel offers an innovative approach to delineate not only the mechanism by which hypoxia regulates cellular responses, but may facilitate the discovery of new pathways involved in the generation of hypoxic and oxygen gradient environments.

Dextran hydrogel scaffolds enhance angiogenic responses and promote complete skin regeneration during burn wound healing.

Neovascularization is a critical determinant of wound-healing outcomes for deep burn injuries. We hypothesize that dextran-based hydrogels can serve as instructive scaffolds to promote neovascularization and skin regeneration in third-degree burn wounds. Dextran hydrogels are soft and pliable, offering opportunities to improve the management of burn wound treatment. We first developed a procedure to treat burn wounds on mice with dextran hydrogels. In this procedure, we followed clinical practice of wound excision to remove full-thickness burned skin, and then covered the wound with the dextran hydrogel and a dressing layer. Our procedure allows the hydrogel to remain intact and securely in place during the entire healing period, thus offering opportunities to simplify the management of burn wound treatment. A 3-week comparative study indicated that dextran hydrogel promoted dermal regeneration with complete skin appendages. The hydrogel scaffold facilitated early inflammatory cell infiltration that led to its rapid degradation, promoting the infiltration of angiogenic cells into the healing wounds. Endothelial cells homed into the hydrogel scaffolds to enable neovascularization by day 7, resulting in an increased blood flow significantly greater than treated and untreated controls. By day 21, burn wounds treated with hydrogel developed a mature epithelial structure with hair follicles and sebaceous glands. After 5 weeks of treatment, the hydrogel scaffolds promoted new hair growth and epidermal morphology and thickness similar to normal mouse skin. Collectively, our evidence shows that customized dextran-based hydrogel alone, with no additional growth factors, cytokines, or cells, promoted remarkable neovascularization and skin regeneration and may lead to novel treatments for dermal wounds.