Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, ...current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.
Cardiovascular defects, injuries, and degenerative diseases often require surgical intervention and the use of implantable replacement material and conduits. Traditional vascular grafts made of ...synthetic polymers, animal and cadaveric tissues, or autologous vasculature have been utilized for almost a century with well-characterized outcomes, leaving areas of unmet need for the patients in terms of durability and long-term patency, susceptibility to infection, immunogenicity associated with the risk of rejection, and inflammation and mechanical failure. Research to address these limitations is exploring avenues as diverse as gene therapy, cell therapy, cell reprogramming, and bioengineering of human tissue and replacement organs. Tissue-engineered vascular conduits, either with viable autologous cells or decellularized, are the forefront of technology in cardiovascular reconstruction and offer many benefits over traditional graft materials, particularly in the potential for the implanted material to be adopted and remodeled into host tissue and thus offer safer, more durable performance. This review discusses the key advances and future directions in the field of surgical vascular repair, replacement, and reconstruction, with a focus on the challenges and expected benefits of bioengineering human tissues and blood vessels.
Vascular smooth muscle cells (VSMCs) can be derived in large numbers from human induced pluripotent stem cells (hiPSCs) for producing tissue-engineered vascular grafts (TEVGs). However, hiPSC-derived ...TEVGs are hampered by low mechanical strength and significant radial dilation after implantation. Here, we report generation of hiPSC-derived TEVGs with mechanical strength comparable to native vessels used in arterial bypass grafts by utilizing biodegradable scaffolds, incremental pulsatile stretching, and optimal culture conditions. Following implantation into a rat aortic model, hiPSC-derived TEVGs show excellent patency without luminal dilation and effectively maintain mechanical and contractile function. This study provides a foundation for future production of non-immunogenic, cellularized hiPSC-derived TEVGs composed of allogenic vascular cells, potentially serving needs to a considerable number of patients whose dysfunctional vascular cells preclude TEVG generation via other methods.
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•Functional VSMCs could be efficiently generated on a large scale from hiPSCs•Optimized biochemical and biophysical conditions were used to generate hiPSC-TEVGs•hiPSC-TEVGs presented mechanical strength comparable to that of saphenous veins•hiPSC-TEVGs maintained patency and mechanical function following rat implantation
Luo et al. generated tissue-engineered vascular grafts (TEVGs) using human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells. These hiPSC-derived TEVGs displayed mechanical strength comparable to that of native vessels used clinically as vascular grafts and maintained excellent patency and mechanical function following implantation into a rat model.
Abstract The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand, FasL. Although Fas is highly expressed in cancer cells, insufficient cell surface Fas expression ...desensitizes cancer cells to Fas-induced apoptosis. Here, we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor, fasudil, that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL), fasudil promoted cancer cell apoptosis, but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis.
Abstract Computational models have the potential to provide precise estimates of stresses and strains associated with sites of coronary plaque rupture. However, lack of adequate mathematical ...description of diseased human vessel wall mechanical properties is hindering computational accuracy. The goal of this study is to characterize the behavior of diseased human coronary and carotid arteries using planar biaxial testing. Diseased coronary specimens exhibit relatively high stiffness (50–210 kPa) and low extensibility (1–10%) at maximum equibiaxial stress (250 kPa) compared to human carotid specimens and values commonly reported for porcine coronary arteries. A thick neointimal layer observed histologically appears to be associated with heightened stiffness and the direction of anisotropy of the specimens. Fung, Choi–Vito and modified Mooney–Rivlin constitutive equations fit the multiaxial data from multiple stress protocols well, and parameters from representative coronary specimens were utilized in a finite element model with fluid–solid interactions. Computed locations of maximal stress and strain are substantially altered, and magnitudes of maximum principal stress (48–65 kPa) and strain (6.5–8%) in the vessel wall are lower than previously predicted using parameters from uniaxial tests. Taken together, the results demonstrate the importance of utilizing disease-matched multiaxial constitutive relationships within patient-specific computational models to accurately predict stress and strain within diseased coronary arteries.
Objective:
Invasive coronary interventions can fail due to intimal hyperplasia and restenosis. Endothelial cell (EC) seeding to the vessel lumen, accelerating re-endothelialization, or local release ...of mTOR pathway inhibitors have helped reduce intimal hyperplasia after vessel injury. While animal models are powerful tools, they are complex and expensive, and not always reflective of human physiology. Therefore, we developed an in vitro 3D vascular model validating previous in vivo animal models and utilizing isolated human arteries to study vascular remodeling after injury. Approach: We utilized a bioreactor that enables the control of intramural pressure and shear stress in vessel conduits to investigate the vascular response in both rat and human arteries to intraluminal injury.
Results:
Culturing rat aorta segments in vitro, we show that vigorous removal of luminal ECs results in vessel injury, causing medial proliferation by Day-4 and neointima formation, with the observation of SCA1+ cells (stem cell antigen-1) in the intima by Day-7, in the absence of flow. Conversely, when endothelial-denuded rat aortae and human umbilical arteries were subjected to arterial shear stress, pre-seeding with human umbilical ECs decreased the number and proliferation of smooth muscle cell (SMC) significantly in the media of both rat and human vessels.
Conclusion:
Our bioreactor system provides a novel platform for correlating ex vivo findings with vascular outcomes in vivo. The present in vitro human arterial injury model can be helpful in the study of EC-SMC interactions and vascular remodeling, by allowing for the separation of mechanical, cellular, and soluble factors.
Novel biological vascular conduits, such as decellularized tissue engineered vascular grafts (TEVGs) are hindered by high thrombogenicity. To mimic the antithrombogenic surface of native vessels with ...a continuous glycosaminoglycan layer that is present on endothelial cells (ECs), a hyaluronic acid (HA) modified surface is established, to effectively shield blood platelets from collagen‐triggered activation. Using the amine groups present on 4 mm diameter decellularized TEVGs, a continuous HA hydrogel coating is built via a bifunctional thiol‐reactive cross‐linker, thereby avoiding nonspecific collagen matrix cross‐linking. The HA hydrogel layer recreates a luminal wall, “hiding” exposed collagen from the bloodstream. In vitro blood tests show that adhered platelets, fibrinogen absorption, and fibrin formation on HA‐coated decellularized TEVGs are significantly lower than on uncoated decellularized TEVGs. The HA surface also inhibits macrophage adhesion in vitro. HA‐coated decellularized syngeneic rat aortae (≈1.5 mm diameter), and TEVGs in rat and canine models, respectively, are protected from aggressive thrombus formation, and preserve normal blood flow. Re‐endothelialization is also observed. HA‐coated TEVGs may be an off‐the‐shelf small‐diameter vascular graft with dual benefits: antithrombogenic protection and promotion of endothelium.
To reduce thrombosis risk on small diameter (<6 mm) vascular grafts, hyaluronic acid is coated on the lumen of decellularized tissue engineered vascular grafts. This layer effectively shields platelets from collagen‐triggered activation, while allowing endothelial repopulation over time in vivo. Hence, hyaluronic acid–coated grafts may be an off‐the‐shelf small‐diameter vascular graft with dual benefits: antithrombogenic protection and promotion of endothelium.
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Myofibroblasts are critical for connective tissue remodeling and wound healing since they can close wound beds and shape tissues rapidly by generating high traction forces and ...secreting abundant extracellular matrix proteins and matrix metalloproteinases. However, their presence in excessive numbers is associated with fibrotic and calcific diseases and tissue thickening in engineered tissues. While activation of the myofibroblast phenotype has been studied extensively, whether myofibroblasts are “cleared” by phenotypic reversal or by apoptosis remains controversial. The goal of this work is to test the hypothesis that mechanical inhibition of myofibroblast force generation leads to de-differentiation or apoptosis depending upon the magnitude of the decrease in tension. To test this hypothesis, we cultured valvular interstitial cells (VICs) in fibrin micro-tissues suspended between flexible posts and dynamically altered the ability of the cells to generate tension by altering boundary stiffness via magnetic forces applied to posts. The flexible posts capped with magnetic beads enable the measurement and modulation of tension generated by the cells within the tissue. As expected, the cell-generated forces were elevated with dynamically increased boundary (post) stiffness, yet surprisingly, the forces continued to increase following dynamic reduction of boundary stiffness back to baseline levels. Increased apoptosis and reduced α-SMA staining were observed with complete freeing of the tissues from the posts but not upon removal of the magnet, resulting in a twofold decrease in post stiffness. Together, these data indicate that an increase in myofibroblast force generation, even if modest and temporary (1day), can have lasting effects on myofibroblast persistence in tissues, and that a significant reduction in the ability of the cells to generate tension is required to trigger dedifferentiation and/or apoptosis. The ability to dedifferentiate myofibroblasts to a quiescent phenotype and to control the percentage of apoptosis would be of great benefit for therapeutic and tissue engineering applications.
Myofibroblasts play an important role in tissue remodeling and wound healing. However, excessive activation of this phenotype is associated with fibrotic diseases and scar formation. Being able to dedifferentiate these cells or controlling their clearance with apoptosis (programmed cell death) would be beneficial. It is known that releasing rigid tissue boundaries trigger apoptosis, while reducing the substrate stiffness can cause myofibroblast dedifferentiation. However, the mechanical tension was not quantified in any of the studies. Here we used micro-cantilever posts at tissue boundaries to measure tension and to regulate boundary stiffness in real time by pulling posts with magnets. We show that temporary stiffening of boundary causes irreversible myofibroblast activation and the magnitude of tension drop controls apoptosis.
Development of autologous tissue-engineered vascular constructs using vascular smooth muscle cells (VSMCs) derived from human induced pluripotent stem cells (iPSCs) holds great potential in treating ...patients with vascular disease. However, preclinical, large animal iPSC-based cellular and tissue models are required to evaluate safety and efficacy prior to clinical application. Herein, swine iPSC (siPSC) lines were established by introducing doxycycline-inducible reprogramming factors into fetal fibroblasts from a line of inbred Massachusetts General Hospital miniature swine that accept tissue and organ transplants without immunosuppression within the line. Highly enriched, functional VSMCs were derived from siPSCs based on addition of ascorbic acid and inactivation of reprogramming factor via doxycycline withdrawal. Moreover, siPSC-VSMCs seeded onto biodegradable polyglycolic acid (PGA) scaffolds readily formed vascular tissues, which were implanted subcutaneously into immunodeficient mice and showed further maturation revealed by expression of the mature VSMC marker, smooth muscle myosin heavy chain. Finally, using a robust cellular self-assembly approach, we developed 3D scaffold-free tissue rings from siPSC-VSMCs that showed comparable mechanical properties and contractile function to those developed from swine primary VSMCs. These engineered vascular constructs, prepared from doxycycline-inducible inbred siPSCs, offer new opportunities for preclinical investigation of autologous human iPSC-based vascular tissues for patient treatment.
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